In order to identify targets for improved therapy and to better elucidate the mechanisms underlying the pathogenesis of this disease, we are performing gene expression profiling of human melanoma biopsies. Using a technique known as the serial analysis of gene expression (SAGE), which allows for the quantification of individual transcripts within a cell, as well as identifies novel transcripts, we can examine the gene expression profiles of melanomas at different stages of disease using minute quantities of tissue. Currently, three of these libraries are available on the public databases (http://cgap.nci.nih.gov), which we generated from three different melanoma tissue samples, with two representing vertical growth phase tumors and one representing a distant tumor metastasis. Studies currently in progress include the generation of libraries from compound nevi, additional metastases and normal melanocytes. We can examine these data using microarray analysis tools rarely utilized to analyze SAGE libraries. These types of multilevel analyses can reveal which genes underlie progression and which genes may be viable targets for future therapy. Thus far, data generated by microarray and SAGE analyses have highlighted the importance of G-protein mediated signaling, resulting in the activation of PKC and rises in intracellular calcium in melanoma progression. These effects can be mediated by the gene WNT5A, the over expression of which can lead to increases in melanoma cell motility and invasion. RNAi inhibition of this pathway, followed by microarray analysis, reveals that Wnt5a may make this contribution to invasion by silencing the expression of metastasis suppressers such as Kiss-1 and NME-1. Furthermore, we have also shown that Wnt5a can mediate melanoma metastasis via the initiation of the epithelial to mesenchymal cell transition, in a PKC-dependent fashion. In addition, Wnt5a can suppress the expression of melanoma antigens, such as MART1, implying it may play a role in escaping immune surveillance. Recent data suggests that this occurs via the activation of STAT3. In the light of recent data from other labs showing that targeting MART1 can cause tumor regression, these results are of significant importance and may provide a means to control MART antigen expression in tumors to facilitate immune responses against or the molecular targeting of such tumors. Because Wnt5A is a secreted ligand, making it a challenging target against which to develop a drug, we wish to identify which receptors might make viable targets for melanoma therapy, and this proposal focuses specifically upon a newly discovered orphan tyrosine kinase receptor, ROR2. Our data indicate that ROR2 inhibition can lead to decreases in melanoma cell motility. Preliminary data from our laboratory indicates that ROR2 is present in high amounts in Wnt5A positive melanoma lines, and lower amounts in Wnt5A- negative lines. It is internalized in the former, but not the latter. Further, in low Wnt5A lines, where the receptor is present at the cell surface, treating these low Wnt5A lines with recombinant Wnt5A causes internalization of ROR2. Inhibiting ROR2 can decrease the effects of Wnt5A signaling and consequently melanoma cell motility in vitro and in vivo. Collectively, these data suggest that Wnt5A, via PKC, results in the phosphorylation of STAT-3 and a subsequent down-regulation of MART1, rendering these cells able to escape from immune surveillance. Modulation of Wnt5A or its receptors may provide beneficial effects for patients, either when coupled with immunotherapy, or as single agents. Another gene implicated by the SAGE expression data is Claudin 1. We have found that claudin 1 is highly expressed in melanomas and that its expression is regulated via PKC. Furthermore, increased expression of claudin 1 results in an increase in melanoma cell invasiveness, with concomitant increase in the matrix metalloproteinase enzymes, MMP9 and MMP2. Staining of a tissue microarray indicates that this protein is upregulated in melanoma as compared to benign nevi. These data provide evidence of both an upstream regulator of PKC (Wnt5a) as well as a downstream target of this enzyme (claudin 1), both of which contribute to the increased invasiveness in melanoma. Analysis of phosphorylation sites in claudin-1 and the contribution of these sites to the motility of melanoma cells are currently underway. It is hoped that identifying important pathways such as these with high throughput gene expression profiling techniques will lead to identifying better molecular targets for the treatment of this disease.
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