Nrf2 role in the BRAFi/MEKi acquired resistance in melanoma

Introduction Melanoma is one of the most aggressive cancers with the poorest prognosis. However, the use of specific inhibitors towards mutant BRAF (BRAFi) and MEK (MEKi) in BRAF-mutated patients has significantly improved progression-free and overall survival. Nevertheless, half of the patients still develop resistance within the first year of therapy. Therefore, understanding the mechanisms of BRAFi/MEKi acquired resistance has become a priority for researchers. In these last few years, scientists have focused on the role of NF-E2-related factor 2 (Nrf2), the master regulator of the cytoprotective and antioxidant response, in acquired chemoresistance. Indeed, its expression and activity are upregulated in various cancer types resistant to several chemotherapic drugs. The aim of this study was to evaluate the contribution of Nrf2 in the BRAFi/MEKi acquired resistance in melanoma, as well as the mechanisms of its activity regulation. Material and Methods Starting from BRAF-mutated murine melanoma cell line D4M, we generated three subclones resistant to BRAFi (dabrafenib, D), MEKi (trametinib, T), andBRAFi/MEKi (double resistance, D+T). Then, we evaluated cell viability (MTT test), anchorage-independent cell growth (Sphere Formation and Soft Agar Assays), apoptosis (Annexin V/IP), Cell invasion (Transwell Boyden chamber), angiogenesis (Tube-Formation assay), intracellular oxidative stress (2′-7′-dichlorodihydrofluorescein diacetate, DCF-DA, assay), glutathione (GSH) levels (Ellman's method), gene expression (western blot, real-time PCR), and gene expression inhibition with specific siRNA. Results and Discussions After nine months of continuous treatments with D, T or D+T, we obtained the three resistant subclones. Compared with the sensitive clone, the resistant sublines showed higher resistance to D, T, or D+T treatments and an enhanced ability to anchorage-independent cell growth with increased migrationand angiogenic capacity. These results allowed us to consider these cell lines good models of resistant melanoma cells toward targeted therapies. These cells showed increased oxidative stress and GSH levels. Nrf2 was upregulated at post-translational levels, with the involvement of deubiquitinase 3 (DUB3). Interestingly, Nrf2 or DUB3 inhibition sensitised cells to targeted therapies.ConclusionNrf2 can contribute to the mechanism of targeted resistancein melanoma. A complete understanding of its role can contribute to developing increasingly effective therapies in advanced melanoma.