Abstract 1599: Elevated expression of ZEB1 upregulates VEGF expression and induces tumor angiogenesis in breast cancer

Abstract Background: Angiogenesis is essential for the growth and metastasis of solid tumors, including breast cancer. Although the zinc finger-homeodomain transcription factor ZEB1 is implied as a key regulatory factor in multistep carcinogenesis of breast cancer, its direct role in the regulation of tumor angiogenesis has not been well elucidated. In this report, we extended our study to examine the potential effect of ZEB1 in breast cancer angiogenesis and its molecular mechanism using the in vitro and in vivo models. Materials and Methods: Quantitative RT-PCR, immunoblotting, and ELISA were used to detect ZEB1-induced expression and/or secretion of VEGF in the absence or presence of the indicated signaling pathway inhibitors (the PI3K, NF-κB, and MAPKs pathways) in MDA-MB-231 breast cancer cells. Tube formation assay was performed to detect ZEB1-driven angiogenesis using human umbilical vein endothelial cells (HUVEC). Luciferase and CHIP assays were used to determine the transcriptional activity of the wild-type and mutant VEGF promoters affected by ZEB1. A nude mouse tumor xenograft model was used to examine ZEB1-driven angiogenesis in vivo. The relationship between the expression of ZEB1 and VEGF protein in human breast cancer specimens was assessed by immunohistochemistry. Results: We found that ectopic expression of ZEB1 promotes the expression of VEGF at both the mRNA and the protein levels in MDA-MB-231 cells, which effect is specifically mediated through the p38 and PI3K pathways. The conditioned medium derived from ZEB1-overexpressing MDA-MB-231 cells significantly induces the tube formation of HUVEC compared with that from control-overexpressing cells. Importantly, elevated expression of ZEB1 resulted in an increased angiogenesis in a nude mouse tumor xenograft model. We further moved on to detect whether ZEB1 is a bona-fide transcriptional regulator of VEGF. The results of luciferase and CHIP assays showed that ZEB1 transcriptionally induces the promoter activity of human VEGF by recruiting Sp1 to its biding sites on the VEGF promoter, thus up-regulating VEGF expression in MDA-MB-231 cells. Through the study of breast cancer specimens, we found a strong positive correlation between ZEB1 and VEGF protein expression, further supporting the contribution of ZEB1/VEGF cascade to tumor angiogenesis in breast cancer. Conclusions: Our data demonstrated that ZEB1 up-regulates VEGF expression and thus induces tumor angiogenesis in breast cancer. Thus, inhibition of VEGF expression by down-regulating ZEB1 provides a potential new strategy for overcoming tumor angiogenesis. Citation Format: Qi Tong, Lingjia Liu, Lin Liang, Yang Gao, Shuang Yang. Elevated expression of ZEB1 upregulates VEGF expression and induces tumor angiogenesis in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1599. doi:10.1158/1538-7445.AM2014-1599