RALP, A NOVEL PROGNOSTIC MOLECULAR MARKER IN MELANOMA, IS INVOLVED IN THE NOTCH PATHWAY

Abstract

Melanoma is an aggressive disease with high metastatic potential and resistance to cytotoxic agents. Early-stage melanomas can be successfully cured by surgery and, as in all solid tumours, morbidity and mortality of melanoma are a consequence of local invasion and metastatic spread. The molecular mechanisms involved in the progression of the malignancy, the genetic markers associated to metastatic melanoma dissemination and the acquisition of chemoresistance are only beginning to be defined. An understanding of the molecular biology of melanoma provides a necessary basis to enable the generation of more effective therapeutic modalities. RaLP, a new member of the SHC family of adaptor proteins was previously characterized in our laboratory as a determinant in the regulation of migration of melanoma cells in short-term assays in vitro. In this study we further characterized the role of RaLP in the progression of melanoma. We have verified that the expression of RaLP significantly correlates with the most important prognostic markers of melanoma (Breslow thickness, Clark\u2019s level of invasion, ulceration, mitotic index and presence of metastasis in lymph nodes) and that patients with RaLP expressing tumours had reduced disease-free survival and overall survival, suggesting that RaLP can be identified as a novel prognostic molecular marker and an independent prediction factor of melanoma progression. We have shown that permanent RaLP silencing does not interfere with the proliferation of three different metastatic melanoma cell lines, while it significantly decreases their migration and this phenomenon was observed even after extended time in culture. The phenotype could be rescued by the overexpression of RaLP in the silenced cells, suggesting that RaLP is a central molecule that positively regulates migration of melanoma cells. Besides regulating migratory abilities of the melanoma cells, RaLP positively influences their invasive potential, by regulating collagen matrix digestion. We also tested cell \u2013 cell and cell \u2013 ECM adhesion abilities of melanoma cells after RaLP ablation. We observed that RaLP decreases adhesion of the cells to each other in cell \u2013 cell adhesion assays and negatively regulates adhesion of melanoma cells to different matrices in cell \u2013 ECM adhesion assay. Analyzing gene expression profiles of RaLP \u2013 proficient and \u2013 deficient cells we have shown that RaLP is involved in the regulation of the NOTCH molecular pathway. Our in vitro studies suggest that RaLP expression in melanoma might facilitate dissociation of metastatic cells from a tumour mass by loosening cell \u2013 cell adhesion, and favour invasion of the surrounding tissues. We still do not know the exact signalling cascade by which RaLP regulates cell motility and adhesion processes and additional studies are necessary to fully understand its role in melanoma progression