Molecular basis of oncogenic transformation and signaling pathways Path 822 October 6, 2005 Bruce Elliott Age-standardized incidence rates for selected cancer sites (Females, Canada, 1970-2004) • 1 in 8 women in their lifetime will be diagnosed with breast cancer. • 1 in 3 will die of metastasis. NCIC Statistics (Cancer.ca) Clonal evolution of cancer • Nowell’s Hypothesis •Vogelstein Model of tumor progression Volgelstein, et al. Nat Rev Cancer. 2003 Sep;3(9):695-701. Characteristics of normal epithelium • Polarity • Adhesion to basement membrane • Transition from basal (stem) cells to differentiated cells (transitional epithelium) • cell-cell contacts Cell-cell contacts 1. Adherens junctions 2. Tight junctions 3. Desmosomes 4. Gap junctions microvilli tight junction Junctional Complex adhesion belt cadherins desmosome keratin filaments gap junctions hemidesmosome basal lamina rd Mammary tumor progression Normal DCIS Invasive Epithelial-mesenchymal transition - E-cadherin +E-cadherin Characteristics: •Loss of epithelial polarity and cell-cell contacts •Aberrant cell-substrate adhesion •Degradation of basement membrane •Increased proliferation and cell migration •Increased cell invasion •Occurs during morphogenesis (transient), and tumor • progression (stable). Architecture of cell-cell contacts Protein Phosphorylation is a key mechanism of regulation of cell function Tyrosine kinases Serine/threonine kinases Phosphatases (eg PTPB1, SHP, PTEN) Balance between phosphorylation and dephosphorylation The WNT/APC pathway Mutation of APC blocks _ degradation of -catenin Nat Med. 2004 Aug;10(8):789-99. Regulation of -catenin signaling (Fodde et al., Nature Reviews Cancer 1, 55 –67, 2001) Regulation of -catenin function Normal Epithelial cells: •APC complexes with GSK and -catenin •GSK mediates tyrosine phosphorylation of -catenin •Phosphorylated -catenin becomes susceptible to degradation by proteosomes. •Cytoplasmic pool of -catenin is low. Malignant epithelial cells: •Wnt/frizzeled pathway is activated •GSK is inhibited. -catenin remains nonphosphorylated and is protected from degradation. •Cytosolic pool of -catenin increaes and -catenin is translocated to the mucleus to facilitate gene transcription (Myc, Waf1, cyclin D1) Mutations in APC Summary of APC effect on catenin signaling 1. APC facilitates degradation of beta catenin in normal Cells resulting in decreased nuclear signaling. 2. The selective advantage provided by loss of APC function resides in the uncontrolled activation of the WNT/-catenin signal transduction pathway. 3. -catenin acts with LEF1 to activate transcription of tumor promoting genes such as Myc and Cyclin D1. ErbB2 IGF1R Met Schlessinger lab. Up-regulated in human breast cancer Nature 411, 355-365 (17 May 2001) Receptor tyrosine kinase pathways Nature Med. 10:789-99, 2004) Cellular signalling Pathways (1995) Hunter T. Cell. 1995 80:225-36, 1995. PTEN Survival Stat3 CELLULAR SIGNALLING NETWORKS (2005) Papin et al. Nat Rev Mol Cell Biol.6:99-111, 2005 ErbB receptor family •There is no known ligand for ErbB-2 (Her-2/neu). •ErbB-2 is activated through transactivation of heterodimers. •Over-expression and activation of erbB-2 correlates with recurrence and poor survival in breast cancer patients. Nature Reviews Cancer 5:342-54, 2005 ErbB-receptor ectodomain structures ErbB2/3 heterodimers are activated Through binding of NRG to ErbB3 Herceptin derivatives bind to ErbB2 Preventing heterodimerization. How do over-expressing ErbB2 cancer Cells excape from herceptin? •Co-ordinate activation of other RTKs (IGF1R, EGFR, Met) •Compensatory downstream pathways Become constitutively activated (PI3K/Akt-induced survival) •Loss of suppressor functions (PTEN) Nature Reviews Cancer 5:342-54, 2005 c-Src is recruited to adherens junctions and focal adhesions Adherens junction Focal adhesions ECM Catenins Cytoskeletal PTPB FAK Proteins Cytoskeletal PTPB c-S rc c-S rc Proteins FAK Src kinase in normal and malignant breast development •Required for normal ductal morphogenesis. •Shows increased activation in most human breast carcinomas. •Required, but not sufficient, in mammary tumourigenesis in transgenic mice. Activated Src induces disruption of cadherin junctions Aggregation c-Src inhibition Weak YES Yes Ca++ dependency No Yes SCATTER No Regulation of cadherin-based adherens junctions by Src kinase 1) Activated Src binds to the cadherin complex. 2) Src causes phosphorylation of -catenin, causing its dissociation from the cadherin complex and endocytosis of the cadherins. 3) Src-induced de-regulation of E-cadherin requires integrin signalling. Frame et al. Nat Rev Cancer 5:505-15, 2005. & Cells Tissues Organs. 179:73-80, 2005. New concept: Cross talk between growth factor and integrin- based cell adhesion signalling is linked to invasive cancer •Focal adhesion kinase (FAK) is a substrate of Src. •Src and FAK link growth factor receptors and integrin adhesion molecules. •Interaction between growth factor receptors and integrins causes increased cell survival and invasion of carcinomas. (Frame et al. Nat Rev Cancer. 5:505-15, 2005. The metastatic cascade Nature Reviews Cancer 4, 448-456 (2004 EMT is a reversible process that occurs at distinct stages of tumor progression (Thiery, Nature Cancer Rev. 2, 442-454, 2002) Intravasation/extravasation model Examples of metastasis genes Enhancers Suppressors Tumor cell Stromal cell Tumor cell HGF/Met Proteinases E-cadherin Her2/neu ECM proteins TIMPs (inhibits Akt2 (survival, (Osteopontin) proteases) invasion) Csk (blocks Src) N-cadherin TGF- (blocks (cross talks with Proliferation) RTKs) Metastasis issues 1) A 1 cm dia. tumor sheds 2.5 x 106 per day into blood. 2) Only 0.01% of tumor cells in blood will metastasize. 3) The majority of tumor cells that arrest in target organs are dormant or will die. 4) Tumor cells arrest by mechanical restruiction in target organs: integrin adhesion does not play a role until after extravasation. 4) BUT, engagement of integrins are required for outgrowth of metastatic foci (Mercurio) Tumor Dormancy Chambers et al. Nature Rev. Cancer 2:563, 2002. Why are the new wonder drugs having only minimal effects in treating human cancer? Leaf, C. Fortune 149: 76-82, 84-6, 88 , 2004. What can we do to improve treatment outcomes? •Develop preventive measures (life styles, screening for susceptibility) •Understand the early stages of cancer and identify early diagnostic markers. (gene expression and signalling networks) •Develop better prognostic indicators (detect the invisible high risk group). •Develop better predictive markers (tailor treatments to individual cancers) •Develop means of controlling the disease. Is it worth while to target tumor dormancy to prevent metastasis?