Reversing molecular cancer phenotypes by targeting epigenetic alterations in prostate cancer

项目概要/摘要 鉴定了导致前列腺癌的可变临床行为和进展的分子改变。 大约20%的前列腺肿瘤包括高级别肿瘤不具有遗传改变（融合， 突变，拷贝数变异），然而，它们确实具有表观遗传改变。药物如地西他滨和 伏立诺他已用于靶向表观遗传改变。然而，这些药物诱导表观遗传变化， 从而改变表观遗传状态需要保持的区域中的表观遗传状态。 一样的因此，可以靶向特定表观遗传改变的治疗方案是至关重要的。通过 使用DNA甲基化分析正常前列腺和前列腺癌中的三维表观基因组， 染色质免疫沉淀测序（ChIP-seq）和Hi-C，我们确定了数千个表观遗传 位于调控元件（启动子、增强子）内的改变。最近的研究表明， 位于非编码区的调节元件可以驱动致癌作用。在监管要素中， 增强子的活性与细胞特性最密切相关。增强子活性的这种独特性可能有助于 为异质性肿瘤类型开发改进的治疗方案。我们现在可以精确地定位20 bp 使用CRISPR/Cas9系统在人类基因组中进行测序。此外，使用融合的表观遗传编辑 抑制结构域的催化失活dCas 9（CRISPR干扰）可以精确靶向和沉默 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， 在前列腺癌中激活的特异性增强子可以逆转分子癌症表型。作为初步 研究中，我们优先选择了30种在前列腺肿瘤中激活的增强子， 与前列腺癌发生有关。在目标1中，为了鉴定前列腺癌中的驱动增强子，我们将 删除从初步研究中鉴定的单个前列腺癌特异性增强子， CRISPR/Cas9敲除系统。下一步，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、集落形成和迁移测定来确定表型。最后，我们将揭开 通过进行RNA-seq、ChIP-seq和捕获Hi-C来鉴定驱动增强子的分子机制。 在目标2中，为了开发使用靶向表观遗传编辑来抑制驱动增强子的技术，我们将 抑制从初步研究中鉴定的单个前列腺癌特异性增强剂的活性， 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、集落形成和迁移测定来确定表型。最后，我们将进一步 进行RNA-seq、ChIP-seq和捕获Hi-C，以表征 鉴定的增强子。这项研究的成功完成不仅有利于改善发展 前列腺癌的治疗工具，但也提供了更好的理解的分子机制， 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.