The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Gene expression profiling identified human melanoma cells demonstrating increased cell motility and invasiveness. The gene WNT5A best determined in vitro invasive behavior. Melanoma cells were transfected with vectors constitutively overexpressing Wnt5a. Consistent changes included actin reorganization and increased cell adhesion. No increase in beta-catenin expression or nuclear translocation was observed. There was, however, a dramatic increase in activated PKC. In direct correlation with Wnt5a expression and PKC activation, there was an increase in melanoma cell invasion. Blocking this pathway using antibodies to Frizzled-5, the receptor for Wnt5a, inhibited PKC activity and cellular invasion. Furthermore, Wnt5a expression in human melanoma biopsies directly correlated to increasing tumor grade. These observations support a role for Wnt5a in human melanoma progression.

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/␤-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner.The molecular mechanisms that govern the motility and metastasis of melanoma cells are not well understood. The prognosis for patients with recurrent melanoma has shown no improvement over the past 50 years. Many of these tumors are histopathologically quite similar but can be subclassified based upon their gene expression profiles (1, 2). In a study by Bittner et al. (1), the gene that best separated highly aggressive tumors from less aggressive tumors was WNT5A, which was consistently underexpressed in the less motile tumors. Wnt5A is a member of the Wnt family of proteins, which were first identified during studies of development in Drosophila (3) and in studies of the mouse mammary tumor virus (4). Unlike its family members Wnt1 and Wnt3A, which signal via the canonical Wnt pathway, resulting in the nuclear translocation of ␤-catenin, Wnt5A acts via G-protein-coupled receptors to activate protein kinase C (PKC) 6 and intracellular calcium (5, 6). The interplay between these two pathways is not well understood, but it does appear that the non-canonical Wnt pathway can inhibit ␤-catenin stabilization both in vitro in human HEK293 cells and in vivo in zebrafish (7,8).In melanoma cells with low motility and low expression of WNT5A, overexpressing WNT5A resulted in an increase in both the activation of PKC and an increase in motility (9). High expression of WNT5A in melanoma patients also correlated to poor outcome in this study. In addition, many studies have highligh...