Abstract
Angiogenesis is required for tumor growth. WT1, a protein that affects both mRNA transcription and splicing, has recently been shown to regulate expression of vascular endothelial growth factor (VEGF), one of the major mediators of angiogenesis. In the present study, we tested the hypothesis that WT1 is a key regulator of tumor angiogenesis in Ewing sarcoma. We expressed exogenous WT1 in the WT1-null Ewing sarcoma cell line, SK-ES-1, and we suppressed WT1 expression using shRNA in the WT1-positive Ewing sarcoma cell line, MHH-ES. Suppression of WT1 in MHH-ES cells impairs angiogenesis, while expression of WT1 in SK-ES-1 cells causes increased angiogenesis. Different WT1 isoforms result in vessels with distinct morphologies, and this correlates with preferential upregulation of particular VEGF isoforms. WT1-expressing tumors show increased expression of pro-angiogenic molecules such as VEGF, MMP9, Ang-1, and Tie-2, supporting the hypothesis that WT1 is a global regulator of angiogenesis. We also demonstrate that WT1 regulates the expression of a panel of pro-angiogenic molecules in Ewing sarcoma cell lines. Finally, we found that WT1 expression is correlated with VEGF expression, MMP9 expression, and microvessel density in samples of primary Ewing sarcoma. Thus, our results demonstrate that WT1 expression directly regulates tumor angiogenesis by controlling the expression of a panel of pro-angiogenic genes.
Highlights
WT1 is a transcriptional regulatory protein that is overexpressed in a wide variety of tumor types, including leukemia, breast cancer, and sarcomas [1,2,3,4]
WT1 mRNA levels were reduced by 58.7 ± 9.33% in MHHshRNA cells (MHH-ES cells stably expressing WT1 shRNA) compared with MHHNC cells (MHH-ES cells transfected with the negative control RNA), and a similar reduction is seen by western blotting
Previous work has demonstrated that WT1 regulates vascular endothelial growth factor (VEGF) expression [10, 24], but this is the first demonstration in an animal model that this translates into an increase in tumor angiogenesis
Summary
WT1 is a transcriptional regulatory protein that is overexpressed in a wide variety of tumor types, including leukemia, breast cancer, and sarcomas [1,2,3,4]. The role of WT1 in sarcoma biology remains unclear, work from our laboratory, and from others, has implicated WT1 in the regulation of angiogenesis. WT1 is upregulated by hypoxia in endothelial cells in a coronary artery ligation model of myocardial infarction [8], and WT1 expression has been demonstrated in tumor endothelial cells [9]. Our laboratory demonstrated that WT1 is upregulated by hypoxia in Ewing sarcoma cells in vitro, and that in these cells, WT1 is a direct positive regulator of vascular endothelial growth factor (VEGF) expression [10]. We further demonstrated that blocking the hypoxia-mediated upregulation of WT1 blunts the hypoxia-mediated upregulation of VEGF in these cells, supporting a functional role of WT1 in the response to hypoxia
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