Earlier studies have shown that microarray analysis of melanoma can distinguish more motile from less motile cells, despite similar histopathology. One key feature of this gene expression signature is that cells that are more motile have high expression of WNT5A and low expression of MART-1. Reconstituting WNT5A expression in cells with low levels of WNT5A increases motility of these cells, via the activation of protein kinase C. In order to better understand the pathways by which WNT5A mediates motility, we performed RNAi knockdown of WNT5A in cells that were transfected with WNT5A. We could establish using Western analysis, and confocal microscopy, that Wnt5a protein expression was downregulated by RNAi, and this contributed to the deactivation of PKC, and its subsequent translocation from the membrane to the cytoplasm. Array analysis of these RNAi treated cells also demonstrated that WNT5A overexpression suppressed the expression of a large amount of genes. To determine if WNT5A could directly affect the expression of MART1 and TYRP1, we treated WNT5A expressing cells with RNAi against WNT5A, and analyzed changes in the expression of these genes using real time PCR. MART1 expression, nearly absent in a number of highly metastatic melanoma lines, was reconstituted as early as 24 hours, as was MITF expression and TYRP, both of which are lost in the presence of high WNT5A. The reverse could also be observed when treating low- WNT5A expressing cells with medium containing Wnt5a protein. In a study of a panel of RNA from melanoma cells derived from patient tissues we found a robust inverse relationship between WNT5A and MART1. Furthermore, preliminary data indicates that these effects may be mediated via a Wnt5a- dependent increase in IL-6 both at the level of secretion, and transcription, resulting in the phosphorylation of STAT-3 and a subsequent down-regulation of PAX3, the presence of which is required, along with MITF, for expression of MART1, TYRP1 and GP100. Experiments to determine if WNT5A over-expression can protect melanoma cells in vitro from cytolysis by tumor-infiltrating lymphocytes (TILs) recognizing MART1 peptide are currently underway.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]