Metastatic melanoma has intrinsic resistance as a central problem to be refractory to almost therapeutic interventions. Despite numerous attempts to treat melanoma, e.g. chemo-, radio- and immunotherapy, the patients survival rate remains with poor prognosis. RAF-type proteins are intermediates in the MAPK and RAS signaling pathways cascade and affect cell proliferation. Mutations in BRAF are prevalent in human melanoma, occurring in about 40-60% of metastatic melanomas. The most prevalent mutation is BRAFV600E. The BRAFV600E mutation active protein downstream of BRAF in signal transduction by MAPK pathway. This results in proliferation and cell survival even in the absence of growth factors that would be needed for these events. Vemurafenib is a molecule that inhibits the action of BRAFV600E mutation and is an option of systemic therapy recommended for the treatment of metastatic melanoma patients. Vemurafenib is a highly selective and potent inhibitor of various mutant forms of BRAFs including BRAFV600E. In vitro, vemurafenib inhibits the MEK and ERK phosphorylation, acting in the cell proliferation inhibition in melanoma cell lines that express BRAFV600E mutation. However, not all melanoma cell lines with BRAFV600E mutation are equally sensitive to vemurafenib due to its intrinsic resistance. Although the initial response to vemurafenib is beneficial in most cases is temporary. While the evidence can suggest that BRAFV600E mutation presence is predictive of response to BRAF inhibitors/MEK, cell signaling pathway involved in the MAPK may be more heterogeneous and locally regulated by the tumor microenvironment. There is also the possibility of other pathways activation in a compensatory mechanism, such as the PI3K/AKT pathway, affecting the response of BRAF/MEK inhibition. This acquired resistance can develop with MAPK reactivation by means of secondary mutations in N-RAS or by other pathways. Although BRAF is an important melanoma oncogene and excellent therapeutic target to MAPK pathway, there may be other changes in the cellular proliferation process that express resistance to BRAF inhibitors. A greater understanding of the genetic basis of response to BRAF inhibitors is extremely necessary for the selection of the most appropriate patient population for clinical studies to develop strategies to overcome this inherent resistance. A growing number of studies suggest that phenotypic and genetic heterogeneity of tumor cell populations are crucial in the resistance to new agents. To identify and characterize the possible routes of melanoma cells resistance with the BRAFV600E mutation and vemurafenib resistant, the parental human melanoma cell lines SK-MEL-28 and UACC62 will be treated with low concentrations of vemurafenib by a long exposure time. Cells resistant to treatment will be isolated and studied opposite the parental cell lines sensitive to this compound. After characterizing the whole profile of the signaling pathways involved with vemurafenib resistance, new therapeutic targets will be studied in order to elucidate possible alternative ways to induce death in these resistant cell lines. The main goal of this project is to generate a vemurafenibe-resistant melanoma cells population to elucidate the mechanisms by which they acquire this resistance to treatment. The Hedgehog pathway (Hh) and epithelial-mesenchymal transition (EMT) show up as important in the metastatic potential control in tumor cells. The mechanisms involved in cell migration and metastasis of vemurafenibe-resistant cell lines will be investigated in the ability of EMT markers expression and possible target genes, such as Gli2, TGF-² and POMC, factors closely related to proliferation, migration and cell invasion. In-depth study of the several genes and proteins expression, we will evaluate new molecular targets related to this resistance profile, targeting the melanoma treatment.
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