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-1α (HIF-1α) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAFV600E increases HIF-1α expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1α gene expression was significantly increased in melanomas harboring BRAFV600E mutation. Stable suppression of mutant BRAFV600E or both wild-type and mutant BRAFV600E by RNA interference in melanoma cells resulted in significantly decreased HIF-1α expression. Knockdown of mutant BRAFV600E induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAFV600E resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAFV600E into tumor cells. Transfection of BRAFV600E into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1α in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1α expression. Although HIF-1α 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 BRAFV600E mutation increases HIF-1α 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-1α pathway. ©2007 American Association for Cancer Research.