Molecular targets for tivantinib (ARQ 197) and vasculogenic mimicry in human melanoma cells

Tivantinib (TivB) was reported previously to target MET and microtubule assembly in different cells resulting in cytotoxicity. However, its other cellular targets remain unknown, especially the proteins involved in focal adhesion and cytoskeletal organization. We studied the effect of TivB on vinculin a focal adhesion protein, and RhoC, a GTPase which promote the reorganization of cytoskeleton. Biomolecules involved in vasculogenic mimicry (VM) previously not reported in melanoma, and their susceptibility to TivB was also evaluated. TivB affects the viability and apoptosis of human melanoma cells depending on the cell type. Vinculin and RhoC were increased in the presence of TivB and affected the integrity of actin filaments and altered the cellular morphology. TivB disrupts the VM exhibited by melanoma cells in 3D matrix. Roundabout Guidance Receptor 4 (Robo4), a receptor protein implicated in axonal guidance and angiogenesis and its ligand Slit2 are expressed in human C8161 and WM793 melanoma cells, but absent in other melanoma cells including normal melanocytes. VM is more prominent in C8161 cells and could be blocked by siRNA mediated silencing of Robo4 mRNA, but TivB does not affect Robo4 in C8161 cells. Immunoblot analysis indicated no changes in Robo4 and Slit2 protein expression, however, both vinculin and RhoC protein increased in TivB treated melanoma cells. These results suggest that TivB affects cell cytoskeleton and morphology by altering proteins such as vinculin and RhoC. Our studies indicate TivB could target molecules other than MET in melanoma cells, which may provide insight into its alternate mechanism of action.