Mitigation of Tumor-Associated Fibroblast-Facilitated Head and Neck Cancer Progression With Anti-Hepatocyte Growth Factor Antibody Ficlatuzumab

Abstract

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.

Figure 1. Inhibition of TAF-Induced HNSCC Migration and Invasion

Head and neck squamous carcinoma (HNSCC) cells were treated with Dulbecco modified Eagle medium alone, tumor-associated fibroblast (TAF)-conditioned medium (TAF-CM) with or without ficlatuzumab (20 µg/mL) or ficlatuzumab alone in transwell migration chambers for 24 hours. Invasion of HNSCC cells through a synthetic hydrogel matrix (PGMatrix) was assessed over 24 hours. Ficlatuzumab significantly inhibited TAF-CM induced migration (A–C) in HN5 (P= .002), UM-SCC-1 (P= .09), and OSC-19 (P= .04) cells and invasion (D–F) in HN5 (P= .047), UM-SCC-1 (P= .03), and OSC-19 (P= .04) cells. The data represent cumulative results from 3 independent experiments. Error bars represent the mean ± SEM

Figure 2. Mitigation of TAF-Induced Vimentin Expression in HNSCC Cells

Head and neck squamous carcinoma (HNSCC) cells treated with Dulbecco modified Eagle medium alone, tumor-associated fibroblast (TAF)-conditioned medium (TAF-CM) with or without ficlatuzumab (20 µg/mL) or ficlatuzumab alone were analyzed by immunofluorescence for vimentin (green) expression. TAF-CM induced vimentin expression was effectively reduced in the presence of ficlatuzumab.

Figure 3. Inhibition of TAF-Induced HNSCC Cell Proliferation

HNSCC cells were treated with Dulbecco’s modified Eagle medium alone (−), TAF-conditioned medium (TAF-CM) (+), with or without ficlatuzumab (0, 1, 10, 50, 100 µg/mL) for 72 hours. Ficlatuzumab significantly inhibited TAF-facilitated (A) HN5 (P< .0001) and (B) UM-SCC-1 (P< .0001) proliferation. Cumulative results from a minimum of 2 independent experiments are graphed. Error bars represent the mean ± standard error of the mean. ^a P= .005. ^b P< .001.

Figure 4. Inhibition of TAF-Induced c-Met and p44/42 MAPK Phosphorylation in HNSCC Cells

Head and neck squamous carcinoma (HNSCC) cells were serum starved for 72 hours and treated with Dulbecco modified Eagle medium alone and recombinant hepatocyte growth factor (HGF) (30 ng/mL) with or without ficlatuzumab (100 µg/mL) for 10 minutes. Cell lysates were analyzed for phospho-c-Met and p44/42 mitogen-activated protein kinase (MAPK) by immunoblotting. Ficlatuzumab inhibited HGF-mediated phosphorylation of c-Met and p44/42 MAPK in (A) HN5, (B) UM-SCC-1, and (C) OSC-19 cells.

Figure 5. Model Summarizing the Antitumor Effects of Ficlatuzumab on Paracrine Stimulation of HNSCC by TAF-Secreted HGF

Hepatocyte growth factor (HGF) binds to the tyrosine kinase receptor. Activation of downstream signal molecules facilitates head and neck squamous carcinoma (HNSCC) cell proliferation, migration, and invasion. Ficlatuzumab, a monoclonal antibody binds HGF, downregulating c-Met and downstream targets, inhibiting HNSCC proliferation, migration and invasion. TAF indicates tumor-associated fibroblast.