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Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC).
MET/phosphorylated-MET and HGF expression were analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry, and 20 HNSCC cell lines by immunoblotting. Effects of MET inhibition using siRNA/two small molecule inhibitors (SU11274/PF-2341066) on signaling, migration, viability, and angiogenesis (in-vivo) were determined. The complete MET gene was sequenced in 66 HNC tissue samples/8 cell lines. MET copy number was determined in 14 cell lines/23 tumor tissues. Drug combination of SU11274 with cisplatin/erlotinib were tested in SCC35/HN5 cell lines. 84% HNSCC samples showed MET overexpression, 18/20 HNSCC cell lines (90%) expressed MET. HGF overexpression was 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). Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the SEMA(T230M/E168D/N375S), the JM(T1010I/R988C), and TK(T1275I/V1333I) domains (13.5%). Increased MET gene copy number was present with >10 copies in 3/23(13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib. EGFR/MET synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression, increased gene copy number, and mutations. MET inhibition abrogated MET functions including proliferation, migration/motility, and angiogenesis. MET is a promising, novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored.
Head and neck cancer (HNC) is the 6th most common cancer worldwide, with an annual incidence of more than 640,000 cases worldwide (47,560 cases in the US(1)). More than 90% of head and neck cancers are of squamous histology (HNSCC). 35–45% 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 2 decades, with the singular exception of cetuximab, an epidermal growth factor receptor (EGFR) antibody, which improved median survival by 2 months when added to standard chemotherapy(2). Overall survival remains poor (median: 6–10months).
In order to improve HNSCC treatment, relevant molecular targets need to be identified. Receptor tyrosine kinases (RTK) appear to play a pivotal role in the pathogenesis of HNC, with prior research focusing on the EGFR RTK. EGFR inhibition has only yielded low response rates (4.3%–13%)(3) despite EGFR overexpression in >90% of tumors(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 receptor tyrosine kinase, using the precedence of EGFR/MET crosstalk and potential cooperation in other diseases(6–9).
MET, located on chromosome 7q31, encodes several functional domains, including the SEMA domain (ligand-binding), juxtamembrane domain (regulatory), and the tyrosine kinase domain(10, 11). The sole ligand for MET is hepatocyte growth factor (HGF, scatter factor), which is produced by stromal and sometimes tumor cells(10, 11). HGF binding activates MET via intracellular phosphorylation initiating RAS-RAF-ERK, and PI3K-AKT-mTOR signaling as well as several other pathways. In-vivo, HGF/MET signaling 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 chemotaxis. Furthermore MET overexpression and activation has transforming properties for normal cells(10, 11).
MET is overexpressed in a number of solid tumors, and 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 mutations is not yet fully understood. MET mutations have been described for HNC, especially in lymph node metastases (relative frequency of up to 25% in some reports)(13) and are located in the TK domain similar to TK domain mutations found for renal cell carcinomas(11) suggesting an important role for MET in HNC(13). However, mutations in the SEMA and JM domains have not been previously investigated for HNSCC. Further highlighting the importance of MET is the observation of MET amplification in several solid tumors including subgroups of lung, and gastric cancers(6, 7, 14, 15).
In our study we employed a large cohort of HNC and normal mucosa tissues as well as cell lines to identify prominent MET expression, increased gene copy number, and mutations in the TK/JM/SEMA domains. Furthermore we demonstrate that MET inhibition alone and in combination with cisplatin or an EGFR inhibitor is a promising target for head and neck cancer.
Tissue microarrays of 97 HNC tissues and 24 normal mucosa samples were built (IRB:#8980). Immunohistochemistry (IHC) was performed for MET (CVD-12V, 1:100), p-MET (pY1003, Invitrogen, 1:25), and HGF (H145, Santa Cruz, 1:50), human CD31 (JC70A, DAKO, 1:40), Ki67 (RM-9106-S, NeoMarkers/Labvision, 1:300) as previously described(12, 15, 16). Appropriate negative controls were prepared. IHC from tumor and adjacent normal tissue were compared semi-quantitatively by a senior pathologist (Grading: 0=negative, 1+=low, 2+=strong, 3+=very strong expression(12)).
Antibodies used for immunoblotting were MET (3D4, Invitrogen/Zymed, C-12, Santa Cruz), phosphorylation site-specific MET pY1003 and pY1230/4/5 (Biosource/Invitrogen, 44-882G, 44-888G), β-actin/p16/ERCC1/Ron-alpha antibodies (H-196/JC-8/8F1/H-170, Santa Cruz), pTyr (4G10, Upstate/Millipore), IGF-1R (#3027, Cell Signaling)(dilution 1:1000) as previously described(12, 15–17).
The following drugs were purchased: SU11274/IGF-1R inhibitor (Calbiochem, #448101/CAS477-47-4), cisplatin (Sigma Aldrich, #479306), erlotinib HCL (ACC, #183319-69-9). PF-2341066 was kindly provided by Pfizer.
Cell lines were obtained from ATCC (SCC9/15/25/68/Cal27/Fadu), Dr. Raph Weichselbaum (University of Chicago, Department of Radiation Oncology)(SQ20B, JSQ3, SCC35/61/294/151), Dr. Gary Clayman (MD Anderson, 1483), the Ludwig Institute for Cancer Research (HN5)(London, UK), Dr. David Raben (University of Colorado Health Sciences Center, Aurora, CO)(MSK921), Dr. Mark Lingen (University of Chicago, Department of Pathology) (OSCC3, SCC17B/28/58)) and maintained in DMEM/F12 or RPMI media and penicillin/streptomycin (Cellgro) with 10% 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.
Cells were grown in antibiotic-free medium to 60% confluency in 96-well (viability)/6-well (immunoblotting) plate. MET siRNA was used at 100uM with Dharmafect transfection reagent (Dharmacon) using the recommended protocol. Controls were treated with transfection agent only. Cells were incubated at 37°C in 5% CO2 for 36–72 hours before viability was assessed or lysate harvested.
Measurement was performed using Alamar blue (Resazurin, R2127, Sigma-Aldrich). Soft-agar colony formation assays were performed as previously described(15). Viability results were evaluated by a fluorescence/absorbance 96-well plate reader Synergy HT (BioTek). Synergy was calculated using Calcusyn as described by Chou et al.(20).
Cells were plated on glass bottom culture dishes (MatTek) in 10% FBS medium and grown for 24h to achieve 20–30% confluency prior to drug treatment. Dishes were placed into a temperature-controlled chamber at 37°C in an atmosphere of 5% CO2.
The cells were imaged on an Olympus IX81 inverted microscope and digitally captured with IPLab software (Scanalytics). Images at 100x were saved every 5min and processed as mpeg4 movies (Sonic DVD). Cell-movement/morphological changes were processed with ImageJ (NIH), Photoshop (Adobe) and MetaMorph (Universal Imaging Corporation/Molecular Devices). The positions of cell nuclei were tracked, and distance/speed calculated over 24h.
Genomic DNA from 63 HNSCC tissues from FFPE tissues was obtained from the University of 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 MET transcript MET-001 (ENST00000318493) was used for identifying genetic changes (e.g. R988C), and MET-002 (ENST00000397752) for identifying phosphorylation sites (e.g. Y1230), which is consistent with common practice(12, 16).
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 cycle 95°C*12min, 45 cycles 95°C*20sec, 58°C *1min), the mean threshold cycle number was used.
Fluorescent in-situ hybridization (FISH) analysis was done utilizing 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 utilizing RP11-144B2(red) together with RP11-163C9(green). Procedure was done as previously described(14) analyzing at least 10 metaphase cells.
HPV testing was performed in cell lines evaluating for p16 expression (JC-8, Santa Cruz) and by PCR using L1 PGMY09/11 primers(21), followed by sequencing. HPV-positive results were confirmed using the Digene HPV test (Qiagen).
Tumor cells were mixed with Matrigel (BD Biosciences) and injected s.c. into the flanks of nude mice (5×106 cells/flank) following institutionally approved protocols (IACUC). The animals were monitored for 2 weeks and subsequently sacrificed. Tissues were fixed in 10% formalin and paraffin embedded.
Data are expressed as mean±SE. Statistical significance was tested with Graphpad Prism5. For comparison between two groups, Student's t test or χ2 test was used. For comparing between >2 groups, one-way ANOVA was used. For evaluation of correlation, Spearman's test was used.
MET immunohistochemistry was done on 121 cores (97 cancers/24 normal mucosa) as well as phosphorylated-MET immunohistochemistry (86 cancers/22 normal mucosa). 85% (N=84) of HNSCC tumors overexpressed (2+/3+) MET and 66% (N=57) overexpressed (2+/3+) activated phosphorylated MET in comparison to adjacent normal mucosa (Figures 1A, 1B). Normal mucosa also expressed MET (21% 1+, 21% 2+), albeit staining was weaker and primarily limited to the basal layer of the mucosa (Figure 1A)(23% 1+/2+ for phosphorylated MET). No cases of 3+ expression were seen for normal mucosa. MET localized primarily to the membrane and the cytoplasm.
Immunoblot analysis confirmed strong MET expression in 16 of 20 HNC cell lines (excluding HNX(derived from HN5) and HaCaT(transformed keratinocytes)); however, SCC17B and SCC151 expressed low levels of MET, which were outside the dynamic range (Figure 1C). SQ20B and SCC294 had low to moderate MET expression. OSCC3 an HPV positive cell line (p16+, PCR positive (HPV18), Digene high-risk HPV positive) showed strong MET expression. EGFR, IGF-1R, RON, ERCC1 expression were prominent in several cell lines. There was no statistical correlation with MET expression.
Analysis of MET gene expression using the publicly available Oncomine database1 and data by Gino et al.(22) showed increased MET gene expression in 41 HNSCC compared to 13 normal controls (Suppl. Figure 1).
HGF expression was evaluated in 68 HNC tumors by immuno-histochemistry. 21% of tumors showed strong (3+), 24% moderate (2+), and 41% weak (1+) HGF expression. 15% of tumors were HGF negative.
Using small molecule MET inhibitors SU11274 (for cell lines, DMSO soluble, Figures 2–3), PF-2341066 (water soluble, clinical candidate, Figure 4)(see Suppl. Table 1) and MET siRNA (Figure 3B), MET activation/expression were suppressed. Figure 2A shows immunoblotting results for phospho-tyrosine, Figure 2B phosphorylated-MET, and downstream signaling effects in 6 HNSCC cell lines: Serum-starved cells lines were pretreated with 0, 2, or 5µM of MET inhibitor SU11274 followed by treatment with HGF for 8 minutes. In cell lines SCC15, SCC28, and to a lesser degree SCC9 and SCC61 HGF stimulation lead to a strong p-Tyr signal, which was suppressed with SU11274 MET inhibitor treatment. SCC17B overall had low p-Tyr expression, suggestive of either a less receptor tyrosine kinase driven phenotype(5) or a more ligand-dependent phenotype. Despite low MET expression, external HGF stimulation and SU11274 pretreatment show typical signaling effects of the HGF/MET axis.
Phosphorylated-MET expression was weak at baseline in most starved cells. Following HGF stimulation in all cell lines a strong phosphorylated-MET response is observed that can be suppressed in a dose dependent fashion (Figure 2A–B). Downstream signaling for phosphorylated-AKT also was increased with HGF and decreased by MET inhibition in cell lines SCC15, SQ20B, SCC28, and to a lesser degree in SCC61 (Figure 2B). Phosphorylated-ERK was only mildly affected by MET inhibition with SU11274.
MET gene silencing with MET specific siRNA was used to validate effects of MET inhibition in HNSCC. MET specific siRNA duplexes were transiently transfected into SCC61 and SQ20B cells (Figure 3A) and protein expression was decreased by >80% 72 hours after transfection.
siRNA down-regulation of MET protein expression in SCC61 and SQ20B cells resulted in inhibition of the serum-stimulated cell growth and viability by more than 62%/55% as determined by MTS assays (Figure 3B).
We used SU11274 to test for its inhibitory effects on 7 HNSCC cell lines (Figure 3C): MET inhibition was effective with IC50 values varying between 1–8µM: SCC61(IC50=1µM), SCC35(IC50=3µM), SCC9(IC50=3.8µM) were the most sensitive lines followed by HN31(IC50=5µM) and MSK921/SCC28(IC50=5.4µM). SQ-20B, which has lower MET expression and strong EGFR expression (EGFR amplification) showed an elevated IC50 of 8µM (extrapolated from Figure 3C). Generally a 50–90% decrease in cell viability compared to control cells was observed.
Furthermore MET inhibition with SU11274 (3.5µM) led to suppression of cell motility/migration. Figure 3D shows a graphical depiction of distances covered by individual cells (SCC61) over a period of 21h. SU11274 treated cells covered significantly shorter distances (P=.0001) than untreated control cells. This effect is consistent during the entire 21h observation period.
In order to study MET inhibition effects on angiogenesis water-soluble PF-2341066 was used in-vivo (Suppl. Table 1). PF-2341066 inhibited HGF dependent MET phosphorylation in a dose-dependent manner at concentrations of 10–100nM in HNC cell lines SCC61, and SCC35 in-vitro (Figure 4A) and also in a soft-agar colony-formation assay (OSCC3) (Figure 4B); no large colonies formed. Comparable results in colony formation assays were observed for SCC61 and SCC35 (data not shown).
Effects on angiogenesis were investigated with an in-vivo Matrigel xenograft tumor model of OSCC3 and SCC35 treated with PF-2341066 (25mg/kg/day) versus control-treated (N=3 in each group). Figure 4C/D shows abundant tumor growth in a vehicle control treated mouse, compared to minimal residual tumor nests in the PF-2341066 group. Staining for the proliferation marker Ki67 shows >80–90% suppression of proliferation in PF-2341066 treated animals. Finally staining of endothelial cells in blood vessels with CD31 shows extensive tumor vessels between tumor nests in control treated animal, versus marked angiogenesis suppression in PF-2341066 treated animals consistent with prior reports using a related MET inhibitor in-vivo(23).
Figure 5 shows four examples of dual treatment with MET inhibitor SU11274 in combination with commonly used agents – cisplatin or erlotinib.
SCC35 and SCC61 which require doses of cisplatin >10µM to achieve IC50 cytotoxicity (Cisplatin 10µM elicited a modest 32% decrease in viability in SCC35 and SCC61 IC50=16µM), were synergistically inhibited by combined treatment of both cisplatin and SU11274 (IC50=1.3µM Cisplatin/SU11274 and IC50=2/1µM Cisplatin/SU11274). Based on the median effect model by Chou(20) the isobologram graph shows combinatorial indices (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 of both at equimolar doses. Both single agents showed efficacy decreasing viability. The combination though was consistently significantly superior to either agent alone. The isobologram shows that 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 and SU11274 (CI <1). Evaluation of downstream signaling in panel C indicates that activation of erbB3 and subsequently AKT are synergistically inhibited.
The entire MET coding region (Schema: Suppl. Figure 2) was sequenced in 66 HNSCC and 8 cell lines. Three mutations in the ligand-binding SEMA domain (T230M/E168D in tumor tissue, N375S in SCC25 cell line) and 2 mutations in 4 tumors samples in the trans-/juxtamembrane domain (R988C, 3×T1010I)(Table 1A/Suppl. Figure 2) were identified (previously reported in other tumor types(12, 17)). Furthermore 2 mutations in the tyrosine kinase domain (T1275I, V1333I) were identified, which have not been described previously. No Y1230C/Y1235D mutations were identified. All mutations were heterozygous. The rate of TK domain mutations was 3% (2/66) and the rate of non-TK domain mutations was 9% (6/66). Overall mutations occurred in 12% of tumors analyzed (8/66). There was no apparent correlation with smoking status or anatomic site, although the sample number was too small to allow sufficient statistical power.
We analyzed a panel of 9 HNSCC cell lines by FISH and followed this up with qPCR due to the ready availability of DNA from HNSCC tumor tissues. Repetition of cell lines previously analyzed by FISH now using qPCR was done (Table 1B). FISH analysis showed 3 cell lines with >4 copies, although qPCR copy number was lower (2.79/1.91). Generally qPCR showed similar or lower copy numbers compared to FISH analysis. We subsequently analyzed 23 HNSCC tumor tissues from patients by qPCR (Table 1C): 3 out of 23 (13%) of tumors showed gene copy numbers of >10 with one sample showing a copy number of 22.1 and two samples of 10.50/10.33. Furthermore 15/23 (65%) HNSCC tumors showed copy numbers of 4–10, and 3/23 (13.0%) showed copy numbers of >10. There was no apparent correlation with smoking status or anatomic site (i.e. an HPV surrogate), although small sample numbers in subgroups do not allow for proper assessment.
In addition to mutations, multiple SNPs in the MET gene were identified, heterozygous (A48A in 2, S178S in 4, Q648Q in 5, I706I in 1, K1250K in 1, D1304D/ A1357A/ P1382P occurred together in 22 samples) and homozygous (Q648Q in 2, D1304D/ A1357A/ P13821 occurred together in 9 samples)(Table 1D).
In this study, we demonstrate that MET is a novel target for HNSCC showing prominent overexpression, mutations, and increased gene copy number. We demonstrate the effectiveness of MET inhibition on cell signaling, migration, and angiogenesis. Our data provide a strong rationale to employ MET inhibition in translational and clinical studies in HNC and suggest studying the integration with established treatments.
MET is activated in HNSCC patient samples and the presence of phosphorylated MET (66%) closely correlated with overall expression (79%): This is consistent with literature reports for HNC (70–90% expression(24–29)) and is comparable to NSCLC(12).
Our study helps to explain the prominent MET overexpression demonstrating increased copy numbers in a subset of tumors. While karyotypic analysis is still considered the gold standard, Bean et al. confirmed the usefulness of qPCR when compared to array CGH analysis(7). While no MET amplified HNC cell lines were identified MET amplification has previously been reported in gastric carcinoma(14) and NSCLC(6, 7) and correlates with sensitivity to MET small molecule inhibitors(6, 14). This may be relevant for predicting HNC sensitivity to MET inhibitors in the future and additional data is necessary to validate.
Normal mucosa weakly expressed MET in the basal mucosa layer (Figure 1A), possibly linked to mucosa turnover/proliferation or field cancerization. Reports by Chen et al. and Ohnishi et al. suggest a role of MET in HNSCC dysplastic lesions(24, 30).
The expression pattern was both cytoplasmic as well as membranous closely resembling data in lung cancer(31); the relative cellular localization is tissue specific, and the functional implications are still being elucidated(31).
Similar to prior reports we confirm elevated HGF expression in 59% of HNSCC (45% strong expression(2+/3+), 15% weak expression(1+)), and in the adjacent stroma suggesting auto- and paracrine signaling loops, which have been described in other tumor types (i.e. gliomas, pancreatic and liver carcinomas(31)(32–34)). This may be another possible predictor of response as recent reports for the EGFR ligand amphiregulin suggest a correlation with sensitivity to EGFR treatments(35).
The correlation MET/HGF expression/amplification status with treatment outcomes is a high priority for future studies. Preclinically Akervall et al. reported higher MET expression based on gene array analysis in cisplatin-resistant HNSCC cell lines compared to sensitive ones(36) and Aebersold et al. reported that MET expression correlated with radioresistance(37, 38). Several studies describe increased MET/HGF expression in more invasive HNSCC(24, 25, 27, 39) as well as metastatic spread(28, 29, 40). Finally the role of epithelial-mesenchymal transition (EMT) has also been implicated with poor prognosis for HNSCC(41) and MET is a known driver of EMT(11).
We report for the first time the identification of novel MET alterations in the SEMA, juxatmembrane (JM), and tyrosine kinase (TK) in human HNSCC. The precise function is part of ongoing studies(13, 17): Previously DiRenzo and Aebersold et al. described TK domain mutations in HNSCC in Y1230C and Y1235D in up to 10.9% of tumors(13, 38). In these studies MET mutations occurred primarily in lymph node metastases, and only could be detected with higher sensitivity SSCP. In a subsequent study though Morello et al. did not identify any MET mutations in HNSCC(26). We employed standard PCR amplification and sequencing technology and identified two novel TK domain mutations in one lymph node metastasis and one primary tumor (T1275I and V1333I). We did not detect Y1230C, Y1235D mutations. TK domain mutations are reported to be somatic mutations in HNSCC and their functional importance is well established in certain papillary renal cell cancers (germline or somatic mutations)(42). The functional role of TK domain mutations in HNSCC remains to be determined and the precise high-sensitivity mutation screening/sequencing may be required as seen for the EGFR T790M mutation in NSCLC(43).
We identify for the first time in HNSCC semaphorin (SEMA) and juxtamembrane (JM) domain mutations/variants. Such mutations/variants have previously been reported in lung cancer. The JM domain changes have been implicated in increased motility and invasiveness in SCLC(12, 17) and appear to have transforming properties(17). Preliminary reports suggest that both SEMA and JM domain mutations/variants can contribute to MET activation and may alter sensitivity to MET inhibitors(44). In contrast to TK domain mutations, SEMA and JM domain mutations/variants may be found in either germline DNA or somatically.
MET inhibition can readily be achieved with small molecule tyrosine kinase inhibitors: PF-2341066 used here for in-vivo studies is currently in phase I clinical testing. SU11274 is poorly water-soluble earlier inhibitor developed by Sugen/Pfizer and clinical development was not pursued.
Various parameters have been suggested as predictors of response to MET kinase inhibitors(31), including strong expression as seen e.g. in NSCLC(12), gene amplification as seen for gastric carcinomas(14), and kinase domain mutations(45), and potentially ligand expression as reported for amphiregulin/EGFR(46). Unlike NSCLC and colon cancer K-Ras mutations are not commonly observed in HNSCC. Our data suggest that generally higher MET expression levels correlate with increased sensitivity to MET inhibition, but is not sufficient to explain the remaining substantial variation in IC50s. Additional factors modulate responsiveness and future studies may include correlation with increased CNV (which may not always be reflected in protein expression levels), HGF expression, use of parallel RTK signaling cascades (Figure 1C), and potentially gene mutations status (e.g. PTEN).
AKT and ERK activation are oftentimes separate events with AKT being more prominently involved in cell survival and ERK in proliferation(47). While sometimes regulated together (i.e. EGFR TK domain mutated NSCLC(47)), it appears that for most HNSCC regulation is separate (Figure 2B). It is possible that concurrent inhibition of the pathways leading to ERK activation will increase therapeutic benefit.
Despite the lack of EGFR mutations in HNSCC in the US(48), most HNSCC are sensitive to EGFR inhibitors and overexpression is abundant(4). Recent evidence suggests a common signaling pathway via HER3/erbB3(5–9). Specifically Engelman et al.(6) implicated erbB3 signaling as the mediator of amplified MET "overtaking" mutant-EGFR signaling control in a NSCLC in-vitro model of acquired gefitinib resistance. On the other hand, the recent study by Tang et al.(9) suggested a central role for erbB3 in mediating the efficacy of dual MET/EGFR inhibition in mediating the efficacy of dual MET/EGFR inhibition against T790M-EGFR mediated resistance in the absence of prior EGFR TKI selection pressure. Our data for HNSCC now also suggests a role of MET/erbB3 in the absence of EGFR selection pressure.
Given the broad use of EGFR inhibition in HNSCC patients, and the limited single agent response rate(3), ways to increase efficacy with dual-/multikinase inhibition are pivotal.
Gene copy numbers appeared to be higher in tumor tissue samples as compared to cell lines, most notably we did not identified an amplified cell line. While the reasons for this are unclear (possible selection, bias of cell line/tumor choice) this may indicated increased sensitivity in such tumors.
Our data also suggests exploring MET inhibition in combination with cisplatin. Interestingly Akervall et al. when comparing cisplatin sensitive and resistant HNSCC cell lines by gene microarray techniques identified MET overexpression in resistant lines(36). Henceforth MET may be involved in mediating cisplatin resistance or could be a general poor prognostic marker. Further studies are indicated.
The pro-angiogenic properties of the MET/HGF axis are well established and MET signaling can initiate VEGF production, a critical angiogenic switch via Shc(49). We provide the first evidence of anti-angiogenic effects of MET inhibition in HNSCC using PF-2341066 in a matrigel plug model. A caveat is that murine HGF does not sufficiently activate human MET(50) therefore use of a human HGF transgenic model is of interest(50). Furthermore in-vivo metastasis modeling of MET overexpression, mutations, and amplification for HNC will provide 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 suppression abrogates a key component of the metastatic cascade.
In summary, we identified MET as a functionally important receptor in HNSCC with activation and overexpression in tumor tissues and cell lines. Furthermore we describe evidence of amplification and the presence of novel TK, SEMA and JM domain mutations. The consistent effects of MET inhibition validate this target further and synergy with cisplatin and erlotinib is therapeutically relevant. Further mechanistic studies into the role of MET mutated/amplified HNC are indicated and will allow us to better use MET specific drugs for selected patients.
The authors would like to acknowledge the support of the entire Salgia laboratory, Ralph Weichselbaum, Stuart Schwartz, Jose Manaligod, Maria Tretiakova, and Thomas Krausz.
Funding: Flight Attendant Medical Research Institute (FAMRI) Young Clinical Scientist Award (T.Y. Seiwert). NIH National Cancer Institute R01 grants CA100750-04 and CA125541-02, American Lung Association, Institutional Cancer Research awards from the University of Chicago Cancer Center with the V-Foundation (R. Salgia), NIH grant DE12322 (M. Lingen)