TheMETReceptorTyrosineKinaseIsaPotentialNovelTherapeutic ... · Head and neck cancer (HNC) is the sixth most common cancer worldwide, with an annual incidence of >640,000 cases worldwide

The MET Receptor Tyrosine Kinase Is a Potential Novel Therapeutic

Target for Head and Neck Squamous Cell Carcinoma

Tanguy Y. Seiwert,1Ramasamy Jagadeeswaran,

1Leonardo Faoro,

1Varalakshmi Janamanchi,

1

Vidya Nallasura,1Mohamed El Dinali,

1Soheil Yala,

1Rajani Kanteti,

1Ezra E.W. Cohen,

1

Mark W. Lingen,2Leslie Martin,

2Soundararajan Krishnaswamy,

1Andres Klein-Szanto,

3

James G. Christensen,4Everett E. Vokes,

1and Ravi Salgia

1

1Section of Hematology/Oncology, Department of Medicine and University of Chicago Cancer Research Center and2Department of Pathology, University of Chicago, Chicago, Illinois; 3Department of Pathology, Fox ChaseCancer Center, Philadelphia, Pennsylvania; and 4Pfizer La Jolla Laboratories, La Jolla, California

Abstract

Recurrent/metastatic head and neck cancer remains a devas-tating disease with insufficient treatment options. We investi-gated the MET receptor tyrosine kinase as a novel target forthe treatment of head and neck squamous cell carcinoma(HNSCC). MET/phosphorylated MET and HGF expression wasanalyzed in 121 tissues (HNSCC/normal) by immunohisto-chemistry, and in 20 HNSCC cell lines by immunoblotting. Theeffects of MET inhibition using small interfering RNA/twosmall-molecule inhibitors (SU11274/PF-2341066) on signaling,migration, viability, and angiogenesis were determined. Thecomplete METgene was sequenced in 66 head and neck cancertissue samples and eight cell lines. METgene copy number wasdetermined in 14 cell lines and 23 tumor tissues. Drugcombinations of SU11274 with cisplatin or erlotinib weretested in SCC35/HN5 cell lines. Eighty-four percent of theHNSCC samples showed MET overexpression, whereas 18 of 20HNSCC cell lines (90%) expressed MET. HGF overexpressionwas present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling, cell viability, motility/migration in vitro , and tumor angiogenesis in vivo. Mutationalanalysis of 66 tumor tissues and 8 cell lines identified novelmutations in the semaphorin (T230M/E168D/N375S), juxta-membrane (T1010I/R988C), and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copynumber was present with >10 copies in 3 of 23 (13%) tumortissues. A greater-than-additive inhibition of cell growth wasobserved when combining a MET inhibitor with cisplatin orerlotinib and synergy may be mediated via erbB3/AKTsignaling. MET is functionally important in HNSCC withprominent overexpression, increased gene copy number, andmutations. MET inhibition abrogated MET functions, includ-ing proliferation, migration/motility, and angiogenesis. MET isa promising, novel target for HNSCC and combinationapproaches with cisplatin or EGFR inhibitors should beexplored. [Cancer Res 2009;69(7):3021–31]

Introduction

Head and neck cancer (HNC) is the sixth most common cancerworldwide, with an annual incidence of >640,000 cases worldwide

(47,560 cases in the United States; ref. 1). More than 90% of head

and neck cancers are of squamous histology (HNSCC). Thirty-fivepercent to 45% of head and neck cancer patients ultimately die

from their disease. Little progress has been made in the treatment

for metastatic/recurrent HNC during the past two decades, withthe singular exception of cetuximab, an epidermal growth factor

receptor (EGFR) antibody, which improves median survival by

2 months when added to standard chemotherapy (2). Overall

survival remains poor (median 6–10 months).To improve HNSCC treatment, relevant molecular targets need

to be identified. Receptor tyrosine kinases (RTK) seem to play a

pivotal role in the pathogenesis of HNC, with prior research

focusing on EGFR. Despite EGFR overexpression in >90% of

tumors, EGFR inhibition has only yielded low response rates of 4.3–

13% in clinical practice (3–4). Multiple lines of evidence indicate

that RTK pathway redundancies/cooperation are common in

RTK-driven malignancies and may account for resistance (5–8).

We studied the MET RTK and also explored EGFR/MET crosstalk

based on reports of cooperation in other diseases (6–9).MET, located on chromosome 7q31, encodes several functional

domains, including the semaphorin (SEMA) domain (ligand-binding), juxtamembrane (JM) domain (regulatory), and thereceptor tyrosine kinase (TK) domain (10, 11). The sole ligand forMET is hepatocyte growth factor (HGF, scatter factor), which isproduced by stromal and sometimes tumor cells (10, 11). HGFbinding activates MET via intracellular phosphorylation initiatingRAS-RAF-ERK, and phosphatidylinositol 3-kinase-AKT-mTOR sig-naling as well as several other pathways. In vivo , HGF/METsignaling leads to increased cell growth, cell motility, invasion/metastasis, angiogenesis, wound healing, and tissue regeneration(10, 11). Studies show that HGF/MET signaling increases motility,epithelial cell dispersion, endothelial cell migration, and chemo-taxis. Furthermore, MET overexpression and activation has trans-forming properties for normal cells (10, 11).

MET is overexpressed in a number of solid tumors, andexpression correlates with an aggressive phenotype and poorprognosis (10, 11). Previously, we had shown that in lung cancer,MET mutations can occur in the JM domain and the SEMAdomain, and not the TK domain (12). The precise function of mostmutations is not yet fully understood. MET mutations have beendescribed for HNC, especially in lymph node metastases (relativefrequency of up to 25% in some reports; ref. 13) and are located inthe TK domain similar to TK domain mutations found for renal cellcarcinomas (11), suggesting an important role for MET in HNC(13). However, mutations in the SEMA and JM domains have notbeen previously investigated for HNSCC. Further highlighting the

Requests for reprints: Tanguy Seiwert, Section of Hematology/Oncology,University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637.Phone: 773-702-2452; Fax: 773-702-3002; E-mail: [email protected].

I2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-2881

www.aacrjournals.org 3021 Cancer Res 2009; 69: (7). April 1, 2009

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importance of MET is the observation of MET amplification inseveral solid tumors, including subgroups of lung and gastriccancers (6, 7, 14, 15).

In our study, we used a large cohort of HNC and normal mucosatissues as well as cell lines to identify prominent MET expression,increased gene copy number, and mutations in the TK/JM/SEMAdomains. Furthermore, we show that MET inhibition alone and incombination with cisplatin or an EGFR inhibitor is a promisingtarget for head and neck cancer.

Materials and Methods

Tumor tissue arrays/immunohistochemistry. Tissue microarrays of97 HNC tissues and 24 normal mucosa samples were built (IRB: 8980).

Immunohistochemistry was performed for MET (C-12, 1:100), p-MET

(pY1003, Invitrogen, 1:25), HGF (H145, Santa Cruz, 1:50), human CD31

(JC70A, DAKO, 1:40), and Ki67 (RM-9106-S, NeoMarkers/Labvision, 1:300)as previously described (12, 15, 16). Appropriate negative controls were

prepared. Immunohistochemistry results from tumor and adjacent normal

tissue were compared semiquantitatively by a senior pathologist (grading:0 = negative, 1+ = low, 2+ = strong, 3+ = very strong expression; ref. 12).

Reagents and antibodies. Antibodies used for immunoblotting were

MET (3D4, Invitrogen/Zymed, C-12, Santa Cruz Biotechnology), phosphor-

ylation site–specific MET pY1003, and pY1230/4/5 (Biosource/Invitrogen,

44-882G, 44-888G), h-actin/p16/ERCC1/Ron-a antibodies (H-196/JC-8/8F1/

H-170, Santa Cruz Biotechnology), pTyr (4G10, Upstate/Millipore), and

insulin-like growth factor-I receptor (IGF-IR; Cell Signaling; dilution 1:1,000)

as previously described (12, 15–17).

The following drugs were purchased: SU11274/IGF-IR inhibitor (Calbio-chem), cisplatin (Sigma Aldrich), and erlotinib HCL (ACC). PF-2341066 was

kindly provided by Pfizer.

Cell lines and culture. Cell lines were obtained from the American Type

Culture Collection (SCC9/15/25/68/Cal27/Fadu), Dr. Ralph Weichselbaum(Department of Radiation Oncology, University of Chicago, Chicago, IL;

SQ20B, JSQ3, SCC35/61/294/151), Dr. Gary Clayman (M.D. Anderson Cancer

Center, Houston, TX; 1483), the Ludwig Institute for Cancer Research(London, United Kingdom; HN5), Dr. David Raben (University of Colorado

Health Sciences Center, Aurora, CO; MSK921), and Dr. Mark Lingen

(Department of Pathology, University of Chicago, Chicago, IL; OSCC3,

SCC17B/28/58) and maintained in DMEM/F12 or RPMI medium andpenicillin/streptomycin (Cellgro) with 10% fetal bovine serum (FBS; Gemini

Bioproducts). HaCaT is a spontaneously transformed human keratinocyte

cell line. HNX was derived from HN5 after prolonged subculture showing

suppressed EGFR and MET expression.Immunoblotting analysis. Immunoblots were done using standard

methodology (12, 15–17).

MET small interfering RNA/small-molecule inhibitors. Cells were

grown in antibiotic-free medium to 60% confluency in 96-well (viability)/6-well (immunoblotting) plates. MET small interfering RNA (siRNA) was used

at 100 Amol/L with Dharmafect transfection reagent (Dharmacon) using the

recommended protocol. Controls were treated with transfection agent only.Cells were incubated at 37jC in 5% CO2 for 36 to 72 h before viability was

assessed or before lysate was harvested.

MET inhibition was achieved using small-molecule MET inhibitors

SU11274 (ACC; ref. 18) and PF-2341066 (Pfizer; ref. 19; SupplementaryTable S1).

Viability. Measurement was performed using Alamar blue (Resazurin,

Sigma-Aldrich) or MTT (R&D Systems). Soft-agar colony formation assays

were performed as previously described (15). Viability results wereevaluated by a fluorescence/absorbance 96-well plate reader Synergy HT

(BioTek). Synergy was calculated using Calcusyn as described by Chou and

colleagues (20).Time-lapse video microscopy. Cells were plated on glass-bottomed

culture dishes (MatTek) in 10% FBS medium and grown for 24 h to achieve

20% to 30% confluency before drug treatment. Dishes were placed into a

temperature-controlled chamber at 37jC in an atmosphere of 5% CO2.

The cells were imaged on an Olympus IX81 inverted microscope anddigitally captured with IPLab software (Scanalytics). Images at �100

magnification were saved every 5 min and processed as mpeg4 movies

(Sonic DVD). Cell movement/morphologic changes were processed with

ImageJ (NIH), Photoshop (Adobe), and MetaMorph (Universal ImagingCorporation/Molecular Devices). The positions of the cell nuclei were

tracked, and distance/speed was calculated over 21 h.

Mutational analysis. Genomic DNA from 63 HNSCC tissues from

formalin-fixed paraffin-embedded tissues was obtained from the Universityof Chicago Head and Neck Cancer tissue bank (IRB: 8980). Genomic MET

reference sequences were obtained from position chr7:116,099,682–

116,225,676 from Ensembl (release 50; July 2008). Please note that the

MET transcript MET-001 (ENST00000318493) was used for identifyinggenetic changes (e.g., R988C) and MET-002 (ENST00000397752) for

identifying phosphorylation sites (e.g., Y1230), which is consistent with

common practice (12, 16).Real-time PCR. Quantitative real-time PCR for gene copy number

measurement was done as previously described (15) using ABI StepOnePlus

(Applied Biosystems) and iQ-SYBR green (Bio-Rad Laboratories). Reactions

were done in triplicates under standard thermocycling conditions (one cycle95jC � 12 min, 45 cycles 95jC � 20 s, 58jC � 1 min). The mean threshold

cycle number was used.

Fluorescence in situ hybridization. Fluorescence in situ hybridization

(FISH) analysis was done using two different BAC probes: RP11-433C10,localized to 7p11.2 ( full-length EGFR gene), and RP11-163C9, localized to

7q31.2 (MET gene). Two-color FISH was done using RP11-144B2 (red)

together with RP11-163C9 (green). The procedure was done as previouslydescribed (14), analyzing at least 10 metaphase cells.

Human papillomavirus testing. Human papillomavirus (HPV) testing

was performed in cell lines evaluating for p16 expression (JC-8, Santa Cruz

Biotechnology) and by PCR using L1 PGMY09/11 primers (21), followed bysequencing. HPV-positive results were confirmed using the Digene HPV test

(Qiagen).

In vivo Matrigel plug nude mouse xenograft modeling. Tumor cells

were mixed with Matrigel (BD Biosciences) and injected s.c. into the flanksof nude mice (5 � 106 cells/flank) following Institutional Animal Care and

Use Committee–approved protocols. The animals were monitored for 2 wk

and subsequently sacrificed. Tissues were fixed in 10% formalin and paraffinembedded.

Statistical analyses. Data are expressed as mean F SE. Statistical

significance was tested with Graphpad Prism5. For comparison between

two groups, Student’s t test or the m2 test was used. For comparing between>2 groups, one-way ANOVA was used. For evaluation of correlation,

Spearman’s test was used.

Results

MET/HGF are expressed in HNSCC tissues and cell lines.MET immunohistochemistry was done on 121 cores (97 cancers/24normal mucosa) as well as in phosphorylated MET (86 cancers/22normal mucosa). Eighty-five percent (n = 84) of HNSCC tumorsoverexpressed (2+/3+) METand 66% (n = 57) overexpressed (2+/3+)activated phosphorylated MET compared with adjacent normalmucosa (Fig. 1A and B). Normal mucosa also expressed MET(21% 1+, 21% 2+), albeit staining was weaker and primarily limited tothe basal layer of the mucosa (Fig. 1A ; 23% 1+/2+ for phosphorylatedMET). No cases of 3+ expression were seen for normal mucosa. METlocalized primarily to the membrane and the cytoplasm.

Immunoblot analysis confirmed strong MET expression in 16 of20 HNC cell lines [excluding HNX (derived from HN5) and HaCaT(transformed keratinocytes)]; however, SCC17B and SCC151expressed low levels of MET, which were outside the dynamic range(Fig. 1C). SQ20B and SCC294 had low to moderate MET expression.OSCC3, a HPV-positive cell line [p16+, PCR positive (HPV18), Digenehigh-risk HPV positive], showed strong MET expression. EGFR,

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Cancer Res 2009; 69: (7). April 1, 2009 3022 www.aacrjournals.org




IGF-IR, RON, and ERCC1 expression were prominent in several celllines. There was no statistical correlation with MET expression.

Analysis of MET gene expression using the publicly availableOncomine database5 and data by Ginos and colleagues (22) showedincreased MET gene expression in 41 HNSCC compared with 13normal controls (Supplementary Fig. S1).

HGF expression was evaluated in 68 HNC tumors by immuno-histochemistry. The tumors showed strong (3+; 21%), moderate (2+;24%), and weak (1+; 41%) HGF expression. Fifteen percent of thetumors were HGF negative.MET-specific small-molecule inhibitors or siRNAs inhibit

MET signaling. Using small-molecule MET inhibitors SU11274( for cell lines, DMSO soluble, Figs. 2 and 3), PF-2341066 (watersoluble, clinical candidate, Fig. 4; see Supplementary Table S1), andMET siRNA (Fig. 3B), MET activation/expression were suppressed.Figure 2A shows immunoblotting results for phosphotyrosine,whereas Fig. 2B shows results for phosphorylated MET anddownstream signaling effects in six HNSCC cell lines. Serum-

starved cells lines were pretreated with 0, 2, or 5 Amol/L of theMET inhibitor SU11274 followed by treatment with HGF for8 minutes. In cell lines SCC15, SCC28, and to a lesser degree SCC9and SCC61, HGF stimulation led to a strong p-Tyr signal, which wassuppressed with SU11274 MET inhibitor treatment. SCC17B overallhad low p-Tyr expression, suggestive of either a less RTK-drivenphenotype (5) or a more ligand-dependent phenotype. Despite lowMET expression, external HGF stimulation and SU11274 pretreat-ment showed typical signaling effects of the HGF/MET axis.

Phosphorylated MET expression was weak at baseline in moststarved cells. Following HGF stimulation in all cell lines, a strongphosphorylated MET response is observed that can be suppressedin a dose-dependent fashion (Fig. 2A and B). Downstream signalingfor phosphorylated AKT was also increased with HGF anddecreased by MET inhibition in cell lines SCC15, SQ20B, SCC28,and to a lesser degree in SCC61 (Fig. 2B). Phosphorylated ERK wasonly mildly affected by MET inhibition with SU11274.MET inhibition decreases viability in HNSCC. MET gene

silencing with MET-specific siRNA was used to validate effects ofMET inhibition in HNSCC. MET-specific siRNA duplexes weretransiently transfected into SCC61 and SQ20B cells (Fig. 3A), and

Figure 1. A, analysis of the frequencyand localization of MET expression byimmunohistochemistry in HNSCC andnormal adjacent mucosa. MET wasstrongly expressed (2+/3+) in 84% oftumors. Normal mucosa had negativeor low MET expression in 79% (0/1+),whereas 21% had 2+ staining (no 3+staining). MET expression wasmembranous and cytoplasmic.B, phospho-MET epitope pY1003immunohistochemistry: The stainingpattern closely resembled MET (A), withstrong expression in 71% of HNSCCsamples. C, MET was expressed in18 of 20 HNSCC cell lines as seen byimmunoblotting, excluding HNX (derivedfrom HN5) and HaCaT (immortalizedkeratinocytes). MET expression inSCC17B/HN5 was very low (outsidethe dynamic range). The 170-kDa(glycosylated MET) and 140-kDa bands(biologically active transmembrane hsubunit) are shown. RON expressionclosely follows MET expression (12 of 15),whereas expression of EGFR and IGF-IRis nonconcordant. ERCC1 (nucleotideexcision repair pathway) is present in mostcell lines. OSCC3 was HPV18+. D, HGFimmunohistochemical staining in HNSCCand normal adjacent mucosa. HGF wasexpressed in 41% of tumors with 2+expression in 21%. There was nosignificant HGF expression in normalmucosa.

5 http://www.oncomine.org

The Role of MET in Head and Neck Cancer





protein expression was decreased by >80% 72 hours aftertransfection.

siRNA down-regulation of MET protein expression in SCC61 andSQ20B cells resulted in inhibition of the serum-stimulated cellgrowth and viability by >62%/55% as determined by MTS assays(Fig. 3B).

We used SU11274 to test for its inhibitory effects on sevenHNSCC cell lines (Fig. 3C). MET inhibition was effective with IC50

values varying between 1 and 8 Amol/L: SCC61 (IC50 1 Amol/L),SCC35 (IC50 3 Amol/L), and SCC9 (IC50 3.8 Amol/L) were the mostsensitive lines followed by HN31 (IC50 5 Amol/L) and MSK921/SCC28 (IC50 5.4 Amol/L). SQ-20B, which has lower MET expressionand strong EGFR expression (EGFR amplification), showed anelevated IC50 of 8 Amol/L (extrapolated from Fig. 3C). Generally, a50% to 90% decrease in cell viability compared with control cellswas observed.

Furthermore, MET inhibition with SU11274 (3.5 Amol/L) led tosuppression of cell motility and migration. Figure 3D shows agraphical depiction of distances covered by individual cells (SCC61)over a period of 21 hours. SU11274-treated cells covered significantlyshorter distances (P = 0.0001) than untreated control cells. Thiseffect is consistent during the entire 21-h observation period.MET inhibition in vivo . To study MET inhibition effects on

angiogenesis, water-soluble PF-2341066 was used in vivo (Supple-mentary Table S1). PF-2341066 inhibited HGF-dependent METphosphorylation in a dose-dependent manner at concentrationsof 10 to 100 nmol/L in HNC cell lines SCC61 and SCC35 in vitro(Fig. 4A) and also in a soft-agar colony formation assay (OSCC3;Fig. 4B); no large colonies formed. Comparable results in colony

formation assays were observed for SCC61 and SCC35 (data notshown).

Effects on angiogenesis were investigated with an in vivoMatrigel xenograft tumor model of OSCC3 and SCC35 treatedwith PF-2341066 (25 mg/kg/d) versus control-treated cells (n = 3 ineach group). Figure 4 (C and D) shows abundant tumor growth in avehicle control–treated mouse, compared with minimal residualtumor nests in the PF-2341066 group. Staining for the proliferationmarker Ki67 shows >80% to 90% suppression of proliferation in PF-2341066–treated animals. Finally, staining of endothelial cells inblood vessels with CD31 shows extensive tumor vessels betweentumor nests in control-treated animal versus marked angiogenesissuppression in PF-2341066–treated animals, consistent with priorreports using a related MET inhibitor in vivo (23).SU11274 can synergize with erlotinib and cisplatin. Figure 5

shows four examples of dual treatment with MET inhibitorSU11274 in combination with commonly used agents—cisplatinor erlotinib.

SCC35 and SCC61, which required doses of >10 Amol/L toapproach IC50 toxicity (SCC35 >10 Amol/L cisplatin; SCC6116 Amol/L cisplatin), were synergistically inhibited by combinedtreatment with SU11274/cisplatin (SCC35 IC50 1.3/1.3 Amol/L,and SCC61 IC50 1/2 Amol/L). Based on the median effect model byChou (20), the isobologram graph shows combinatorial index (CI)values below 1 for the ED50 and ED75 (values <1 indicate synergy).

For combination with erlotinib, HN5 and SCC35 were chosen.Cells were treated with either agent alone or with a combination ofboth at equimolar doses. Both single agents showed efficacy,decreasing viability. The combination, however, was consistently

Figure 2. A, phosphorylated tyrosine(p-Tyr ) immunoblot of six HNSCC cell lineswith or without HGF stimulation andinhibition with SU11274. Expression ofphosphotyrosine is seen in all cell lines inresponse to HGF treatment. SCC9 andSQ20B have the highest backgroundp-Tyr expression. SU11274 pretreatmentwith 2 Amol/L and 5 Amol/L SU11274affected phosphorylated tyrosine levels.B, stimulation of MET phosphorylationwith 2 Amol/L and downstream signaling infive HNSCC cell lines is completelyabrogated by pretreatment withSU11274. Downstream AKT and ERKphosphorylation is partially affected incertain cell lines.

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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 notsuppress 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 (�62%/55%). C, SU11274 treatment led to a dose-dependentdecrease in cell viability compared with untreated control (DMSO solvent). In decreasing order of sensitivity (IC50), the following HNSCC cell lines responded toincreasing SU11274 concentrations: SCC61, SCC35, SCC9, HN31, MSK921, SCC28 and SQ20B. D, migration was significantly decreased after treatment withSU11274 over a tracking period of 21 h. Colored lines, individual cell movement over 21 h. Cells treated with SU11274 moved significantly shorter distances(�38%; P < 0.0001).






significantly superior to either agent alone. The isobologram showsthat at ED25 (CI = 0.73/035), ED50 (CI = 0.32/0.36), and ED75(CI = 0.21/0.36), there was synergistic activity between erlotinib andSU11274 (CI <1). Evaluation of downstream signaling in Fig. 5Cindicated that activation of erbB3 and subsequently AKT aresynergistically inhibited.MET mutations in HNSCC tumor tissues and cell lines. The

entire MET coding region (schema in Supplementary Fig. S2) wassequenced in 66 HNSCC and 8 cell lines. Three mutations in theligand-binding SEMA domain (T230M/E168D in the tumor tissue;N375S in the SCC25 cell line) and two mutations in four tumorsamples in the transmembrane or JM domain (R988C, 3xT1010I;Table 1A; Supplementary Fig. S2) were identified (previouslyreported in other tumor types; refs. 12, 17). Furthermore, twomutations in the TK domain (T1275I, V1333I) were identified,which have not been described previously. No classic Y1230C/Y1235D mutations were identified. All mutations were heterozy-gous. The rate of TK domain mutations was 3% (2 of 66) and therate of non-TK domain mutations was 9% (6 of 66). Overall,

mutations occurred in 12% of tumors analyzed (8 of 66). There wasno apparent correlation with smoking status or anatomic site,although the sample number was too small to allow sufficientstatistical power.MET gene copy number. We analyzed a panel of nine HNSCC

cell lines by FISH and followed this up with qPCR due to the readyavailability of DNA from HNSCC tumor tissues. Repetition of celllines previously analyzed by FISH now using qPCR was done(Table 1B). FISH analysis showed three cell lines with >4 copies,although qPCR copy number was lower (2.79 and 1.91). Generally,qPCR showed similar or lower copy numbers compared with FISHanalysis. We subsequently analyzed 23 HNSCC tumor tissues frompatients by qPCR (Table 1C): 3 of 23 (13%) tumors showed genecopy numbers of >10 with one sample showing a copy number of22.1 and two samples 10.50/10.33 respectively. Furthermore, 15 of23 (65%) HNSCC tumors showed copy numbers of 4 to 10. Therewas no apparent correlation with smoking status or anatomic site,although small sample numbers in subgroups do not allow forproper assessment.

Figure 4. A, immunoblot of twoHNSCC cell lines, SCC61 and SCC35,after treatment with PF-2341066 atdoses ranging from 0 to 500 nmol/L.PF-2341066 led to a dose-dependentabrogation of HGF-induced METphosphorylation. B, soft agar colonyformation assay of OSCC3 HNSCC withand without PF-2341066 treatment(0–1,000 nmol/L). Colonies were stainedwith crystal violet and counted. Photoshows comparison of 0 and 1,000 nmol/L.There was marked suppression of colonyformation by 33%/53%. RepresentativeH&E-stained images (originalmagnification, �20) of OSCC3 (C ) andSCC35 (D ) xenograft tumors from micetreated with vehicle or PF-2341066(25 mg/kg body weight). Animals weresacrificed before development ofmacroscopic tumors. Middle and bottom,Ki67/CD31 immunohistochemistry.PF-2341066 reduced cell proliferation(Ki67) and reduced blood vessel density(CD31), demonstrating in vivo activity ofPF-2341066 on angiogenesis.

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MET SNPs in HNSCC tumor tissues and cell lines. In additionto mutations, multiple SNPs in the MET gene were identified asheterozygous (A48A in 2, S178S in 4, Q648Q in 5, I706I in 1, K1250Kin 1, and D1304D/A1357A/P1382P occurred together in 22 samples)and homozygous (Q648Q in 2, D1304D/A1357A/P13821P occurredtogether in 9 samples; Table 1D).

Discussion

In this study, we show that MET is a novel target for HNSCCshowing prominent overexpression, mutations, and increased genecopy number. We show the effectiveness of MET inhibition on cellsignaling, viability, migration, and angiogenesis. Our data provide astrong rationale to use MET inhibition in translational and clinicalstudies in HNC and suggest studying the integration withestablished treatments.

MET is activated in HNSCC patient samples and the presence ofphosphorylated MET (66%) closely correlated with overall expres-sion (79%): This is consistent with literature reports for HNC(70–90% expression; refs. 24–29) and is comparable with NSCLC (12).

Our study helps to explain the prominent MET overexpressiondemonstrating increased copy numbers in a subset of tumors.Although karyotypic analysis is still considered the gold standard,Bean and colleagues confirmed the usefulness of qPCR whencompared with array CGH analysis (7). Although no MET-amplifiedHNC cell lines were identified, MET amplification has previouslybeen reported in gastric carcinoma (14) and NSCLC (6, 7) andcorrelates with sensitivity to MET small-molecule inhibitors (6, 14).This may be relevant for predicting HNC sensitivity to METinhibitors in future studies.

Gene array data was also consistent in showing overexpressionin HNC (Supplementary Fig. S1); furthermore, Ginos andcolleagues reported a link to an increased rate of locoregionalHNC recurrence (22).

Normal mucosa weakly expressed MET in the basal mucosa layer(Fig. 1A), possibly linked to mucosa turnover/proliferation or fieldcancerization. Reports by Chen and colleagues and Ohnishi andcolleagues suggest a role of MET in HNSCC dysplastic lesions(24, 30).

The expression pattern was both cytoplasmic and membranous,closely resembling data in lung cancer (31); the relative cellularlocalization seems to be tissue specific and the functionalimplications are still being elucidated (31).

Similar to prior reports, we confirm elevated HGF expressionin 59% of HNSCC [45% strong expression (2+/3+); 15% weakexpression (1+)] including the adjacent stroma, suggesting auto-crine and/or paracrine signaling loops, which have been describedin other tumor types (gliomas, pancreatic and liver carcinomas;refs. 31–34). This may be another possible predictor of responseand e.g. recent reports for the EGFR ligand amphiregulin suggest acorrelation with sensitivity to EGFR treatments (35).

The correlation MET/HGF expression/amplification status withtreatment outcomes is a high priority for future studies. Preclini-cally, Akervall and colleagues reported higher MET expressionbased on gene array analysis in cisplatin-resistant HNSCC cell linescompared with sensitive ones (36) and Aebersold and colleaguesreported that MET expression correlated with radioresistance(37, 38). Several studies describe increased MET/HGF expression inmore invasive HNSCC (24, 25, 27, 39) as well as metastatic spread(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 atindicated 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. Bothsingle agents showed efficacy, but the combination was consistently superior to either agent alone. The isobologramsindicate synergy (CI <1). C, immunoblotting of untreated and 4 h SU11274-, erlotinib-, and combination-treatedcells. Whereas both SU11274 and erlotinib suppress erbB3 and AKT phosphorylation, the combination achievesincreased suppression levels.






Table 1. MET sequencing and gene copy number analysis in HNSCC

A

Mutation Domain n n Zygosity

Tumor samples analyzed — — 66 —Cell lines analyzed — — — 8Mutations T230M SEMA 1 — Hetero

E168D SEMA 1 — HeteroN375S SEMA — 1 HeteroR988C JM 1 — HeteroT1010I JM 4 — HeteroT1275I TK 1 — HeteroV1333I TK 1 — Hetero

B

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 — — NormalSQ20B 2 1.1 0.18 NormalSCC28 2 — — NormalSCC15 2 — — NormalSCC25 2 2.03 0.49 NormalSCC68 2 2.11 0.14 NormalSCC35 — 3.56 0.98 NormalHN5 — 1.83 0.19 NormalMSK921 — 2.08 0.36 NormalSCC17B — 2.98 0.89 NormalOSCC3 — 2.73 0.84 Normal

C

Tumor site (n = 23) Tobacco Gene copy number (qPCR) SD Assessment

1. Larynx Yes 22.10 2.58 Increased copy number2. BOT No 10.50 0.38 Increased copy number3. BOT Yes 10.33 0.81 Increased copy number4. Tonsil Yes 7.93 2.46 Increased copy number5. Hypopharynx Yes 7.90 0.44 Increased copy number6. Larynx Yes 7.85 1.33 Increased copy number7. Unknown Pr. Yes 7.55 1.09 Increased copy number8. Larynx Yes 7.35 0.38 Increased copy number9. BOT Yes 7.27 0.42 Increased copy number10. BOT Yes 6.09 0.19 Increased copy number11. Larynx Yes 5.89 2.18 Increased copy number12. Tongue Yes 5.67 0.33 Increased copy number13. BOT Yes 5.42 2.48 Increased copy number14. Buccal Yes 4.99 0.33 Increased copy number15. Unknown Pr. Yes 4.96 0.36 Increased copy number16. FOM Yes 4.96 0.18 Increased copy number17. Larynx Yes 4.63 0.24 Increased copy number18. Pharynx Yes 4.51 1.86 Increased copy number19. Tonsil No 3.92 0.5420. Tongue Yes 3.72 0.2821. Larynx Yes 3.10 0.2522. Tongue No 2.93 0.5923. Larynx Yes 2.52 0.65Summary >10 Copies: 3/23 = 13.0%

4–10 Copies: 15/23 = 65.2%

(Continued on the following page)

Cancer Research





has also been implicated with poor prognosis for HNSCC (41) andMET is a known driver of epithelial to mesenchymal transition (11).

We report for the first time the identification of novel METalterations in the SEMA, JM, and TK domains in human HNSCC.The precise function is part of ongoing studies (13, 17): Previously,DiRenzo and colleagues and Aebersold and colleagues describedTK domain mutations in HNSCC in Y1230C and Y1235D in up to10.9% of tumors (13, 38). In these studies, MET mutations primarilyoccurred in lymph node metastases and could only be detectedwith a higher sensitivity method (single-strand conformationalpolymorphism). In a subsequent study, however, Morello andcolleagues did not identify any MET mutations in HNSCC (26). Weused standard PCR amplification and sequencing technology andidentified two novel TK domain mutations in one lymph nodemetastasis and one primary tumor (T1275I and V1333I). We did notdetect Y1230C and Y1235D mutations. TK domain mutations arereported to be somatic mutations in HNSCC and their functionalimportance is well established in certain papillary renal cellcancers (germline or somatic mutations; ref. 42). The functionalrole of TK domain mutations in HNSCC remains to be determinedand high-sensitivity mutation screening/sequencing may berequired as seen for the EGFR T790M mutation in NSCLC (43).

We identify for the first time in HNSCC SEMA and JM domainmutations/variants. Such mutations/variants have previously beenreported in lung cancer. The JM domain changes have beenimplicated in increased motility and invasiveness in SCLC (12, 17)and may have transforming properties (17). Preliminary reportssuggest that both SEMA and JM domain mutations/variants cancontribute to MET activation and may alter sensitivity to METinhibitors (44). In contrast to TK domain mutations, SEMA and JMdomain mutations/variants may be found in either germline DNAor somatically.

MET inhibition can readily be achieved with small-molecule TKinhibitors: PF-2341066 used here for in vivo studies is currently inphase I clinical testing. SU11274 is a poorly water-soluble earlier

inhibitor developed by Sugen/Pfizer and clinical development wasnot pursued.

Various parameters have been suggested as predictors of responseto MET kinase inhibitors (31), including strong expression as seen,for example, in NSCLC (12), gene amplification as seen for gastriccarcinomas (14), and kinase domain mutations (45) and potentiallyligand expression as reported for amphiregulin/EGFR (46). UnlikeNSCLC and colon cancer, K-Ras mutations are not commonlyobserved in HNSCC. Our data suggest that generally higher METexpression levels correlate with increased sensitivity to METinhibition but are not sufficient to explain the remaining substantialvariation in IC50 values. Additional factors modulate responsivenessand future studies may include correlation with increased gene copynumber, HGF expression, use of parallel RTK signaling cascades(Fig. 1C), and potentially gene mutation status (e.g., PTEN).

AKT activation and ERK activation are oftentimes separateevents, with AKT being more prominently involved in cell survivaland ERK in proliferation (47). Although sometimes regulatedtogether (i.e., EGFR TK domain mutated NSCLC; ref. 47), it seemsthat, for most, HNSCC regulation is separate (Fig. 2B). It is possiblethat concurrent inhibition of the pathways leading to ERKactivation will increase therapeutic benefit.

Despite the lack of EGFR mutations in HNSCC in the UnitedStates (48), most HNSCC are sensitive to EGFR inhibitors andoverexpression is abundant (4). Recent evidence in NSCLC suggestsa common signaling pathway via HER3/erbB3 (5–9). SpecificallyEngelman and colleagues (6) implicated erbB3 signaling as themediator of amplified MET ‘‘overtaking’’ mutant-EGFR signaling ina NSCLC in vitro model of acquired gefitinib resistance. On theother hand, the recent study by Tang and colleagues (9) suggested acentral role for erbB3 in mediating the efficacy of dual MET/EGFRinhibition against T790M-EGFR–mediated resistance in theabsence of prior EGFR TKI selection pressure. Our data for HNSCCnow also suggest a similar role of MET/erbB3 in the absence ofEGFR selection pressure.

Table 1. MET sequencing and gene copy number analysis in HNSCC (Cont’d)

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 genedepicting 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. Inaddition 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.






Given the broad use of EGFR inhibition in HNSCC patients andthe limited single-agent response rate (3), ways to increase efficacywith dual-kinase or multikinase inhibition are pivotal.

Gene copy numbers seemed to be higher in tumor tissuesamples compared with cell lines; most notably, we did not identifyany amplified cell lines. Although the reasons for this are unclear(possible selection pressure, bias of cell line/tumor choice), suchtumors with higher gene copy numbers may be more sensitive toMET inhibition.

Our data also suggest exploring MET inhibition in combinationwith cisplatin. Interestingly, Akervall and colleagues when com-paring cisplatin-sensitive and cisplatin-resistant HNSCC cell linesby gene microarray techniques identified MET overexpression inresistant lines (36). Henceforth, MET may be involved in mediatingcisplatin resistance or could be a general poor prognostic marker.Further studies are indicated.

The proangiogenic properties of the MET/HGF axis are wellestablished and MET signaling can initiate vascular endothelialgrowth factor production, a critical angiogenic switch via Shc (49).We provide the first evidence of antiangiogenic effects of METinhibition in HNSCC in vivo using PF-2341066 in a Matrigel plugmodel. A caveat is that murine HGF does not sufficiently activatehuman MET (50); therefore, the use of a human HGF transgenicmodel is of interest (50). Furthermore, in vivo metastasis modelingof MET overexpression, mutations, and amplification for HNC willprovide additional insight into the role of MET for HNC metastasis.Migration/motility is a surrogate metastasis marker, and we

provide the first evidence for HNSCC that MET suppressionabrogates a key component of the metastatic cascade.

In summary, we identified MET as a functionally importantreceptor in HNSCC with activation and overexpression in tumortissues and cell lines. Furthermore, we describe evidence ofamplification and the presence of novel TK, SEMA, and JM domainmutations. The consistent effects of MET inhibition validate thistarget further and synergy with cisplatin and erlotinib istherapeutically relevant. Further mechanistic studies into the roleof MET-mutated/amplified HNC are indicated and will allow us tobetter use MET-specific drugs for selected patient groups.

Disclosure of Potential Conflicts of Interest

E.E.W. Cohen and R. Salgia received a major commercial research grant fromPfizer; E.E.W. Cohen, R. Salgia, and E. Vokes are consultants for Pfizer. The otherauthors disclosed no potential conflicts of interest.

Acknowledgments

Received 7/30/08; revised 11/18/08; accepted 2/2/09; published OnlineFirst 3/24/09.Grant support: Flight Attendant Medical Research Institute Young Clinical

Scientist Award, IASLC Fellowship Award, and Cancer Research Foundation YoungInvestigator Award (T.Y. Seiwert), NIH National Cancer Institute R01 grants CA100750-04 and CA125541-02, American Lung Association, Institutional Cancer Researchawards 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.Cohen), and MARF Research Grant Award (R. Jagadeeswaran).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the support of the entire Salgia laboratory, Ralph Weichselbaum, StuartSchwartz, Jose Manaligod, Maria Tretiakova, and Thomas Krausz.

Cancer Research


References

1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008.CA Cancer J Clin 2008;58:71–96.2. Vermorken JB, Mesia R, Rivera F, et al. Platinum-basedchemotherapy plus cetuximab in head and neck cancer.N Engl J Med 2008;359:1116–27.3. Vermorken JB, Trigo J, Hitt R, et al. Open-label,uncontrolled, multicenter phase II study to evaluate theefficacy and toxicity of cetuximab as a single agent inpatients with recurrent and/or metastatic squamouscell carcinoma of the head and neck who failed torespond to platinum-based therapy. J Clin Oncol 2007;25:2171–7.4. Grandis JR, Tweardy DJ. Elevated levels of trans-forming growth factor a and epidermal growth factorreceptor messenger RNA are early markers of carcino-genesis in head and neck cancer. Cancer Res 1993;53:3579–84.5. Guo A, Villen J, Kornhauser J, et al. Signaling networksassembled by oncogenic EGFR and c-Met. Proc NatlAcad Sci U S A 2008;105:692–7.6. Engelman JA, Zejnullahu K, Mitsudomi T, et al. METamplification leads to gefitinib resistance in lungcancer by activating ERBB3 signaling. Science 2007;316:1039–43.7. Bean J, Brennan C, Shih JY, et al. MET amplificationoccurs with or without T790M mutations in EGFRmutant lung tumors with acquired resistance togefitinib or erlotinib. Proc Natl Acad Sci U S A 2007;104:20932–7.8. Wheeler DL, Huang S, Kruser TJ, et al. Mechanisms ofacquired resistance to cetuximab: role of HER (ErbB)family members. Oncogene 2008;27:3944–56.9. Tang Z, Du R, Jiang S, et al. Dual MET-EGFRcombinatorial inhibition against T790M-EGFR-mediatederlotinib-resistant lung cancer. Br J Cancer 2008;99:911–922.10. Ma PC, Maulik G, Christensen J, Salgia R. c-Met:structure, functions and potential for therapeuticinhibition. Cancer Metastasis Rev 2003;22:309–25.

11. Peruzzi B, Bottaro DP. Targeting the c-Metsignaling pathway in cancer. Clin Cancer Res 2006;12:3657–60.12. Ma PC, Jagadeeswaran R, Jagadeesh S, et al.Functional expression and mutations of c-Met and itstherapeutic inhibition with SU11274 and small interfer-ing RNA in non-small cell lung cancer. Cancer Res 2005;65:1479–88.13. Di Renzo MF, Olivero M, Martone T, et al. Somaticmutations of the MET oncogene are selected duringmetastatic spread of human HNSC carcinomas. Onco-gene 2000;19:1547–55.14. Smolen GA, Sordella R, Muir B, et al. Amplificationof MET may identify a subset of cancers with extremesensitivity to the selective tyrosine kinase inhibitorPHA-665752. Proc Natl Acad Sci U S A 2006;103:2316–21.15. Jagadeeswaran R, Surawska H, Krishnaswamy S, et al.Paxillin is a target for somatic mutations in lung cancer:implications for cell growth and invasion. Cancer Res2008;68:132–42.16. Jagadeeswaran R, Ma PC, Seiwert TY, et al. Func-tional analysis of c-Met/hepatocyte growth factorpathway in malignant pleural mesothelioma. CancerRes 2006;66:352–61.17. Ma PC, Kijima T, Maulik G, et al. c-MET mutationalanalysis in small cell lung cancer: novel juxtamembranedomain mutations regulating cytoskeletal functions.Cancer Res 2003;63:6272–81.18. Sattler M, Pride YB, Ma P, et al. A novel smallmolecule met inhibitor induces apoptosis in cellstransformed by the oncogenic TPR-MET tyrosine kinase.Cancer Res 2003;63:5462–9.19. Zou HY, Li Q, Lee JH, et al. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibitscytoreductive antitumor efficacy through antiprolifer-ative and antiangiogenic mechanisms. Cancer Res 2007;67:4408–17.20. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multipledrugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55.

21. Coutlee F, Gravitt P, Kornegay J, et al. Use of PGMYprimers in L1 consensus PCR improves detection ofhuman papillomavirus DNA in genital samples. J ClinMicrobiol 2002;40:902–7.22. Ginos MA, Page GP, Michalowicz BS, et al. Identifi-cation of a gene expression signature associated withrecurrent disease in squamous cell carcinoma of thehead and neck. Cancer Res 2004;64:55–63.23. Puri N, Khramtsov A, Ahmed S, et al. A selectivesmall molecule inhibitor of c-Met, PHA665752, inhibitstumorigenicity and angiogenesis in mouse lung cancerxenografts. Cancer Res 2007;67:3529–34.24. Chen YS, Wang JT, Chang YF, et al. Expression ofhepatocyte growth factor and c-met protein is signifi-cantly associated with the progression of oral squamouscell carcinoma in Taiwan. J Oral Pathol Med 2004;33:209–17.25. Lo Muzio L, Leonardi R, Mignogna MD, et al. Scatterfactor receptor (c-Met) as possible prognostic factor inpatients with oral squamous cell carcinoma. AnticancerRes 2004;24:1063–9.26. Morello S, Olivero M, Aimetti M, et al. MET receptoris overexpressed but not mutated in oral squamous cellcarcinomas. J Cell Physiol 2001;189:285–90.27. Murai M, Shen X, Huang L, et al. Overexpression ofc-met in oral SCC promotes hepatocyte growth factor-induced disruption of cadherin junctions and invasion.Int J Oncol 2004;25:831–40.28. Kim CH, Moon SK, Bae JH, et al. Expression ofhepatocyte growth factor and c-Met in hypopharyngealsquamous cell carcinoma. Acta Otolaryngol 2006;126:88–94.29. Yucel OT, Sungur A, Kaya S. c-met overexpression insupraglottic laryngeal squamous cell carcinoma and itsrelation to lymph node metastases. Otolaryngol HeadNeck Surg 2004;130:698–703.30. Ohnishi T, Daikuhara Y. Hepatocyte growth factor/scatter factor in development, inflammation andcarcinogenesis: its expression and role in oral tissues.Arch Oral Biol 2003;48:797–804.31. Ma PC, Tretiakova MS, MacKinnon AC, et al.






Expression and mutational analysis of MET in humansolid cancers. Genes Chromosomes Cancer 2008;47:1025–37.32. Xie Q, Liu KD, Hu MY, Zhou K. SF/HGF-c-Metautocrine and paracrine promote metastasis ofhepatocellular carcinoma. World J Gastroenterol 2001;7:816–20.33. Rosen EM, Laterra J, Joseph A, et al. Scatter factorexpression and regulation in human glial tumors. Int JCancer 1996;67:248–55.34. Jin H, Yang R, Zheng Z, et al. MetMAb, the one-armed5D5 anti-c-Met antibody, inhibits orthotopic pancreatictumor growth and improves survival. Cancer Res 2008;68:4360–8.35. Yonesaka K, Zejnullahu K, Homes AJ, Johnson BE,Janne PA. Presence of amphiregulin autocrine-looppredicts sensitivity of EGFR wild type cancers togefitinib and cetuximab. Proceedings of the 99th AnnualMeeting of the American Association for CancerResearch; 2008 Apr 12-16. San Diego, CA. Philadelphia(PA): AACR; 2008. Abstract: 4958.36. Akervall J, Guo X, Qian CN, et al. Genetic andexpression profiles of squamous cell carcinoma of thehead and neck correlate with cisplatin sensitivity andresistance in cell lines and patients. Clin Cancer Res 2004;10:8204–13.37. Aebersold DM, Kollar A, Beer KT, Laissue J, GreinerRH, Djonov V. Involvement of the hepatocyte growthfactor/scatter factor receptor c-met and of Bcl-xL in the

resistance of oropharyngeal cancer to ionizing radiation.Int J Cancer 2001;96:41–54.38. Aebersold DM, Landt O, Berthou S, et al.Prevalence and clinical impact of Met Y1253D-activating point mutation in radiotherapy-treatedsquamous cell cancer of the oropharynx. Oncogene2003;22:8519–23.39. Uchida D, Kawamata H, Omotehara F, et al. Role ofHGF/c-met system in invasion and metastasis of oralsquamous cell carcinoma cells in vitro and its clinicalsignificance. Int J Cancer 2001;93:489–96.40. Cortesina G, Martone T, Galeazzi E, et al. Staging ofhead and neck squamous cell carcinoma using the METoncogene product as marker of tumor cells in lymphnode metastases. Int J Cancer 2000;89:286–92.41. Chung CH, Parker JS, Karaca G, et al. Molecular clas-sificationofhead andneck squamous cell carcinomasusingpatterns of gene expression. Cancer Cell 2004;5:489–500.42. Jeffers M, Schmidt L, Nakaigawa N, et al. Activatingmutations for the met tyrosine kinase receptor in humancancer. Proc Natl Acad Sci U S A 1997;94:11445–50.43. Pao W, Miller VA, Politi KA, et al. Acquired resistanceof lung adenocarcinomas to gefitinib or erlotinib isassociated with a second mutation in the EGFR kinasedomain. PLoS Med 2005;2:e73.44. Jiang S, Du R, Tang Z, Kern J, Ma P. Targetedinhibition of wild type and mutated MET receptorvariants in the sema, juxtamembrane and kinasedomain. J Thorac Oncol 2007;2:S546, P2–139.

45. Berthou S, Aebersold DM, Schmidt LS, et al. The Metkinase inhibitor SU11274 exhibits a selective inhibitionpattern toward different receptor mutated variants.Oncogene 2004;23:5387–93.46. Yonesaka K, Zejnullahu K, Homes AJ, Johnson BE,Janne PA. Presence of amphiregulin autocrine-looppredicts sensitivity of EGFR wild type cancers togefitinib and cetuximab. Proceedings of the 99th AnnualMeeting of the American Association for CancerResearch; 2008 Apr 12-16;San Diego, CA Philadelphia(PA): AACR; 2008. Abstract: 4958.47. Engelman JA. The role of phosphoinositide 3-kinasepathway inhibitors in the treatment of lung cancer. ClinCancer Res 2007;13:s4637–40.48. Cohen EE, Lingen MW, Martin LE, et al. Response ofsome head and neck cancers to epidermal growth factorreceptor tyrosine kinase inhibitors may be linked tomutation of ERBB2 rather than EGFR. Clin Cancer Res2005;11:8105–8.49. Saucier C, Khoury H, Lai KM, et al. The Shc adaptorprotein is critical for VEGF induction by Met/HGFand ErbB2 receptors and for early onset of tumorangiogenesis. Proc Natl Acad Sci U S A 2004;101:2345–50.50. Zhang YW, Su Y, Lanning N, et al. Enhanced growthof human met-expressing xenografts in a new strain ofimmunocompromised mice transgenic for humanhepatocyte growth factor/scatter factor. Oncogene2005;24:101–6.




2009;69:3021-3031. Published OnlineFirst March 24, 2009.Cancer Res Tanguy Y. Seiwert, Ramasamy Jagadeeswaran, Leonardo Faoro, et al. CarcinomaTherapeutic Target for Head and Neck Squamous Cell The MET Receptor Tyrosine Kinase Is a Potential Novel

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TheMETReceptorTyrosineKinaseIsaPotentialNovelTherapeutic ... · Head and neck cancer (HNC) is the sixth most common cancer worldwide, with an annual incidence of >640,000 cases worldwide