COMPARATIVE ANALYSIS OF ENDOCANNABINOID SYSTEM GENE EXPRESSION ON METASTATIC VS PRIMITIVE MELANOMA CELL LINES.

The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in cell proliferation, differentiation and survival is well recognized, while its involvement in malignancy is actively investigated. In order to characterize the endocannabinoid system in melanoma cells with progressive levels of malignancy, we investigated endocannabinoid receptor expression by RT-PCR in four melanoma cell lines. To investigate possible differences in the expression levels of endocannabinoid system genes that could be related to melanoma progression, we used as experimental model two pairs of primitive-metastatic melanoma cell lines, LCP/LCM and WM115/WM266, each pair deriving from two melanoma lesions at different stage of a single patient. Additionally, the metastatic melanoma cell line A375 was also investigated. We evidenced significant results regarding TRPV2 and TRPV4 receptors, which were respectively overexpressed in both the metastatic cell lines LCM and WM266, with respect to their primitive counterparts LCP and WM115. Instead, we observed low levels of expression of CB1 and CB2 receptors in all cell lines tested. Moreover, we found no significant differences of expression levels between primitive and metastatic cell lines in all the other endocannabinoid system genes investigated (GPR18, GPR55, TRPA1, TRPV1). Levels of endocannabinoids AEA, 2AG, PEA and OEA were also quantified by LC-MS analysis evidencing high levels of OEA, mostly in primitive cells. AEA, PEA showed also higher levels in the primitive cell lines with respect to the metastatic ones. Based on the results of qPCR, we investigated the role of TRPV2 and TRPV4 channels in melanoma growth and proliferation. We measured the effect on cell viability of the TRPV2 agonist lysophosphatidylcholine (LPC) on LCP/LCM melanoma cells and the TRPV4 agonist 4?-Phorbol 12,13-didecanoate (4?PDP) on WM115/WM266 cells using a MTT test. Preliminary results pointed out similar effect of LPC on both LCP and LCM melanoma cell lines, not confirming the significance of the TRPV2 differential expression observed between primitive and metastatic cells. On the other hand, 4?PDP did not show significant effect on the WM115/WM266 pair of melanoma cells, discouraging our hypothesis of TRPV4 possible involvement in melanoma progression. Further analyses to identify potential molecular targets related to endocannabinoid system in advanced melanoma are ongoing.