Reversing molecular cancer phenotypes by targeting epigenetic alterations in prostate cancer

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Project Summary/Abstract Molecular alterations that lead to variable clinical behavior and progression of prostate cancer are identified. Approximately 20% of prostate tumors including high-grade tumors do not possess genetic alterations (fusion, mutation, copy number variation), however, they do have epigenetic alterations. Drugs such as Decitabine and Vorinostat have been used to target epigenetic alterations. However, these drugs induce epigenetic changes in an untargeted manner and thus alter epigenetic states in regions where epigenetic states need to remain the same. Therefore, treatment regimens that can target specific epigenetic alterations are crucially needed. By profiling the three-dimensional epigenomes in normal prostate and prostate cancer using DNA methylation, chromatin immunoprecipitation with sequencing (ChIP-seq), and Hi-C, we identified thousands of epigenetic alterations that are located within regulatory elements (promoters, enhancers). Recent studies showed that regulatory elements located in noncoding regions can drive carcinogenesis. Among regulatory elements, the activity of enhancers is most closely linked to cell identity. This uniqueness of enhancer activity may facilitate developing improved treatment regimens for heterogeneous tumor types. We can now precisely target a 20-bp sequence in the human genome using the CRISPR/Cas9 system. Moreover, epigenetic editing using fusions of repressor domains to the catalytically inactive dCas9 (CRISPR interference) can precisely target and silence the genomic regions. Here we propose to test the hypotheses that targeted genetic or epigenetic editing at specific enhancers activated in prostate cancer can reverse molecular cancer phenotypes. As preliminary studies, we have prioritized and selected 30 enhancers that are activated in prostate tumors and potentially associated with prostate carcinogenesis. In Aim 1, to identify driver enhancers in prostate cancer, we will delete individual prostate cancer-specific enhancers identified from preliminary studies using the CRISPR/Cas9 knockout system. Next, we will determine if any of enhancer deletion can reverse tumor phenotypes by performing cell proliferation, colony formation, and migration assays. Lastly, we will unravel the molecular mechanisms of the identified driver enhancers by performing RNA-seq, ChIP-seq and capture Hi-C. In Aim 2, to develop technologies to inactivate driver enhancers using targeted epigenetic editing, we will repress the activity of individual prostate cancer-specific enhancers identified from preliminary studies using the CRISPR interference system. Next, we will determine if repression of an enhancer can reverse tumor phenotypes by performing cell proliferation, colony formation, and migration assays. Lastly, we will further perform RNA-seq, ChIP-seq and capture Hi-C to characterize the molecular mechanisms underlying the identified enhancers. Successful completion of this study will not only facilitate the development of improved therapeutic tools for prostate cancer but also provide a better understanding of the molecular mechanisms of noncoding drivers.