Dual targeting of C-kit and Integrin Linked Kinase abrogates the phosphorylation of S6 ribosomal protein and reduces melanoma cell growth

Abstract

Background: Activating mutations in c-kit have been observed in distinct subtypes of melanoma and are associated with disease progression and the development of resistance to treatment. Mutant c-kit uses mainly the phosphatidylinositol 3 kinase (PI3k)/Akt pathway and to a lesser extent the mitogen activated protein kinase (MAPK) pathway in order to promote characteristics of the cancer phenotype, such as excessive proliferation, angiogenesis and invasiveness. However, clinical studies on the inhibition of c-kit in melanoma patients have yielded disappointing results. The reactivation of these pathways have been reported in treatment resistant melanoma cells. Previously we showed that downregulation of integrin linked kinase (ILK), an intracellular adapter molecule that promotes tumor progression and angiogenesis when dysregulated, suppressed Akt phosphorylation and inhibited tumor growth. Therefore we investigated the effects of dual inhbition of c-kit and ILK on tumor cell growth and angiogenesis. Methods: The effects of dual blocking of c-kit and ILK-down regulation using siRNA on tumor cell growth were evaluated in an in vitro co-culture system that contained human vein endothelial cells (HUVECs) and melanoma cells. To investigate the effects of blocking c-kit and ILK on angiogenesis, the migration and invasion of HUVECs in response to conditioned medium from melanoma cells was studied using the xCELLigence system, while endothelial cell tube formation was studied using a collagen-based assay. Protein array studies were employed to investigate the expression patterns of angiogenic proteins in mono- and co-cultured cells. Further marker expression was determined using immunocytochemistry. Results: Significant inhibition of melanoma cell growth was observed in treated co-cultures, and the inhibition correlated with reduced expression of S6 ribosomal protein and S6 kinase. ILK-knockdown inhibited endothelial cell migration and invasion, as well as endothelial cell morphogenesis induced by melanoma conditioned medium. Results also revealed an increase in the expression of Platelet Factor 4 (PF4), and a suppression of proangiogenic factors, including leptin, vascular endothelial growth factor and basic fibroblast growth factor. Conclusions: Findings from this study suggest that the cross-talk between melanoma and endothelial cells which supports tumor cell growth, can be disrupted by dual targeting of c-kit and ILK. Furthermore, dual targeting of c-kit and ILK which also resulted in the altered expression profile of angiogenesis markers to favor antiangiogenesis, may imply that such dual targeting could contribute to enhancing complimentary approaches for melanoma therapy. Legal entity responsible for the study: Angiogenesis Laboratory, University of Pretoria Funding: National Research Foundation, University of Pretoria Disclosure: All authors have declared no conflicts of interest.