Hypoxia increases the heterogeneity of melanoma cell populations and affects the response to vemurafenib

Abstract

A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib-treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen-activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3-kinase signaling pathway proteins revealed a cell-type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p-)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p-extracellular-signal-regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type-specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non-proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.