In vitro models for pre clinical studies of melanoma Chemoresistant

Abstract

Human melanoma presents extreme chemoresistance and poor prognosis with a high risk of metastasis. The overexpression of MAPK/ERK pathway proteins is closely related to uncontrolled melanoma cells survival, and this route has been studied for the development of targeted therapies. Thus, the recent chemotherapeutics Vemurafenib (BRAF V600E inhibitor) and Trametinib (MEK inhibitor) have been considered a hope for patients with melanoma, who have presented very high rates responses few weeks after treatment. Despite the clinical success of the advanced melanoma treatment, most of the observed responses is transient, with relapse and resistance in the majority of cases, about after 7 months of treatment. The current treatment is therefore still based on the same used in the past decades, even in spite of the evolution therapeutic technology. Understanding the resistance mechanism can provide clues both for developing improved versions of a drug and for guiding the selection of appropriate drug combinations for therapy. Recent data from our laboratory has shown that the novel compounds 4-NC as well as DM-1 induce cell death for melanoma cells in a specific manner. The 4-NC is a potent proteasome inhibitor, and DM-1, a caspase 3-dependent apoptosis inductor. Here, we aim to evaluate and overcome the melanoma chemoresistance to BRAF and MEK inhibitors, by use of combinatorial therapies using 4-NC or DM-1 with BRAF/MEK inhibitors on melanoma resistant cells resistant to such therapeutics currently established. For this, Vemurafenib and Trametinib resistant from established melanoma cultures cells will be generate in our lab. In addition, we will also isolate primary cells from chemoresistant tumors samples of patients at the Cancer Hospital of Barretos. Their resistance profile will be confirmed by cell viability, DNA fragmentation, cell migration and invasion mechanisms, gene expression, and protein expression focusing on ERK reactivation, proteasome activity, autophagy and senescence profiles. Furthermore, we will compare organotypical models of human reconstructed skin in vitro with melanoma cells to monolayer cultures for characterization the importance of the tumor microenvironment in chemoresistance overcoming. Thus, drugs combinations with 4-NC and DM-1 will be determinated according to the results obtained, and collaborate on improving of current melanoma therapeutics. (AU)