Abstract 5281: Regulation of tumor growth and angiogenesis by Wilms tumor 1(WT1)

Angiogenesis is required for the growth and metastasis of cancers. Although hypoxia is the major driver of angiogenesis, there is clear variation in angiogenesis between tumor types, and even between different tumors of the same histology, suggesting that there may be other factors that regulate tumor angiogenesis. One molecule that has been implicated in angiogenesis is a transcription factor known as WT1. WT1 was originally identified as a tumor suppressor gene, is overexpressed in tumors of varied origins including sarcomas, carcinomas, and leukemias, and high level WT1 expression often correlates with poor patient survival. The role of WT1 in tumorigenesis and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. Here, we show that expression of WT1 promotes clonogenic growth in soft agar and a migratory phenotype in vitro. Tumors arising fromWT1-expressing SK-ES-1 (SKWT1A and SKWT1D) cells grew faster than control (SKNC), while tumors arising from WT1-silenced MHH-ES (MHshRNA) cells grew more slowly than control (MHNC). Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31. Quantification of CD31 positive area in tumors grown from SKWT1A and SKWT1D showed 70 −80 % more staining compared to the controls. In contrast, CD31 expression is abolished in tumors arising from MHshRNA cells. Also, immunohistochemical (IHC) analysis showed enhanced expression of WT1 in tumor sections correlates with increased expression of pro-angiogenic molecules such as VEGF, MMP9, angiopoietin-1 (Ang1), and Tie2, further supporting our hypothesis that WT1 plays a major role in regulating tumor angiogenesis. Interestingly, MHHshRNA tumors exhibited decreased expression of VEGF, MMP9, Ang1, and Tie2 proteins when compared to the MHHNC tumors. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays demonstrated that WT1 can regulate the activity of the MMP9 promoter, and chromatin immunoprecipitation assays showed that WT1 can bind directly to the MMP9 promoter. Finally, IHC analysis of primary Ewing9s sarcoma samples demonstrate that, as was seen in cell lines and xenografts, WT1, VEGF, CD31 and MMP9 expression levels in primary Ewing sarcoma tissues are correlated with each other. In summary, our data identifies MMP9 as a novel WT1 target gene and demonstrates the WT1 expression directly regulates tumor growth through an effect on angiogenesis and supports the notion that development of therapeutic strategies that target WT1 will provide effective treatment options for WT1-expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5281. doi:1538-7445.AM2012-5281