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.