title: Mutant V600E BRAF increases hypoxia inducible factor-1alpha 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine and The Wistar Institute, Philadelphia, Pennsylvania, USA.
authors.forename: Suresh M
authors.initials: SM
authors.lastname: Kumar
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.forename: Robin
authors.initials: R
authors.lastname: Edwards
authors.forename: Lianjun
authors.initials: L
authors.lastname: Chen
authors.forename: Steven
authors.initials: S
authors.lastname: Kazianis
authors.forename: Patricia
authors.initials: P
authors.lastname: Brafford
authors.forename: Geza
authors.initials: G
authors.lastname: Acs
authors.forename: Meenhard
authors.initials: M
authors.lastname: Herlyn
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Xu
mesh_terms: D001557:Benzenesulfonates / Q000494:pharmacology; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D000077157:Sorafenib; D014162:Transfection; D015854:Up-Regulation
chemical_list: D001557:Benzenesulfonates; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010671:Phenylurea Compounds; D011725:Pyridines; D034741:RNA, Small Interfering; D009536:Niacinamide; D000077157:Sorafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf