Mutant V600E BRAF Increases Hypoxia Inducible Factor-1α Expression in Melanoma

Abstract

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1alpha (HIF-1alpha) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAF(V600E) increases HIF-1alpha expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1alpha gene expression was significantly increased in melanomas harboring BRAF(V600E) mutation. Stable suppression of mutant BRAF(V600E) or both wild-type and mutant BRAF(V600E) by RNA interference in melanoma cells resulted in significantly decreased HIF-1alpha expression. Knockdown of mutant BRAF(V600E) induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAF(V600E) resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAF(V600E) into tumor cells. Transfection of BRAF(V600E) into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1alpha in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1alpha expression. Although HIF-1alpha translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAF(V600E) mutation increases HIF-1alpha expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1alpha pathway.