The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous cell carcinoma

Abstract

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 was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry, and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling, migration, viability, and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression, whereas 18 of 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 semaphorin (T230M/E168D/N375S), juxtamembrane (T1010I/R988C), and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with >10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and 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.

Fig. 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+), while 21% had 2+ staining (no 3+ staining). MET expression was membranous and cytoplasmic. B, Phospho-MET epitope pY1003 Immunhistochemistry: Staining pattern resembles closely MET(Fig.1A) with strong expression in 71% of HNSCC samples. C, MET was expressed in 18 out 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). 170-kDa (glycosylated MET) and the 140-kDa band (biologically active transmembrane β subunit) are shown. RON expression closely follows MET expression (12/15), whereas expression of EGFR and IGF-1R 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 42% of tumor with 2+ expression in 21%. There was no HGF expression in normal mucosa.

Fig. 2

A, Phosphorylated Tyrosine (p-Tyr) immunblot of six HNSCC cell lines +/−HGF stimulation and inhibition with SU11274. Expression of phospho-tyrosine is seen in all cell lines in response to HGF treatment. SCC9 and SQ20B have the highest background p-Tyr expression. SU11274 pretreatment with 2/5µM and 5µM SU11274 affected phosphorylated tyrosine levels. B, Stimulation of MET phosphorylation with HGF and downstream signaling in five HNSCC cell lines is completely abrogated by pretreatment with SU11274. Downstream AKT and ERK phosphorylation is partially impacted in certain cell lines.

Fig. 3

A, In SCC61 and SQ20B MET specific siRNA (100µM) lead to a significant decrease MET protein expression, whereas control siRNA did not suppress MET expression. B, SCC61 and SQ20B cells 72 hours after transfection with MET specific siRNA and control siRNA were analyzed by MTT and showed significant decreases in viability in comparison to control (−62%/55%). C, SU11274 treatment led to a dose-dependant decrease in cell viability in comparison to untreated control (DMSO solvent). In decreasing order of sensitivity (IC50) the following HNSCC cell lines responded to increasing SU11274 concentrations: SCC61, SCC9, HN31/SCC35, MSK921/SCC28, SQ20B. D, Migration was significantly decreased after treatment with SU11274 over a tracking period of 21 hours. Colored lines depict individual cell movement over 21h. Cells treated with SU11274 moved significantly shorter distances (−38%)(P<.0001).

Fig. 4

A, Immunoblot of two HNSCC cell lines SCC61 and SCC35 after treatment with PF-2341066 at doses ranging from 0–500nM. PF-12340166 led to dose dependent abrogation of HGF-induced MET phosphorylation. B, Soft Agar colony formation assay of OSCC3 HNSCC with and without PF-2341066 treatment (0–1000nM). Colonies were stained with crystal violet and counted. Photo shows comparison of 0nM and 1000nM. There was marked suppression of colony formation by 33%/53%. C/D, Representative hematoxylin and eosine (HE) stained images (original magnification ×20) of OSCC3(C), SCC35(D) xenograft tumors from mice treated with vehicle or PF-2341066 (25mg/kg body weight). Animals were sacrificed prior to development of macroscopic tumors. Middle and lower panel show Ki67/CD31immunohistochemistry. PF-2341066 reduced cell proliferation (Ki67) and reduced blood vessel density (CD31) demonstrating in-vivo activity of PF-2341066 on angiogenesis.

Fig. 5

A, HNSCC cell lines SCC35/SCC61 were treated with cisplatin, SU11274, or combination at indicated doses (ratio:1:1/1:2). Both single agents showed efficacy decreasing viability between 25–70%. The combination was consistently superior to either agent alone. The isobologram indicated synergy (CI values<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 4h SU11274, erlotinib, and combination-treated cells. While both SU11274 and erlotinib suppress erbB3 and AKT phosphorylation the combination achieves increased suppression levels.