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1 Colon cancer microenvironment that helps invasion and


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Colon cancer microenvironment that helps invasion and metastasis: studies using mouse
Makoto Mark Taketo1
1. Kyoto Univ. Grad. Sch. Medicine, Kyoto, Japan
Owing to early detection and intervention, most patients of early stage colon cancer are now cured completely. However, colon
cancer is still lethal for many patients whose disease has progressed to the metastatic stage. Colon cancer appears to progress
through a series of multi-step genetic/epigenetic changes via the adenoma-carcinoma sequence.
We earlier constructed ApcD716 knockout mice, a model for human FAP. Using the model, we found the polyp adenoma
morphogenetic mechanisms, and demonstrated the role of COX-2 in adenoma expansion. Importantly, we found that COX-2 is
expressed exclusively in the fibroblasts of tumor stroma rather than in the epithelial cells at this early stage of tumorigenesis.
To study the role of TGF-b signaling in colon cancer, we introduced a Smad4 knockout mutation into the ApcD716 knockout mice,
and found that their intestinal polyp adenomas become locally invasive adenocarcinomas. This was caused by recruitment of
bone marrow-derived CCR1+ cells that produce MMP9. Furthermore, essentially the same mechanism appears to help
disseminated colon cancer cells to colonize in the liver. Interestingly, treatment of the Apc/Smad4 mice with a CCR1 inhibitor
suppressed the metastatic colonization of colon cancer cells in the liver.
To investigate the mechanisms that stimulate metastasis by colon cancer epithelium, we have screened for and found a novel
metastasis suppressor Aes that has turned out to be an endogenous inhibitor of Notch signaling. In colon cancer tissues,
cancer cells express abundant Notch receptors whereas their ligands such as Jagged1 and Delta4 are expressed by the tumor
stroma on vascular endothelial cells, smooth muscle layers and infiltrating macrophages.
These results collectively indicate that from early stages of colon cancer progression, tumors expand, invade and metastasize
through the interactions with their microenvironment in multiple modes.


Targeting the HIF/VHL pathway therapeutically to treat metastatic disease
Amato Giaccia1 , Yu Maio1 , Errin Rankin1 , Katherin Fuh2 , Mihalis Kariolis3 , Jennifer Cochrane3
1. Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, United States
2. Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, United States
3. Department of Bioengineering, Stanford University School of Medicine, Stanford, CA, United States

The receptor tyrosine kinase AXL has recently been identified as a critical factor driving tumor cell invasion and migration. AXL
is the founding member of the TAM family of receptor tyrosine kinases, which include Tyro3 (or SKY), AXL, and MER. The
ligand growth arrest specific gene-6 (GAS6) is the common ligand for all three receptors. We recently discovered that AXL is a
hypoxia inducible gene and a HIF target gene. In addition, it is both a biomarker and genetic driver for several types of
metastatic cancer. While AXL plays an important biologic role in metastasis, there are currently no therapeutic agents directed
against GAS6/AXL signaling available that can be used to inhibit tumor progression and/or metastasis in clinical trials. We have
developed a novel screening strategy to identify inhibitors of the GAS/AXL signaling axis. This strategy takes advantage of the
fact that unlike other proto-oncogenic receptor tyrosine kinases (RTKs), AXL appears to be a suitable target for inhibition
through modulation of ligand binding, given the lack of compelling data for ligand independent activation in cancer. We have
engineered and produced wild type and ultra-high affinity soluble AXL FC-fusion proteins. In our studies, we demonstrate
increased thermostability, affinity for GAS6, and therapeutic efficacy in vivo of the engineered AXL-S-1 soluble receptor
compared to the wild type soluble AXL receptor. Furthermore, soluble AXL therapy has an added benefit over AXL monoclonal
antibodies in being able to block GAS6 activation of the AXL family receptor tyrosine kinases MER and TYRO3, and can be
engineered to increase its affinity and stability.


Targeting Src family kinases as novel therapies for breast cancer brain metastases
Dihua Yu1
1. The University of Texas MD Anderson Cancer Center, Houston, TX, United States
Among 1.3 million women diagnosed with breast cancer every year, about 10-16% develop brain metastases. Despite the
better control of primary tumor and improved overall survival of breast cancer patients, the incident of developing life-
threatening brain metastasis is increasing. At present, no effective drug treatment exists for patients with refractory breast
cancer metastatic to the brain. Therefore, novel and effective therapeutic approaches are urgently needed for this population.
Unfortunately, developing effective therapeutics for brain metastasis is largely hampered by a lack of in-depth understanding of
the basic mechanisms of brain metastasis. Here, we reveal a critical role of Src activation in promoting breast cancer brain
metastasis and tested Src-targeting combinatorial regimen for preventing and treating brain metastases. Src is hyper-activated
in brain-seeking human breast cancer cell lines and patients’ brain metastasis tumors. Mechanistically, Src activation in tumor
cells promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood-brain barrier. When
coupled with an EGFR/HER2-dual-targeting agent lapatinib, a clinically applicable Src-targeting exhibited a significant pre-
clinical efficacy in preventing the outgrowth of disseminated breast cancer cells through induction of cell cycle arrest. More
importantly, the combinatorial treatment also significantly inhibited established experimental brain metastases and prolonged
survival of metastases-bearing mice (p=0.004, log-rank survival test). Our results highlight a highly promising, clinically
applicable, Src-targeting regimen for breast cancer patients suffering brain metastasis. Ultimately, our findings could be
translated to clinical trials as new therapies for breast cancer patients with brain metastasis.
Siyuan Zhang1,2, Wen-Chien Huang1,2, Chenyu Zhang1, Frank J. Lowery1, Lin Zhang1, Patricia S. Steeg3, Dihua Yu1
1Departments of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030,
USA, 3Women’s Cancers Section, National Cancer Institute, USA, 2These authors contributed equally to this work.


Osteopontin: a promoter and a potential therapeutic target for hepatocellular carcinoma
Lun-Xiu Qin1
1. Liver Cancer Institute & Zhongshan Hospital, Fudan University, Shanghai, China

Osteopontin (OPN) is an extracellular matrix (ECM) protein that binds to αvβ integrins and receptors of CD44 family to
propagate cellular signals, and promotes induction of cell adhesion, chemotaxis, ECM degradation, angiogenesis, prevention of
apoptosis, and indolent tumor growth. Our previous gene expression profiling study found OPN was overexpressed in
metastatic HCC and identified OPN as one of the leading genes associated with metastasis of HCC.A significantly elevated
plasma OPN level was found in patients with HCC recurrence during the follow-up time compared with those without
recurrence, and was one of the leading independent prognostic factors for HCC patients, even in those with early stage of
HCC. Serum OPN levels significantly decrease after resection of HCCs. It could serve as a surrogate serologic biomarker for
monitoring treatment response and tumor recurrence after HCC resection, including those AFP-negative ones.
To determine the effects of genetic variants of OPN on the progression and prognosis of HCC, OPN promoter polymorphisms
were assessed in HCC tissues of 856 patients who underwent radical liver resection. Single nucleotide polymorphisms (SNPs)
were identified at four loci (-1748, -616, -443, and -155). Only SNPs at -443 and their related haplotypes (Ht2, Ht3) were
significantly associated with HCC prognosis. Ht2 and -443TT genotype could significantly increase the promoter transcriptional
activity and expression level of OPN compared with Ht3 or -443CC genotype, and lead to an obvious increase both tumor
growth and lung metastasis of HCC.We also used microarray-based comparative genomic hybridization (array-CGH) to identify
the difference of intratumor genomic aberrations between OPN-positive and OPN-negative HCC cells, and found that OPN-
positive HCC cells have much more amplifications of chromosomal regions including 4q13.1-q13.3, 4q21.23-q22.1, and
13q32.1-q32.3 compared with the OPN-negative ones.The abundance of clinical and experimental evidence regarding the link
between OPN and HCC metastasis makes OPN an attractive potential therapeutic target for combating HCC metastasis.
Based on both in vitro and in vivo investigations using antibody or Lentiviral-mediated miRNA against OPN, we found that OPN
plays an important role in metastasis as well as HCC tumor growth. Moreover, the necessary level of OPN for tumor growth is
much lower than that for HCC metastasis, and a very low level of OPN is sufficient for tumor growth. OPN might regulate tumor
growth through activation of the MAPK pathway. Furthermore, inducing the NF-kB translocation and the production/activation of
MMP-2 may be an important mechanism by which OPN is involved in HCC metastasis. The serine protease thrombin interacts
with OPN and can modify the biological activity of this molecule. We found that the thrombin level was strongly associated with
the metastatic potential of HCC cells, and that thrombin was remarkably overexpressed in HCC tissues, and associated with
tumor recurrence after HCC resection, particularly in those with elevated OPN levels. In vitro and in vivo assays demonstrated
that thrombin promotes the proliferation and pulmonary metastases of OPN+ HCC cells, which were significantly suppressed by
treatment with thrombin inhibitor. In conclusions: OPN is an attractive potential therapeutic target for combating HCC
metastasis, and thrombin may be an alternative target to inhibit HCC metastasis in OPN+ patients.


The role of RAD51 in the biology and therapeutic response of high-grade metastatic breast
Adrian P Wiegmans1 , Fares Al-Ejeh1 , Nicole Chee1 , Leonard DaSilva2 , Sarah Song2 , Georgia Chenevix-Trench1 ,
Robin Anderson3 , Peter Simpson2 , Sunil Lakhanni2 , Kum Kum Khanna1
1. Queensland Institute of Medical Research, Brisbane, QLD, Australia
2. UQ Centre for Clinical Research, University of Queensland, Brisbane, QLD, Australia
3. Metastasis Research, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
Among women with breast cancer, 30-40% will develop metastatic disease and only achieve an overall survival of less than 5
years. Despite new-targeted therapy, breast tumors that harbour similar histology or molecular phenotype differ in their
response to treatment. To uncover potential new therapeutic targets and improve outcome, we performed systematic analysis
of published gene expression profiling data to interrogate the expression of DNA repair genes in breast cancers. We found that
high expression of the homologous recombination protein, RAD51, was significantly associated with high-grade breast cancer,
aggressive subtypes and increased risk of metastasis, which was confirmed by immunohistochemistry. This provided a
rationale for targeting RAD51 in high-grade, therapy-resistant breast cancers. We report for the first time a preclinical
evaluation of RAD51 as a therapeutic target. In vitro that high RAD51 expressing cell lines were resistant to PARPi via
enhanced DNA repair capability, while knockdown of RAD51 reversed this resistance and repressed metastatic gene
expression. In-vivo, knockdown of RAD51 inhibited metastatic progression using a syngeneic breast cancer model and the
seeding of human xenografts to distant sites, including brain and lung. We are currently dissecting the molecular mechanisms
for RAD51-mediated pro-metastatic tumour growth that provide pre-clinical data demonstrating RAD51 as a new biomarker for
metastatic progression and targeted therapy against aggressive metastatic breast cancer.

EMT in the progression of carcinoma
Jean Paul Thiery1 2 3 , Virgile Viasnoff4 , Wilfried Engl4 , Sylvie Dufour5 , Wen Jing Sim2 , Kelvin Chua3 , Seiichi Mori3 ,
Ruby Huang3
1. National University, Singapore
2. Institute of Molecular and Cell Biology, Proteos, Republic of Singapore
3. Department of Biochemistry, Cancer Science Institute, A*STAR, Singapore, Republic of Singapore
4. National University of Singapore, Mechanobiology Institute, Singapore , Republic of Singapore
5. Institut Curie Paris, Paris, France
Epithelial-Mesenchymal Transition (EMT) is a fundamental mechanism governing morphogenesis, employing multiple signaling
pathways to shape the embryo. The intriguing possibility that similar mechanisms operate in carcinomas has been documented
repeatedly in experimental models, characterizing mesenchymal phenotypes in tumor subsets. The transition from an epithelial
carcinoma to a mesenchymal-like state is potentially associated with increased stemness, therapeutic resistance, and immune
escape. We shall first present recent data on the adhesive mechanisms involved in the maintenance of the epithelial state or
conferring mesenchymal-like phenotypes. We shall then discuss studies supporting the concept that EMT contributes to tumor
progression. In ovarian serous adenocarcinoma, two out of five molecular subtypes exhibit a strong EMT phenotype associated
with a significantly worse prognosis. We have also identified an EMT spectrum in ovarian carcinoma lines. Mesenchymal-like
cell lines exhibit enhanced invasive properties and clonogenicity as compared with epithelial-like cell lines. Our current strategy
is aimed at designing new therapeutic approaches that interfere with the plasticity of ovarian carcinoma cells using a
combination of drugs, with the ultimate hope that the reversal to a more epithelial phenotype will improve patient response to
conventional cytotoxics.


Cellular plasticity as an important mechanism of drug-resistance to cancer therapies
John Haley1 , S. Thomson1 , D. Epstein1 , E. Buck1 , J. Bean1 , E. Ullman1
1. OSI Pharmaceuticals LLC, Farmingdale, NY, United States
Cellular plasticity in epithelial cancers has been associated with progression and resistant to anti-cancer therapies. Several
forms of plasticity have been documented, including epithelial mesenchymal transition (EMT), endothelial-mesenchymal
transition and neuroendocrine-epithelial transition. Cellular plasticity plays a major role in the progression of cancer and the
acquisition of mesenchymal cancer stem cell-like phenotypes has been correlated with poor prognosis. The ability of cancer
cells to undergo an EMT has been implicated as a major factor driving metastasis, through the acquisition of enhanced
migratory and invasive properties. However it is also clear that by undergoing this process the cancer cells become resistant to
a number of targeted therapies. Recent retrospective analysis of phase 3 clinical trial samples has revealed that a poorer
response to erlotinib in the 2/3rd line setting in NSCLC was associated with a loss of E-cadherin (an epithelial tumor marker),
suggesting that tumors that had undergone EMT were less responsive to EGFR-directed therapy. In addition an EMT
phenotype has been reported in a number of EGFR-mutant NSCLC patients who have progressed while on erlotinib therapy.
In order to understand the full impact of these clinical observations and identify mechanisms of resistance in mesenchymal
tumor cells we have modeled EMT in a number of different ways in vitro. We have used panels of NSCLC cell lines that are in
a fixed epithelial or mesenchymal state, induced an EMT with TGFb, or driven an EMT through prolonged exposure to EGFR-
TKi targeted therapy (erlotinib). Using large-scale phosphoproteomic and transcriptomic datasets we used a systems biology
approach to uncover important observations relating to the role of EMT as a drug-resistance mechanism. Firstly, these models
confirm the clinical observations and show that tumor cells that have undergone EMT are less responsive to a number of
targeted agents including EGFR and IGF1R-IR directed agents. Secondly, they reveal the plasticity of the EMT process where
three distinct stages of EMT: epithelial, ‘metastable’ mesenchymal and ‘epigenetically-fixed’ mesenchymal are observed.
Thirdly, upon undergoing EMT tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial
counterparts. These include receptor tyrosine kinase (RTK) autocrine and paracrine loops, such as PDGFR, FGFR, AXL and
integrin a5b1 and up regulation of IL-6 and IL-11 mediated JAK-STAT signaling. Reciprocal activation of PDGFR signaling
through EGFR inhibition was observed in the mesenchymal state. Lastly, these models indicate that as part of the EMT process
the tumor cells display a CD44high/CD24low cancer stem cell phenotype and show enhanced colony formation.

These observations reinforce the important role that EMT can have in driving drug resistance in tumor cells and highlight the
wide diversity of mechanisms that can be used by tumor cells to evade targeted drug therapy. An understanding of these
mechanisms and the contexts in which they are most likely to arise will have important implications in driving combinatorial drug
therapy in cancer patients in the future.


ARHGAP8 is a marker of differentiated epithelium whose loss during breast cancer
progression is tightly linked to epithelial-to-mesenchymal transition (EMT), the claudin-low
molecular subtype, and a cancer stem cell phenotype
Cameron Johnstone1 2 3 , Catherin Q. Pan4 , Laura M Chang5 , Mark J Bowser5 , Sophie A Rich5 , Lewis A Chodosh5 ,
Philip Gregory6 , Anil K Rustgi5 , Boon Chuan Low4 , Robin L Anderson1 2 7
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Department of Pathology, University of Melbourne, Parkville, Vic, Australia
3. Department of Pharmacology, University of Melbourne, Parkville, VIC, Australia
4. Mechanobiology Institute, National University of Singapore, Singapore
5. GI Division, University of Pennsylvania, Philadelphia, PA, USA
6. Centre for Cancer Biology, SA Pathology, Adelaide, Australia
7. Sir Peter MacCallum Department of Oncology, University of Melbourne, VIC, Australia
ARHGAP8 encodes a GTPase activating protein (GAP) for RHO, RAC, and CDC42 in vitro, although the target(s) of its GAP
activity have not yet been defined in vivo. ARHGAP8 is structurally related to ARHGAP1 and they both contain a protein- and
lipid-interacting BCH domain at their N-termini, a central proline-rich SH3 binding motif, and a C-terminal GAP domain.1,2 The
gene encoding human ARHGAP8 resides on chromosome 22q13.31, within a region commonly deleted in breast cancer.3We
analyzed the ARHGAP8 gene sequence and expression in human and mouse breast tumors. While somatic mutations were
not found, mRNA levels were dramatically down-regulated in approximately 1/3 of human ductal carcinomas analyzed (n=31),
and in invasive breast carcinoma cell lines displaying epithelial-to-mesenchymal transition (EMT) including MDA-MB-231,
SUM159, Hs578T, and BT549. Unlike the situation in melanoma cells, a histone deacetylase inhibitor was more potent than
demethylating agents in inducing expression of ARHGAP8 in EMT-positive human breast carcinoma cell lines suggesting
silencing of ARHGAP8 expression may not occur through methylation in breast cancer. Expression of mouse Arhgap8 was
dramatically down-regulated in a transgenic mouse model of EMT in breast cancer.4 Finally, Arhgap8 was induced along with
other epithelial markers such as E-cadherin in 4T1.2 mouse breast cancer cells stably expressing miR-200b, a microRNA that
promotes the epithelial phenotype.5 Induction of EMT through short- or long-term treatment of NMuMG normal mouse
mammary epithelial cells with TGF-beta1 reduced Arhgap8 expression levels by 80%, whereas expression of the gene
encoding E-cadherin was completely silenced. The functional role of ARHGAP8 loss in breast cancer progression is now
being evaluated through overexpression or silencing in human breast cancer cell lines. Stable shRNA-mediated knockdown of
ARHGAP8 was achieved in the estrogen receptor positive lines MCF7 and BT474 using the pLV3 lentiviral vector. Doxycycline-
inducible re-expression in MDA-MB-231 and SUM159 cells is also being pursued.

1.        Johnstone C.N. et al., (2004) Gene 336:59-71
2.        Shang X. et al., (2003) JBC 278:45903-45914
3.        Castells A. et al., (2000) Cancer Res. 60:2836-2839
4.        Moody S.E. et al., (2002) Cancer Cell 2:451-461
5.        Gregory P.A. et al., (2008) 10:593-601


miR-27 regulates endothelial differentiation of breast cancer stem-like cells
Qiang Liu1 , Wei Tang1 , Fengyan Yu1 , Erwei Song1
1. Sun Yat-Sen memorial hospital, Guangzhou, GD, China
Recent studies suggest that cancer stem cells are capable of differentiating into endothelial cells and tumor endothelium may
be derived from cancer stem cells. But the mechanism remains unclear. We showed that VEGF-treated Breast tumor initiating
cells (BT-ICs) expressed endothelial markers, VEGFR2, CD31 and vWF. The level of miR-27a was also highly elevated in
these cells. Antagonizing mir-27a in VEGF-treated BT-ICs by mir-27a inhibitors led to the reduction of VEGFR2 and disruption
of reticular structure in matrigel, while increasing mir-27a in BT-IC resulted in enhanced endothelial formation. Increased mir-
27a paralleled reduced ZBTB10 protein, a known mir-27a target and Sp1 repressor. Infecting BT-ICs with miR-27a-lentivirus or
ZBTB10-shRNA-lentivirus promoted tumor vessel formation, vascular permeability and tumor metastasis. Further,
we demonstrated that VEGF-treated BT-ICs secreted more endogenous VEGF compared to undifferentiated BT-ICs. Thus,
miR-27a promotes tumor vessel formation by mediating endothelial differentiation of BT-IC and it may be a new target for anti-
angiogenesis cancer therapy.


Tumor Propagating cells in breast cancer tumorigenesis and metastasis
Radhika Nair1 , Wee Sang Teo1 , Sunny Ye1 , Andrea McFarland1 , Alexander Swarbrick 1
1. The Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
Breast cancer is a heterogenous disease. It is becoming increasingly apparent that in many breast cancers, a minority of
neoplastic cells comprising the tumor are drivers of the malignant, metastatic seeding ability and inherent chemoresistance of a
tumor. We term these cells “Tumor Propagating Cells” (TPCs), referring only to their functional activity and not to their cell of
origin. The characterization of TPCs has been largely hindered by the lack of reliable methods for the isolation and purification
of these cells including the current best practice of using cell surface markers. Our strategy to isolate TPCs is to develop
“molecular probes” that report on the activity of pathways controlling the TPC phenotype. We have identified a transcription
factor, Id1 (Inhibitor of Differentiation 1) which is expressed by a rare cell in ~ 50% of Hormone receptor negative (HR-) breast
cancers. The Id family (Id1-4) are basic Helix Loop Helix (bHLH) proteins which hetero-dimerize with other bHLH transcription
factors and control their transcriptional activity. Id1 has a general role in the inhibition of differentiation and lineage commitment
and its expression is required for the maintenance of self renewal and multi-potency in numerous stem cells (like
 hematopoietic, neuronal and embryonic). Work from our lab and others have shown that in a cancer context, Id1 is required
and sufficient to drive breast cancer cell proliferation, migration and invasion.
Based on their role in mammary gland development and tumorigenesis, we hypothesize that Id1 expressing cells are TPCs in
the HR- subtype. Using a lentiviral vector encoding the Id1 proximal promoter upstream of the EGFP cDNA to mark Id1 + cells,
we are able to prospectively isolate the rare Id1+ cellular fraction in the p53-/- mouse model of Triple Negative breast cancer.
Id1+ tumour cells have high in vivo tumour reconstituting activity as well as increased in vitro self renewal capacity
(tumorsphere assay). We have demonstrated that knock down of Id1 leads to diminished primary tumor formation in vivo using
the 4T1 syngeneic mouse model and are investigating Id1’s role in a metastatic context. Current directions include defining the
transcriptional and proteomic landscape of the rare cells which are critical for tumorigenesis and metastasis, with the ultimate
aim of identifying and targeting these cells in a therapeutic context.


Fascin is required for neural crest migration and brain tumor invasion in zebrafish
Elena Boer1 , Katarzyna Modzelewska1 , Cicely A Jette1 , Rodney A Stewart1
1. Dept. Oncological Sciences, Huntsman Cancer Institute/University of Utah, Salt Lake City, UT, USA
Metastasis is the leading cause of death in cancer patients. Current models support a multistep process for metastasis
progression, including escape from the primary tumor, migration and survival in the circulatory system, and colonization of
distant organs. The genetic mechanisms that govern each of these steps remain incompletely understood due to a lack of
appropriate genetic models of metastasis. We use neural crest development to model metastatic dissemination because both
metastatic cancer cells and neural crest cells undergo EMT, migrate collectively, and colonize distant organs. Specifically, our
research focuses on the genetic mechanisms that regulate collective cell migration, which requires the coordination of cell-cell
and cell-ECM interactions through cell surface receptors and actin-rich protrusions such as filopodia. A critical regulator of
filopodia formation is the actin bundling protein Fascin, which is upregulated in many human cancers and associated with
clinically aggressive tumors, metastasis, and poor survival. We combined imaging and genetic approaches in zebrafish to study
the role of Fascin in collective neural crest cell migration and cancer metastasis in vivo. We found that fascin-deficiency
results in abnormal filopodia formation in neural crest cells, which causes loss of cell-cell adherence within the cranial streams,
decreased cell velocity and invasion of cells into neighboring tissues. Consistent with these defects, fascin-deficient embryos
display abnormal development of cranial neural crest derivatives. We are also using a novel zebrafish brain tumor model
developed in our lab to study the requirement for Fascin in tumor invasion and metastasis. These tumors express high levels
of Fascin at the invasive front and we are using genetic and pharmacological approaches to determine if Fascin is necessary
and sufficient for brain tumor cell invasion and/or metastasis.


Imaging the metastatic process
Erik Sahai1
1. Cancer Research UK, London, tbc, United Kingdom
The acquisition of invasive behaviour enables the tumour cells to move into either the surrounding tissue or the vasculature and
thereby spread to other parts of the body. The focus of our research is investigating why cancer cells become invasive and how
they move. To study cell motility in this environment we perform intravital multi-photon confocal imaging of tumours in
anaesthetised mice. This enables the heterogeneous behaviour of cancer cells to be studied as they transit between primary
and secondary sites. We have used a range of transcriptional reporters to evaluate the signalling and differentiation status of
invasive and metastatic melanoma cells. This work demonstrates that differentiation hierarchies remain even in aggressive
melanoma models. By using transgenic hosts we can also visualise the interplay between tumour cells, leukocytes, stromal
fibroblasts and endothelial cells. We will present new data about endothelial cell organisation and dynamics in different
melanoma micro-environments and the relationship to invasive cancer cell behaviour.


Modeling and visualizing metastasis
Robert M. Hoffman1
1. AntiCancer Inc., San Diego, CA, United States
The introduction of nude mice to cancer research in 1969 by Rygaard allowed successful transplantation of human cancers.
Initial models using nude mice involved subcutaneous transplantation of human tumors which did not metastasize. The
development of orthotopic models in the late 1970s and early 1980s by Heppner, Sordat, and others demonstrated that
spontaneous metastasis of human tumors could occur in nude mice. The work of Fidler in the late 1980s and 1990s put
orthotopic models on a solid scientific basis and demonstrated their great potential for understanding metastasis. At the
beginning of the 1990s, our laboratory introduced surgical orthotopic implantation (SOI) models, whereby tumor fragments are
implanted orthotopically by surgical methods and enabled patient-like orthotopic nude mouse models to be developed for every
major cancer type. With the SOI model, we demonstrated in 1995 that organ colonization is the governing step of metastasis.
The discovery of green fluorescent protein (GFP) by Shimomura in 1962 and its cloning by Prasher, 30 years later, provided
the opportunity for our laboratory to label metastatic cancer cells in vivo with GFP in 1997. With the use of GFP and other
colored fluorescent proteins such as red fluorescent protein (RFP) discussed by Labas, metastasis could be visualized in living
mice in orthotopic models, at cellular and subcellular resolution, in real time. The use of GFP- and RFP-labeled cancer cells
enabled our laboratory, in collaboration with Glinsky in 2003, to observe that orthotopic tumor models produced highly-
metastatic viable circulating tumor cells (CTC) and subcutaneous tumor models did not. These results may explain why
orthotopic tumor models metastasize and subcutaneous tumor models do not. The introduction of GFP, RFP, and cyan
fluorescent protein (CFP) transgenic nude mice by our laboratory, beginning in 2004, enabled color-coded imaging of cancer
cell/stromal cell interaction during metastasis in orthotopic models. With these models, our laboratory demonstrated that tumor-
associated stromal cells are necessary for metastasis in 2006 and in collaboration with Verma in 2011, demonstrated that
cancer cells can transdifferentiate into vascular endothelial cells. The clinical potential of these models for metastatic cancer
was demonstrated by our laboratory in 2009 with the use of a telomerase-dependent adenovirus to label metastatic cancer in
situ which enabled fluorescence-guided resection of the metastatic nodules. Powerful technologies of orthotopic transplantation
and fluorescent-protein imaging have brought metastasis research to a level that can enable the visualization and
understanding of metastasis at each step of the process.


Contribution of cancer stem cells to angiogenesis and invasion of cancer
Xiu-Wu Bian1 , Yi-fang Ping1 , Xiao-hong Yao1 , Shi-cang Yu1
1. Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing,
Cancer stem cells (CSCs) possess a greater invasiveness than their differentiated descendants with unknown mechanism. This
study aimed to invastigate the mechanism of glioma stem cells (GSCs) participating in angiogenesis and invasion of maligmant
glioma. We developed four novel methods for enrichment and isolation of GSCs from human glioblastoma cell lines and
primary human glioma tissues, and studied the mechanism of their self-renewal, multipotency, tumorigenicity, drug resistance,
angigenesis and invasion. Techniques of cell sorting, immunostaining, laser cofocal and electron microscopy, chemotaxis,
miRNA microarray, DNA methylation and siRNA, etc. were used in this study. We found that: (1) Activation of either CXCR4 or
FPR preferentially expressed on GSCs increased calcium mobilization and the production of pro-angiogenic factors VEGF and
CXCL8. Tumor xenografts in SCID mice derived from GSCs cells grew faster with more abundant microvessels. (2) GSCs
showed reduced gap junctional intercellular communication (GJIC). CSCs expressed markedly reduced connexins, Cx43 in
particular, which are key components of gap junction. We also observed hypermethylation in the promoter of gap junction
protein α1 (GJA1), which encodes Cx43) gene promoter and increased expression of microRNAs targeting GJA1 transcripts in
GSCs. Reconstitution of Cx43 partially restored GJIC between GSCs and decreased their capacity of self-renewal,
invasiveness, angiogenesis and tumorigenicity. This was associated with upregulation of E-cadherin and inactivation of SDF-
1alpha/CXCR4 signaling pathway. Our results indicate that (1) GSCs contribute to tumor angiogenesis through, at least in part,
the activation of CXCL12/CXCR4 and/or fMLF/FPR axes, which might be potential targets for glioma therapy, and (2) Cx43 is
essential for normal GJIC and is capable of reversing the malignant phenotype of GSCs.(This study was supported by grants
from the National Basic Research Program (973 Program, No.2006CB708503) and National Natural Science Foundation of
China (NSFC, No. 30725035).


Innate immune system mediates focal metastasis
Sachie Hiratsuka-Nakamura1
1. Tokyo Women's Medical University, Tokyo, Japan
Distant metastatic process includes a cascade of sequential events, including tumor cell intravasation at the primary site, tumor
cell circulation, homing to distant organs, extravasation into these organs, and subsequent colonization, growth and
angiogenesis. The “premetastatic phase” describes a period during which a distant organ is rendered more hospitable towards
circulating tumor cells with metastatic potential, facilitated by the presence of a distant primary tumor. Primary tumor-derived
factors, such as cytokines, chemokines, amine oxidases and exsozome, can modify the lung environment before tumor cell
arrival. Spontaneous lung metastasis often presents as a discrete series of lesions separated by large areas of unaffected lung
tissue. Last year, I showed that primary tumors induce distinct foci of vascular hyperpermeability in premetastatic lung, which
co-localize with increased tumor cell homing. Endothelial cell-specific, inducible FAK-knockdown suppresses hyperpermeability
and reduces tumor cell homing to these foci. This year, we show that innate immune system is involved in the generation of
focal permeability regions in premetastatic lungs, and contributes focal metastases.


Blocking Ron kinase prevents metastasis by boosting anti-tumor immunity
Henok Eyob, Yoko S DeRose, Huseyin A Ekiz, Alana L Welm1
1. Huntsman Cancer Institute/University of Utah, Salt Lake City, UT, United States
Many stage I-III cancers have spawned clinically undetectable metastatic colonies even prior to diagnosis and surgical removal
of the primary tumor. It is eventual outgrowth of these microscopic lesions that causes metastatic relapse and death, yet events
that dictate when and how disseminated cells convert to overt metastases are largely unknown. Suppression of immune
function within the tumor microenvironment is a recognized phenomenon during tumor progression; however, the mechanisms
by which tumors maintain immune suppression during systemic metastasis remain enigmatic. Our data implicate the Ron
receptor tyrosine kinase pathway as a key mediator of CD8+ T cell suppression during conversion of micro-metastases to bona
fide metastatic lesions. Loss of Ron function through genetic manipulation or pharmacological inhibition allows an effective anti-
tumor CD8+ T cell response, specifically inhibiting outgrowth of seeded metastatic tumor colonies. Our findings unveil a new
therapeutic opportunity for preventing outgrowth of micro-metastases in cancer patients, thus potentially reducing cancer


MiR-106b is regulated by transforming growth factor β signaling and promotes metastasis in
breast cancer
Chang Gong1 , Shaohua Qu1 , Shujuan Pan1 , Yu Jiao1 , Bodu Liu1 , Erwei Song1
1. Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou, GD, China
Exposure of tumor cells to Transforming Growth Factor β (TGF-β) induces epithelial-mesenchymal transition (EMT) and
generates cells with a stable cancer stem cell (CSC) phenotype, which promotes metastasis in advanced malignancy. TGF-β
promotes EMT by a combination of Smad-dependent transcriptional events and Smad-independent effects. A role for the
microRNA (miRNA) in TGF-β/Smad signaling has recently been appreciated. However, the role of microRNAs in the
noncanonical TGF-βsignalings remains illusive. Our previous study found that breast CSCs had elevated miR-106b, a member
of miR-106b family,thus, the role of miR-106b in TGF-βinduced EMT was further investigated. Here, we profiled that miRNA
signature of EMT induced by TGF-βin normal human mammary gland epitherial cells. We further demonstrated that TGF-β1
transcriptionally upregulates miR-106b via c-jun. miR-106b mediates TGF-β-induced proliferation, EMT, migration and invasion
as well as self-renew. Morever, breast cancer metastasis-suppressor 1-like (BRMS1L) and Retinoblastoma1(RB1) are
negatively regulated by miR-106b and contribute to TGF-β-induced phenotypes. In addition, oncogeneic function of miR-106b
was also validated in mouse models and clinical correlation studies.


Tumour cell induced immune suppression dictates bone metastasis in breast cancer
Clare Slaney1 2 , Bradley Bidwell1 , Jai Rautela1 2 , Paul Hertzog3 , Robin Anderson1 2 , Belinda Parker1 2
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia
3. Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton,
The molecular mechanisms of breast cancer spread to distant sites and the influence of immune surveillance in preventing
metastatic outgrowth are largely unknown. We recently combined the use of our unique syngeneic 4T1.2 model of
spontaneous metastasis to bone with expression profiling of immunopurified tumour cells, to analyse tumour cell-specific
alterations that occur when tumours spread from the primary site to the bone in an immunocompetent system. This study
revealed that primary breast tumour cells express an innate immune pathway that is regulated by interferon regulatory factor 7
(Irf7), and that upon spread to the bone there is a dramatic decrease in the expression of this pathway. Restoration of tumour
cell Irf7 signalling suppresses metastasis to bone in an immune dependent manner suggesting that tumour cells directly
stimulate anti-tumour immune responses via secretion of type I IFNs. Additionally, loss of the type I IFN receptor in the host
stroma (Ifnar1-/- Balb/c mice) accelerated metastasis to bone in orthotropic and spontaneous mammary tumour models,
suggesting that cross-talk between tumour cells and the immune system (by secretion of type I IFN) dictates bone metastasis in
breast cancer. Our work is now focused on identifying the immune responses that are stimulated by tumour cell interferon
secretion and are critical for metastasis suppression.


Neural regulation of metastasis: New avenues for therapeutic intervention
Caroline P Le1 , Matthew A Pimentel1 , Ming G Chai1 , Corina Kim-Fuchs1 , Donald Lamkin2 , Steven W Cole2 , Erica K
1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
2. Cousins Center for PNI, University of California, Los Angeles, USA
Recent clinical and experimental studies have demonstrated that the neuroendocrine system regulates cancer development
and progression. Using advanced in vivo imaging technologies we found that chronic activation of the sympathetic nervous
system accelerates breast cancer metastasis to distant tissues. Increased metastasis was dependent on beta-adrenergic
signaling which induced macrophage recruitment to primary tumors and induced a switch to pro-metastatic and pro-
inflammatory gene expression. Our recent studies in orthotopic xenograft models of pancreatic cancer and leukemia showed
that activation of neural stress-response pathways similarly accelerated progression, suggesting that adrenergic signaling may
be a general physiological regulator of cancer progression. These findings raise the possibility that beta-blockade of neural
stress response pathways may be a novel therapeutic strategy to slow or prevent metastasis. In addition, we will discuss
findings that targeting peripheral nerve fibers may provide therapeutic leverage against metastasis.


Bioenergetic requirements of metastatic dissemination
Valerie LeBleu1
1. Harvard Medical School / BIDMC, Boston, MA, United States

Cancer cells divert glucose and glutamine into anabolic pathways to support their energy demand for rapid proliferation and
accumulation of cellular building blocks required for tumor growth. Here we probed the energetic requirement of metastatic
invasion using gene expression profiling and biological validation combined with metabolomic analysis. Migratory cancer cells
exhibit a unique glucose metabolism signature that functionally and dynamically support their invasive properties. Interestingly,
modulating such metabolic signature did not impact primary tumor kinetics, however it profoundly altered their potential for
dissemination. Our experiments identified specific gene targets that informed us about the bioenergetic switch that fuels
invasion and metastasis.

Endoplasmic reticulum stress regulator GRP78 and cancer
Amy Lee1
1. University of Southern California School of Medicine, Los Angeles, California, CA, United States
Mammalian cells have evolved multiple adaptive pathways, referred to as the unfolded protein response (UPR), that allow them
to respond to perturbations in endoplasmic reticulum (ER) homeostasis. One major pro-survival factor is the ER chaperone
GRP78, also referred to as HSPA5 or BiP. In addition to assisting in the assembly and quality control of ER proteins, GRP78
regulates UPR signaling in response to ER stress. Mechanisms for the cytoprotective effects of GRP78 include suppression of
pro-apoptotic pathways, maintenance of ER structural integrity and stress-induced autophagy. GRP78 is overexpressed in a
wide variety of human cancers and is associated with poor prognosis, metastasis and resistance to therapy. Mouse models
with genetically altered GRP78 provide valuable systems to interrogate the role of GRP78 in tumorigenesis and metastasis.
Targeted knockout of the GRP78 allele in mouse models of cancer revealed that GRP78 is critical for tumor proliferation,
survival and tumor angiogenesis. Conditional knockout of GRP78 in endothelial cells further establishes that GRP78
haploinsufficiency impedes neovascularization during tumor growth and metastasis, with minimal effect on normal tissue
microvessel density. Knockdown of GRP78 in human endothelial cells suppresses proliferation, survival and migration. These
studies underscore the potential value of GRP78 as a novel therapeutic target for dual anti-tumor and anti-angiogenesis
activity. Stress induction of GRP78 in tumors leads to resistance to a wide variety of therapeutic agents, whereas suppression
of GRP78 sensitizes them to the treatment. Furthermore, evidence is emerging that GRP78, traditionally regarded as an ER
resident protein, is preferentially expressed on the surface of tumor cells but not in normal organs. Cell surface GRP78 serves
as an upstream regulator of the PI3K/AKT oncogenic pathway which is widely implicated in cancer proliferation, survival,
migration and resistance. Thus, GRP78 as cell signaling regulator and protector of ER homeostasis, is a prime candidate for
therapeutic targeting.


Metabolism of cancer cells in hypoxia
Masahiro Inoue1
1. Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan
Response of the cancer cells to the hypoxic challenge can result in manifestation of malignant features of cancer, including
angiogenesis, metabolic change, invasion/metastasis, and chemo- or radio-resistance. Acute hypoxic response of the cultured
cancer cell lines has been intensively studied, but what happens in the cancer cells in patient tumors is poorly understood. We
previously reported that glycolytic enzymes were not induced in hypoxic region in vivo, while the glycolytic enzymes were
remarkably up-regulated when the cells were challenged by acute hypoxia in vitro. Thus, cancer cells might be in metabolically
inactive status under chronic hypoxic conditions in vivo.
The energy-consuming process is often activated in cancer cells but the energy consumption of cancer cells in hypoxic region
in vivo was not clear. We found that expression of c-Myc and phosphorylation of mTOR, both of which regulate energy
consuming process, were suppressed in hypoxic region in vivo. Suppressing energy consuming processes might be a strategy
for cancer cells to survive under metabolic stress.
Most of the cancer cell lines in vitro failed to survive under chronic hypoxic conditions (1% oxygen, two weeks). AsPC-1, a
pancreatic cancer cell line, was exceptionally able to survive under chronic hypoxic conditions. The ATP turnover was
suppressed under chronic but not acute hypoxic conditions. We recently developed a novel culture system for primary cancer
cells. The primary cancer cells were able to survive without increase of cell death under chronic hypoxic conditions,
suppressing the energy consuming signals, including c-Myc and mTOR. After re-oxygenation, they grew as well as the cells
maintained in normoxic conditions. Thus, the primary culture of cancer cells will provide a useful tool for studying the
mechanism of cancer dormancy in hypoxia.


The role of germline genetic variation in the regulation of metastasis-related transcriptional
Nigel Crawford1
1. NHGRI/NIH, Bethesda, MD, United States
Germline genetic variation is becoming increasingly recognized for its capacity to regulate metastasis.


The role of molecular chaperones in metastasis
Cara Lomas1 , Marissa Powers1 , Paul Workman1 , Sue Eccles1
1. The Institute of Cancer Research, London, United Kingdom
Molecular chaperones are essential for the function of multiple ‘client’ proteins, particularly oncoproteins. Adverse tumour
microenvironmental conditions (e.g. hypoxia, glycolytic metabolism), have been linked to chaperone activation, metastasis and
drug resistance.
Highly metastatic MDA-MB-231 and MDA-MB-435 cells were profiled for chaperone expression and response to inhibitors.
HSP90, HSP70, GRP78 and GRP94 were abundant, whilst small chaperones (HSP27 and αB crystallin) were variable.
  Chaperone inhibitors 17-AAG or NVP-AUY922 differentially affected cellular functions. MDA-MB-231 proliferation was
relatively resistant to 17-AAG (due to low DT-diaphorase levels) but was sensitive to NVP-AUY922. MDA-MB-231 chemotaxis
was inhibited by both compounds, and reduced in hypoxia. Basal HSP27 was minimal, but robustly induced following HSP90
inhibition and in hypoxia.
MDA-MB-435 cell proliferation was sensitive to 17-AAG and NVP-AUY922 although chemotaxis was unaffected; furthermore,
hypoxia enhanced chemotaxis. HSP27 was again up-regulated by HSP90 inhibition and hypoxia. αB crystallin was higher in a
metastatic sublines and further induced by HSP90 inhibition and hypoxia. Interestingly, both HSP27 and αβ crystallin were also
induced by 3D spheroid culture and this was again associated with reduced response to chaperone inhibitors. Thus
upregulation of small (ATP-independent) chaperones under certain conditions may reduce the efficacy of HSP90 inhibitors.
MDA-MB-231 orthotopic xenografts responded to 17-AAG, but bone metastases did not. The reduced sensitivity of bone
metastases (or indeed growth enhancement according to some reports) may be due to the hypoxic nature of the marrow
Stress-inducible chaperones were explored further as potential confounders of response to inhibitors in 3D and under hypoxia.
Stable knockdown of GRP78 or 94 by shRNA had no discernable phenotypic effects in vitro or in vivo. However, since
knockdown induced compensatory reciprocal induction, dual depletion is required. Overall, it is clear that chaperones constitute
a highly interdependent survival system allowing tumour cells to successfully achieve metastasis and circumvent therapeutic


Overexpression of the GTP-binding protein rab31 induces a switch from an invasive to a
proliferative phenotype in breast cancer cells
Susanne Sölch1 , Bettina Grimayer1 , Bastian Seubert2 , Thomas Kirchner3 , Sonja Schäfer1 , Manfred Schmitt1 ,
Thomas Luther3 , Achim Krüger2 , Matthias Kotzsch3 , Viktor Magdolen1
1. Clinical Research Unit, Klinikum rechts der Isar TUM, Munich, Germany
2. Experimental Oncology and Therapy Research, Klinikum rechts der Isar TUM, Munich, Germany
3. Institute of Pathology, University of Technology, Dresden, Germany
Monomeric GTP-binding Rab proteins regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been
shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer.
Previously, we found a significant association of high rab31 mRNA expression levels with poor prognosis in node-negative
breast cancer patients. In the present study, we aimed at investigating the impact of rab31 overexpression in breast cancer
cells on important aspects of tumor progression in vitro and in vivo.
Breast cancer cell transfectants, with different rab31 expression levels, were analyzed in vitro using proliferation, adhesion, and
invasion assays. Subsequently, the impact of rab31 overexpression on experimental metastasis in a xenograft tumor model in
mice was monitored. Increased rab31 protein levels were associated with enhanced proliferation of breast cancer cells, a
reduced adhesion of cells towards extracellular matrix proteins and decreased invasive capacity through Matrigel TM.
Overexpression of an inactive rab31-mutant neither stimulated proliferation nor reduced the adhesive capacity of the cells
whereas a constitutively active rab31-mutant showed the same phenotype in MDA-MB-231 cells as rab31 overexpressing cells.
Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing
MDA-MB-231 cells to the lung. Taken together, our results demonstrate that rab31 overexpression leads to a switch from an
invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced cell adhesion, and decreased
cellular invasion in vitro and in vivo.


Breast Tumor Kinase (Brk/PTK6) Mediates Increased HGF/MET-Induced Breast Cancer Cell
Signaling and Migration
Carol A Lange1
1. University of Minnesota, Minneapolis, MN, United States
Breast tumor kinase (Brk) is highly expressed in a majority of human breast cancers, but is absent from normal breast tissues.
Enforced Brk expression transforms human mammary epithelial cells in vitro, in part by conferring survival and proliferative
advantages. Recently, we discovered that HGF-induced MET receptor signaling (a hypoxic stress-induced signaling pathway
commonly associated with triple-negative/basal-type breast cancers) results in robust Brk activation. Notably, Brk mRNA and
protein were rapidly upregulated in both normal mammary epithelial cells and breast cancer cells in response to stressful
conditions associated with tumor development and progression such as hypoxia, oxidative stress, and low glucose. MET and
Brk are co-expressed in invasive breast cancers adapted to survive under hypoxic conditions, due to their constitutive
expression of HIF1, a transcription factor mediator of cellular responses to stress. Remarkably, once expressed, Brk activates
stress-associated protein kinases such as Erk5 and p38 MAPK, two key effectors of pro-survival/chemoresistance. New data
demonstrate that Brk-mediated activation of MAPK family members results in the direct phosphorylation of Sam68, an RNA-
binding protein and splicing factor member of the STAR (Signal Transduction Activator of RNA) family that is required for breast
cancer cell migration. In RNAi-based studies, knock-down of Brk, ERK5, or Sam68 blocked HGF-induced breast cancer cell
migration. Expression of phospho-mutant but not wild-type Sam68 blocked HGF-induced cell migration, indicating an essential
role for MAPK-dependent Sam68 phosphorylation. In in vivo studies, WAP-Brk-transgenic mice exhibited heightened stress-
activated protein kinase signaling and delayed involution, while aged multi-parious mice developed infrequent mammary
tumors. When these mice were crossed with mutant MET receptor knock-in animals, tumor development occurred with
shortened latency; early tumors expressed increased levels of Brk and Sik (the mouse homologue of Brk). These data
collectively support a model in which tumor-associated hypoxia/nutrient starvation mediates HIF1-induced Brk expression and
function, resulting in hyperactivation of MET-driven stress-activated signaling pathways (ERK5 and p38) that lead to increased
breast cancer cell survival and migration/invasion. We hypothesize that targeting Brk expression and/or function will inhibit
these advanced tumor phenotypes allowing (in the long term) for improved management of triple-negative breast cancer
patients. Treatments aimed at cell surface receptors are easily circumvented by activation of downstream effectors or “signaling
nodes” in resistant cancers. Brk or Brk-activated signaling molecules (ERK5, p38, Sam68) are potentially druggable targets that
could be exploited in breast cancer therapy, thus offering more selective/non-toxic options for patients with deadly basal-type
breast cancer who may become resistant to small-molecule inhibitors or antibodies aimed at cell surface receptors (these
studies were supported by NIH/NCI grant R01 CA107547).


Selection and adaptation during metastatic cancer progression
Christoph A Klein1
1. University of Regensburg, Regensburg, Germany
Metastasis is increasingly gaining scientific attention, while the traditional focus on the primary tumor is still predominant. This
can be easily observed in several experimental models as well as in current attempts to select therapy targets for systemic
cancer after mutational or molecular profiling of primary tumors. The shared characteristic of such primary tumor-centric
concepts is the exclusion of selective adaptation outside the primary tumor. They generally hold that most metastasis-
associated molecular traits and therapy targets are acquired or mutated within the primary tumor and then transmitted to
disseminating tumor cells. By direct ex-vivo analysis of disseminated cancer cells we found little support for these hypotheses.
In general, we document frequently early dissemination and observe that disseminated cancer cells (DCCs), which we isolated
from bone marrow or lymph nodes, and primary tumor cells display disparate changes on all levels of genomic resolutions,
including point mutations, allelic losses, and genome wide chromosomal rearrangements, providing evidence for ectopic
selective adaptation. As DCCs were isolated at the time of surgery or months to years after, they are representative (i) for cells
that have disseminated until resection of their source or (ii) for cells that have ectopically survived at least for the period
between resection of the primary tumor and bone marrow sampling. DCCs, detected by epithelial markers such as cytokeratin
or EpCAM, in bone marrow and lymph nodes are extremely rare (frequency 10-5 - 10-6) but associated with metastatic relapse
and poor outcome and therefore apparently comprise the metastatic precursor cells. While the genomic characterization of
DCCs has generated important insights into the time point of dissemination and provided evidence of selection and mutation
outside the primary tumor, it will be important to describe their phenotype and determine the molecular mechanisms of ectopic
survival and outgrowth.


Cancer micrometastasis and circulating tumour cells
Klaus Pantel1
1. Institute of Tumour Biology, Center of Experimental Medicine, University Medical Center Hamburg Eppendorf, Hamburg,
Blood-borne tumour cell dissemination to distant organs can start early in cancer patients and micrometastatic spread of cancer
cells is usually undetected by current imaging technologies. Therefore, sensitive methods have been developed to detect
circulating tumor cells (CTC) in the peripheral blood and disseminated tumour cells (DTC) in the bone marrow at the single cell
level (Alix-Panabieres et al, ARM 2012). Interestingly, the bone marrow seems to be a common homing organ for cells derived
from various epithelial tumours including breast and prostate cancer (Braun et al., NEJM 2005; Koellermann et al., JCO 2008).
However, a significant fraction of DTC remain over years in a “dormant” stage, and little is known about the conditions required
for the persistence of dormancy or the escape from the dormant phase into the active phase of metastasis formation (Pantel et
al., Nat Rev Cancer 2008, Uhr & Pantel, PNAS 2011; Lu et al., Cancer Cell 2011). Sequential peripheral blood analyses,
however, are more convenient for patients than BM analyses and many research groups are currently assessing the clinical
utility of CTC for assessment of prognosis and monitoring of systemic therapy (Riethdorf et al., CCR 2010). There is an unmet
need for biomarkers for real-time monitoring of the efficacy of systemic adjuvant therapy in individual patients. The monitoring
of CTC as “liquid biopsy” will provide new insights into the selection of tumor cells under biological therapies. CTC analyses are
therefore incorporated into many current clinical trials testing new anti-cancer agents as companion diagnostics. However,
current CTC assays need to be improved: CTC undergoing epithelial-to-mesenchymal transition may escape detection by
epithelial markers (Bednarz et al., CCR 2010; Joosse et al, CCR 2012) and benign inflammatory bowel diseases release
epithelial cells into the blood that may lead to false-positive findings (Pantel et al., Clin Chem 2012). Interestingly, cell-free
nucleic acids released by CTC might become valuable biomarkers of micrometastatic disease in the future (Schwarzenbach et
al., Nat Rev Cancer 2011). In conclusion, molecular characterization of DTC and CTC opens a new avenue for detecting,
understanding and fighting early metastatic spread of tumour cells with important implications for future therapies.


Combating breast cancer brain metastasis: the CTC signature
Lixin Zhang1 , Lon D. Ridgway1 , Michael D. Wetzel1 , Jason A. Ngo1 , Jerry C. Goodman1 , Wendy Schober2 , Morris D.
Groves3 , Dario Marchetti1
1. Pathology & Immunology, Baylor College of Medicine, Houston, Texas, USA
2. Leukemia, UT MD Anderson Cancer Center, Houston, Texas, USA
3. Neuro-oncology, UT M.D. Anderson Cancer Center, Houston, Texas, USA
Mechanisms of fatal brain metastatic breast cancer (BMBC) are largely unknown. Similarly, properties and biomarker
identification of circulating tumor cells (CTCs), the “seeds” of metastasis, remain elusive. Here we report novel strategies
investigating CTCs isolated from peripheral blood mononuclear cells (PBMCs) of patients with BMBC, including the
development       and     characterization  of   CTC     lines.   We    identified  a     unique    BMBC    CTC      signature
(HER2+/EGFR+/HPSE+/Notch1+/EpCAM-) by investigating CTCs that could not be captured by the US Federal Drug
Administration-approved Veridex CellSearch platform (EpCAM - negative CTCs). Second, we analyzed the invasive and
metastatic competencies of isolated CTCs. Established CTC lines expressing the BMBC signature were highly invasive and
capable to form brain metastasis in xenografts. Third, tumor cell morphologies of CTC - induced metastases closely resembled
ones of pathologically assessed tumors of patients whose blood was source of CTCs. Fourth, the presence of BMBC signature
protein – expressing cells was detected in CTC - induced BMBC. Collectively, we provide first-time evidence of human CTCs
isolation and long-term growth, and the establishment of CTC lines. Further, we prove the metastatic competency of these CTC
lines and necessity of the BMBC CTC signature to promote brain metastasis. These strategies and results can be of
significance to develop new therapies against breast cancer metastasis in general, BMBC in particular.


Disseminated cancer cells from non-metastatic patients display tumorigenic potential in
immunodeficient mice
Melanie Werner-Klein1 , Steffi Treitschke2 , Christian Werno2 , Christoph Klein2 3 , Lahiri Kanth Nanduri3 , Sebastian
Scheitler3 , Petra Rummele4 , Hans-Stefan Hofmann5 , Christian Hafner6 , Claus Lattrich7 , Brigitte Rack8
1. Institute for Immunology, University of Regensburg, Regensburg, Germany
2. ITEM Regensburg/Project Group: Personalized Tumor Therapy, Fraunhofer Regensburg, Regensburg, Germany
3. Chair of Experimental Medicine, University of Regensburg, Regensburg, Germany
4. Institute for Pathology, University of Regensburg, Regensburg, Germany
5. Clinic for Thoracic Surgery, University of Regensburg, Regensburg, Germany
6. Clinic for Dermatology, University of Regensburg, Regensburg, Germany
7. Clinic for Gyneology, University of Regensburg, Regensburg, Germany
8. Clinic for Gyneology, LMU Munich, Munich, Germany
In patients without metastatic disease, disseminated cancer cells (DCC) belong to the rarest cells in the human body and are
found in about 20-50% of M0 stage patients at frequencies of 1-2 cells per million bone marrow cells (BM). Despite their
rareness, their detection in BM or lymph nodes (LN) has been shown to be a strong predictor of outcome, which suggests that
DCC comprise metastases founder cells. However, it has not been possible to generate xenograft tumors from DCC and
consequently their relevance has been repeatedly questioned. Here we show that DCC can form tumors in NodScidIL2Rg-/-
(NSG) mice and therefore provide direct evidence for their metastasiogenic potential. The DCC were isolated from diagnostic
BM-aspirates or LN-samples of melanoma or Non-Small-Cell-Lung-Cancer (NSCLC) patients. Xenograft-tumors could be
established when cells were transplanted either directly, even at low cell numbers (<10 cells), or after short in vitro culture and
serially transplanted. To date, 6 out of 9 melanoma LN-samples and 4 out of 11 NSCLC-samples from BM or LN have formed
tumors. Molecular genomic comparisons between ex vivo isolated DCC and xenografts are currently underway and will be
presented. DCC expansion may pave the way for molecular and functional studies, which will eventually enable improved
systemic therapies at the dark stage of minimal residual cancer to prevent lethal metastasis.


Fibrosis and Cancer Progression
Raghu Kalluri1
1. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States

Chronic tissue fibrosis involves fibroblast activation and inflammation that leads to deposition of type I collagen and eventual
organ failure. There is a strong experimental and clinical correlation between tissue fibrosis and incidence of cancer. But it
remains unclear whether it is the chronic inflammation or activated myofibroblasts that contribute to the emergence of cancer.
This lecture will highlight the molecular pathways associated with emergence, activation and proliferation of fibroblasts in organ
fibrosis and strategies for reversing fibrosis. Additionally, the molecular connection between organ fibrosis and
the emergence of cancer will be discussed.


Vegf-D promotes tumor metastasis by regulating prostaglandins produced by the collecting
lymphatic endothelium
Steven Stacker1 2 , Tara Karnezis1 2 , Ramin Shayan1 2 3 4 5 , Carol Caesar1 2 , Sally Roufail1 2 , Nicole C Harris1 2 6 , Katie
Ardipradja1 2 , Steven P Williams1 2 3 , Rae H Farnsworth1 2 , You Fang Zang2 , Ming G Chai7 , Erica K Sloan7 , Thusitha
Rupasinghe8 , Dedreia Tull8 , Megan Baldwin9 , Stephen Fox2 , Marc G Achen1 2
1. Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Melbourne, Victoria, Australia
2. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
3. Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia
4.   Jack Brockhoff Reconstructive Plastic Surgery Research Unit, Royal Melbourne Hospital, Melbourne, Victoria, Australia
5.   Department of Anatomy Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria,
6. Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia
7. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia
8. Bio21 Institute for Molecular Science and Biotechnology, Metabolomics Australia, Melbourne, Victoria, Australia
9. Vegenics Pty Ltd, Circadian Technologies Limited, Melbourne, Victoria, Australia
Lymphatic metastasis, an important initial step in tumor dissemination and is facilitated by lymphangiogenic growth factors such
as VEGF-D and VEGF-C which are secreted by some primary tumors. Elevated levels of these proteins in some human tumors
correlate with lymph node metastasis and poor patient prognosis. We have used a tumor model driven by VEGF-D over-
expression to examine changes in gene expression within the collecting lymphatic vessels (1) during VEGF-D-driven tumor
metastasis and in regional lymph nodes (2). Our study identified tissue-specific regulation of PGDH, the key enzyme in the
prostaglandin degradation pathway, in endothelial cells of collecting lymphatic’s. The VEGF-D-dependent regulation of the
prostaglandin pathway was supported by the finding that the dilation of collecting lymphatic vessels and subsequent ability to
support metastasis was affected by NSAIDS, known inhibitors of prostaglandin synthesis. Our data suggests a key control point
for cancer metastasis within the collecting lymphatic endothelium, which links the lymphangiogenic growth factors and the
prostaglandin signaling network and has implications for anti-metastatic therapies. Collecting lymphatic vessels may therefore
constitute a novel target for both NSAID and anti-VEGF-C/D therapeutics.
1. Karnezis et al., Cancer Cell 21:181-195, 2012
2. Farnsworth et al., Cancer Res, 71: 6547-6557, 2011


A novel small noncoding RNA regulated pathway controlling EPC biology and tumor
Prue N Plummer1 , Brittany Umer1 , Jelena Vider1 , Vivek Mittal2 , Albert S. Mellick1
1. School of Medical Science, Griffith University, Gold Coast, QLD, Australia
2. Lung Cancer Centre, Weill Cornell Medical College, New York, United States
Endothelial progenitor cells (EPCs) are adult bone marrow derived vascular progenitors, which promote rapid blood vessel
formation and tumor spread. Since they were proposed more than ten years ago, the role they play in controlling vascular
development in a growing tumor and metastases has made them the subject of extensive study, and controversy 1-3 . In recent
years, it has become increasingly clear that not only do EPCs form a distinct vascular bone marrow derived lineage, but that
their role in promoting adaptive resistance in tumors, following anti-angiogenic therapies, must be considered when designing
future anti-cancer strategies. Using novel strategies in retroviral based delivery of transgenes we report for the first time on the
characterization of a clinically significant/miRNA pathway regulating both EPC and tumor vascular biology. In work in review in
Cancer Research4 , we also demonstrate the use of a strategy for the peptide directed delivery of liposome encapsulated anti-
miRs, to modulate vascular miRNA signaling in situ, with consequent effects on tumor growth and vascularization.

1.       Mellick AS*, et al (2010) Using Transcription Factor Id1 to Selectively Target Endothelial Progenitor Cells Offers
     Novel Strategies to Inhibit Tumor Angiogenesis and Growth. Cancer Res 70:7273-82
2.       DingChen G, et al (2008) EPCs control the angiogenic switch in mouse lung metastasis. Science 319:195-8.
3.       Nolan D, et al (2007) BM-derived endothelial progenitor cells are a major determinant of nascent tumour
     neovascularization. Genes Dev 21,1546-58.
4.       Plummer PN, et al (2012) MicroRNAs Regulate Endothelial Progenitor Cell-Mediated Tumor Angiogenesis. Cancer
     Research, Decision Pending.


A novel molecular mechanism mediating vascular metastasis
Yaowu He1 , Ying Dong2 , Berta Casar3 , Andreas Wortmann1 , Mark Adams1 , Judith Clements2 , James Quigley3 , John
1. Mater Medical Research Institute, South Brisbane, Qld, Australia
2. Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Qld, Australia
3. The Scripps Research Institute, La Jolla, California, USA
Vasculature dissemination of cancer requires a series of cellular events including extravasation, the egress of cells across an
endothelial cell layer. We have recently shown that a cell surface glycoprotein called CDCP1, that is upregulated in kidney, lung
and pancreatic cancers, promotes this process (1). Our data indicate that CDCP1 is proteolytically processed at the cell surface
in vitro (2) and in vivo (3) by serine proteases thereby initiating an intracellular signalling cascade involving activation and
docking to CDCP1 of the kinases Src and PKCδ and downstream activation of Akt. Interestingly, we have shown that this
cascade protects cancer cells as they extravasate in vivo and we have identified the protease essential for initiating this
protection in a mouse model of metastasis (3). Our findings further demonstrate roles for CDCP1 in cell migration in vitro
including its competition as a Src substrate with a critical mediator of cell adhesion FAK and its role in migration induced by
EGFR signalling (4, 5). We speculate that targeting CDCP1 or CDCP1-induced signalling may be a rational approach to treat
cancer patients at risk of recurrent disease.

1.        Deryugina EI et al Mol Can Res 2009
2.        He Y et al JBC 2010
3.        Casar B et al Oncogene 2012
4.        Wortmann A et al JBC 2011
5.        Dong Y et al JBC 2012


Degradomics analyses of dynamic proteolytic networks in the 4T1 tumor and lung
metastases model of breast cancer
Chris Overall1 , Philipp Lange1 , Yuanmei Lou2 , Caroline Bellac1 , Ling Yi3 , Antoine Dufour1 , Ulrich auf dem Keller1 ,
Anna Prudova1 , Reinhild Kappelhoff1 , Amanda Starr1 , Paul McDonald2 , David Perrin3 , Shoukat Dedhar2
1. Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada
2. BC Cancer Research Centre, University of British Columbia, Vancouver, BC, Canada
3. Department of Chemistry, University of British Columbia, Vancouver, BC, Canada
Proteomics technologies are revolutionizing the way in which cancer pathways and drug targets can be identified, validated and
assessed. To understand the roles of proteases in metastasis and in tumor associated macrophages we applied quantitative
proteomics in multiplex analyses to identify the proteome, the N-terminome, and cleaved protease substrates on a global scale.
In the 4T1 syngeneic mouse model of breast cancer we analyzed protease expression by our CLIP-CHIPTM, a complete
dedicated human and murine protease and inhibitor oligonucleotide microarray. Matrix metalloproteinases (MMPs) 10 and 13
and matriptase-2 were highly expressed. MMP proteases were imaged spatially in 3D and temporally by F18 coupled to
Marimastat, a reversible nM MMP inhibitor drug. To elucidate the function of these proteases in vivo by identifying their cleaved
substrates we developed a new technique called Terminal Amine Isotope Labeling of Substrates (TAILS) (Nature
Biotechnology 28, 281-288, 2010). MS/MS both identifies the substrate and sequence of the cleavage site in the same
experiment. We identified numerous novel MMP-10 and 13 substrate candidates including members of the insulin-like growth
factor binding protein family in breast cancer secretomes. Proteomic evidence of proteolytic processing of novel substrates was
found including Insulin-like growth factor binding protein-4 and 6, follistatin-like 1 and cystatin C, heparin affin regulatory peptide
(HARP/pleiotrophin) and connective tissue growth factor (CTGF), which released vascular endothelial growth factor (VEGF)
from angiogenic inhibitory complexes. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of
their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. This underscores the potential
of TAILS to quantitatively analyze the proteolytic fingerprint of noninvasive 67NR, the metastatic 66cl and the highly malignant
4T1 tumors in vivo in ongoing studies.


Molecular and cellular crosstalk between tumor and inflammatory cells in the tumor
microenvironment: role in metastasis
Ibrahim H. Younos, Alicia Dafferner, Holly Britton, James E. Talmadge1
1. University of Nebraska Medical Center, Omaha, NE, United States
Tumors and their infiltrating cells form a complex and heterogeneous (focal and cellular) microenvironment with paracrine and
exocrine regulatory activities. The nature and mediators of the resulting molecular crosstalk are poorly defined and a better
understanding is critical to controlling tumor progression and metastasis. Molecular and cellular signatures of tumors and
peripheral blood can predict clinical outcomes; however, the origin and regulation of these signatures is obscure. To improve
our understanding of this crosstalk, we examined tumor/infiltrating cell regulation of cellular and molecular targets in the
regulation of the microenvironment, hematopoietic homeostasis, and molecular expression. These experiments used
orthotopic metastatic (4T1) and non-metastatic (168-FARN) murine mammary tumors and revealed significant differences in
the transcripts of metastatic and benign tumor cells (in vitro and in vivo) including growth factors (G-CSF, SCF), enzymes
(NOS-2, MMP-13), interleukins (IL-23a), and chemokines (CXCL-5, CXCL-1, CCL-2). On a cellular level; differences between
tumors in the frequency and number of splenic, blood and tumor-infiltrating T-, dendritic-, and myeloid-derived suppressor cells
were observed. Unexpectedly, we also report differences in transcript levels between primary tumors and cultured tumor cells
with significant increases in tumor expression of ARG-1 and VEGFa and decreased G-CSF, suggesting micro-environmental
crosstalk regulation or contamination by infiltrating inflammatory cells. This was clarified using isolated infiltrating and splenic
myeloid cells (>99% pure) demonstrating tumor induced changes to transcripts from infiltrating myeloid cells (VEGF-a, IL23a)
and host regulation of tumor transcripts (ARG-1, G-CSF, CXCL5). Cellular studies revealed that metastatic tumors stimulated
myelopoiesis and cellular trafficking independent of host cytokines, which based on NOS2 knockout mouse studies dampened
rather than increased the inflammatory response. Together, these observations support a critical role for tumor
microenvironmental-host crosstalk in the regulation of cellular and molecular phenotypes and tumor progression.


Tissue Factor, VCAM-1 and VAP-1 orchestrate the recruitment of monocytes/macrophages to
tumour cells in pulmonary metastasis
Ana M. Gil-Bernabe1 , Spela Ferjancic1 , Peter Richardson2 , Tim Sparey2 , Sally A. Hill1 , Ruth J. Muschel1
1. Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford, United Kingdom
2. Proximagen Group, plc., London, United Kingdom
Coagulation is required for spontaneous and experimental metastases. Tissue factor (TF) is a key molecule in the coagulation
cascade. TF expression by tumour cells correlates with metastasis in clinical and experimental settings. We show that clot
formation by TF enhances tumour cell survival after arrest, during experimental lung metastasis, by recruiting macrophages
(CD11b+, CD68+, F4/80+ and CX3CR1+, but CD11c-). Genetic (expression of TFPI) or pharmacological (hirudin) inhibition of
coagulation abrogated macrophage recruitment and tumour cell survival. Furthermore, impairment of macrophage function
(Mac1 KO and CD11b-DTR mice) decreased tumour cell survival without altering clot formation, demonstrating that the
recruitment of functional macrophages was essential for tumour cell survival. Moreover, a similar population of macrophages
was also recruited to the lung during the formation of a premetastatic niche. Anticoagulation inhibited their accumulation and
prevented the enhanced metastasis associated with the formation of the niche.
Leukocyte homing during inflammation is mediated by endothelial activation. We examined the role of endothelial activation in
myeloid cell recruitment during early pulmonary metastasis. VCAM-1 and VAP-1 were upregulated after tumour cell arrest and
associated with tumour cell-clot formation and myeloid cell recruitment. VCAM-1 expression was independent of functional
monocytes/macrophages, but decreased if coagulation was inhibited. Inhibition of VCAM-1 (blocking antibody), VAP-1 (small
molecule inhibitor) or both reduced myeloid cell recruitment and subsequent tumour cell survival without altering clot formation.
Thus, TF-expressing tumour cells form platelet clots on their surface soon after arrest in the pulmonary vasculature. Endothelial
activation is induced after tumour cell arrest, and enhanced by tumour cell-clot formation, resulting in the recruitment and
retention of monocytes/macrophages that are essential for tumour cell survival. Interference with clot formation,
monocytes/macrophages function or endothelial activation leads to reduced metastasis. These results suggest a means for
therapeutic intervention to combat early metastasis, for example in relapse after treatment.


Co-evolution of breast to brain metastatic and neural progenitor cells
Josh Neman1 , Vincent Duenas1 , Claudia Kowlik1 , Cecilia Choy1 , Sarah Waliany1 , Mike Chen1 , Rahul Jandial1
1. City of Hope Medical Center, Los Angels, Calif, United States
Metastases are responsible for 90% of deaths from cancer, and patients diagnosed with brain metastasis have only a dismal
20% probability of surviving for more than 1 year. In the case of breast cancer (BC), the incidence of brain metastasis has
increased dramatically, possibly as an indirect result of improved therapies that effectively control extracranial disease. Due to
the grave consequences of brain colonization by metastatic BC cells, we wished to elucidate the brain microenvironmental cues
that promote secondary tumor formation.
 We report that surgical specimens from a patient with breast-to-brain metastasis exhibit increased active gliosis (GFAP+,
J131+, GLAST+/CD44+) around the peritumoral brain region, accompanied by infiltration of the tumor by neural progenitor cells
(NPCs) (Emx2+, Sox2+, Pax6+, Numb+, Sox9+, Nestin+). Because signaling by the TGF-b superfamily is involved in
gliogenesis and also exerts tumor-suppressive effects on BC cells, we investigated potential roles for these proteins in the brain
microenvironment in the context of metastatic lesions. We found that metastatic BC cells exhibit increased bone morphogenic
protein-2 (BMP-2) expression compared with primary breast tumors, and hypothesized that an interaction between BC cells
that have metastasized to the brain and the resident peritumoral NPCs may be mediated by BMP-2. To examine the effect of
BMP-2 overexpression, we created an in vitro model that utilized co-cultures of NPCs and primary or metastatic BC cells. We
observed that during early co-culture (1-5 days) NPCs inhibited the proliferation of metastatic BC cells but later in co-culture (5-
15 day) NPCs differentiated into astrocytes with a concomitant loss of their ability to inhibit metastatic BC growth. This result
suggested an adaptive strategy adopted by metastatic BC cells for colonization of the brain may be the BMP-2-mediated
differentiation of NPCs into reactive astrocytes. Inhibition of BMP-2 signaling by Noggin or the knockdown of BMP-2 expression
in the metastatic breast tumor cells prevented NPC differentiation into astrocytes and maintained their ability to inhibit tumor cell
proliferation. Conversely, BMP-2 overexpression in the primary tumor cells enhanced NPC differentiation into astrocytes and
promoted tumor cell growth. Together, the results suggest that breast-brain metastatic tumor cells and NPCs co-evolve,
creating a more permissive tumor microenvironment favoring tumor proliferation through increased expression of BMP-2.


A furin resistant form of semaphorin-3E as a potential treatment for metastatic disease
Gera Neufeld1 , Andrea Casazza2 , Boaz Kigel1 , Massimiliano Mazzone3 , Luca Tamagnone2
1. Technion, Israel Institute of Technology, Haifa, Israel
2. Institute for Cancer Research and Treatment (IRCC), University of Torino, Torino, Italy
3. Vesalius Research Center, VIB, Leuven, Belgium
Secreted semaphorin 3E (Sema3E) promotes cancer cell invasiveness and metastatic spreading. The pro-metastatic activity of
Sema3E is due to its proteolytic fragment p61, which is capable of trans-activating the oncogenic tyrosine kinase ErbB2 that
associates with the Sema3E receptor PlexinD1 in cancer cells. Here, we show that a mutated, uncleavable variant of Sema3E
(Uncl-Sema3E) binds to PlexinD1 like p61-Sema3E, but does not promote the association of PlexinD1 with ErbB2 nor activates
the ensuing signaling cascade leading to metastatic spreading. Furthermore, Uncl-Sema3E competes with endogenous p61-
Sema3E produced by tumour cells, thereby hampering their metastatic ability. Uncl-Sema3E also acts independently as a
potent anti-angiogenic factor. It activates a PlexinD1-mediated signaling cascade in endothelial cells that leads to the inhibition
of adhesion to extracellular matrix, directional migration and cell survival. The putative therapeutic potential of Uncl- Sema3E
was validated in multiple orthotopic or spontaneous tumour models in-vivo, where either local or systemic delivery of Uncl-
Sema3E-reduced angiogenesis, growth and metastasis, even in the case of tumours refractory to treatment with a soluble
vascular endothelial growth factor trap. In summary, we conclude that Uncl-Sema3E is a novel inhibitor of tumour angiogenesis
and growth that concomitantly hampers metastatic spreading.

Roles of membrane-bound serine protease inhibitor, HAI-1, in invasion and metastasis of
Tsuyoshi Fukushima1 , Hiroaki Kataoka1
1. Faculty of Medicine, University of Miyazaki, Miyazaki, JPN, Japan
Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1)
gene, is a transmembrane protease inhibitor that is believed to regulate the activities of various membrane-associated serine
proteases. HAI-1 has two Kunitz-type inhibitor domains with the N-terminal Kunitz domain (KD1) responsible for inhibiting
known target proteases. To analyze the roles for HAI-1 in cancer, we performed stable knockdown of HAI-1 in human cancer
cell lines HAI-1 knockdown resulted in morphological changes suggesting epithelial to mesenchymal transition (EMT) in certain
cell lines such as SUIT-2 pancreatic cancer and HLC-1 lung cancer cell lines with up-regulation of smad-interacting protein 1
(SIP1/ZEB2) and reduced E-cadherin expression. These changes are mediated partly by type-II transmembrane serine
proteases, matriptase and TMPRSS4. Next, we examined the effect of HAI-1 knockdown on metastasis of SUIT-2 cells. In a
nude mouse subcutaneous transplantation model, the tumor growth was rather slower in the knockdown SUIT-2 cells and the
incidence of pulmonary metastasis was comparable between knockdown and control cells. However, the number of metastatic
colonies per mouse was higher in the knockdown cells as long as metastasis arose. In an experimental pulmonary metastasis
model with tail vein injection, metastatic colonization was apparently increased in the knockdown cells. One week after the
injection, metastatic colonization was observed in 36% (4/11) of mice injected with HAI-1-knockdown SUIT-2, whereas none
(0/11) of the control mice shows metastatic lesion. The metastasis ratios were 80% (4/5) and 40% (2/5) after two weeks, and
82% (9/11) and 45% (5/11) after four weeks, for HAI-1-knockdown and control SUIT-2 cells, respectively. Moreover,
pretreatment with recombinant KD1 reduced metastasis. Therefore, the loss of HAI-1 promoted pulmonary colonization during
the early phase of metastasis. In conclusion, HAI-1 has a suppressing role in cancer invasion and metastasis, and KD1 may
serve as a therapeutic modality for inhibiting metastasis.

1.         Fukushima T, Kawaguchi M, Yamasaki M, Tanaka H, Yorita K, Kataoka H: Hepatocyte growth factor activator
     inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells. Cancer Sci. 102:407-13,
2.         Cheng H, Fukushima T, Takahashi N, Tanaka H, Kataoka H: Hepatocyte growth factor activator inhibitor type 1
     regulates the epithelial to mesenchymal transition through membrane-bound serine proteinases. Cancer Res.69:1828-35,


 Cancer immune-escalation process and the role of inflammatory tumor microenvironment for
promoting metastasis
Yoshihiro Hayakawa1 2 , Yoshitaka Kimura2 , Naoki Tsunekawa2 , Marimo Sato-Matsushita3 , Hideaki Tahara3 , Ikuo
Saiki1 , Tatsuro Irimura2
1. Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan
2. Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of
      Tokyo, Bunkyo-ku, Tokyo, Japan
3. Department of Surgery and Bioengineering, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo,
Mounting evidences strongly supports the existence of an effective cancer immune surveillance process that prevents cancer
development in both mouse and humans. It has been demonstrated that the importance of anti-cancer innate and adoptive
effector cells and their anti-cancer effector molecules in the cancer immune-editing process. In addition to such positive role of
immune responses, the indications of a possible link between inflammation and cancer has been extended in the last decade
that the immune responses may play a positive role at different stage of tumor development including carcinogenesis process,
malignant progression and disseminating metastasis. Although an inflammatory microenvironment is recognized as an
essential component of all tumors, the precise mechanism remains to be clarified by which type of immune response can
promote cancer disease in such microenvironment. In the present study, we have studied inflammatory immune responses and
the role of cytokines in malignant progression of cancer. To examine the regulatory mechanism of host inflammatory responses
in cancer malignant progression, we employ a very unique in vivo mouse model in which low tumorigenic original cancer cells
acquire highly malignant metastatic phenotype after exposure to host inflammatory microenvironment. By using this unique in
vivo cancer progression model combined with gene-targeted knockout mice, we examined the role of inflammatory mediators,
specifically IFN-γ and IL-17, in the process of inflammation -associated cancer malignant progression. We have also
characterized the precise immunological tumor microenvironment by which pre-malignant cells progress into malignant
metastatic tumor cells, and found that a critical contribution of distinct inflammatory type γδ T cells in this immune-escalation
process by producing IL-17. We would like to further discuss the potential of inflammatory IL-17-producing γδ T cells as a
conductor of subsequent chronic inflammatory responses to orchestrate pro-tumor immunological microenvironment.


Toward a human platform for the tumor-targeted IFN-alpha delivery by genetically modified
Roberta Mazzieri1 2 , Giulia Escobar2 , Davide Moi2 , Anna Ranghetti2 , Erika Zonari2 , Bernhard Gentner2 , Mario
Squadrito2 3 , Michele De Palma2 3 , Luigi Naldini2
1. University of Queensland, Wooloongabba, Brisbane, Qld, Australia
2. Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
3. ISREC, EPFL, Lausanne, Switzerland
We have previously shown effective delivery of a transgene-encoded IFN-alpha protein at the tumor site by combining the use
of lentiviral vectors (LVs), capable of efficient gene transfer in hematopoietic stem/progenitor cells (HS/PCs), and the selective
activation of the Tie2 promoter in a subset of tumor-infiltrating monocytes (Tie2-expressing monocytes, TEMs), providing the
proof of principle of its antitumor efficacy in mice1 . We now aim to increase the safety and feasibility of this strategy toward the
goal of a clinical translation. Being TIE2 also expressed by primitive HSPCs, where sustained IFN-alpha expression might be
detrimental, we have developed new LVs with de-targeted transgene expression from HS/PCs via microRNA-mediated post-
transcriptional regulation. We are currently investigating the safety of this IFN-alpha delivery platform monitoring the HSC long-
term repopulating activity in murine and human hematochimeric NSG mice transplanted with gene modified HS/PCs. Moreover
we have validated the efficacy of this approach in vivo. Indeed, detargeting of the transgene expression from HS/PCs did not
affect the ability of TEM-mediated IFN-alpha delivery to inhibit both primary and secondary spontaneous mammary tumors. By
FACS analysis on peripheral blood of transplanted human hematochimeric NSG mice, we showed specific GFP expression in
the myeloid compartment, as expected. Most importantly, human Tie2-IFN-alpha myeloid cells were recruited to tumors
orthotopically injected into NSG mice, where they effectively targeted IFN-alpha, as shown by the local upregulation of IFN-
inducible genes. Altogether our data support the feasibility of a clinical translation of this strategy, which bear the potential of
providing a safe and more effective adjuvant treatment against cancer.

1.        De Palma, Mazzieri et al., Cancer Cell 2008


FOXC2 as prognostic factor and mediator of colorectal cancer progression
Wen-Ting Liao1 2 3 , Yan-Mei Cui1 2 3 , Yan-qing Ding1 2 3
1. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
2. Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, China
3. Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Southern Medical University, Guangzhou, China
Purpose: The present study was to investigate the clinicopathologic significance and potential role of FOXC2 in the
progression of colorectal cancer (CRC).
Methods: The relationship between FOXC2 expression and clinical characteristics of CRC was analyzed in 205 paraffin-
embedded archived CRC specimens by immunohistochemistry (IHC).
The effects of FOXC2 on EMT, migration and invasion, as well as on metastasis, were examined both in vitro and in vivo, using
Western bot, immunofluorescent staining and migration assay and experimental metastasis assay in nude mice. Western blot
and immunofluorescent staining were performed to examine the impact of FOXC2 on the PI3K/Akt, MAPK and Wnt/b-Catenin
signaling pathways. TOP/FOP luciferase assay was using to detect Wnt/b-Catenin signaling acitvity changes under expression
of FOXC2.
Results: FOXC2 protein level was significantly correlated with advanced Dukes stage (P=0.036), N stage (P=0.013), distant
metastasis (P=0.001), and poor survival of patients (P=0.005). Enforced expression of FOXC2 induced a EMT-like phenotype
in SW480 cells, enhanced cell migration in vitro and metastasis in vivo. Conversely, knockdown of FOXC2 caused an
increased expression of epithelial markers and decreased expression of mesenchymal markers, accompanied by inhibition
effects of cell migration in vitro and metastasis in vivo. Overexpression of FOXC2 upregulated p-AKT, p-GSK3b, and p-ERK,
while silence of FOXC2 downregulated p-AKT, p-GSK3b, and p-ERK. Additionally, TOP/FOP luciferase acitvity was dramaticly
increased in cells overexpressing FOXC2. Moreover, overexpression of FOXC2 significantly promoted the nuclear b-Catenin
expression, which indicate that the Wnt/b-Catenin signaling pathway was activited. Thus, these results suggested that FOXC2
could enhance PI3K/AKT, MAPK and Wnt/b-Catenin pathway activities in CRC cells.
Conclusions: Our findings suggest that FOXC2 protein, as a valuable marker of CRC prognosis, plays an important role in the
progression of human CRC.


Matrix metalloproteinase control of the metastatic prostate cancer bone-microenvironment
Jeremy S Freiling1 , Leah M Cook1 , Lizzie Atomi-Pamen1 , Marilena Tauro2 , Conor C Lynch1
1. Moffitt Cancer Center, Tampa, FL, United States
2. University of Bari, Bari, Italy
The paradigm for tumor-host interaction in the context of prostate to bone metastases is the “vicious cycle” in which PTHrP,
RANKL and TGFβ play key roles. In animal models and human specimens, we have found that matrix metalloproteinases
(MMPs) are highly expressed in the metastatic bone microenvironment. Although MMPs are implicated in extracellular matrix
turnover, emerging results from our group define MMPs as principle mediators of vicious cycle via their ability to regulate
bioactivity and bioavailability of factors such as PTHrP, RANKL and TGFβ. We have found that individual MMPs expressed in
the bone microenvironment; 1) can process mature PTHrP into a number of distinct fragments that impact osteoblast cell
signaling; 2) generate soluble RANKL that acts as a factor in the recruitment of osteoclast precursors and; 3) are critical for the
generation of bioavailable TGFβ. We posit that the selective inhibition of MMPs by novel bone seeking MMP inhibitors or that
targeting MMP substrate neo-epitopes can lead to the generation of therapies which will halt the vicious cycle and ultimately
cure prostate to bone metastases

Identification and characterisation of histological growth patterns of colorectal cancer liver
metastases: (potential) role of the micro-environment
Gert G Van den Eynden1 2 , Nigel Bird1 3 , Martin Illemann1 4 , Rikke L Eefsen1 4 , Andrew Reynolds1 5 , Pnina Brodt1 6 ,
Ali W Majeed1 3 , Gunilla Hoyer-Hansen1 4 , Peter B Vermeulen1 2
1. on behalf of the Liver Metastasis Research Network,, International, collaborative research network
2. Oncology Center - Augustinus Hospital, Wilrijk, Belgium
3. Liver Research Group, University of Sheffield, Sheffield, UK
4. Finsen Lab, Copenhagen, Denmark
5. Tumour Biology Team, The Breakthrough Breast Cancer Research Center, London, UK
6. Surgery, Oncology and Medicine, McGill University Health Center - Royal Victoria Hospital, Montreal, Canada
Worldwide, colorectal cancer (CRC) is the 4th and 3rd most common cancer in men and women, respectively. Of all patients,
25% present with liver metastases (LMs) and a further 25-50% will develop LMs during the 2 years following resection of the
primary tumour. Remarkably little is known about the biological characteristics that determine the heterogeneity and prognosis
of patients with LMs.
We have identified three different histological growth patterns (GPs) of LMs of solid tumours in general and of CRC in
particular1 2 . In LMs with a desmoplastic GP the tumour is separated from the liver parenchyma by desmoplastic,
inflammatory stroma. In the pushing type, the liver plates are compressed, running parallel to the tumour-liver interface without
desmoplastic stroma and with only a mild inflammatory infiltrate. In the replacement GP tumour cells replace the hepatocytes
without destruction of the liver architecture. There is no compression, no desmoplastic stroma or inflammatory infiltrate.
We have demonstrated significant biological differences associated with these GPs. Angiogenesis and hypoxia are significantly
increased in pushing type LMs1 2 . The GP also differs according to the biological characteristics of the primary tumour,
including the histological subtype and the differentiation grade2 . We also demonstrated different expression of the components
of the plasminogen system in LMs with different GPs3 . Furthermore, we observed a remarkable tendency toward GP uniformity
in multiple LMs arising in the same patient4 . Recently, we have demonstrated that the presence of a pushing component in the
GP was an independent predictor for overall survival5 .
Collectively, these data provide compelling evidence that the GPs in LMs of CRC are not a random phenomenon, but the
apparent result of different tumour-micro-environment interactions. Current efforts focus on the molecular pathways underlying
these GPs and the identification of GP-related biomarkers to guide the treatment of patients with CRC LMs.

1.        Liver metastases from colorectal adenocarcinomas grow in three patterns with different angiogenesis and
     desmoplasia. Vermeulen PB, Colpaert C, Salgado R, Royers R, Hellemans H, Van Den Heuvel E, Goovaerts G, Dirix LY,
     Van Marck E. J Pathol. 2001 Oct;195
2.        Breast adenocarcinoma liver metastases, in contrast to colorectal cancer liver metastases, display a non-angiogenic
     growth pattern that preserves the stroma and lacks hypoxia. Stessels F, Van den Eynden G, Van der Auwera I, Salgado
     R, Van den Heuvel E, H
3.        Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon
     cancer liver metastases. Illemann M, Bird N, Majeed A, Laerum OD, Lund LR, Danø K, Nielsen BS. Int J Cancer. 2009 Apr
4.        Histopathological growth pattern, proteolysis and angiogenesis in chemonaive patients resected for multiple
     colorectal liver metastases. Eefsen RL, Van den Eynden GG, Høyer-Hansen G, Brodt P, Laerum OD, Vermeulen PB,
     Christensen IJ, Wettergren A, Federsp
5.        The histological growth pattern of colorectal cancer liver metastases has prognostic value. Van den Eynden GG, Bird
     NC, Majeed AW, Van Laere S, Dirix LY, Vermeulen PB. Clin Exp Metastasis. 2012 Apr 3. Epub ahead of print


Latent metastasis seeds and niches
Joan Massague1
1. Memorial Sloan-Kettering Cancer Center, tba, NY, United States
The signals and niches that allow disseminated tumor cells (DTCs) to stay viable as metastasis-initiating seeds are largely
unknown. An understanding of these mechanisms would help in developing treatments to eradicate DTCs and thus prevent
metastasis. Recently we have identified signals that prime breast cancer stem cells for survival in distant organs. Activated
SRC primes DTCs for a robust PI3K-Akt survival response to CXCL12 in the bone marrow. The leukocyte-tethering receptor
VCAM-1 primes DTCs for PI3K-Akt activation by macrophage contacts in the lungs. The extracellular matrix protein tenascin-C
(TNC) forms micrometastatic niches that promote WNT and NOTCH signaling in cancer stem cells. Lastly, a CXCL1-S100A8
paracrine loop protects DTCs from the stresses of metastasis and chemotherapy, providing a link for these two clinically related
phenomena. In preclinical models, SRC kinase inhibitors, VCAM-1 blocking antibodies, and CXCL1 receptor inhibitors
suppress metastasis and augment the efficacy of chemotherapy. SRC, VCAM-1, TNC and CXCL1 previously emerged in
“metastasis gene signatures” that predict breast cancer relapse to distant organs. How is it that adaptive advantage traits for
distant sites pre-exist in primary tumors? A newly identified “metastasis seed pre-selection” phenomenon addresses this old
conundrum. We find that when the stroma of a breast tumor resembles that of a distant organ, it selects for cancer cells that are
predisposed to thrive in that organ. In sum, niches and signals for DTC fitness can be identified, molecularly deconstructed,
conceptualized, and therapeutically targeted.


Breast cancer bone metastasis — from molecular insights to novel therapeutics
Yibin Kang1
1. Princeton University, Princeton, NJ, United States
Metastasis represents the most devastating stage of cancer progression. In particular, bone metastasis affects more than 70%
of late stage breast cancer patients. Discovering bone metastasis genes that are clinically relevant and functionally important
are critical for the development of novel therapeutics for high-risk breast cancer patients. We apply a multidisciplinary approach
to analyze the molecular basis of breast cancer bone metastasis, combining functional genomics tools with animal models and
clinical analysis of cancer metastasis. Candidate bone metastasis genes were identified from gene expression profiling of
highly bone metastatic cells derived after in vivo selection in animal models, and from human breast tumor samples with clinical
associations with skeletal relapse. Functional characterization of these genes revealed their novel role in mediating tumor-
stromal interactions essential for the formation of osteolytic bone metastasis. Using in vivo imaging technology, we showed that
TGFβ is released from bone matrix upon bone destruction, and signals to breast cancer to further enhance their malignancy in
developing bone metastasis. We furthered identified Jagged1 as a TGFβ target genes in tumor cells that engaged bone
stromal cells through the activation of Notch signaling to provide a positive feedback to promote tumor growth and to activate
osteoclast differentiation. Analysis of miRNAs involved in osteoclast differentiation also revealed miRNAs that can effectively
serve as biomarkers and therapeutic agents for osteolytic bone metastasis. These discoveries have led to the development of
novel biomarkers and therapeutic agents with the great potential for more effective clinical management of skeletal
complications of breast cancer.


A functional in vivo shRNA screen for regulators of breast cancer metastasis
Richard P Redvers1 , Kaylene J Simpson1 , Izhak Haviv1 , Robin L Anderson1
1. Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
Approximately 1.5 million women worldwide are diagnosed with breast cancer annually, many of whom will have occult
metastases at first diagnosis. Despite significant improvements in early diagnosis and treatment, these metastatic outgrowths
will progress to life threatening lesions in about 20% of patients, for whom few cures exist. The molecular mechanisms
underlying metastasis remain poorly understood but are known to include the loss of a class of genes termed metastasis
suppressors. Thus far, only a handful of these critical determinants of breast cancer metastasis have been identified. We
hypothesised that the loss of one of these genes in a poorly metastatic tumour line would lead to the genesis of a cell with
significantly improved metastatic capabilities and the spread of such transmogrified variants to distant organs would provide
sufficient selective pressure to enrich for those cells deficient in a specific bona fide metastasis suppressor transcript.
Using our clinically relevant model of spontaneous breast cancer metastasis, we initiated a genome-wide in vivo shRNA screen
to identify genes involved in spontaneous dissemination of tumour cells to distant organs. Significantly, our preliminary screen
yielded three candidates whose loss permitted metastasis to spine, namely Hsd17b13, an enzyme involved in steroid
metabolism, Muc15, a cell surface glycoprotein known to suppress invasion of trophoblasts, and Pdlim1, one of the PDZ-LIM
proteins involved in stabilising focal adhesions. Remarkably, all three are significantly diminished at the DNA and
transcript levels in large human cohorts of aggressive breast carcinomas versus normal breast tissue (Oncomine). We are
continuing our screen using a next-generation sequencing approach to rigorously evaluate candidates on a genome-wide basis
and will validate these in our in vivo model.
It is of paramount importance that we identify functionally relevant metastasis suppressors and understand their biology to
provide novel therapeutic opportunities for those with incurable disease.


Malignant tumor growth in the bone microenvironment; crosstalk between TGFb and
ERK/AKT/PTEN signaling pathway
Mitsuru Futakuchi1 , Yuto Sakai1 , Katsumi Fukamachi1 , Rakesh K Singh2 , Masumi Suzui1
1. Graduate School of Medical Sciences, Nagoya City University, Nagoya City, Aichi, Japan
2. Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA
Understanding how metastatic cancer cells co-opt bone microenvironment to establish as bone metastases is essential for the
identification of new therapies to control bone metastasis. TGF-beta, released as the osteolysis in the bone metastatic lesion
has been shown to promote tumor growth in the bone microenvironment. In the present study, we examined the crosstalk
signaling among TGF-beta, ERK, PTEN and AKT in the bone microenvironment. Murine mammary tumor cell lines were
transplanted in two different sites, on the cranial bone and subcutaneous lesions, and the effect of TGF-beta receptor1 kinase
inhibitor (R1-KI) on tumor growth in the bone microenvironment was compared with that in the subcutaneous
microenvironment. R1-KI treatment significantly suppressed tumor growth and cell proliferation in the bone but not in the
subcutaneous microenvironment. This treatment also suppressed phosphorylated Smad2 in tumor cell in the bone but not in
the subcutaneous microenvironment. Our data demonstrate that R1-KI treatment decreased the expression of phosphorylated
ERK in the tumor cells and increased PTEN expression in the stromal cells in the bone microenvironment.


Investigating breast cancer cell colonization of bone using engineered microenvironments
Anna V Taubenberger1 2 , Verena C Quent1 3 , Laure Thibaudeau1 , Judith A Clements1 2 , Dietmar W Hutmacher1 2
1. Queensland University of Technology, Kelvin Grove, QLD, Australia
2. Australian Prostate Cancer Research Centre Queensland, Brisbane, QLD, Australia
3. Obstetrics and Gynecology, University Hospital Erlangen, Erlangen , Germany
About 70 % of women with advanced breast cancer (BCa) develop bone metastases. The mechanisms leading to colonization
of BCa cells in the skeleton are still incompletely understood. Here, we demonstrate that mineralized extracellular matrices
secreted by primary human osteoblasts (OBM) modulate cellular processes associated with BCa colonization of bone. A panel
of four BCa cell lines of different bone-metastatic potential, T47D, SUM1315, MDA-MB-231 and the bone-seeking subline
MDA-MB-231BO, was cultured on OBM. After 3 days, the metastatic BCa cells had undergone morphological changes on OBM
and were aligned along the OBM fibers, while the non-metastatic cells showed a random orientation. Atomic Force Microscopy-
based Single Cell Force Spectroscopy revealed that the metastatic cell lines adhered more strongly to OBM compared to non-
metastatic cells. Function-blocking experiments indicated that b1-integrins mediated cell adhesion to OBM. Metastatic BCa cells
migrated directionally and invaded OBM, which was accompanied by enhanced MMP-2 and -9 secretion. In addition, we
observed gene expression changes associated with osteomimickry in BCa cultured on OBM. We further introduce a novel
humanized in vivo model of bone colonization, in which polymeric electrospun tubes served as scaffolds for ectopic bone
formation. Together, we have developed an experimental platform consisting of in vitro and in vivo models that will allow to
further dissect the mechanisms underlying bone colonization by BCa cells.


Melanoma-derived exosomes educate bone marrow progenitor cells toward a pro-metastatic
phenotype through MET
David Lyden1 , Hector Peinado1 , Jacqueline Bromberg1
1. Weill Cornell Medical College, New York, NY, United States
Tumor-derived exosomes are emerging as mediators of tumorigenesis with a tissue-specific address and message. We
explored the function of melanoma-derived exosomes in formation of primary tumors and metastatic progression in both murine
models and patients. Whereas exosomes from highly metastatic melanoma cells increased the metastatic behavior of primary
tumor cells by ‘educating’ bone marrow (BM) progenitor cells via the MET receptor, exosomes from low metastatic melanoma
cells did not alter the incidence of metastases. Melanoma-derived exosomes induced vascular leakiness at pre-metastatic
sites, and reprogrammed BM progenitor cells towards a pro-vasculogenic phenotype (c-Kit+Tie2+MET+). Reducing MET
expression in tumor-derived exosomes diminished the pro-metastatic behavior of BM cells. Importantly, MET expression was
upregulated in circulating BM progenitor cells (CD45-CD117low and CD45-CD117lowTIE2+) isolated from stage III and stage
IV melanoma patients. Rab1a, Rab5b, Rab7, and Rab27a were highly expressed in melanoma and Rab27a RNA interference
decreased exosome production and/or soluble angiogenic factors in melanoma cells, thereby preventing mobilization of BM
progenitor cells, tumor growth and metastasis. Finally, we identified a ‘melanoma signature’ in exosomes isolated from
metastatic melanoma patients, comprised of TYRP2, VLA-4, Hsp70, an Hsp90 isoform and MET oncoprotein, which together
with Rab proteins, appear to represent exosome-specific proteins with prognostic potential, and may provide new therapeutic


Lysyl oxidase in pre-metastatic niche formation
Janine Erler1
1. Biotech Research and Innovation Centre (BRIC), Copenhagen, DK, Denmark
Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidases that catalyzes the cross-linking of collagens and elastin in
the extracellular matrix (ECM). Expression of LOX is clinically correlated with metastasis and poor patient survival. Genetic,
chemical or antibody inhibition of LOX significantly reduces metastases in various models of cancer. We have shown this
occurs through preventing ECM remodeling at the primary site enhancing tumor cell invasion, and also through ECM
remodeling at distant sites of future metastasis. This remodeling at pre-metastatic sites results in recruitment of bone marrow-
derived cells and activation of host cells, creating niches permissive to metastasizing tumor cell colonization and growth.
Moreover, we have found LOX to be responsible for fibrosis-enhanced metastasis, providing an important link between
extracellular matrix homeostasis, fibrosis and cancer.


Formation of an immunosuppressive pre-metastatic niche in breast cancer triggered by
hypoxic tumour-derived factors
Jaclyn Sceneay1 2 , Melvyn T Chow1 2 , Mark J Smyth1 2 3 , Andreas M1 2 3
1. Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
2. Department of Pathology, The University of Melbourne, Melbourne, Victoria, Australia
3. Department of Biochemistry, The University of Melbourne, Melbourne, Victoria, Australia
Hypoxia is a common feature and poor prognostic factor in many solid cancers including breast cancer. Within the primary
tumour, hypoxia acts as a strong selective pressure that promotes angiogenesis, invasion and metastatic spread of tumour
cells. The pro-tumourigenic properties of future metastatic sites are determined by the interactions between factors secreted by
the primary tumour and bone marrow-derived cell lineages, which drives formation of pre-metastatic niches in distant organs
before the arrival of tumour cells. Individually, various hypoxic tumour-derived factors have been associated with pre-metastatic
niche formation, which suggests hypoxia may be the unifying process crucial in creating these permissive microenvironments
for disseminating tumour cells.
Here, we demonstrate in different immune competent, syngeneic orthotopic breast cancer models, that primary tumour hypoxia
promotes pre-metastatic niche formation. Cell-free supernatant derived from hypoxic breast tumour cells results in increased
bone marrow-derived cell infiltration into the lungs in the absence of a primary tumour, and leads to increased metastatic
burden in breast and melanoma experimental metastasis models.
We furthermore define bone marrow-derived CD11b+/Ly6Cmed/Ly6G+ and CD3-/NK1.1+ NK cell lineages as main constituents of
the pre-metastatic niche, and show that NK cell cytotoxicity and maturity is decreased. This subset of myeloid cells suppresses
NK cell function to create an immune-suppressed environment allowing for increased tumour cell expansion and metastatic
growth. Additionally, secretion of the systemic soluble factor MCP-1/CCL2 by hypoxic breast cancer cells, controls myeloid cell
mobilisation and recruitment to the pre-metastatic niche. This data demonstrates a pivotal role for factors produced by hypoxic
primary tumour cells in orchestrating the recruitment and modulation of bone marrow-derived cells in the pre-metastatic niche in
breast cancer.


Mechanisms of colorectal liver metastasis and novel molecular targets.
Hirotoshi Kikuchi1 , Ichirota Iino1 , Masayoshi Yamamoto1 , Shinichiro Miyazaki1 , Yoshihiro Hiramatsu1 , Manabu Ohta1
, Kinji Kamiya1 , Hiroyuki Konno1
1. Hamamatsu University School of Medicine, Hamamatsu, Japan
Introduction: In the treatment of colorectal cancers, the control of liver metastasis is an important issue. Here we present our
data showing the importance of tumor microenvironment and premetastatic niche in the process of colorectal liver metastasis
and      discuss      possibility   of     novel    molecular      targeting     therapy     to    inhibit    liver   metastasis.
Methods: 1) We implanted the highly metastatic human colon cancer TK-4 orthotopically into the cecal walls of nude mice,
followed by twice-daily administration of TSU68, an antiangiogenic receptor tyrosine kinase inhibitor, or vehicle. Gene
expression profile in premetastatic liver was analyzed using microarrays. 2) Cancer tissues were collected from FFPE samples
of human primary colorectal cancer and colorectal liver metastasis using laser-capture microdissection methods. Total RNA
was     extracted    and     expression     profile of microRNA was             analyzed     by TaqMan         microRNA   Array.
Results: 1) Five weeks of treatment with TSU68 significantly inhibited liver metastasis compared with the control group (P <
0.001). mRNA levels for the chemokine CXCL1 were significantly increased in premetastatic liver but not in lung of
tumorbearing mice compared with non–tumor-bearing mice. CXCR2 expression was detected predominantly in orthotopic
tumors compared with ectopic tumors. The amount of IL-12 p40 subunit protein in the portal vein was elevated in tumor-bearing
mice. Blockade of both CXCR2 and IL-12 p40 with a neutralizing antibody and TSU68 treatment significantly inhibited liver
metastasis. 2) miR-122 was the best upregulated in colorectal liver metastasis compared with primary tumor.
Immunohistochemical analysis revealed that the expression levels of cationic amino acid transporter 1 (CAT1) protein which
has been reported as a negative target gene of miR-122 were lower in liver metastases than primary tumors. Expression levels
of CAT1 in 132 primary tumors were correlated with the existence of synchronous liver metastasis. In the analysis of 121 colon
cancer patients without synchronous liver metastasis, patients with CAT1-low colon cancer had significantly shorter liver
metastasis-free                   survival               but                  not                 overall               survival.
Conclusions: Tumor microenvironment in the primary site plays important roles in cancer metastasis by forming premetastatic
niche in the target organ through the CXCL1/CXCR2 axis. Overexpression of miR-122 and concomitant suppression of CAT1 in
primary tumor appears to be involved in the process of colorectal liver metastasis.


The pivotal role of astrocytes in the biology and therapy of brain metastasis
Isaiah (Josh) Fidler1
1. U.T. M.D. Anderson Cancer Center, Houston, TX, United States
The pathogenesis of metastasis depends on the interaction of unique tumor cells (“seed”) with specific host organ
microenvironment (“soil”). In fact, metastases develop and survive because tumor cells usurp homeostatic mechanisms. A
prime example is brain metastasis. Brain metastases and glioblastoma are densely surrounded and infiltrated by activated
astrocytes expressing glial fibrillary acidic protein. The role of astrocytes is to support the function and viability of neurons, and
we therefore questioned whether astrocytes can also protect tumor cells from cytotoxic drugs. Astrocytes cocultured with tumor
cells established a gap-junction communication network and protected the tumor cells from all tested chemotherapeutic drugs.
We have identified the signal that astrocytes send to tumor cells as endothelin-1, -2. Phosphorylation of endothelin receptors A
and B on tumor cells was followed by activation of Akt, MAPK, and upregulation of survival genes in tumor cells. Luciferase-
tagged breast cancer, lung cancer, and glioblastoma cells were implanted in the brains of nude mice. Administration of the
dual endothelin receptor inhibitor Macitentan® combined with Taxotere or Temozolomide produced complete destruction of
established metastasis and glioblastoma in nude mice. These data clearly demonstrate that astrocytes influence the biological
behavior of tumor cells in the brain and reinforce the conclusion that the organ microenvironment must be taken into
consideration for the design of effective therapy. The studies described here began with a hypothesis that astrocytes protect
tumor cells from cytotoxic agents. In vitro studies using cocultures of astrocytes and tumor cells were followed by successful in
vivo therapy studies. These translational studies stimulated ongoing phase I clinical trials of glioblastoma.


Correlations between biomarkers for invasion/metastasis and patient clinical outcomes in
rectal cancer
Seong Beom Ahn1 , Charles Chan2 , Sun Young Kwun2 , Betty Pai Chun Lin2 , Owen F. Dent 3 , Pierre H. Chapuis3 , Mark
S. Baker1
1. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia
2. Anatomical Pathology, Concord Hospital , University of Sydney, Sydney, NSW, Australia
3. Colorectal Surgery, Concord Hospital, University of Sydney, Sydney, NSW, Australia
Colon and rectal cancer (CRC) is one of most common malignancies by incidence and cause of death. Five-year survival rates
are dramatically lower in stage C (metastasised to lymph nodes) than in stage B (non-metastasized). In other words, tumour
metastasis is directly responsible for the majority of cancer deaths. Therefore it is important to understand the relationship
between biomarkers of invasion/metastasis and patient clinical outcomes (e.g., recurrence and 5-year survival) to minimise
metastasis-related deaths. Urokinase plasminogen activator receptor (uPAR) plays a number of critical roles in cancer
metastasis and is overexpressed in many cancers including CRC. The Integrin β6 subunit is an epithelial cell-surface restricted
integrin and is also significantly overexpressed in CRC. These data suggest that both uPAR and integrin β6 may be specific,
sensitive metastasis-related protein biomarkers in CRC. Furthermore, there is considerable evidence (i.e., cross-over IPs,
proximity ligation assays and peptide arrays) that these two cell-surface proteins can interact with each other.

In this study, ~396 stage B and C resected rectal cancer specimens with paired normal tissues were examined for expression
levels of uPAR, integrin β6 and the uPAR. β6 complex. The expression levels of antigens were correlated with various clinico-
pathological outcomes including 5-year overall survival. In addition, we utilised two differentially discriminatory epitope specific
MAbs; the first (MAb R4) identifying a uPAR epitope expressed on stromal associated cells (e.g., macrophages and fibroblasts)
and the second (MAb ADI#3936) identifying uPAR expression levels on CRC epithelial cells. Given the considerable discussion
in the research community regarding where uPAR is actually expressed in human CRC, we examined correlations of uPAR
expression with patient outcomes using both MAbs in both locations.


Prognostic factor for the patients with bone-only metastasis in breast cancer
Sung Gwe Ahn1 , Hak Min Lee1 , Sang Hoon Cho2 , Seung Hyun Hwang1 , Joon Jeong1 , Hy-De Lee1
1. Gangnam Severance Hospital, Yonsei University Medical College, Gangnamgu, Seoul, South Korea
2. 2Department of Statistics and Actuarial Science, Soongsil University, Seoul, South Korea
Bone is the most frequent metastasis site of the patients with breast cancer. We investigated to identify prognostic factors
affecting survival following bone-only metastasis for breast cancer patients.
Patients and method
The medical records of breast cancer patients, who were treated and followed at Gangnam Severance Hospital, Yonsei
University, Seoul, Korea, were retrospectively reviewed to identify patients with bone metastasis. Among them, patients who
developed bone metastasis during follow up periods and patients with bone-only metastasis at the initial diagnosis were
included in this study.
91 patients (82.7%) had bone-only recurrence and 19 (17.3%) showed bone metastasis at the initial diagnosis (De Novo bone
metastasis). The Kaplan-Meier overall survival estimate at 5 year for the breast cancer patients with De Novo bone metastasis
was higher than that for the patients with bone-only recurrence (60.2 vs. 44.1 %); however, there was no statistically significant
difference between their overall survival estimates (log-rank test, P = 0.136). In the multivariate Cox regression model selected
by Akaike Information Criteria, bisphosphonate treatment (hazard ratio (HR) 0.18, 95% CI 0.07 - 0.43), presence of estrogen
receptor (HR 0.51, 95% CI 0.28 – 0.94), and solitary bone metastasis (HR 0.32, 95% CI 0.14 – 0.72) were the significant
prognostic factors associated with longer overall survival in the bone-only recurrence group.
Our finding of the identification of the prognostic factors having an effect on breast cancer mortality after bone-only metastasis
will help to understand the clinical course and improve the treatment outcome of bone-only metastasis from breast cancer.
Bisphosphonate treatment identified as one of the most significant prognostic factors in our study warrants further investigation


Rational combination therapy against triple-negative breast cancer
Fares Al-Ejeh1 , Mariska Miranda1 , Peter T Simpson2 , Georgia Chenevix-Trench1 , Sunil R Lakhani2 3 , Kum Kum
1. Queensland Institute of Medical Research, Herston, QLD, Australia
2. Molecular & Cellular Pathology, The University of Queensland Centre for Clinical Research, Brisbane, QLD, Australia
3. Pathology Queensland, The Royal Brisbane & Women’s Hospital, Brisbane, QLD, Australia
Background: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with higher incidence of
recurrence, more distant metastasis, and poorer survival. This subtype is also characterized by complex genomes where little
of their genomes remain at normal copy number but without high, focal copy number amplifications. At the transciptome level,
the majority of TNBC (~75%) are classified as basal-like breast cancer (BLBC) according to the five intrinsic subtypes. Despite
considerable genomic and gene expression characterization of TNBC, proteomic and phospho-proteomic investigations of this
disease are limited with no available targeted therapies in clinical use.
Methods & Results: We used the KinexTM antibody array ( to interrogate protein/phosphoproteins levels
in 43 primary breast cancer biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines.
Unsupervised hierarchical clustering of protein/phosphoprotein levels revealed two subgroups of TNBC in comparison to other
subtypes. Western blotting and Proteome ProfilerTM Arrays (R&D Systems) were used to validate deregulated
proteins/phosphoproteins in TNBC. Pathway analysis revealed that one subgroup of TNBC exploits overlapping and cross-
talking networks for survival. These signaling networks are downstream from elevated activation of EGFR, integrins and Insulin-
like growth factor 1 receptor (IGF1R). To overcome compensatory downstream signaling that would limit the inhibition of a
given pathway; we developed EGFR-targeted radioimmunotherapy (RIT) strategy to systemically deliver cytotoxic loads of beta
particles (177Lu) that would kill targeted cells and surrounding cells by crossfire effect. The combination of EGFR-directed RIT
with chemotherapy and PARP inhibition successfully treated orthotopic and metastatic TNBC models established from cell lines
and patient-derived xenografts. The superior efficacy of this triple-agent combination therapy is explained by enhanced DNA
damage and reduced DNA repair response, higher apoptotic cell death and the elimination of putative breast cancer stem cells.
Conclusion: Proteomic analysis of TNBC provides a powerful tool to elucidate druggable signaling networks with therapeutic
potential. TNBC utilizes complex interacting signaling networks and rational combination therapies are required for effective


Antibodies binding the ADAM10 substrate recognition pocket inhibit Eph function
Lakmali Atapattu1 , Peter W Janes1 , Nayanendu Saha2 , Carmen Llerena1 , Dimitar Nikolov2 , Andrew M Scott3 , Martin
1. Monash University, Clayton, VIC, Australia
2. Structural Biology Program, Memorial Sloan-Kettering Cancer Centre, New York, USA
3. Austin Branch, Ludwig Institute for Cancer Research, Heidelberg, Victoria, Australia
The function of a number of cell surface receptors implicated in oncogenesis relies on shedding of their ligands by members of
the ADAM metalloprotease family. Important ADAM substrates include ligands for Eph and EGFR (erbB) receptor tyrosine
kinases (RTKs), which are active in a wide range of chronic diseases, including cancer. ADAMs are thus considered promising
targets for multi-pathway therapeutic intervention strategies1,2. However, to date considerable effort to develop specific
inhibitors of ADAM protease activity has failed to yield specific and effective agonists.
Previously, our discovery that ADAM substrate recognition occurs within a non-catalytic, extracellular domain of ADAM10
allowed elaborating the mechanism regulating the release of Eph-RTK ligands (ephrins), which specifies that only ligands
bound to catalytically-active RTKs are cleaved3,4. This regulatory motif thus provides a promising molecular target to inhibit key
pathways in tumour progression, and prompted us to develop α-ADAM10 monoclonal antibodies (mAbs) that may block
substrate access to this site. From a panel of α-ADAM10 mAbs, the specificity of ‘8C7’ and ‘3A8’ for the substrate recognition
domain was confirmed by Biomolecular Interaction Analysis with recombinant ADAM10 fragments and Western blot analysis of
ADAM10 KO mouse embryonic fibroblasts expressing human ADAM10 and derived substrate recognition site mutants.
Functional analysis demonstrates that mAb 8C7 is rapidly internalised into ADAM10 expressing cells, and reveals 8C7 dose-
dependent attenuation of Eph+ cell segregation away from surfaces of high ephrin density, a model considered a paradigm of
Eph function5. In addition, 8C7 treatment of tumour cells dampens Eph and EGF receptor phosphorylation, suggesting its
capacity to modulate RTK downstream signalling. Together, our results illustrate the potential of our mAbs for further
development as novel anti-cancer therapeutics.

1.        Moss, M. L., White, J. M., Lambert, M. H. & Andrews, R. C. TACE and other ADAM proteases as targets for drug
     discovery. Drug Discov Today 6, 417-426 (2001).
2.        Murphy, G. The ADAMs: signalling scissors in the tumour microenvironment. Nat Rev Cancer 8, 929-941 (2008)
3.        Janes, P. W. et al. Adam Meets Eph: An ADAM Substrate Recognition Module Acts as a Molecular Switch for Ephrin
     Cleavage In trans. Cell 123, 291-304 (2005)
4.        Janes, P. W. et al. Cytoplasmic relaxation of active Eph controls ephrin shedding by ADAM10. PLoS Biol 7,
     e1000215 (2009)
5.        Knoll, B., Weinl, C., Nordheim, A., and Bonhoeffer, F. (2007) Stripe assay to examine axonal guidance and cell
     migration, Nat. Protocols 2, 1216-1224


Can Dicer explain decreased microRNA expression in lymph node metastases of triple
negative breast cancer?
Kelly A Avery-Kiejda1 , Stephen G Braye2 , John F Forbes3 4 , Rodney J Scott1 2
1. Hunter Medical Research Institute, University of Newcastle, New Lambton Heights , NSW, Australia
2. Hunter Area Pathology Service, John Hunter Hospital, New Lambton Heights, NSW, Australia
3. Australian New Zealand Breast Cancer Trials Group, Department of Surgical Oncology, Calvary Mater Newcastle Hospital,
      Waratah, NSW, Australia
4. School of Medicine and Public Health, University of Newcastle, Newcastle, NSW, Australia
Breast cancer is the most common malignancy that develops in women, responsible for the highest cancer-related death rates
worldwide. Triple negative breast cancers represent a clinically important breast cancer subtype that have an aggressive
clinical phenotype, are associated with a higher likelihood of metastasis and are not responsive to current targeted therapies.
miRNAs have emerged as an attractive candidate for biomarkers and treatment targets in breast cancer, but their role in the
progression of triple negative breast cancer remains largely unexplored. In addition, reduced expression of the enzyme
responsible for miRNA production, Dicer, has been associated with shorter metastasis-free survival in breast cancer and with
the triple negative subtype. However, the role of Dicer in the progression of triple negative breast cancer is unknown.
Our previous results have shown that the repertoire of miRNAs expressed in lymph node negative and lymph node positive
breast cancers are largely distinct from one another. In particular, miRNA profiles associated with lymph node negativity tended
to be up-regulated, while those associated with lymph node positivity were down-regulated and largely overlapped with the
profiles of their matched lymph node metastases. This study has investigated if Dicer expression is altered in the progression of
triple negative breast cancer from normal to primary breast cancer to lymph node metastases. For this analysis, we have
examined the mRNA expression of Dicer by real-time PCR in 31 primary triple negative breast cancers (with known lymph node
status) and in 13 matched lymph node metastases compared with 23 matched normal breast tissues. The results of this study
are currently being analysed and will be presented. This study will provide novel insight into the regulation of miRNAs that
contribute to the progression of a triple negative breast cancer to a metastasis.


Association of Kallikrein gene variants with metastatic prostate cancer
Jyotsna Batra1 , Louise Marquart2 , Felicity Lose2 , Srilakshmi Srinivasan1 , Rosalind A Eeles3 , Douglas F Easton4 , The
Australian Prostate Cancer Bioresource5 , The PRACTICAL consortium, Amanda B Spurdle2 , Judith A Clements1
1. Australian Prostate Cancer Research Centre-Qld and Cancer Program, Queensland University of Technology, Brisbane,
      QLD, Australia
2. Queensland Institute of Medical Research, Brisbane, QLD, Australia
3. Section of Cancer Genetics, The Institute of Cancer Research & Cancer Genetics Unit, The Royal Marsden NHS
      Foundation Trust, London and Surrey, UK
4. Cancer Research UK Genetic Epidemiology Unit, Strangeways Laboratory, Worts Causeway, Cambridge, UK
5. QUT, Kelvin Grove, QLD, Australia
Human tissue kallikreins (KLK), encoded by the largest contiguous cluster of protease genes located on chromosome 19q13.4,
are secreted serine proteases with diverse expression patterns and physiological roles. Certain KLKs are implicated in many
cancer-related processes such as invasion, proliferation, and metastasis, by acting individually or in cascades with other KLKs
or proteases. In addition, they have potential or known applicability as biomarkers e.g. PSA/KLK3 for prostate cancer. We
participated in a three stage genome-wide association study as a part of the PRACTICAL consortium, where a SNP
(rs2735839) located close to KLK3 was found to be significantly associated with PrCa risk. Fine-mapping studies identified
KLK3 nonsynonymous exonic SNP rs17632542 as the causal SNP underlying the association, and a large validation study of
25,000 PrCa patients and 25,000 healthy male controls carried out with the COGS consortium has refined the risk associated
with the SNP to be OR=0.74, 95%CI=0.70-0.78. In addition, we have previously reported a significant association of KLK3,
KLK14 and KLK15 SNPs with PrCa aggressiveness as defined by Gleason scores. To analyse the association of KLK SNPs
with PrCa metastasis, we undertook a case-case analysis. Out of 17,680 patients with available data on PrCa seer-staging, 883
were identified with metastatic disease. Twenty-four SNPs within the KLK region previously found to be associated with risk or
aggressiveness of PrCa were genotyped using Illumina infinium assay as a part of COGS chips. We identified rs1058205,
rs17632542 and rs2735839 in KLK3 to be significantly associated with metastasis (p<0.01), where most significant results were
observed for the rs17632542 (OR=1.5, 95%CI=1.3-1.8). These results are interesting as they indicate that while KLK3 minor
allele has a protective role in PrCa predisposition, it is more prevalent in metastatic disease. We are now undertaking the
suitable functional assays to validate the results of our prediction modelling. Our preliminary data suggested that the KLK3 non-
synonymous SNP might affect protein stability, and thus could be the causal variant at the kallikrein locus to be associated with
PrCa risk, aggressiveness and metastasis.


Chromosomal Instability in BRAF Mutant, Microsatellite Stable Colorectal Cancers
Catherine E Bond1 , Aarti Umapathy1 , Ron L Buttenshaw1 , Leesa Wockner1 , Barbara A Leggett1 , Vicki LJ Whitehall1
1. Queensland Institute of Medical Research, Herston, QLD, Australia
BRAF mutant colorectal cancers can be stratified according to the presence of microsatellite instability (MSI). BRAF mutant
cancers that are MSI, confer an excellent prognosis and are well described. In contrast, microsatellite stable (MSS), BRAF
mutant cancers are aggressive and correlate with a poor prognosis, however the molecular mechanisms underlying this are not
well understood. Chromosomal instability (CIN) is commonly observed in BRAF wild type, MSS cancers and associates with a
worse prognosis, whilst BRAF mutant, MSI cancers are diploid. We hypothesized that CIN would be frequent in BRAF mutant,
MSS                                                                                                                  cancers.
Presence of CIN was investigated in 60 BRAF mutant/MSS (BRAFmut/MSS), and 90 BRAF wild type/MSS (BRAFwt/MSS)
using loss of heterozygosity (LOH) analysis encompassing chromosomal regions 5q, 8p, 17p and 18q. Genome-wide SNP
arrays (Illumina CytoSNP-12) were conducted on a subset of MSS cancers and compared to 30 BRAFmutant/MSI cancers.
LOH analysis demonstrated comparably high rates of CIN in the MSS subgroups (72% BRAFmut/MSS, 82% BRAFwt/MSS). In
BRAFmut/MSS cancers, CIN correlated with advanced stage, lymph node involvement, distant metastases and worse survival
at specific chromosomal regions. SNP arrays confirmed similarly frequent rates of CIN in both MSS cohorts, with 60%
BRAFmut/MSS and 62% BRAFwt/MSS chromosomal arms affected. BRAFmut/MSI cancers had a low degree of CIN (13%). Of
the regions with copy number variation, loss events were predominant over gain in both MSS cohorts (BRAFmut/MSS
533/736,72%; BRAFwt/MSS 279/389,72%). BRAFwt/MSS cancers mostly demonstrated whole chromosomal arm loss events
(185/279,66%), whereas BRAFmut/MSS cancers showed primarily regional loss events (336/533,63%)(p<0.0001).
BRAFmut/MSS cancers had significantly greater rates of loss at 17q, 6p and 6q compared to BRAFwt/MSS cancers (p=0.002,
p=0.009,                                                 p=0.04                                                 respectively).
This study has identified a novel form of genetic instability associated with BRAF mutant/MSS cancers primarily involving
regional rather than whole arm deletions. High rates of loss were identified in regions not typically implicated in colorectal
cancer, which may contain putative tumour suppressor genes associated with the aggressiveness of this cancer type.

Nanoparticles, cobalt complexes and tumour activatable pro-drug peptides: Hypoxia targeting
strategies to inhibit tumour growth and metastasis
Nicole S Bryce1 , Binh Pham1 , Byung J Kim1 , Jenny Z Zhang1 , Natsuho Yamamoto1 , Nicole Fong1 , Nirmesh Jain1 ,
Brian S Hawkett1 , Trevor W Hambley1
1. School of Chemistry, The University of Sydney, Sydney, NSW, Australia
The cellular environment in a solid tumour promotes the growth of cancer cells that have adapted to the unique conditions
present in tumours, such as low levels of oxygen (hypoxia). Cells exposed to this harsh environment over time are more
migratory, invasive and can also develop both radio and chemo-resistance. Effectively killing cancer cells or inhibiting cell
migration within the hypoxic tumour microenvironment is expected to reduce the metastatic potential of the solid tumour. By
specifically targeting the hypoxic cells, we aim to reduce the metastatic potential of the solid tumour and provide new
therapeutic agents to be used in conjunction with other anti-cancer compounds that target the proliferative cells.
We have developed a 3-dimensional cell culture spheroid model of an avascular colon tumour that develops hypoxic regions
that are easily distinguishable by live cell fluorescent microscopy. Several approaches have been developed to target hypoxic
areas in the tumour model including: cobalt based hypoxia targeted compounds, peptides that are not taken up by cells until
cleaved by a tumour specific enzyme and enhancement of drug diffusion by nanoparticles. We show through a combination of
live cell microscopy, fluorescence lifetime imaging and cellular assays, that we can successfully manipulate the physico-
chemical properties of the molecule/nanoparticle to enhance drug accumulation within hypoxic areas of the 3D spheroid. The
development of hypoxia-targeted compounds represents an alternate and complementary use of chemotherapy which is to
target the cancer cells with a high potential to metastasize rather than the most proliferative.


Association of β-catenin with HPV Surrogate Marker p16 and Lymph Node Metastasis in
Oropharyngeal Carcinoma
Zhuo Georgia Chen1 , Zhong Liang Hu1 , Susan Muller1 , Jing Xu1 , Dongsheng Wang1 , Dong M Shin1
1. Emory University, Atlanta, GA, United States
Incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has increased in
recent years. HPV-associated OPSCC follows a distinct carcinogenic pathway from the HPV negative OPSCC. The molecular
basis of disease progression due to HPV remains to be elucidated. Since clinic observation has revealed frequent and early
lymph node metastasis and unusual distant metastasis in HPV-associated OPSCC as compared with HPV negative OPSCC,
we would like to study whether β-catenin, one of the major proteins in the Wnt pathway and in regulation of epithelial-
mesenchymal transition (EMT), is involved in HPV-associated OPSCC metastasis. The study samples (n = 209) were collected
from formalin-fixed and paraffin-embedded or fine-needle aspiration OPSCC tissues. Expressions of p16 as a surrogate marker
for HPV-associated cancer and β-catenin in these samples were determined by immunohistochemistry (IHC) analyses. Positive
expression was defined as greater than 70% of tumor cells showing immunoreactivity for p16. Membrane expression of β-
catenin was scored as a weight index (WI) = intensity (0, 1+, 2+, and 3+) x positive staining (%), where the membrane intensity
on normal epithelium served as an internal positive control (3+). Nuclear staining of β-catenin was quantified as either positive
or negative. Our results showed that p16 expression in OPSCC was significantly correlated with males (p<0.001), Stage III and
IV (p = 0.009), undifferentiated phenotype (p=0.05), and positive lymph node (p<0.001). Membrane WI of β-catenin was
inversely correlated with p16 positive OPSCC (p < 0.001) and positive lymph nodes (p = 0.035), while nuclear staining of β-
catenin was associated with p16 positive OPSCC (p = 0.007). Low level of membrane β-catenin (cut-off by the median value)
was significantly associated with disease free survival and overall survival (p < 0.05 in both cases). Our study suggested that
cytoplasmic and nuclear localization of β-catenin is associated with p16 and therefore, HPV status and lymph node metastasis.
Molecular study on the regulation of β-catenin by HPV oncogenes E6 or E7 and the role of β-catenin in metastasis of OPSCC is
ongoing in our laboratory. (This study was supported by US National Institutes of Health grant HHSN2610201000125C and
Georgia Cancer Coalition Distinguished Cancer Scholar Award to GZC).


Inhibition of EGFR/Her-2 affects growth of ascitic ovarian cancer in vitro study
Kenny Chitcholtan1 , Peter Sykes1 , John Evans1
1. University of Otago, Christchurch, Chch, New Zealand
Elevated expression of epidermal growth factor receptor (EGFR) and Her-2 has been shown to correlate with poor survival
outcomes in women with ovarian cancers. Gefitinib (EGFR inhibitor) and canertinib (EGFR/Her-2 inhibitor) were studied in
clinical trials with recurrent and refractory ovarian cancers, but clinical responses were limited. Therefore, the use of anti-EGFR
inhibitors was not considered the frontline treatment in ovarian cancers. Advanced ovarian cancers often cause the
accumulation of body fluid (ascites) in the abdominal cavity. Ascites carrying ovarian cancer cells is a main route of cancer cell
metastasis. Therefore, inhibition of ascitic ovarian cancer may reduce the metastatic potential and secondary growth. We are
particularly interested in growth inhibition by gefitinib and canertinib in ascitic ovarian cancer cells. We hypothesized that
ovarian cancer cells that over-expressed EGFR and Her-2 were susceptible to gefitinib and canertinib. Therefore, the cells
would exhibit significant reduction in the growth and survival in the presence of the drugs compared with cancer cells that had
low expressions of the protein receptors. OVCAR-5, SKOV-3, and OVCAR-4 cell lines were cultured in culture plates with non-
adherent surface to mimic ascitic ovarian cancer cells. Cancer cells were exposed to inhibitors for 48 hours. Cell proliferation,
apoptosis, glucose uptake, activation of EGFR, Her-2, Akt, Erk, and cell adhesion were investigated. Results showed that
gefitinib and canertinib significantly inhibited cell growth. Canertinib was a more potent inhibitor than gefitinib for reduction of
proliferation, glucose uptake and adhesion, and canertinib induced an increase of apoptosis in SKOV-3 cell line. The responses
to gefitinib and canertinib were strongly associated with elevated expression of EGFR and Her-2. In conclusion, we suggest
that targeting both EGFR and Her-2 can produce profound inhibitory effects in ascitic ovarian cancer. In addition, pre-screening
the status of EGFR/Her-2 expression in a patient is essential to obtain clinical benefits from anti-EGFR inhibitors.


Investigating the anti-proliferative activity of ginger-derived substances
James A da Silva1 , Amanda B Becceneri1 , Angelina M Fuzer1 , Júlio César C Filho1 , Ana Carolina Baptista M Martin1 ,
João B Fernandes1 , Paulo C Vieira1 , Marcia R Cominetti1
1. UFSCar, São Carlos, SP, Brazil
Several population-based studies indicate that people in Asian countries have a much lower risk of different cancer types when
compared to their Western counterparts. It is widely claimed that constituents of their diet such as ginger, garlic, soy, curcumin,
onion, tomatoes, cruciferous vegetables, chillies, and green tea contribute to that lower incidence 1. Therefore, there is
increasing interest in naturally occurring cancer chemopreventive agents. Ginger (Zingiber officinale Roscoe) is widely used
worldwide as a food, spice and herb2. Gingerols comprise a series of homologue substances differentiated by the length of
their alkyl chains with [6]-, [8]- and [10]-gingerol having 10, 12 and 14 carbons in their unbranched alkyl chains, respectively 3.
The aim of this study was to perform an optimization of [6]-, [8]- and [10]-gingerol isolation and purification and to determine
their anti-proliferative activities against tumor cells (MDA-MB-231) and non-tumor cells (Human Fibroblasts – HF). [6]-, [8]- and
[10]-gingerol inhibited the proliferation of MDA-MB-231 tumor cell line with IC50 of 666.2±134.6 µM, 135.6±22.6 µM and
12.1±0.3 µM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations
starting from 500 µM.
Conclusion: Our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against
tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side
effects when compared to conventional chemotherapy. Assays to investigate the function of these substances in processes of
cell adhesion, migration and invasion are currently being performed. Migration and invasion inhibition of MDA-MD-231 and FH
cells in the presence of gingerols will be tested in standard transwell migration chambers and/or in a wound healing assay.
Financial Support: FAPESP, CAPES

1.       T. Dorai, B.B. Aggarwal, Cancer Lett, 215 (2004) 129-140.
2.       S. Dugasani, M.R. Pichika, V.D. Nadarajah, M.K. Balijepalli, S. Tandra, J.N. Korlakunta, J Ethnopharmacol, 127
     (2010) 515-520.
3.       S. Sang, J. Hong, H. Wu, J. Liu, C.S. Yang, M.H. Pan, V. Badmaev, C.T. Ho, J Agric Food Chem, 57 (2009) 10645-


Understanding metastatic breast cancer through an autopsy series
Margaret C Cummings1 2 , Peter T Simpson2 , Lynne Reid2 , Janani Jayanthan2 , Louise Marquart3 , Peter O'Rourke3 ,
Sunil R Lakhani1 2
1. Pathology Queensland, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia
2. University of Queensland, UQ Centre for Clinical Research, Herston, Queensland , Australia
3. Queensland Institute of Medical Research, Herston, Queensland, Australia
Patients who die of breast cancer almost always die of metastatic disease rather than of complications of the primary tumour.
Understanding the biology of metastasis is therefore fundamental to treating these patients, although it has long been assumed
that treatment of the secondary tumours should be determined by characteristics of the primary tumour. Using material
obtained from autopsies from a series of 204 patients who died of metastatic breast cancer and for whom treatment details are
available, we have been able to learn about the natural history of metastatic breast cancer, particularly the heterogeneity of the
disease, and its clonal and phenotypic evolution. We have evaluated clinicopathological variables associated with metastatic
disease, including the nature of the primary tumour compared with tumours from different metastatic sites within individuals.
With nearly 1,200 metastases available to study, we have analysed the distribution of disease, including the frequency of
metastases at various sites, with lung, liver and bone being the most common, and an average of 5 - 6 metastatic sites per
patient. Immunoprofiling for ER, PR, Her2, Ki67, c-kit, EGFR and p53 was performed on all patients for whom the primary
breast cancer (57) was available. Relative stability of Her2 status was observed between the primary tumour and metastases at
different sites, whereas ER and PR expression was more variable. Genomic analysis was undertaken on a subset of patients
and identified DNA copy number aberrations that are consistently altered in all lesions of a case (primary and metastases), and
alterations found in metastases only, providing evidence of clonal evolution during metastatic progression. In summary,
analysis of this extraordinarily rare and well characterised clinicopathological autopsy resource is providing substantial insight
into the nature of metastatic spread of breast cancer and will be important for informing treatment decisions.


SRF methylation in myofibroblasts predicts less metastasis of non-cardiac gastric carcinomas
in China, Japan, and Korea
Zhaojun Liu1 , Chen Yang1 , Jiafu Ji1 , Wooho Kim2 , Toshikazu Ushijima3 , Dajun Deng1
1. Peking University Cancer Hospital and Institute, Beijing, China
2. Pathology, Seoul National University College of Medicine, Seoul, Korea
3. Carcinogenesis, National Cancer Center Research Institute, Tokyo, Japan
[Background & Aims] SRF promotes growth and metastasis of cancer cells. The aim is to determine whether SRF is inactivated
by methylation of CpG islands and can be used as a biomarker to predict metastasis of gastric carcinoma (GC).
[Methods] 356 Chinese, 78 Japanese, and 152 Korean GC patients followed up for at least 3 years were included in the
present study. Methylation status of SRF CpG islands in GC and the corresponding surgical margin (SM) samples was
determined with DHPLC, MethyLight, and bisulfite sequencing. Myofibroblasts were identified with aSMA-immunostaining.
Relationships between SRF methylation and GC metastasis and patients’ overall survival were analyzed. [Results] Methylated-
CpG cluster was mainly characterized within the SRF exon-1 in myofibroblasts in GC or SM. Proportion of the methylated SRF
inversely correlated with its transcription activity (P=0.021). More SRF methylation-positive SM samples were seen in non-
metastatic-GC patients than metastatic-GC patients in the training cohort (n=199), and consistently confirmed in the testing
cohort in China (n=157) and two independent validation cohorts in Japan and Korea, respectively. Moreover, SRF methylation-
positive patients with non-cardiac GC had longer overall survival than SRF methylation-negative cases not only in the training
cohort [multivariate analysis, hazard ratio=0.479 (P=0.023)], but also in the testing and validation cohorts among non-cardiac
GC patients from China [0.469 (P=0.016)], Japan [0.547 (P=0.045)], and Korea [0.461 (P=0.031)]. The protective effect of SRF
methylation on the followup- metastasis was prospectively observed among 144 baseline-non-metastatic-GC patients
(P=0.045). [Conclusion] DNA methylation inactivates SRF expression in stromal myofibroblasts and is a useful predictor of
metastasis and overall survival of non-cardiac GC patients.


The highly selective focal adhesion kinase inhibitor CTx-0294945 reduces tumour cell growth
in an experimental metastasis model.
J Doherty1 2 , A Natoli1 2 , J Schreuders1 2 , L Allan2 3 , R Anderson1 2 , Y Bergman2 4 , M Camerino2 3 , S Charman2 4 , N
Choi2 4 , T Connor2 3 , M de Silva2 3 , H Falk2 3 , R Foitzik2 4 , D Ganame2 3 , M Gorman2 5 , A Gregg2 4 , C Hemley2 3 , G
Holloway2 3 , W Kersten2 3 , K Lackovic2 3 , R Lessene2 3 , K Leuchowius2 3 , G Lovrecz2 6 , G Lunniss2 4 , G McArthur1 2 ,
N McKern2 5 , B Monahan2 4 , B Morrow2 4 , M Nikac2 4 , P Novello2 3 , M Parker2 5 , T Peat2 6 , C Scott2 7 , M Tiong2 3 , K
Visser1 2 , S Walker2 4 , H Yang2 3 , I Holmes2 , M Devlin1 2 , I Street2 3
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Cancer Therapeutics CRC Pty Ltd, Melbourne, Vic, Australia
3. Division of Chemical Biology, The Walter & Eliza Hall Institute of Medical Research, Melbourne, Vic, Australia
4. Monash Institute of Pharmaceutical Sciences, Melbourne, Vic, Australia
5. St Vincent's Institute, Melbourne, Vic, Australia
6. CSIRO MSE, Melbourne, Victoria, Australia
7. Molecular Genetics of Cancer Division , The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria,
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that represents a central point of convergence for many
signalling pathways implicated in cancer progression and metastasis. Evidence from pre-clinical models suggests that inhibition
of FAK activity in both stromal and tumour cells will aid in the prevention of the growth and metastasis of human cancers. CTx-
0294945 is an orally bioavailable small molecule ATP competitive inhibitor of focal adhesion kinase (FAK KD=0.21 nM). It
exhibits high selectivity against a diverse panel of 125 kinases including the closely related Pyk2 (Pyk2 IC50=550 nM). CTx-
0294945 inhibits autophosphorylation of 397Y-FAK in MDA-MB-231 cells with an IC50 = 7 nM. The compound exhibits low
general cellular toxicity (IC50 = 6.6 µM) against MDA-MB-231 cells grown under standard conditions. CTx-0294945 is suitable
for       oral      administration       (%F=58        and          t1/2=5.1     h      at     20       mg/Kg       in       rat).
The ability of CTx-0294945 to prevent the outgrowth of micro metastases was assessed in a model of disseminated basal
breast cancer. Luciferase tagged MDA-MB-231 cells (105) were injected into the left ventricle of the heart of BalbC SCID mice
(n = 9 per group) and treatment with vehicle (hydroxypropylmethylcellulose), or CTx-0294945 (20 mg/kg, 40 mg/kg or 80 mg/kg
p.o. QD) was commenced the day after injection of cells. The distribution and growth of metastatic lesions was quantitated by
bioluminescent imaging over the course of the experiment. The model exhibited prominent metastasis to the jaw as well as
knee joints and internal organs and mice exhibited marked body weight loss that correlated with the strength of the whole body
BLI signal. Mice were euthanized when weight loss was ≥ 20% of starting weight or when they displayed overt signs of
metastatic disease. CTx-0294945 dose-dependently decreased the rate of metastasis growth based on whole body BLI and
increased survival in this aggressive model of tripe-negative breast cancer metastasis. The results recommend further
investigation of CTx-0294945 alone and in combination with other targeted and chemotherapeutic agents for the prevention
and treatment of breast cancer metastasis.


Breast Cancer Network Australia - Working with you for results
Jennifer Johnston1 , Niki Aravanis1 , Kathy Wells1 , Nicca Grant1
1. Breast Cancer Network, Kew East, VIC, Australia
Breast Cancer Network Australia (BCNA) is the peak national organisation for Australians personally affected by breast cancer.
We empower, inform, represent and link together people whose lives have been affected by breast cancer. BCNA represents
more than 70,000 individual members and 300 Member Groups from across Australia.
BCNA’s Seat at the Table program is an internationally recognised program that recruits, appoints, trains and supports breast
cancer survivors as consumer representatives. Seat at the Table Consumer Representatives are actively supported by BCNA
throughout their appointment. Feedback is sought from the women, and clinicians involved, to inform evaluation and program
BCNA Consumer Representatives have worked together with scientists, researchers, clinicians, health service providers and
planners since 2000 to improve the provision of services to those affected by breast cancer. The program currently has more
than 75 trained Consumer Representatives including women with secondary breast cancer.
BCNA’s Consumer Representatives can contribute to the work of committees by:
• representing women with breast cancer to ensure their voices are heard
• assisting with trial ethics and consent documents
• supporting applications for research funding
• assisting with recruitment for scientific trials
• raising awareness of research amongst women and the public
Key program successes include:
• the assistance of a BCNA consumer representative in the writing of information for patients considering participation in the
Royal Australasian College of Breast Surgeons SNAC1 trial, which helped to make the trial a fantastic success
• BCNA Consumer Representative on the Royal Australian and New Zealand College of Radiologists – Quality Use of
Diagnostic Imaging Program Management Committee received the College’s ‘Distinguished QUDI Service Award’
Current appointments include:
• National Breast Cancer Foundation EMPathy Breast Cancer Network Management Committee
• Cancer Australia Working Group on the management of central nervous metastases in women with secondary breast cancer
• Peter MacCallum Cancer Centre NHMRC Translation of PALB2 genetic information into breast cancer genetics services
This poster will present information on how BCNA’s Seat at the Table program operates, key successful Consumer
Representative appointments, and current appointments.


Evaluation of Surgical treatment of metastatic spinal tumors
Wei Guo1
1. Peking University,People' Hospital, Xicheng District, Beiji, China
Objective Evaluation of the indication and results of surgical treatment of metastatic spinal tumors. Method From July 2000
through July 2005, 167 patients (71 women and 96 men) with metastatic spinal tumors had been treated by authors. The
thoracic vertebra was involved in 99 of them, lumbar vertebra in 53, cervical region in 15. Among 112 of 167 patients who
presented with neurological dysfunction, 28 patients were completely paralyzed and the others were incompletely paralyzed. All
patients in this group, were performed surgical treatment by anterior or poterior approach. Result Pain relief was obtained in
155 of 167 patients (92.8%) and neurological improvement was obtained in 67 of the112 patients. The follow-up time ranges
from 12 to 60 months. The over all one-year survival rate was 70% (117 cases). Conclusion In order to alleviate paralysis,
improve spinal stability and the quality of life, urgent decompression should be performed on the patient with spinal metastasis.


YKL40 as a therapeutic target for the treatment of castration resistant prostate cancer
Varinder Jeet1 , Colleen Nelson1
1. Queensland University of Technology, Woolloongabba, Qld, Australia
Prostate cancer (PCa) is the most commonly diagnosed lethal cancer and the second leading cause of cancer-related deaths in
Australian men with a total of 20,000 new cases and 3,300 deaths annually. Androgen Deprivation Therapy (ADT) is the
mainstay therapy for advanced PCa and leads to 1-3 years of remission, but the disease ultimately progresses to a stage of
castration resistant PCa (CRPC) which is currently incurable. Angiogenesis plays a fundamental role in promoting tumour
growth and thereby anti-angiogenesis therapy has been seen as a promising approach for treating patients with CRPC disease.
Our focus in this project is to explore the biological significance of a potential angiogenic target, YKL40. Methods: YKL40
mRNA and protein expression profiles were analysed in different PCa cell lines representing progressive stages of human
disease. siRNA mediated YKL40 specific knockdown was performed in LNCaP, C42, and C42-B cell lines and the effects on
markers of angiogenesis, metastasis, and radioresistance were analysed by real time PCR and western blotting. Results: The
results show that gene and protein expression of YKL40 increases in cell lines representative of advanced disease as
compared to less aggressive cancer. Moreover, cells treated with YKL40 siRNA show downregulation of classical markers of
angiogenesis and metastasis, such as VEGF-A, angiogenin and MMP-9. However, the expression of E-cadherin increases
after YKL40 knockdown. We have also observed that YKL40 regulates the expression of NNMT, a gene overexpressed in a
number of cancers, and is involved in radioresistance. Conclusion: Our preliminary results suggest the potential role of YKL40
in processes related to cell survival and growth and warrants further investigation. This has the potential to lead to a new
targeting approach for therapeutic intervention in advanced therapy-resistant prostate cancers that have thus far proved difficult
to treat.


Fibroblast Growth Factor Receptor-2 Mutations in Endometrial Cancer
Yvette Jeske1 , Sara Byron2 , Mike Gartside2 , Amy Schmidt3 , Nilsa Ramirez4 , Paul Goodfellow3 , Pamela Pollock1
1. Institute of Health and Biomedical Innovation (IHBI) , kelvin grove, QLD, Australia
2. Translational Genomics Institute (TGEN), Phoenix, Arizona, USA
3. Siteman Cancer Centre and Washington University School of Medicine, St Louis, Missouri, USA
4. Nationwide Children’s Hospital, Columbus, Ohio, USA
Germ line gain-of-function FGFR mutations lead to a variety of developmental skeletal disorders. Many of these same gain-of-
function mutations lead to various types of cancer when arising somatically. Mutations in FGFR2 have been identified in ~12%
of endometrial carcinomas (EC) and many of these mutations have been shown to be oncogenic when examined in vitro (1-3).
Our group previously sequenced FGFR2 in 466 primary tumours obtained from Washington University School of Medicine and
report that in early stage patients, mutations are associated with shorter Progression Free Survival (4). To validate these
findings we performed PCR-Sequencing of the known hotspot exons (7, 8, 10, 13, 15) in 933 clinically annotated ECs obtained
from the multi-institutional Gynaecological Oncology Group (GOG) tissue bank. We identified a total of 147 mutations in 143
patients (14.4%). The most common mutations were S252W, N550K, C383R and K660E known to be frequently mutated in EC
(1-4). We identified novel mutations in 14 patients:- V274I, F276E, V294M, V311I, A380S, A380T, Y382D, G385R, D627Y,
A629E, K660R, D651Y, Del.I388-M391 and Del.E378-C383.Ins.R. Four patients had known activating mutations in addition to
a novel mutation (S252W/ V294L, K660E/N653S and N550K/L551F x2). Preliminary analyses of novel mutations using known
FGFR2 3D-Structure and Online Mutation Assessment Software tools (Mutation Assessor, SIFT, Polyphen) indicate a likely
effect on protein function for all mutations except V311I and A380S. Further functional analysis of these novel mutations (cell
lines, 3D-modelling) is required to show that they are gain-of-function. Outcome analysis by the GOG statistical data centre is
1. Dutt, A., et al., Proc Natl Acad Sci U S A 2008, 105, (25), 8713-7.
2. Pollock, P. M., et al., Oncogene 2007, 26, (50), 7158-62.
3. Mohammadi, M., et al., Cytokine Growth Factor Rev 2005, 16, (2), 107-37.
4. Byron, S., et al., PLoS One 2012, 7, (2), e30801.


Engineering receptor based antagonists of the Gas6/Axl for therapeutic applications
Yu Miao1 , Mihalis S Kariolis2 , Douglas S Jones2 , Jennifer R Cochran2 , Amato J Giaccia1
1. Radiation Oncology, Stanford University, Stanford, California, United States
2. Bioengineering, Stanford University, Stanford, California, United States
AXL is a receptor tyrosine kinase that has been shown to be a major player during tumor metastasis. Recently, many studies
have defined AXL as a therapeutic target for metastatic disease. However, despite several therapeutic approaches including
small molecules and antibodies have been developed to inhibit AXL signaling, only moderate therapeutic efficacy in metastatic
in vivo models has been reported. In this study, by using directed in vitro evolution approach, we engineered a “super-binder”
AXL mutant protein with a superior binding affinity to the only known AXL receptor ligand GAS6, thus preventing ligand-
mediated activation of AXL receptor signaling. Compared to wild-type AXL/GAS6 binding, the super-binder mutant has a
enhanced improvement in the binding affinity to GAS6. When tested in vivo, this improvement in binding affinity was translated
into a significantly improved antitumor responses and survival in many metastatic tumor models including the SKOV3 and
OVCAR8 metastatic ovarian models as well as the 4T1 spontaneous metastatic breast cancer model. Subsequent analysis
also showed reduction in pAXL and pAKT expressions in tumors treated with the super-binder, indicative of successful
inhibition of the AXL signaling cascade. Furthermore, no tissue toxicity was observed in animals treated with the super-binder
protein. Collectively, we have engineered a mutant AXL protein with enhanced GAS6 binding affinity and improved therapeutic
outcome, which has implications for a number of metastatic diseases that currently lack effective treatments.


Antitumor Effects of Anti-podoplanin Antibody NZ-1 against Malignant Mesothelioma
Yasuhiko Nishioka1 , Shinji Abe2 , Yuki Morita3 , Mika kato Kaneko4 , Masaki Hanibuchi1 , Hisatsugu Goto1 , Soji
Kakiuchi1 , Yoshinori Aono1 , Jun Huang1 , Atsushi Mitsuhashi1 , Seidai Sato1 , Kazuo Minakuchi3 , Yukinari Kato4
1. Department of Respiratory Medicine and Rheumatology, Institute of Health Biosciences, The University of Tokushima
      Graduate School, Tokushima, Japan
2. Practice Room for Clinical Pharmacy, Institute of Health Biosciences, The University of Tokushima Graduate School,
      Tokushima, Japan
3. Department of Pharmacy, Tokushima University Hospital, Tokushima, Japan
4. Molecular Tumor Marker Research Team, Global COE Program, The Oncology Research Center, Advanced Molecular
      Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata, Japan
Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural
mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin antibody, NZ-1. However, the
direct anti-tumor activity against MPM has not been investigated. In the present study, we examined whether anti-podoplanin
antibody NZ-1 showed antitumor effects mediated by antibody-dependent cellular cytotoxicity (ADCC). We used human MPM
cell lines and tissues. Expression of podoplanin was examined by using flow cytometry and immunohistochemistry. ADCC
activity was measured by 51Cr-release assay. In vivo antitumor effects was examined in a xenograft model of human MPM cells
in SCID mice. MPM cell lines in 12/15 (80%) expressed podoplanin and MPM tissues also expressed high level of podoplanin.
NZ-1 showed high ADCC activity when rat NK cells were used as effector cells. Administration of NZ-1 with rat NK cells
significantly suppressed the growth of human MPM cells expressing podoplanin. These results suggest that podoplanin is a
promising target for immunotherapy of mesothelioma with anti-podoplanin antibody having ADCC activity.


The Role of IQGAP1 in Ovarian Cancer Progression
Sheri Nixdorf1 , Imad Ben Hmeda1 , James P Scurry2 , Jake Olivier3 , Neville F Hacker4 , Kerrie L McDonald5 , Viola A
Heinzelmann-Schwarz1 6
1. Ovarian Cancer Group, University of New South Wales, Kensington, NSW, Australia
2. Department of Anatomical Pathology, John Hunter Hospital, Newcastle, NSW, Australia
3. Biostatistics Group, University of New South Wales, Kensington, NSW, Australia
4. Gynaecological Cancer Centre, Royal Hospital for Women, Randwick, NSW, Australia
5. Cure for Life Foundation Neuro-Oncology Laboratory, University of New South Wales, Kensington, NSW, Australia
6. Gynaecological Oncology Research Group, University Hospital Basel, Basel, Switzerland
Rationale: Ovarian cancer has the highest mortality rate within all gynaecological cancers. Most undifferentiated serous
cancers (75%) present with advanced FIGO stage, characterised by a 5-year survival rate of only 30%. To improve this
outcome, there is an urgent need to further elucidate the underlying molecular processes of ovarian cancer development and
progression to aid in the identification of reliable biomarkers with diagnostic, prognostic and therapeutic potential. The IQ motif
containing GTPase activating protein 1 (IQGAP1) is involved in cell proliferation, adhesion, migration and angiogenesis.
Dysregulated protein expression was observed in various cancers including gastric, colon and endometrial cancer. Additionally,
we previously identified IQGAP1 as a marker of progression and survival in high grade glioma patients. We hypothesised that
in ovarian cancer, IQGAP1 expression would correlate with disease progression.
Methods: IQGAP1 protein expression and cellular localisation was examined in a cohort of 721 ovarian cancer patients and
healthy controls using immunohistochemistry (IHC). Additionally, we screened a panel of ovarian cancer cell lines by RT-qPCR,
Western Blotting and IHC to ascertain IQGAP1 expression in vitro.
Results: Strong IQGAP1 protein expression was observed within the cell membrane and was also present to a lesser degree
within the cytoplasm, in both tissues and cell lines. Over-expression of IQGAP1 was noted in metastatic tumours when
compared to their matched primary lesion. Within the cell lines, no significant alteration was observed at the mRNA or total
protein level; however, increased plasma membrane expression of IQGAP1 was associated with the more aggressive serous
subtype. IQGAP1 expression was also found to correlate significantly to members of the Wnt-signalling pathway.
Conclusions: This study indicates that IQGAP1 is a potential new marker for ovarian cancer progression to metastasis, and
suggests that IQGAP1-targeted therapies may be useful for treatment of advanced ovarian cancers.


Zoledronic acid facilitates large scale ex-vivo expansion of human γδ T cells for use in
immunotherapy targeting bone metastases
Vasilios Panagopoulos1 , Irene Zinonos1 , Vasilios Liapis1 , Shelley Hay1 , Mark O DeNichilo1 , Vladimir Ponomarev2 ,
Andrew CW Zannettino3 , Andreas Evdokiou1
1. Discipline of Surgery, Breast Cancer Research Unit, Basil Hetzel Institute and Centre for Personalised Cancer Medicine,
      University of Adelaide, Adelaide, South Australia, Australia
2. Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, USA
3. Myeloma and Mesenchymal Research Laboratory, Bone and Cancer Laboratories, Division of Haematology, Hanson
      Institute, Adelaide, South Australia, Australia
Human γδ T cells comprise a small percentage (1-5%) of circulating lymphocytes and exert potent cytotoxicity against a broad
range of tumour cells in a major histocompatibility complex unrestricted manner, thus creating an attractive candidate for
cancer                                                                                                             immunotherapy.
Gamma-delta T cells can be activated and expanded upon treatment with nitrogen containing bisphosphonates such as
zoledronic acid (ZOL), an agent widely used clinically to treat malignancy-associated bone loss. We and others have shown
that ZOL inhibits the mevalonate pathway leading to the accumulation of phosphoantigens which are recognized by γδ T cells
resulting in a significant increase to γδ T cell mediated cytotoxicity of tumour cells. Human γδ T cells expressing CD16 (FcγRIII)
may be used in conjunction with the tumour targeting monoclonal antibody drozitumab, which targets the signalling death
receptor DR5 for apoptosis induction. In this study we facilitated large scale ex-vivo expansion of cytotoxic γδ T cells using ZOL
for use in adoptive immunotherapy and assessed the potential for γδ T cells to enhance antibody dependent cellular cytotoxicity
(ADCC). Peripheral blood mononuclear cells from patients were cultured with ZOL and IL-2 for 14 days to expand γδ T cells ex-
vivo. Phenotypic analysis and cytotoxicity assays were measured by flow cytometry and LDH release, respectively. Our results
demonstrate that ZOL facilitated ex-vivo expansion of γδ T cells up to 90% and exerted potent cytotoxicity against a variety of
cancer cells including breast, prostate, osteosarcoma, and multiple myeloma in vitro. In addition, pre-treatment of cancer cells
with low concentration of ZOL sensitized tumour cells to rapid killing by γδ T cells and cooperated with drozitumab for
enhanced cytotoxicity to levels reaching more than 90%. Adoptive transfer of γδ T cells has the potential for cell-based
immunotherapy, when used alone and in combination with drozitumab especially in cancer patients treated with ZOL.


Uncovering molecular mechanisms involved in metastasis to the brain
Jodi M Saunus1 , Majid Momeny1 , Leonard da Silva1 2 , Ana Cristina Vargas1 , Fares Al-Ejeh3 , Brian Morrison3 , Kum
Kum Khanna3 , Sean Grimmond4 , Georgia Chenevix-Trench3 , Rosalind L Jeffree5 , Nic Waddell4 , Peter T Simpson1 ,
Sunil R Lakhani1 2 6
1. The University of Queensland, UQ Centre for Clinical Research, Herston 4029, Qld, AUSTRALIA
2. University of Queensland School of Medicine, The University of Queensland, Herston 4029, Qld , AUSTRALIA
3. Queensland Institute of Medical Research, Herston 4029, Qld, AUSTRALIA
4. Queensland Centre for Medical Genomics, The Institute for Molecular Bioscience, University of Queensland, St Lucia
     4072, Qld, AUSTRALIA
5. Department of Neurosurgery, The Royal Brisbane and Women's Hospital, Herston 4029, Qld, AUSTRALIA
6. Pathology Queensland, The Royal Brisbane and Women's Hospital, Herston 4029, Qld, AUSTRALIA
Brain metastasis is a fatal complication of invasive cancer, affecting 25-40% of patients with advanced solid tumours (most
commonly lung, breast, melanoma, renal, and colon cancers). This is a major unmet clinical need associated with morbidity,
neurological decline and 100% mortality1. It is also a major economic burden (~$7B USD/year2), mainly due to the high costs of
clinical management and palliative care.
No targeted agents have been developed based on the direct study of clinical or experimental brain metastasis samples. Drug
development has been hampered by a lack of clinical specimens, the impermeability of the blood-brain-barrier (BBB) and an
historic view that development of brain metastases marks end-stage, incurable disease. In order to expand the available
therapeutic options, it is essential to understand more about the molecular pathways that enable the metastatic process. There
is a growing body of evidence that brain metastasis can be an early event in progression, implying parallel clonal evolution in
primary and secondary tumours, consistent with our previous observations that brain metastases are divergent from matched
primary breast tumours3. Focussing on processes that promote colonisation and regulate dormancy may therefore be the most
promising in terms of clinical translation.
Using gene expression array analysis of archival matched pairs of primary breast tumours and brain metastases, we
discovered that some metastases achieve activation of ErbB signalling by up-regulating HER33, raising the possibility that this
pathway could be targeted therapeutically. We are now performing functional validation of these findings using in vitro and
mouse models. We are also extending the breadth of the original studies to characterise the genomes and transcriptomes of a
discovery cohort of prospectively collected, fresh frozen human brain metastasis tissues from melanoma, breast and lung
cancer patients using next generation sequencing. Latest developments will be presented.

1.        Maher, E.A., et al. Cancer Res, 2009. 69(15): p. 6015-20.
2.        Nieder, C., et al. Oncology, 2010. 78(5-6): p. 348-55.
3.        Da Silva, L., et al. Breast Cancer Research, 2010. 12(4): p. R46.


Sub1 is a marker of aggressive breast cancer
Wei Shi1 , Fares Al-Ejeh1 , Jessie Jeffery1 , Peiyi Yap1 , Mariska Miranda1 , Kum Kum Khanna1
1. Signal Transduction Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia
RNA polymerase II cofactor PC4 was found highly expressed in more invasive breast cancer cell lines and up-regulated during
clinical progression of carcinoma in situ to invasive and metastatic carcinomas. Here we investigate the functional role of PC4
in breast cancer cell invasion and breast cancer progression. PC4 protein levels were elevated in invasive breast carcinoma
compared with normal breast epithelia. Analysis of microarray datasets for breast cancer survival cohorts showed that higher
SUB1 gene expression (mRNA) was associated with higher relapses in basal tumours with emphasis on brain relapses.
Consistently, we have observed up-regulated expression of PC4 in metastatic samples compared to primary tumours in
matched primary and brain metastasis breast cancer pairs. Depletion of PC4 in invasive breast cancer cell lines abolished cell
invasion. Collectively, these data identify PC4 as a potentially new marker in breast cancer progression and metastasis and
provide a rationale for novel therapeutic target.

1.        Financial Support: This work was supported by the 2011 Rio Tinto Ride to Conquer Cancer funding [to W.S.];
     National Health & Medical Research Council Program Grant [442903 to S.R.L and KK.K.] and Australian Research
     Council Grant [DP0880372 to KK.K.].


Prediction of Postoperative Venous Thromboembolism in Patients with Malignancy of the
Lower Extremity
Sang-Min Kim1 2 , Joong-Min Park1 , Seung Han Shin1 , Han-Soo Kim3 , Hwan Seong Cho4 , Sung Wook Seo1
1. Department of Orthopaedic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul,
2. Department of Orthopaedic Surgery, Pusan National University School of Medicine, Yangsan, Korea
3. Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Hospital,
     Seoul, Korea
4. Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Bundang
     Hospital, Seongnam, Korea
Background: Determination of patients at an elevated risk for VTE is important to prevent thromboembolic events. This study
aimed to investigate the incidence and risk factors of postoperative VTE in patients with malignancy of the lower extremity. Our
secondary aim was to examine the rates of VTE before and after introduction of chemical thromboprophylaxis.
Methods: One hundred and sixty-eight consecutive patients with bone or soft tissue malignancy of the lower extremity were
included in this study. The study period of 22 months included 10 months prior to and 12 months following the introduction of
chemical thromboprophylaxis. All information on potential risk factors were retrieved and classified into 3 types (patients-,
surgery, and tumor-related). The outcome measure was thromboembolic events within 90 days of surgery.
Results: Of the 168 patients, 8 (4.8%) had a confirmed symptomatic deep vein thrombosis and 1 (0.6%) had a fatal pulmonary
embolism. Of the twenty-eight variables tested for an association with venous thromboembolism, advanced age more than 60
years, higher American Society of Anesthesiologists grade, and metastatic tumor were independent risk factors. The overall
rates of symptomatic venous thromboembolism were not significantly different between the patients receiving chemical
thromboprophylaxis and those not receiving chemical thromboprophylaxis.
Conclusions: Increasing age, low physical status and metastatic tumor are at a particularly high risk of thromboembolism in
patients with bone or soft tissue malignancy of the lower extremity. We believe that knowledge of the above factors could
provide a valuable basis to improve surgical outcome of orthopedic oncologic patients.


Evidence for activation of signaling pathways that define the aggressive biology of TNBrCA in
atypical breast lesions from high-risk African American women
Victoria Seewaldt1 , Adria Suarez, Lee Wilke, Catherine Ibarra-Drenall
1. Duke University, Durham, NC, United States
Background: Triple-negative breast cancers [ER/PR-/-, HER2/neu wt] are frequently metastatic at diagnosis and at 5-years only
14% of African American women are alive. In addition to being poorly differentiated, triple-negative breast cancers frequently
have little or no associated DCIS. As a result, the precursor lesion for triple-negative breast cancer is poorly understood. Here
we aim to investigate whether the aggressive biology that defines triple-negative breast cancer exists in pre-malignant breast
lesions from high-risk African American women.
     Epithelial plasticity (EMT and the reverse process, MET) is hypothesized to play a key role in defining the aggressive
behavior of triple-negative breast cancer. The prevailing model is that epithelial triple-negative breast cancer undergoes EMT,
invades into the peripheral vasculature, and then undergoes the reverse process, MET, in order to develop distal metastasis.

     Stat3 is an inflammatory-kinase that predicts poor prognosis in triple-negative breast cancer, and is also regulated by ECM
signaling. Stat3 is known to promote EMT, stem cell regeneration, and induces a subclass of microRNAs [miR200/Let-
7/miR203/mi149] that promote EMT and invasion. Activated Stat3-pTyr705 heterodimerizes and transcriptionally regulates
Results: We performed high-resolution proteomic-profiling and immunohistochemical staining of premalignant breast lesions
from high-risk African American women that had contralateral triple-negative breast cancer; 100 cores were
tested/breast. Activation of Stat3/vimentin-signaling (proteomic profiling) and expression of vimentin/nuclear Snail-1 (IHC) was
first observed in the earliest identifiable lesions (columnar change and atypical hyperplasia). Combined miRNA and
proteomic profiling of mammary atypia from high-risk African American women identified activation of Stat3 network signaling
(IL6/Stat3/Akt/vimentin) and modulation of Stat3-regulated miRNAs.
Conclusions: We observe activation of Stat3/vimentin/miRNA-network signaling in mammary atypia from high-risk
women. These observations provide evidence that the signaling pathways that promote the aggressive phenotype of triple-
negative breast cancer may be activated in mammary atypia.


The Chemokine Receptor CXCR4 in Small Cell Lung Cancer (SCLC) and its role in metastasis
Sanaz Taromi1 , Julie Catusse, Stefan Wollner, Meike Burger
1. University Clinic, Freiburg in Breisgau, Germany
Small cell lung cancer (SCLC) is a highly aggressive and metastatic neuroendocrine carcinoma which progresses rapidly.
There is growing evidence that the chemokine receptor CXCR4 and its ligand CXCL12 are involved in migration and metastasis
of SCLC. As STAT3 and Rho GTPases have been shown to be involved in the CXCR4 signaling pathway, we are investigating
these pathways in SCLC cell lines and the potential therapeutic use of inhibitors of CXCR4 and/or downstream signaling
pathway components in SCLC metastasis processes in vitro and in vivo.
Analysing the CXCR4/CXCL12 axis we could demonstrate different STAT3 activation types in SCLC cell lines. STAT3 was
constitutively phosphorylated on both tyrosine and serine residues, which could be further increased upon CXCL12 stimulation
and was completely inhibited by TN14003, a CXCR4 antagonist and/or by CDDO-Me, a synthetic triterpenoid which inhibits
STAT3 directly. STAT3 tyrosine phosphorylation could also be inhibited by AG490, a Jak2 inhibitor. In addition to tyrosine
phosphorylation, STAT3 acetylation on a lysine residue has also been associated in the initiation of STAT3 dimerization.
CXCL12 stimulation of SCLC cells led to an increased STAT3 acetylation specific for the nuclear fraction.
STAT3 may also be involved in Rho GTPases mediated signalling cascades regulating cellular responses like actin
organization, proliferation and cell migration. We could show a strong CXCR4 mediated RhoA and Rac1 activation as potential
upstream signaling of STAT3 activation in SCLC cell lines. Further functional investigations (e.g. chemotaxis,
pseudoemperipolesis, and cell viability assessment) will reveal the involvement of STAT3 and Rho GTPases in CXCR4
mediated metastatic processes in SCLC.
To investigate the effects of CXCR4 inhibitors and inhibitors of downstream pathways on metastasis formation in vivo, an
orthotopic tumor mouse model is being established. Human SCLC cells are injected intrathoracically into the pleural space of
Rag2γc mice. Tumor engraftment can be observed using BL-Imaging, MRI and PET. Six weeks after inoculation of tumor cells
the animals are sacrificed in order to investigate metastasis formation in specific CXCL12 expressing organs.
To summarise, we have shown constitutive serine and tyrosine phosphorylation of STAT3 and CXCL12 induced regulation.
CXCL12 also induced acetylation of STAT3 and activation of RhoA and Rac1. These results are clarifying the CXCR4
downstream cascade in small cell lung cancer and underline the interest in inhibitors for this receptor and its targets in this
pathway for potential therapeutic use.

B7-H3 - a new metastasis-promoting protein
Vibeke Anett Ingebrigtsen1 , Christina Tekle1 , Marit Kveine Nygren1 , Cathrine Pedersen1 , Suvi K. Leivonen1 , Kristine
Kleivi Sahlberg1 , Øystein Fodstad1
1. Oslo University Hospital , Oslo, Norway
B7-H3 is a cell surface protein with immunoglobulin like structure and its high expression is associated with advanced disease
and/or poor prognosis in several cancer types. We have focused on its non-immunological role and found that in an
experimental metastasis model in nude rodents, silencing of B7-H3 protein resulted in reduced metastatic capacity and a
significant increase in the symptom-free survival of the animals. Importantly, the expression of metastasis-associated proteins
like IL-8, MMP-2, TIMPs, and phospho-Stat-3 was modulated by B7-H3 in a way that may explain the observed attenuated
metastatic potential of melanoma cells1. Metastasis is closely linked to treatment resistance and we have previously shown that
B7-H3 silencing increased the effect of paclitaxel by interfering with the Jak2/Stat3 pathway2. We are further studying the effect
of B7-H3 on resistance by performing a drug screen to evaluate the effect of 22 anti-cancer compounds. In this screen we
found that API-2 and everolimus, two small molecule inhibitors known to target proteins in the PI3K/Akt/mTOR pathway,
showed a significant better effect in B7-H3 silenced metastatic breast cancer cells compared to their B7-H3 expressing
counterparts. A link between B7-H3 and the PI3K/Akt/mTOR pathway was also supported by Western blot analysis
demonstrating reduced phosphorylation of Akt and p70S6K in B7-H3 knockdown cells. Furthermore, preliminary data indicates
that CXCR4, which is known to activate the PI3K/Akt pathway, is downregulated in B7-H3 silenced metastatic melanoma cells.
Also the expression of PTEN, which negatively regulates the PI3K/Akt pathway, was reduced in B7-H3 silenced cells, probably
reflecting a general lower activity in this pathway upon B7-H3 knockdown. By our efforts to map in detail the signaling
molecules affected by B7-H3 we anticipate to identify novel ways to reduce metastasis formation and increase the effect of anti-
cancer drugs by inhibiting B7-H3 expression.


Esophageal squamous carcinoma and normal squamous epithelia express differentially
glycosylated mucin 21
Yuan Tian1 , Kaori Denda-Nagai1 , Mika Kamata-Sakurai1 , Shoji Nakamori2 , Tatsuro Irimura1
1. The University of Tokyo, Bunkyo-ku, Tokyo, Japan
2. National Hospital Organization Osaka National Hospital, Osaka, Japan
Aberrant glycosylation occurs in essentially all types of experimental and human cancers. Many glycosyl epitopes constitute
tumor-associated antigens, and mucin with aberrant glycoforms has been known to involve cancer progression and metastasis.
However, there are few reports of the association between the glycosylation of mucins and squamous carcinoma. Here, we
show that human mucin 21 (MUC21) could be used as a marker of squamous carcinoma in esophagus. Monoclonal antibodies
(mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic
kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry,
immunoprecipitation, and Western blotting, focusing on the differential glycosylation of MUC21 expressed in variant Chinese
hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the
unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies,
including mAb heM21C, bound to MUC21 with Tn, T, or sialyl-T epitopes but not the unmodified core polypeptide of MUC21.
Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of
these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed
in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with
mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first
report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia
and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial
differentiation and carcinogenesis. This work was supported in part by Global COE Program “Medical System Innovation on
Multidisciplinary Integration” from MEXT, Japan.


Paraneoplastic polyarteritis nodosa masquerading as widespread metastatic disease: A case
David Veitch1
1. Prince of Wales Hospital, sydney, NSW, Australia
Polyarteritis nodosa (PAN) as a paraneoplastic vasculitis is rarely described, especially in association with metastatic
squamous cell carcinoma (SCC). Furthermore, only 5% of all PAN patients have central nervous system (CNS) involvement -
almost       exclusively    in     the     form     of     cerebral    infarction    or     intracerebral     haemorrhage.
We report a patient presenting with a spectrum of neurological symptoms found to have multiple cerebral lesions on CT head.
A subsequent whole body PET scan revealed widespread glucose avid lesions thought to represent metastatic disease from an
unknown primary malignancy. In preparation for a brain biopsy steroid therapy was commenced, but after a dramatic
improvement in symptoms, a repeat PET was performed. There was almost complete resolution of the widespread lesions. The
remaining base of tongue lesion later proved to represent SCC. A diagnosis of paraneoplastic PAN was made.
We believe this is a world-first case of PAN in association with systemic and cerebral vasculitic lesions presumed to be
metastatic malignancy, with steroid-induced resolution demonstrated on serial magnetic-resonance imaging (MRI) and
positron-emission tomography (PET) scans.

Differential expression of Kindlin-1 and Kindlin-2 during lung cancer progression
JUN ZHAN1 , Hongquan Zhang1
1. Peking University, Haidian, China
Lung cancer is highly heterogeneous and is composed of various subtypes that are in different differential stages. The newly
identified integrin-interacting proteins Kindlin-1 and Kindlin-2 are the activators of transmembrane receptor integrins, which play
important roles in cancer progression. In this report we showed the expression profile of Kindlin-1 and Kindlin-2 in various types
of lung cancer using patients’ specimen and established their correlation with lung cancer progression. We found that Kindlin-1
is expressed in differentiated non-small-cell lung cancer (NSCLC), especially in squamous cell cancer (SCC) but expressed low
in undifferentiated large cell lung cancer, whereas Kindlin-2 is highly expressed in large cell lung cancer. Both of Kindlin-1 and
Kindlin-2 are low or not expressed in neurocrine-derived small cell lung cancer (SCLC). Importantly, Kindlin-1 expression level
is correlated with the differentiation degree of SCC. Functionally, overexpression of Kindlin-1 in NSCLC cells inhibits in vitro cell
migration and in vivo tumor growth, whereas Kindlin-2 functions in the opposite way. Mechanistically, Kindlin-1 prohibits while
Kindlin-2 enhancse epithelial to mesenchymal transition in lung cancer cells. Taken together, we identified that Kindlin-1 and
Kindlin-2 differentially regulate lung cancer cell progression and the relative expression levels of Kindlin-1 can be potentially
used as a marker for lung cancer differentiation.


Screening for drugs targeting metastatic breast cancer
Karl-Johan Leuchowius1 , Anannya Chakrabarti2 , Kurt Lackovic1 , Robin Anderson2 , Ian Street1
1. Walter and Eliza Hall Institute of Medical Research, Bundoora, VIC, Australia
2. Metastasis Research Laboratory, Peter MacCallum Cancer Centre , Melbourne, Australia
Metastasis is the leading cause of death among breast cancer patients, and drugs targeting metastatic processes are much
needed. Screening for compounds to inhibit such processes in an assay format reminiscent of in vivo conditions should
increase the likelihood of finding suitable compounds. We have established a three-dimensional in vitro cellular assay to
simulate conditions of disseminated tumour cells, and have used it to screen a library of kinase inhibitors for compounds that
inhibit growth of metastatic human breast cancer cells. As a reference, we have also screened the same compounds in a
standard two-dimensional assay. From our screen, a set of compounds was found that had an increased inhibitory effect on
cells growing in a three-dimensional matrix compared to cells growing directly on a plastic surface. Of particular interest was a
set of compounds targeting focal adhesion kinase (FAK), Src and PI-3K/mTOR, showing significantly increased potency in the
three-dimensional assay compared with normal tissue culture conditions. Contact between cells and extracellular matrix is
mediated by cell surface integrins to the intracellular signalling networks through adapter proteins such as Src and FAK. This
signalling can affect many other pathways, e.g. pathways activated by receptor tyrosine kinases, and can influence cellular
processes relevant to metastasis such as growth, invasiveness and motility. Thus, inhibition of this pathway, either on its own
or in combination with other pathways, could be an attractive strategy to target metastatic disease.


Molecular analysis of circulating (CTC) and disseminated tumour cells (DTC) in human breast
cancer xenograft models.
Anthony Tachtsidis1 2 , Tony Blick1 , Devika Gunasinghe2 , Peter F.M Choong2 3 , Mark Waltham1 , Alex Dobrovic4 , Erik
W Thompson1 2
1. St. Vincent's Institute, Fitzroy, VIC, Australia
2. Surgery, University of Melbourne, Melbourne, VIC, Australia
3. Orthopaedics, St Vincent's Hospital, Melbourne, VIC, Australia
4. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
Our goal is to establish reproducible and robust mouse models for use in breast cancer DTC/CTC research. Blood samples,
bone marrow and tumour tissue were collected from mice xenografted with the MDA-MB-468 cell line and also using metastatic
breast cancer material that was successfully grafted and passaged in vivo (ED-03). Using a species-specific tandem nested
qRT-PCR approach, we are able to detect and measure a panel of human specific markers relating to: detection of CTC/DTC,
prognostic markers of breast cancer, the ‘cancer stem cell’ phenotype and epithelial-mesenchymal plasticity (EMP). EMP is
hypothesised to be involved in the generation and function of DTC/CTC. All samples from each xenograft were assessed and
comparisons made between blood, bone marrow and tumour. Analysis of relative expression levels in CTC and DTC compared
to tumour of the MDA-MB-468 model revealed statistically significant changes in CD24, CD44, OAZ1, SNAI1, CLDN3 and
CLDN4. In the ED03 model, significant changes in relative levels of CDH1, OAZ1, SNAI1, and CK20 were determined. Several
other markers between the two models trended towards significance but did not reach a p-value <0.05. Immunohistochemical
analysis on cytospin slides of bone marrow from the MDA-MB-468 and ED-03 xenografts has also been conducted and visual
confirmation of DTC presence was achieved. These are the first such analyses of human xenograft CTC and DTC.


Dynamic analysis of lung metastasis by mouse osteosarcoma LM8 – VEGF is a candidate for
anti-metastasis therapy
Takaaki Tanaka1 2 , Yoshihiro Yui1 , Hideki Yoshikawa2 , Kazuyuki Itoh1
1. Osaka Medical Center of Cancer and Cardiovascular Diseases, Osaka, Japan
2.   Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine , Osaka, Japan

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis depends on pulmonary metastases, which
arise from multi-step progression of malignant tumors. We aimed to clarify the critical step of pulmonary metastasis using the
syngeneic mouse spontaneous highly metastatic OS LM8 and parental Dunn cell lines, to identify new candidate molecules to
suppress pulmonary metastasis. We first investigated the chronological detection of circulating tumor cells (CTCs) from mice
with either cell line. LM8 CTCs appeared faster, at a higher rate and with a greater number of cultured colonies compared to
Dunn CTCs. Cultured cells from CTCs showed higher proliferative ability than cells from the primary site in suspension culture,
which mimicked the environment of the bloodstream for CTCs. The proliferative ability of LM8 cells was also higher than that of
Dunn cells in 3D collagen culture with low stiffness (~150 Pa; close to conditions in the lung). We next focused on the
extravasation step. LM8 showed higher migration ability compared to Dunn with transendothelial migration assay. We also
found a disruption in endothelial barrier function throughout co-culture with LM8 using time-lapse imaging. In addition, LM8
secreted high levels of vascular endothelial growth factor (VEGF), while VEGF signal inhibition with a small molecule tyrosine
kinase inhibitor (pazopanib) decreased disruption of the vascular barrier and transendothelial migration of LM8. Finally, daily
oral administration of pazopanib reduced the rate and size of pulmonary metastasis in vivo. Collectively, these results show
anti-VEGF therapy as a candidate for pulmonary metastasis of OS.


Genome wide profiling of lung metastases in the MMTV-PyMT model
Stephanie L Allerdice1 , David Gallego-Ortega1 , Christopher J Ormandy1
1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
The ETS transcription factor E74-like-factor 5 (Elf5) is an epithelial restricted protein found in the mammary gland, kidney,
salivary gland and stomach. In the mammary gland, Elf5 expression dramatically increases during pregnancy and high levels of
Elf5 are maintained throughout lactation. Elf5 is downstream to the prolactin receptor and is necessary for lobuloalveolar
development and milk production. It has been found that Elf5 drives the production of alveolar cells from the luminal progenitor
cell in the mammary gland cell hierarchy. We have pursued a developmental understanding of the normal growth and
development that occurs in the mammary gland, which encompasses proliferation, apoptosis and invasion, in order to
understand molecular pathways which breast cancer cells utilise. In human breast cancers it has been shown that, when
compared to patient-matched microdissected controls, Elf5 is one of the most consistently downregulated genes at all stages of
breast cancer. Elf5 expression is consistently lower in the luminal subgroups relative to basal subgroups of breast cancer,
suggesting a crucial role for Elf5 in both tumour initiation and subgroup determination. By using the metastatic breast cancer
mouse model MMTV-PyMT we have found that inducing the overexpression of Elf5 leads to a change in the proliferative ability
of the primary tumour cells. We hypothesised that overexpression of Elf5 may also change the metastatic phenotype of breast
cancer cells.
Using differentially expressed surface proteins in lung and mammary epithelium we are able to purify breast cancer metastatic
cells from the lung of PyMT animals. Following a genome wide expression profiling approach, we are studying the cellular
effect of Elf5 on lung metastasis in comparison with the primary tumour cells in the MMTV-PyMT mouse model. In addition, we
have developed an inducible model for Elf5 overexpression coupled with luciferase. Metastatic human cancer cell lines carrying
this construct are used to create mouse xenografts, allowing us to explore the role of Elf5 on metastasis in vivo using a
combination of in vivo imaging and cell sorting. This will allow study of key genes involved in the progression to metastasis.
While Elf5 has previously been identified to be involved in mouse mammary gland development, this is the first look at its role in
cancer and progression to metastasis.


Carcinoembryonic antigen promotes metastasis via disruption of E-cadherin adherens
junction complexes.
Olga Bajenova1 , Nina Chaika2 , Peter Thomas2
1. Department of Genetics and Selection, St. Petersburg State University, St Petersburg, Russia
2. Department of Surgery and Biomedical Sciences, Creighton University, Omaha, USA
Carcinoembryonic antigen (CEA) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in
monitoring metastatic colorectal cancer. In this study we investigated the mechanism of CEA tumorigenesis, particularly the
effect of CEA production on E-cadherin adherens junction protein complexes. As a model we used CEA-producing and non-
producing MIP101 colorectal carcinoma cell lines. By immunoprecipitation we discovered the novel interactions between CEA
and -catenin in the CEA excreting cells. No direct interaction has been detected between E-cadherin and CEA and no
downregulation of neither -catenin nor E-cadherin in the CEA overexpressing cells. In addition, the functional interactions
between E-cadherin and its binding protein- -catenin as well as between cateninand catenin have been impared in the
CEA-producing cell lines. β-catenin is also a key factor in the Wnt signaling pathway that has essential functions in the
regulation of cell growth and differentiation. Aberrant β-catenin signaling has been linked to the various disease pathologies,
including an important role in metastasis. We have previously identified and cloned a novel CEA-binding protein (CEAR) from
the liver macrophages, Kupffer cells. It has been shown that CEA-CEAR binding produces an inflammatory response in the
liver by altering the microenvironment such that implantation and survival of tumor cells increases. This study also identified
novel interactions of CEAR with α-catenin that may interfere with the  catenin and its signaling pathways. Our findings
indicate that the CEA production in tumors contributes to EMT and metastatic dissemination by interfering with the function of
E-cadherin adherens junction complexes. This may explain the link between the elevated levels of CEA and an increase in the
soluble E-cadherin during the progression of colorectal cancer. This work has been supported by The Russian Foundation for
Fundamental Research and by the Health and Future Foundation of Creighton University.

Loss of TGFBR3 in ovarian cancers reduces sensitivity to chemotherapeutics and enhances
cancer stem cell marker expression
Maree Bilandzic1 , Yao Wang1 , Jock K Findlay1 , Kaye L Stenvers 1
1. Prince Henry's Insitute , Clayton, VIC, Australia
Type III TGF-beta receptor (TGFBR3; betaglycan) expression is significantly reduced in the majority of advanced stage serous
epithelial ovarian cancers and granulosa cell tumours (GCTs), with increasing loss correlated with disease progression
(Bilandzic et al., 2009, Mol Endocrinol 23:539-548; Hempel et al., 2007, Cancer Res 67:5231-5238). In this study, we tested the
hypothesis that loss of TGFBR3 by GCTs contributes to their acquisition of chemoresistance. Using MTT cell viability assays,
we examined the effect of TGFBR3 on the response of the KGN GCT line to the chemotherapeutic, cisplatin. Comparison of
IC50 values for cisplatin showed that monolayer cultures of TGFBR3-expressing KGN were 4-fold (p<0.0001) more sensitive to
cisplatin compared to controls. Similarly, spheroid culture experiments demonstrated a significant 2-fold decrease in viability
from 2-5 d in TGFBR3-expressing spheroids compared to controls (p<0.05). Acquisition of chemoresistance by ovarian cancer
cells has been previously associated with the presence of a 'cancer stem cell' (CSC) population, which is capable of surviving
initial chemotherapy (Bapat SA et al., Cancer Research 65 3025–3029.). Therefore, we determined the levels of the stem cell
markers NANOG, WNT1, CD44, and OCT4 in the KGN cell line by RT-PCR analysis in response to cisplatin treatment. Under
basal conditions, TGFBR3 and control cells maintained similar levels of the CSC markers. Following 5 d cisplatin treatment,
significant increases (p<0.0001) in WNT1, OCT4, NANOG, and CD44 were observed in the control cells, but not the TGFBR3-
expressing cells. Collectively, these data suggest that the loss of TGFBR3 expression in advanced, metastatic ovarian cancers
is linked to the acquisition of chemoresistance. Additionally, loss of TGFBR3 expression in ovarian cancers may enhance stem
cell-like features, which underlies their reduced sensitivity to chemotherapeutics and their increased capacity for tumour
recurrence. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government
Infrastructure funds.


Is there a clinical relevance of EMT in metastatic progression?- investigations of colorectal
and breast cancer patient materials
Linda Bojmar1 , Elin Karlsson1 , Sander Ellegård2 , Hans Olsson3 , Marie Larsson4 , Olof Hallböök5 , Olle Stål1 , Per
1. Dept of Clinical and Experimental Research/Division of Surgery and Clinical Oncology, Linkoping University, Linkoping,
2. Dept of Oncology, County Council of Ostergotland, Linköping, Sweden
3. Dept of Pathology, County Council of Ostergotland, Linköping, Sweden
4. Linkoping University, Linkoping, Swede, Sweden
5. Dept of Surgery, County Council of Ostergotland, Linköping, Sweden
In vitro, the epithelial-mesenchymal transition (EMT) in relation to cancer is widely studied. However in vivo results, especially
from human materials, are rare and lack clear proof. Several microRNAs have been associated with EMT and metastasis. The
microRNA 200 (mir-200) family is one of the most commonly reported, and acting negatively on ZEB, which is a repressor of
CDH1 (E-cadherin) thereby supporting the epithelial maintenance. We aimed to investigate the role of microRNAs, especially
mir-200, and associated EMT/metastasis markers in human breast (BC) and colorectal cancer (CRC) materials. Frozen and
FFPE matched microdissected tissues from primary tumours and liver metastasis, and larger cohorts of colorectal liver
metastases, and primary BC with more than 10 years of follow-up were used. Laser-capture microdissection (LCM), microRNA
and mRNA extraction and expression analysis, microRNA in situ hybridization (ISH), immunohistochemistry, microRNA array
profiling and primary cell culturing were performed. Correlations were found between the signalling pathway-members of the
EMT process; both in primary tumours and metastatic lesions. Altered expression of EMT-players was associated with
decreased survival from BC, and secondary recurrence in metastatic CRC in liver. Radiotherapy response, in terms of
metastasis from BC, was significantly altered in relation to the expression of the mir-141/200c cluster. Correlations to many
important factors in BC progression, eg. p53, ER and HER2 status and activation of the PI3K/AKT pathway could be possible
explanations. ISH against the mir-200 confirmed selective expression to epithelial cells and microRNAs was shown to be more
stable compared to mRNAs. LCM of CRC showed a significant decrease in mir-200 from normal mucosa to malignant cells,
and when comparing metastatic CRC to non-metastatic. Correlations to FGF expression further strengthen that plasticity of
cells is an important feature in the metastatic process and related factors could be useful prognostic and treatment predictive
factors in metastatic cancers.


Anna Chen1 , Jaclyn Sceneay1 , Mira CP Liu1 , Christina SF Wong1 , Erik W Thompson2 , Andreas Möller1
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
Metastasis is the major cause of breast cancer-related morbidity and mortality. The molecular mechanisms of metastasis, a
complex and multi-step process, are yet to be fully elucidated, but epithelial-mesenchymal transition (EMT) is proposed to play
a role in enabling metastasis. Cells that undergo EMT have a greater capacity to migrate, to invade and to resist therapies.
Zeb1, the most apical EMT transcription factor, is a very powerful inducer of EMT via its transcriptional suppression of E-
cadherin and of the microRNA-200 family expression. While the control of Zeb1 gene expression is currently being unravelled,
the mechanisms of post-transcriptional Zeb1 regulation are not yet fully understood.
Siah, a family of E3 ubiquitin ligases, has recently been shown to have a role in tumourigenesis and metastasis. We found that
Siah protein levels are reduced in breast cancer cells undergoing EMT. To assess whether Siah plays a functional role in the
process of EMT, we generated and analysed murine Siah2-/- breast cancer cell lines. These cells display a mesenchymal
phenotype in 2D and 3D cultures, despite unchanged levels in the gene expression of EMT transcription factors. Interestingly,
Siah2-/- cells have increased Zeb1 protein abundance.
Mechanistically, we demonstrate that Zeb1 is a novel Siah-interacting protein, and that Zeb1 is polyubiquinated and targeted for
proteasomal degradation by Siah. Human breast cancer cells undergo spontaneous EMT, associated with increased Zeb1
abundance, when Siah proteins are blocked by inhibitors or knocked down. This inverse correlation between Siah and Zeb1
proteins levels, as well as Siah expression and a mesenchymal gene signature, is also seen in breast and ovarian cancer
patient samples.
Considering the importance of Zeb1 in controlling EMT, these findings of a mechanism of post-transcriptional regulation of
Zeb1 by Siah ubiquitin ligases will greatly enhance our current understanding of EMT-inducing signalling networks.


Changes in tumour infiltrating cells following vascular disruptive agent treatment may
contribute to therapy resistance
Theodora Fifis1 , Linh Nguyen , Caterina Malcontenti-Wilson , Christopher Christophi
1. University of Melbourne, Heidelberg, Vic, Australia
Colorectal cancer (CRC) is the second most frequent cancer in Australia. It results in over 4,000 deaths annually, with liver
metastasis being the predominant cause. Apart from a small group amenable to surgery the majority of patients with CRC liver
tumours are treated with palliative chemotherapy. Alternative strategies are focussing on selective targeting of the tumour blood
supply, either alone or in combination with chemotherapy. We have previously tested the effect of a novel vascular disruptive
agent (VDA) Oxi4503 (Oxigene), in an established mouse model of colorectal liver metastases and have shown that it reduces
the tumour mass by about 90%. However, a small population of cancer cells persist at the tumour periphery, resulting in tumour
regrowth. The mechanisms by which these tumour cells evade treatment are poorly understood. We have demonstrated local
and systemic changes in pro-angiogenic growth factors and tumour cell morphology changes including epithelial to
mesenchymal transition and the acquisition of stem cell markers after Oxi4503 treatment. In this study we investigated the
kinetics and expression profiles of infiltrating immune cells including macrophages and T-cells into the residual tumour at
various time points following treatment. We found significant changes in the pattern of infiltration of these cells compared to
untreated controls.
Infiltrating cells modulate tumour progression through secreted growth factors and cytokines and may contribute to the survival
of tumours in the tumour periphery following treatment.
This work was supported by the Austin Health Medical Research Foundation and Australian Rotary Health


The Rho Pathway and Hormone Resistance and/or Metastasis of ER alpha-Positive Breast
Cancer Cells
Suzanne AW Fuqua1 , Covington R Kyle1 , Brusco Lauren1 , Ciupek Andrew1
1. Baylor College of Medicine, Houston, TX, United States
Mortality from breast cancer results from the ability of some tumors to metastasize to distant sites. Unfortunately, there are only
a few preclinical breast cancer models that metastasize to distant sites to study the complex process of metastasis, and almost
all of these are estrogen receptor (ER)-negative. We have discovered that a cytoskeletal regulatory gene called Rho guanine
disassociation inhibitor alpha (Rho GDI alpha) is expressed at lower levels in metastatic, hormone-resistant ER-positive human
breast tumors, suggesting that it potentially might be a biomarker of resistance and/or metastasis. We have data suggesting
that Rho GDI alpha may indeed be a metastasis suppressor, because when we knockdown its expression in ER-positive breast
cancer cells to simulate our clinical tumor data, we generated a phenotype that was resistant to the antiestrogen tamoxifen
(Tam), and which metastasized to lungs with a high frequency when grown as xenografts in athymic nude mice. We have also
discovered that Rho GDI alpha knockdown activates the Rho signaling pathway, increasing downstream p21-activating kinase
(PAK1) activity, which then increased phosphorylation of ER alpha at serine 305. Concomitantly, levels of the metastasis
associated (MTA2) protein and the androgen receptor (AR) were enhanced with Rho GDI alpha knockdown. MTA2
overexpression in ER-positive breast cancer cells rendered them hormone dependent, and in silico analysis of published
datasets show that high MTA2 levels are associated with poor clinical outcome in patients treated with Tam. Treatment of Rho
GDIα knockdown cells with the AR antagonist bicalutamide blocked tamoxifen’s agonist effects, suggesting that AR
overerxpression may play a role in the Tam-resistant phenotype. The role of Rho GDIα, MTA2, and AR in metastasis of Rho
knockdown cells is being explored and will be presented.


Uncovering the metastatic potential of the transcription factor Elf5 in breast cancer
David Gallego Ortega1 , Anita Ledger1 , Stephanie Allerdice1 , Heather Lee1 2 , Daniel Roden1 , Wendy Au1 , Christopher
1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
2. Babraham Institute, Cambridge, United Kingdom
The role of the ets transcription factor Elf5 in the mammary gland development has been well established. Elf5 is a hormone
target; it specifies the development of the alveolar secretory epithelium during pregnancy by forcing cell fate decisions within
the CD61+ progenitor population. Forced Elf5 expression in virgin mice produces a precocious development of the mammary
alveoli and milk secretion. During breast carcinogenesis, Elf5 is one of the most consistently down-regulated genes, with a
large loss of Elf5 expression observed in luminal cancers and more moderate loss by the basal subtype. Thus higher Elf5
expression is associated with high-grade tumours, lymphocytic infiltration and with BRCA1 mutation, reflecting the basal-
luminal differences in these features. Furthermore, since the CD61+ progenitor cell is the cell of origin for basal breast cancers,
a role for Elf5 in the development of basal over luminal breast cancer by forcing similar cell fate decisions, may occur.
Using an in vitro inducible model for over-expression of Elf5, we have recently demonstrated that Elf5-driven transcriptional
network suppresses estrogen action, and reduces cell proliferation and tumour growth of luminal breast cancer cells. We also
identified enhancement of basal subtype characteristics in these cells. In this study, we use the polyoma middle-T oncoprotein
(PyMT) mouse mammary tumour model to study the effect of Elf5-overexpression in luminal subtype mammary cancer initiation
and progression. Acute over-expression of Elf5 in npre-existing PyMT tumours reduced cell proliferation, as observed in
human luminal models. Chronic Elf5 over-expression during carcinogenesis and progression dramatically increased the
number and size of lung metastasis. Investigation of transcriptional events showed Elf5 influenced the balance between
epithelial and mesenchymal phenotypes in both acute and chronic scenarios.
This study shows for the first time the role of the alveolar fate regulator Elf5 in breast cancer initiation and progression,
demonstrating the role of normal developmental mechanisms in specifying the subtype of breast cancer, knowledge that will
inform the development of novel therapeutic strategies.


The microRNA-200 family as regulators of breast and colorectal tumour metastasis
Philip A Gregory1 2 , Xiaochun Li1 , Emily L Paterson1 , Suraya Roslan1 , Cameron P Bracken1 2 , Josephine A Wright1 2 ,
Andrew G Bert1 , Matthew J Anderson1 , Cameron N Johnstone3 , Robin L Anderson3 , Gregory J Goodall1 2 4 , Yeesim
Khew-Goodall1 4
1. Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia
2. Discipline of Medicine, The University of Adelaide, Adelaide, South Australia, Australia
3. Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
4. School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia
The miR-200 family plays a critical role in maintaining the epithelial phenotype through repression of the E-cadherin regulating
transcription factors ZEB1 and ZEB2 (1,2,3). Loss of the miR-200 family has been shown to lead to an epithelial-mesenchymal
transition (EMT) with an associated increase in invasive capacity suggesting it may play a regulatory role in tumour metastasis.
We have examined the expression and function of individual miR-200 family members in two distinct models of tumour
metastasis– (1) A xenograft model of spontaneous breast cancer metastasis and (2) Human colorectal adenocarcinomas.
Stable expression of miR-200b or miR-200c, but not miR-141, in the MDA-MB-231 (LM2) subline attenuated the ability of these
cells to metastasise from the mammary fat pad. Interestingly, restoration of ZEB1 expression in miR-200b stable cells was not
sufficient to alleviate metastatic repression, but instead several cytoskeletal associated miR-200 targets appear to also
contribute to cell invasion from the primary tumour. In human colorectal adenocarcinomas, in situ hybridization and laser
capture microscopy revealed the expression of miR-200b and miR-200c is specifically reduced at the invasive front of
colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. Examination of regional lymph
node metastases in these samples show strong expression of miR-200, indicating this family of miRNAs can induce a
mesenchymal to epithelial transition (MET) and recapitulate the primary tumour phenotype at metastatic sites. Taken together,
these data support a regulatory role for the miR-200 family in controlling EMT/MET and tumour metastasis in breast and
colorectal carcinomas.

1.        Gregory PA et al (2008) Nature Cell Biol. 10, 593-601.
2.        Bracken CP et al (2008) Cancer Res. 68, 7846-7854.
3.        Gregory PA et al (2011) Mol. Biol. Cell. 22, 1686-1698.


Snail up-regulates Dnmt1 and sequesters it in cytoplasm: An epigenetic alteration by Snail in
ovarian cancer
Hongyan Jin1 , Jiayi Zhou1 , Luoqi Jia1 , Yilin Lu2 , Yinhua Yu1
1. Obstet@Gyneco Hospital Fudan University, Shanghai, China
2. The University of Texas, M.D. Anderson Cancer Center, Houston , U.S.A.
Snail plays an important role in metastasis of ovarian cancer; our previous works confirmed that Snail expression correlated
with the stage of ovarian cancer. In early-stage tumors, Snail was localized in both the cytoplasm and nucleus. In late stage
and metastatic lesions, the level of Snail was elevated, and Snail was mainly localized in the nucleus. To further illustrate the
mechanisms of Snail regulation in ovarian cancer, we performed the Reverse Phase Protein Assay (RPPA) using the SKOv3-
Snail inducible cell line. RPPA analysis revealed that Dnmt1, the maintenance DNA methylation enzyme, was extremely high in
Snail induced cells compared to the control. Snail-Dnmt1 correlation was validated by Western blot in SKOv3-Snail and
HO8910PM-Snail inducible cells. Most interesting finding is that compare to non-induced cells, the protein level of Dnmt1 was
enhanced in the cytoplasm after Snail induced, while there is no change in nucleus both in Snail-induced cells and its control.
We therefore investigated whether Dnmt1 expression was associated with Snail, Dnmt1 was examined in an ovarian tissue
array by immunohistochemistry, it shows that the expression of Dnmt1 was extremely higher in metastasis tumor compared
with its primary tumor. Most intriguingly, we observed that Dnmt1 was mainly localized in the nucleus of primary tumors;
whereas it exhibited a higher level in both nucleus and cytoplasm of metastasis lesions. All these results suggested that Snail
could up-regulate Dnmt1, and sequester it in cytoplasm when metastasis occurred. As we know, sequestration of Dnmt1 in the
cytoplasm may result global DNA hypomethylation. To understand the epigenetic alteration by Snail, SKOv3-Snail inducible
cells were used to perform methylation array analysis. DNAs were immunoprecipitated by anti-5’-methylcytidine antibody,
labeled with Cy5 or Cy3, then hybridized to NimbleGen Multiplex CpG Island Plus RefSeq Promoter Array. Chip analysis found
methylation level of more then 40 genes were decreased after Snail induction, these genes were involved in cell transcription
and adhesion. Further studies are going on to characterize these candidates. Our results inferred that Snail might regulate
gene methylation through sequestering Dnmt1 in cytoplasm.


Mesenchymal-transitioned epithelial cancer cells instigate EMT via paracrine WNT signal
Shinichiro Kato1 , Satoru Yokoyama1 , Yoshihiro Hayakawa1 , Ikuo Saiki1
1. Pathogenic Biochemistry, Institute of Natural Medicine , University of Toyama, Toyama, Japan
Epithelial-to-mesenchymal transition (EMT) plays an important role in cancer cell malignancy and metastatic potential, and
indeed epithelial and mesenchymal cancer cells are often heterogeneously interspersed in tumor invasive front. Although such
intra-tumor heterogeneity may account for the malignant behavior of cancer cells, it is not known whether mesenchymal
transitioned cancer cells (M-cells) can cross-talk with epithelial cancer cells (E-cells) within the heterogeneous tumor
microenvironment. In this study, we tested the potential of M-cells to instigate EMT of surrounding cancer cells by using human
epithelial Panc-1 or A549 cancer cells treated with TGF-β to induce EMT. In co-culture experiments of E- and M-cells in both
cell lines, we have found that the conditioned medium (CM) of M-cells induced EMT in E-cells and further demonstrated WNT
inhibitors, IWP-2 and rhDKK1, inhibited such M-cells induced EMT. The critical contribution of WNT pathway was confirmed by
siWNT3 and siWNT5B transfection into M-cells. Furthermore, CM of M-cells also has unexpected role for induction of MET in
M-cells itself. Importantly, the interaction between E- and M-cells could dynamically reinforce tumor in vivo metastatic potential.
These results implicate that mesenchymal-transitioned cancer cells could control intratumoral EMT and also MET, and in
particular, instigate EMT of surrounding cancer cells through paracrine WNT signal pathway including WNT3, to enhance
metastatic spread to distant organs.


Acidic extracellular pH induces epithelial-mesenchymal transition in Lewis lung carcinoma
Yasumasa Kato1 , Atsuko Suzuki1 , Toyonobu Maeda1 , Kazuhiro Shimamura
1. Ohu University School of Dentistry, Koriyama, Japan
Acidic extracellular pH is a hallmark phenotype of solid tumor. We have shown that acidic pH induced matrix metalloproteinase-
9 (MMP-9) expression in B16 melanoma cells through Ca2+-triggered phopholipase D - MAP kinases - NFκB signaling. Here,
we investigated whether acidic extracellular pH is a candidate microenvironment to induce epithelial-mesenchymal transition
(EMT) using the Lewis lung carcinoma (LLC) model. First, we established two LLC variants with differential metastatic ability by
repeated tail vein injection of tumor cells from metastasized foci in lung, named LLC-meta-I (low metastatic) and LLC-meta-IV
(high metastatic). Morphology of LLC-meta-I and LLC-meta-IV cells was a cobble-stone like and fibroblastic, respectively.
When LLC-meta-I cells were cultured with acidic pH medium, cell shape became fibroblastic which was similar to LLC-meta-IV.
Then, we checked whether matrix metalloproteinase-9 (MMP-9) production was induced by acidic pH. In good accordance with
the B16 melanoma model, MMP-9 was extremely induced in LLC-meta-IV at acidic pH as compared with LLC-meta-I cells. This
was also confirmed by RT-qPCR technology. RT-qPCR analysis showed that acidic pH induced vimentin expression but
reduced E-cadherin expression, which are typical features of EMT. These data suggested acidic extracellular pH is a
microenvironmental niche to induce EMT in some kinds of cancer.


ZEB2 and Sp1 have cooperative roles as transcriptional activators of mesenchymal genes
during EMT
Eun-Hee Nam1 , Young-Kyu Park, Semi Kim1
1. Korea Research Institute of Bioscience and Biotechnology , Daejon , South Korea
Epithelial-mesenchymal transition (EMT) is a process implicated in tumor invasion, metastasis, embryonic development, and
wound healing. ZEB2 is a transcription factor involved in EMT that represses E-cadherin transcription. Although E-cadherin
downregulation is a major event during EMT and tumor progression, E-cadherin reduction is probably not sufficient for full
invasiveness. The mechanisms by which E-cadherin transcriptional repressors induce mesenchymal genes during EMT remain
largely unknown. Here, we investigated the role of ZEB2 in the induction of integrin α5 during cancer EMT and its underlying
mechanism. In human cancer cells, ZEB2 was found to directly upregulate integrin α5 transcription in a manner that is
independent of the regulation of E-cadherin expression. Conversely, depletion of ZEB2 by small interfering RNA suppressed
integrin α5 expression, leading to reduced invasion. Suppression of integrin α5 inhibited cancer cell invasion, suggesting an
important role for integrin α5 in cancer progression. Furthermore, ZEB2 was found to activate the integrin α5 and vimentin
promoters by interacting with and activating the transcription factor Sp1, suggesting that cooperation between ZEB2 and Sp1
represents a novel mechanism of mesenchymal gene activation during EMT.

Gene silencing of c-Met leads to brain metastasis inhibitory effects
Se Jeong Lee1 , Hye Won Lee2 , Yu Jin Cho1 , Do-Hyun Nam3 , Kyeung Min Joo1
1. Anatomy, Seoul National University College of Medicine, Seoul, South Korea
2. Samsung Advanced Institute for Health Sciences & Technology, Samsung Medical Center, Sungkyunkwan University
      School of Medicine, Seoul, South Korea
3. Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
An unfortunate consequence of improvements in the treatments of advanced primary cancers is the concurrent increase of
metastatic brain tumors. Despite of unfavorable clinical prognosis, radiation therapy is still the only viable treatment option for
brain metastases. Expression of c-Met induces cell migration and invasion in many cancers, which are indispensable steps for
metastasis. Accordingly, we examined the effects of gene silencing of c-Met on brain metastasis to evaluate the possibility of c-
Met as a potential target. MDA-MB-435 cells were transfected with c-Met targeting short hairpin RNAs (shRNAs). Effects of c-
Met shRNAs on the expression of epithelial mesenchymal transition (EMT) related proteins, in vitro migration, and in vivo brain
metastasis were examined. Expression of mesenchymal markers and in vitro migration of MDA-MB-435 cells were significantly
inhibited by introduction of c-Met shRNAs. When c-Met-silenced MDA-MB-435 cells were stereotactically implanted into the
brains of immune-compromised mice or injected into the right internal carotid arteries, c-Met-silenced MDA-MB-435 cells
produced significantly smaller tumor masses or survival time was significantly prolonged, respectively, compared with MDA-
MB-435 cells transfected with control shRNA. The data reveal the novel function of c-Met in the process of brain metastasis
and its potential as a preventive and/or therapeutic target in this disease.


Protein Tyrosine Phosphatase Pez - a novel regulator of Protein Trafficking and Secretion
X. Li1 , L Belle1 2 , N. Ali3 , A. Lonic1 , S. Roslan1 , F. Gehling1 , R. Daly3 , Y. Khew-Goodall1 2
1. Center for Cancer biology, SA pathology, Adelaide, SA, Australia
2. Discipline of Biochemistry, School of Molecular and Biomedical Sciences, the University of Adelaide, Adelaide, SA,
3. Cancer Research Programme, Garvan Institute of Medical Research, Sydney, NSW, Australia
We recently identified the protein tyrosine phosphatase Pez as a novel regulator of TGF-beta signalling and epithelial-
mesenchymal transition (EMT)(Wyatt et al 2007). TGF-beta is a pleiotropic cytokine that plays opposing roles in cancer
progression, acting early as a tumour suppressor, but promoting invasion and metastasis at later stages of carcinogenesis.
Cancer-associated mutations in Pez have been identified in breast (Sjoblom et al, 2006) and colorectal cancers (Wang et al,
2004), however the role of such mutations in cancer progression remains unclear. Our recent data suggests that Pez is present
in golgi / endosomal compartments in breast and other epithelial cells, modulates the secretion of multiple cytokines in addition
to TGF-beta, and influences the trafficking of various signalling receptors. In an experimental mouse model of metastasis,
reduced Pez expression leads to a specific increase in breast cancer metastasis to bone, suggesting Pez is a novel suppressor
of bone metastasis. We hypothesise that deregulation of Pez expression or function in human cancers may lead to aberrant
secretion of multiple factors into the tumour microenvironment to influence metastatic outcome. Ongoing studies aim to
elucidate the specific molecular mechanisms by which Pez functions to exert these effects on secretion and cancer metastasis.

1.        Wyatt et al (2007) J. Cell Biol. 178: 1223-1235
2.        Sjoblom et al (2006) Science 314:268-74
3.        Wang et al (2004) Science 304:1164-6


A mathematical model to simulate tumour cell interactions, motility and invasion
J. Guy Lyons1 2
1. University of Sydney, CAMPERDOWN, NSW, Australia
2. Sydney Head & Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
Background: Cancer progression is an evolutionary process. As in whole organisms, there is the potential for cancer cells to
evolve through a process of mutual benefit, or cooperation. This can occur when distinct mutations in different cells are
mutually beneficial, enhancing the growth of both populations through cell-cell interactions. Carcinoma cells often undergo an
epithelial-mesenchymal transition (EMT) during malignant progression, but this usually occurs within a predominantly epithelial
cancer cell environment. We hypothesised that EMT carcinoma cells might be able to cooperate with well-differentiated
carcinoma cells to produce an emergent behaviour that is more malignant than each population on its own.
Methods: To simulate carcinogenesis, we have produced an agent based mathematical model based on Voronoi tesselations.
This enables us to visualise cells in a dynamic way, producing realistic representations of cell shape and motility in an animated
form. At the same time, it enables us to quantitatively predict the effects of mutation rate, proliferation, adhesion, motility,
extracellular matrix breakdown and cell-cell communication on tumour progression and response to therapies. Input biological
parameters are chosen from data obtained empirically from live cell imaging and other observations of squamous cell
Results: We have used the model to compare the progression of a squamous cell carcinoma in 6 distinct scenarios of cancer
evolution, including the traditional view in which the effects of all mutations are cell-autonomous and must accumulate within a
clone for malignancy to be achieved. The endpoint for malignancy being reached was invasion of the underlying extracellular
matrix. Late occurring mutations induce EMT-like changes in cell behaviour and uncontrolled active proliferation, which were
complementary. Scenarios in which mutations are not cell-autonomous gave higher probabilities of malignancy and recurrence
following treatment than the traditional, cell-autonomous scenario.
Conclusion: Cooperation between EMT cells and non-EMT cells is a plausible mechanism for the evolution of primary and
recurrent carcinomas.


Overexpression of the urokinase receptor splice variant uPAR-del4/5 in breast cancer cells
modulates gene expression of other tumor-relevant proteins
Bettina Grismayer1 , Sumito Sato1 , Charlotte Kopitz2 , Christian Ries3 , Manfred Schmitt1 , Achim Krüger2 , Thomas
Luther4 , Matthias Kotzsch4 , Viktor Magdolen1
1. Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany
2. Institute of Experimental Oncology and Therapy Research, Technical University of Munich, Munich, Germany
3. Institute for Cardiovascular Prevention, Ludwig-Maximilians-University of Munich, Munich, Germany
4. Institute of Pathology, Dresden University of Technology, Munich, Germany
uPAR, the three-domain membrane receptor of the serine protease urokinase-type plasminogen activator, plays an important
role in tumor growth and metastasis. uPAR-del4/5 is a splice variant lacking domain DII and has been shown to be an
independent prognostic marker for distant metastasis-free survival in node-negative breast cancer patients. Moreover, uPAR-
del4/5 overexpression in breast cancer cells affects tumor biologically relevant processes such as proliferation, cellular
adhesion to extracellular matrix proteins and the invasive capacity of the cells in vitro, as well as lung colonization in an
experimental metastasis xenograft mouse mode. The aim of the present study was to investigate whether overexpression of
uPAR-del4/5 in breast cancer cells modulates expression of other tumor-relevant proteins.
Gene expression of a series of tumor-associated proteases, inhibitors and extracellular matrix proteins was analyzed in stably
transfected vector control versus uPAR-del4/5 overexpressing MDA-MB-231 breast cancer cells using a low density microarray.
Several genes were identified to be up-regulated or repressed in uPAR-del4/5 overexpressing compared to vector control cells.
One of these genes encodes the matrix metalloproteinase MMP-9 and was found to be more than 10-fold up-regulated in
uPAR-del4/5 overexpressing cells. Zymograms as well as Western blot analysis confirmed enhanced secretion of MMP-9 into
the supernatant of cultured breast cancer cells compared to vector control. uPAR-del4/5 may thus play an important role in the
regulation of the extracellular proteolytic network of breast cancer cells and, by this, influence the metastatic potential of breast
cancer cells.


A novel role for the E3 -ligase E6AP in the control of cell invasion and migration
Mariam Mansour1 , Ai-leen Chan1 , Sue Haupt1 , Cameron Johnstone1 , Mark Bishton1 , Nathan Godde 1 , Kamil
Wolyniec1 , Patrick Humbert1 , Ora Bernard 2 , Ygal Haupt1
1. PeterMac Callum Cancer center, Australia, VIC, Australia
2. St. Vincent's Institute of Medical Research, Melbourne, Victoria 3065, Australia
The E6AP E3 ligase has been implicated in HPV-related cancers and in Angelman syndrome1. We have previously
demonstrated a key role for E6AP in cell growth and in cellular stress response 2. More recently we found that mouse embryos
fibroblasts deficient for E6AP overcome cellular senescence and show a transformed phenotype in vitro and vivo 3. We
therefore investigated the role of E6AP in cell invasion and migration. Abnormal cell migration and invasion are key
components of the metastatic phenotype. The Rho GTPase (RhoA, Rac1 and cdc42) are key regulators of the actin
cytoskeleton and control cell migration, cell polarity, contractility, and invasion. Here we demonstrate for the first time a novel
role for E6AP in controlling cell invasion and migration via the regulation of Rho signalling. A loss or downregulation of E6AP
expression in human and mouse cells promotes elongated morphology with enhanced membrane protrusion, filopodia, and
actin spikes formation. Downregulation of E6AP in tumour and primary cells increases stress fibers formation and focal
adhesion turnover. Deficiency in E6AP promotes direct cell migration in-vitro and wound closure in-vivo. Likewise, E6AP
deficiency promotes cell invasion in-vitro. Importantly, reconstitution of E6AP expression decreases considerably tumour cell
invasion as demonstrated in a 3D invasion assays. In search for the mechanism underlying these effects of E6AP, we found
that Rac1 is accumulated and stabilised in cells deficient for E6AP. Our results suggest that E6AP plays an important role in
restraining cell invasion and motility.

1.         1-UBE3A/E6-AP mutations cause Angelman syndrome.Kishino T, Lalande M, Wagstaff J.; Nat Genet 1997
2.         2- E6AP promotes the degradation of the PML tumor suppressor . Louria-Hayon I, Alsheich-Bartok O, Levav-Cohen
     Y, Silberman I, Berger M, Grossman T, Matentzoglu K, Jiang YH, Muller S, Scheffner M, Haupt S, Haupt Y. Cell Death
     Differ. 2009 Aug;16(8):1156-66
3.         3-E6AP is required for replicative and oncogene-induced senescence in mouse embryo fibroblasts. Levav-Cohen Y,
     Wolyniec K, Alsheich-Bartok O, Chan AL, Woods SJ, Jiang YH, Haupt S, Haupt Y; Oncogene. 2011 Sep 19.


Mechanisms of TGFß1 signaling and repression of E-cadherin in Oral Cancer
Ali Nawshad1 , Azadeh Jalali, ChangChih Liu
1. College Of Dentistry, Lincoln, NE, United States
Epithelial Mesenchymal Transitions (EMT) creates invasive cell types from relatively sedentary epithelial cells during
embryogenesis, carcinoma invasiveness, and metastasis. The primary traits of mesenchymal cells are cell motility, migration
and invasion into the extra cellular matrix. Transforming Growth Factor ß1 (TGF1) is a potent inducer of EMT in cancer
metastasis. Cell-cell adhesion molecule, E-cadherin acts as powerful tumor suppressors in all epithelia. Loss of E-cadherin,
facilitates cell migration and invasion, and is a hallmark of EMT. TGFß1 is known to cause loss of cell-cell adhesion and loss of
cell polarity during metastasis of Oral Squamous Cell Carcinomas (OSCC). But the molecular mechanisms that control the
onset of EMT by repressing E-cadherin remain largely unexplored. Objectives: We proposed to investigate the mechanisms of
transcriptional down regulation of E-cadherin in response to TGFß1 signaling. Methods: We used cells derived from OSCC
(UMSSC-38) and treated them with exogenous recombinant TGFß1 to evaluate cellular response and EMT. We dissected the
TGFß1 pathways in detail to demonstrate the activation of kinases and transcription factors downstream to TGFß1 to show the
mechanisms of TGFß1 signaling during OSCC EMT. Results: We have demonstrated that TGFß1 signals via both Smad
dependent and Smad independent pathways to activate several EMT related transcription factors such as, Snail and SIP1. All
these transcription factors directly downregulate E-cadherin gene activity and thereby repress cell-cell adhesion. Our results
also showed that TGFß1 signaling promotes acquisition of mesenchymal markers such as Fibronectin and Vimentin to facilitate
cell migration and invasion of UMSCC-38 cells. Conclusion: Our results define the pathways of TGFß1 that alters E-cadherin
affecting cellular motility, invasiveness in OSCC. Collectively, our results suggest that combinatorial detection of these proteins
could serve as a new tool for EMT analysis in oral cancer patients.


New avenues to inhibit mutant p53-driven metastasis
Paul M Neilsen1 , Rachel J Suetani1 , David F Callen1
1. Centre for Personalised Cancer Medicine, University of Adelaide, Adelaide, SA 5005, Australia
The p53 tumor suppressor plays a pivotal role in the prevention of neoplastic transformation, with half of all human tumours
harbouring mutations in TP53. Such mutations not only lead to the expression of a protein unable to impart its normal tumour
suppressor ability, but also endow the mutant p53 protein with newly acquired oncogenic properties that drive invasion and
metastasis. Although it is widely established that mutant p53-expressing tumours have a high capacity to metastasize and are
associated with poor outcomes, the key molecular mechanisms utilised by mutant p53 are largely unknown.
We have developed a panel of H1299 carcinoma derivatives that can inducibly re-express various mutant p53 proteins into a
p53 null background. In vivo induction of mutant p53 in the primary H1299 xenograft tumour was associated with increased
distant metastases. Induction of mutant p53 drove an EMT and was associated with an increase invasive capacity in vitro;
properties consistent with the pro-metastatic phenotype1,2. We show that mutant p53 functions as an aberrant transcription
factor to selectively alter the expression of key cancer-related genes3 . Remarkably, the majority of mutant p53 target genes
identified from this study are also direct targets of the metastasis suppressor, p63. We show that mutant p53 forms an
oncogenic complex with p63 at the promoters of p63 target genes, resulting in disruption of the anti-metastatic p63-signalling
pathway. Key direct targets of mutant p53-p63 include Dicer2 , miR-1554 and DKK13 . Surprisingly, the majority of mutant p53
target genes were secreted products. This aberrant ‘secretome’ driven by mutant p53 was predicted to promote bone
destruction, as exposure of normal primary human osteoblasts to this mutant p53 secretome altered the expression of key
genes that maintains bone homestasis. As such, we predict that the secreted products from mutant p53-expressing tumours
may create a ‘pre-metastatic niche’ in the bone.

1.        Noll JE, Jeffery J, Al-Ejeh F, Kumar R, Khanna KK, Callen DF and Neilsen PM. Mutant p53 drives invasion and
     multinucleation through a process that is suppressed by ANKRD11. Oncogene. 2011. Oct 10. PMID 21986947.
2.        Muller PAJ, Trinidad AG, Timpson P, et al, Lane DP, Sansom O, Neilsen PM, Norman JC and Vousden KH. Mutant
     p53 induces MET signalling to drive cell scattering and invasion by inhibiting TAp63 and Dicer. Oncogene. In press
     (accepted 18th March 2012).
3.        Neilsen PM, Noll JE, Suetani RJ, Ho K, Schulz RB, Al-Ejeh F, Evdokiou A, Lane DP and Callen DF. Mutant p53 uses
     p63 as a molecular chaperone to alter gene expression and induce a pro-invasive secretome. Oncotarget. 2011;
4.        Neilsen PM, Noll JE, Mattiske S, Bracken CP, Gregory PA, Schulz RB, Lim SP, Kumar R, Suetani RJ, Goodall GJ
     and Callen DF. Mutant p53 drives invasion in breast tumors through up-regulation of miR-155. Oncogene. 2012. In press
     (accepted June 2012).


Identification & characterisation of EMT-regulatory miRNAs in mammary development and
Akira Nguyen1 , Yuwei Phua1 2 , Hosein Kouros-Mehr3 , Alexander Swarbrick1 2
1. Cancer Research Program, Garvan Institute of Medical Research, Sydney, N.S.W., Australia
2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, N.S.W., Australia
3. Tumour Biology and Angiogenesis Department, Genentech, Inc., South San Francisco, California, United States of
Metastasis is the major underlying cause of breast-cancer deaths and at present is incurable. The molecular pathways which
endow cancer cells with the ability to disseminate from the primary tumour, invade through stroma, and re-establish themselves
in distal tissues are not well understood. At the cellular level, self-renewal, migration and invasion are recognised as key
cellular processes driving the metastatic phenotype. During the epithelial-to-mesenchymal transition (EMT), epithelial cells lose
polarity and gain mesenchymal traits, including the capacity to migrate and self-renew. As such, re-activation of the EMT
programme in tumours is believed to be a major component of metastatic progression. We utilised an innovative ex-vivo
microarray technique to identify miRNAs that distinguish epithelial cells from mesenchymal cells in the developing mammary
gland, and hence may regulate EMT in the breast. Comparison of the miRNA expression profiles of epithelium and stroma
revealed an epithelium-specific expression pattern for miR-183 and miR-200c; both miRNAs have previously been implicated in
the regulation of EMT-associated self-renewal and invasion. Significantly, as we have identified these miRNAs in the
physiologically relevant milieu of the mammary gland, they likely represent pathways of EMT regulation native to the breast.
Consistent with a role in EMT regulation, we found that miR-183 and miR-200c expression is lost in triple negative patient
samples, compared to matched normal tissue. As miR-200c function is already well characterised, our efforts are currently
focused on investigating the potential role of miR-183 in regulation of EMT in mammary epithelium. We have generated normal
mammary epithelial and breast cancer cell lines with doxycycline-inducible expression of miR-183 which have enabled us to
investigate the effect of miR-183 in in vitro models of EMT and metastasis, and also in vivo using pre-clinical mouse models of
breast cancer.


Sunitinib/OXi4503 combination therapy leads to induction of EMT
Linh Nguyen1 , Fifis Theodora1 , Malcontenti-Wilson Cathy1 , Muralidharan Vijayaragavan1 , Christophi Christopher 1
1. The University of Melbourne, Heidelberg, vic, Australia
Introduction: Colorectal cancer (CRC) is the second most frequently occurring form of cancer worldwide. More than one
million people are diagnosed annually and over half a million die per year, with liver metastasis being the predominant cause or
mortality. Apart from a small group amenable to surgery, the majority of patients are treated with palliative chemotherapy.
Previously, we have tested the effect of a novel vascular disruptive agent (VDA) OXi4503 (Oxigene), in a clinically relevant
mouse model of colorectal liver metastases and have shown that it reduces the tumour mass by about 90%. However,
complete eradication of the tumour is not achieved following OXi4503 monotherapy. A small rim of tumour cells survives in the
tumour periphery and resumes growth as soon as treatment ceases. Tumour regrowth is characterised with robust
We hypothesize that combining OXi4503 treatment with Sunitinib, a potent angioinhibitory agent (AIA) which inhibits new
vessel formation, would prevent or delay tumour recovery
Methods: Male CBA mice are induced with colorectal liver metastases. Mice received daily Sunitinib (40mg/kg) intraperitoneal
(IP) injections beginning on day 14 post tumour induction until day 21 post tumour induction. A single IP injection of OXi4503
(100mg/kg) was administered 16 days post tumour induction.
Tissues were collected at several timepoints during the treatment process, fixed in formalin and embedded in paraffin. The
effects of the treatment were investigated using stereology, histopathology and immunohistochemistry. Tumour reduction and
changes in vascularity, proliferation, and cell morphology (EMT markers and stem cell markers) were investigated.
Results: Combination therapy demonstrated significant reduction in the percentage of viable tumour compared to either
monotherapy. Additionally there were significantly fewer tumour vessels in the combination treatment than either therapy alone.
Following combination therapy the surviving tumours were characterized by High expression of ZEB1 and Vimentin, nuclear
accumulation of E-cadherin and β-catenin and increased stem marker expression.
Conclusions: Our results demonstrate that combination treatment is more effective in reducing tumour burden than either
treatment alone; however the strong upregulation of EMT and stem cell morphology suggests development of resistance that
may contribute to metastases.


Epithelial Mesenchymal Plasticity associated loss of EpCAM in Breast Cancer – A Functional
Genomics Approach
Cletus A Pinto1 2 , Tony Blick1 , Izhak Haviv3 , Kaylene J Simpson4 , Mark Waltham1 , Erik W Thompson1 2
1. St Vincent's Institute, Fitzroy, VIC, Australia
2. Surgery, University of Melbourne, Melbourne, Victoria, Australia
3. Bar Ilan University, Ramat Gan, Israel
4. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
Epithelial Mesenchymal Transition (EMT) provide tumour cells with the ability to migrate and invade secondary sites while
Mesenchymal Epithelial Transition (MET), may then allow establishment and proliferation of the malignant tumour cells. The
term, Epithelial Mesenchymal Plasticity (EMP), encompasses the interconversions between epithelial and mesenchymal states.
We have been able to demonstrate marked similarities between the aggressive basal breast cancer cell lines and cells that
have undergone an EMT. Epithelial cell adhesion molecule (EpCAM) is widely used clinically to isolate circulating (CTC) and
disseminated (DTC) tumour cells from patient samples. EpCAM is significantly downregulated in basal breast cancer cell lines
and studies show EpCAM downregulation following an EMT.
The basal like EpCAM +ve PMC42LA cells have been transduced with a boutique shRNA library targeting the genes
responsible for maintenance of cell polarity and markers of Breast Cancer Stem Cells, EMP and metastasis. EpCAM low cells
isolated from this transduced population display a mesenchymal morphology while EpCAM high cells are more proliferative.
Using FACS, these populations have been sorted and currently awaiting next generation sequencing for identification of
hairpins enriched within these phenotypes. Further studies would incorporate the utilisation of individual hairpins and
subsequent functional studies in vitro and in vivo to identify to role of these genes in cancer biology.

The Embryonic Morphogen Nodal Promotes EMT, Invasion and Metastasis via a Non-
Canonical ERK-Dependent Pathway
Daniela F Quail1 , Guihua Zhang1 , David A Hess1 , Lynne-Marie Postovit1
1. Western University, London, ON, Canada
Breast cancer progression from a localized lesion to an invasive metastatic disease is characterized by alterations in cellular
migration, and epithelial-to-mesenchymal transition (EMT). Studies have shown that this transition is associated with an up-
regulation of embryonic stem cell-associated genes, resulting in a dedifferentiated phenotype and poor patient prognosis. Nodal
is an embryonic factor that plays a critical role in promoting early invasive events during development. Nodal is silenced as
stem cells differentiate; however, it plays a specialized role in adult life during placentation, and an aberrant role during cancer
progression. Here, we show that Nodal over-expression in poorly invasive breast cancer and choriocarcinoma cell lines causes
increased invasion and migration in vitro concomitant with the induction of EMT-associated phenomena. We show that Nodal
promotes these invasive events via an Extracellular Regulated Kinase (ERK) dependent pathway. Since Nodal normally signals
through SMADs, these findings lend insight into alternative pathways that are hijacked by this protein in cancer. To evaluate the
clinical implications of our results for cancer, we show that Nodal inhibition reduces liver tumor burden in a model of
spontaneous breast cancer metastasis in vivo, and that Nodal loss-of-function in aggressive breast cancer lines reduces
invasion and causes a Mesenchymal-to-Epithelial-like Transition (MET) concomitant with a mitigation of ERK activation. Our
results demonstrate that Nodal is involved in promoting invasion in multiple cellular contexts, and that Nodal inhibition may be
useful as a therapeutic target for patients with metastatic disease.


Wnt-5a is over-expressed in epithelial ovarian cancer and is associated with poor patient
Gaya Punnia-Moorthy1 , Caroline Ford1 , Sheri Nixdorf2 , Jake Olivier3 , Viola Heinzelmann-Schwarz2 , Robyn Ward4
1. Wnt Signalling and Metastasis Group, Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical
     School, University of New South Wales, Sydney, Australia
2. Ovarian Cancer Research Group, Adult Cancer Program, Lowy Cancer Research Centre, School of Women's and
     Children's Health and Prince of Wales Clinical School, UNSW, Sydney, Australia
3. Biostatistics Group, Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical School, UNSW ,
     Sydney, Australia
4. Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical School, UNSW , Sydney, Australia
Epithelial ovarian cancer (EOC) is the ninth most common cancer diagnosed in Australian women, but the fifth most common
cause of cancer related death. One of the pathways involved in EOC is the Wnt signalling pathway, a key developmental
pathway which is often dysregulated in cancer and is thought to play a role in epithelial to mesenchymal transition (EMT) and

In this study we investigated the expression of the Wnt ligand, Wnt-5a in a large cohort of 721 patients with benign, borderline
and EOC disease. Wnt-5a expression was significantly higher in EOC compared to benign and borderline patients (p<0.0001).
Patients with high Wnt-5a expression had a shorter relapse-free and disease-specific survival compared to patients with low or
absent Wnt-5a. There was no difference in Wnt-5a expression amongst the histological subtypes of EOC (serous,
endometrioid, mucinous and clear cell).

Wnt-5a expression was correlated with clinico-pathological parameters of EOC; positive correlation was found between Wnt-5a
and tumour marker CA-125 (p=0.01). Wnt-5a expression was also significantly associated with patient age (p<0.0001), ascites
(p<0.0001), residual disease (p<0.0001) and advanced FIGO stage (p=0.0075). In addition, there was a significant negative
correlation between Wnt-5a and sFRP4 expression, an antagonist of the Wnt signalling pathway (p<0.0001).
This study demonstrated for the first time an association between Wnt-5a, sFRP4 and aggressive ovarian cancer. We are
currently undertaking a series of in vitro studies investigating the effects of Wnt-5a modulation in the serous ovarian cancer cell
line OVCAR3 and the normal ovarian surface cell line HOSE 6.3. Preliminary results using siRNA against Wnt-5a and
recombinant Wnt-5a indicate that modulation of this Wnt ligand can effect downstream Wnt signalling and EMT.

In conclusion, we have shown in a large patient cohort that Wnt-5a plays an oncogenic role and is a predictor of poor survival
for EOC patients.

1.        Loss of secreted frizzled-related protein 4 correlates with an aggressive phenotype and predicts poor outcome in
     ovarian cancer patients. Jacob F, Ukegjini K, Nixdorf S, Ford CE, Olivier J, Caduff R, Scurry JP, Guertler R, Hornung D,
     Mueller R, Fink DA, Hacker NF, Heinzelmann-Schwarz VA. Translational Research Group, University Hospital Zurich,
     Zurich, Switzerland. PLoS One. 2012;7(2):e31885. Epub 2012 Feb 21


Inhibiting p90 ribosomal S6 kinase (RSK)/Y-box binding protein-1 (YB-1) signaling eliminates
tumor-initiating cells (TICs) in triple-negative breast cancer.
Kristen Reipas1 2 , Anna Stratford2 , Nicole Couto1 2 , Kaiji Hu2 , Amina Zoubeidi3 , Sandra E Dunn1 2
1. Experimental Medicine, University of British Columbia, Vancouver, British Columbia, Canada
2. Pediatrics, Child and Family Research Institute, Vancouver, British Columbia, Canada
3. Vancouver Prostate Center, Vancouver, British Columbia, Canada
Despite advances in the treatment of breast cancer, disease recurrence rates remain high and secondary tumors are often
incurable. The concept of tumor-initiating cells (TICs) is an emerging model to explain cancer relapse. Breast TICs express
CD44+/CD24- surface markers, have the capacity to self-renew, differentiate and indefinitely perpetuate tumor initiation1.
Recently, a link between epithelial-mesenchymal transition (EMT) and stemness has also been identified 2. Due to their
inherent resistance to conventional chemotherapeutics, TICs survive and can go on to drive relapse3.
Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor consistently associated with disease recurrence
in breast cancer. YB-1 is activated predominantly by phosphorylation via p90 ribosomal S6 kinase (RSK)4. Once activated it up-
regulates expression of CD445. YB-1 is expressed in ~70% of one of the most aggressive subtypes – triple-negative breast
cancer (TNBC). Currently, the treatment for TNBC relies upon conventional chemotherapies and although these tumors often
initially respond well, they paradoxically have the highest relapse rates.
We observe that residual cells after paclitaxel or epirubicin treatment have increased P-RSKS221/7, P-YB-1S102 and CD44 and
retain the ability to form mammospheres. Moreover, cells expressing high levels of P-YB-1S102 proliferate in the presence of
paclitaxel. Inhibiting YB-1 in residual cells using siRNA suppresses their growth and induces apoptosis. Cell death can also be
induced in CD44+/CD24--sorted cells by inhibiting YB-1 directly, or by blocking its activation through RSK inhibition (siRNA or
small molecule BI-D1870). The dependence of TICs on YB-1 may be, in part, through regulation of Notch4 as the latter
maintains mammary stem cells. Inhibiting YB-1 decreases Notch4 mRNA, which may affect TIC self-renewal. Conversely,
expressing constitutively active YB-1S102D increases Notch4. Interestingly, inhibiting RSK in post-EMT MDA-MB-231 cells
blocks Notch4 expression, reduces spindle-like morphology and induces E-cadherin re-expression.
Collectively, these data indicate that RSK/YB-1 signaling is critical for the survival of TICs and that inhibiting this pathway has
the potential to reduce relapse in TNBC.

1.         Al-Hajj, M. et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003.
2.         May, C.D. et al. Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast
     cancer progression. Breast Cancer Res. 2011.
3.         Li, X. et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008.
4.         Stratford, A.L. et al. Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-
     like breast cancer cells. Breast Cancer Res. 2008.
5.         To, K. et al. Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal,
     mammosphere growth, and drug resistance. Cancer Res. 2010.


MicroRNA-200 family members differentially regulate spontaneous breast cancer metastasis
Phillip A Gregory1 2 , Xiaochun Li1 , Suraya Roslan1 , Josephine A Wright1 , Cameron P Bracken1 , Andrew G Bert1 ,
Robin L Anderson1 3 , Gregory J Goodall1 2 , Yeesim Khew-Goodall1 4
1. Centre for Cancer Biology, Adelaide, SA, Australia
2. Discipline of Medicine , The University of Adelaide, Adelaide, SA, Australia
3. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
4. School of Molecular and Biomedical Sciences, The University of Adelaide, Adelaide, Adelaide, SA, Australia
A major cause of cancer related death for women in Australia is breast cancer metastasis. For a tumour to metastasise, it first
must invade from the primary tumour, extravasate and survive in the bloodstream and then germinate at a distant site to form a
secondary tumour. An important component of this process is epithelial-mesenchymal transition (EMT), characterised by
tumour cells losing their epithelial non-invasive properties and gaining mesenchymal properties, including loss of cell–cell
adhesions which make them more motile and have the ability to invade. We have recently demonstrated that the miR-200
family play a crucial role in maintaining the epithelial phenotype via repression of the ZEB transcription factors 1 2 3 . The miR-
200 family is divided into two functional clusters miR-141/200a and miR-200b/200c/429, which are clustered depending on their
“seed” sequence. The aim of this study is to investigate the individual roles of miR-200 family members in regulating
spontaneous breast cancer metastasis using a xenograft animal model. We found that stable over-expression of miR-200b or
miR-200c, but not miR-141, in the highly metastatic MDA-MB-231 (LM2) subline, led to a loss of ZEB1 expression and a
reduction in the ability of these cells to spontaneously metastasise from the mammary fat pad. In experimental metastasis
assays, miR-200b expressing cells did not enhance lung colonisation indicating miR-200b was specifically influencing cell
invasion from the primary tumour. Re-expression of ZEB1 in the miR-200b stable cell line could not alleviate metastatic
repression, suggesting miR-200b suppresses tumour cell invasion by targeting multiple genes. Together, this data implies that
miR-200 family members can differentially regulate spontaneous breast cancer metastasis and can operate through ZEB1-
independent pathways.

1.        Gregory PA et al (2008) Nature Cell Biol. 10, 593-601.
2.        Bracken CP et al (2008) Cancer Res. 68, 7846-7854
3.        Gregory PA et al (2011) Mol. Biol. Cell. 22, 1686-1698.


3D quantitative analysis of local invasiveness and lymphovascular dissemination of human
oral squamous cell carcinoma xenografts in mouse tongue microenvironment
Yoshihito Shimazu1 , Yuuichi Soeno1 , Kazuya Fujita 1 , Yuji Taya1 , Kayo Nakau1 , Kaori Sato1 , Takaaki Aoba1
1.   Department of Pathology, School of Life Dentistry at Tokyo, Nippon Dental University School of Life Dentistry at Tokyo,
       Tokyo, Japan
Squamous cell carcinoma (SCC) in tongue exhibits aggressive progression and early-stage lymphnode metastasis. The
present study aimed at elucidating a putative linkage between SCC cell phenotypes and lymphovascular metastasis in a mouse
xenograft model. Five tongue and oral floor-derived SCC cell-lines (OSC19, OSC20, HSC2, KOSC2, HO-1u1) were used. Each
cell-line grown in culture was transplanted into BALB/c nude mouse tongue. Phenotypic plasticity of SCC cells with growth of
primary xenograft tumors and nodal metastasis were examined immunohistochemically. We also conducted 3D reconstruction
of the primary tumors using serial histological images to assess quantitatively the local invasiveness of SCC cells, the density
of blood/lymphatic vasculature, and the location and frequency of lymphovascular tumor invasion. All five cell-lines gave rise to
visible tumor masses in the tongue within 2-3 weeks, but individual tumor cells showed diversities in Ki67-positive proliferation
activity, intra- and peritumor vascular densities, and EMT-like phenotypes. 3D visualization of the primary tumor architecture
disclosed discrete invasion modes from massive growth with pushing border (OSC20, OSC19) to branching (HSC2, KOSC2)
and fingering infiltration (HO-1u1). EMT-like cells showing loss of E-cadherin and up-regulation of vimentin accumulated at the
peripheral region of tumor mass, resulting in segregation of individual tumor cells from mother cell population, although
lymphovascular invasion by single and/or a few EMT-like cells were only occasionally seen. In contrast, the majority of
lymphovascular invasion took place in a form of penetration of cohesive tumor cell clusters through the lymphatic vessel wall,
giving rise to large tumor emboli retaining epithelial phenotypes (cytokeratins and E-cadherin) and Ki67-positive proliferation
activity. Similar SCC phenotypes including persistent E-cadherin expression were also seen in nodal metastasis. The results
indicate that EMT-like phenotypic plasticity of SCC cells is responsible for their local invasive properties but not prerequisite for
lymphovascular dissemination.


Eliza T.L Soo1 2 , Tony Blick1 , Mark Waltham1 , Gregory J Goodall3 , Angels Fabra-Fres4 , Izhak Haviv5 , Kaylene J
Simpson6 , Erik W Thompson1 2
1. St Vincent's Institute, Fitzroy, VIC, Australia
2. Surgery, University of Melbourne, Melbourne, Victoria, Australia
3. Center for Cancer Biology, SA Pathology, Adelaide, South Australia, Victoria
4. IDIBELL, Barcelona, Spain
5. Bar Ilan University, Ramat Gan, Israel
6. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
MicroRNAs (miRs) are a class of small RNA molecules (18-25 nucleotides) that regulate gene transcript stability and
processing by binding to discreet motifs in the 3’ and 5’ UTRs of mRNAs. They play important roles in development, including
epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have been associated with
cancer metastasis. Epithelial mesenchymal plasticity (EMP) encompasses the dynamic interconversion between epithelial and
mesenchymal states. Mesenchymal PMC42-ET (ET) human breast cancer cells and its’ epithelial-like PMC42-LA (LA) sub-line
provide an ideal tool to study miR expression profiles associated with EMP. miR profiling of control and EMT-induced ET and
LA cells using the mirVana probe set V1 and Next Generation Sequencing (miRSeq) was undertaken. Several miRs were
reproducibly up- or down-regulated between untreated ET and LA cells, as well as in response to EGF. Variations in miR
expression were also assessed bioinformatically in public data from >50 human breast cancer cell lines. Whilst a number of
these have already been implicated in cancer (e.g. miR-200 family), other novel miRs consistently associated with EMP were
also identified. The expression levels of >20 miRs were validated using TaqMan® miR assays in 6 breast cancer cell lines.
Stable over-expression of miRs in cell lines with low endogenous expression was achieved by lentiviral transduction. Stable
breast cancer cell lines over-expressing the miRs of interest were produced and examined by qRT-PCR for EMP-associated
gene expression. Following this, miR over-expressing cell lines with EMP-related gene signatures were tested for changes in in
vitro migratory potential using a monolayer wound healing assay on the Cellomics platform. miRs with the most compelling
functional changes will be examined in the MDA-MB-468 xenograft model of in vivo EMP.


Functional validation of a novel prostate cancer susceptibility variant in the KLK3 gene
Srilakshmi Srinivasan1 , Batra Jyotsna1 2 , Carson Stephens1 , Amanda B Spurdle2 , Judith A Clements1
1. Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland
     University of Technology, Brisbane, Queensland, Australia
2. Molecular Cancer Epidemiology Laboratory, Genetics and Population Health Division, Queensland Institute of Medical
     Research, Brisbane, Queensland, Australia
Prostate specific antigen (PSA) is the only known biomarker for prostate cancer (PCa). Elevated levels are seen in PCa
patients owing to PSA leakage into the circulation, due to the loss of glandular architecture and breakdown of basement
membrane within the tumour. PSA/Kallikrein related peptidase 3 (KLK3) by cleaving proteins with known roles in invasion and
metastases (like transforming growth factor β2, parathyroid hormone-related protein etc.) is also suggested to facilitate PCa
progression and metastasis (1, 2). Considerable evidence suggests that genetic factors can be linked to PCa susceptibility or
tumour aggressiveness. Thus we hypothesize that, transcriptional or protein alterations due to single nucleotide polymorphisms
(SNPs) may be determinants of the differential expression or activity of PSA/kallikrein 3 (KLK3). Fine-mapping studies have
identified a missense SNP, rs17632542 (Ile179Thr) in the KLK3 gene to be strongly associated with PCa risk compared to the
previously identified KLK3 variants (3, 4, 5). In silico analysis for this missense SNP suggests an alteration in a splice site (6)
and/or protein stability (7) for the risk allele. To validate the biological function of the rs17632542 SNP, we have performed
gene expression studies correlating the genotype with KLK3 expression levels and protein stability studies to verify the
thermodynamic stability difference of the variant protein encoded by the risk and wild-type alleles. Further, the protein allele
isoforms displayed a significant difference in their thermal stability, which suggests a possible functional difference between the
variant and wild-type alleles in PCa. These results indicate that SNPs in the KLK3 gene may be important in regulating the
expression and/or activity of KLK3/PSA, an important mediator of prostate carcinogenesis and metastasis.


A significant subpopulation of tumor cells in sentinel lymph nodes from breast cancer patients
express mesenchymal characteristics and are not detected by conventional methods
Siri Tveito1 , Kristin Andersen1 , Rolf Kaaresen2 , Oystein Fodstad1
1. Oslo University hospital, The Radium hospital, Oslo, Norway
2. Oslo University hospital, Ullevaal hospital, Oslo, Norway
  The presence or absence of tumor cells in the sentinel lymph node (SLN) is the most important prognostic factor in early
stage breast cancer. However, the ideal method for SLN examination is still being sought and currently many different protocols
are employed.
We have used two methods for tumor cell detection in SLNs; immunomagnetic selection (IMS) using antibodies recognizing the
surface proteins EpCAM and Mucin 1, and RT-PCR targeting breast epithelial transcripts (hMAM, AGR2, TFF1 and SBEM).
The two methods were used on the same SLN samples, enabling the comparison of results based on the expression of
external cell-surface markers (IMS) with those based on intracellular markers (RT-PCR). EpCAM-IMS proved to have highest
sensitivity and identified more positive SLNs than the RT-PCR targeting breast epithelial transcripts. Further examination
showed that all RT-PCR marker-expressing cells were also positive with EpCAM, but also that EpCAM positive cells lacking
expression of epithelial transcripts had increased expression of MMPs, repressors of E-Cadherin, vimentin, EGFR and
members of the Wnt signaling pathway (1). Notably, these cells could not be identified using epithelial cytokeratins or
mammaglobin as RT-PCR targets, and might represent a subpopulation of mesenchymal like tumor cells with a putative more
aggressive phenotype. The results raise important questions regarding existing methods for detection of micrometastatic cells
and may have significant clinical implications.
To further evaluate the temporal relationship between the expression of external protein markers (e.g. EpCAM) and internal
epithelial/mesenchymal transcriptional markers, we are currently working with model systems using breast epithelial cell lines
that are induced to undergo EMT. We aim to establish a timeline for the expression of specific markers used for tumor cell
detection, in particular EpCAM but also transcriptional markers.

1.       1. Tveito S, Andersen K, Kåresen R, Fodstad Ø. ”Analysis of EpCAM positive cells isolated from sentinel lymph
     nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles.” Breast Cancer Res.
     2011 Aug 4;13 (4):R7


Alternative pre-messenger RNA splicing events in the PMC42 model of Epithelial Mesenchymal
Edwin Widodo1 2 , Honor Hugo1 , Kimberly Dittmar3 , Russ P Carstens3 , Bryce van Denderen4 5 , Erik W Thompson1 4 ,
Eva Tomaskovic-Crook1 4
1. Surgery, University of Melbourne, Melbourne, VIC, Australia
2. Faculty of Medicine, Brawijaya University , Malang, East Java, Indonesia
3. School of Medicine, University of Pennsylvania , Philadelphia, USA
4. IMU, St Vincent's Institute of Medical Research, Melbourne, VIC, Australia
5. Medicine, University of Melbourne, St Vincent's Hospital, Melbourne, Australia

   Alternative pre-messenger RNA splicing (AS) events are regulated when breast cancer cells acquire metastatic features
through epithelial mesenchymal transition (EMT). ASE following EMT in the PMC42 human breast cancer cell lines were
investigated by (i) comparisons between the more mesenchymal parental PMC42-ET (ET) cells and the more epithelial
PMC42-LA (LA) subline, and (ii) in response to epidermal growth factor (EGF), which stimulates EMT-like changes at the
mRNA and protein level in both PMC42 variants. We examined Epithelial Splicing Regulatory Protein (ESRP)-associated AS in
the PMC42 system and other human breast cancer cell lines, using 2D and 3D cultures, as well as upon ZEB1-knockdown in
ET cells. AS events in the PMC42 model showed an epithelial vs. mesenchymal splicing pattern that was directly correlated
with expression of ESRP 1 and 2. ESRP1 was significantly higher (p<0.001) in LA than ET. ESRP1 levels were much higher
than ESRP2, implicating ESRP1 rather than ESRP2 was the primary regulator of these AS events. The ESRP1-regulated
epithelial-specific exon FGFR2-IIIb was higher in PMC42-LA than ET (p<0.001), and reduced by EGF in LA (p<0.01). ESRP
regulation was also reflected in increased levels of 6 other ESRP-enhanced exons (ENAH, FLNB, RALGPS2, SLK, BAIAP2,
FNIP1) and reduced levels of 5 other ESRP-silenced exons (OSBPL3, MAGI1, SCRIB, ARHGAP17, MBNL1) in PMC42-LA
compared to PMC42-ET. CD44 standard isoform (CD44s) was elevated from Luminal to Basal B cells, and increased post EGF
treatment in both PMC42 variants. CD44 variable regions (exon 5-6 and 8-10) exhibited higher levels in PMC42-LA than ET.
ZEB1-knockdown in PMC42-ET cells caused increased levels of ESRP1 and the FGFR2-IIIb splice variant.
In 3D environments (Matrigel or collagen (Vitrogen)), ESRP1 and FGFR2-IIIb were significantly higher in PMC42-LA than
PMC42-ET irrespective of EGF treatment. ENAH expression in Matrigel was similar in both PMC42 variants, irrespective of
EGF, but increased by EGF in Vitrogen. CD44 was regulated more in Vitrogen than in Matrigel. These results suggest that 3D
environment influence ESRP-related AS


Whole genome multiparametric screening using morphological change and vimentin reporter
activity to identify modulators of epithelial-mesenchymal transition
Nur Akmarina BM Said1 , Cathryn M Gould2 , Kaylene J Simpson2 3 , Elizabeth D Williams4
1. Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
2. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
3. Department of Pathology, University of Melbourne, Parkville, Victoria, Australia
4. Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Metastasis accounts for the majority of treatment-refractory cancers and poor prognosis of the disease. The phenotypic
transition of non-motile epithelial tumour cells to migratory and invasive mensenchymal cells (epithelial-mesenchymal transition
(EMT)) enables the escape of cells from primary tumor site into the circulation. Morphological change from cobblestone to
elongated and spindle-shaped like cells is a primary feature of this process. This transition is also marked by upregulation of
biomarker vimentin, an intermediate filament found in mesenchymal cells. The reverse process, mesenchymal to epithelial
transition (MET) is postulated to allow these disseminated mesenchymal-like cells to establish macrometastasis at secondary
sites. Our lab has previously developed the TSU-Pr1 bladder cancer model, which we have used to demonstrate the
relationship between epithelial/mesenchymal state and metastasis (1). Given the need to elucidate the pathways and assign
functions to particular genes involved in this process, we conducted a high content screening assay using TSU-Pr1-B1 cells
together with genome wide siRNA, miRNA and kinase inhibitor libraries to systematically identify modulators of EMT/MET.
Using morphological change and a vimentin promoter-reporter system (Vimentin/dsRed) that is responsive to alterations in
cellular epithelial and mesenchymal state as primary readouts, screening in this model has identified candidate genes and
miRNAs involved in the regulation of EMT/MET. Interestingly, a number of miRNA hits (z-score ≥ 2) predicted target genes
overlap with siRNA hits (morphological change z-score ≥ 4 and/or Vimentin/dsRed z-score ≥ 5). These genes (n=310), together
with an additional ninety genes identified using the siRNA and kinase libraries, are currently being validated. This robust
multiparameter assay has the power to generate high confidence hits that will enable the identification of novel molecules
involved in this process, in addition to confirming the involvement of some established genes in regulating EMT/MET. This
unbiased genome wide approach will contribute to our understanding of this essential process.
Project supported by the National Breast Cancer Foundation (Australia), a NHMRC Career Development Award (EDW) and a
Malaysia Government scholarship (NABMS).
(1) Chaffer et al. (2005) Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in
transitional cell carcinoma of the bladder. Clin Exp Metastasis 22: 115-25.


Integrin linked kinase complexes with p38 to regulate bladder cancer cell migration and actin
cytoskeleton remodelling
Liang Yu1 , Badia Barakat1 , Die Wang1 , Georgina Ryland2 , Elizabeth D Williams1 , Gregory E Hannigan 1
1. Monash Institute of Medical Research, clayton, vic, Australia
2. Peter MacCallum Cancer Centre , East Melbourne, Victoria, Australia
Integrin linked kinase (ILK) is an essential transducer of integrin and growth factor signals, and its protein levels and activity are
often elevated in solid tumours. ILK physically links integrins and receptor tyrosine kinases to the actin cytoskeleton, and
genetic or pharmacologic inhibition of ILK activity blocks cell migration, making it an attractive target for development of anti-
cancer therapeutics. Many of the functions of ILK rely on its protein adaptor role to regulate various oncogenic signals. In this
study we found that levels of activated p38β, the predominant p38 isoform in bladder cancer cells, directly correlated with the
migratory capacity of bladder cancer cells. RNAi knockdown of either p38β or ILK markedly inhibited migration of cells toward
serum. Consistent with this, depletion of either ILK or p38β in bladder cancer cell lines resulted in disruption of the actin
cytoskeleton. However, p38 knockdown did not affect cellular ILK levels, suggesting that p38 is an effector of ILK-dependent
migration in these cells. Using co-immunoprecipitation, proximity ligation and bimolecular fluorescent complementation assays,
we found that ILK selectively forms complexes with p38β, indicating that interaction with ILK plays a critical role in stabilizing
p38β in these cells. Treatment of ILK-depleted cells with the 26S proteosomal inhibitor MG132 partially rescued p38 levels,
suggesting a key role for ILK is the protection of p38b from proteosomal degradation. Interestingly, a canonical target of ILK
signalling, Akt Ser473 phosphorylation, was not affected by ILK depletion in these experiments. These results identify a novel
ILK-p38β axis that plays important role in cancer cell migration. Thus targeting the ILK-p38 pathway is a potential strategy to
inhibit cancer metastasis.


Kindlin-2 forms a transcriptional complex with β-catenin and TCF4 to enhance Wnt signaling
and promotes tumor invasion
Hongquan Zhang1
1. Department of Anatomy, Histology and Embryology,, Beijing, China
Kindlin-2, as a focal adhesion protein, controls integrin activation. However, the association of Kindlin-2 with cancer-related
signaling pathways is unknown. Here, we identified a novel direct interaction between Kindlin-2 and the active β-catenin.
Importantly, Kindlin-2 forms a tripartite complex with β-catenin and TCF4. Mechanistically, Kindlin-2 selectively strengthens the
occupancy of β-catenin on the Wnt target gene Axin2 and enhances Axin2 gene expression. Functionally, the β-catenin–Axin2–
Snail cascade is required for Kindlin-2-induced tumor cell invasion. Our data reveal that Kindlin-2 is a new regulator of Wnt
signaling, providing a mechanistic insight into the role of Kindlin-2 in cancer progression.


Beta-blocker therapy reduces breast cancer metastasis.
Ming Chai1 , Caroline Le1 , Ian Holmes1 2 , Erica Sloan1
1. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia
2. CRC for Cancer Therapeutics, Monash University, Melbourne, Australia
Our recent experimental data demonstrated a protective role of beta-blockers against sympathetic nervous system-enhanced
breast cancer metastasis. Consistent with these findings, human epidemiological data suggests women with breast cancer who
also received beta-blocker treatment for hypertension exhibited better cancer related outcomes including reduced metastasis.
However, the relative importance of beta-1 versus beta-2 adrenergic pathways remains unclear. To investigate the role of
specific beta-adrenergic pathways in improving breast cancer outcomes, we used beta antagonists CGP20712A and ICI118551
to selectively inhibit beta-1 and beta 2 pathways, respectively, in an orthotopic mouse model of breast cancer metastasis.
Selective antagonism of the beta-2 adrenergic pathway reduced distant metastasis to lung and lymph node by 47% (p = 0.03),
consistent with the effects of the non-selective beta-blocker propranolol. In contrast, selective beta-1 antagonism alone did not
reduce metastasis. Beta-2 antagonism reduced the expression of prometastatic genes including Vegfa and Mmp9 and genes
associated with an M2 differentiated macrophage phenotype including Arg1 and Tgfb. These findings identify the beta-2
adrenergic pathway as a potential driver of breast cancer progression. This suggests that pharmacological agents that non-
specifically target beta-adrenergic pathways such as propranolol may be more appropriate adjuvant therapies in reducing
breast cancer metastasis compared to their more commonly prescribed beta-1 selective blockers such as atenolol and


Stable and potent suppression of TUBB3 modulates the expression of proteins involved in
tumourigenesis and metastases in non-small cell lung cancer
Rafael B Erlich 1 2 , Joshua A McCarroll1 2 , Pei Pei Gan1 , Tanya Dwarte1 , Frances L Byrne1 , Maria Kavallaris1 2
1. Childrens Cancer Institute Australia, Randwick, NSW , Australia
2. Australian Centre for Nanomedicine-UNSW, Sydney, NSW, Australia
Non-small cell lung cancer (NSCLC) is the most common and deadliest form of lung cancer. Despite aggressive treatment the
survival rates remain dismal. High levels of the microtubule protein, βIII-tubulin (TUBB3), correlate with drug resistant and
aggressive tumours in the clinic. Recently, we showed that silencing TUBB3 using RNA interference sensitises NSCLC cells to
chemotherapy and plays a role in regulating tumour growth. However, how TUBB3 is mediating these effects in NSCLC is
unknown. The aims of this study were to examine protein pathways regulated by TUBB3 using differential and functional
Methods: NSCLC cells stably expressing shRNA against TUBB3 or control shRNA were developed. Cytosolic and nuclear
protein fractions were prepared from these cells and 2-D DIGE was performed. Differences in protein expression between
TUBB3 knockdown (KD) and control cellswere quantitatively assessed and protein spots of interest were excised and identified
by mass spectrometry.
Results: Eleven out of a total of 963 proteins (pI 4-7) and 53/753 proteins (pI 4.5-5.5) were significantly altered in the
cytoplasmic fractions of TUBB3 KD cells. In addition, 33 out of 1331 proteins (pI 4-7) and 42/502 proteins (pI 4.5-5.5) were
significantly altered in the nuclear fractions of TUBB3 KD cells. Importantly, a number of proteins involved in regulating tumour
growth and metastases were differentially expressed in the TUBB3 KD cells, including a significant increase in the tumour
suppressor protein maspin. Differential expression of maspin was validated using western blotting and qPCR. Rescue of
TUBB3 in the KD cells restored maspin levels to control levels, indicating a potential interaction between TUBB3 and maspin.
Conclusions: This is the first study to show that TUBB3 regulates the expression of proteins involved in tumourigenesis and
metastasis in NSCLC. Targeting TUBB3 could be a promising strategy for inhibiting tumour growth and metastasis in lung


Breast cancer progression and metastasis regulated by SIN3 chromatin modification
Douglas R. Hurst1 , John W. Thomas1 , Jianzhong Liu1
1. University of Alabama at Birmingham, Birmingham, AL, United States
Epigenetic regulation of gene transcription by histone modification and chromatin remodeling has been linked to many
biological and pathological events including breast cancer metastasis. SIN3 epigenetically regulates the expression of multiple
genes by recruiting protein and DNA modifying enzymes. Altering the protein composition of these large SIN3 complexes leads
to suppression of breast cancer metastasis in mouse models without preventing orthotopic tumor growth. We hypothesized that
composition of these complexes regulates the metastatic ability of breast cancer cells. Many studies have analyzed SIN3
associated proteins in human cells using systems with a protein of interest ectopically expressed. While these studies have
been useful to identify many interacting proteins and possible functions, the complexes are altered with over-expression
systems. In order to understand how the complex differs between metastatic and non-metastatic cells, we characterized the
endogenous composition of SIN3 complexes. Size exclusion FPLC was used to isolate complexes > 1 MDa in both the nuclear
and cytoplasmic lysates. These large complexes were co-immunoprecipitated with anti-SIN3A and all associated proteins were
identified by MALDI-TOF mass spectrometry. The intracellular location of these interactions was also determined in situ using a
proximity ligation assay (DuoLink). HDAC1 and 2 interactions with SIN3A and B were found to be differentially localized
between metastatic MCF10CA cell lines compared to the normal MCF10A breast cell line. Also, the metastasis suppressor
BRMS1 was found to associate with SIN3A in the cytoplasm that contributes to functional metastasis suppression. These
results identify new roles for the SIN3 chromatin modification complexes that are distinct from chromatin structure and may be
critical for breast cancer progression and metastasis.


Neural regulation of pancreatic cancer: Beta-adrenergic signaling increases primary tumor
growth and metastasis
Corina Kim-Fuchs1 2 , Ming Chai1 , Eliane Angst2 , Erica Sloan1
1. Monash Institute of Pharmaceutical Science, Monash University, Melbourne, Australia
2. Department of Visceral Surgery and Medicine, University Hospital, Bern, Switzerland
Pancreatic cancer is the 4th leading cause of cancer death. New therapeutic strategies to treat pancreatic cancer and to
prevent recurrence and metastasis are critically needed. The sympathetic nervous system (SNS) innervates the pancreas and
key metastatic target organs including lymph nodes and liver, and releases stress neurotransmitters in response to activation.
However the impact of SNS signalling on pancreatic cancer progression is currently unknown. To investigate the effect of SNS
signalling in an orthotopic mouse model of pancreatic cancer progression we used 2 hr daily restraint stress to physiologically
activate the SNS. To track tumor progression, 1 mm3 tumor pieces were derived in vivo from luciferase-tagged PANC-1
pancreatic carcinoma cells and then implanted into the pancreatic tail. Bioluminescence imaging was used to track primary
tumour growth, metastasis and tumor recurrence after surgical resection. Physiological SNS activation increased primary tumor
size by 19-fold (p = 0.05). Pharmacological SNS activation with the beta-agonist isoprenaline increased primary tumor burden
by 4-fold. RT-PCR confirmed expression of beta1- and beta2-adrenergic receptors on PANC-1 cells. However, isoprenaline did
not modulate tumor cell proliferation in vitro, suggesting that chronic stress accelerated cancer progression through an indirect
mechanism. Stress increased metastasis to liver, local regional lymph nodes and adrenal gland. Consistent with beta-
adrenergic regulation of metastasis, isoprenaline increased invasion of PANC-1 cells through matrigel by 7.3-fold (p <0.01). We
are now conducting proof-of-principle studies to investigate if blocking beta-adrenergic signalling slows pancreatic cancer
progression. These studies will pave the way for new therapeutic interventions that slow or block pancreatic cancer progression
by targeting peripheral neural pathways.


Heat Stress Induces Epithelial Plasticity and Cell Migration Independent of Heat Shock Factor
Benjamin J Lang1 , Linh Nguyen2 , Chau Hoang Nguyen1 , Jessica L Vieusseux1 , Ryan C.C. Chai1 , Christopher
Christophi2 , Theodora Fifis2 , Michelle M Kouspou1 , John T Price1
1. Monash University, Clayton, VIC, Australia
2. Department of Surgery, The University of Melbourne, Heidelberg, Victoria, Australia
The Heat Shock Response (HSR) is a major cellular stress pathway activated by protein-damaging (proteotoxic) stress. Both
proteotoxic stress and HSR activation are inherent to cancer cells and cancer therapies including cytotoxic chemotherapy,
radiation and hyperthermic therapy. The current study examines the concept that tumour cell exposure to acute proteotoxic
stress results in the acquisition of a more advanced and aggressive cancer cell phenotype through activation of the
transcriptional regulator of the HSR, Heat Shock Factor-1 (HSF1). Specifically, we determined whether heat stress resulted in
an epithelial-to-mesenchymal transition (EMT) and/or the enhancement of cell migration, components of advanced and
therapeutically resistant cancer phenotypes. We identified that heat stress enhanced cell migration in the lung A549, and breast
MDA-MB-468 human adenocarcinoma cell lines, with A549 cells also undergoing a partial EMT. Moreover, in an in vivo model
of thermally ablated liver metastases of the mouse colorectal MoCR cell line, immunohistological analysis of classical EMT
markers demonstrated a shift to a mesenchymal phenotype in the surviving tumour fraction, further demonstrating that thermal
stress can induce epithelial plasticity. However, despite HSF1 being the master regulator of the HSR, knockdown of HSF1 in
the A549 model did not prevent the associated morphological changes or enhanced migratory profile of heat stressed cells.
Therefore, this study provides evidence that heat stress significantly impacts upon cancer cell epithelial plasticity and the
migratory phenotype independent of HSF1. These findings further our understanding of novel biological downstream effects of
heat stress and their potential independence from the classical heat shock pathway.


Neural signaling promotes lymphangiogenesis and increases lymphatic metastasis.
Caroline P Le1 , Ming G Chai1 , Matthew A Pimentel1 , Erica K Sloan1
1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
Lymphangiogenesis facilitates tumour cell escape from primary tumours to promote lymphatic metastasis. However, little is
known about the physiological dynamics that regulate lymphangiogenesis. We previously showed that chronic physiological
stress increases breast cancer metastasis through the peripheral sympathetic nervous system that signals via β-adrenergic
receptors. This study investigated the impact of chronic stress on lymphangiogenesis to promote breast cancer progression. To
determine the effect of stress-induced SNS activation on lymphatic metastases, mice were inoculated into the fourth mammary
fat pad with 2 x 10^5 MDA-MB-231/HM breast cancer cells and tumour cell dissemination was tracked by bioluminescence
imaging. Chronic stress increased lymph node metastasis by 14-fold (p < .001). Similarly, pharmacological activation of SNS
beta-adrenergic signalling increased lymphatic metastasis by 42-fold (p < .001). To investigate if increased lymphatic
metastasis was linked to increased lymphatic density in primary mammary tumours, we used immunostaining for lyve-1
lymphatic endothelial cell marker and found that chronic physiological stress increased lymph vessel density by 3-fold (p = .03).
RT-PCR was used to quantify the effects of SNS activation on genes involved in lymphangiogenesis, and found that chronic
stress increased VEGF-D expression in primary mammary tumours by 60% (p = .02). Blocking SNS signalling through β2-
adrenergic receptors using the β2-specific antagonist ICI118551 reduced stress-induced VEGF-D expression by 55% (p <
.001). β1-adrenergic receptor-specific antagonist CGP20712A did not block stress-induced VEGF-D gene expression,
suggesting the effect of stress on lymphangiogenesis may be mediated through the β2-adrenergic signalling pathway. β-
adrenergic agonists had no effect on VEGF-D expression in cultured tumour cells, suggesting increased VEGF-D expression
observed within primary tumours of animals subjected to restraint stress may be due to SNS signalling to tumour-associated
stromal cells such as macrophages or lymphatic endothelial cells. These studies suggest that SNS signaling promotes
lymphatic metastasis through β2-adrenergic regulation of primary tumour lymphangiogenesis. Ongoing studies are identifying
the cellular mediators of these effects and defining the impact of blocking VEGF-D activity. Findings of these studies will
provide a mechanistic foundation for translation of β-blockers and other SNS antagonists into the cancer clinic.


Over-expression of either CD44 or CD147 modulates in vitro and in vivo prostate cancer
metastasis and chemoresistance
Yong Li1 , Jingli Hao1 2 , Michele C Madigan3 , Aparajita Khatri4 , Carl A Power5 , Tzong-Tyng Hung4 5 , Julia Beretov1 2 6 ,
Jie Nil1 , Paul J Cozzi2 7 , Peter H Graham1 2 , John H Kearsley1 2
1. Cancer Care Centre, St George Hospital, Kogarah, Australia
2. St George Clinical School, University of New South Wales, Kensington, Australia
3. School of Optometry and Visual Science, University of New South Wales, Kensington, Australia
4. Price of Wales Clinical School, University of New South Wales, Kensington, Australia
5. Biological Resources Imaging Laboratory, University of New South Wales, Kensington, Australia
6. Department of Anatomical Pathology, St George Hospital, Kogarah, Australia
7. Department of Surgery, St George Hospital, Kogarah, Australia
AIM: CD44 and CD147 are associated with cancer metastasis and progression. We aimed to investigate the effects of CD44 or
CD147 down-regulation on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and
potential mechanisms involved, using vitro and in vivo models.
METHODS: CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA. Expression of CD44,
CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using
immunofluorescence and Western blotting. Cell proliferation and the DTX dose-response was measured by MTT and colony
assays. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in the PI3K/Akt and
MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to
assess CaP tumorigenecity, lymph node metastases and DTX response.
RESULTS: CD44 and CD147 interact in CaP cells. KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced
CaP proliferation and invasive potential and enhanced DTX sensitivity. KD of CD44 or CD147 down-regulated p-Akt and p-Erk,
the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts
displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and
CD147 enhanced metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and
MAPK                                                                                                               pathways.
CONCLUSION: Selective targeting of CD44 or CD147 alone, or combined with DTX, may limit CaP metastasis and increase
chemosensitivity, with promise for future CaP treatment.


MicroRNA-137, a HMGA1 target, suppresses cell invasion and metastasis of colorectal
carcinoma by directly targeting FMNL2
Li Liang1 , Xianzheng Li1 , Xiaojing Zhang1 , Zhenbing Lv1 , Yanqing Ding1
1. Southern Medical University, Guangzhou city, guang, China
Background & Aims: FMNL2, a member of diaphanous-related formins, has been strongly associated with tumor progression
but the post-transcriptional regulatory mechanism of FMNL2 remains unknown. Our study aimed to investigate whether
increased FMNL2 expression was mediated by microRNAs in colorectal carcinoma (CRC). Methods: Real-time PCR or
Western blot was used to detect expression levels of miR-137, HMGA1 and FMNL2 in CRC cells and tissues. The in vitro and
in vivo functional effect of miR-137 was examined further. Luciferase reporter assay was conducted to confirm the associations
between miR-137 and FMNL2 3’UTR, HMGA1 and miR-137 promoter. ChIP was used to assess direct binding of HMGA1 to
miR-137 promoter. Results: miR-137 and miR-142-3p were initially selected as potential miRNAs targeting FMNL2 based on
bioinformatic predictions. But only miR-137 demonstrated significant inverse correlation with FMNL2 protein level in CRC cell
lines and tissues. FMNL2 was a target gene of miR-137, and miR-137 could inhibit cell proliferation, invasion in vitro, hepatic
and intestinal metastases in vivo by targeting FMNL2. The study further proved that HMGA1 enhanced miR-137 transcription
by binding its promoter, subsequently down-regulating FMNL2 expression. miR-137 also inhibited the activation of p-MAPK and
p-Akt, followed by the suppression of MMP2, MMP9 and VEGF. MiR-137 decreased cell invasiveness by inhibiting PI3K/Akt
and MAPK/ERK pathways. Conclusions: Our findings reveal a novel mechanism of post-transcriptional regulation of FMNL2
expression. miR-137, induced by its upstream transcription factor HMGA1, can suppress invasion and metastasis of CRC by
regulating its direct target FMNL2, at least in part through inhibiting PI3K/Akt and MAPK/ERK pathways.


Molecular pathways and therapeutic targets for triple-negative breast cancer
Mariska Miranda1 2 , Fares Al-Ejeh1 , Peter Simpson3 , Georgia Trench4 , Sunil Lakhani3 5 , Kum Kum Khanna1
1. Signal Transduction Laboratory, Queensland Institute of Medical Research, Brisbane, Qld, Australia
2. UQ School of Medicine, University of Queensland, Brisbane, Queensland, Australia
3. The University of Queensland, UQ Centre for Clinical Research (UQCCR), Brisbane, Qld, Australia
4. Cancer Genetics, Queensland Institute of Medical Research, Brisbane, Qld, Australia
5. Pathology Queensland, The Royal Brisbane & Women’s Hospital, Brisbane, Qld, Australia
Breast cancer is the leading cause of cancer-related mortality among women worldwide. Triple negative breast cancer is a
subtype of breast cancer known for its aggressive behaviour and poor prognosis. Currently there has been a shift from using
empirically derived agents that inhibit tumor cell growth and/or survival, to molecular therapies that target specific molecules
that regulate these and other important biological processes. Despite the success of some new-targeted therapies to treat
breast cancer, the outlook for the majority of patients with triple negative breast cancer (TNBC) remains poor. Therefore we
have utilized the KinexTM antibody array to resolve potential new targetable “nodes”. We screened 43 primary breast cancer
biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines for protein/phosphoprotein levels.
Unsupervised hierarchical clustering of protein/phosphoprotein levels revealed two distinct subgroups of TNBC. Western
blotting and Proteome ProfilerTM Arrays (R&D Systems) were used to validate deregulated proteins/phosphoproteins in TNBC.
Pathway analysis revealed that one subgroup of TNBC exploits overlapping and cross-talking networks (FAK, MAPK/ERK,
PLCg1/PDK1, p38aMAPK/JNK and S6 kinase signaling) for survival. These signaling networks are downstream from elevated
activation of integrins, EGFR, and Insulin-like growth factor 1 receptor (IGF1R). We have then investigated the effect of
inhibition of these pathways in a panel of breast cancer cell lines that represent different subtypes. Functional evaluation of
defined pathway alterations in vitro and in vivo could lead to the development of more effective therapies for TNBC and improve
morbidity by targeting new proteins/pathways that drive tumor progression and growth in TNBC patients.


Silencing Heat Shock Proteins 27 and 47 Inhibits Platelet-Derived Growth Factor (PDGF)-
Induced Pancreatic Stellate Cell Proliferation: Implication in Pancreatic Cancer Progression.
Janet Youkhana1 , Jie Liu1 , Narada Kiriella1 , Yang Lu2 , Biankin Andrew3 , Goldstein David1 , Apte Minoti2 , Phoebe
1. Pancreatic Cancer Translational Research Group, University of New South Wales, Sydney, Australia
2. Pancreatic Research Group, University of New South Wales, Sydney, Australia
3. Garvan Institute of Medical Research, Sydney, Australia
Heat shock proteins (HSPs) can regulate cell migration and proliferation and are known to be overexpressed in human
pancreatic cancer (PC) tissue. The stromal reaction of PC is produced by activated pancreatic stellate cells (PSCs) which
interact with PC cells to facilitate cancer progression and metastasis. Growth factors (including PDGF) produced by tumour
cells induce proliferation and migration of PSCs. We have also previously shown that PDGF induces the expression of several
HSPs (27, 47, 70 and 90) in human cancer associated-PSCs (CA-hPSC). However, little is known about the role of HSPs in
PSC function. Aim: To determine the effect of silencing HSPs (27, 47, 70 or 90) in CA-hPSCs on cell migration and

Methods: CA-hPSC were isolated from resected pancreatic tissue from PC patients and treated (n=4) with siRNA targeting
HSP27 (protein 1 or 2), HSP47, HSP70, HSP90 (>90% protein silencing achieved) or non-silencing siRNA (ns-siRNA). 48h
post transfection CA-hPSCs were incubated ± PDGF (10ng/ml) for 48h. Cell proliferation assessed by the cell counting kit-8
and migration assessed using modified Boyden chambers with PDGF as a chemotactic agent.

Results: PDGF stimulated CA-hPSC proliferation and migration in ns-siRNA treated PSCs (#p<0.001). Notably, silencing
HSP27 (p1 and p2) or HSP47 inhibited PDGF-induced proliferation by 55.4±18.5%, 50.4±7.7%, and 72.8±14% (*p<0.01 vs ns-
siRNA+PDGF) respectively. In contrast, HSP70 and HSP90 siRNA had no effect on PDGF-induced proliferation. However,
silencing HSP27 (p1 and p2) and HSP47 had no effect on basal or PDGF-induced PSC migration.

Conclusion: This is the first study to show that suppression of HSP27 or HSP47 inhibits PDGF-induced PSC proliferation.
Implication: Modulation of HSPs in PSCs may represent a novel approach to influence PSC function and PSC interactions with
pancreatic cancer cells.


Regulation of metastasis via CDK-mediated phosphorylation of BRMS1 and RBP1
Randy Suryadinata1 , Maria Soetjipto1 , Danny Welch2 , Boris Sarcevic1
1. St. Vincent's Institute of Medical Research, Fitzroy, Vic, Australia
2. Departmet of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas, USA
Human cancers arise from defects in regulatory pathways that control the normal proliferation and differentiation of cells. The
predominant cause of cancer mortality (~90%) is metastatic tumours inducing pathology in vital organs. Pivotal to
carcinogenesis is deregulation of the cell cycle machinery leading to tumour growth at the primary and metastatic sites. Cell
division is mediated by the cyclin-dependent kinases (CDKs), which phosphorylate substrate proteins to promote cell cycle
progression and it is clear that deregulated CDK activity is important in many human cancers. A critical CDK substrate is the
retinoblastoma tumour suppressor protein, pRb, which inhibits cell cycle progression. Recently published studies from our
laboratory have shown that CDK-mediated phosphorylation pRb and its binding partner, termed the retinoblastoma binding
protein 1 (RBP1) is important for overcoming the cell cycle inhibitory functions of these proteins. In addition to binding pRb,
RBP1 binds to the breast cancer metastasis suppressor protein 1, BRMS1. BRMS1 suppresses metastasis of various cancer
cells. Our data now show that BRMS1 is also phosphorylated by CDKs and this reduces the ability of this protein to inhibit the
migration of breast cancer cells. These data for the first time suggests that deregulated CDKs may promote two fundamental
aspects of carcinogenesis, proliferation and metastasis.


ERK5 regulates invasiveness of osteosarcoma by inducing MMP-9
Sang-Min Kim1 2 , Hyewon Lee1 , Youn-Soo Park1 , Youbin Lee1 , Seung Han Shin1 , Han-Soo Kim3 , Hwan Seong Cho4 ,
Sung Wook Seo1
1. Department of Orthopaedic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul,
2. Department of Orthopaedic Surgery, Pusan National University School of Medicine, Yangsan, Korea
3. Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Hospital,
     Seoul, Korea
4. Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Bundang
     Hospital, Seongnam, Korea
The purpose of this study is to determine the role of ERK5 in cellular invasion of osteosarcoma (OS). The Human OS cell lines
(MG63, SaOS, and U2OS) and primary OS cells were used for the study. The expression of ERK5 and MMP-9 in each cell was
examined by western blot or RT-PCR. To evaluate the biological role of ERK5, proliferation assay (MTT) and invasion assay
(BD Matrigel™) were performed after silencing ERK5 using siRNA. MMPs expressions were analyzed using RT-PCR and
zymography after silencing ERK5. ERK5 was distinctly overexpressed in U2OS and primary OS cell. Both of them also
expressed MMP-9, which was not shown in MG63 and SaOS in RT-PCR. ERK5 silencing did not suppress the proliferation of
OS cells. However, ERK5 silencing significantly reduced the number of invading cells in invasion assays. The expression of
MMP-9 was specifically reduced after silencing ERK5. The zymography showed that the enzyme activity of MMP-9 was also
reduced after ERK5 suppression. The expression of ERK5 regulates the invasion of OS cells by inducing MMP-9 expression.
Therefore, ERK5 may be a new therapeutic target in invasive OS expressing MMP-9.


Sodium butyrate inhibited invasion of cancer cells
Kiyoko Yoshioka1 , Kazuyuki Itoh1
1. Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan
Sodium butyrate (SB)-C-4 saturated fatty acids are present in the human bowel membrane in high concentration (2 mM) as
food metabolites. Here, we focused on the role of SB on cancer cell motility and invasion. SB inhibited human fibrosarcoma
(HT1080) and human glioblastoma (A172) cell motility and invasiveness, decreased cell growth in a dose-dependent fashion.
SB also affected the morphology of the HT1080 and the A172 cells, namely spread out. Phosphorylation levels of focal
adhesion kinase (FAK, pY577 and pY397 sites) were increased. All of these biological effects of SB were reversible, and
recovered after withdrawal. In addition, HT1080 cells and A172 cells treated with SB showed positivity for senescence-
associated b-gal (SA-b-gal) staining with elevated expression levels of p53 and p21 proteins in a dose-dependent manner.
Further, the proteosome inhibitor, MG-132 decreased 2D cell growth and inhibited motility and, invasiveness of the HT1080 and
A172 cells, and showed similar of all these biological effects of SB. Collectively, SB induced cellular senescence, inhibited
invasion and growth, and would be a good candidate for anti-invasive therapy without severe adverse effects.


Tumour-microenvironment interaction: the effect of analgesics.
Banafsheh Afshar Imani1 , Peter J cabot1 , Marie-Odile Parat1
1. University of Queensland, Woolloongabba, Qld, Australia
Surgery is the mainstay of breast cancer treatment; however, there are concerns about the possible effect of the anaesthetic
regimen including analgesic medications on postoperative tumour recurrence and metastasis. A proposed contributor to tumour
recurrence is the existence of micrometastases at the time of surgery. Whether these small malignant foci could lead to new
tumours is determined in part by their microenvironment. This includes non-malignant cells such as macrophages and
endothelial cells and the extracellular matrix (ECM). We hypothesized that perioperative analgesic drugs could modulate the
interaction between tumour and non-tumour cells and thus affect the growth and invasiveness of micrometastases. In a mouse
syngeneic breast cancer model we observed a dramatic increase in serum levels of the two ECM proteolytic enzymes, matrix
metalloproteinase-9 (MMP-9) and urokinase-like plasminogen activator (uPA) as well as tissue inhibitor of metalloproteinase
(TIMP)-1 in tumour-bearing mice. The tumour and its surrounding tissue differentially contributed to the overproduction of these
enzymes. In in vitro co-culture models, direct contact between tumour cells and either macrophages or endothelial cells
resulted in a surge of proteolytic enzymes MMP-9, MMP-2 and uPA as well as TIMPs. In these models, we examined the effect
of two opioid analgesics widely used in the perioperative period, morphine and fentanyl and two non-steroidal anti-inflammatory
drugs, aspirin and ketorolac, on the production of proteases and their inhibitors. Exposure to aspirin and ketorolac caused
significant decrease in MMP-9, MMP-2 and uPA levels and treatment with morphine and fentanyl increased the level of TIMP-1.
The results of our study suggest that the interactions between breast tumour cells, macrophages and endothelial cells induce
changes in ECM degrading potential and that short-term exposure to analgesic drugs, commonly used in anaesthetic regimens,
could modulate this interaction by either reducing the level of the enzymes or increasing their endogenous inhibitors.


The inflammation-resolving trihydroxy-docosahexaenoic acid derivative, resolvin D2 is an
estrogen receptor ligand that supports MCF-7 cell proliferation
Nuha Alzaubai1 , Trudi Harris1 , May Leong2 , Mark Rizzacasa2 , Alastair Stewart1
1. University of Melbourne, Melbourne, VIC, Australia
2. school of chemistry, university of melbourne, melbourne , victoria, australia
Introduction: Resolvin D2 (RvD2) is an inflammation-resolving tri-hydroxy lipid mediator generated endogenously from the
omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) (Serhan et al., 2011). Formyl peptide receptors (FPRs) are
G-protein coupled receptors. FPRs can bind a wide range of structurally diverse ligands including proteins, small peptides and
bioactive lipid mediators. They are expressed in both estrogen receptor-positive (MCF-7) and estrogen receptor-negative
(MDA-MB-231) breast cancer cell lines. Some of the endogenous inflammation-resolving FPR ligands (annexin A1 and its N-
terminal peptide Ac2-26, and the tri-hydroxy lipid mediator lipoxin A4) stimulate the proliferation of both MCF-7 and MDA-MB-
231 cells through activation of FPRs (Khau et al., 2011). Aim: To investigate the role of FPR in the effect of RvD2 on breast
tumour cell proliferation. Methods: Resolvin D2 was obtained by total chemical synthesis. Both MCF-7 and MDA-MB-231 were
seeded in 24-well plates in 10% FCS media at 50,000 cells/well for 24 hours. Cells were then incubated with serum free media
for another 24 hours before treatment with FCS 5% (v/v) or RvD2 0.1-100 nM. After 48 hours viable cells were enumerated.
MCF-7 cells were transfected with estrogen response element (ERE)-controlled secretory alkaline phosphatase (SEAP) and
pGL3 luciferase vector to account for transfection efficiencies. ERE activity was measured using a SEAP detection kit. Rat
uterine cytosol was incubated with increasing concentrations of estradiol or RvD2 in the presence of 3H-E2. Results: RvD2
stimulated the proliferation of MCF-7 cells (P<0.0001), but not MDA-MB-231 cells. The proliferative effect of 100nM RvD2 was
prevented by incubation with estrogen receptor antagonist, ICI 182,780 (100nM) (P<0.0001). RvD2 stimulated ERE activity and
completely displaced 3H-E2 (at 1µM) from rat uterine cytosol binding sites. Conclusion: The data suggest that RvD2 induces
the growth of MCF-7, but not MDA-MB-231 cells, via activation of estrogen receptor.

1.        Khau, T., Langenbach, S. Y., Schuliga, M., Harris, T., Johnstone, C. N., Anderson, R. L., and Stewart, A. G. (2011).
     Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1
     and 2. FASEB J
2.        Serhan, C. N., Krishnamoorthy, S., Recchiuti, A., and Chiang, N. (2011). Novel anti-inflammatory--pro-resolving
     mediators and their receptors. Curr Top Med Chem 11, 629-647.


The inflammatory cytokine oncostatin M is a potent inducer of mammary tumor metastasis
Celeste Bolin1 , Ken Tawara1 , Jim Moselhy1 , Joel Garbow2 , Robin Anderson3 , Cheryl L. Jorcyk1
1. Department of Biological Sciences, Boise State University, , USA
2. Division of Radiological Sciences, Washington University School of Medicine, , USA
3. The Sir Peter MacCallum Department of Oncology, The University of Melbourne, , Australia
The propensity of primary breast cancer cells to metastasize to bone and lung has been partially explained by high levels of
endogenous inflammation. However, it is not fully understood how inflammatory mediators expressed either by the primary
breast cancer cells or cells of the microenvironment specifically contribute to metastasis. Our lab has shown that oncostatin M
(OSM), an interleukin-6 (IL-6)-family inflammatory cytokine, has distinct metastatic properties from IL-6. In this study, we
investigated the role of tumor cell-produced OSM in bone and lung metastasis from a primary mammary tumor using a
syngeneic, 4T1.2 mouse mammary tumor cell model. Specifically, 4T1.2 cells in which OSM expression was knocked down
using shRNA (4T1.2-OSM) or control cells were implanted into the mammary fat pad of Balb/c mice, and spontaneous
metastasis to the lung was monitored in vivo with magnetic resonance imaging (MRI) or to both bone and lung ex vivo by qPCR
and histology. At each experimental endpoint, 4T1.2-OSM injected mice showed significantly reduced metastasis.
Furthermore, in a tumor resection model, mice bearing 4T1.2-OSM tumors showed a statistically significant increase in survival,
and intratibial injection of these same 4T1.2-OSM cells dramatically reduced osteolytic destruction compared to control cells.
The specific, OSM-regulated mechanism promoting this osteolysis will be presented. Additional experiments are underway to
test the role of OSM produced by cells of the metastatic microenvironment by orthotopically injecting 4T1.2 cells into OSM
knockout mice. Results from these experiments delineating the contribution of OSM in the bone and lung microenvironment
versus the metastatic cells themselves will be presented. In sum, the results of this study provide evidence of a novel, OSM-
mediated mechanism of primary breast cancer metastasis to bone and lung with implications for pharmaceutical intervention.

Enhanced expression of MDA-9/syntenin induced through the tissue-factor pathway in
melanoma promotes tumor cell migration and metastasis
Habib Boukerche1 , Hanaa Aissaoui1 , Jean-luc Plantier1 , Claude Négrier1
1. University Claude Bernard Lyon I- Faculty of Mediciine René Laennec - INSERM, Lyon, France

The hemostatic system is frequently activated in cancer patients, but how tumor cells exploit a normal physiologic response in a
way that allows tumor growth and dissemination is not well understood. Tissue factor (TF), the cellular receptor for factor VIIa
(FVIIa), besides initiating blood coagulation, plays an important role in tissue repair, inflammation, angiogenesis, and tumor
metastasis. Subtraction hybridization identified melanoma differentiation associated gene-9 (MDA-9), also known as syntenin,
as a novel gene that positively regulates cancer cell motility through distinct biochemical and signaling pathways. A notable
feature of MDA-9/ syntenin is the presence of a tandem PDZ domains that are mandatory for the assembly and organization of
diverse cell signaling processes occurring at the plasma membrane. Using a melanoma model, we now demonstrate that MDA-
9/syntenin is dramatically up-regulated by the combination of plasma concentrations of rFVIIa (10nM) and factor F(X) in these
cells. We used anti-TF antibodies, competitive inhibitors of TF-FVIIa-mediated catalytic activity and genetic approaches to block
TF, FVIIa, FXa, and PAR-1 to demonstrate that up-regulation of MDA-9/syntenin expression by tumor cells is induced through
the TF/FVIIa/Xa pathway. We also document that MDA-9/syntenin physically interacts with c-Src through its PDZ binding motif
following stimulation of melanoma cells with rFVIIa and FX. By specifically blocking the functionality or expression of the
pertinent signaling molecules, we demonstrate that the signaling pathway consisting of PAR-1/c-Src/Cdc42/Rac1/JNK/paxillin
mediates TF/FVIIa/Xa-induced MDA-9/syntenin that leads to NF-κB activation and subsequent expression of MMP-2, thereby
promoting migration/invasion and metastatic potential of human melanoma cells. The present finding uncover a novel role of
MDA-9/syntenin as an important TF/FVIIa/Xa/PAR-1-regulated gene that initiates a novel signaling circuit essential for cell
motility and metastasis of melanoma. In these contexts, targeting TF-FVIIa-Xa and its relevant downstream targets such as
MDA-9/syntenin, may be useful for preventing tumor growth and dissemination but also for managing thrombotic complications
associated with malignancy.


The contribution of stromal caveolin-1 to breast cancer metastasis
Allan D Burrows1 , Robin L Anderson1
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary
breast cancer is a powerful prognostic indicator of subsequent metastatic disease. Whilst reports linking expression of CAV1
within breast tumour cells to clinical outcome have not led to any clear conclusions, our finding of a strong positive correlation
between loss of CAV1 expression in breast tumour stroma and poor prognosis (p<0.0001) (Sloan et al., 2009, AJP 174: 2035-
2043), is novel and exciting as it offers the potential of a reliable prognostic indicator of metastatic disease.
Tumours arising from non- or weakly metastatic mammary cells grow at a faster or similar rate when co-injected with CAV1 null
mammary fibroblasts. In contrast, co-injection with CAV1 expressing mammary fibroblasts has a suppressive effect on tumour
growth. To mimic this phenotype observed in vivo, 3D co-culture assays are being developed, and although they have
demonstrated a positive effect of fibroblasts on tumour cell migration and proliferation, no stromal CAV1 specific effect has
been observed.
To further understand the consequences of the loss of stromal CAV1, profiling of CAV1 expressing and null mouse mammary
fibroblasts was conducted using cytokine arrays and cDNA microarrays. Differences in the secretion of known CAV1
associated cytokines such as RANTES, Gas6 and IL-6 were observed, with no significant changes in their expression.
Based on these findings, CAV1 may be contributing directly to the progression of breast cancer and thus provide the basis of a
stromal targeted therapy. An understanding of the mechanism by which CAV1 expression is lost from host cells, and how this
loss leads to progressive disease, could allow a therapy that restores or substitutes for CAV1 activity in stromal cells or that
targets the key cytokines involved in this paracrine signalling.


Suppression of breast cancer metastasis by BMP4
Yuan Cao1 2 , Bedrich L Eckhardt1 3 , Robin L Anderson1 4
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Biochemistry, The University of Melbourne, Melbourne, VIC, Australia
3. Genitourinary Medical Oncology, MD Anderson Cancer Center, Houston, Texas, USA
4. Sir Peter MacCallum Department of Oncology, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
Breast cancer is one of the leading causes of cancer related death in women, as a result of metastatic disease in vital organs.
In the course of our studies into the mechanisms of breast metastasis, our group has identified a number of factors that
regulate the process. Bone morphogenetic protein 4 (BMP4) is one of factors that potently suppresses breast cancer
metastasis. The mechanism by which BMP4 suppresses metastasis is the focus of current research.
Sensitivity to anoikis, an important natural barrier of metastasis, is related to metastatic capacity. BMP4 is able to sensitise
highly metastatic cancer cells to anoikis in a concentration dependent manner, indicating that BMP4 can act in an autocrine
manner to reduce metastatic capacity.
Among mice bearing tumours from the syngeneic 4T1 model, splenomegaly and leukocytosis are observed in mice bearing the
highly metastatic 4T1.2 tumour to a greater extent than in mice bearing 4T1.2 tumours with enforced BMP4 expression. BMP4
suppresses both expression and secretion of G-CSF in 4T1.2 cells, and thereby restricts the splenomegaly and leukocytosis of
the tumour bearing mice. Recombinant G-CSF treatment expand the CD11b+Gr1+ myeloid derived suppressor cells (MDSC)
in mice, facilitating the metastasis of weakly metastatic tumours. Forced expression of G-CSF in 4T1.2-BMP4 cells partially
restores their metastatic capacity. These data indicate that BMP4 is able to modulate immune cytokine secretion and immune
responses towards tumours, which may in turn prevent tumour cell dissemination.


Stanniocalcin 1 in mammary tumorigenesis
Andy Chang1 , Lily Huschtscha1 , Judy Doherty2 , Christina Restall2 , Richard Redvers2 , Robin Anderson2 , Roger
1. Children's Medical Research Institute, Westmead, NSW, Australia
2. Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
Stanniocalcin (STC) is a secreted glycoprotein first discovered in bony fish. Two homologues, STC1 and STC2, have been
identified in mammals and both are expressed in a variety of tissues including kidney, bone, mammary gland, testes and ovary.
There is now a considerable body of evidence to indicate that STCs have a role in cancer, and several studies have indicated
that their expression may be useful for cancer diagnosis and/or classification. To investigate the role of STCs in breast cancer,
we examined STC1/2 expression using quantitative RT-PCR on a series of isogenic mammary tumour lines with varying
degrees of metastatic capacity when injected into Balb/c mice. We found that high STC1 expression was associated with the
highly metastatic ‘triple negative’ (ER-/PR-/Her2-) 4T1.2 and 4T.13 lines, with very low levels in the 67NR (non-metastatic) and
66cl4 (weakly metastatic) lines. This was confirmed with subsequent northern and western blot analysis. STC2 expression is
not altered in these cell lines. To explore the function of STC1, we down-regulated its expression using lentiviral vectors
encoding STC1 shRNA. Cells transduced with non-targeting shRNA were used as a negative control. These cells were then
injected into the mammary gland of female Balb/c mice and mammary tumour growth was monitored. Cells with STC1 down-
regulation were found to develop primary mammary tumours at a much slower rate than control cells. In vitro experiments on
cell migration and invasiveness through Matrigel associated STC1 expression with a more migratory and invasive phenotype.
Bioinformatic analysis of human breast cancer data sets indicated that STC1 is a metastasis-associated gene within the poor
prognosis triple negative subtype. Our preclinical study indicates that STC1 has a positive role in primary breast tumour growth
and possibly in metastasis as well, although this still needs to be clearly demonstrated.


Melaleuca alternifolia Concentrate (MAC): a novel plant-derived anticancer agent
Amanda M Clark1 , Koichi Ito1 , Max Reynolds2 , Steve J Ralph1
1. Griffith University, Southport, QLD, Australia
2. Griffith University, Nathan, Queensland, Australia
Natural products historically represent a source of clinically approved drugs that have contributed significantly to anticancer
drug development. Melaleuca alternifolia Concentration (MAC) is an extract prepared from the native Australian plant M.
alternifolia (also a common source of Tea Tree Oil). Initial investigations analysing the in vitro cytotoxicity of Melaleuca oils and
its main components against tumour cells revealed the presence of compounds with strong anticancer activities. In vitro, MAC
induced cell death through the intrinsic mitochondrial pathway, while at lower cytostatic concentrations induced autophagy and
cell cycle arrest in G1 phase. In vivo, the anticancer properties of MAC were examined using a spontaneous FVB/N c-neu
murine model of breast cancer, with mice receiving intratumoural treatments every 3 days. MAC suppressed tumour growth
over a 30 day period and induction of cell death through the intrinsic mitochondrial pathway was also replicated in vivo, as
determined by the presence of both intramitochondrial superoxide and TUNEL positive regions within MAC treated tumours.
Potential immunomodulatory activity of MAC was examined in the 4T1 breast cancer model. MAC was associated with
increased CD3+CD4+ and CD3+CD8+ T cell populations as well as a reduction in CD3+CD4+CD25+Foxp3+ regulatory T cells in
both tumour and blood. Furthermore, there was an increased presence of Ly6GhiCD11b+ neutrophils in treated tumours and
bone marrow. Intratumourally, MAC also elevated the expression of CD80 on Ly6GhiCD11b+ cells, indicating the presence of a
potential antigen presenting-type neutrophil population. In both models, MAC treatment was not associated with any observable
adverse effects. In conclusion, MAC represents a promising anticancer agent through its ability to induce apoptosis via the
intrinsic mitochondrial pathway as well as by reducing tumour growth and promoting an anti-tumour immune response in mice
with no observable adverse effects. Additionally, the immunostimulatory response may also aid in combating the prevalence of
metastasis due to the increased immune cell presence.


Tetraspanins as metastatic regulators of prostate cancer
Ben T Copeland1 , Matthew J Bowman1 , Claude Boucheix2 , Ashman K Leonie1
1. School of Biomedical Sciences and Pharmacy, University of Newcastle and Cancer Research Program, Hunter Medical
     Research Institute, Newcastle, N.S.W, Australia
2. INSERM U602, Hôpital Paul Brousse, Villejuif, Cedex, France
Prostate cancer accounts for the second highest cause of cancer related deaths in men in most western countries, including
Australia and the United States. The majority of deaths are attributed to late stage metastatic forms of the disease.
Tetraspanins are integral membrane bound proteins that associate with motility related molecules such as integrins.
Experimental studies have indicated tetraspanins may be important regulators of tumour invasion and metastasis. High
expression levels of the tetraspanin CD9 have been linked to good prognosis, while in contrast increased expression of the
tetraspanin CD151 has been associated with poor outcomes. In this study, for the first time the effects of knocking-out the pro-
and anti-tumorigenic/metastatic tetraspanins on development of prostate cancer have been evaluated in a spontaneously
developing model of prostate cancer.
The Cd9 and Cd151 knock-out mouse models were independently crossed onto the TRansgenic Adenocarcinoma of Mouse
Prostate (TRAMP) mouse model. Development of primary prostate tumours were not affected by knocking out either Cd9 or
Cd151. Knocking out the Cd9 gene resulted in a significant increase of metastatic lesions, number of foci and total area of
metastatic lesions, to the liver. While knocking out the Cd151 gene resulted in a significant decrease of metastatic lesions,
number of metastatic foci and total area of metastatic lesions, to the lungs.
In summary, in the TRAMP model, tetraspanins CD9 and CD151 have no significant effect on the development of primary
prostate tumours. CD9 has a significant anti-metastatic effect while conversely CD151 has a significant pro-metastatic effect.
CD9 and CD151 may prove to be therapeutic targets that benefit prostate cancer patients.


Microenvironmental regulation of kinase signalling dynamics as a critical mediator of tumour
Thomas R Cox1 , Erwin M Schoof2 , Rune Linding2 , Janine T Erler1
1. Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark
2. Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark
Tumour metastasis is a highly complex, dynamic and inefficient process involving multiple steps, yet it accounts for over 90% of
cancer patient deaths. The tumour microenvironment and in particular the extracellular matrix is a key component in driving this
process at multiple stages. In order to respond to changes in the environment, a cell has to integrate multiple input-cues and
modulate its signalling networks accordingly. Both the biochemical and biomechanical properties of tumour extracellular matrix
(ECM) are important in determining cell behaviour, and phosphorylation events play a major role in translating these
environmental cues into cellular messages.
Metastatic tumours often show elevated ECM remodelling and increased stiffness in comparison to their non-metastatic
counterparts and these changes in stiffness are known to drive metastatic cell behaviour although the underlying molecular
mechanisms remain elusive. Utilising multiple approaches we evaluate both the molecular and behavioural changes occurring
in tumour cells leading to metastasis, focussing on the phosphorylation dynamics behind metastatic phenotypes. By
computationally integrating molecular and phenotypic data, we elucidate the molecular networks associated with tumour
progression and identify key enablers of kinase signalling leading to metastatic progression.
Using breast and colorectal cancer models, we show that metastatic tumours are stiffer than matched non-metastatic tumours,
and that increased ECM stiffness can drive the invasive behaviour of non-metastatic cancer cells. We observe changes in both
dynamic cell signalling events and gene expression, leading to global shifts in molecular networks associated with enhanced
metastasis in response to environmental cues.
Our ultimate goal is to predict and test novel network-based therapeutic strategies for the treatment of metastatic disease.


CD151, a novel candidate for regulation of prostate cancer cell motility, invasion and
Sujitra Detchokul1 , Bradley Newell2 , Elizabeth D. Williams3 , Albert G. Frauman1
1. Department of Medicine (Austin Health), The University of Melbourne, Heidelberg, VIC, Australia
2. Department of Surgery and Urology, The University of Melbourne, Heidelberg, VIC, Australia
3. Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia
Tumour metastasis is a multi-factorial process and tumour cells are sensitive to local microenvironmental cues that regulate
normal physiological functions such as wound healing and epithelial morphogenetic changes leading to malignant behaviour1.
CD151, a member of the tetraspanin superfamily, has been identified in our laboratory as a promoter of prostate cancer (PCa)
migration and invasion and involves association with integrins on both cell-cell and cell-stroma levels2, 3. Furthermore, CD151
plays a role in endothelial cell motility4-6. We investigated this protein further for its potential role in PCa metastasis.
CD151 is heterogeneously expressed across different PCa cell lines and the level of CD151 expression was significantly higher
in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP
(P<0.001). This supports the previous findings by our laboratory7 that CD151 has value as a prognostic indicator for human
PCa. Also, we found that CD151 has a role in the promotion of motility and invasion of PCa cell lines. Orthotopic implantation of
human PC-3 cells into SCID mice prostate was investigated and the majority developed pelvic lymph node metastases. The
level of CD151 expression in metastatic lesions was significantly increased compared to matched primary xenografts (P=0.04).
Specimens from primary and secondary sites were investigated for CD31 (angiogenesis marker) expression; primary
xenografts from mice forming secondary metastases had higher microvessel density (MVD) than xenografts from those without
any metastases (P=0.029). MVD were not correlated with tumour size. These findings underscore the potential role of CD151
and angiogenesis in the metastatic potential of PCa, as suggested by a previous report in which CD151 knock-out animals had
impaired pathologic angiogenesis8.
CD151 is likely to be an important regulator of cancer cell communication with the surrounding microenvironment, as an
increase in tumor cell CD151 expression was associated with enhanced motility/metastasis and angiogenesis.

1.      Quaranta V. Motility cues in the tumor microenvironment. Differentiation 2002; 70: 590-8.
2.      Ang J, Fang B-L, Ashman LK, Frauman AG. The migration and invasion of human prostate cancer cell lines involves
     CD151 expression. Oncol Rep 2010; 24(6): 1593-7.
3.        Nishiuchi R, Sanzen N, Nada S, et al. Potentiation of the ligand-binding activity of integrin alpha 3 beta 1 via
     association with tetraspanin CD151. Proc Natl Acad Sci U S A 2005; 102(6): 1939-44.
4.        Y
5.        Longo N, Yanez-Mo M, Mittelbrunn M, et al. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction
     during transendothelial invasion of melanoma cells. Blood 2001; 98(13): 3717-26.
6.        Sadej R, Romanska H, Baldwin G, et al. CD151 regulates tumorigenesis by modulating the communication between
     tumor cells and endothelium. Mol Cancer Res 2009; 7(6): 787-98.
7.        Ang J, Lijovic M, Ashman LK, Kan K, Frauman AG. CD151 protein expression predicts the clinical outcome of low-
     grade primary prostate cancer better than histologic grading: a new prognostic indicator? Cancer Epidemiol Biomarkers
     Prevent 2004; 13(11): 1717-
8.        Takeda Y, Li QL, Kazarov AR, et al. Diminished metastasis in tetraspanin CD151-knockout mice. Blood 2011; 118(2):


Metastasis in the ovarian cancer microenvironment is induced by kallikrein proteases
Ying Dong1 , Carson Stephen1 , Glen M Boyle2 , Peter G Parsons2 , Michael A McGuckin3 , Jonathan M Harris1 , Judith A
1. QUT, Kelvin Grove, QLD, Australia
2. QIMR, Herston, QLD, Australia
3. MMRI, South Brisbane, QLD, Australia
     Epithelial ovarian cancer (EOC) is commonly diagnosed when tumours have metastasised to the abdominal cavity with an
accumulation of fluid (ascites) (1). We have shown that high levels of kallikrein (KLK)4 and KLK7 proteases are associated with
a poor outcome for women with the most lethal histotype, serous EOC. Metastasis and chemoresistance are key events in the
ovarian cancer microenvironment that lead to poor outcome. We examined KLK4- and KLK7-transfected SKOV3 EOC cells in
3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV3 cells formed multicellular
aggregates (MCAs) as seen in ascites, as did SKOV3 cells treated with active KLK4. A high level of KLK4 is expressed by
ascitic EOC cells compared to matched primary tumour cells. KLK4-MCAs formed larger cancer cell foci in mesothelial cell
monolayers than those formed by vector and native SKOV3 cells, suggesting a role for KLK4 in metastasis in the peritoneal
microenvironment. Additionally, KLK4-SKOV3 cells expressed high levels of urokinase plasminogen activator (uPA) and high
levels of both KLK4 and uPA were observed in cells from patient ascites. Importantly, Taxol resistance of KLK4-MCAs was
reversed by the selective sunflower trypsin KLK4-inhibitor SFTI-FCQR. KLK7-transfected SKOV3 cells also formed large,
compact Taxol-resistant MCAs in 3D (2). Different from KLK4-, KLK7-SKOV3 cells showed increased levels of α5/β1 integrins,
as did the EOC cells derived from ascites compared to their matched primary tumour cells, suggesting an integrin-mediated
pathway induced by this protease. Both KLK4 and KLK7 promote EOC peritoneal metastasis and Taxol resistance despite via
different molecular mechanisms. Our data may help design efficacious approaches to treat women with EOCs, in particular
those with high tumour KLK levels.

1.        Bast RC, Jr., et al. Nat Rev Cancer. 2009. 9:415-28.
2.        Dong Y, … Clements JA, Cancer Res. 2010. 70:2624-33.


Genetic ablation of SOX18 function suppresses tumor lymphangiogenesis and metastasis of
melanoma in mice
Tam Duong1 , Steven Proulx2 , Paola Luciani2 , Jean-Christophe Leroux2 , Michael Detmar2 , Peter Koopman1 , Mathias
1. University of Queensland, Brisbane, QLD, Australia
2. 2. Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology ETH, Zurich, Switzerland
The lymphatic vasculature provides a major route for tumor metastasis and inhibiting neo-lymphangiogenesis induced by
tumors can reduce metastasis in animal models. Developmental biology studies have identified the transcription factor SOX18
as a critical switch for lymphangiogenesis in the mouse embryo. Here, we show that SOX18 is also critical for tumor-induced
lymphangiogenesis and we demonstrate that suppressing SOX18 function is sufficient to impede tumor metastasis.
Immunofluorescence analysis of murine tumor xenografts showed that SOX18 is re-expressed during tumor-induced neo-
lymphangiogenesis. Tumors generated by implantation of firefly luciferase-expressing B16-F10 melanoma cells exhibited a
reduced rate of metastasis to the regional draining lymph node in Sox18-deficient mice, as assessed by live bioluminescence
imaging. Lower metastatic rates correlated with reduced tumoral lymphatic vessel density and diameter, and with impaired
drainage of peri-tumoral injected liposomes specific for lymph vessels from the sentinel lymph nodes. Overall, our findings
suggested that SOX18 induction is a key step in mediating tumor lymphangiogenesis and metastasis, and they identify SOX18
as a potential therapeutic target for metastatic blockade.


Loss of osteoclasts contributes to the development of osteosarcoma pulmonary metastasis
Liliana Endo-Munoz1 , Andrew Cumming1 , Geoff Strutton2 , Andreas Evdokiou3 , Scott Sommerville4 5 , Ian Dickinson4 5
, Alexander Guminski6 , Nicholas A Saunders1
1. University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia
2. Department of Pathology, Princess Alexandra Hospital, QLD, Australia
3. Department of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia
4. Department of Orthopaedics, The Wesley Hospital, QLD, Australia
5. Department of Orthopaedics, Princess Alexndra Hospital, QLD, Australia
6. Department of Medical Oncology, Royal North Shore Hospital, NSW, Australia
Osteosarcoma (OS) is a highly aggressive primary bone tumour in children and adolescents but the basis for its metastatic
properties is unclear. Pulmonary metastases can occur in up to 50% of cases and result in a long-term survival rate of only 10-
20%. In a transcriptomic screen of primary OS biopsies, we found a significant correlation between OS and impaired
osteoclastogenesis (Endo-Munoz et al. British J Cancer 103:73-81, 2010). Specifically, lesions from OS patients with
pulmonary metastases had 2-fold less expression of the osteoclast-specific enzyme, ACP5, than lesions from patients without
pulmonary metastases, and there was a direct correlation between ACP5 expression and time to metastasis. Based on these
findings, we tested whether metastasis-competent OS cells could induce the loss of osteoclasts in the bone microenvironment
as a mechanism to drive metastasis. We found that these cells could inhibit osteoclastogenesis in vivo and in vitro. In addition,
osteoclasts inhibited the migration of metastasis-competent OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid
increased the number of metastatic lung lesions in an orthotopic mouse model of OS, while treatment with fulvestrant, at a
clinically-relevant dose, increased osteoclast numbers and reduced metastatic lesions (Endo-Munoz et al. Cancer Res
70:7063-7072, 2010). Our data indicates that the osteoclast is a key regulator of OS metastasis and that osteoclast-preserving
therapies may be of clinical value in preventing OS metastasis.


Glucocorticoids inhibit breast tumour cell migration
Ebony R Fietz1 , Cameron N Johnstone2 , Alastair G Stewart1
1. Department of Pharmacology, The University of Melbourne, Parkville, VIC, Australia
2. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
Introduction. Glucocorticoids are a commonly used adjuvant treatment for breast cancer, alleviating side effects of
chemotherapy. However, glucocorticoids also alter tumour cell behaviour. We have previously identified a drug class effect of
glucocorticoids in inhibiting serum-induced migration in the breast tumour cell line, MDA-MB-231, in a 2-dimensional scrape
wound healing assay and a 3-dimensional modified Boyden chamber assay.
Aim. To characterise the mechanism of glucocorticoid inhibition of breast tumour cell migration.
Methods. Scrape wound healing assays were performed on MDA-MB-231 cells incubated with fetal calf serum (FCS), following
pre-treatment with glucocorticoids, the glucocorticoid receptor antagonist, RU486 or protein synthesis inhibitor, cycloheximide.
Time-lapse microscopy images of the wound were taken every 30min for 16 hours. MDA-MB-231 cells were transiently
transfected with a Glucocorticoid Response Element (GRE)-SEAP reporter construct to examine the effect of glucocorticoids on
GRE activity, as a measure of transactivation.
Results. The transactivation antagonist RU486 completely prevents glucocorticoid inhibition of FCS-induced MDA-MB-231 cell
migration in the scrape wound healing assay. Transactivation-selective glucocorticoid RU24782 and non-steroidal
glucocorticoid receptor agonist GSK9027 also inhibited FCS-induced tumour cell migration. The protein synthesis inhibitor,
cycloheximide, reduced the extent to which dexamethasone inhibited FCS-induced migration by 57 ± 10%, (n=3, P<0.05).
Dexamethasone induced GRE activity at migration-inhibiting concentrations; these responses were blocked by RU486 (n=3,
P<0.05). GSK9027 and RU24782 showed increased GRE activity at concentrations that inhibit FCS-induced migration of MDA-
MB-231 cells.
Discussion. Inhibition of migration is prevented by the transactivation antagonist, RU486, and by the protein synthesis
inhibitor, cycloheximide. The inhibitory effects are also associated with an increase in GRE activity. These results suggest the
inhibitory effects of glucocorticoids on tumour cell migration are mediated through altered transcription of genes containing


Endogenous interleukin-4 receptor expression confers a distinct survival advantage to
mammary and colon tumor cells in vitro and in vivo.
Katherine T Venmar1 , Miranda A Hallett1 , Daniel G Hwang1 , Kathy J Carter1 , Ashley Dozier1 , Felicitas L Koller1 ,
Barbara Fingleton1
1. Vanderbilt University Med Ctr, Nashville, TN, United States
The TH2 cytokines, interleukins-4 and -13 (IL4 & IL13), are important determinants of immune system activation and polarity.
 In addition to T cell stimulation, these cytokines are drivers of the 'M2' pro-tumorigenic macrophage phenotype. Additionally,
both cytokines can bind a heterodimeric receptor found on tumor cells of several types. Our goal is to to define the function of
IL4/IL13 signaling in tumor behavior and determine if this pathway represents a therapeutic target, particularly for metastatic
cancers. We have used stable knockdown of il4ra, the signaling competent component of the heterodimeric IL4/IL13 receptor,
in human and murine mammary and colon tumor cell lines to assess the contributions of IL4 and IL13 signaling to proliferation,
survival and invasion of tumor cells in vitro. These lines were also implanted in vivo. Additionally, de novo carcinogenesis
studies in il4ra- deficient mice were performed. Finally, we did a pilot study with an IL4 neutralizing agent to test therapeutic
potential. Expression of il4ra was reduced by 50-90% in the murine colon tumor lines MC38 and CT26 and mammary lines 4T1
and PyvT-R221A. Knockdown clones grew more slowly than controls, but EdU assays failed to detect any differences in DNA
synthesis amongst clones. MTT assays as well as direct cell counting indicated fewer cells over time, and this was
corroborated by clonogenic assays which showed fewer and smaller colonies with knockdown lines compared to controls.
 These effects were most likely due to a difference in basal survival. When cell lines were injected in vivo, either orthotopically
or intravenously or intrasplenically for experimental metastases, significant attenuation of tumor growth was observed. Cecal
tumors indicated effects on proliferation as determined by immunostaining for phosphohistone H3, with marginal effects on
apoptosis. Il4ra-null mice, when exposed to azoxymethane and dextran sulfate sodium, developed fewer and smaller colonic
tumors compared to wild type controls. Further, these tumors were less progressed as assessed by nuclear beta-catenin. In
addition to reduced tumorigenicity when il4ra was genetically ablated, treatment of mice with an il4-neutralizing antibody also
led to reduced tumor burden. Tumor cell IL4 receptor appears to be a strong survival factor for breast and colon tumors,
impacting both primary tumor progression and metastatic growth. Therapeutic approaches targeting IL4/IL13 should be
considered for metastatic carcinomas


The activity of the urokinase receptor in promoting tumor growth requires its interaction with
the extracellular matrix protein vitronectin
Sonu Gandhi1
1. IFOM-IEO Campus, Milan, MI, Italy
Extensive evidence implicates the urokinase plasminogen activator (uPA) and its receptor (uPAR) in the malignant processes
of tumor growth, invasion and metastasis. Mechanistically, uPAR modulates these processes by promoting extracellular
proteolysis and through its activity as a signal transduction receptor inducing migratory and proliferative signaling. Recent
studies have underscored the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN)
for the signaling activity of the receptor in vitro. However, the possible relevance of this interaction for the activity of uPAR in
promoting tumor growth and metastasis in vivo has not been assessed. With this aim in mind we generated a panel of HEK293
cell lines expressing either wild-type mouse uPAR (muPARWT), an uPAR mutant specifically deficient in VN binding
(muPARW32A) and an uPAR variant lacking domain 1 (muPARΔD1) that is required for both VN and uPA binding. In vitro cells
expressing muPARWT display increased cell adhesion, spreading, migration and proliferation associated with increased
p130Cas and MAPK signaling. Specific disruption of VN binding, or combined ablation of both VN and uPA binding, abrogates
these activities of uPAR. When xenografted into SCID mice the expression of muPARWT, but not muPARW32A or muPARΔD1,
causes a highly significant reduction of tumor latency demonstrating that VN-binding is responsible for the tumor promoting
activity of uPAR in vivo. Consistent with the predicted importance of VN for tumor growth we find that in an orthotopic xenograft
model using MDA-MB-231 cells in RAG1-/-/VN-/- mice host deficiency of VN strongly delay or prevents tumor formation. To drive
the above findings towards the bedside we have developed highly potent and specific first-in-class antagonists of the
uPAR/VN-interaction that display promising activity in a mouse model of prostate cancer.


Surfactant protein A suppresses progression of human lung adenocarcinoma in an
experimental lung metastasis model.
Hisatsugu Goto1 , Atsushi Mitsuhashi1 , Takuya Kuramoto1 , Masaki Hanibuchi1 , Soji Kakiuchi1 , Sho Tabata1 , Atsuro
Saijo1 , Jo Rae Wright2 , Saburo Sone1 , Yasuhiko Nishioka1
1. The University of Tokushima Graduate School, Tokushima, Japan
2. Duke University Medical Center, Durham, NC, USA
RATIONALE: Surfactant protein A (SP-A) is a large multimeric protein found in the airways and alveoli of the lungs. SP-A is a
member of the collectin family of proteins, characterized by NH2-terminal collagen-like regions and COOH-terminal lectin
domains. Although other surfactant proteins such as SP-B function to reduce surface tension in the lungs, SP-A (as well as SP-
D) is known to regulate the pulmonary immune response against the variety of pathogens. On the other hand, it is shown that
SP-A can be used as a marker of lung adenocarcinoma, suggesting that SP-A may also play important role in lung cancer
progression as well as pulmonary immune response. METHODS: In this study, a human SP-A gene was introduced into the
human lung adenocarcinoma cell line PC14PE6 (PC14PE6/SP-A). In vivo effect of SP-A over-expression on cancer
progression was examined in experimental lung metastasis model using NUDE mice. Tumor sections were used for
immunohistochemistry to determine the tumor cell death and the host cell recruitment. RESULTS: PC14PE6/SP-A cells
produced significantly fewer lung metastatic colonies and pleural effusion compared to vector transfected cells in vivo.
Immunohistochemical analysis showed that the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL)-positive tumor cells and macrophage recruitment were increased in the lung metastatic colonies
produced by PC14PE6/SP-A cells. Among the recruited macrophages in the tumor, M1 antitumor macrophages were
increased, while M2 tumor-promoting macrophages were decreased in the tumor produced by PC14PE6/SP-A cells. Moreover,
RT-PCR of metastatic lung tissue revealed that the expression of multiple M1 macrophage markers such as IFN-gamma and
TNF-alfa was up-regulated in the tumor produced by PC14PE6/SP-A cells, whereas the expression of M2 macrophage markers
was not changed. CONCLUSION: In addition to its protective effect in infectious diseases, SP-A may also have a protective
role in non-infectious lung diseases such as lung cancer through modulating host immune response.


The heparan sulfate mimetic PG545 acts against heparanase to reduce metastasis and prolong
survival in 4T1 breast carcinoma model
Edward Hammond1 , Paul Handley1 , Keith Dredge1
1. Progen Pharmaceuticals, Darra, QLD, Australia
PG545 is a dual angiogenesis and heparanase inhibitor that recently entered clinical trials in advanced cancer. This compound
has been shown to potently inhibit metastasis in several experimental and spontaneously arising metastatic tumour models, an
effect that is purportedly due to inhibition of the enzymatic function of heparanase by PG545 [1]. Heparanase is the only
endoglycosidase known to cleave heparan sulfate (HS), an important component of the extracellular matrix (ECM). The
degradation of HS by heparanase has been strongly implicated in tumour modification of the microenvironment, cell
dissemination and the metastatic process. The limited number of studies using overall survival as an endpoint in mouse
metastasis models has been raised as a factor affecting the success of new cancer treatments [2]. This led us to examine the
effect of PG545 on the progression of both primary tumour growth and spontaneously metastasizing disease in the 4T1
syngeneic breast carcinoma model in a non-surgical and surgical (mastectomy) setting. PG545 significantly inhibited primary
tumour growth but also inhibited lung metastasis in treated mice, an effect not observed with the tyrosine kinase inhibitor
sorafenib. Importantly, PG545 significantly enhanced overall survival compared to vehicle control or the sorafenib group,
suggesting PG545’s inhibitory effect on heparanase could be critical for this anti-metastatic activity. In support of these
observations, the levels of heparanase in the primary tumour and lung was significantly reduced by PG545 treatment. This
reduction in heparanase appeared to be related to PG545 inhibition of growth factor signalling as indicated by the reduced level
of ERK 1/2 signalling in the PG545 treated tumours. These results support the ongoing development of PG545 and highlight
the potential utility in metastatic disease settings.

1.        Dredge K, Hammond E, Handley P, Gonda TJ, Smith MT, Vincent C, et al. PG545, a dual heparanase and
     angiogenesis inhibitor, induces potent anti-tumour and anti-metastatic efficacy in preclinical models. Br J Cancer 2011;
2.        Ebos JML, Kerbel RS. Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat Rev
     Clin Oncol 2011; 8:210–221.


Potential involvement of ASC in cancer progression and metastasis.
Chifumi Fujii1 , Shigeaki Hida2 , Kensuke Ito2 , Masato Kitazawa2 , Shun'ichiro Taniguchi2
1. Development of Integrative Physiology & Bio-System Control, Shinshu University Graduate School of Medicine, Matsumoto
      city, Nagano, Japan
2. Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto city, Nagano, Japan
Apoptosis-associated spec-like protein containing a caspase recruitment domain (ASC), found in our lab, is an adaptor protein
that is a main mediator to form various inflammasomes, and has a crucial role such as maturation of interleukin (IL)-1 and IL-18
by activating caspase-1. ASC has been also known as a pro-apoptotic molecule that is epigenetically silenced in several
human cancers, including melanoma, during their progression. Although they are well-known phenomena, little information is
available for the signaling pathway regulated by ASC in tumor cells. Here, in order to investigate the roles of ASC in cancer
progression, i.e. acquisition of metastatic ability of cancer cells, we used several cell lines with differential endogenous
expression of ASC. In particular, we artificially overexpressed ASC in HT1080 cell line, where ASC is epigenetically
suppressed, and/or ablated ASC mRNA by RNA interference in three melanoma cell lines expressing ASC. The retroviral
reconstitution of ASC suppressed cellular proliferation and tumorigenesis of HT1080 in the xenograft model. When we did ASC
knockdown in two human and one murine melanoma cell lines, the enhancement of snail mRNA expression was observed.
Furthermore, endogenous snail mRNA expression levels were inversely correlated with ASC expression between two human
melanoma cell lines. Snail has been reported as a key molecule of epithelial-mesenchymal transition (EMT). Recent evidence
has highlighted a link between EMT and the cancer stem cells that initiate and maintain tumors, and EMT has also been
implicated in invasion and metastasis. Our findings suggested that a potential involvement of ASC in cancer progression and
metastasis by the regulation of snail expression level. Further analyses remain to be done to clarify the roles of ASC in
metastasis more in detail, trying to examine the effect of ASC on metastatic potential with animal models.


Cathepsin D is required for tumour stroma development.
Floriane M Imhoff1 , Chloe Squires1 , Mitchell Clarke1 , Sarah K Baird1
1. University of Otago, Dunedin, Otago, New Zealand
Formation of tumour stroma involves the migration and homing of bone marrow mesenchymal stem cells (MSCs) to the tumour
site1. The migratory process requires proteolysis of the extracellular matrix to allow MSCs to exit the bloodstream and reach the
tumour2. Cathepsin D is the most widely expressed member of the aspartic acid protease family and is upregulated in invasive
breast cancer cells. It has therefore been indirectly linked to an increase in metastasis 3. This raises the possibility that
cathepsin D protease activity may be involved in the migration of MSCs towards tumours. We have shown that pepstatin A, an
aspartic acid protease inhibitor, decreases the migration and invasion of the immortalized MSC line RCB2157 towards the
breast cancer line MDA-MB-231 through Boyden chambers as measured by MTT assay. Cathepsin D is synthesized as a pre-
pro-enzyme, which is cleaved to give rise to procathepsin D, the immature form of cathepsin D. Finally, the cleavage of
procathepsin D releases the mature cathepsin D4. Our Western blot analysis showed high levels of all three forms of cathepsin
D in MDA-MB-231 cells whereas expression in MSCs was only weak. Furthermore, migration of MSCs towards MDA-MB-231
conditioned medium was reduced by 30% when compared to the migration towards MDA-MB-231 cells themselves. These
results suggest that cathepsin D, synthesized by cancer cells as a result of crosstalk during co-culture is involved in the
migration of MSCs towards tumours. Targeting cathepsin D inhibition may therefore allow the prevention of tumour stroma

1.        Kidd S. et al., PLoS One. 2012; 7(2):e30563
2.        Hess DA. and Allan AL., Methods Mol Biol., 2011; 750:25-44
3.        Rochefort H, Acta Oncol., 1992; 31(2):125-30
4.        Koelsh G et al., FEBS Lett. 1994 Apr 18;343(1):6-10


Anti-adhesive and anti-apoptotic effect of epiglycanin/mucin 21
Jessica Zhao1 , Yuri Yi1 , Katrin Ishii-Schrade1 , Yuan Tian1 , Kaori Denda-Nagai1 , Yoshihiro Hayakawa1 , Tatsuro
1. The University of Tokyo, Tokyo, Japan
Cell surface expression of epiglycanin, a mucin-like molecule, is implicated with malignant behavior of carcinoma
cells, as suggested by its presence on TA3-Ha but not on TA3-St mammary carcinoma cells. However, the mechanism
was not clear due to lack of molecular identification. We cloned the gene for the core polypeptide of epiglycanin and
renamed it as mucin 21 (Muc21). Muc21 is a membrane-type mucin with tandem repeats of 15 amino acids extremely
rich in Ser/Thr. A cDNA of Muc21 having 84 tandem repeats was constructed and transfected using a Venus vector
into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were non-adherent. The
transfectants were less adherent to ECM components than mock cells. Antibody binding to the cell surface integrin
subunits was reduced in Muc21 transfectants in a tandem repeat-dependent manner. We also tested whether Muc21
modulated cell survival by interfering with the cellular apoptotic pathways. By using Muc21 expressing CHO-K1 cells,
we observed the resistance of Muc21 CHO-K1 cells to apoptotic cell death induced by actinomycin D and the
functional significance of this anti-apoptotic effect was supported by lower caspase activities compared to control
CHO-K1 cells. The inhibitory effect of Muc21 expression in caspase activation was confirmed in TA3-St mammary
carcinoma cells. These results indicate that Muc21 inhibit actinomycin D-induced caspase activation thereby
reducing apoptotic cell death. Because Muc21 expression is seen on highly malignant cancer cells, the anti-adherent
and anti-apoptotic activities of Muc21 might be exploited to therapeutic resistance and/or facilitation of tumor growth
and metastasis.
(References: Codington et al, Carbohydrate Res, 40, 171, 1975; Itoh et al, Glycobiology, 18:74, 2008; Yi et al, J Biol Chem,
285:21233, 2010)


Galectin-1 as a Potent Target for Cancer and Metastasis Therapy: Role in the Tumour
Koichi Ito1 2 , Selda Onturk1 , Michael Lousick1 , Shi-Yong Neo1 , Helen Blanchard2 , Stephen J. Ralph1
1. Griffith Health Institute, Southport, QLD, Australia
2. Institute for Glycomics, Griffith University, Southport, QLD, Australia
Galectin-1 is a hypoxia-inducible beta-galactoside binding lectin protein produced by a wide range of cancer cells and plays a
number of significant roles within the tumour microenvironment. This includes immunosuppression, pro-angiogenesis,
promotion of cell adhesion and metastasis. Higher levels of galectin-1 and galectin-1 bound glycoproteins in plasma of cancer
patients were reported to correspond with poor diagnosis and high risk of metastasis. It was first demonstrated that blocking
galectin-1 carbohydrate recognition domains (CRDs) using thiodigalactoside (TDG) or knocking down the expression of
galectin-1 (G1KD) by shRNA exhibited tumour suppressive effects in primary B16F10 melanoma, 4T1 breast cancer and CT26
colon cancer models in mice1. This was associated with reduced tumour angiogenic activities and increased T cell infiltration
into the tumours. Anti-metastatic effects of targeting galectin-1 were also investigated by using the 4T1.2 mCherry+ and CT26
cell lines. The numbers and size of metastatic foci were significantly reduced by either TDG or G1KD, associated with
increased CD4+ and CD8+ T cell levels in the peripheral blood and TUNEL+mCherry+ cells in the lung, indicating higher levels of
targeted metastatic cell death2 . Moreover, galectin-1 was found to bind to cancer-initiating cell (CIC) markers, CD44 and
CD326, in 4T1 cells by co-immunoprecipitation assay2. Inhibiting galectin-1 significantly reduced its binding to CD44 and
CD326, leading to suppressed 4T1 cell attachment onto endothelium substrata, including laminin, Matrigel and human
endothelial EAhy926 cells2. More recent data implicate a further significant role of galectin-1 in CIC biology, which is important
to understand a metastatic process. In conclusion, the present study confirms that targeting galectin-1 represents a novel and
effective anti-cancer and anti-metastatic strategy in different types of cancers.

1.        K.Ito et al, Angiogenesis, 2011.
2.        K.Ito et al, Clin Exp Metastasis, 2012.
3.        K.Ito et al, Cancer Metastasis Rev, 2012.


Analysis of the role of the G protein-coupled receptors formyl peptide receptor-1, -2, and -3 in
progression of human and murine breast cancer
Cameron N Johnstone1 2 3 , Jay Rautela1 3 , Shenna Y. Langenbach3 , Thippadey Khau3 , Robin L. Anderson1 2 4 ,
Alastair G Stewart3
1. Cancer Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
2. Department of Pathology, University of Melbourne, Parkville, VIC, Australia
3. Department of Pharmacology, University of Melbourne, Parkville, VIC, Australia
4. Sir Peter MacCallum Department of Oncology, University of Melbourne, VIC, Australia
Human formyl peptide receptors (FPRs) 1, 2 and 3 comprise a small family of seven transmembrane domain G protein-coupled
receptors that have a well established role in innate immunity and inflammation. Recognition of bacteria-derived N-formyl
peptides by the FPRs activates the microbicidal, secretory, and chemotactic functions of phagocytic cells in vitro. Extracellular
Annexin-A1, and peptides derived from it, also act as agonists of FPR2. FPR1 and FPR2 are primarily expressed by
monocytes, neutrophils and dendritic cells whereas FPR3 is almost exclusively expressed by activated dendritic cells.
 However, FPRs can also be ectopically expressed in certain tumour types such as breast cancer, and our laboratory recently
showed that well-characterized agonists for FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin
A(4), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MDA-MB-231 breast cancer
cells in vitro that was attenuated by incubation with FPR2 antagonists WRW(4) (1μM) or Boc2 (100nM) or by siRNA against
FPR2, together suggesting that FPR2 may promote epithelial tumourigenesis. 1 TaqMan qPCR demonstrated that both FPR1
and FPR2 mRNAs were expressed by the estrogen receptor negative basal-like cell lines MDA-MB-231 and Hs578T, and that
FPR1 was also expressed by the immortal mammary epithelial cell line, MCF10A. To study the role of FPRs in human breast
cancer cells in vivo, stable shRNA-mediated knockdown of FPR1 was produced in MCF10A and MDA-MB-231 cells and of
FPR2 in MDA-MB-231 cells using the lentiviral pGIPZ vector, which also encodes GFP. Our laboratory has also acquired
C57Bl/6 mice null for murine Fpr2 (Fpr2-/-) or its endogenous ligand Anxa1 (Anxa1-/-). This will permit dissection of the roles of
both host-derived and tumour-derived molecules through orthotopic implantation of murine mammary tumour lines such as
EO7712 and AT33 , engineered to over-express formyl peptide receptors.

1.        Khau T, Langenbach SY, Schuliga M, Harris T, Johnstone CN, Anderson RL, and Stewart AG. FASEB J. 2011
2.        CASEY AE, LASTER WR Jr, and ROSS GL. Proc. Soc. Exp. Biol. Med. 1951 77(2):358-362
3.        Stewart TJ, and Abrams SI. J Immunol. 2007 179(5):2851-2859


Three-dimensional (3D) modelling of the prostate cancer microenvironment
Chambers F Karen1 , Michael R Doran1 , Pamela J Russell2 , Judith A Clements1
1. Institute of Health and Biomedical Innovation, QUT, Kelvin Grove, QLD, Australia
2. The Australian Prostate Cancer Research Centre, Princess Alexandra Hospital, Brisbane, QLD, Australia
Introduction: Monolayer cultures on tissue culture plastic do not accurately mimic the cancer environment and often provide a
poor prediction of whether a drug will yield clinical benefit. By contrast, when prostate cancer cells are cultured as 3D
aggregates their response to chemotherapy agents more realistically parallels the in vivo response. In addition, the
microenvironment in which prostate cancer cells reside, particularly the stromal cell populations, has the potential to
significantly influence prostate cell behaviour and contribute to cancer progression. We have developed a high-throughput 3D
prostate cancer-stromal cell micro-aggregate co-culture system suitable for drug testing.
Methods: Micro-aggregates of uniform diameter (100-200µm) are cultured on a PDMS micro-well surface. Prior to cell
seeding, the PDMS micro-well surface is coated with poly-lysine and multi-layers of hyaluronan and chitosan, to minimize
protein adsorption and subsequent cell attachment to the micro-well surface. The prostate cancer cells lines, LNCaP and
RWPE-2 were seeded either in mono-culture or in co-culture with the fibroblast cell line, WPMY-1. The normal prostate cell
line, RWPE-1, was used as a comparison.
Results and Discussion: In mono-culture, the micro-aggregates do not form a dead cell core, as shown by a live/dead stain.
In addition, apical (E-Cad, β-catenin, aPKC) and basal (α6integrin, lam5) polarity markers show a random distribution
throughout the aggregates. This poor cell organisation and lack of defined polarity is also seen in high grade cancer tissue
samples. In co-culture, the RWPE-1 and-2 cells from an epithelial cell cluster surrounded by the WPMY-1 cells, and LNCaP
cells have the inverse cell distribution. In summary, the prostate micro-aggregates can be used to model a prostate cancer
stage prior to angiogenesis, before a dead cell core has formed. Furthermore, direct co-culture with stroma provides a means
to study stromal-epithelial interactions in response to drug treatment.


NBRI16716A inhibits tumor growth through modulating tumor-stromal cell interactions
Manabu Kawada1 , Hiroyuki Inoue1 , Ihomi Usami1 , Shuichi Sakamoto1 , Masahide Amemiya1 , Shun-ichi Ohba1 , Tohru
Masuda1 , Akio Nomoto1
1. Institute of Microbial Chemistry, Numazu, Microbial Chemistry Research Foundation, Shizuoka, Japan
Tumor microenvironment is now getting much attention as antitumor strategy. Tumor cells interact with many other surrounding
cells such as immune, endothelial, and fibroblast-like stromal cells. Among them the stromal cells can modulate tumor growth
and metastasis positively and negatively through direct adhesion and secreted factors. We have been studying such tumor
microenviroment, especially focusing on stromal cells, to innovate a new antitumor strategy. We previously developed an in
vitro coculture system of human prostate cancer cells and prostate stromal cells, and found a novel antitumor compound
NBRI16716A from a fungus metabolite. Our preliminary experiments suggested that NBRI16716A modulates tumor-stromal cell
interactions, we therefore have studied the action of NBRI16716A using new coculture systems of various types of cancer. We
established green fluorescence protein-expressing cells of lung, gastric, colon, pancreatic, breast, and prostate cancer cell
lines, and developed various coculture systems using each organ-derived stromal cells. Using these coculture systems, we
have found that NBRI16716A inhibits the growth of A549 lung cancer cells in coculture more strongly than cancer cells alone as
well as the coculture system of DU-145 prostate cancer cells. The conditioned medium prepared from the stromal cells
pretreated with NBRI16716A also showed stronger growth inhibitory activity suggesting possible involvement of some secreted
factors from the stromal cells. We are now trying to identify the secreted factors from the stromal cells. Collaborators: T Kato, Y
Aritoku, MicroBiopharma Japan.


Expression profiling of ECM proteins in Indian Patients with Urothelial carcinoma of bladder
Rehan Khan1 , Manoj Sharma2 , Amlesh Seth3 , Alpana Sharma4
1. Biochemistry, All India Institute of Medical Sciences, New Delhi, Delhi, India
2. Radiation Oncology, Maulana Azad Medical College, New Delhi, Delhi, India
3. Urology, All India Institute of Medical Sciences, New Delhi, Delhi, India
4. Biochemistry, All India Institute of Medical Sciences, New Delhi, Delhi, India
Bladder cancer is the 2nd most common urologic malignancy amongst the males after prostate cancer. Migration is one of the
important processes fundamental to cell invasion and dissemination. Role of extracellular matrix (ECM) proteins is also very
important in this crucial process and cannot be ignored. ECM proteins are involved in cell migration, tissue remodeling,
angiogenesis and cell adhesion.
Fibulin-1 is cysteine-rich, calcium-binding extracellular matrix and plasma protein that has been implicated as playing a role in
tumour progression by modulating cell morphology, growth, adhesion, and motility. Increased plasminogen activator (PA)
secretion has been observed in malignant cells and tissue and PA is thought to be involved in the processes of tumorigenesis,
cancer invasion and metastasis. Laminins are also important for proliferation, differentiation, growth, migration & angiogenesis.
Besides the structural function of nidogens, it has long been suggested that nidogens play a role in cell attachment and tumor
This study was aimed to investigate the expression and significance of ECM proteins (Fibulin-1, uPA, Laminin and Nidogen) in
Urothelial carcinoma of bladder.
Material & Methods
The study cohort consisted of 90 subjects; 45 patients with Urothelial carcinoma of bladder (High grade Muscle Invasive MI
(T2): 14, High grade Non-Muscle Invasive NMI (T1): 12, Low grade LG: 19; According to the WHO/ISUP 2004 classification
and T staging) and 45 healthy controls. Circulatory levels of Fibulin-1, Laminin and Nidogen were measured using high
sensitivity ELISA kits. uPA activity was measured by activity assay kit. The mRNA expressions of these molecules in the
Peripheral blood mononuclear cells (PBMC) were further analyzed by Real Time RT-PCR. The data was analyzed statistically
and correlated with the severity of the disease.
Statistical significant (p < 0.001) increase was observed in Fibulin-1, Nidogen, uPA and Laminin levels as compared to controls
and found to be significantly correlated with each other. Increased mRNA expression for all these molecules has also been
observed in the isolated PBMC by qRT-PCR.
The elevated serum/plasma levels and increased quantitative expression of these ECM proteins suggests their involvement in
the pathogenesis of the disease. This study suggests that rise in ECM proteins might contribute to the complicated and
unregulated process of metastasis in Urothelial carcinoma of bladder. The association of extracellular matrix proteins and the
prognosis of the disease may lead to have a better approach in treating the bladder cancer patients with a combination


Testing the efficacy of novel HDAC inhibitors for the treatment of breast cancer brain
Soo Hyun Kim1 , Chloe Georgiou1 , Delphine Denoyer1 2 , David Fairlie3 , Robin L. Anderson1 2 , Normand Pouliot1 2
1. Research Division, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia
3. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
Introduction: Of the 12,000 patients diagnosed with breast cancer in Australia annually, nearly 3,000 will die due to metastatic
disease, most commonly to bone, liver, lung and brain. Once brain metastases are established, prognosis for these patients is
extremely poor, with a median survival of ~ 4-6 months if treated with radiotherapy. The poor efficacy of current therapies
against brain metastases is contributing to the recent surge in the incidence of advanced breast cancer patients developing
brain metastases. We have developed a unique mouse model of triple negative breast cancer metastasis to brain (4T1Br4) that
is suited for testing the efficacy of anti-metastatic drugs. Recent in vitro and animal studies have shown that brain permeable
histone deacetylases (HDAC) inhibitors such as vorinostat enhance the radiosensitivity of brain metastatic breast cancer and
reduce brain metastasis in experimental models.
Aim: To test the inhibitory and radiosensitising properties of novel HDAC inhibitors against 4T1Br4 brain metastatic cells in vitro
using proliferation and colony forming assays.
Results: Compounds derived from 2-aminosuberic acid [1], were tested and their efficacy compared to that of vorinostat against
4T1Br4 tumour cells and neonatal foreskin fibroblasts. One of these, 1179.4b, was significantly more potent (IC 50 = 72 nM) than
vorinostat (IC50 >400 nM) at inhibiting 4T1Br4 cell proliferation. Importantly, inhibition by 1179.4b was 5.6-fold more selective
for 4T1Br4 tumour cells compared to normal fibroblasts (IC50 ~ 400 nM). In a colony forming assay, vorinostat (1 μM) did not
inhibit 4T1Br4 colony formation but at the same concentration, 1179.4b completely blocked colony formation. In combination
with irradiation, 1179.4b (50 nM) even at a concentration 10 times lower than vorinostat enhanced the effect of irradiation.
Other 1179.4b-related compounds will be tested and the results presented.
Conclusion: These results demonstrate the potency of aminosuberic acid-derived compounds against brain metastatic cells and
warrant further investigation in vivo.


3,3-diindolylmethane plus LY294002 suppress the oncogenic activity of YAP in colon
carcinoma cancer cells
Xiu Juan Li1 , Hwan Hee Lee1 , Soo Mi Kim1
1. Chonbuk National University, Joen Ju, JB, South Korea
Inhibition of PI3K/Akt pathway might be the best way to overcome colon cancer and small molecules [e.g., 2-(4-morpholinyl)-8-
phenyl-chromone (LY294002)] are being developed for cancer therapy. Recent studies revealed that 3,3-diindolylmethane
(DIM) has antitumor effects both in vivo and in vitro models of colon cancer. Therefore, this study aims to investigate the
antitumor effects of DIM plus LY294002 in human colon carcinoma cancer cells. Two human colon cancer cell lines (HCT-116,
HT-29) were used to test the response to DIM plus LY294002. MTT assay, flow cytometry, and western blotting were
conducted. DIM plus LY294002 significantly inhibited the proliferation of human colon cancer cells in a dose dependent manner
compared to when DIM or LY294002 is treated alone. The percentages of cells in the G1 phase were significantly increased
12h after being treated with DIM plus LY294002. DIM plus LY294002 significantly induced cleaved poly (ADP-ribose)
polymerase and cleaved caspase9 levels. In addition, DIM plus LY294002 significantly induced p-YAP and suppressed YAP
expression. Therefore, these results suggest that DIM may have more dramatic therapeutic effects when it is combined with
PI3K inhibitors in the treatment of colon cancer cells.


Overxpression of Angiomodulin (AGM/IGFBP-rP1) in Tumor Stroma and Vasculature:
Differential Regulation and Functions.
Eriko Komiya1 , Hiroki Sato1 , Shouichi Higashi1 , Yohei Miyagi2 , Kaoru Miyazaki1
1. Division of Cell Biology, Yokohama-city University, Yokohama-city, Japan
2. Devision of Cancr Therapy, Kanagawa Cancer Center Reseach Institute, Yokohama-city, Japan
Angiomodulin (AGM/IGFBP-rP1) is a member of IGFBP superfamily. We previously reported that AGM is highly accumulated in
blood vessels of human cancer tissues. Contradictory results have been reported with respect to the roles of AGM in tumor
progression. To elucidate its roles in tumor growth, we analyzed the distribution of AGM in four major types of human cancers
by immunohistochemistry in detail. In colon, lung, uterus and breast cancers, AGM was overexpressed in stromal fibroblasts as
well as in vasculature near tumor cells, but its expression in tumor cells was evident only in a few cases of cancers. AGM
expression in stromal fibroblasts was often colocalized with alpha-smooth muscle actin (SMA), an activated fibroblast
(myofibroblast) marker. These results suggested that AGM may play some roles in cancer-associated fibroblasts (CAFs).
Among different stages of breast cancers, AGM was highly overexpressed in vasculature even in benign tumors (DCIS), while
its expression in fibroblasts was more prominent in invasive carcinomas than DCIS. AGM was induced by TGF-beta in
fibroblasts but by VEGF in endothelial cells. In vitro analyses showed that AGM stimulated strongly the expression of
fibronectin and weakly that of SMA in normal fibroblasts and induced their morphological change to a myofibroblast-like shape.
AGM also significantly stimulated the proliferation of fibroblasts. The induction of protein expression and morphological change
were blocked by TGF-beta signal inhibitor, but its growth activity was not. On the other hand, AGM did not stimulate the growth
of human umbilical endothelial cells (HUVEC). However, it significantly promoted their adhesion through integrins. These
results indicate that AGM is overexpressed by different mechanisms and exerts different functions between stromal fibroblasts
and endothelial cells in tumor tissues. It is supposed that such expression and activities of AGM contribute to tumor

1.        Komiya E., et al. Elevated expression of angiomodulin (AGM/IGFBP-rP1) in tumor stroma and its roles in fibroblast
     activation. Cancer Sci 4: 691-9, 2012.


Dll4-Fc, an inhibitor of Dll4-notch signaling, suppresses liver metastasis of small cell lung
cancer cells through down-regulation of NF-kappa-B activity
Takuya Kuramoto1 , Hisatsugu Goto1 , Atsushi Mitsuhashi1 , Sho Tabata1 , Hirohisa Ogawa2 , Hisanori Uehara2 , Atsuro
Sijo1 , Soji Kakiuchi1 , Masaki Hanibuchi1 , Yoichi Maekawa3 , Koji Yasutomo3 , Shin-ichi Akiyama1 , Saburo Sone1 ,
Yasuhiko Nishioka1
1. Departments of Respiratory Medicine & Rheumatology, The University of Tokushima Graduate School, Tokushima, Japan
2. Departments of Molecular and Environmental Pathology, The University of Tokushima Graduate School., Tokushima,
3. Departments of?Immunology & Parasitology, The University of Tokushima Graduate School, Tokushima, Japan
Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of
Dll4-Notch signaling between cancer cells, and how it affects cancer metastasis. We therefore assessed role of Dll4-Notch
signaling in cancer metastasis. We generated a soluble Dll4 fused to the human IgG1 Fc constant region (Dll4-Fc) that acts as
a blocker of Dll4-Notch interactions, and introduced it into human SCLC cell lines expressing high level (SBC-3 and H1048) and
low level (SBC-5) of Dll4. The effect of Dll4-Fc on metastasis of SCLC was evaluated using a mouse model. Although Dll4-Fc
has no effect on liver metastasis of SBC-5, the number of macroscopic metastatic nodules in the livers of mice inoculated with
SBC-3 and H1048 cells expressing Dll4-Fc were significantly lower than that in the livers of mice injected with control cells. In
order to study the molecular mechanism of the effect of Dll4-Fc on liver metastasis, PCR array analysis was performed. Since
expression of NFkB target genes were affected by Dll4-Fc, we explored whether NFkB is involved in Dll4-Notch signaling in the
cancer cells. We observed by electrophoretic mobility shift assay that both with and without TNF-a-stimulated NFkB activities
were down-regulated in Dll4-Fc-overexpressing SBC-3 and H1048 cells, compared with control cells. Moreover, interaction of
Notch1 intracellular domain with p65 enhanced the NFkB signaling. These results suggest that Dll4-Notch signaling in cancer
cells plays a critical role in liver metastasis of small cell lung cancer by regulating the NFkB signaling.


Annexin A2 released during ovarian cancer-peritoneal cell interaction promotes a pro-
metastatic cancer cell behaviour
Noor A Lokman1 , Miranda P Ween2 , Peter Hoffmann3 , Martin K Oehler1 4 , Carmela Ricciardelli1
1. Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA, Australia
2. Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide,
      SA, Australia
3. Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia
4. Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, SA, Australia
Ovarian cancer metastasis is characterized by the spread of cancer cells from the ovarian surface and their implantation onto
the peritoneum, a layer of cells that surrounds the abdominal organs. We conducted a proteomic study investigating the
interaction between peritoneal and ovarian cancer cells and identified annexin A2 to be upregulated in the conditioned media of
co-cultured ovarian cancer (OVCAR-5) and peritoneal (LP-9) cells. Annexin A2, a calcium dependent phospholipid-binding
protein, has been shown to be over expressed in many cancers but its role has not been investigated previously in ovarian
Our study characterized the expression of annexin A2 in human ovarian cancer tissues and cell lines. Western blot and real
time-PCR showed annexin A2 was expressed in ovarian cancer cell lines (OVCAR-3, OVCAR-5, OV-90 and SKOV-3).
Immunohistochemistry demonstrated annexin A2 expression in epithelial cancer cells, the cancer associated stromal cells and
peritoneal mesothelial cells. Stromal annexin A2 levels were significantly increased in ovarian cancer tissues compared to the
stromal surrounding non-invasive borderline ovarian tumours and benign ovarian tumors. Increased annexin A2 expression
was also observed in ovarian cancer cells adjacent to peritoneal cells.
We also investigated the role of annexin A2 in ovarian cancer invasion. In vitro functional assays showed that neutralizing
antibodies against annexin A2 could inhibit OVCAR-5 and OV-90 cell motility, invasion and adhesion to peritoneal cells.
Moreover, knockdown of annexin A2 expression using siRNA decreased OVCAR-3, OVCAR-5, OV-90 and SKOV-3 cancer cell
motility and invasion in vitro. We assessed ovarian cancer invasion in vivo using a matrigel graft in a chick embryo
chorioallantoic membrane (CAM) model. Treatment of chick embryos with neutralizing antibodies against annexin A2
significantly blocked OV-90 ovarian cancer cells invasion (p=0.004, Mann-Whitney U test).
Our findings indicate that annexin A2, which is increased as a result of ovarian cancer-peritoneal cell interactions, plays an
important role in promoting ovarian cancer metastasis.


Resistance to anoikis alters the adhesion and promotes the increase of syndecan-4 and
heparanase expression in endothelial cells in culture.
Paulo Pernambuco Filho1 2 , Bruna Carneiro1 2 , Douglas Silva1 2 , Ana Paula Vasconcelos1 2 , Maria Aparecida Pinhal1 ,
Helena Bonciani Nader1 , Carla Cristina Lopes1 2
1. Bioquímica, UNIFESP, São Paulo, SP, Brasil
2. Ciências Biológicas, UNIFESP, São Paulo, SP, Brasil
Tumor cells present important characteristics about the changes in the mechanism of adhesion, which is related to changes in
cell-cell and cell-extracellular matrix (ECM) contacts. The proliferation, invasion and maintenance of the tumors are strictly
correlated to proteoglycans. Syndecan-4, a heparan sulfate proteoglycan (HSPG), has an important role in the mechanism of
cell adhesion by its interaction with extracellular ligands. Changes in its expression have been observed in tumor cells,
indicating its involvement in cancer. The acquisition of resistance to cell death induced by blockade of adhesion to the
substratume (anoikis resistance) is a feature of neoplastic transformation and a critical step during the metastatic process. In
this study, endothelial cells derived from rabbit aorta (EC) were subjected to transformation induced by blockade of adhesion to
the substratume (adh-EC). After one deadhesion cycle, phenotypic alterations were observed in the few surviving cells, which
became more numerous and showed progressive alterations after each adhesion impediment step. EC cells and adh-EC
clones obtained were tested in nude mice to observe their tumorigenic capacity. Tumor development was observed in mice
injected with adh-EC clones. Sulfated glycosaminoglycans (SGAG) synthesized by the cells were metabolically labeled with
[35S] sulfate (150µCi/ml). SGAG from both medium and cells were extracted by proteolysis and identified by radioautography
of the agarose gel electrophoresis. An increase in heparan sulfate (HS) synthesis was detected in the adh-EC clones. Analysis
of the expression of the syndecan-4 protein core by RT-PCR and heparanase expression by Real Time PCR suggests this
proteoglycan is related to HS chains increase. Also, an increase in adhesion to fibronectin, laminin and collagen IV was
observed in adh-EC clones. These findings suggest that syndecan-4 is involved in acquisition of tumor cell resistance induced
by anchorage independence in EC cells, thus contributing to neoplastic transformation. Supported by FAPESP, CNPq and

Host-derived CCL2 plays a critical role in prostate cancer growth and skeletal metastasis
Lihui Wang1 , Xin Yang1 , Jiejun Fu1 , Wenchu Wang1 , YI LU1
1. Guangxi Medical University, Nanning, GX, China
Prostate cancer (PCa) is the most common cancer in men in Western countries. In China, the incidence has sharply increased
for the past 20 years. Prostate cancer preferentially metastasizes to bone resulting in high mortality. We have previously
reported that the presence of CC chemokine ligand 2 (CCL2) produced by prostate cancer (PCa) epithelial cells,
stroma/osteoblasts, and bone marrow endothelial cells, is pro-tumorigenic for prostate cancer cells. CCL2 increases prostate
cancer cell proliferation, migration, and survival and induces osteoclast formation. Moreover, the key role of CCL2 in the tumor
growth and metastasis has been linked to its regulatory role in mediating monocyte/macrophage infiltration into the tumor
microenvironment. Using a model of human PCa cells implanted in mice, Loberg R. et al. reported that anti-murine CCL2
neutralizing antibodies resulted in greater reduction of overall tumor burden compared to the use of anti-human antibody (anti-
tumor cell-derived). This suggests that host-derived CCL2 may play a key role in prostate tumor growth and metastasis.
Therefore, in this study, we have employed CCL2 knockouts which were interbred to SCID mice to generate colonies of
homozygous SCID/CCL2-/- mice. PCa cells (PC3Luc) were injected into the left ventricle (Intracardiac-injection) of SCID/CCL2-
/- and wild-type controls (n=20/group). The effects of deletion of CCL2 in the host on prostate cancer cell homing, growth in
bone and bone destruction were evaluated for 4 weeks. In a parallel experiment, PC3Luc cells were subcutaneously implanted
and tumor size was monitored weekly for 4 weeks. This parallel study addressed bone-specific vs. generalized influences of
host-derived CCL2 on prostate cancer growth. We found that skeletal metastases occurred in 4/20 SCID/CCL2-/- mice vs 14/20
WT mice. In addition, X-ray photographs showed significantly fewer bone resorptive lesions in SCID/CCL2-/- mice vs. WT mice.
Histology examinations confirmed x-ray findings. In the parallel experiment, PC3Luc tumor grew slower in SCID/CCL2-/- mice
vs. WT mice. These results indicate that host-derived CCL2 production defines a permissive microenvironment for prostate
cancer growth and skeletal metastasis. These findings may significantly impact the therapeutic field through targeting “the host”
instead of “the tumor cells” in prostate cancer. Supported by U.S. Department of Defense PC061231 (J. Zhang), National
Natural Sciences Foundation Key Project (81130046, J. Zhang) and National Natural Science Foundation Project (81171993,
Y. Lu).


Investigating the anti-metastatic activity of the recombinant disintegrin domain of ADAM9 in
breast cancer cell lines
Ana Carolina B.M. Martin1 , Normand Pouliot2 , Kelli C. Micocci1 , Marcia R. Cominetti3 , Heloisa S. Selistre-de-Araújo1
1. Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, São Carlos, São Paulo, Brasil
2. Department of Oncology, The Sir Peter MacCallum, Melbourne, Victoria, Australia
3. Departamento de Enfermagem, Universidade Federal de São Carlos, São Carlos, São Paulo, Brasil
Metastasis is the major cause of death in cancer patients and more effective therapies are required. ADAMs (A Disintegrin And
Metalloprotease) are a family of proteins known that regulate several cellular functions required for metastasis such as cell
adhesion, migration, invasion and survival. ADAMs are composed of a set of conserved domains including a disintegrin
domain, which interacts with cell surface integrins. We have recently reported on the cloning and purification of soluble and
active form of the recombinant disintegrin domain of human ADAM9 (ADAM9D) and showed that ADAM9D binds multiple
integrins including β1, αvβ5, αvβ3, α2 and a6 [(1) and unpublished data). The aim of this study was to investigate the inhibitory
activity of ADAM9D in vitro against two metastatic breast cancer lines, the human MDA-MB-231 and the mouse 4T1BM2 [2].
Migration and invasion inhibition of MDA-MD-231 and 4T1BM2 cells in the presence of ADAM9D were tested in standard
Transwell migration chambers and/or in a wound healing assay. Supernatants from wound healing assays were also analyzed
for matrix metalloproteinases (MMPs) activity by zymography. The impact of ADAM9D treatment of the colony forming ability of
4T1BM2 cells was also tested.
ADAM9D significantly decreased cell invasion and migration. Results from zymography showed that the expression of MMP2
and MMP9 was inhibited by ADAM9D. Treatment of 4T1BM2 metastatic cells with ADAM9D inhibited colony formation on
laminin-511 substrate. However, ADAM9D did not further inhibit colony formation in 4T1BM2 with suppressed b4 integrin
suggesting that ADAM9D inhibition may be mediated in part through binding to a6b4 integrin.
Conclusion: Our results demonstrate that ADAM9D inhibits multiple cellular function associated with metastasis. The
development of bioactive recombinant ADAM9D represents a useful tool for investigating the role of ADAMs in metastasis and
for the design of selective inhibitors against metastatic breast tumours.
Financial Support: FAPESP, CNPq, Capes and NHMRC


A role for the disintegrin domain of adam9 in tumor cell transmigration and cell adhesion
Kelli C. Micocci1 , Ana Carolina B.M. Martin1 , Marcia R. Cominetti1 , Araceli C. Durante1 , Laila R. Fernandes2 , Antonio
G.F. Lima2 , Verônica Morandi2 , Camila C. Figueiredo2 , Heloisa S. Selistre-de-Araujo.1
1. Universidade Federal de São Carlos, São Carlos, SP, Brazil
2. Universidade do Estado do Rio de Janeiro, Rio de Janeiro , RJ, Brazil
ADAM9 is a member of the metalloprotease protein family with a disintegrin domain that plays key roles in many physiological
processes such as in fertilization, migration, and cell survival, and in diseases as well, including cancer (1). ADAM9 is highly
expressed in metastatic cancer tissues and so it was suggested to be involved in metastatic spreading (2). We have previously
demonstrated that the recombinant disintegrin domain of ADAM9 (ADAM9D) inhibited the adhesion of both platelets and MDA-
MB-231 breast tumor cells to type I collagen, a major component of the extracellular matrix (3). Here we provide additional
evidence for a role of ADAM9D in metastatic spreading. ADAM9 silencing inhibited MDA-MB-231 tumor cell invasion in matrigel
in approximately 72% when compared to control, without affecting the rate of cell proliferation. For transmigration endothelial
assay, MDA-MB-231 cells (7x104 cells/chamber) were placed in the upper chamber covered with a monolayer of HMEC
(Human Dermal Microvascular Endothelial Cells) or HUVEC (Human Umbilical Vein Endothelial Cells) and incubated for 16
hours at 37oC and 5% CO2. The effect of siADAM9 and ADAM9D on the adhesion of tumor cells was also analyzed by flow
adhesion assay. 2x106 MDA-MB-231 cells (siADAM9 or control) were stimulated in a dynamic flow (5 dynes - 5 minutes)
through a monolayer of HMEC cells. Transient ADAM9 silencing reduced MDA-MB-231 cells transmigration through HUVEC or
HMEC. In addition, ADAM9D (1000nM) inhibited tumor cells transmigration through endothelial cells. These results suggested
that ADAM9 can be involved in the processes of adhesion and transmigration and also that its disintegrin domain could be an
important target for anti-metastatic therapy.

Financial Support: FAPESP, CNPq and CAPES (Brazil).
1- SEALS, D. F.; COURTNEIDGE, S. S. The ADAMs family of metalloproteases: multidomain proteins with multiple functions.
Gen. Dev., 17: 7-30, 2003.
2- PEDUTO, L.; REUTER, V. E.; SHAFFER, D. R.; SCHER, H. I.; BLOBEL, C. P. Critical Function for ADAM9 in Mouse
Prostate Cancer. Cancer Research, 65: 9312-9319, 2005.
ARAUJO, H. S. Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under
dynamic flow conditions. Biochimie., 91 (8): 1045-1052, 2009.


Alteration of tumor microenvironment during antiangiogenic therapy in colorectal cancer
Shinichiro Miyazaki1 , Hirotoshi Kikuchi1 , Ichirota Iino1 , Yoshihiro Hiramatsu1 , Manabu Ohta1 , Kinji Kamiya1 ,
Hiroyuki Konno1
1. Hamamatsu University School of Medicine, Second Department of Surgery, Hamamatsu, Japan
Introduction: Antiangiogenic therapy has been thought to hold significant potential for the treatment of cancer. Inhibition of
vascular endothelial growth factor (VEGF) pathway impedes tumor growth and, clinically, the VEGF-neutralizing antibody has
been used as anticancer treatments in several tumor types including colorectal cancer. However, several studies indicate
antiangiogenic therapy may have limited clinical benefit. Here we investigated the alteration of colorectal cancer phenotype in
vivo during antiangiogenic therapy. Methods: TK-4, a solid tumor strain, derived from human colon cancer was utilized. TK-4
was implanted orthotopically into cecal walls of BALB/c nu/nu mice. The animals were treated with anti-human VEGF
monoclonal antibody (treated group, n=14, 10μg/body, i.p, thrice a week) or control IgG (Control group, n=14), The animals
were sacrificed and tumors were removed at two time points, day21 (n=4, each group) and day42 (n=10, each group). The
tumors removed at day42 were examined for tumor volume and microvessel density (MVD) using CD31 immunohistochemistry
and gene expression was analyzed using microarrays (Human Gene 1.0ST Array, Affymetrix). Mitosis and apoptosis were
evaluated using H&E staining and TUNEL assay, respectively in the tumors at day21 and day42. Hypoxia in tumors was
evaluated by HIF-1α immunohistochemistry. Results: Tumor volume and MVD were significantly lower in treated group than
those in control (p=0.013, p=0.003, respectively). Mitosis was significantly lower in treated tumors than control at day21
(p=0.049), but not at day42. Whereas TUNEL positivity was comparable between the two groups at day21, it was lower in
treated tumors at day42 (p=0.003). Gene set enrichment analysis revealed that the gene set “HYPOXIA_REG_UP” was
upregulated in treated group. HIF-1α positivity was significantly higher in treated tumors (p=0.010). Conclusion: Tumor hypoxia
appears to play important roles in enhancing or selecting tumor cells with high malignant potential.


SSeCKS modulates metastasis through multiple organ- and route-specific tumor-
microenvironment interactions.
Masashi Muramatsu1 , Shin Akakura1 , Hyun-Kyung Ko1 , Irwin H Gelman1
1. Roswell Park Cancer Institute, Buffalo, NY, United States
The metastatic microenvironment is composed of a complex interaction between tumor, mural and bone marrow-derived stem
cells as well as soluble factors, such as chemoattractants and ligands that trigger signaling receptors. We previously
characterized a role for SSeCKS/Gravin/AKAP12 (“SSeCKS”) as a metastasis suppressor based on its ability to inhibit
neovascularization in the metastatic niche through the downregulation of tumor-encoded pro-angiogenic factors such as
VEGF1-2. SSeCKS, whose downregulation in many solid and liquid tumors correlates with poor prognosis and increased
malignancy, encodes a scaffolding protein for multiple signaling mediators such as PKC, PKA, calmodulin, cyclins and Src, and
thus, is thought to normalize proliferative and cytoskeletal signaling3-5. Here, we report an additional metastasis suppressing
role for SSeCKS in host microenvironment based on the increased level of metastasis formation in the peritoneum and lung by
B16F10 mouse melanoma cells in SSeCKS-null (KO) vs. WT syngeneic mice. For example, the orthotopic injection of B16F10
resulted in higher formation of spontaneous peritoneal metastases in KO hosts, whereas i.v. injection led to increased lung
metastases in KO hosts. Peritoneal cell-free fluid (PF) from naïve KO mice, but not PF from WT mice, could transfer sensitivity
to peritoneal metastasis formation. Protein profiling showed a large increase in chemokines and cytokines in PF from KO vs.
WT mice, correlating with increased in vitro B16F10 chemotaxis to the KO-PF. KO mice also exhibited increased numbers of
CD11b+ monocyte-lineage cells, known to facilitate tumor cell recruitment and survival in the metastatic niche. In contrast,
SSeCKS seems to control lung metastasis following i.v. injection by modulating tumor cell attachment in the lung. Specifically,
the rate of B16F10-luc-G5 colonization in the first 6h after injection was 2- to 3-fold higher in KO vs. WT hosts. Our results
suggest that SSeCKS controls metastasis by modulating multiple organ- and route-specific tumor-microenvironment

1.        Sae-Won L, et al., Nat Med, 9(7), 900-906 (2003)
2.        Su B, et al., Cancer Res, 66(11), 5599-5607 (2006)
3.        Lin X. et al., Mol Cell Biol, 15(5), 2754-2762 (1995)
4.        Gelman IH., Genes Cancer, 1(11), 1147-1156 (2010)
5.        Akakura S, et al., Cell Cycle, 9(23), 4656-4665 (2010)


N-glycosylation regulates cathepsin L2 functions
Yuki Niwa1 , Takehiro Suzuki2 , Naoshi Dohmae2 , Kazuo Umezawa1 , Siro Simizu1
1. Keio University, Kohoku-ku, Yokohama, Japan
2. RIKEN, Wako, Japan
Glycosylation is one of the post-translational modifications, and known to be important for protein folding, stability, and
secretion. When nascent glycoproteins enter the ER, a preformed oligosaccharide known as dolichol-phosphate precursor is
often attached co-translationally to certain Asn residue(s) involved in the consensus sequence Asn-Xaa-Ser/Thr (where Xaa
represents any amino acid except Pro). Changes in glycosylation are early indicators of cellular changes in many diseases,
most notably cancer, providing useful diagnostic markers and insights into disease progression and pathogenesis. Cathepsin
L2, also known as cathepsin V, is a lysosomal cysteine protease that belongs to cathepsin family, and related to cancer
invasion and metastasis. All cathepsin family members have potential N-glycosylation site(s), but role of N-glycosylation on
cathepsin functions has not been fully understood. Cathepsin L2 contains two predicted N-glycosylation sites, Asn221 and
Asn292, but glycosylation of cathepsin L2 has not been reported. We demonstrated that cathepsin L2 was N-glycosylated at both
Asn221 and Asn292 sites using mass spectrometry and site-directed mutagenesis analyses. N-glycosylation of cathepsin L2 was
found to be important for transportation to lysosome and secretion. Furthermore, N-glycosylation of cathepsin L2 was required
for the enzymatic activity of cathepsin L2. These data demonstrated that functions of cathepsin L2 were controlled by N-
glycosylation. Thus, inhibition of cathepsin L2 N-glycosylation may suppress cancer invasion and metastasis.


Characterization of malignant melanoma growth triggered by the brain microenvironment in
experimental metastasis models
Vigdis Nygaard1 , Lina Prasmickaite1 , Kotryna Vasiliauskaite1 , Trevor Clancy1 , Hanne H1 , Fodstad1 , Gunhild M1 ,
Eivind Hovig1
1. Dept. of Tumor Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
Melanomas are particularly renowned for their plasticity in response to microenvironmental signals. Uncovering organ-specific
regulation of melanoma plasticity is important in developing anti-metastatic therapy aimed to tackle issues of resistance and
evasiveness. The aim of our study was to reveal characteristics of metastatic growth of malignant melanoma in different
anatomical sites, particularly brain, by in vivo imaging, cellular characterization and molecular approaches. The study was
based on experimental metastasis models in vivo established from melanoma cell lines, Melmet 1 and Melmet 5 with opposing
phenotypes (invasive/proliferative, respectively) in the in vitro setting. In vivo imaging indicated that Melmet 5 establishes
metastases in multiple organs and at a faster rate than Melmet 1, which preferred the brain. Gene expression profiling of
melanoma cells isolated from different metastatic growth phases, revealed a dynamic nature of the phenotypes transitioning
from an invasive gene signature to a proliferative signature as the metastases passed from early to late phase. The presence of
a molecular signature characteristic of neural cells in melanoma from brain metastases indicates activation of neurogenetic
transcriptional programs. The neural-like signature was more pronounced in early versus late phase. To uncover
microenvironmental factors that could stimulate reprogramming of melanoma cells, we analyzed gene expression in stroma
cells from metastatic versus healthy brain. Genes implicated in immune-/inflammatory-response were up-regulated, suggesting
an involvement of immunity-related cells. Indeed, cellular characterization of metastatic brain tissue revealed the emergence
and accumulation of cells of bone-marrow origin like microglia (F4/80+, CD11b+) that correlated with “active growth” phase
initiation. Altogether, this suggests that acquisition of the neuronal-invasive phenotype might be critical for the establishment of
metastatic growth in the brain microenvironment. Host factors that appear to be involved in creating a permissive
microenvironmental niche for melanoma cells to express such a neuronal-invasive phenotype, may represent focal points for
targeting metastasis.


Drug Transporters are Differentially Expressed in Cancer-Associated Pancreatic Stellate Cells
(Key Cells Involved in Enhancing Pancreatic Cancer Progression and Chemoresistance).
Nelson Russia1 , Narada Kiriella1 , Julia Lee1 , Jie Liu1 , Janet Youkhana1 , Andrew Biankin2 , David Goldstein1 , Phoebe
1. Pancreatic Cancer Translational Research Group, University of New South Wales, Sydney, Australia
2. Garvan Institute of Medical Research, Sydney, Australia
Pancreatic cancer has a dismal prognosis and is highly resistant to chemotherapy. It is now known that the extensive
desmoplastic reaction, principally produced by pancreatic stellate cells (PSCs), plays a role in chemoresistance via impairment
of drug delivery and the formation of a hypoxic microenvironment. Interestingly despite high concentrations of cytotoxic drugs
sequestered in the stromal compartment, hypoxia and low nutrient levels, PSCs thrive and proliferate. Membrane drug
transporters associated with chemoresistance and cell survival are known to be altered in pancreatic cancer, yet the expression
of drug transporters in PSCs and the impact of the hypoxic microenvironment on drug transporter expression in pancreatic
cancer cells, have not been examined.
Aims: i) To characterise the expression of drug transporters in PSCs; and ii) To investigate the effect of hypoxia on drug
transporter expression in pancreatic cancer cells.
Methods: i) PSCs were isolated from surgically resected normal pancreas and pancreatic cancer tissue from human patients. ii)
Pancreatic cancer cells (MiaPaCa-2) were exposed ± hypoxia for 48h. mRNA expression of 84 human drug transporter genes
were analysed in PSCs and pancreatic cancer cells using a PCR Array and differentially regulated genes in PSCs were
validated by real-time PCR.
Results: Comparison between normal and cancer-associated PSCs revealed 11 differentially regulated genes with functions
relating to drug-resistance, nutrient uptake and survival. Additionally in pancreatic cancer cells exposed to hypoxia, 25 drug
transporter genes, which may regulate pro-survival and chemoresistant mechanisms, were significantly altered.
Conclusions: Drug transporters are: i) expressed in PSCs and differentially regulated in cancer-associated PSCs compared to
normal PSCs; and ii) altered in pancreatic cancer cells under hypoxic conditions.
Implication: Characterisation of expression and alterations of drug transporters in PSCs and pancreatic cancer cells has the
potential to identify new therapeutic targets that may improve the clinical outcome of pancreatic cancer patients.


Sympathetic nervous system regulation of tumor associated macrophages in metastasis
Matthew A Pimentel1 , Saul J Priceman2 , Erica K Sloan1
1. Monash Institute of Pharmaceutical Sciences, Melbourne, VIC, Australia
2. Beckman Research Institute and City of Hope Comprehensive Cancer Center, Duarte, CA, United States
Macrophage recruitment has been associated with poor prognosis in solid cancers by promoting angiogenesis and suppressing
anti-tumor immune responses. Using a mouse model of breast cancer, we have shown that chronic stress increased
recruitment of macrophages to the primary tumor. Stress increased metastasis to lymph node and lung and was dependent on
beta-adrenergic signaling to tumor-associated macrophages. To begin to understand the role of macrophages in neural
regulation of metastasis, we carried out global gene expression profiling using Illumina mouse Ref8 beadarrays on tumor-
associated macrophages to identify SNS-responsive macrophage genes. To confirm a role for adrenergic signaling we also
investigated the effect of norepinephrine on bone marrow derived macrophages. Consistent with the functional changes in pro-
inflammatory responses, proliferation and invasive properties, gene ontology analyses of upregulated genes found
overrepresentation of neural-responsive genes with functional roles in arginine metabolism (ARG1, ARG2), cell cycle regulation
(CCND1), reorganization of the actin cytoskeleton (DIAPH1, WASF1), and pro-inflammatory cytokines, chemokines, and
receptors for inflammatory mediators (IL1B, IL-15, TLR2, CD14, HIF1A VEGFA, IL22RA, CXCL14). Neural regulation of
macrophages resulted in down regulation of genes involved in cell cycle, DNA replication, and base excision repair (CCND2,
PCNA, MCM5, MCM6, UNG) and the lysosome compartment (CTSZ, HEXB, ATP6V0A1), all p < 0.05. Current studies are
elucidating the signaling pathways required for macrophages to induce primary tumor cell dissemination, and to investigate
therapeutic strategies for blocking neural regulation of macrophage-tumor interactions.


Targeting CSF-1 induced macrophage migration to inhibit tumour invasion and metastasis
Fiona J Pixley1 , Kellie A Mouchemore1 , Natalia G Sampaio1 , Richard Stanley2
1. University of Western Australia, Crawley, WA, Australia
2. Albert Einstein College of Medicine, Bronx, NY, USA
Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility as well as mononuclear phagocytic cell
proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces the pleiotropic signals of CSF-1 via
autophosphorylation of up to 8 intracellular tyrosine residues. Using a novel bone marrow-derived macrophage cell line system
to examine specific CSF-1R phosphotyrosine-activated signaling pathways, we demonstrate that macrophages expressing a
Y721F mutant CSF-1R display striking morphological alterations. Signalling downstream of pY721 regulates adhesion and actin
polymerisation to control macrophage spreading, motility and invasive capacity as well as their ability to enhance carcinoma
cell invasion. The pY721 motif is the primary activator of CSF-1 induced macrophage motility via mediation of the direct
association of PI3K with the CSF-1R. Work is now focussed on identifying specific inhibitors for this motility pathway to reduce
macrophage infiltrative capacity and the promotion of tumour invasion and metastasis.


Is β3 integrin a good therapeutic target for bone-metastatic breast tumours?
Normand Pouliot1 2 , Anthony Natoli1 , Rachel Z Carter1 , Sophie Paquet-Fifield1 , Delphine Denoyer1 , Heloisa S Selistre
de Araújo3 , Robin L Anderson1 2
1. Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia
3. Universidade Federal de São Carlos, Brazil, São Carlos, Brazil
Few studies have documented the role of β3 integrin in the spontaneous spread of breast tumours to bone due to the lack of
clinically relevant animal models. Published evidence for its role in regulating tumour growth and angiogenesis is controversial.
Moreover, many questions remain regarding the relative contribution of tumour versus stromal β3 integrin to metastasis to
bone. To address this, we measured the impact of stable downregulation of tumour β3 expression on spontaneous and
experimental metastasis using aggressive bone metastatic lines isolated in our laboratory (4T1.2 and 4T1BM2). The role of
stomal β3 integrin in metastasis was evaluated in integrin β3 wild-type and null mice.
Interestingly, suppression of tumour β3 dramatically reduced spontaneous but not experimental (intracardiac tumour injection)
metastasis to spine and femurs indicating that tumour β3 is required early during metastasis. In addition, suppression of
tumour β3 or treatment of the cells with a β3 inhibitor (DisBa-01) unexpectedly resulted in a visible increase in the incidence of
experimental metastasis to soft tissues. Thus, suppression or pharmacological inhibition of tumour β3 integrin may alter the
metastatic pattern of disseminated breast tumour cells and lead to enhanced soft tissue metastasis.
Contrary to previous studies in melanoma, the lack of stromal β3 expression did not impact significantly on 4T1.2 or 4T1BM2
spontaneous metastasis to bone ruling out a critical role for stromal β3 in this process. Moreover, we found that the loss of
stromal β3 expression did not impact on 4T1BM2 primary tumour growth or vascularisation when implanted orthotopically. In
contrast, subcutaneous tumour growth and angiogenesis was enhanced in β3-null mice indicating that regulation of tumour
growth and vascularisation by β3-expressing stromal cells is site-specific. Taken together, these observations have important
clinical implications for the design of anti-metastatic therapies targeting β3 integrin in advanced breast tumours.


The microenvironment factor S100A4 as a modulator of tumor-stroma crosstalk and
aggressive phenotype in malignant melanoma
Lina Prasmickaite1 , Ingrid Bettum1 , Kotryna Vasiliauskaite1 , Solveig Pettersen1 , Hari Prasad Dhakal1 , Lars Ahrlund-
Richter2 , Gunhild Mælandsmo1
1. Oslo University Hospital, Radium Hospital, Oslo, Norway
2. Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
The association of S100-family proteins with metastasis is well known, and recent data links soluble factors from this family to
formation of a metastatic niche. One of the family members, S100A4 is a known metastasis-promoting protein, but the
molecular mechanisms involved are not completely elucidated. S100A4 is secreted from cancer and stroma cells, and can
modulate the microenvironment through induction of matrix-modifying enzymes and effects on stroma cells. Here we
hypothesize that S100A4 can execute its pro-metastatic function via modulation of tumor-stroma cell crosstalk, which
eventually potentiates metastatic functions and provide tumor cells with survival advantage. By analyzing metastatic melanoma
lesions growing in different microenvironments in vivo, we have shown that S100A4-expressing cells localize in the invasive
front, where tumor cells interact with the stroma. This suggests that S100A4 participates in the crosstalk between malignant
and non-malignant cells in vivo. When soluble S100A4 was added to melanoma cells growing in cultures in vitro or inside lung
tissue ex vivo it triggered secretion of pro-inflammatory cytokines like IL-8. The effect was confirmed on many melanoma and
non-melanoma cancer cell lines. IL-8 is a significant regulator within tumor microenvironment, able to affect various stroma
cells and render therapeutic resistance. To investigate how S100A4-triggered melanoma cells and/or secreted cytokines may
modulate tumor-stroma cell crosstalk, we are currently analyzing effects on endothelial cells and macrophages, stroma cells
known to be involved in metastases development.


Type 1 interferons as potent suppressors of breast cancer metastasis to bone
Jai Rautela1 , Clare Slaney1 , Belinda Parker1
1. Sir Peter MacCallum Cancer Institute, East Melbourne, Vic, Australia
Breast cancer is the most commonly diagnosed non-skin cancer among Western women. Early diagnosis and treatment result
in good long-term survival, however, women that succumb usually do so due to the cancer metastasising to vital organs.
Approximately 70% of breast cancer cases metastasise to the bone, creating painful and untreatable loss of bone density
leading to fractures and paralysis.
Recent work in the laboratory has shown that breast cancer cells spread to bone by suppressing tumour cell induced type I
interferon (IFN) signalling. Restoration of this signalling in tumour cells by enforced expression of the master regulator
interferon regulatory factor 7 (Irf7) significantly reduces metastasis to bone by stimulating anti-tumour immune responses.
Our current work will explore the direct role of tumour cell IFN in regulating metastatic spread and its' potential as a therapeutic
for patients likely to develop bone metastasis. We will do this by enforced expression of two type I IFNs (IFN alpha and IFN
beta) in tumour cells, to examine the impact on bone metastasis in the 4T1.2 syngeneic model. Additionally, we will treat mice
bearing 4T1.2 tumours systemically with type I IFNs to assess the therapeutic benefit. Our preliminary experiments show that
IFN significantly reduces metastasis to bone. We will now explore the best therapeutic window for treatment by mimicking the
adjuvant and metastatic treatment setting in mouse models of metastasis.
This will allow assessment of the future utility of IFN therapies in breast cancer patients.

Laminin Gamma2 Chain Promotes Invasion of Tumor Cells into Vascular Endothelial Cell
Layer In Vitro
Hiroki Sato1 , Takashi Ogawa1 2 , Eriko Komiya1 , Jun Oyanagi1 , Shouichi Higashi1 , Kaoru Miyazaki1
1. Yokohama City University, Totsuka-ku, yokohama, kanag, Japan
2. Kagawa University, 1750-1 ikeda, Miki-cho, kida-gun, Kagawa, Japan
Laminin gamma2 chain (Lm gamma2), a subunit of the basement membrane protein laminin-332, is known to be a
representative tumor invasion marker, and its expression correlates with poor prognosis of the cancers. We recently reported
that Lm gamma2 is induced in cancer cells by some cytokines such as TNF-alpha and TGF-beta and a human bladder
carcinoma cell line over-expressing Lm gamma2 (gamma2-T-24) shows enhanced invasive growth in vivo. However, the
mechanism of the pro-invasive activity of Lm gamma2 remains unclear. To understand the role of Lm gamma2 in tumor growth,
we analyzed interaction of Lm gamma2 with vascular endothelial cells. When a purified proteolytic fragment of Lm gamma2
(gamma2pf) was added to the monolayer of human umbilical vein endothelial cells (HUVECs), those cells became markedly
retracted or shrunk. In addition, gamma2-T-24 cells or the cells expressing the gamma2 short arm (gamma2SA-T-24) more
effectively invaded into the HUVEC monolayer, extending protrusions, than control T-24 cells. Similar morphology was found
when control T-24 cells were treated with purified gamma2pf protein. To show the mechanisms for these activities of Lm
gamma2, we next examined cytoskeletal change of endothelial cells. This analysis revealed that gamma2pf induced
rearrangement of actin cytoskeleton in HUVECs, and the gamma2pf-induced formation of actin stress fiber was blocked by the
ROCK inhibitor, Y-27632. In addition, the gamma2pf protein stimulated the phosphorylation of ERK and p38MAPK but
suppressed that of Akt in HUVECs. These data suggest that the gamma2 N-terminus induces destabilization of endothelial
barrier function and promotes transendothelial migration of tumor cells by inducing cytoskeletal change of endothelial cells via
the Rho/ROCK pathway. Such activities of Lm gamma2 may enhance tumor metastasis in vivo.


The tumour-stroma microenvironment of late stage ovarian cancer in 3D
Jana Schulz1 , Daniela Loessner, Jeremy Baldwin, Helen Irving-Rodgers, Anja Rockstroh, Melanie Lehman, Colleen
Nelson, Viktor Magdolen, Dietmar W Hutmacher, Judith A Clements
1. QUT, Kelvin Grove, QLD, Australia
Ovarian cancer is the most lethal of gynaecological diseases with most women diagnosed at an advanced stage when tumour
cells have spread into the peritoneum. The tumour-associated stroma promotes cancer progression, yet the precise
interactions between cancer cells and their stromal compartments are poorly defined. Cancer-associated proteases like
kallikrein-related (KLK) peptidases initiate peritoneal invasion via proteolytic cascades, degradation of extracellular matrix
(ECM) proteins and integrin signalling implicated in cell survival and proliferation. We discovered mediators of metastasis by
employing a bioengineered 3D approach mimiking the tumour-stroma microenvironment of late stage ovarian cancer. We
established an integrated 3D co-culture model of ovarian cancer and stromal cells using imaging and proliferation analyses. In
patients, ovarian cancer cells form multicellular spheroids that accumulate in the tumour fluid and attach to the stromal
peritoneal layer. Replicating this interaction, spheroids were grown within biomimetic polyethylene glycol-based hydrogels that
comprise ECM features due to incorporation of protease cleavage sites and integrin-binding motifs and layered onto
electrospun-fabricated polycaprolactone meshes that allowed attachment of stromal cells representing the peritoneal lining. A
whole human genome microarray was conducted to identify genes differentially regulated upon 3D co-culture and KLK
expression. Genes were grouped by biological processes using Gene Ontology, and pathways mapped using Ingenuity.
Spheroid growth was enhanced and more genes were differentially expressed upon 3D co-cultures and KLK expression.
Regulation of biosynthesis and transcription were altered in cancer cells, while inflammatory and migratory responses changed
in stromal cells after 3D co-culture. The prostaglandin-endoperoxide synthase 2 network, including fibroblast growth factor 2,
fibroblast growth factor receptor 1 and vascular endothelial growth factor C, was upregulated in cancer cells upon 3D co-
culture. Using this integrated 3D approach, we unraveled pathways that may be crucial in the aetiology of ovarian cancer
metastasis and highlight the role of tumour-stromal interactions in disease progression.


Paracrine Hedgehog signaling: Roles in tumor microenvironment and metastasis
Lalita A Shevde1 , Shamik Das1 , Shikha Khullar2 , J Allan Tucker3 , Rajeev S Samant1
1. Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA
2. Department of Radiology, University of South Alabama, Mobile, Alabama, USA
3. Department of Pathology, University of South Alabama, Mobile, Alabama, USA
The Hedgehog (Hh) signaling pathway is aberrantly activated in multiple malignancies. This pathway controls cell proliferation
and differentiation during embryonic development and when mis-regulated, contributes to tumorigenesis. Our investigations
show that the Hedgehog (Hh) developmental pathway is aberrantly activated in breast cancer and melanoma. Our studies have
revealed that Hh signaling promotes tumor vascularity and metastasis by promoting expression of CYR61 (cysteine-rich
angiogenic inducer 61) and the oncogenic protein, OPN (osteopontin). Moreover, active Hh signaling facilitated the
development of osteolytic metastasis characterized by increased tumor biomass in femur and tibia in experimental animals. In
contrast, inhibiting Hh signaling in tumor cells by stable silencing of the transcription factor GLI1 reduced the intensity and
incidence of osteolysis in an intracardiac model of bone colonization concomitant with decreased tumor biomass in the bone.
As such, our results demonstrate that the Hh pathway plays an important role in the metastatic behavior of breast cancer cells
and regulates their ability to cause osteolytic metastasis. Our findings suggest that administration of pharmacological Hh
inhibitors can inhibit Hh signaling in both, breast cancer cells and osteoclasts and may reduce breast cancer-mediated bone
loss in metastatic disease. This strategy targets the tumor cells as well as the bone microenvironment and thus can reduce
tumor burden and tumor-derived bone lesions.

Immune regulation of breast cancer metastasis
Clare Y Slaney1 , Bradley N Bidwell1 , Jai Rautela1 , Robin L Anderson1 , Paul J Hertzog2 , Belinda S Parker1
1. Peter MacCallum Cancer Centre, East Melbourne, Vic, Australia
2. Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia
Breast cancer is the most common cancer diagnosed in women. The leading cause of death from breast cancer is due to the
metastatic spread. Although it has been widely recognized the immune system plays an important role in preventing the
establishment of the primary tumor, it remains a question whether the immune system regulates the metastatic process.
In this study, we discovered that tumor cell intrinsic type I interferon pathway stimulates host anti-tumor immune responses and
demonstrate the critical function of these anti-tumor immune responses in controlling breast cancer metastasis. In the
syngeneic 4T1.2 metastasis model, we utilized mice deficient in adaptive immune system and NK cell function (NOD SCID IL-2
gamma knockout mice) or lacking the Type I IFN signaling pathway (Ifnar1 knockout mice). The ablation of these immune
responses greatly reduced the metastasis-free survival rates and enhanced metastasis in mice. In addition, we identified that
NK and CD8+ T cells play a key role in the metastatic immunosurvillance. Our data support a breast cancer metastatic immune
editing process that provides the fundamental basis for further study of immune surveillance mechanisms that regulate cancer


Degradation products of hyaluronan produced in the context of tumors are novel regulators of
Jonathan P Sleeman1 2 , Anja Schmaus2 , Jochen Bauer2 , Melanie Rothley1 , David Jackson3 , Wilko Thiele1 2
1. University of Heidelberg, Mannheim, Baden, Germany
2. Institute of Toxicology and Genetics, KIT, Karlsruhe, Baden, Germany
3. University of Oxford, Oxford, Oxfordshire, United Kingdom
Tumor lymphatics attract increasing interest as a possible target for cancer therapy. During the formation of metastases,
lymphatics serve as a conduit for disseminating tumor cells. The first metastases often form in regional lymph nodes and they
are highly clinically significant for the prognostic evaluation of different types of tumors. Thus, it is important to understand how
tumors interact with the lymphatic vasculature. A molecule involved in cancer progression is the extracellular matrix component
hyaluronic acid. It exists as a high molecular weight polymer which can be degraded progressively in the tumor context by
hyaluronidases or by reactive oxygen species. The resulting small fragments of HA (sHA) are highly bioactive molecules. In
contrast to high molecular weight HA (HMW HA), for example, sHA can potently induce angiogenesis. Here we show that sHA
is also a novel regulator of lymphangiogenesis. Low concentrations of sHA increase the proliferation rate of primary lymphatic
endothelial and act additively with VEGF-C and FGF-2. In addition, sHA promotes outgrowth of lymphatic capillaries in ex-vivo
lymphangiogenesis assays. The sHA induced lymphangiogenesis is dependent on Lyve-1, a hyaluronan receptor that is widely
used as a marker of lymphatic endothelial cells. In contrast to this, we observed that high sHA concentrations inhibit
lymphangiogenesis. This anti-proliferative effect is mediated by TGF-β, an inhibitor of lymphangiogenesis. Together, these
results show that sHA regulated lymphangiogenesis is highly concentration dependent. In order to determine the
pathophysiological sHA concentrations in the context of tumors we established a novel method that allows analysis of sHA
levels in interstitial fluid derived from tumor samples. In tumor interstitial fluid from syngeneic rodent tumors and human
colorectal tumors, most of the HA content is HMW HA. However, interstitial fluid from some tumors also contains sHA
concentrations in the pro-lymphangiogenic range. These results suggest that tumor-produced sHA may contribute to
lymphangiogenesis and metastasis. To provide functional data to support this conclusion, we have begun to investigate the role
of hyaluronidases in sHA production in tumors and their significance for metastasis. Among different expressed hyaluronidases,
Hyal1 is the prime suspect for being responsible for sHA production, but functional evidence for a role in metastasis is lacking.
We found that manipulation of the expression of Hyal1 is not sufficient to influence sHA levels in tumor interstitial fluid but
nevertheless promoted metastasis formation. These results allow novel conclusions concerning the function of Hyal1 in the
context of tumors to be made.


Critical roles of Notch signaling pathway in colon cancer metastasis
Masahiro Sonoshita1 , Makoto M. Taketo1
1. Dept. Pharmacology, Kyoto Univ. Grad. Sch. Med., Kyoto, Japan
Metastasis is responsible for most cancer deaths. Recently, we have found that Notch signaling stimulates colon cancer
metastasis. In tumor cells, Notch signaling is triggered by ligands on adjoining blood vessels, and stimulates transendothelial
migration. We have also found that Aes (or Grg5) inhibits the Notch signaling pathway in colon cancer cells, resulting in
metastasis suppression. Genetic depletion of Aes in ApcΔ716 intestinal polyposis mice has caused marked tumor invasion and
intravasation. Consistently, the malignant phenotypes have been suppressed by treatment with a gamma-secretase inhibitor or
additional knockout of Rbpj. These results suggest that inhibition of Notch signaling can be a promising strategy for prevention
and treatment of colon cancer metastasis. As a model for colon cancer invasion and intravasation, compound mutant mice for
the Apc and Aes genes should be useful to evaluate upcoming therapeutics including Notch signaling inhibitors against colon
cancer metastasis.

TGFBR3 blocks human ovarian cancer spheroid adhesion and invasion in a three-dimensional
culture model of intraperitoneal metastasis
Maree Bilandzic1 , Yao Wang1 , Jock K. Findlay1 , Kaye L. Stenvers1
1. Prince Henry's Institute, Clayton, VIC, Australia
Ovarian cancer metastasis progresses through a series of stages, each of which occurs in a distinct microenvironment.
Notably, metastasising ovarian cancer cells, shed from the primary ovarian tumour, form multicellular structures (spheroids)
within the peritoneal fluid, which contribute to tumour dissemination.We have previously shown that the type III TGFβ receptor
(TGFBR3, or betaglycan) is lost from the surface of the majority of metastatic granulosa cell tumours (GCTs) and blocks
migration and invasion of GCT cell lines in monolayer cultures (Bilandzic et al., 2009, Mol Endocrinol 23:539-548). In the
current work, we utilised the KGN GCT cell line and three-dimensional non-adherent spheroid culture to test the hypothesis that
TGFBR3 blocks GCT metastasis at the spheroid stage. This approach demonstrated that TGFBR3-expressing spheroids form
more slowly than control spheroids when grown suspended in methylcellulose (2 d vs 1 d). Furthermore, using the
xCELLigence Real-Time Cell Analyzer, we show that, compared to control spheroids, TGFBR3-expressing spheroids exhibit
decreased attachment to fibronectin and collagen IV, two matrix components of the peritoneum. Furthermore, while control
spheroids rapidly disaggregated overnight following replating onto plastic or glass, TGFBR3-expressing spheroids exhibited no
disaggregation during the same time period. Finally, TGFBR3-expressing spheroids were 2-fold less invasive through Matrigel
or a monolayer of human stromal cells (p<0.05). Western blot analysis of total cell lysates revealed that MMP2, MMP15,
MMP16, and N-cadherin, markers of motile, invasive ovarian cancer cells, were significantly downregulated in the TGFBR3-
expressing cells compared to control cells. Collectively, our data indicate that loss of TGFBR3 during ovarian cancer
progression contributes to intraperitoneal metastasis by enhancing spheroid formation and invasion of the peritoneum.
Conversely, retention of TGFBR3 by GCT cells may hamper tumour dissemination by an MMP-dependent mechanism.
Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds.


Metastatic tumours promote monocyte mobilisation
Agnieszka Swierczak1 , Andrew D Cook1 , Christina M Restall2 , Robin L Anderson2 , John A Hamilton1
1. University of Melbourne, Parkville, VIC, Australia
2. Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
Metastasis is the main cause of morbidity and mortality in breast cancer patients. Treatment options remain limited for women
presenting with advanced disease in part because of our incomplete understanding of the cellular processes involved in
metastasis. The association between inflammation and cancer initiation is well recognised, but the link between inflammation
and metastasis is not yet clear. Macrophages are the main immune cell type found in breast tumours. Tumour associated
macrophages (TAMs) have been implicated in a number of key steps required for metastasis. The aim of this project is to
determine if systemic blood monocyte and/or primary tumour macrophage numbers are associated with tumour growth and
metastatic capacity. We utilised three isogenic murine mammary tumour variants, 4T1.2 (highly metastatic), 66cl4 (weakly
metastatic) and 67NR (non-metastatic). These cells were inoculated into the mammary glands of Balb/C mice and primary
tumour growth monitored. Mice were culled at various times during primary tumour growth and monocyte/macrophage
subpopulations in the peripheral blood and primary tumours were assessed by flow cytometry. Ly6C was used as a marker for
monocyte subpopulations. We found that Ly6Clo macrophage numbers were increased in metastatic primary tumours vs. non-
metastatic even though no difference was seen in tumour volume or weight. Systemic blood Ly6C hi (immature) monocyte
numbers were also associated with metastatic capacity, but again not with tumour volume or weight. Depletion of Ly6C lo
(mature) monocytes by clodronate liposomes reduced metastasis to lung. A model is proposed in which metastatic tumours
promote mobilisation of cells from the bone marrow resulting in infiltration of Ly6C hi monocytes into the primary tumour, where
they mature into Ly6Clo cells and assist in the process of tumour cell dissemination to distant tissues such as the lung.


Aloe vera and honey solution and ethanolic fraction decrease metastatic process in Walker
256 tumour-bearing rats.
Rebeka Tomasin1 , Maria CC Gomes-Marcondes1
1. State University of Campinas, Campinas, SP, Brazil
Metastasis is responsible for up than 90% of all cancer deaths (WHO, 2008). The cancer cells ability to detach from the primary
tumour depends on several cell features, such as matrix degradation capacity and motility and also tumour microenvironment
features, including vascularization (OPPENHEIMER, 2006; FURUYA et al., 2009). Researches have shown that both Aloe vera
and honey have anticancer properties, including metastasis inhibition (AKEV et al., 2007; HE et al., 2008; SWELLAM et al.,
2003). Since our previous results have shown that treatment with Aloe vera and honey solution (AH) or ethanolic fraction (F)
can increase the survival time in tumour-bearing rats, the present study aims to evaluate whether these treatments could
modulate the pro-invasive features of primary tumours in rats. Wistar rats received tumour implant, subcutaneously (2x10e6
tumour viable cells/inoculation) and AH or F intragastric administration; following three groups: W=tumour-bearing untreated,
WAH=tumour-bearing treated with AH daily, WF=tumour-bearing treated with F daily. After 21 days, the rats were sacrificed.
The analysis showed that tumour size (relative weight, %) decreased in WAH tumours compared to W, while WF presented
intermediary values (W=18.01±1.49, WAH=10.46±2.06, F=13.96±1.81). Although the presence of invadopodia (% of cells
coexpressing MT1-MMP and Integrinβ1, accessed by immunofluorescence) had shown no difference between the groups
(W=23.75±6.43, WAH=25.43±6.79, F=17.01±4.09), the number of blood vessels and also their diameter significantly decreased
in WAH and WF compared to W (number of vessels in 12μm 2: W=68.67±16.85, WAH=10.67±1.79, F=23.44±5.83; vessel
diameter (μm): W=29.50±2.04, WAH=12.70±0.09, F=21.35±2.47). The metalloproteinase-2 activity, accessed by gelatin
zymography, showed strong activity in W group, but lacked in both treated WAH and WF tumours. These data suggest that AH
or F treatment could reduce the metastatic process, especially modulating the neoangiogenesis process. Studies are now
undergoing to elucidate additional mechanisms and also the role of AH and F on metastasis establishment.

1.        Akev N, Turkay G, Can A, Gurel A, Yildiz F, Yardibi H, Ekiz EE, Uzun H. Effect of Aloe vera leaf pulp extract on
     Ehrlich ascites tumours in mice.Eur J Cancer Prev. Apr;16(2):151-7, 2007.
2.        He TP, Yan WH, Mo LE, Liang NC. Inhibitory effect of aloe-emodin on metastasis potential in HO-8910PM cell line. J
     Asian Nat Prod Res. May-Jun;10(5-6):383-90, 2008.
3.        Furuya M, Yonemitsu Y, Aoki I. III. Angiogenesis: complexity of tumor vasculature and microenvironment. Curr
     Pharm Des. 15(16):1854-67, 2009.
4.        Oppenheimer SB. Cellular basis of cancer metastasis: A review of fundamentals and new advances. Acta
     Histochem. 108(5):327-34, 2006.
5.        Swellam T, Miyanaga N, Onozawa M, Hattori K, Kawai K, Shimazui T, Akaza H.Antineoplastic activity of honey in an
     experimental bladder cancer implantati
6.        World Health Organization (WHO)., accessed on March 22nd, 2012.


The antimicrobial peptide LL-37 inhibits migration of the prostate cancer PC-3 and DU145 cell
Yan Tu1 , Cameron Johnstone2 , Alastair G Stewart1
1. University of Melbourne, Parkville, VIC, Australia
2. Peter MacCallum Cancer Centre, East Melbourne , VIC, Australia
Introduction, Prostate cancer is the most commonly diagnosed cancer in Australia. The progression of the disease is usually
protracted and most men diagnosed ultimately die of other causes. However, when metastases occur, metastatic progression
is rapid and often resistant to therapeutic intervention. The human cathelicidin-derived antimicrobial peptide (hCAP18), LL-37
exhibits potent immune-regulatory activities independently of its anti-microbial actions and is found in prostatic fluid in high
concentrations. hCAP18/LL-37 is implicated in lung, breast, and ovarian cancer cell migration.
Aims, To characterise the impact of LL-37 on prostate cancer cell migration.
Methods, The effect of LL-37 on the prostate cancer PC-3 and DU145 cell migration was assessed using an in vitro scrape
injury wound closure assay, and a modified Boyden chamber assay. The receptor dependence of the LL-37 effects on the
wound closure assay was probed using agonists and antagonists of the formyl peptide receptor (FPR2), purinoceptors and the
epidermal growth factor receptor (EGFR) kinase. The impact of LL-37 on proliferation was investigated to ascertain whether the
impact of the LL-37 on wound closure was secondary to proliferation.
Results, LL-37 (0.1nM to 1000nM) alone had no influence on the migration of PC-3 and DU145 cells. However, LL-37 (0.1nM
to 10μM) inhibited serum-induced wound closure and chemotaxis. LL-37 had no impact on PC-3 proliferation, indicating that
proliferation is not a contributing factor to the LL-37 effect on wound closure. A high concentration of LL-37 (10μM) exerted
cytotoxic effect on DU145 cells, but not PC-3 cells. The effect of LL-37 on wound closure was mimicked by purinoceptor
agonist, ATP and FPR2 peptide ANXA2-26, but not by EGF. However, attenuation of serum-induced wound closure by LL-37
was insensitive to pre-treatment by the FPR2 receptor antagonist BOC2 (100nM). Moreover, PCR analysis showed only very
low levels of FPR2 receptor expression in PC-3 cells, further reducing the likelihood of involvement of FPR2 in the actions of
Discussion, Our findings are consistent with the possibility that LL-37 reduces PC-3 migration by actions at a purinoceptor.
Impairment of prostate cancer cell migration in vivo may engender an anti-metastatic effect of LL-37.


Anti-tumor effect of VEGF-targeted therapy on Synovial sarcoma
Toru Wakamatsu1 2 , Norifumi Naka1 , Hideki Yoshikawa1 , Kazuyuki Itoh2
1. Osaka University Graduate School of Medicine, Department of Orthopaedics, Osaka, Japan
2. Biology Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan
Synovial sarcoma (SS) has high metastatic potential and poor prognosis, due to their chemo- and radio-resistance, thus a
novel therapy is needed. In order to examine the anti-tumor effect of VEGF targeted therapy against SS, we herein used two
human SS cell lines established in our laboratory. These SS cells produced markedly higher level of vascular endothelial
growth factor (VEGF) in 3 dimensional (3D) compare to adhesion (2D) cultures. Bevacizumab (Bev), a humanized monoclonal
antibody against VEGF, has been approved to clinically improve the prognosis with several malignancies by inhibiting
angiogenesis and proliferation of primary and metastatic sites. On the contrary, several studies reported that inhibition of VEGF
signal increased tumor invasion and lung metastasis, depending on HIF1α. In soft agar assay, treatment with Bev inhibited the
colony formation of these two SS cells, and combination treatment of Bev with Ifosfamide showed higher efficiency. However,
proliferation of the two SS cell lines on 2D cultures couldn’t be inhibited by the treatment with Bev. In xenograft models, the
combination therapy also effectively reduced the tumor volume without any adverse effect. We are currently focusing on how
Bev inhibited tumor growth in 3D but not on 2D. Collectively VEGF-targeted therapy can be proposed as a novel therapy for SS
by combination with current chemotherapy.


Targeting L-type amino acid transporter 1 (LAT1) to suppress metastasis in prostate cancer
Qian Wang1 2 , Jessamy Tiffen1 3 , Chuck Bailey1 3 , John Rasko1 3 , Jeff Holst1 2 3
1. Centenary Institute, Newtown, NSW, Australia
2. Origins of Cancer Laboratory, Sydney, NSW, Australia
3. Sydney Medical School, University of Sydney, Sydney, NSW, Australia
Prostate cancer is the most common malignancy in men, and the second leading cause of male cancer-related deaths in the
Western world. Approximately one in 30 men currently die from metastatic prostate cancer. The discovery of novel treatments
for metastasis is a priority. L-type amino acid transporter (LAT1; SLC7A5) transports large neutral amino acids such as
leucine, in exchange for more abundant amino acids such as glutamine. We have shown that LAT1 is highly expressed after
hormone ablation therapy and in metastatic prostate cancer, in order to maintain intracellular leucine levels, enabling protein
synthesis and cell growth through the mTORC1 pathway. Recent studies have shown that mTOR regulates mRNA translation
of genes related to cell invasion and metastasis. Therefore, LAT1 may be a novel target for therapeutic intervention in
metastatic prostate cancer. LAT1 is expressed on the plasma membrane in a complex with CD98, which in turn binds 1-
integrin. This complex is present at the leading edge of PC-3 cells in 2D culture. PC-3 cells adopt either an elongated or
cuboidal shape in 2D culture, however LAT1 shRNA knockdown, resulted in the majority of PC-3 cells assuming a more
cuboidal shape. LAT1 overexpressing cells, in contrast, maintain a more elongated shape. This result suggests that LAT1 is
important in cell spreading and/or attachment, and may therefore be important in metastasis. In order to determine whether
LAT1 plays a role in metastasis in vivo, we stably transduced PC-3-luc cells with a LAT1 shRNA, and used these cells in a sub-
cutaneous xenograft tumour model. Knockdown of LAT1 decreased tumour growth (sub-cutaneous) as well as the numbers of
metastases in the liver and lymph nodes. Our study suggests that targeting LAT1 may provide a novel therapy for metastatic
prostate cancer.


Phospholipase A2 activities in cancers
Yan Xu1 , Qingchun Cai1 , Zhenwen Zhao1 , Caryl Antalis1 , Libo Yan1 , Giuseppe Del Priore1 , Ali H Hamed1 , Frederick
B Stehman1 , Jeanne M Schilder1 , Hui Cai1
1. Indiana University School of Medicine, Indianapolis, IN, United States
Ascites production is characteristic of late stage human epithelial ovarian cancer (EOC) and it correlates with tumor spread
clinically. Ascites contributes to the microenvironment of EOC cells and promotes tumor development by mechanisms that are
incompletely understood. Lysophosphatidic acid (LPA), a major tumor-promoting factor in EOC ascites, is an enzymatic product
of autotaxin (ATX) and phospholipase A2 (PLA2) enzymes. The contribution of PLA2 activities to ovarian tumorigenesis was
investigated. The quantitative measurement of PLA2 activities in ascites and tissues as well as assay conditions selective for
PLA2 subtypes were optimized and validated. PLA2 activities correlated with tumor-promoting activates in cell-based and in
vivo assays. High activities consistent with both cytosolic and calcium-independent PLA2 were found in human EOC ascites for
the first time. Elevated PLA2 and ATX activities were also observed in EOC compared to benign tumors and normal tissues.
Cell-free and vesicle-free (S4) human EOC ascites potently promoted proliferation, migration, and invasion of human EOC cells
in a PLA2-dependent manner. LPA mediated a significant part of the cell-stimulating effects of ascites. S4 ascites stimulated
tumorigenesis/metastasis in vivo, and methyl arachidonyl fluorophosphonate was highly effective in inhibiting EOC metastasis
in mouse xenograft models. PLA2 activity was found in conditioned media from both EOC cells and macrophages. In addition,
elevated PLA2 activities were detected in blood samples of several cancers, including ovarian, lung, and pancreatic cancers.
Collectively, our work implies that PLA2 activity is a potential marker and therapeutic target in EOC, and possibly other cancers.


Hepatocyte Growth Factor: A potential therapeutic target in pancreatic cancer
Zhihong Xu1 , Arun S Jayappa1 , Eva Fiala-Beer1 , Sam Abraham1 , Vadim Dedov1 , David Goldstein2 , Andrew V
Biankin3 , Romano C Pirola1 , Jeremy S Wilson1 , Minoti V Apte1
1. Pancreatic Research Group, The University of New South Wales, Sydney, NSW, Australia
2. Prince of Wales Hospital, Sydney, NSW, Australia
3. Garvan Institute of Medical Research, Sydney, NSW, Australia

Pancreatic stellate cells (PSCs, which produce the desmoplastic reaction of pancreatic cancer) interact with pancreatic cancer
(PC) cells to potentiate PC progression. A candidate factor that may mediate this interaction is the hepatocyte growth factor
(HGF). High serum HGF levels correlate with poor clinical outcome, yet little is known about the role of HGF in PC.
Aims: To i) determine whether human PSCs (hPSCs) and PC cell lines express HGF; ii) assess the effects of HGF inhibition on
PC progression in vitro; and iii) in vivo.
Methods: i) HGF expression in PSCs and PC cells was assessed by RT-PCR, immunoblotting/immunocytochemistry; ii)
Proliferation of PC cells incubated in PSC secretions pre-treated with or without anti-HGF monoclonal antibody rilotumumab
[AMG 102, Amgen Inc.] was assessed; iii) Orthotopic model: AsPC-1 (human PC cell line) + hPSCs were implanted into mouse
pancreas, and mice were treated with AMG 102 or IgG. Seven weeks later, tumour and metastasis were assessed.
Results: i) At mRNA and protein levels, hPSCs express HGF, while PC cells exhibit negligible expression. On the other hand,
the receptor for HGF (c-Met) is expressed by cancer cells but not by hPSCs. ii) PSC-induced cancer cell proliferation
(29.3±5.1% over basal level) was significantly inhibited by HGF antibody to 12.1±3.9% over basal level (p<0.03;n=3). iii)
Orthotopic model: a) 300 and 600μg AMG 102 treatments significantly inhibited tumour growth compared to IgG treatment
(1312.63±175.27mm3 vs 532.47±91.83* and 382.85±42.70* respectively, *p<0.001;n=8/group); b) The number of proliferating
cancer cells in AMG 102-treated tumours was significantly reduced to 79.3±6.4% and 66.5±1.1% of that in IgG-treated tumours
(p<0.005;n=4); c) 600μg AMG 102 significantly inhibited metastasis (liver, diaphragm, mediastinum, p<0.05) in mice compared
to IgG treatment.
Conclusions: We have shown for the first time that i) human PSCs synthesise HGF, but do not express its receptor; ii) HGF
inhibition reduces cancer cells proliferation in vitro, the growth and metastasis of cancer in vivo. Implication: Targeting the
stromal reaction with relevant specific inhibitors may represent a novel therapeutic approach PC.


Osteopontin promotes lung cancer invasion and metastasis by improve the invadopodia
Qinghua Zhou, Meng Luo, Jiacong You, Bing Liu, Yang Li
Invadopodia are detected in various types of human cancer and Src-transformed fibroblasts. It is unclear, however, whether the
invadopodia involved in lung cancer invasion and metastasis. To find out the presence of invadopodia in lung cancer cells and
the regulation mechanisms of invadopodia, we carried out immunofluorescence and FITC-gelatin degradation assay and
transplanted lung cancer mouse model to demonstrate the presence of invadopodia in human lung cancer cell lines, L9981,
NL9980 and A549. In addition, we detected the expression of osteopontin, cortactin, Arp2, and MMP9 in NL9980 and NL9980-
OPN. Our investigations indeed demonstrated that invadopodia are present in lung cancer cells A549 and L9981. Invadopodia
enable increased invasion and metastasis of A549 and L9981 cells . Moreover, A549 and NL9980 cells forced overexpressing
osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity accompany with elevated
expression of cortactin, Arp2, in addition, OPN can remarkably up-regulated the expression of cortactin, Arp2 and MMP9 of
A549 cells in a time-dependent manner. Furthermore, we found that forced overexpressing of OPN activate the Src, ERK1/2
phosphorylation. Thus, we conclude that invadopodia participates in the process of invasion and metastasis of human lung
cancer, and OPN can improve the invadopodia formation by activate the Src, ERK1/2 pathway.


Local release of Osteoprotegerin by breast cancer cells inhibits cancer-induced osteolysis and
intra-osseous tumour burden but promotes pulmonary metastasis.
Irene Zinonos1 , Agatha Labrinidis1 , Vasilios Liapis1 , Shelley Hay1 , Vladimir Ponomarev2 , Gerald J Atkins3 , Peter
Diamond4 , David M Findlay3 , Andrew CW Zannettino4 , Andreas Evdokiou1
1. Discipline of Surgery, Breast Cancer Research Unit, Basil Hetzel Institute and Centre for Personalised Cancer Medicine,
     University of Adelaide, Adelaide, South Australia, Australia
2. Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, USA
3. Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia
4. Myeloma and Mesenchymal Research Laboratory, Bone and Cancer Laboratories, Division of Haematology, Hanson
     Institute, Adelaide, South Australia, Australia
Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily which binds to receptor activator of nuclear
factor kB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was
previously shown to inhibit tumour growth in bone and prevent cancer-induced osteolysis. In this study we examined the effect
of OPG when produced locally by breast cancer cells in bone using a mouse model of osteolytic breast cancer. MDA-MB-231-
TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human
OPG were transplanted directly into the tibial marrow cavity of nude mice. Tumour progression was monitored in live animals
using bioluminescence imaging, whereas the development of breast cancer-induced osteolysis was measured using high
resolution micro-computer tomography and histology. Animals transplanted with empty vector-transfected cells developed large
osteolytic lesions involving the cortices and led to tumour growth extending into the surrounding soft tissues. In contrast, over-
expression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis, diminished intra-osseous
tumour growth but was without effect on extra-skeletal tumour growth. This effect was associated with a significant reduction in
the number of osteoclast lining the bone surface. This demonstrates that tumour production of OPG exerts indirect effects on
tumour growth by inhibiting osteoclast-mediated bone loss, which in turn restricts tumour cell growth in the diaphysis. Despite
the bone protection, OPG over-expression resulted in a significant increase in the incidence of pulmonary metastasis. Our
results demonstrate for the first time that OPG secreted by tumours in bone may affect the behaviour of cancer cells within the
bone microenvironment and their likelihood of spreading and establishing metastases elsewhere in the body.


The Effect of StarD13 on Colorectal Cancer Proliferation and Invasion
Mirvat El-Sibai1 , Anita Nasrallah1
1. Lebanese American University, Beirut, Lebanon
Colon cancer, also considered as colorectal cancer or bowel cancer, is the cancer of the epithelial cells lining the colon. There
are many risk factors and many prophylactic measurements against this cancer. 95% of colon cancer is known as
adenocarcinoma, mainly divided into different stages according to invasiveness and metastatic ability of the tumor. Many
mutations are acquired, leading to this malignancy. Mainly, these occur in entities that greatly alter and affect the cell cycle, cell
signaling pathways, cell movement, which all involve the action of Rho GTPases. The protein of our interest is DLC2, also
known as StarD13 or START-GAP2, a GAP for Rho and Cdc42. Literature states that this protein is considered a tumor-
suppressor in hepatocellular carcinoma. Previous work in our lab proved StarD13 to be a tumor suppressor in brain tumor and
in breast cancer. In this work, we studied the role of StarD13 in colon cancer.
When overexpressed, StarD13 led to a decrease in cell proliferation in colon cancer cells. This was measured by WST MTT
kits. Knocking down StarD13 using StarD13 siRNA led to an increase in cell proliferation. This showed that, similarly to its role
in astrocytoma and breast cancer, StarD13 seems to be a tumor suppressor in colon cancer as well. We were also interested in
examining the role of StarD13 in cell motility. StarD13 knock down resulted in an inhibition of cell motility, as seen by time lapse
microscopy. This might be due to the inhibition of Rho, thus Rac-dependant focal complexes are not formed nor detached for
the cells to move forward. future work is still needed to determine the exact mechanism of inhibition.


Novel targets for kallikrein-related peptidase 4 in the prostate cancer microenvironment
establish its role in tumour progression
Ruth A Fuhrman-Luck1 2 , Scott H Stansfield1 2 , Carson R Stephens1 2 , Daniela Loessner1 , Judith A Clements1 2
1. Institute for Health and Biomedical Innovation, Queensland University, KELVIN GROVE, QLD, Australia
2. Australian Prostate Cancer Research Centre, Brisbane, QLD, Australia

Prostate cancer is a leading cause of male cancer-related deaths, worldwide. Kallikrein-related peptidase 4 (KLK4) is a serine
protease which is over-expressed in prostate cancer, versus benign disease, and further increased in fatal bone metastatic
prostate cancer. In vitro, KLK4 induces prostate cancer cell proliferation, migration and invasion, as well as promoting an
epithelial-to-mesenchymal transition. However, KLK4 substrates in primary and metastatic prostate cancer are not well
understood and must be identified to understand the mechanism of KLK4 action in prostate cancer.

To elucidate KLK4 targets on a proteome-wide scale, we employed a novel proteomic approach, called the PROtein
Topography and Migration Analysis Platform (PROTOMAP1). Proteins secreted by PC-3 cells, derived from a patient with bone
metastatic prostate cancer, were harvested, concentrated and treated with active, recombinant KLK4, or an inactive mutant
KLK4 control. Protein digests were separated by SDS-PAGE and gel lanes sliced into 32 horizontal sections, prior to
identification of proteins in each section by LC-MS/MS. Those proteins detected in gel sections of lower molecular weight in
KLK4-treated samples, versus controls, were deemed KLK4 substrates.

In total, 452 PC-3 cell-secreted proteins were identified, of which 20 were potential novel KLK4 substrates. These included
proteases, protease inhibitors, extracellular matrix proteins, cell adhesion molecules and cytokines. Of particular interest was
matrix metalloproteinase-1 (MMP-1), as KLK4 was shown to activate MMP-1, a protein involved in prostate cancer growth and
metastasis in vivo2. In addition, the anti-angiogenic thrombospondin-1 (TSP-1) was cleaved by KLK4, potentially as a
mechanism to increase blood vessel formation, favouring tumour progression.

We are currently using 2- and 3-dimensional cell culture models of prostate cancer to delineate the biological consequences of
KLK4 proteolysis of these and other novel targets identified in this study, with the aim of identifying pathways amenable to
therapeutic intervention.

1.        Dix MM, Simon GM, Cravatt BF. Cell. 2008; 134: 679-91.
2.        Pulukuri, SMK, Rao, JS. Int J Oncol. 2008; 32(4): 757-765.


The Role Of Megakaryocytes In Breast Cancer Metastasis To Bone
Andrea M Mastro1 , Walter Jackson III1
1. Penn State University, University Park, PA, United States
The mechanisms responsible for the metastasis of breast cancer to bone remain ambiguous. While analyzing femur sections
from mice that had been inoculated with metastatic MDA-MB-231 breast cancer cells, we saw an increased number of
megakaryocytes (MKs) in the bone marrow. We hypothesized that either MKs aid in the growth of cancer cells by preparing a
niche for the metastases or that the increase in MKs resulted from the altered microenvironment of the marrow due to the
presence of cancer. MKs are the pre-cursors to platelets. Thrombocytosis, high platelet count, is a poor prognostic factor for
metastasis; many cancer patients die from thromboembolisms. MKs also play a crucial role in bone metabolism and skeletal
Objectives: to determine (1) if the increase in MKs precedes the growth of cancer cells in the bone; (2) the role that the cancer
cell-osteoblast/stromal interactions play in the increase in megakaryopoiesis. For aim 1, we compared MK numbers in the
femurs of athymic mice inoculated with MDA-MB-231-luc intracardially (metastasis) with those inoculated in the mammary
gland (no metastasis). Serum was assayed for key MK maturation factors thrombopoietin (TPO) and stromal-derived factor-1
(SDF-1). We found an increase in MKs in femurs of mice with metastatic disease but not with localized growth. The increase
mirrored the increase in metastatic tumor burden. There was no increase in platelet count, SDF-1, or TPO even when MK
numbers were high. We concluded that the cancer cells precede the increase of MKs in the bone marrow in a xenograft model.
Next we will use a syngeneic model of Balb/c with 4T1.2 cancer cells that metastasize from the mammary gland. Finally, Balb/c
mice lacking TPO and thus having low MKs will be tested for metastasis. We will also add TPO to increase MKs and determine
how metastasis is affected. (Support: DOD W81XWH-10-0253)

miR-193b regulates breast cancer proliferation and metastasis in vivo
Alexandra Rizzitelli1 , Cameron N. Johnstone 1 2 3 , Lauren L. Burton 1 , Erin Lucas 1 , Francesca M. Buffa 4 , Adrian L.
Harris 4 , Ioannis Ragoussis 4 , Philip A. Gregory5 , Yeesim Khew Goodall5 , Gregory J. Goodall 5 , Robin L. Anderson 1 2

1. Cancer Metastasis Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
2. Department of Pathology, University of Melbourne, Parkville, VIC, Australia
3. Department of Pharmacology, University of Melbourne, Parkville, Vic, Australia
4. The Weatherall Institute of Molecular Medicine, Department of Oncology,, University of Oxford, Oxford, UK
5. Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia
6. Sir Peter MacCallum Department of oncology, University of Melbourne, Melbourne, VIC, Australia
Small non-coding RNA molecules called microRNAs (miRs) have been identified as key regulators of cancer progression. In
our laboratory, we used microRNA arrays to identify microRNAs that were associated with metastatic ability in the 4T1
syngeneic mouse model of breast cancer1 . mCherry expressing cells were grown as primary tumours, sorted by flow cytometry
and subjected to microRNA array profiling using an Affymetrix platform.
Known oncogenic (eg. miR-155) and tumour suppressor (eg. miR-143~miR-145 cluster) were deregulated in metastatic 4T1.2
and 4T1.13 cells as expected. Of the less well studied microRNAs, we found that miR-193b was up-regulated in the epithelial
cells of 4T1.2 and 4T1.13 tumours compared to weakly- and non-metastatic tumours. This was confirmed by quantitative stem-
loop PCR in mCherry sorted tumour cells and in cell lines cultured in vitro.
Lentiviral-mediated knockdown of miR-193b activity in metastatic 4T1.13 cells reduced in vitro cell proliferation and migration.
4T1.13_miR-193b knockdown (KD) implanted orthotopically into Balb/c mice displayed reduced primary tumour growth rate
with an associated reduction in distant metastasis. Reduced lung metastasis was also observed when 4T1.13_miR-193b KD
cells were used in experimental metastasis (tail-vein) assays.
Transient over-expression of miR-193b in weakly metastatic 168FARN and 66cl4 cells dramatically reduced mRNA levels of
putative target genes including cyclin-D1, DDB1 and CUL4-associated factor-7 (Dcaf7), urokinase plasminogen activator (uPa),
and cell adhesion molecule-1 (Cadm1) / tumour suppressor in lung cancer-1 (Tslc1).
Finally, the analysis of a large human breast cancer dataset with over 10 years of clinical follow-up showed miR-193b
expression was correlated with estrogen receptor positivity (ER+) and associated with reduced survival in both ER+ and ER-
tumours (univariate analysis)2 .
  Taken together, our data suggest that over-expression of miR-193b regulates both primary tumour proliferation and metastatic
dissemination in vivo.

1.        1. Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, Tavaria MD, Stanley KL, Sloan EK, Moseley JM, and
     Anderson RL. Mol. Cancer Res. (2005) 3(1):1-13.
2.        2. Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, Taylor M, Harris AL, and Ragoussis J. Cancer
     Res. (2011) 71(17):5635-5645


Identification and validation of two novel metastatic markers in Osteosarcoma
Eleni Topkas1 , Na Cai1 , Andrew Cumming1 , Nicholas A Saunders1 , Liliana B Endo-Munoz1
1. The University of Queensland, Diamantina Institute, Woolloongabba, QLD, Australia
Osteosarcoma (OS) accounts for 56% of malignant bone cancers and 6% of all cancer cases in children and adolescents 1.
Pulmonary metastasis occurs in approximately 50% of patients with a 5 year survival rate of only 20%, while non-metastatic OS
has an expected 5 year survival rate of 70%. In order to improve survival in the metastatic cohort of patients it is crucial to
identify genes and pathways that drive the metastatic behaviour of OS for the identification of therapeutic targets.
To identify markers that may define inherent metastatic OS we conducted microarray-based comparative profiling analysis of
clonal variants from an inherently metastatic cell line, KHOS. Two highly metastatic (C1 and C6) and two poorly metastatic
clones         (C4       and         C5)       were         compared           in         the        transcriptomic    screen.

Vascular endothelial growth factor A (VEGFA) and two other novel genes were identified as potential markers for OS
metastasis with 2-4 fold increased expression in highly metastatic clonal variants when compared to poorly metastatic clonal
variants. The transcriptomic expression of VEGFA and the two new markers was also investigated in non-malignant bone (NB),
OS patients with non-metastatic (NM) and metastatic (M) disease. All three markers were found to be highly expressed in 29-
42% of M-OS with little to no expression seen in NB and NM-OS. VEGFA has also been shown to be a promising target in the
treatment of OS development and metastasis2. This further supports the identification of the two novel OS metastasis markers.

VEGFA and two novel markers were found to be up-regulated in highly metastatic KHOS clonal variants and their identification
was further supported by expression levels in metastatic OS patient biopsies. These markers are promising targets which will
require further validation as possible drivers of OS metastasis and therapeutic targets.

1.       Ottaviani, G. and N. Jaffe, The epidemiology of osteosarcoma. Cancer treatment and research, 2009. 152: p. 3-13.
2.       Quan, G.M. and P.F. Choong, Anti-angiogenic therapy for osteosarcoma. Cancer metastasis reviews, 2006. 25(4): p.

Monitoring early efficacy of PI3K/mTOR targeted cancer therapies using F-FPHCys, a new
potential imaging biomarker
Delphine Denoyer1 , Peter Roselt1 , Thomas Bourdier2 , Andrew Katsifis2 , Rodney J Hicks1
1. Molecular Imaging and Targeted Therapeutics Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC,
2. Department PET and nuclear medicine, Royal Prince Alfred Hospital, Sydney, NSW, Australia
Background: A major challenge facing clinicians who treat cancer patients with new molecular targeted drugs, such as
PI3K/mTOR inhibitors is to assess early response to treatment. Positron Emission Tomography (PET) has emerged as an
excellent imaging modality to assess early drug efficacy to such agents. However, there are currently no reliable imaging
biomarkers available to monitor PI3K/mTOR targeting treatment by PET. Our team developed a new 18F-labelled methionine
derivative, 18F-FPHCys with excellent potential for imaging solid tumours and uptake in cancer cells mediated primarily by the
LAT1 system-L amino acid transporter. Given the well-characterised role of PI3K/mTOR in the regulation of protein synthesis,
we hypothesised that inhibition of PI3K/mTOR may impact on amino acid uptake by modulating LAT1 activity and/or
expression. If so, PI3K/mTOR inhibitors would be expected to decrease 18F-FPHCys accumulation in cancer cells.
Aim: The aim of this study is to investigate the potential of 18F-FPHCys as a non-invasive biomarker to demonstrate the anti-
tumour activity of PI3K/mTOR inhibitors.
Material and Methods: 18F-FPHCys uptake was assessed in various human cancer cell lines following 24-, 48- or 72-hours
treatment with an mTORC1 inhibitor, a dual mTORC1/mTORC2 inhibitor or a dual PI3K/mTOR inhibitor. LAT1 expression was
measured by immunoblot.
Results: The preliminary results obtained with PC3 cells show a marked inhibition of 18F-FPHCys uptake as early as 24 hours
following treatment with either of the inhibitors, which is even more pronounced after 72 hours. Decreased 18F-FPHCys
accumulation is associated with decreased LAT1 expression but not with cell proliferation status. None of the inhibitors tested
impacted on cell viability. The results with other cell lines will be further discussed at the meeting.
Conclusion: These results suggest the importance of mTOR for the regulation of amino acid transport and that 18F-FPHCys
has the potential to monitor the early effects of PI3K/mTOR inhibition on tumour metabolism.


ITGBL1 is a Transcriptional Target of RUNX2 and Promotes Bone Metastasis of Breast Cancer
Yu-Mei Feng1 , Xiao-Qing Li1 , Peng-Zhou Kong1 , Xin Du1
1. Tianjin Medical University Cancer Institute and Hospotal, Tianjin, China
To clarify the role of ITGBL1 in breast cancer metastasis and especially in organ-specific bone metastasis, we investigated the
relationship of ITGBL1 mRNA expression levels in primary breast cancers with disease-free survival and bone metastasis of
breast cancer patients. The results showed that low level of ITGBL1 mRNA correlated with tumor progression and metastasis
and indicated shorter disease-free survival of patients, whereas high level of ITGBL1 mRNA increased the risk of excluded
bone metastasis. Furthermore, by transwell experiments in vitro, we observed that MDA-MB-231/ITGBL1 shRNA subclones
presented increased invasion and migration capability, whereas their chemotaxis capacity to osteoblast was reduced. By
blocking secretory ITGBL1 of MDA-MB-231 cells with anti-ITGBL1 antibody, we also observed the decline of chemotaxis
capacity to osteoblast. These results indicated that ITGBL1 may function as a secreted signaling molecule can regulate the
biological processes of breast cancer cells and promote organ-specific bone metastasis. To exploring the functional mechanism
of ITGBL1 in breast cancer metastasis and organ-specific bone metastasis, we compared the differential transcriptional
profiling among the MDA-MB-231/ITGBL1 shRNA subclone cells, parental cells and negative control cells, and found that the
osteomimetic phenotype of the cells with decreased ITGBL1 expression were reversed because of changed gene expression of
the “Bone Metastasis Signature” and “Osteomimetic Signature”. Meanwhile, the genes coding key proteins in Wnt signaling
pathway were down-regulated, the specific marker of breast cancer stem cells CD44 and CD24 was up- and down-regulated
respectively, the genes related to TGF-β signaling pathway were down-regulated, these presented that ITGBL1 can inactivate
Wnt signaling pathway and cancer stem cells, and activate TGF-β signaling pathway. The RUNX2 mRNA and ITGBL1 mRNA
were co-expressed in breast cancer tissues and dependent on the TGF-β signaling. Chromatin immunoprecitation and dual-
luciferase reporter assay confirmed that RUNX2 regulated ITGBL1 expression positively. Wnt signaling also regulated the
ITGBL1 expression in a RUNX2 independent manner. These results indicated that TGFβ-RUNX2-ITGBL1-TGFβ positive-
feedback loop regulates the formation of osteomimetic breast cancer and which can facilitate its chemotaxis capacity to bone
and metestatic surviving in bone microenvironment. In another hand, ITGBL1 regulates Wnt signaling pathway negatively to
inhibit the proliferation of metestatic cancer stem cells, and reduces the recurrent growth and visceral metastasis accordingly.


CXCR4 chemokine signalling in neuroblastoma bone marrow metastasis
Benjamin R Wilkinson1 , Zillan Neiron1 , Amanda Tivnan1 , Toby N Trahair1 , Jamie I Fletcher1
1. Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, NSW, Australia
Background: Neuroblastoma is the most common cancer diagnosed during the first year of life, and accounts for 15% of all
paediatric oncology deaths. Almost half of all patients have metastatic disease at diagnosis and metastases most frequently
occur in the bone marrow, which is also the most common site of relapse. While expression of the chemokine receptor CXCR4
in primary neuroblastoma correlates with the presence of bone marrow disease, a causative role for CXCR4 in neuroblastoma
metastasis has not been clearly demonstrated. It is also unknown whether inhibition of CXCR4 can slow metastatic growth or
sensitize neuroblastoma cells to chemotherapy.
Methods & Results: We have used in vivo selection techniques to generate human neuroblastoma cell lines variants with
differing tropism for bone marrow in immunodeficient mice. Comparative gene expression studies using these variants indicate
that higher CXCR4 expression correlates with a preference for bone marrow colonization. In transwell migration assays, these
neuroblastoma cells migrate toward the CXCR4 ligand SDF-1 and chemotaxis can be inhibited by the highly specific small
molecule CXCR4 inhibitor AMD3100. We are currently generating neuroblastoma cell lines with specific down-regulation of
CXCR4 to determine whether there is a causative link between CXCR4 expression and bone marrow metastasis and whether
inhibition of CXCR4 can sensitize neuroblastoma cells to cytotoxic drugs or reduce their growth in the bone marrow.
Conclusions: CXCR4 is likely to be involved in metastasis of neuroblastoma to bone marrow in mouse xenograft models and
further preclinical studies of CXCR4 inhibition are warranted.


Characterisation of a novel breast cancer cell line, BCBM1, derived from a bone metastatic
lesion of a breast cancer patient
Julie K Johnson1 2 , Fares Al-Ejeh1 , Jodi Saunus3 , Chanel Smart3 , Sarah Song3 , Rebecca Johnston3 , Sibylle
Cocciardi1 , Cameron N Johnstone4 , Peter MacCallum Cancer Centre Tissue Bank5 , Kum Kum Khanna1 , Sunil
Lakhani3 , Georgia Chenevix-Trench1 , Nic Waddell6
1. Queensland Institute of Medical Research, Brisbane, Queensland, Australia
2. School of Medicine, University of Queensland, Brisbane, Queensland, Australia
3. University of Queensland Centre for Clinical Research, Brisbane, Queensland, Australia
4. Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
5. Peter MacCallum Cancer Centre Tissue Bank, Melbourne, Victoria, Australia
6. Queensland Centre for Medical Genomics, The Institute for Molecular Bioscience, The University of Queensland, Brisbane,
      Queensland, Australia
Metastases at distant sites are the main cause of death from breast cancer but there are few cell lines derived from distant
organs that represent the spectrum of normal breast cancer metastasis patterns. We have successfully established a breast
cancer cell line from a bone metastatic lesion of a breast cancer patient, which we have designated as BCBM1 (Breast Cancer
Bone Metastasis 1), and have performed expression and copy number profiling on it, and the matched primary tumour.
The BCBM1 cell line grows as an adherent monolayer and can be maintained continuously in vitro. Phase contrast microscopy
identifies a heterogeneous population where most cells have a spindle-like epithelial morphology, some have fibroblast-like
morphology and others are giant, multinucleated cells. Immunofluorescence shows BCBM1 cells are triple negative for
oestrogen receptor, progesterone receptor and HER2. Cells are heterogeneously positive for epidermal growth factor, have low
expression of cytokeratins 8/18 and 19 but are 100% positive for vimentin. Flow cytometry analysis shows BCBM1 cultures are
predominantly CD44+/CD49f+/EpCAM-, consistent with a primitive, mesenchymal-like phenotype. BCBM1 retains copy number
alterations that were present in the matched primary tumour but also contains additional alterations, some of which may be
involved in metastasis. Homozygous deletions detected in the cell line that were also evident in the primary tumour were found
in regions that contained known tumour suppressor genes (CDKN2A, CDKN2B and CDKN1B) and 23 putative tumour
suppressors: MTAP, C9orf53, CDKN2B-AS1, DMRTA1, ELAVL2, TUSC1, C9orf82, IFT74, PLAA, LRRC19, TEK, C9orf11,
IFNK, MOBKL2B, C9orf72, LINGO2, ACO1, CREBL2, GPR19, APOLD1, APAF1, HTA and ZFHX3. Cell line specific gene
amplification, coupled with over-expression, identified 23 genes with potential pro-metastatic function: PDCD10, TMEM106B,
CDC123, HSPA14, ARL5B, MLLT10, MASTL, MKX and CDK8. Tail-vein injection of BCBM1 cells into BALBc/nude mice
produced metastases in the bone, brain and lung.
The BCBM1 is a new addition to the breast cancer cell line resource that can be used in pre-clinical therapeutic trials and to
improve understanding of breast cancer cell biology.


Elucidation of Genomic Copy Number Alterations Associated with the Metastasis of Non Small
Cell Lung Cancer (NSCLC) to Brain by Comparative Molecular Inversion Probe (MIP)
Hye Won Lee1 2 3 4 , Se Jeong Lee1 2 3 5 , Yu Jin Cho5 , Kyeung Min Joo5 , Do-Hyun Nam1 2 3
1. Department of Neurosurgery, Seoul, South Korea
2. Samsung Biomedical Research Institute, Seoul , South Korea
3. Cancer Stem Cell Research Center, Seoul, South Korea
4. Samsung Advanced Institute for Health Sciences & Technology (SAIHST) Samsung Medical Center, Sungkyunkwan
     University School of Medicine, Seoul, South Korea
5. Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea
Non-small cell lung cancer (NSCLC) is the major determinant of overall cancer mortality in the developed world. Although the
brain metastasis (BM), one of the major relapse in NSCLC, contributes significantly to the morbidity and mortality of lung
cancer, relevant driver mechanisms associated are largely unknown. The elucidation of pre-determining signature which is
highly prognostic for the development of brain or general metastasis and overall survival may enable better stratification of
NSCLC patients at high risk of BM and identify novel therapeutic candidates targeting BM. In this study, first of all, we
evaluated copy number alterations (CNA) in 12 ADCs adenocarcinomas (ADCs) and 6 squamous cell carcinomas (SQCCs)
formaldehyde fixed and paraffin embedded (FFPE) samples to identify distinct pattern of chromosomal aberrations depending
on histologic-subtype. The most frequent CNAs detected in primary ADCs were gains of 3q, 5p, 5q, 6p, 8q, 9p, 11p, 15q, 17q,
17q, and losses of 10q and 22q whereas MIP analysis on primary SQCCs revealed gains in 4q and 12q and loss in 9q. In
addition, the patterns of CNAs in 11 matched NSCLC cases composed of primary tumor and BM were analyzed with MIP array.
The BM carried the majority of genetic alterations present in the corresponding primary tumor but with a significantly higher
frequency including gain of 9p11.2 and loss of 2p11.2-11.1 in ADC cases. Finally, to uncover predetermining genetic signatures
that can predict the risk of BM in especially ADC which is characterized by the early development of BM, comparative MIP was
performed in primary ADC of 4 cases with synchronous and 8 cases with metachronous BM. Selectively amplified regions of
5q35, 10q23 and 17q23-24 were found to contain the putative candidate genes including NeurL1B, ACTA, FAS and ICAM2.
These genomic signatures elucidated in our study may help to generate useful prognostic biomarkers associated NSCLC
metastasis to brain.


Anticancer efficacy of the hypoxia–activated prodrug TH-302 in a preclinical model of
osteosarcoma development and metastasis.
Vasilios Liapis1 , Agatha Labrinidis1 , Shelley Hay1 , Irene Zinonos1 , Vasilios Panagopoulos1 , Mark O DeNichilo1 ,
Vladimir Ponomarev2 , Gerald J Atkins3 , David M Findlay3 , Andrew CW Zannettino4 , Andreas Evdokiou1
1. Discipline of Surgery, Breast Cancer Research Unit, Basil Hetzel Institute and Centre for Personalised Cancer Medicine,
      University of Adelaide, Adelaide, South Australia, Australia
2. Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, USA
3. Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia
4. Myeloma and Mesenchymal Research Laboratory, Bone and Cancer Laboratories, Division of Haematology, Hanson
      Institute, Adelaide, South Australia, Australia
Tumour hypoxia is a major cause of treatment failure for a wide variety of malignancies. Tumour hypoxia leads to resistance to
anticancer chemotherapy and radiotherapy and predisposes to increased malignancy and tumour metastasis. However, despite
its adverse effects upon tumour response to treatment, hypoxia offers several opportunities for the development of new cancer
therapeutics. TH-302 is a hypoxia-activated pro-drug that displays potent hypoxia-dependent cytotoxicity. In this study, we
investigated the cytotoxic activity of TH-302 in a panel of osteosarcoma (OS) cell lines (K-HOS, BTK-143, MSK-8G) in vitro and
evaluated its anticancer efficacy in a preclinical model of osteosarcoma development progression and metastasis. In vitro, TH-
302 treatment exerted significant dose-dependent cytotoxicity, only under hypoxic conditions whereas; normal primary human
osteoblasts                                  were                              relatively                              resistant.
To test the efficacy of TH-302 in vivo, K-HOS human OS cells, tagged with a luciferase reporter construct, were transplanted
directly into the tibial marrow cavity of nude mice. Tumour development in bone and subsequent lung metastases with and
without TH-302 treatment was monitored in live animals and in real time using bioluminescence imaging and histology,
whereas the development of OS induced bone destruction was measured using high resolution micro-computer tomography.
TH-302 monotherapy reduced tumour burden in bone and protected the bone from OS-induced bone destruction. Importantly,
this translated to a 50% reduction in the incidence of lung metastases. TH-302 treatment was selectively toxic to cancer cells in
bone, as evidenced by strong TUNEL positivity in a large portion of the tumour mass whereas osteoblasts, osteocytes,
osteoclasts                        or                     chondrocytes                      were                     unaffected.
These results show that TH-302 is a potent agent with strong activity against the development, progression and metastatic
spread of OS and provide supporting evidence that targeting the intrinsically hypoxic bone marrow niche is a useful strategy for
the treatment of skeletal malignancy.


An integrated in vivo genomics screen implicates long non-coding RNAs H19 and Neat1 in
breast cancer metastasis
Richard P Redvers1 , Izhak Haviv1 , Normand Pouliot1 , Joshy George1 , Christina Restall1 , Robin L Anderson1
1. Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
Despite recent advances in molecular characterisation of breast cancer, the underlying mechanisms of dissemination of
primary tumour cells remain elusive. By utilising an integrated genomics and expression profiling approach with multiple
independent breast cancer isolates encompassing a spectrum of metastatic capabilities, we sought to identify genes relevant to
metastasis with greater confidence.
We have generated multiple sublines of the clinically relevant 4T1 model of breast cancer metastasis with a spectrum of
spontaneous metastatic phenotypes when transplanted orthotopically into syngeneic hosts. Tumour cells were purified from
primary tumours using FACS and interrogated for DNA copy number variations with the Agilent 105K mouse CGH array, and
expression profiled with the Affymetrix exon array for gene and exon level analysis.
Significantly, our top upregulated candidate from the exon array in the aggressive metastatic tumour lines was the imprinted
long non-coding RNA (lncRNA) H19. While H19 can act epigenetically to repress transcription, it can also associate with
polyribosomes and therefore be capable of regulating translation. Hence, the precise mechanism by which H19 modulates
metastatic potential remains unresolved and is under investigation.
We also identified DNA amplification of a region harbouring Neat1, an architectural lncRNA critical in the biogenesis of
subnuclear organelles known as paraspeckles. Subsequent analyses revealed significant upregulation of this lncRNA transcript
in highly metastatic tumours. In addition to its role as a molecular scaffold, Neat1 participates in the selective retention of
hyperedited transcripts within the nucleus to prevent their translation. It is therefore possible that Neat1 facilitates metastasis by
preventing the translation of yet to be discovered metastasis suppressors that we are seeking to identify through an RNA-seq
approach by comparing total and nuclear RNA fractions in tumours with and without elevated Neat1 expression.
To compensate for the paucity of annotated lncRNA probesets on the exon array, we also interrogated our raw data using a
custom ncRNA chip definition file devised by Risueño and colleagues (GATExplorer) to unambiguously re-map all intronic and
un-assigned probes to bona fide lncRNA entities with accessions in RNAdb. We discovered over 400 unique long non-coding
RNA transcripts associated with metastasis, nine of which exhibited corresponding DNA copy number variations.
In summary, we have implicated H19 and Neat1 and revealed a large set of long non-coding RNAs associated with breast
cancer metastasis. Efforts are underway to explore the functional role of these lncRNAs in our metastasis models.


Development of a novel orthotopic transplantation model of human small cell lung cancer
Shuichi Sakamoto1 , Manabu Kawada1 , Hiroyuki Inoue1 , Shun-ichi Ohba1 , Ihomi Usami1 , Akio Nomoto1
1. Institute of Microbial Chemistry, Numazu, Microbial Chemistry Research Foundation, Shizuoka, Japan
Novel strategies against metastasis are urgently needed to improve lung cancer treatment. However, the molecular mechanism
of metastasis of lung cancer, which is important for the development of the effective treatment, remains largely to be elucidated.
For further studies of lung cancer metastasis, we are trying to develop a novel orthotopic transplantation model in the mouse.
We established GFP-labeled clones from human lung cancer cell lines and transplanted them orthotopically into the lung of
Balb/c nude mice with matrigel. Tumor formation at the orthotopic site and metastasis to distant organs were evaluated by GFP
fluorescence and histologically. A clone derived from human small cell lung cancer cell line showed significant metastatic
activity and formed metastatic foci mainly in the bone, with a few incidences in adrenal gland, brain, lymph, and kidney. The
metastatic characteristics of the clone are similar to the clinical aspects of small cell lung cancer.
Furthermore, through in vivo selection of this metastatic clone, we have succeeded to establish a series of variant clones with
different metastatic potential. These clones have nearly identical tumorigenicity at the orthotopic site, adhesion activities to
extracellular matrix, and anchorage-independent growth activities. Interestingly, these clones showed different invasion
activities in a Matrigel chamber using conditioned medium as chemoattractant. Comprehensive analysis such as DNA
microarray and cytokine antibody array revealed that there are some genes/factors differentially expressed among these


CEP55; a new target for prevention of breast cancer progression and metastasis
Jacinta Simmons1 2 , Fares Al-Ejeh1 , Robin Anderson3 , Winnie Fernando4 , Mariska Miranda1 , Kevin Spring1 , Kum
Kum Khanna1
1. Signal Transduction Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia
2. University of Queensland, St Lucia, QLD, Australia
3. Metastasis Research Laboratory, The Peter MacCallum Cancer Institute, East Melbourne, Vic, Australia
4. Conjoint Gastroenterology Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia
Over expression of the centrosomal protein Cep55 is often cited in gene expression profiles of a variety of tumour types
including breast cancer signatures of late stage disease, which have shown to correlate with aggressive subtypes and poor
patient outcome. High expression of Cep55 has been associated with increased rates of metastasis and has also been shown
to contribute to migration and invasion of lung cancer cells in vitro. To examine Cep55’s role in metastatic progression we
depleted Cep55 in the metastatic breast cancer cell line MDA-MB-231. We found Cep55 to be required for cell migration in
vitro. Interestingly in mouse xenograft models, Cep55 depletion reduces lung metastatic growth but not primary mammary
tumour growth. We hypothesize that Cep55 is likely to be required to interact and activate the PI3K/AKT pathway resulting in
increased cellular survival, proliferation and migration in the metastatic setting which is a crucial step in disease progression of
carcinomas. Further insights gained into Cep55 function in tumour progression and metastasis will provide potential avenues
for novel treatment options for metastatic disease in the future.

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