Screening And Functional Study Of Transcription Factors Regulating VEGF Expression

Angiogenesis is essential for cancer development and progression since adequate blood supply is necessary for cancer cell growth and metastasis. Without capillaries, the tumor will be limited to about 1~2 mm, and prone to necrosis or apoptosis. Vascular endothelial growth factor(VEGF), originally known as vascular permeability factor, is the most important regulator of tumor angiogenesis. It specifically binds to the receptors of vascular endothelial cells and participates in tumor angiogenesis, promoting tumor growth and metastasis. Currently, the VEGF family includes seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor. Since VEGF-A plays the most important function in angiogenesis, it is also referred to as VEGF. The VEGF expression is mainly regulated at the transcriptional level. Therefore, the regulation of VEGF through the binding of transcription factors to VEGF promoter is the major mechanism. Indeed, VEGF promoter contains many transcription factor binding sites, such as SP1, HIF-1, Stat3 and AP1. These transcription factors not only bind to VEGF promoter directly but also interact with growth factors, oncogenes and tumor suppressor genes to promote VEGF transcription. In addition to the already known transcription factors, identification of previously unreported transcription factors regulating VEGF transcription and their regulatory mechanisms have yet to be studied in depth.To identify previously unreported transcription factors regulating VEGF transcription, we constructed a VEGF promoter(from-2304 to +73 bp)-luciferase reporter and used a luciferase reporter assay to screen the transcription factors regulating VEGF transcription. Our data suggested that, based on the screening for transcription factors library in ZR75-1 breast cancer cells, we identified some transcription factors that could regulate VEGF transcription, such as GATA1 and DEK. Therefore, we further studied the function and mechanism of transcription factors GATA1 and DEK regulating VEGF expression and angiogenesis in breast cancer cells.Our data showed that, using luciferase reporter assay, real-time PCR and ELISA assay in breast cancer cells, GATA1 stimulated VEGF-Luc reporter activity, and increased VEGF mRNA expression and VEGF secretion level, and GATA1 knockdown decreased VEGF-Luc reporter activity, VEGF mRNA expression and VEGF secretion level. In terms of biological function, GATA1-overexpressing breast cancer cell promoted VEGF secretion level, and then increased HUVEC proliferation, migration, and tube formation in vitro, and enhanced the formation of new blood vessels on the CAM in vivo. In contrast, GATA1 knockdown led to the opposite results. Mechanistically, Ch IP assay and EMSA assay indicated that GATA1 was recruited to the region containing the GATC site, but not the GATA site, and GATA1 recruited the histone methyltransferase SET7, RNA polymerase II and TFIIB to the GATC site of VEGF promoter. SET7 can monomethylate H3K4, leading to activation of gene transcription. Our results showed that GATA1 and SET7 could synergistically enhance VEGF transcription. Reciprocal coimmunoprecipitation experiment indicated that GATA1, SET7, RNA Pol II, and TFIIB interacted with each other, and formed a preinitiation complex on the GATC site of VEGF promoter. Furthermore, GATA1 regulated VEGF expression and VEGF-induced HUVEC proliferation, migration and tube formation as well as angiogenesis via SET7. Breast cancer cell proliferation assay and animal experiment showed that GATA1 promoted breast cancer cell growth and tumor angiogenesis through SET7. The IHC results showed that expression of both GATA1 and SET7 positively correlated with VEGF expression and microvessel number, and was independent prognostic marker for breast cancer, implicating the significance of GATA1 and SET7 in breast tumor angiogenesis and providing new concepts and strategies for breast cancer treatment.Moreover, DEK also increased VEGF expression. DEK-overexpressing breast cancer cell enhanced VEGF secretion level, and increased HUVEC proliferation, migration, tube formation as well as angiogenesis. Co-immunoprecipitation(co-IP) assays showed that DEK interacted with HIF-1α(hypoxia-inducible factor-1α. Since HIF-1α is a master regulator of VEGF expression in response to hypoxia, we detected the function of HIF-1α in DEK-mediated VEGF expression and angiogenesis. Our results showed that DEK enhanced VEGF expression in HIF-1α-dependent and-independent manners. Furthermore, DEK was recruited to the regions containing DRE and HRE of VEGF promoter, as well as p300, and DEK enhanced the recruitment of HIF-1α and p300. Reciprocal coimmunoprecipitation experiment demonstrated that endogenous DEK, HIF-1α, and p300 proteins interacted with each other, and formed a complex to regulate VEGF expression. Animal experiment with MDA-MB-231 breast cancer cells showed that DEK enhanced tumor angiogenesis and growth in HIF-1α-dependent and-independent manners. The IHC results indicated that DEK positively correlated with VEGF expression and microvessel number in breast cancer patients.In conclusion, we identified GATA1 and DEK, two unreported transcription factors that promoted VEGF transcription. They regulate VEGF expression and VEGF-induced HUVEC proliferation, migration and tube formation, and enhance breast tumor growth and angiogenesis, through interacting with SET7 or partially with HIF-1α. Our data suggest that GATA1 and DEK play important roles in breast cancer development and progression.