The Role of Redox Sensitive NRF2 Transcription Factor in Melanoma Drug Resistance

Human melanocytes induces cytoprotective gene expression in response to UV induced oxidative stress. NRF2, a cap'n' collar basic-region leucine zipper protein is an oxidative and electrophilic stress sensitive transcription factor responsible for the expression of antioxidants and expulsion of xenobiotic drugs. NRF2 targets a key cis-acting element called the antioxidant response elements (ARE) found at the promoter of cytoprotective genes. However, constitutive activation of NRF2 in cancer increases tumorigenicity, resistance to chemotherapy and proliferation. We hypothesized, that constitutive NRF2 activity in metastatic melanoma cells mediates resistance to chemotherapy.;The basal transcript expression of NRF2 with respect to human melanocytes is expression in all 8 melanoma cell lines. Immunoblots of NRF2 displayed abundant expression in 5 of 8 human malignant melanoma lines compared to human melanocytes. A surrogate reporter of NRF2's transcriptional activity is NQO1, a gene closely regulated by NRF2 through ARE elements at its promoter. NQO1 expression was increased in 5 of 6 metastatic melanoma; similar results in immunoblots for NQO1.;Next, to determine if NRF2 in melanoma cells leads to chemosensitivity we developed stable NRF2 knockdowns in melanoma cell lines. The shRNA targeting (mRNA)NRF2 expression, resulted in 70% reduction of NRF2 transcripts in 4 of 6 constructs in 3 metastatic melanoma cell lines, and immunoblots for NRF2 resulted in protein levels below detection in 2 transgenic lines. Loss of NRF2 in SK-MEL-28KD1 sensitized these cell lines to vemurafenib, cisplatin and DHA treatment, with a calculated fold reduction of -5.2-fold, -1.7-fold, and -2.4-fold, respectively. These results demonstrated that NRF2 loss sensitized metastatic melanoma lines to vemurafenib, cisplatin, and DHA treatment, in spite of their wide difference in mechanism that lead to apoptosis.;BRAF mutation frequency in human metastatic melanoma is about 50%, vemurafenib is an inhibitor of mutant BRAF kinase that produces an effective clinical response. However, patients eventually develop resistance to vemurafenib through a mechanism that reactivates the MAPK pathway resulting in decreased overall survival. Here, we investigated the possibility of NRF2 induction contributing to a mechanism of vemurafenib resistance in BRAF mutant human metastatic melanoma cells and found vemurafenib inhibited NRF2 expression and activity in melanoma cells. Also, MEK1/2 inhibitor induced loss of NRF2 expression. An attractive strategy to sensitize metastatic melanoma to drug treatment is to inhibit NRF2 expression, in combination with a chemotherapeutic agent. DNA damaging agent (cisplatin) and vemurafenib synergistically (CI value of 0.51 +/- .11) enhanced the cell-killing effects of the drugs. Finally, the current availability of FDA approved vemurafenib when used in combination with other drugs, offers a novel strategy for augmenting the potency of previously ineffective therapies, through the pharmacological inhibition of NRF2.;The expression of NRF2 in human melanocytes cells is highly regulated and maintains an important function in protecting cells from the oxidative effects of environmental stressors and xenobiotics. However, in human melanoma cells NRF2 is aberrantly regulated, enhancing resistance to drug treatments and overall survival. Surprisingly, vemurafenib is a novel inhibitor of NRF2, and demonstrated that NRF2 is downstream of the MAPK cascade pathway. Furthermore, acquired resistance to vemurafenib contributed to increased expression of NRF2, an effect leading to drug resistance. This study provides evidence of a therapeutic potential of a pharmacological inhibitor, which sensitized melanoma to drug treatments. Finally, the current availability of FDA approved vemurafenib when used in combination with other chemotherapies, offers a novel strategy for augmenting the potency of previously ineffective therapies, through the pharmacological inhibition of NRF2.