ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is a highly morbid disease. Recent developments including Food and Drug Administration (FDA) approved molecular targeted agent's pembrolizumab and cetuximab show promise but did not improve the five-year survival which is currently less than 40%. The hepatocyte growth factor receptor; also known as mesenchymal-epithelial transition factor (c-Met) and its ligand hepatocyte growth factor (HGF) are overexpressed in head and neck squamous cell carcinoma (HNSCC); and regulates tumor progression and response to therapy. The c-Met pathway has been shown to regulate many cellular processes such as cell proliferation, invasion, and angiogenesis. The c-Met pathway is involved in cross-talk, activation, and perpetuation of other signaling pathways, curbing the cogency of a blockade molecule on a single pathway. The receptor and its ligand act on several downstream effectors including phospholipase C gamma (PLC?), cellular Src kinase (c-Src), phosphotidylinsitol-3-OH kinase (PI3K) alpha serine/threonine-protein kinase (Akt), mitogen activate protein kinase (MAPK), and wingless-related integration site (Wnt) pathways. They are also known to cross-talk with other receptors; namely epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) and specifically contribute to treatment resistance. Clinical trials targeting the c-Met axis in HNSCC have been undertaken because of significant preclinical work demonstrating a relationship between HGF/c-Met signaling and cancer cell survival. Here we focus on HGF/c-Met impact on cellular signaling in HNSCC to potentiate tumor growth and disrupt therapeutic efficacy. Herein we summarize the current understanding of HGF/c-Met signaling and its effects on HNSCC. The intertwining of c-Met signaling with other signaling pathways provides opportunities for more robust and specific therapies, leading to better clinical outcomes.
Project description:Head and neck cancer is the sixth most common cancer across the globe. This is generally associated with tobacco and alcohol consumption. Cancer in the pharynx majorly arises through human papillomavirus (HPV) infection, thus classifying head and neck squamous cell carcinoma (HNSCC) into HPV-positive and HPV-negative HNSCCs. Aberrant, mesenchymal-epithelial transition factor (c-MET) signal transduction favors HNSCC progression by stimulating proliferation, motility, invasiveness, morphogenesis, and angiogenesis. c-MET upregulation can be found in the majority of head and neck squamous cell carcinomas. c-MET pathway acts on several downstream effectors including phospholipase C gamma (PLCγ), cellular Src kinase (c-Src), phosphotidylinsitol-3-OH kinase (PI3K), alpha serine/threonine-protein kinase (Akt), mitogen-activated protein kinase (MAPK), and wingless-related integration site (Wnt) pathways. c-MET also establishes a crosstalk pathway with epidermal growth factor receptor (EGFR) and contributes towards chemoresistance in HNSCC. In recent years, the signaling communications of c-MET/HGF in metabolic dysregulation, tumor-microenvironment and immune modulation in HNSCC have emerged. Several clinical trials have been established against c-MET/ hepatocyte growth factor (HGF) signaling network to bring up targeted and effective therapeutic strategies against HNSCC. In this review, we discuss the molecular mechanism(s) and current understanding of c-MET/HGF signaling and its effect on HNSCC.
Project description:We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
Project description:IMPORTANCE:Ficlatuzumab can be used to treat head and neck squamous cell carcinoma (HNSCC) by inhibiting c-Met receptor-mediated cell proliferation, migration, and invasion. OBJECTIVE:To understand the effect of ficlatuzumab on HNSCC proliferation, migration, and invasion. DESIGN, SETTING, AND PARTICIPANTS:The effects of ficlatuzumab on HNSCC proliferation, invasion, and migration were tested. Mitigation of c-Met and downstream signaling was assessed by immunoblotting. The tumor microenvironment has emerged as an important factor in HNSCC tumor progression. The most abundant stromal cells in HNSCC tumor microenvironment are tumor-associated fibroblasts (TAFs). We previously reported that TAFs facilitate HNSCC growth and metastasis. Furthermore, activation of the c-Met tyrosine kinase receptor by TAF-secreted hepatocyte growth factor (HGF) facilitates tumor invasion. Ficlatuzumab is a humanized monoclonal antibody that sequesters HGF, preventing it from binding to and activating c-Met. We hypothesized that targeting the c-Met pathway with ficlatuzumab will mitigate TAF-mediated HNSCC proliferation, migration, and invasion. Representative HNSCC cell lines HN5, UM-SCC-1, and OSC-19 were used in these studies. EXPOSURES FOR OBSERVATIONAL STUDIES:The HNSCC cell lines were treated with ficlatuzumab, 0 to 100 µg/mL, for 24 to 72 hours. MAIN OUTCOMES AND MEASURES:Ficlatuzumab inhibited HNSCC progression through c-Met and mitogen-activated protein kinase (MAPK) signaling pathway. RESULTS:Ficlatuzumab significantly reduced TAF-facilitated HNSCC cell proliferation (HN5, P <?.001; UM-SCC-1, P <?.001), migration (HN5, P =?.002; UM-SCC-1, P =?.01; and OSC-19, P =?.04), and invasion (HN5, P =?.047; UM-SCC-1, P =?.03; and OSC-19, P =?.04) through a 3-dimensional peptide-based hydrogel (PGmatrix). In addition, ficlatuzumab also inhibited the phosphorylation of c-Met at Tyr1234/1235 and p44/42 MAPK in HNSCC cells exposed to recombinant HGF. CONCLUSIONS AND RELEVANCE:We demonstrate that neutralizing TAF-derived HGF with ficlatuzumab effectively mitigates c-Met signaling and decreases HNSCC proliferation, migration, and invasion. Thus, ficlatuzumab effectively mitigates stromal influences on HNSCC progression.
Project description:Aberrant signaling of the hepatocyte growth factor (HGF)/c-Met pathway has been identified as a promoter of tumorigenesis in several tumor types including head and neck squamous cell carcinoma (HNSCC). Despite a relatively low c-Met mutation frequency, overexpression of HGF and its receptor c-Met has been observed in more than 80% of HNSCC tumors, with preclinical and clinical studies linking overexpression with cellular proliferation, invasion, migration, and poor prognosis. c-Met is activated by HGF through a paracrine mechanism to promote cellular morphogenesis enabling cells to acquire mesenchymal phenotypes in part through the epithelial-mesenchymal transition, contributing to metastasis. The HGF/c-Met pathway may also act as a resistance mechanism against epidermal growth factor receptor (EGFR) inhibition in advanced HNSCC. Furthermore, with the identification of a biologically distinct subset of HNSCC tumors acquired from human papillomavirus (HPV) infection that generally portends a good prognosis, high expression of HGF or c-Met in HPV-negative tumors has been associated with worse prognosis. Dysregulated HGF/c-Met signaling results in an aggressive HNSCC phenotype which has led to clinical investigations for targeted inhibition of this pathway. In this review, HGF/c-Met signaling, pathway alterations, associations with clinical outcomes, and preclinical and clinical therapeutic strategies for targeting HGF/c-Met signaling in HNSCC are discussed.
Project description:Hepatocyte growth factor (HGF)/Met signaling has critical roles in pancreatic ductal adenocarcinoma (PDA) development and progression and is considered a potential therapeutic target for this disease. However, the mechanism of aberrant activation of HGF/Met signaling and resistance to Met inhibition in PDA remains unclear. The mechanistic role of cross talk between Forkhead box M1 (FOXM1) and HGF/Met signaling in promotion of PDA growth and resistance to Met inhibition was examined using cell culture, molecular biology and mouse models; and the relevance of our experimental and mechanistic findings were validated using human PDA tissues. Met was markedly overexpressed in both PDA cell lines and pancreatic tumor specimens, and the expression of Met correlated directly with that of FOXM1 in human tumor specimens. Mechanistically, FOXM1 bound to the promoter region of the Met gene and transcriptionally increased the expression of Met. Increased expression of FOXM1 enhanced the activation of HGF/Met signaling and its downstream pathways, including retrovirus-associated DNA sequences/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/AKT and signal transducer and activator of transcription 3. Furthermore, activation of HGF/Met signaling increased the expression and transcriptional activity of FOXM1, and the cross talk between FOXM1 and HGF/Met signaling promoted PDA growth and resistance to Met inhibition. Collectively, our findings identified a positive feedback loop formed by FOXM1 and HGF/Met and revealed that this loop is a potentially effective therapeutic target for PDA.
Project description:The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.
Project description:PURPOSE:Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade. EXPERIMENTAL DESIGN:Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways. RESULTS:Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-?, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-? can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo. CONCLUSIONS:Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.
Project description: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.
Project description:Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Methigh-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.
Project description:The hepatocyte growth factor receptor (Met) is frequently overexpressed in Head and Neck Squamous Cell Carcinoma (HNSCC), correlating positively with high-grade tumors and shortened patient survival. As such, Met may represent an important therapeutic target. The purpose of this study was to explore the role of Met signaling for HNSCC growth and locoregional dissemination.Using a lentiviral system for RNA interference, we knocked down Met in established HNSCC cell lines that express high levels of the endogenous receptor. The effect of Met silencing on in vitro proliferation, cell survival and migration was examined using western analysis, immunohistochemistry and live cell imaging. In vivo tumor growth, dissemination and mouse survival was assessed using an orthotopic tongue mouse model for HNSCC.We show that Met knockdown (1) impaired activation of downstream MAPK signaling; (2) reduced cell viability and anchorage independent growth; (3) abrogated HGF-induced cell motility on laminin; (4) reduced in vivo tumor growth by increased cell apoptosis; (5) caused reduced incidence of tumor dissemination to regional lymph nodes and (6) increased the survival of nude mice with orthotopic xenografts.Met signaling is important for HNSCC growth and locoregional dissemination in vivo and that targeting Met may be an important strategy for therapy.