Tetraspanins and Melanoma: Role of CD63 in the control of cell motility, invasiveness and proliferation

Premises: Melanoma is one of the most aggressive tumors with high metastatic potential and rate of mortality. Over the last decades, the increase in incidence have mainly been reported for thin melanomas (£1mm) associated with a good prognosis. Whereas the rate for thick melanomas (>1mm), with unfavourable prognosis and associated with a higher rate of distant metastases and worse survival, seems to be fairly stable. Hence, there is a need to identify new prognostic markers of progression and the new potential drug targets. A possible negative marker of metastasis is the CD63 protein, belonging to the Tetraspanins superfamily. It is highly expressed in several normal tissues as well as in the early stage of melanoma. We have previously observed a progressive decrease of CD63 protein levels in tissue samples of human melanocytic nevi, dysplastic nevi, thin melanomas, thick melanomas and metastasizing melanomas in advanced stages of melanoma progression. This suggests that the gene coding CD63 could be a potential metastasis suppressor. Working hypothesis: We hypothesized that CD63 could play a functional role in controlling cell proliferation, motility and invasion, probably by means of functional interactions with other proteins playing key role in these processes. Based on data reported in literature, the pro-metastatic proteins uPAR, MMP-2, MMP-9 and the tissue inhibitor of metalloproteinases-1 (TIMP-1) seemed the best candidates. We also supposed that CD63 could play a role in the Epithelial-Mesenchymal transition (EMT), acting as negative marker of this process. Results: We demonstrated that the exogenous modulation of CD63 in human melanoma cell line A375 was correlated with cell proliferation, invasiveness and motility. In particular, A375 cells overexpressing CD63 shown a significant reduction in cell proliferation, motility and invasiveness compared to untreated control and compared to cell lines where CD63 has been reduced with siRNA. We also demonstrated that CD63 was inversely correlated with the pro-metastatic proteins uPAR, MMP-2 and MMP-9, whose levels were reduced in cells overexpressing CD63. We also observed that B16 murine melanoma cells stimulated by inflammatory cytokines shown a reduction of CD63 tetraspanin expression and this was associated with the promotion of a metastatic phenotype, characterized by a high capacity to colonize host lungs and expressing a high level of uPAR and MMP-9 (Bianchini F et al., Onc Rep 2006). The reduction of CD63 expression was dose-dependent and proportional to the increase of IFNγ stimulation. However the molecular mechanism awaits to be unrevealed. Recently, many studies reported the role of epithelial-mesenchymal transition in matrix degradation and cell invasiveness. We observed that CD63 expression in A375M6, a lung colony derived from A375 cells that shown a non-EMT profile, was higher compared to A375 and significantly decreased after treatment with the EMT inducer TGFβ. To explain and confirm this observation, we performed an invasion assay. We observed an increased invasiveness of clones overexpressing CD63 stimulated with medium derived from silenced cells as compared to invasion capacity of non-stimulated cells, probably due to a major content of MMPs. Conclusions: In summary, these results strongly point the high expression of CD63 as a major contributor to a non-invasive phenotype in melanoma. As the invasive phenotype of cells involves induction of molecular changes associated with EMT, we have reason to believe that CD63 may play a key role in these processes, acting as a negative marker of EMT. Finally, we conclude that CD63 has a potential to be a suppressor gene playing an important role in inhibition of melanoma progression, and as such could be a target for pharmacological and drug developing strategies.