Resensitizing therapy-resistant anoxic metastatic melanoma cells

Introduction: Metastatic melanoma significantly reduces patient survival. Metastatic growth can outpace vascularization creating areas of extreme hypoxia or anoxia. HIF-1alpha is a master transcriptional regulator of cellular survival response to hypoxia that can facilitate the selection of more stem cell-like, drug-resistant cancer cells. BRAF inhibitors (BRAFi), commonly used to treat melanoma, targets cell signaling involving pERK-dependent melanoma growth. Resistance to BRAFi can lead to melanoma recurrence and poor survival. Dual specificity phosphatases (DUSP), such as DUSP6 can reduce ERK activity; moreover, DUSP6 has been shown to be upregulated by HIF-1alpha during cellular responses to hypoxia. We hypothesize that HIF-1alpha levels increase in metastatic melanoma cells as these adapt to the anoxic metastatic niche, which then increases DUSP6 expression, which in turn reduces ERK activity that causes resistance to BRAFi. Methods: Western blot (WB) was used to compare the expression of HIF-1alpha, pERK, and DUSP6 in lysates from A375 melanoma cells grown in anoxia or normoxia. An MTT cell proliferation, and a cell apoptosis assay were used to compare the effect of the BRAFi, Dabrafenib between treated normoxic and anoxic A375 cells. Lysates were collected for WB to assess effects on pERK between anoxic A375 treated with the DUSP6 inhibitor, BCI, and untreated control. Finally, to see if BCI can increase drug sensitivity, MTT and cell apoptosis assays were performed on anoxic A375 treated with both BCI and Dabrafenib. Results: WB results show that anoxic A375 cells express higher levels of HIF-1alpha, and lower levels of pERK compared to normoxic control cells. Dabrafenib treatment reduced cell proliferation of normoxic but not anoxic A375 cells, presumably due to reduced expression of the pERK target in these cells. Inhibition of DUSP6 with BCI significantly increased pERK expression and when combined with Dabrafenib significantly reduced proliferation and increased cell death of the anoxic A375 cells. Conclusion: We show that melanoma cells cultured in anoxia express higher levels of HIF-1alpha. This in turn led to an increase in DUSP6 expression. In these anoxic cells, DUSP6, in turn, reduces pERK level, which is an important component of the signaling cascade targeted by Dabrafenib. Finally, our results from the combination treatment with both BCI and Dabrafenib suggest that by inhibiting DUSP6 it may be possible to re-express pERK in drug-resistant anoxic metastatic melanoma cells and reestablish drug sensitivity.