Characterising the therapeutic potential of c-MET inhibitor crizotinib in metastatic melanoma

Abstract

Despite the success seen with BRAF and MEK inhibitors against cutaneous malignant melanoma, the emergence of drug resistance is almost inevitable. One of the resistance mechanisms involves the HGF/c-MET cross-talk between melanoma and fibroblasts in the tumour microenvironment. Studies have shown that fibroblast-derived HGF activates c-MET signalling in melanoma cells, thereby providing an alternative pathway for proliferation and survival in the presence of BRAF and MEK inhibition. Therefore, targeting the HGF/c-MET axis offers an attractive strategy to combat resistance against BRAF and MEK inhibitors. Sulforhodamine B colorimetric assays and western immunoblotting were used to characterise early passage metastatic melanoma cell lines for their sensitivity to ALK/c-MET inhibitor, crizotinib, both as a single-agent and in combinations with BRAF inhibitor, vemurafenib and MEK inhibitor, CI-1040. A co-culture system with melanoma lines stably expressing pEGFP-C1 was developed to investigate melanoma response to vemurafenib and CI-1040 in the direct presence of human dermal fibroblasts. Confocal microscopy was used to investigate the spatial arrangement of melanoma cells, fibroblasts and fibronectin in a co-culture ellipsoid. Crizotinib demonstrated strong inhibitory effects on the growth of melanoma cells at sub-micromolar concentrations in virtually all cell lines irrespective of BRAF, NRAS or WT status. Importantly, combinations of crizotinib with vemurafenib and CI-1040 exhibited synergistic effects when compared to single-agent exposure. Fibroblasts in co-cultures induced innate resistance to vemurafenib and CI-1040 treatment. This drug protective effect was not abrogated when treatment was combined with crizotinib. High cell density co-cultures formed singular ellipsoid-like structures with distinctive fibroblast and fibronectin localization. These findings describe the therapeutic potential of crizotinib as a single-agent and with BRAF and MEK inhibitors in an adjuvant setting. In addition, the HGF/c-MET crosstalk between melanoma cells and fibroblasts creates a potential for a niche that protects melanoma cells from small molecule inhibitor-mediated cytotoxicity.