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

¹ Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine and ² The Wistar Institute, Philadelphia, Pennsylvania

Requests for reprints: Xiaowei Xu, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104. Phone: 215-662-6503; Fax: 215-349-5910; E-mail: xug{at}mail.med.upenn.edu.

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1 (HIF-1) 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-1 expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1 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-1 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-1 in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1 expression. Although HIF-1 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-1 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-1 pathway. [Cancer Res 2007;67(7):3177–84]

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