Role of Methylation in Modulating MAPK Signaling. Implications in Melanoma. Papel de la metilación como modulador de la señalización en MAPK. Implicaciones en melanoma.

1. General Overview MTAP is a key enzyme in the methionine salvage pathway and controls the levels of methylthioadenosine (MTA). Lost of MTAP, which is frequent in melanoma, results in higher intra and extracellular MTA levels. As MTA is an inhibitor of methylation reactions within the cell, we decided to further investigate the role of methylation in the progression of cancer and how the main signaling pathway involved in melanoma (RAS-RAF-MEK-ERK pathway) can be affected in the presence or absence of methylation inhibitors. 2. Results • Exogenous addition of MTA (and therefore blocking of methylation reactions) to melanoma cells increased total activity of the RAS-ERK pathway, highlighting a new role of methylation into controlling the amplitude of the signal transduction. • We found out that PRMT5, a protein arginine methyl transferase enzyme, binds to CRAF, a key component of the RAS-ERK pathway. In vitro and in vivo experiments showed that PRMT5 methylates CRAF and thus modifies the pathway’s output. We identified CRAF arginine 563 as the target of PRMT5 methyl transferase activity. Furthermore, in vitro kinase assays and siRNA technology experiments showed that methylation at arginine 563 can modify the kinase activity of CRAF protein, therefore modulating the total activity of the RAS-ERK pathway. • The previous results provided the first known evidence of methylation as a key modulator of the activity in MAPKs pathways, so we looked for a biological significance of this new regulatory mechanism. We used the well-established PC12 cell line system (EGF growth factor triggers proliferation whereas NGF promotes differentiation): as we had shown that PRMT5-catalyzed methylation can modify the kinase activity of the RAS-ERK pathway, we wondered whether this regulation could be part of a general mechanism controlling cell behavior. In order to test this, we tried to mimic the differentiating effect of NGF using EGF to activate the pathway in the presence of MTA or after PRMT5 siRNA. Interestingly, when triggering the cells with EGF after PRMT5 depletion or MTA treatment, they differentiated in a manner indistinguishable from that induced by NGF. Moreover, we observed that cells transfected with CRAFR563K mutant underwent differentiation, instead proliferation, when treating the cells with EGF. Together these data demonstrate a central role of methylation in the growth factors-elicited biological responses. • RAS-ERK pathway plays a key role into controlling development and maintenance of melanoma. There is a clear correlation between the activity of the pathway and melanoma growth and proliferation, so we wondered how methylation reactions could affect melanoma progression. In order to address this question we performed in vitro assays in a variety of melanoma cell lines and we found out that MTA inhibited cell proliferation and viability. Next, we injected these cells in a melanoma xenograft mouse model to induce tumoral growth: mice treated with MTA showed a significant decrease (47%) in the tumor volume with no apparent toxic effects, indicating that MTA is effective blocking melanoma in vivo tumor growth. Besides, we have been able to proof that MTA inhibitory effect was due to citostatic MTA capability rather than a pro-apoptotic effect. 3. Discussion In our reports we demonstrate that methylation is an important mechanism modulating signal transduction through the RAS-ERK pathway in response to specific growth factors. Our data implicate a specific PRMT5 methylation motif (GRG) that is conserved in all RAF proteins and we demonstrate that methylation appears to be a critical posttranslational modification controlling the RAF input to the downstream activity. We have shown that methylation is controlling ERK signal amplitude in response to specific growth factors in cells from different species. We also have identified PRMT5 as the protein performing the methylation reaction. Our results provide clues about the possible fine-tuning mechanism by which methylation regulates the total signal, indicating that PRMT5 methylates CRAF at arginine 563. Our research shows that protein arginine methylation is a universal mechanism that modulates signal transduction in response to specific growth factors and we demonstrate that PRMT5 is the methyltransferase involved in the process regulating the total CRAF kinase activity within the pathway. The mechanism we describe provides the means by which cells can modulate the time profile of ERK activation for which a particular biological response is evoked. More importantly, because cancer cells tend to heavily rely on oncogenic signalling through the RAS-RAF-MEK-ERK pathway, this additional level of signal transduction regulation identifies novel candidate targets for therapeutical intervention. Finally, we have tested the potential of the methylation inhibitor MTA regarding to tumour progression: our experiments have demonstrated that MTA inhibits, in vitro, cell proliferation and viability in a dose dependent manner in a variety of mouse and human melanoma cell lines. Importantly, MTA was also effective inhibiting in vivo tumour growth in a mouse melanoma xenograft model. In summary, here we show the therapeutic potential of the natural occurring nucleoside MTA and we demonstrate that MTA can inhibit melanoma cell proliferation and in vivo tumour growth at non-toxic rates, supporting the use of MTA in antitumoral therapies and presenting protein methylation reactions as potential targets when developing new antitumoral strategies