Abstract 5585: DNA methylation and transcription factors regulate overexpression of PVT1 in prostate cancer

Abstract Population-level genomic analysis of the PVT1 gene, in the 1000 Genomes Project, revealed genetic differentiation between African and non-African populations at PVT1 exons 4A and 4B. Also, PVT1 exons 4A and 4B showed increased expression in prostate cancer (PCa) tissues relative to normal prostate tissues and benign prostatic hyperplasia tissues in men of African ancestry (moAA). We demonstrated that overexpression of PVT1 exons 4A, 4B and 9 caused increased migration and proliferation of prostate epithelial cells. Further, overexpression of PVT1 exon 9 transformed non-tumorigenic prostate epithelial cells into tumorigenic cells that form tumors with neuroendocrine phenotype when implanted in mice. Gene expression is modulated by DNA methylation (DMT), so we investigated the role of DMT as a mechanism of regulating PVT1 expression. Using qPCR analysis, we tested this hypothesis by treating five prostate epithelial cell lines (RWPE-1, WPE1-NA22, DU145, PC-3 and MDA-PCa-2b) with 5-azacytidine (a global DMT inhibitor) and found significantly (p-value <0.05) increased expression of PVT1 exons 1A, 4A, 4B and 9 in the PCa cell lines when compared to RWPE-1 and no treatment. We further identified 13 distinct promoter regions on the PVT1 gene annotated on Ensembl database and compared the strength of the PVT1 exon 9 - associated promoter with the canonical promoter using a luciferase reporter assay. We found that the canonical promoter was stronger in driving the expression of luciferase when compared to the PVT1 exon 9 - associated promoter and the ubiquitin C promoter, a well-established weak promoter. Further, EM-seq of human prostate tissues from men of African ancestry confirmed differential methylation peaks in tumor versus adjacent normal tissues within the PVT1 canonical and PVT1 exon 9 - associated promoter. Additionally, we investigated the role of MYC-PVT1 co-amplification regulation and PVT1 associated extra chromosomal DNAs (ecDNAs) using fluorescence in-situ hybridization. We found that MYC had no regulatory function on the expression of PVT1 exon 9 and there were no PVT1 associated ecDNAs in all the prostate epithelial cell lines used in this study. Lastly, we used bioinformatics to probe the PVT1 canonical and exon 9 - associated promoters for transcription factors. We found NKX2.5 and CDX1 to have the highest binding scores and significantly overexpressed in aggressive PCa. We will further characterize their function in the transcriptional regulation of PVT1. In conclusion, DMT regulates PVT1 exon 9 overexpression; the canonical PVT1 promoter is stronger than the PVT1 exon 9 - associated promoter, PCa in moAA is characterized by hypomethylation at PVT1, and NKX2.5 and CDX1 are putative transcriptional regulators of PVT1 expression in PCa. Understanding of these epigenetic regulatory mechanisms of PVT1 overexpression in PCa in moAA may lead to uncovering novel therapeutic targets in aggressive PCa. Citation Format: Chinedum C. Udekwu, Colin Finnegan, Murtaza Barkarar, Oluwabusola Ajagbe, Rachel E. Bonacci, Olorunseun O. Ogunwobi. DNA methylation and transcription factors regulate overexpression of PVT1 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5585.