Project Summary/Abstract There is an urgent clinical need to understand and distinguish indolent from aggressive prostate cancers to inform treatment. The thesis work to date revealed DNA hypermethylation that distinguishes indolent from aggressive prostate cancer. DNA hypermethylation in promoters correlates with reduced expression in aggressive disease for genes where reduced expression correlates with poor outcomes. However, the majority of alterations occur outside promoters and are enriched for enhancer-like chromatin. The hypothesis of this proposal is that differentially methylated regions (DMRs) in non-promoter regions function to regulate enhancer and insulator elements which increases tumor aggressiveness by changes in oncogene and tumor suppressor expression within topologically associated chromatin domains. The F99 phase will perform a computational analysis to identify aggressiveness-associated DMRs which occur inside or at the borders of topologically associated domains (TADs) that contain aberrantly expressed genes in aggressive disease. Using existing data, DMRs within TADs will be classified as enhancer-associated or insulator-associated through integration with histone modification and CTCF ChIP-seq data from prostate cancer tissues and cell lines. Initial experimental verification of enhancer function will be performed by luciferase reporters. Using prostate cancer cell lines, chromatin conformation capture (3C) will be used to confirm the existence of chromatin loops between the putative enhancer and target gene(s). Finally, genome editing will be performed to delete key motifs in the enhancer. The effect on gene expression of the target will be analyzed with qPCR, and assays will be performed to measure changes in proliferation and aggressive cellular behavior. For the K00 phase, the applicant will identify mentorship to extend this approach into other cancer types, types of epigenetic modifications, and/or model systems (such as organoids and xenografts) and generate new data using genome-wide conformation capture assays (such as capture Hi-C) and histone mark/transcription factor ChIP-seq to inform the interplay between these phenomena and cancer DMRs. This will establish novel functional activities of DNA methylation in cancer and connect these changes to gene expression and aggressiveness to augment our understanding of the specific mechanisms of how cancer aggressiveness is regulated by DNA methylation, leading to potential tests and treatment targets to modify the course of this disease and improve diagnostic and treatment outcomes. " |