The MET Receptor Tyrosine Kinase Is a Potential Novel Therapeutic
Target for Head and Neck Squamous Cell Carcinoma
1 1 1 1
Tanguy Y. Seiwert, Ramasamy Jagadeeswaran, Leonardo Faoro, Varalakshmi Janamanchi,
1 1 1 1 1
Vidya Nallasura, Mohamed El Dinali, Soheil Yala, Rajani Kanteti, Ezra E.W. Cohen,
2 2 1 3
Mark W. Lingen, Leslie Martin, Soundararajan Krishnaswamy, Andres Klein-Szanto,
4 1 1
James G. Christensen, Everett E. Vokes, and Ravi Salgia
Section of Hematology/Oncology, Department of Medicine and University of Chicago Cancer Research Center and
Department of Pathology, University of Chicago, Chicago, Illinois; 3Department of Pathology, Fox Chase
Cancer Center, Philadelphia, Pennsylvania; and 4Pfizer La Jolla Laboratories, La Jolla, California
Abstract (47,560 cases in the United States; ref. 1). More than 90% of head
Recurrent/metastatic head and neck cancer remains a devas- and neck cancers are of squamous histology (HNSCC). Thirty-five
tating disease with insufficient treatment options. We investi- percent to 45% of head and neck cancer patients ultimately die
gated the MET receptor tyrosine kinase as a novel target for from their disease. Little progress has been made in the treatment
the treatment of head and neck squamous cell carcinoma for metastatic/recurrent HNC during the past two decades, with
(HNSCC). MET/phosphorylated MET and HGF expression was the singular exception of cetuximab, an epidermal growth factor
analyzed in 121 tissues (HNSCC/normal) by immunohisto- receptor (EGFR) antibody, which improves median survival by
chemistry, and in 20 HNSCC cell lines by immunoblotting. The 2 months when added to standard chemotherapy (2). Overall
effects of MET inhibition using small interfering RNA/two survival remains poor (median 6–10 months).
small-molecule inhibitors (SU11274/PF-2341066) on signaling, To improve HNSCC treatment, relevant molecular targets need
migration, viability, and angiogenesis were determined. The to be identified. Receptor tyrosine kinases (RTK) seem to play a
complete MET gene was sequenced in 66 head and neck cancer pivotal role in the pathogenesis of HNC, with prior research
tissue samples and eight cell lines. MET gene copy number was focusing on EGFR. Despite EGFR overexpression in >90% of
determined in 14 cell lines and 23 tumor tissues. Drug tumors, EGFR inhibition has only yielded low response rates of 4.3–
combinations of SU11274 with cisplatin or erlotinib were 13% in clinical practice (3–4). Multiple lines of evidence indicate
tested in SCC35/HN5 cell lines. Eighty-four percent of the that RTK pathway redundancies/cooperation are common in
HNSCC samples showed MET overexpression, whereas 18 of 20 RTK-driven malignancies and may account for resistance (5–8).
HNSCC cell lines (90%) expressed MET. HGF overexpression We studied the MET RTK and also explored EGFR/MET crosstalk
was present in 45% of HNSCC. MET inhibition with SU11274/ based on reports of cooperation in other diseases (6–9).
PF-2341066 abrogated MET signaling, cell viability, motility/ MET, located on chromosome 7q31, encodes several functional
migration in vitro, and tumor angiogenesis in vivo. Mutational domains, including the semaphorin (SEMA) domain (ligand-
analysis of 66 tumor tissues and 8 cell lines identified novel binding), juxtamembrane (JM) domain (regulatory), and the
mutations in the semaphorin (T230M/E168D/N375S), juxta- receptor tyrosine kinase (TK) domain (10, 11). The sole ligand for
membrane (T1010I/R988C), and tyrosine kinase (T1275I/ MET is hepatocyte growth factor (HGF, scatter factor), which is
V1333I) domains (incidence: 13.5%). Increased MET gene copy produced by stromal and sometimes tumor cells (10, 11). HGF
number was present with >10 copies in 3 of 23 (13%) tumor binding activates MET via intracellular phosphorylation initiating
tissues. A greater-than-additive inhibition of cell growth was RAS-RAF-ERK, and phosphatidylinositol 3-kinase-AKT-mTOR sig-
observed when combining a MET inhibitor with cisplatin or naling as well as several other pathways. In vivo, HGF/MET
erlotinib and synergy may be mediated via erbB3/AKT signaling leads to increased cell growth, cell motility, invasion/
signaling. MET is functionally important in HNSCC with metastasis, angiogenesis, wound healing, and tissue regeneration
prominent overexpression, increased gene copy number, and (10, 11). Studies show that HGF/MET signaling increases motility,
mutations. MET inhibition abrogated MET functions, includ- epithelial cell dispersion, endothelial cell migration, and chemo-
ing proliferation, migration/motility, and angiogenesis. MET is taxis. Furthermore, MET overexpression and activation has trans-
a promising, novel target for HNSCC and combination forming properties for normal cells (10, 11).
approaches with cisplatin or EGFR inhibitors should be MET is overexpressed in a number of solid tumors, and
explored. [Cancer Res 2009;69(7):3021–31] expression correlates with an aggressive phenotype and poor
prognosis (10, 11). Previously, we had shown that in lung cancer,
MET mutations can occur in the JM domain and the SEMA
domain, and not the TK domain (12). The precise function of most
Head and neck cancer (HNC) is the sixth most common cancer mutations is not yet fully understood. MET mutations have been
worldwide, with an annual incidence of >640,000 cases worldwide described for HNC, especially in lymph node metastases (relative
frequency of up to 25% in some reports; ref. 13) and are located in
Requests for reprints: Tanguy Seiwert, Section of Hematology/Oncology, the TK domain similar to TK domain mutations found for renal cell
University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. carcinomas (11), suggesting an important role for MET in HNC
Phone: 773-702-2452; Fax: 773-702-3002; E-mail: email@example.com.
I2009 American Association for Cancer Research. (13). However, mutations in the SEMA and JM domains have not
doi:10.1158/0008-5472.CAN-08-2881 been previously investigated for HNSCC. Further highlighting the
www.aacrjournals.org 3021 Cancer Res 2009; 69: (7). April 1, 2009
importance of MET is the observation of MET amplification in The cells were imaged on an Olympus IX81 inverted microscope and
several solid tumors, including subgroups of lung and gastric digitally captured with IPLab software (Scanalytics). Images at Â100
cancers (6, 7, 14, 15). magnification were saved every 5 min and processed as mpeg4 movies
In our study, we used a large cohort of HNC and normal mucosa (Sonic DVD). Cell movement/morphologic changes were processed with
ImageJ (NIH), Photoshop (Adobe), and MetaMorph (Universal Imaging
tissues as well as cell lines to identify prominent MET expression,
Corporation/Molecular Devices). The positions of the cell nuclei were
increased gene copy number, and mutations in the TK/JM/SEMA tracked, and distance/speed was calculated over 21 h.
domains. Furthermore, we show that MET inhibition alone and in Mutational analysis. Genomic DNA from 63 HNSCC tissues from
combination with cisplatin or an EGFR inhibitor is a promising formalin-fixed paraffin-embedded tissues was obtained from the University
target for head and neck cancer. of Chicago Head and Neck Cancer tissue bank (IRB: 8980). Genomic MET
reference sequences were obtained from position chr7:116,099,682–
Materials and Methods 116,225,676 from Ensembl (release 50; July 2008). Please note that the
MET transcript MET-001 (ENST00000318493) was used for identifying
Tumor tissue arrays/immunohistochemistry. Tissue microarrays of genetic changes (e.g., R988C) and MET-002 (ENST00000397752) for
97 HNC tissues and 24 normal mucosa samples were built (IRB: 8980). identifying phosphorylation sites (e.g., Y1230), which is consistent with
Immunohistochemistry was performed for MET (C-12, 1:100), p-MET common practice (12, 16).
(pY1003, Invitrogen, 1:25), HGF (H145, Santa Cruz, 1:50), human CD31 Real-time PCR. Quantitative real-time PCR for gene copy number
(JC70A, DAKO, 1:40), and Ki67 (RM-9106-S, NeoMarkers/Labvision, 1:300) measurement was done as previously described (15) using ABI StepOnePlus
as previously described (12, 15, 16). Appropriate negative controls were (Applied Biosystems) and iQ-SYBR green (Bio-Rad Laboratories). Reactions
prepared. Immunohistochemistry results from tumor and adjacent normal were done in triplicates under standard thermocycling conditions (one cycle
tissue were compared semiquantitatively by a senior pathologist (grading: 95jC Â 12 min, 45 cycles 95jC Â 20 s, 58jC Â 1 min). The mean threshold
0 = negative, 1+ = low, 2+ = strong, 3+ = very strong expression; ref. 12). cycle number was used.
Reagents and antibodies. Antibodies used for immunoblotting were Fluorescence in situ hybridization. Fluorescence in situ hybridization
MET (3D4, Invitrogen/Zymed, C-12, Santa Cruz Biotechnology), phosphor- (FISH) analysis was done using two different BAC probes: RP11-433C10,
ylation site–specific MET pY1003, and pY1230/4/5 (Biosource/Invitrogen, localized to 7p11.2 ( full-length EGFR gene), and RP11-163C9, localized to
44-882G, 44-888G), h-actin/p16/ERCC1/Ron-a antibodies (H-196/JC-8/8F1/ 7q31.2 (MET gene). Two-color FISH was done using RP11-144B2 (red)
H-170, Santa Cruz Biotechnology), pTyr (4G10, Upstate/Millipore), and together with RP11-163C9 (green). The procedure was done as previously
insulin-like growth factor-I receptor (IGF-IR; Cell Signaling; dilution 1:1,000) described (14), analyzing at least 10 metaphase cells.
as previously described (12, 15–17). Human papillomavirus testing. Human papillomavirus (HPV) testing
The following drugs were purchased: SU11274/IGF-IR inhibitor (Calbio- was performed in cell lines evaluating for p16 expression (JC-8, Santa Cruz
chem), cisplatin (Sigma Aldrich), and erlotinib HCL (ACC). PF-2341066 was Biotechnology) and by PCR using L1 PGMY09/11 primers (21), followed by
kindly provided by Pfizer. sequencing. HPV-positive results were confirmed using the Digene HPV test
Cell lines and culture. Cell lines were obtained from the American Type (Qiagen).
Culture Collection (SCC9/15/25/68/Cal27/Fadu), Dr. Ralph Weichselbaum In vivo Matrigel plug nude mouse xenograft modeling. Tumor cells
(Department of Radiation Oncology, University of Chicago, Chicago, IL; were mixed with Matrigel (BD Biosciences) and injected s.c. into the flanks
SQ20B, JSQ3, SCC35/61/294/151), Dr. Gary Clayman (M.D. Anderson Cancer of nude mice (5 Â 106 cells/flank) following Institutional Animal Care and
Center, Houston, TX; 1483), the Ludwig Institute for Cancer Research Use Committee–approved protocols. The animals were monitored for 2 wk
(London, United Kingdom; HN5), Dr. David Raben (University of Colorado and subsequently sacrificed. Tissues were fixed in 10% formalin and paraffin
Health Sciences Center, Aurora, CO; MSK921), and Dr. Mark Lingen embedded.
(Department of Pathology, University of Chicago, Chicago, IL; OSCC3, Statistical analyses. Data are expressed as mean F SE. Statistical
SCC17B/28/58) and maintained in DMEM/F12 or RPMI medium and significance was tested with Graphpad Prism5. For comparison between
penicillin/streptomycin (Cellgro) with 10% fetal bovine serum (FBS; Gemini two groups, Student’s t test or the m2 test was used. For comparing between
Bioproducts). HaCaT is a spontaneously transformed human keratinocyte >2 groups, one-way ANOVA was used. For evaluation of correlation,
cell line. HNX was derived from HN5 after prolonged subculture showing Spearman’s test was used.
suppressed EGFR and MET expression.
Immunoblotting analysis. Immunoblots were done using standard
methodology (12, 15–17). Results
MET small interfering RNA/small-molecule inhibitors. Cells were MET/HGF are expressed in HNSCC tissues and cell lines.
grown in antibiotic-free medium to 60% confluency in 96-well (viability)/6- MET immunohistochemistry was done on 121 cores (97 cancers/24
well (immunoblotting) plates. MET small interfering RNA (siRNA) was used
normal mucosa) as well as in phosphorylated MET (86 cancers/22
at 100 Amol/L with Dharmafect transfection reagent (Dharmacon) using the
recommended protocol. Controls were treated with transfection agent only.
normal mucosa). Eighty-five percent (n = 84) of HNSCC tumors
Cells were incubated at 37jC in 5% CO2 for 36 to 72 h before viability was overexpressed (2+/3+) MET and 66% (n = 57) overexpressed (2+/3+)
assessed or before lysate was harvested. activated phosphorylated MET compared with adjacent normal
MET inhibition was achieved using small-molecule MET inhibitors mucosa (Fig. 1A and B). Normal mucosa also expressed MET
SU11274 (ACC; ref. 18) and PF-2341066 (Pfizer; ref. 19; Supplementary (21% 1+, 21% 2+), albeit staining was weaker and primarily limited to
Table S1). the basal layer of the mucosa (Fig. 1A; 23% 1+/2+ for phosphorylated
Viability. Measurement was performed using Alamar blue (Resazurin, MET). No cases of 3+ expression were seen for normal mucosa. MET
Sigma-Aldrich) or MTT (R&D Systems). Soft-agar colony formation assays localized primarily to the membrane and the cytoplasm.
were performed as previously described (15). Viability results were Immunoblot analysis confirmed strong MET expression in 16 of
evaluated by a fluorescence/absorbance 96-well plate reader Synergy HT
20 HNC cell lines [excluding HNX (derived from HN5) and HaCaT
(BioTek). Synergy was calculated using Calcusyn as described by Chou and
(transformed keratinocytes)]; however, SCC17B and SCC151
Time-lapse video microscopy. Cells were plated on glass-bottomed expressed low levels of MET, which were outside the dynamic range
culture dishes (MatTek) in 10% FBS medium and grown for 24 h to achieve (Fig. 1C). SQ20B and SCC294 had low to moderate MET expression.
20% to 30% confluency before drug treatment. Dishes were placed into a OSCC3, a HPV-positive cell line [p16+, PCR positive (HPV18), Digene
temperature-controlled chamber at 37jC in an atmosphere of 5% CO2. high-risk HPV positive], showed strong MET expression. EGFR,
Cancer Res 2009; 69: (7). April 1, 2009 3022 www.aacrjournals.org
The Role of MET in Head and Neck Cancer
Figure 1. A, analysis of the frequency
and localization of MET expression by
immunohistochemistry in HNSCC and
normal adjacent mucosa. MET was
strongly expressed (2+/3+) in 84% of
tumors. Normal mucosa had negative
or low MET expression in 79% (0/1+),
whereas 21% had 2+ staining (no 3+
staining). MET expression was
membranous and cytoplasmic.
B, phospho-MET epitope pY1003
immunohistochemistry: The staining
pattern closely resembled MET (A), with
strong expression in 71% of HNSCC
samples. C, MET was expressed in
18 of 20 HNSCC cell lines as seen by
immunoblotting, excluding HNX (derived
from HN5) and HaCaT (immortalized
keratinocytes). MET expression in
SCC17B/HN5 was very low (outside
the dynamic range). The 170-kDa
(glycosylated MET) and 140-kDa bands
(biologically active transmembrane h
subunit) are shown. RON expression
closely follows MET expression (12 of 15),
whereas expression of EGFR and IGF-IR
is nonconcordant. ERCC1 (nucleotide
excision repair pathway) is present in most
cell lines. OSCC3 was HPV18+. D, HGF
immunohistochemical staining in HNSCC
and normal adjacent mucosa. HGF was
expressed in 41% of tumors with 2+
expression in 21%. There was no
significant HGF expression in normal
IGF-IR, RON, and ERCC1 expression were prominent in several cell starved cells lines were pretreated with 0, 2, or 5 Amol/L of the
lines. There was no statistical correlation with MET expression. MET inhibitor SU11274 followed by treatment with HGF for
Analysis of MET gene expression using the publicly available 8 minutes. In cell lines SCC15, SCC28, and to a lesser degree SCC9
Oncomine database5 and data by Ginos and colleagues (22) showed and SCC61, HGF stimulation led to a strong p-Tyr signal, which was
increased MET gene expression in 41 HNSCC compared with 13 suppressed with SU11274 MET inhibitor treatment. SCC17B overall
normal controls (Supplementary Fig. S1). had low p-Tyr expression, suggestive of either a less RTK-driven
HGF expression was evaluated in 68 HNC tumors by immuno- phenotype (5) or a more ligand-dependent phenotype. Despite low
histochemistry. The tumors showed strong (3+; 21%), moderate (2+; MET expression, external HGF stimulation and SU11274 pretreat-
24%), and weak (1+; 41%) HGF expression. Fifteen percent of the ment showed typical signaling effects of the HGF/MET axis.
tumors were HGF negative. Phosphorylated MET expression was weak at baseline in most
MET-specific small-molecule inhibitors or siRNAs inhibit starved cells. Following HGF stimulation in all cell lines, a strong
MET signaling. Using small-molecule MET inhibitors SU11274 phosphorylated MET response is observed that can be suppressed
( for cell lines, DMSO soluble, Figs. 2 and 3), PF-2341066 (water in a dose-dependent fashion (Fig. 2A and B). Downstream signaling
soluble, clinical candidate, Fig. 4; see Supplementary Table S1), and for phosphorylated AKT was also increased with HGF and
MET siRNA (Fig. 3B), MET activation/expression were suppressed. decreased by MET inhibition in cell lines SCC15, SQ20B, SCC28,
Figure 2A shows immunoblotting results for phosphotyrosine, and to a lesser degree in SCC61 (Fig. 2B). Phosphorylated ERK was
whereas Fig. 2B shows results for phosphorylated MET and only mildly affected by MET inhibition with SU11274.
downstream signaling effects in six HNSCC cell lines. Serum- MET inhibition decreases viability in HNSCC. MET gene
silencing with MET-specific siRNA was used to validate effects of
MET inhibition in HNSCC. MET-specific siRNA duplexes were
http://www.oncomine.org transiently transfected into SCC61 and SQ20B cells (Fig. 3A), and
www.aacrjournals.org 3023 Cancer Res 2009; 69: (7). April 1, 2009
Figure 2. A, phosphorylated tyrosine
(p-Tyr ) immunoblot of six HNSCC cell lines
with or without HGF stimulation and
inhibition with SU11274. Expression of
phosphotyrosine is seen in all cell lines in
response to HGF treatment. SCC9 and
SQ20B have the highest background
p-Tyr expression. SU11274 pretreatment
with 2 Amol/L and 5 Amol/L SU11274
affected phosphorylated tyrosine levels.
B, stimulation of MET phosphorylation
with 2 Amol/L and downstream signaling in
five HNSCC cell lines is completely
abrogated by pretreatment with
SU11274. Downstream AKT and ERK
phosphorylation is partially affected in
certain cell lines.
protein expression was decreased by >80% 72 hours after formation assays were observed for SCC61 and SCC35 (data not
siRNA down-regulation of MET protein expression in SCC61 and Effects on angiogenesis were investigated with an in vivo
SQ20B cells resulted in inhibition of the serum-stimulated cell Matrigel xenograft tumor model of OSCC3 and SCC35 treated
growth and viability by >62%/55% as determined by MTS assays with PF-2341066 (25 mg/kg/d) versus control-treated cells (n = 3 in
(Fig. 3B). each group). Figure 4 (C and D) shows abundant tumor growth in a
We used SU11274 to test for its inhibitory effects on seven vehicle control–treated mouse, compared with minimal residual
HNSCC cell lines (Fig. 3C). MET inhibition was effective with IC50 tumor nests in the PF-2341066 group. Staining for the proliferation
values varying between 1 and 8 Amol/L: SCC61 (IC50 1 Amol/L), marker Ki67 shows >80% to 90% suppression of proliferation in PF-
SCC35 (IC50 3 Amol/L), and SCC9 (IC50 3.8 Amol/L) were the most 2341066–treated animals. Finally, staining of endothelial cells in
sensitive lines followed by HN31 (IC50 5 Amol/L) and MSK921/ blood vessels with CD31 shows extensive tumor vessels between
SCC28 (IC50 5.4 Amol/L). SQ-20B, which has lower MET expression tumor nests in control-treated animal versus marked angiogenesis
and strong EGFR expression (EGFR amplification), showed an suppression in PF-2341066–treated animals, consistent with prior
elevated IC50 of 8 Amol/L (extrapolated from Fig. 3C). Generally, a reports using a related MET inhibitor in vivo (23).
50% to 90% decrease in cell viability compared with control cells SU11274 can synergize with erlotinib and cisplatin. Figure 5
was observed. shows four examples of dual treatment with MET inhibitor
Furthermore, MET inhibition with SU11274 (3.5 Amol/L) led to SU11274 in combination with commonly used agents—cisplatin
suppression of cell motility and migration. Figure 3D shows a or erlotinib.
graphical depiction of distances covered by individual cells (SCC61) SCC35 and SCC61, which required doses of >10 Amol/L to
over a period of 21 hours. SU11274-treated cells covered significantly approach IC50 toxicity (SCC35 >10 Amol/L cisplatin; SCC61
shorter distances (P = 0.0001) than untreated control cells. This 16 Amol/L cisplatin), were synergistically inhibited by combined
effect is consistent during the entire 21-h observation period. treatment with SU11274/cisplatin (SCC35 IC50 1.3/1.3 Amol/L,
MET inhibition in vivo. To study MET inhibition effects on and SCC61 IC50 1/2 Amol/L). Based on the median effect model by
angiogenesis, water-soluble PF-2341066 was used in vivo (Supple- Chou (20), the isobologram graph shows combinatorial index (CI)
mentary Table S1). PF-2341066 inhibited HGF-dependent MET values below 1 for the ED50 and ED75 (values <1 indicate synergy).
phosphorylation in a dose-dependent manner at concentrations For combination with erlotinib, HN5 and SCC35 were chosen.
of 10 to 100 nmol/L in HNC cell lines SCC61 and SCC35 in vitro Cells were treated with either agent alone or with a combination of
(Fig. 4A) and also in a soft-agar colony formation assay (OSCC3; both at equimolar doses. Both single agents showed efficacy,
Fig. 4B); no large colonies formed. Comparable results in colony decreasing viability. The combination, however, was consistently
Cancer Res 2009; 69: (7). April 1, 2009 3024 www.aacrjournals.org
The Role of MET in Head and Neck Cancer
Figure 3. A, in SCC61 and SQ20B, MET-specific siRNA (100 Amol/L) led to a significant decrease in MET protein expression, whereas control siRNA did not
suppress MET expression. B, SCC61 and SQ20B cells 72 h after transfection with MET-specific siRNA and control siRNA were analyzed by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide and showed significant decreases in viability compared with control (À62%/55%). C, SU11274 treatment led to a dose-dependent
decrease in cell viability compared with untreated control (DMSO solvent). In decreasing order of sensitivity (IC50), the following HNSCC cell lines responded to
increasing SU11274 concentrations: SCC61, SCC35, SCC9, HN31, MSK921, SCC28 and SQ20B. D, migration was significantly decreased after treatment with
SU11274 over a tracking period of 21 h. Colored lines, individual cell movement over 21 h. Cells treated with SU11274 moved significantly shorter distances
(À38%; P < 0.0001).
www.aacrjournals.org 3025 Cancer Res 2009; 69: (7). April 1, 2009
Figure 4. A, immunoblot of two
HNSCC cell lines, SCC61 and SCC35,
after treatment with PF-2341066 at
doses ranging from 0 to 500 nmol/L.
PF-2341066 led to a dose-dependent
abrogation of HGF-induced MET
phosphorylation. B, soft agar colony
formation assay of OSCC3 HNSCC with
and without PF-2341066 treatment
(0–1,000 nmol/L). Colonies were stained
with crystal violet and counted. Photo
shows comparison of 0 and 1,000 nmol/L.
There was marked suppression of colony
formation by 33%/53%. Representative
H&E-stained images (original
magnification, Â20) of OSCC3 (C ) and
SCC35 (D ) xenograft tumors from mice
treated with vehicle or PF-2341066
(25 mg/kg body weight). Animals were
sacrificed before development of
macroscopic tumors. Middle and bottom,
PF-2341066 reduced cell proliferation
(Ki67) and reduced blood vessel density
(CD31), demonstrating in vivo activity of
PF-2341066 on angiogenesis.
significantly superior to either agent alone. The isobologram shows mutations occurred in 12% of tumors analyzed (8 of 66). There was
that at ED25 (CI = 0.73/035), ED50 (CI = 0.32/0.36), and ED75 no apparent correlation with smoking status or anatomic site,
(CI = 0.21/0.36), there was synergistic activity between erlotinib and although the sample number was too small to allow sufficient
SU11274 (CI <1). Evaluation of downstream signaling in Fig. 5C statistical power.
indicated that activation of erbB3 and subsequently AKT are MET gene copy number. We analyzed a panel of nine HNSCC
synergistically inhibited. cell lines by FISH and followed this up with qPCR due to the ready
MET mutations in HNSCC tumor tissues and cell lines. The availability of DNA from HNSCC tumor tissues. Repetition of cell
entire MET coding region (schema in Supplementary Fig. S2) was lines previously analyzed by FISH now using qPCR was done
sequenced in 66 HNSCC and 8 cell lines. Three mutations in the (Table 1B). FISH analysis showed three cell lines with >4 copies,
ligand-binding SEMA domain (T230M/E168D in the tumor tissue; although qPCR copy number was lower (2.79 and 1.91). Generally,
N375S in the SCC25 cell line) and two mutations in four tumor qPCR showed similar or lower copy numbers compared with FISH
samples in the transmembrane or JM domain (R988C, 3xT1010I; analysis. We subsequently analyzed 23 HNSCC tumor tissues from
Table 1A; Supplementary Fig. S2) were identified (previously patients by qPCR (Table 1C): 3 of 23 (13%) tumors showed gene
reported in other tumor types; refs. 12, 17). Furthermore, two copy numbers of >10 with one sample showing a copy number of
mutations in the TK domain (T1275I, V1333I) were identified, 22.1 and two samples 10.50/10.33 respectively. Furthermore, 15 of
which have not been described previously. No classic Y1230C/ 23 (65%) HNSCC tumors showed copy numbers of 4 to 10. There
Y1235D mutations were identified. All mutations were heterozy- was no apparent correlation with smoking status or anatomic site,
gous. The rate of TK domain mutations was 3% (2 of 66) and the although small sample numbers in subgroups do not allow for
rate of non-TK domain mutations was 9% (6 of 66). Overall, proper assessment.
Cancer Res 2009; 69: (7). April 1, 2009 3026 www.aacrjournals.org
The Role of MET in Head and Neck Cancer
MET SNPs in HNSCC tumor tissues and cell lines. In addition Gene array data was also consistent in showing overexpression
to mutations, multiple SNPs in the MET gene were identified as in HNC (Supplementary Fig. S1); furthermore, Ginos and
heterozygous (A48A in 2, S178S in 4, Q648Q in 5, I706I in 1, K1250K colleagues reported a link to an increased rate of locoregional
in 1, and D1304D/A1357A/P1382P occurred together in 22 samples) HNC recurrence (22).
and homozygous (Q648Q in 2, D1304D/A1357A/P13821P occurred Normal mucosa weakly expressed MET in the basal mucosa layer
together in 9 samples; Table 1D). (Fig. 1A), possibly linked to mucosa turnover/proliferation or field
cancerization. Reports by Chen and colleagues and Ohnishi and
colleagues suggest a role of MET in HNSCC dysplastic lesions
Discussion (24, 30).
In this study, we show that MET is a novel target for HNSCC The expression pattern was both cytoplasmic and membranous,
showing prominent overexpression, mutations, and increased gene closely resembling data in lung cancer (31); the relative cellular
copy number. We show the effectiveness of MET inhibition on cell localization seems to be tissue specific and the functional
signaling, viability, migration, and angiogenesis. Our data provide a implications are still being elucidated (31).
strong rationale to use MET inhibition in translational and clinical Similar to prior reports, we confirm elevated HGF expression
studies in HNC and suggest studying the integration with in 59% of HNSCC [45% strong expression (2+/3+); 15% weak
established treatments. expression (1+)] including the adjacent stroma, suggesting auto-
MET is activated in HNSCC patient samples and the presence of crine and/or paracrine signaling loops, which have been described
phosphorylated MET (66%) closely correlated with overall expres- in other tumor types (gliomas, pancreatic and liver carcinomas;
sion (79%): This is consistent with literature reports for HNC refs. 31–34). This may be another possible predictor of response
(70–90% expression; refs. 24–29) and is comparable with NSCLC (12). and e.g. recent reports for the EGFR ligand amphiregulin suggest a
Our study helps to explain the prominent MET overexpression correlation with sensitivity to EGFR treatments (35).
demonstrating increased copy numbers in a subset of tumors. The correlation MET/HGF expression/amplification status with
Although karyotypic analysis is still considered the gold standard, treatment outcomes is a high priority for future studies. Preclini-
Bean and colleagues confirmed the usefulness of qPCR when cally, Akervall and colleagues reported higher MET expression
compared with array CGH analysis (7). Although no MET-amplified based on gene array analysis in cisplatin-resistant HNSCC cell lines
HNC cell lines were identified, MET amplification has previously compared with sensitive ones (36) and Aebersold and colleagues
been reported in gastric carcinoma (14) and NSCLC (6, 7) and reported that MET expression correlated with radioresistance
correlates with sensitivity to MET small-molecule inhibitors (6, 14). (37, 38). Several studies describe increased MET/HGF expression in
This may be relevant for predicting HNC sensitivity to MET more invasive HNSCC (24, 25, 27, 39) as well as metastatic spread
inhibitors in future studies. (28, 29, 40). Finally, the role of epithelial to mesenchymal transition
Figure 5. A, HNSCC cell lines SCC35 and SCC61 were treated with cisplatin, SU11274, or combination at
indicated doses (ratio 1:1/1:2). Both single agents showed efficacy, decreasing viability between 40% and 70%.
The combination was consistently superior to either agent alone. The isobologram indicated synergy (CI <1).
B, HNSCC cell lines HN5 and SCC35 were treated with erlotinib, SU11274, or combination at equimolar doses. Both
single agents showed efficacy, but the combination was consistently superior to either agent alone. The isobolograms
indicate synergy (CI <1). C, immunoblotting of untreated and 4 h SU11274-, erlotinib-, and combination-treated
cells. Whereas both SU11274 and erlotinib suppress erbB3 and AKT phosphorylation, the combination achieves
increased suppression levels.
www.aacrjournals.org 3027 Cancer Res 2009; 69: (7). April 1, 2009
Table 1. MET sequencing and gene copy number analysis in HNSCC
Mutation Domain n n Zygosity
Tumor samples analyzed — — 66 —
Cell lines analyzed — — — 8
Mutations T230M SEMA 1 — Hetero
E168D SEMA 1 — Hetero
N375S SEMA — 1 Hetero
R988C JM 1 — Hetero
T1010I JM 4 — Hetero
T1275I TK 1 — Hetero
V1333I TK 1 — Hetero
Cell line (n = 14) Gene copy number Assessment
FISH qPCR SD
SCC61 >4 — — Increased copy number (by FISH)
JSQ3 >4 1.91 0.41 Increased copy number (by FISH)
HN31 >4 2.79 0.57 Increased copy number (by FISH)
SCC9 2 — — Normal
SQ20B 2 1.1 0.18 Normal
SCC28 2 — — Normal
SCC15 2 — — Normal
SCC25 2 2.03 0.49 Normal
SCC68 2 2.11 0.14 Normal
SCC35 — 3.56 0.98 Normal
HN5 — 1.83 0.19 Normal
MSK921 — 2.08 0.36 Normal
SCC17B — 2.98 0.89 Normal
OSCC3 — 2.73 0.84 Normal
Tumor site (n = 23) Tobacco Gene copy number (qPCR) SD Assessment
1. Larynx Yes 22.10 2.58 Increased copy number
2. BOT No 10.50 0.38 Increased copy number
3. BOT Yes 10.33 0.81 Increased copy number
4. Tonsil Yes 7.93 2.46 Increased copy number
5. Hypopharynx Yes 7.90 0.44 Increased copy number
6. Larynx Yes 7.85 1.33 Increased copy number
7. Unknown Pr. Yes 7.55 1.09 Increased copy number
8. Larynx Yes 7.35 0.38 Increased copy number
9. BOT Yes 7.27 0.42 Increased copy number
10. BOT Yes 6.09 0.19 Increased copy number
11. Larynx Yes 5.89 2.18 Increased copy number
12. Tongue Yes 5.67 0.33 Increased copy number
13. BOT Yes 5.42 2.48 Increased copy number
14. Buccal Yes 4.99 0.33 Increased copy number
15. Unknown Pr. Yes 4.96 0.36 Increased copy number
16. FOM Yes 4.96 0.18 Increased copy number
17. Larynx Yes 4.63 0.24 Increased copy number
18. Pharynx Yes 4.51 1.86 Increased copy number
19. Tonsil No 3.92 0.54
20. Tongue Yes 3.72 0.28
21. Larynx Yes 3.10 0.25
22. Tongue No 2.93 0.59
23. Larynx Yes 2.52 0.65
Summary >10 Copies: 3/23 = 13.0%
4–10 Copies: 15/23 = 65.2%
(Continued on the following page)
Cancer Res 2009; 69: (7). April 1, 2009 3028 www.aacrjournals.org
The Role of MET in Head and Neck Cancer
Table 1. MET sequencing and gene copy number analysis in HNSCC (Cont’d)
SNP n Zygosity
Tumor samples analyzed 66
A48A 2 Hetero
S178S 4 Hetero
Q648Q 7 5 Hetero/2 homo
I706I 1 Hetero
K1250K 1 Hetero
D1340D* 31 22 Hetero/9 homo
A1357A* 31 22 Hetero/9 homo
P1382P* 31 22 Hetero/9 homo
NOTE: A. Table of MET mutations/variants. T1010I JM mutations have been reported to increase MET-related functions (signaling, tumorigenicity,
motility; ref. 12). Somatic MET TK domain mutations have been described in HNSCC as gain-of-function mutations (13). The schema of MET gene
depicting mutations is provided in Supplementary Fig. S2. Fourteen cell lines (B) and 23 HNSCC tumor tissues (C) were analyzed by FISH and/or qPCR.
Increased gene copy number was identified in three tumor samples with >10 copies and 15 tumor samples with 4 to 10 copies (qPCR). Comparison of
FISH and qPCR results showed reasonable correlation, with qPCR underestimating copy number. All tissue samples were analyzed by qPCR. D. In
addition to mutations, sequencing of 66 HNSCC tumor tissues identified eight SNPs. Three SNPs clustered together (indicated by *) and were present in
31 samples (47.0%). Other SNPs were repeated in seven, four, and two separate tumor samples.
has also been implicated with poor prognosis for HNSCC (41) and inhibitor developed by Sugen/Pfizer and clinical development was
MET is a known driver of epithelial to mesenchymal transition (11). not pursued.
We report for the first time the identification of novel MET Various parameters have been suggested as predictors of response
alterations in the SEMA, JM, and TK domains in human HNSCC. to MET kinase inhibitors (31), including strong expression as seen,
The precise function is part of ongoing studies (13, 17): Previously, for example, in NSCLC (12), gene amplification as seen for gastric
DiRenzo and colleagues and Aebersold and colleagues described carcinomas (14), and kinase domain mutations (45) and potentially
TK domain mutations in HNSCC in Y1230C and Y1235D in up to ligand expression as reported for amphiregulin/EGFR (46). Unlike
10.9% of tumors (13, 38). In these studies, MET mutations primarily NSCLC and colon cancer, K-Ras mutations are not commonly
occurred in lymph node metastases and could only be detected observed in HNSCC. Our data suggest that generally higher MET
with a higher sensitivity method (single-strand conformational expression levels correlate with increased sensitivity to MET
polymorphism). In a subsequent study, however, Morello and inhibition but are not sufficient to explain the remaining substantial
colleagues did not identify any MET mutations in HNSCC (26). We variation in IC50 values. Additional factors modulate responsiveness
used standard PCR amplification and sequencing technology and and future studies may include correlation with increased gene copy
identified two novel TK domain mutations in one lymph node number, HGF expression, use of parallel RTK signaling cascades
metastasis and one primary tumor (T1275I and V1333I). We did not (Fig. 1C), and potentially gene mutation status (e.g., PTEN).
detect Y1230C and Y1235D mutations. TK domain mutations are AKT activation and ERK activation are oftentimes separate
reported to be somatic mutations in HNSCC and their functional events, with AKT being more prominently involved in cell survival
importance is well established in certain papillary renal cell and ERK in proliferation (47). Although sometimes regulated
cancers (germline or somatic mutations; ref. 42). The functional together (i.e., EGFR TK domain mutated NSCLC; ref. 47), it seems
role of TK domain mutations in HNSCC remains to be determined that, for most, HNSCC regulation is separate (Fig. 2B). It is possible
and high-sensitivity mutation screening/sequencing may be that concurrent inhibition of the pathways leading to ERK
required as seen for the EGFR T790M mutation in NSCLC (43). activation will increase therapeutic benefit.
We identify for the first time in HNSCC SEMA and JM domain Despite the lack of EGFR mutations in HNSCC in the United
mutations/variants. Such mutations/variants have previously been States (48), most HNSCC are sensitive to EGFR inhibitors and
reported in lung cancer. The JM domain changes have been overexpression is abundant (4). Recent evidence in NSCLC suggests
implicated in increased motility and invasiveness in SCLC (12, 17) a common signaling pathway via HER3/erbB3 (5–9). Specifically
and may have transforming properties (17). Preliminary reports Engelman and colleagues (6) implicated erbB3 signaling as the
suggest that both SEMA and JM domain mutations/variants can mediator of amplified MET ‘‘overtaking’’ mutant-EGFR signaling in
contribute to MET activation and may alter sensitivity to MET a NSCLC in vitro model of acquired gefitinib resistance. On the
inhibitors (44). In contrast to TK domain mutations, SEMA and JM other hand, the recent study by Tang and colleagues (9) suggested a
domain mutations/variants may be found in either germline DNA central role for erbB3 in mediating the efficacy of dual MET/EGFR
or somatically. inhibition against T790M-EGFR–mediated resistance in the
MET inhibition can readily be achieved with small-molecule TK absence of prior EGFR TKI selection pressure. Our data for HNSCC
inhibitors: PF-2341066 used here for in vivo studies is currently in now also suggest a similar role of MET/erbB3 in the absence of
phase I clinical testing. SU11274 is a poorly water-soluble earlier EGFR selection pressure.
www.aacrjournals.org 3029 Cancer Res 2009; 69: (7). April 1, 2009
Given the broad use of EGFR inhibition in HNSCC patients and provide the first evidence for HNSCC that MET suppression
the limited single-agent response rate (3), ways to increase efficacy abrogates a key component of the metastatic cascade.
with dual-kinase or multikinase inhibition are pivotal. In summary, we identified MET as a functionally important
Gene copy numbers seemed to be higher in tumor tissue receptor in HNSCC with activation and overexpression in tumor
samples compared with cell lines; most notably, we did not identify tissues and cell lines. Furthermore, we describe evidence of
any amplified cell lines. Although the reasons for this are unclear amplification and the presence of novel TK, SEMA, and JM domain
(possible selection pressure, bias of cell line/tumor choice), such mutations. The consistent effects of MET inhibition validate this
tumors with higher gene copy numbers may be more sensitive to target further and synergy with cisplatin and erlotinib is
MET inhibition. therapeutically relevant. Further mechanistic studies into the role
Our data also suggest exploring MET inhibition in combination of MET-mutated/amplified HNC are indicated and will allow us to
with cisplatin. Interestingly, Akervall and colleagues when com- better use MET-specific drugs for selected patient groups.
paring cisplatin-sensitive and cisplatin-resistant HNSCC cell lines
by gene microarray techniques identified MET overexpression in Disclosure of Potential Conflicts of Interest
resistant lines (36). Henceforth, MET may be involved in mediating E.E.W. Cohen and R. Salgia received a major commercial research grant from
Pfizer; E.E.W. Cohen, R. Salgia, and E. Vokes are consultants for Pfizer. The other
cisplatin resistance or could be a general poor prognostic marker. authors disclosed no potential conflicts of interest.
Further studies are indicated.
The proangiogenic properties of the MET/HGF axis are well Acknowledgments
established and MET signaling can initiate vascular endothelial Received 7/30/08; revised 11/18/08; accepted 2/2/09; published OnlineFirst 3/24/09.
growth factor production, a critical angiogenic switch via Shc (49). Grant support: Flight Attendant Medical Research Institute Young Clinical
We provide the first evidence of antiangiogenic effects of MET Scientist Award, IASLC Fellowship Award, and Cancer Research Foundation Young
Investigator Award (T.Y. Seiwert), NIH National Cancer Institute R01 grants CA100750-
inhibition in HNSCC in vivo using PF-2341066 in a Matrigel plug 04 and CA125541-02, American Lung Association, Institutional Cancer Research
model. A caveat is that murine HGF does not sufficiently activate awards from the University of Chicago Cancer Center with the V-Foundation
(R. Salgia), NIH grant DE12322 (M. Lingen), ASCO Career Development Award (E.E.W.
human MET (50); therefore, the use of a human HGF transgenic Cohen), and MARF Research Grant Award (R. Jagadeeswaran).
model is of interest (50). Furthermore, in vivo metastasis modeling The costs of publication of this article were defrayed in part by the payment of page
of MET overexpression, mutations, and amplification for HNC will charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
provide additional insight into the role of MET for HNC metastasis. We thank the support of the entire Salgia laboratory, Ralph Weichselbaum, Stuart
Migration/motility is a surrogate metastasis marker, and we Schwartz, Jose Manaligod, Maria Tretiakova, and Thomas Krausz.
References 11. Peruzzi B, Bottaro DP. Targeting the c-Met 21. Coutlee F, Gravitt P, Kornegay J, et al. Use of PGMY
signaling pathway in cancer. Clin Cancer Res 2006; primers in L1 consensus PCR improves detection of
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. 12:3657–60. human papillomavirus DNA in genital samples. J Clin
CA Cancer J Clin 2008;58:71–96. 12. Ma PC, Jagadeeswaran R, Jagadeesh S, et al. Microbiol 2002;40:902–7.
2. Vermorken JB, Mesia R, Rivera F, et al. Platinum-based Functional expression and mutations of c-Met and its 22. Ginos MA, Page GP, Michalowicz BS, et al. Identifi-
chemotherapy plus cetuximab in head and neck cancer. therapeutic inhibition with SU11274 and small interfer- cation of a gene expression signature associated with
N Engl J Med 2008;359:1116–27. ing RNA in non-small cell lung cancer. Cancer Res 2005; recurrent disease in squamous cell carcinoma of the
3. Vermorken JB, Trigo J, Hitt R, et al. Open-label, 65:1479–88. head and neck. Cancer Res 2004;64:55–63.
uncontrolled, multicenter phase II study to evaluate the 13. Di Renzo MF, Olivero M, Martone T, et al. Somatic 23. Puri N, Khramtsov A, Ahmed S, et al. A selective
efficacy and toxicity of cetuximab as a single agent in mutations of the MET oncogene are selected during small molecule inhibitor of c-Met, PHA665752, inhibits
patients with recurrent and/or metastatic squamous metastatic spread of human HNSC carcinomas. Onco- tumorigenicity and angiogenesis in mouse lung cancer
cell carcinoma of the head and neck who failed to gene 2000;19:1547–55. xenografts. Cancer Res 2007;67:3529–34.
respond to platinum-based therapy. J Clin Oncol 2007; 14. Smolen GA, Sordella R, Muir B, et al. Amplification 24. Chen YS, Wang JT, Chang YF, et al. Expression of
25:2171–7. of MET may identify a subset of cancers with extreme hepatocyte growth factor and c-met protein is signifi-
4. Grandis JR, Tweardy DJ. Elevated levels of trans- sensitivity to the selective tyrosine kinase inhibitor cantly associated with the progression of oral squamous
forming growth factor a and epidermal growth factor PHA-665752. Proc Natl Acad Sci U S A 2006;103:2316–21. cell carcinoma in Taiwan. J Oral Pathol Med 2004;33:
receptor messenger RNA are early markers of carcino- 15. Jagadeeswaran R, Surawska H, Krishnaswamy S, et al. 209–17.
genesis in head and neck cancer. Cancer Res 1993;53: Paxillin is a target for somatic mutations in lung cancer: 25. Lo Muzio L, Leonardi R, Mignogna MD, et al. Scatter
3579–84. implications for cell growth and invasion. Cancer Res factor receptor (c-Met) as possible prognostic factor in
5. Guo A, Villen J, Kornhauser J, et al. Signaling networks 2008;68:132–42. patients with oral squamous cell carcinoma. Anticancer
assembled by oncogenic EGFR and c-Met. Proc Natl 16. Jagadeeswaran R, Ma PC, Seiwert TY, et al. Func- Res 2004;24:1063–9.
Acad Sci U S A 2008;105:692–7. tional analysis of c-Met/hepatocyte growth factor 26. Morello S, Olivero M, Aimetti M, et al. MET receptor
6. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET pathway in malignant pleural mesothelioma. Cancer is overexpressed but not mutated in oral squamous cell
amplification leads to gefitinib resistance in lung Res 2006;66:352–61. carcinomas. J Cell Physiol 2001;189:285–90.
cancer by activating ERBB3 signaling. Science 2007; 17. Ma PC, Kijima T, Maulik G, et al. c-MET mutational 27. Murai M, Shen X, Huang L, et al. Overexpression of
316:1039–43. analysis in small cell lung cancer: novel juxtamembrane c-met in oral SCC promotes hepatocyte growth factor-
7. Bean J, Brennan C, Shih JY, et al. MET amplification domain mutations regulating cytoskeletal functions. induced disruption of cadherin junctions and invasion.
occurs with or without T790M mutations in EGFR Cancer Res 2003;63:6272–81. Int J Oncol 2004;25:831–40.
mutant lung tumors with acquired resistance to 18. Sattler M, Pride YB, Ma P, et al. A novel small 28. Kim CH, Moon SK, Bae JH, et al. Expression of
gefitinib or erlotinib. Proc Natl Acad Sci U S A 2007; molecule met inhibitor induces apoptosis in cells hepatocyte growth factor and c-Met in hypopharyngeal
104:20932–7. transformed by the oncogenic TPR-MET tyrosine kinase. squamous cell carcinoma. Acta Otolaryngol 2006;126:
8. Wheeler DL, Huang S, Kruser TJ, et al. Mechanisms of Cancer Res 2003;63:5462–9. 88–94.
acquired resistance to cetuximab: role of HER (ErbB) 19. Zou HY, Li Q, Lee JH, et al. An orally available small- 29. Yucel OT, Sungur A, Kaya S. c-met overexpression in
family members. Oncogene 2008;27:3944–56. molecule inhibitor of c-Met, PF-2341066, exhibits supraglottic laryngeal squamous cell carcinoma and its
9. Tang Z, Du R, Jiang S, et al. Dual MET-EGFR cytoreductive antitumor efficacy through antiprolifer- relation to lymph node metastases. Otolaryngol Head
combinatorial inhibition against T790M-EGFR-mediated ative and antiangiogenic mechanisms. Cancer Res 2007; Neck Surg 2004;130:698–703.
erlotinib-resistant lung cancer. Br J Cancer 2008;99: 67:4408–17. 30. Ohnishi T, Daikuhara Y. Hepatocyte growth factor/
911–922. 20. Chou TC, Talalay P. Quantitative analysis of dose- scatter factor in development, inflammation and
10. Ma PC, Maulik G, Christensen J, Salgia R. c-Met: effect relationships: the combined effects of multiple carcinogenesis: its expression and role in oral tissues.
structure, functions and potential for therapeutic drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22: Arch Oral Biol 2003;48:797–804.
inhibition. Cancer Metastasis Rev 2003;22:309–25. 27–55. 31. Ma PC, Tretiakova MS, MacKinnon AC, et al.
Cancer Res 2009; 69: (7). April 1, 2009 3030 www.aacrjournals.org
The Role of MET in Head and Neck Cancer
Expression and mutational analysis of MET in human resistance of oropharyngeal cancer to ionizing radiation. 45. Berthou S, Aebersold DM, Schmidt LS, et al. The Met
solid cancers. Genes Chromosomes Cancer 2008;47: Int J Cancer 2001;96:41–54. kinase inhibitor SU11274 exhibits a selective inhibition
1025–37. 38. Aebersold DM, Landt O, Berthou S, et al. pattern toward different receptor mutated variants.
32. Xie Q, Liu KD, Hu MY, Zhou K. SF/HGF-c-Met Prevalence and clinical impact of Met Y1253D- Oncogene 2004;23:5387–93.
autocrine and paracrine promote metastasis of activating point mutation in radiotherapy-treated 46. Yonesaka K, Zejnullahu K, Homes AJ, Johnson BE,
hepatocellular carcinoma. World J Gastroenterol 2001; squamous cell cancer of the oropharynx. Oncogene Janne PA. Presence of amphiregulin autocrine-loop
7:816–20. 2003;22:8519–23. predicts sensitivity of EGFR wild type cancers to
33. Rosen EM, Laterra J, Joseph A, et al. Scatter factor 39. Uchida D, Kawamata H, Omotehara F, et al. Role of gefitinib and cetuximab. Proceedings of the 99th Annual
expression and regulation in human glial tumors. Int J HGF/c-met system in invasion and metastasis of oral Meeting of the American Association for Cancer
Cancer 1996;67:248–55. squamous cell carcinoma cells in vitro and its clinical Research; 2008 Apr 12-16;San Diego, CA Philadelphia
34. Jin H, Yang R, Zheng Z, et al. MetMAb, the one-armed significance. Int J Cancer 2001;93:489–96. (PA): AACR; 2008. Abstract: 4958.
5D5 anti-c-Met antibody, inhibits orthotopic pancreatic 40. Cortesina G, Martone T, Galeazzi E, et al. Staging of 47. Engelman JA. The role of phosphoinositide 3-kinase
tumor growth and improves survival. Cancer Res 2008; head and neck squamous cell carcinoma using the MET pathway inhibitors in the treatment of lung cancer. Clin
68:4360–8. oncogene product as marker of tumor cells in lymph Cancer Res 2007;13:s4637–40.
35. Yonesaka K, Zejnullahu K, Homes AJ, Johnson BE, node metastases. Int J Cancer 2000;89:286–92. 48. Cohen EE, Lingen MW, Martin LE, et al. Response of
Janne PA. Presence of amphiregulin autocrine-loop 41. Chung CH, Parker JS, Karaca G, et al. Molecular clas- some head and neck cancers to epidermal growth factor
predicts sensitivity of EGFR wild type cancers to sification of head and neck squamous cell carcinomas using receptor tyrosine kinase inhibitors may be linked to
gefitinib and cetuximab. Proceedings of the 99th Annual patterns of gene expression. Cancer Cell 2004;5:489–500. mutation of ERBB2 rather than EGFR. Clin Cancer Res
Meeting of the American Association for Cancer 42. Jeffers M, Schmidt L, Nakaigawa N, et al. Activating 2005;11:8105–8.
Research; 2008 Apr 12-16. San Diego, CA. Philadelphia mutations for the met tyrosine kinase receptor in human 49. Saucier C, Khoury H, Lai KM, et al. The Shc adaptor
(PA): AACR; 2008. Abstract: 4958. cancer. Proc Natl Acad Sci U S A 1997;94:11445–50. protein is critical for VEGF induction by Met/HGF
36. Akervall J, Guo X, Qian CN, et al. Genetic and 43. Pao W, Miller VA, Politi KA, et al. Acquired resistance and ErbB2 receptors and for early onset of tumor
expression profiles of squamous cell carcinoma of the of lung adenocarcinomas to gefitinib or erlotinib is angiogenesis. Proc Natl Acad Sci U S A 2004;101:
head and neck correlate with cisplatin sensitivity and associated with a second mutation in the EGFR kinase 2345–50.
resistance in cell lines and patients. Clin Cancer Res 2004; domain. PLoS Med 2005;2:e73. 50. Zhang YW, Su Y, Lanning N, et al. Enhanced growth
10:8204–13. 44. Jiang S, Du R, Tang Z, Kern J, Ma P. Targeted of human met-expressing xenografts in a new strain of
37. Aebersold DM, Kollar A, Beer KT, Laissue J, Greiner inhibition of wild type and mutated MET receptor immunocompromised mice transgenic for human
RH, Djonov V. Involvement of the hepatocyte growth variants in the sema, juxtamembrane and kinase hepatocyte growth factor/scatter factor. Oncogene
factor/scatter factor receptor c-met and of Bcl-xL in the domain. J Thorac Oncol 2007;2:S546, P2–139. 2005;24:101–6.
www.aacrjournals.org 3031 Cancer Res 2009; 69: (7). April 1, 2009