Abstract
Hypoxia stimulates a number of pathways critical to cancer cell survival, including the activation of vascular endothelial growth factor (VEGF) transcription. In normal fibroblasts, hypoxia-induced activation of the protein tyrosine kinase, Src, is required for VEGF expression. We show here in both pancreatic and prostate carcinoma cell lines cobalt chloride (used to mimic hypoxia) -induced VEGF expression requires Src activation and leads to increased steady-state levels of HIF-1alpha and increased phosphorylation of signal and transducer of transcription 3 (STAT3). STAT3 and hypoxia-inducible factor (HIF)-1alpha bind simultaneously to the VEGF promoter, where they form a molecular complex with the transcription coactivators CBP/p300 and Ref-1/APE. Expression of activated Src from an inducible promoter is sufficient to increase VEGF expression and form these STAT3/HIF-1alpha-containing promoter complexes. Inhibition of DNA binding by expression of either STAT3 or HIF-1alpha dominant negative mutants significantly reduces VEGF expression. These data suggest that the binding of both STAT3 and HIF-1alpha to the VEGF promoter is required for maximum transcription of VEGF mRNA following hypoxia.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Autoradiography
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Cell Line, Tumor
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Chromatin Immunoprecipitation
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Cobalt / pharmacology
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DNA-(Apurinic or Apyrimidinic Site) Lyase / biosynthesis*
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DNA-Binding Proteins / metabolism*
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Enzyme Activation
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Enzyme-Linked Immunosorbent Assay
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Female
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Fibroblasts / metabolism
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Gene Expression Regulation, Neoplastic*
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Humans
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Hypoxia / metabolism
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Hypoxia-Inducible Factor 1, alpha Subunit
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Immunoblotting
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Immunoprecipitation
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Luciferases / metabolism
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Male
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Microscopy, Fluorescence
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Neoplasm Metastasis
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Neoplasms / metabolism
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Neovascularization, Pathologic
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Nuclear Proteins / biosynthesis*
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Pancreatic Neoplasms / metabolism
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Phosphorylation
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Promoter Regions, Genetic
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Prostatic Neoplasms / metabolism
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Protein Binding
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RNA, Messenger / metabolism
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STAT3 Transcription Factor
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Signal Transduction
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Trans-Activators / biosynthesis*
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Trans-Activators / metabolism*
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Transcription Factors / biosynthesis*
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Transcription, Genetic
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Vascular Endothelial Growth Factor A / metabolism*
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src-Family Kinases / metabolism
Substances
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DNA-Binding Proteins
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HIF1A protein, human
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Hypoxia-Inducible Factor 1, alpha Subunit
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Nuclear Proteins
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RNA, Messenger
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STAT3 Transcription Factor
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STAT3 protein, human
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Trans-Activators
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Transcription Factors
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Vascular Endothelial Growth Factor A
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Cobalt
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Luciferases
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src-Family Kinases
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APEX1 protein, human
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DNA-(Apurinic or Apyrimidinic Site) Lyase
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cobaltous chloride