Reversing molecular cancer phenotypes by targeting epigenetic alterations in prostate cancer

项目摘要/摘要 导致前列腺癌不同临床行为和进展的分子改变被确定。 包括高级别肿瘤在内的大约20%的前列腺癌没有基因改变(融合， 突变，拷贝数变化)，然而，它们确实有表观遗传变化。药物，如地西他滨和 涡旋器已被用于靶向表观遗传改变。然而，这些药物会导致表观遗传学的变化 一种非靶向的方式，从而改变表观遗传状态需要保持 一样的。因此，迫切需要针对特定表观遗传学改变的治疗方案。通过 用DNA甲基化分析正常前列腺癌和前列腺癌的三维表观基因组， 染色质免疫沉淀测序(CHIP-SEQ)和Hi-C，我们鉴定了数千个表观遗传学 位于监管要素(推动者、增强者)内的变更。最近的研究表明， 位于非编码区的调控元件可以推动癌症的发生。在监管要素中， 增强剂的活性与细胞特性关系最为密切。增强子活性的这种独特性可以促进 针对不同类型的肿瘤开发改进的治疗方案。我们现在可以精确地瞄准20-BP 使用CRISPR/Cas9系统对人类基因组进行测序。此外，使用融合的表观遗传编辑 对催化失活的dCas9的抑制域(CRISPR干扰)可以精确地靶向和沉默 基因组区域。在这里，我们建议测试针对遗传或表观遗传编辑的假设 前列腺癌中激活的特定增强子可以逆转分子癌症的表型。作为初步准备 研究中，我们优先选择了30种在前列腺癌和潜在的前列腺癌中被激活的增强剂。 与前列腺癌的发生有关。在目标1中，为了确定前列腺癌的驱动程序增强剂，我们将 删除从初步研究中确定的个别前列腺癌特异性增强子 CRISPR/Cas9基因敲除系统。接下来，我们将确定是否有任何增强子缺失可以逆转肿瘤 通过进行细胞增殖、克隆形成和迁移分析进行表型分析。最后，我们将揭开 通过执行RNA-SEQ、CHIP-SEQ和捕获Hi-C来鉴定所鉴定的驱动增强子的分子机制。 在目标2中，为了开发使用有针对性的表观遗传编辑来灭活驱动程序增强剂的技术，我们将 抑制从初步研究中确定的单个前列腺癌特异性增强子的活性 CRISPR干扰系统。接下来，我们将确定抑制增强子是否可以逆转肿瘤 通过进行细胞增殖、克隆形成和迁移分析进行表型分析。最后，我们将进一步 进行RNA-SEQ、CHIP-SEQ和捕获Hi-C以表征潜在的分子机制 确定了增强剂。这项研究的顺利完成不仅将促进改进的发展 前列腺癌的治疗手段也提供了对前列腺癌分子机制的更好的理解 非编码驱动程序。

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.