WT1 protein directly regulates expression of vascular endothelial growth factor and is a mediator of tumor response to hypoxia.
WT1 is a zinc finger transcription factor expressed at high levels in many types of solid tumors, and high WT1 expression is an adverse prognostic factor. How WT1 contributes to tumor growth and influences prognosis remains unclear. We investigated the hypothesis that WT1 up-regulates VEGF in solid tumors, augmenting the response to hypoxia. We found a correlation between levels of WT1 expression and VEGF expression in Ewing sarcoma cell lines. Transfecting WT1-null SK-ES-1 cells with WT1 up-regulated VEGF mRNA expression and resulted in increased angiogenic activity in vitro. Conversely, diminishing WT1 expression in WT1-positive cell lines using WT1-specific shRNA down-regulated VEGF mRNA expression and decreased angiogenic activity in vitro. Transient transfection assays demonstrated that WT1 can regulate the activity of the VEGF promoter, and chromatin immunoprecipitation assays showed that WT1 can bind directly to the VEGF promoter in intact cells. WT1 expression in Ewing sarcoma cells is up-regulated by hypoxia. Importantly, using shRNA to inhibit this up-regulation blunted the hypoxia-mediated increase in VEGF expression. Taken together, these data demonstrate that VEGF is a direct, bona fide WT1 target gene in sarcoma and that WT1 plays a key role in optimizing the response of tumor cells to hypoxia.
Division of Pediatric Oncology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21231.
Gregory
G
McCarty
Division of Pediatric Oncology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21231.
Ola
O
Awad
Division of Pediatric Oncology, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21231. Electronic address: loebda@jhmi.edu.
David M
DM
Loeb
D000818:Animals; D018450:Disease Progression; D042783:Endothelial Cells / Q000166:cytology; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D000860:Hypoxia; D051379:Mice; D041681:NIH 3T3 Cells; D009389:Neovascularization, Pathologic; D011379:Prognosis; D017434:Protein Structure, Tertiary; D012512:Sarcoma, Ewing / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D025721:WT1 Proteins / Q000378:metabolism*
D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D025721:WT1 Proteins