Epigenetic regulation in conjunction with signaling pathways, play crucial roles in pluripotency, cell differentiation, and transcriptional plasticity in cancer. Indeed, a third of all cancers aberrantly express pluripotency genes and retain stemness, allowing for cancer recurrence following chemo- or radiotherapy. Vascular Endothelial Zinc Finger 1 (VEZF1) is a transcription factor expressed ubiquitously in adult tissues. VEZF1 null ESCs retain alkaline phosphatase staining postdifferentiation, suggesting a potential role of VEZF1 in pluripotency exit. However, the role of VEZF1 in pluripotency maintenance and exit is novel. Using mouse embryonic stem cells as a model system for pluripotency maintenance and exit, we sought to determine the regulatory role of VEZF1 in pluripotency. Our data indicate that ESCs derived from Vezf1 knockout mice (Vezf1 KO) show higher expression of pluripotency genes than Wildtype (WT) cells. Increased expression of pluripotency genes was confirmed upon the temporal ablation of Vezf1 in ESCs using a Doxycycline-inducible knockdown system. Interestingly, we observed that Vezf1 KO ESCs show severe defects in the repression of the pluripotency program postdifferentiation. Our analysis of VEZF1 ChIP-Seq data in ESCs revealed the binding of VEZF1 to promoters of multiple genes involved in canonical WNT and MAPK signaling. The WNT and MAPK signaling pathways are crucial for pluripotency maintenance and exit. Therefore, we proposed that VEZF1 enhances the inducible expression of WNT signaling genes in normal and cancer cells. To test this hypothesis, we performed RNA-Seq analysis which showed decreased expression of WNT signaling genes in Vezf1 KO ESCs, supporting the role of VEZF1 in Wnt signaling. Previous data indicate a significant overlap of VEZF1 and CTCF genome-wide. Therefore, to gain insights into the mechanism of gene regulation by VEZF1, we performed CTCF ChIP-Seq in WT and Vezf1 null cells. Our data revealed an increase in CTCF binding in the absence of VEZF1 compared to WT cells. The unique CTCF binding profile in Vezf1 KO cells was associated with genes involved in WNT and MAPK signaling pathways, both of which regulate pluripotency. This observation suggests that Vezf1 occludes CTCF from repressing WNT signaling genes. Our study of VEZF1 in ESCs suggests that the reduced expression of Vezf1 in cancer cells will potentially support the formation of cancer stem cells. Indeed, we have observed reduced expression of Vezf1 in F9 embryonal carcinoma cells (ECCs). Notably, F9 ECCs exhibit incomplete repression of pluripotency genes akin to Vezf1 KO ESCs. Collectively, this novel study emphasizes the importance of VEZF1 in regulating pluripotency and will expand our understanding of the development of stemness in cancer cells toward developing effective therapeutics against cancer. Citation Format: Isaiah K. Mensah, Martin Emerson, Hern Tan, Ming He, Humaira Gowher. Vascular endothelial zinc finger 1 promotes stemness by regulating key signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 329.
Cardiovascular diseases (CVDs) are the leading cause of high morbidity rates and death worldwide. The heart is the first organ that is formed during mammalian development. Progenitor cells that form the cardiovascular system (heart and blood vessels) originate from the mesoderm post gastrulation. To date, there remain considerable gaps in the molecular mechanisms that govern the differentiation of cardiac progenitor cells into a functional heart, knowledge of which will provide advancements in cardiac regenerative medicine. Vascular endothelial zinc finger 1 (Vezf1) is a nuclear protein expressed predominantly in the mesoderm of a developing mouse embryo. A recent study showed that diseased human myocardia had reduced expression of Vezf1 when compared to healthy hearts. However, the function of Vezf1 in heart development is largely uncharacterized and understudied. Using murine embryonic stem cells (mESCs), we sought to investigate the early role(s) of Vezf1 in cardiovascular development. We show that genetic ablation or conditional knockdown of Vezf1 in mESCs impairs cardiomyocyte differentiation, as determined by the absence of contractile activity in these mutants. The absence of cardiomyocytes from Vezf1 mutant cell lines was confirmed using gene expression analysis and immunofluorescence. We observed impaired mesoderm induction in Vezf1 mutants despite external stimulation of Wnt signaling using small molecule inhibitors, suggesting a potential role of Vezf1 in the modulation of genes in the signaling pathway. Indeed, our gene expression analysis and ChIP-qPCR indicate that Vezf1 potentially associates with the promoters of Wnt signaling genes to activate their expression. These studies point towards a model where Vezf1 regulates the formation of cardiac progenitor cells by modulating the Wnt signaling pathway. Our observations suggest that Vezf1 may sit at the center of determining the fate of mesodermal cells into cardiovascular progenitors, the absence of which impairs cardiomyocyte differentiation. This study is the first to delineate the functional role of Vezf1 in the early development of cardiomyocytes and may form the foundation for future cardiac regenerative medicine.
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