Activation of the HGF/c-Met signaling axis produces significant biological effects in human HNSCC. These effects include stimulation of signaling molecules important in cell proliferation and survival, release of cytokines that induce inflammation and angiogenesis, cell migration, and invasion. Overexpression of either c-Met or HGF, or both, was found in HNSCC tumor tissues, and evidence for a paracrine mechanism of action was found in cells isolated from HNSCC tumors, with production of HGF observed in TDFs derived from HNSCC tumor tissue and c-Met signaling activated in HNSCC epithelial tumor cells. These findings suggest that the signaling cascade induced by the c-Met pathway plays an important role in the growth and survival of human HNSCC, and contributes to invasion into normal tissues, neovascularization, and metastasis. All of these attributes suggest the HGF/c-Met pathway may replace or augment signaling pathways induced by other tyrosine kinase receptors, such as the EGFR, making it a good target for therapy in HNSCC. Inhibitor studies showed that c-Met TKIs could inhibit HGF-induced phosphorylation, signal transduction, wound closure, and IL-8 release. These observations are similar to recently reported effects with a different c-Met TKI in papillary thyroid carcinoma (28
). We show for the first time that a clinically relevant c-Met TKI, PF-2341066, can inhibit HNSCC tumor growth in a preclinical animal model.
HGF and/or c-Met overexpression has been described in a wide variety of human tumors including carcinomas of the breast (29
), lung (30
), head and neck (31
), stomach (33
), and pancreas (34
). Expression of c-Met was found to be even more highly increased in lymph node metastases in HNSCC patients compared with levels in the corresponding primary tumor, which were both elevated compared with normal tissue and unaffected lymph nodes (35
). Tumor content of c-Met and HGF has also been associated by others with poor outcome in oral cancer (9
). We and others have reported that elevated c-Met and/or HGF expression in human tumors such as lung and breast is often associated with poor clinical outcomes (38
). Increased HGF serum levels in HNSCC patients were also significantly correlated with higher tumor stage progression (41
). The results reported here with regard to clinical outcome represent a very small population compared with previous studies. A trend towards better overall survival was observed only when both HGF and c-Met expression was low in the tumors. Further studies using a larger number of patients will be necessary to establish the clinical usefulness of HGF in either tumor tissue or circulating blood as a biomarker for patients with HNSCC.
In addition to HGF and c-Met overexpression, this pathway can also be activated though genetic alterations such as c-Met–activating mutations. c-Met–activating mutations seem to be rare in all tumor types. Di Renzo et al. identified two somatic constitutively active c-Met mutations in lymph node metastases of HNSCC whereas it was barely detectable in the primary tumors from the same patients, suggesting that these mutations are selected during the metastatic process (42
). However, there have been no other reports of c-Met mutations in primary HNSCC tumors.
The HGF pathway has been implicated in clinical resistance to chemotherapy and targeted therapy in HNSCC patients and cell lines. In this regard, Akervall et al. (43
) identified low c-Met expression as a predictive factor for favorable response to cis-platin in HNSCC patients. Increased c-Met activation was also recently reported to be correlated with resistance to cetuximab, an EGFR inhibitor, in HNSCC cells (44
). Recently, it has been reported that overexpression of cortactin in HNSCC stabilizes c-Met and enhances HGF-induced properties leading to resistance to the EGFR TKI gefitinib (45
). Others have reported that EGFR and c-Met are linked via HER3, which has been implicated in clinical resistance to EGFR inhibitors in lung cancer (46
). These later findings suggest that c-Met pathway activation can substitute for or act in consort with the EGFR pathway in HNSCC to stimulate growth in the setting of EGFR inhibition.
Molecularly targeted therapies that inhibit the action of pathogenic tyrosine kinases and growth factor pathways have been extensively pursued in recent years. Due to its strong involvement in human cancer, the HGF/c-Met pathway is considered one of the most attractive drug targets for cancer therapy. As such, there are several different therapeutic strategies available to inhibit HGF/c-Met signaling, including small molecule tyrosine kinase inhibitors as well as antagonistic antibodies to either HGF or c-Met that are currently being evaluated in phase I and/or phase II clinical trials for treatment of various types of cancers (47
). PF-2341066 is in phase I trials for patients with advanced cancers, including head and neck cancer.4
PF-2341066 also has been reported to inhibit the nucleophosmin anaplastic lymphoma kinase (ALK) oncongenic fusion variant of the ALK tyrosine kinase (23
). We cannot discount the possibility that the effects that we observed with PF-2341066 are due solely to inhibition of c-Met if these cell populations express ALK. To our knowledge, however, ALK is not expressed in HNSCC cells.
, we have shown that HGF can induce IL-8 production. Elevated IL-8 tumor levels have been reported to be correlated with more aggressive disease, more recurrences, and shorter disease-free intervals in HNSCC patients (48
). In HNSCC tumors, IL-8 and HGF were found to be coexpressed at high levels in a subset of tumors, suggesting that common mechanisms regulate their expression in HNSCC (49
). HGF-induced IL-8 production in tumor cells may be an important downstream consequence of the HGF paracrine loop observed in HNSCC tumors.
We report here that close to 80% of primary HNSCC tumors examined in this study express either HGF, c-Met, or both. In addition to HGF binding to c-Met, HGF also binds to heparin binding domains on the cell surface (50
). Although the underlying stroma is actually producing HGF, HGF can also be detected in tumor tissue in immunohistochemical staining because it can bind to the tumor cell surface through heparin binding domain interactions. In preparing the tissue microarrays, tissues were cored from tumor epithelium whereas stroma was excluded. Thus, stromal production of HGF could not be evaluated within HNSCC tumors from this tissue microarray. HGF can induce a wide range of biological functions, including invasive growth, in all HNSCC cell lines examined in vitro
, and these functions can be inhibited by c-Met TKIs. HGF seems to be necessary for invasion; enhanced invasion due to HGF release by TDFs in coculture can be completely inhibited with a HGF NA. Daly et al. (19
) recently reported that invasion of oral squamous cell carcinoma depends on both HGF and stromal derived factor-1 found in conditional medium of stromal fibroblasts. One limitation of the above mentioned study is that fibroblasts from the oral cavity of healthy donors were used whereas our study utilized tumor-derived fibroblasts. In addition, use of a HGF NA in the previous study did not decrease tumor cell invasion in response to fibroblast medium back to control levels whereas we report here complete inhibition with HGF NA. HGF may be the major molecule released by TDFs that promotes tumor invasion. Our data suggest that both an antibody strategy directed at the ligand and a TKI approach directed at the receptor have potential to treat HNSCC. The clinically relevant TKI used here produced significant inhibition of HNSCC xenograft growth and caused a high level of apoptosis and accumulation of cellular debris within tumor xenografts.
Head and neck cancer has been particularly resistant to systemic therapies, and new treatments such as the EGFR monoclonal antibody cetuximab have shown little improvement. Due to either intrinsic or acquired resistance to EGFR inhibitors, the clinical utility of this class of drugs is limited. Thus, new strategies are greatly needed to combat this disease. Together these results suggest that therapeutic targeting of the HGF/c-Met pathway may offer new possibilities in the treatment of HNSCC patients either alone or in combination with other treatment strategies. Future research should focus on the identification of a population of patients most likely to benefit from this treatment. The rational design of combinations of molecularly targeted agents which may have a survival benefit to single-agent therapies in the treatment of head and neck cancers is necessary.