Role of EZH2 in Epithelial Ovarian Cancer

描述（由申请人提供）：上皮性卵巢癌（EOC）仍然是美国妇科恶性肿瘤死亡的主要原因。因此，迫切需要新的EOC治疗剂。zeste同源物2增强子（EZH 2）是一种组蛋白甲基转移酶，通过在组蛋白H3（H3 K27 Me 3）的赖氨酸27残基上产生三甲基化来沉默基因表达。我们最近发表了EZH 2在原代人EOCs中频繁过表达。然而，EOC中EZH 2上调的潜在机制知之甚少。我们有证据表明CCAAT结合转录因子NF-Y在调节EOC细胞中EZH 2表达中起关键作用。值得注意的是，我们已经表明EZH 2表达调节EOC细胞的增殖、凋亡和侵袭。本提案的目的是研究EZH 2促进EOC细胞恶性行为的分子基础。我们进行了全基因组染色质免疫沉淀（ChIP），然后进行下一代测序分析和基因表达谱分析。综合分析揭示了60个直接EZH 2靶基因的列表，其表达在EOC细胞中通过EZH 2敲低而上调。为了对鉴定的基因进行优先级排序以进行验证，我们在癌症基因组图谱EOC数据库中检查了它们的表达，并发现60个基因中的3个下调>2倍，同时在>75%的EOC病例中EZH 2上调。有趣的是，3个优先基因涉及调节细胞生长、侵袭、凋亡和/或癌症干细胞。我们通过ChIP验证了EZH 2/H3 K27 Me 3与其启动子的结合，并证实了它们在EZH 2敲低细胞中的上调。我们的中心假设是EZH 2通过表观遗传学沉默我们已经鉴定的靶基因来调节EOC细胞的恶性行为。我们的长期目标是靶向EZH 2以开发新的EOC治疗方法。在这里，我们发现两种新的EZH 2甲基转移酶活性特异性抑制剂抑制EOC细胞的增殖和侵袭。提出了三个具体目标。目标1：研究CCAAT结合转录因子NF-Y在调节人EOC细胞中EZH 2表达中的作用;目的2：研究经验证的EZH 2靶基因在人卵巢肿瘤中的表达模式和功能;目的3：研究在用EZH 2甲基转移酶活性抑制剂处理的EOC细胞中观察到的作用是否由经验证的EZH 2靶基因介导。这项研究具有创新性，因为它不仅阐明了EZH 2上调的新分子机制，还为EZH 2如何促进EOC的恶性行为提供了新的机制见解。此外，拟议的研究将揭示两种新型EZH 2甲基转移酶抑制剂的作用机制。这些研究是重要的，因为它有可能建立EZH 2作为开发EOC治疗的新靶点。此外，从目前的研究中获得的机制见解对许多不同类型的癌症也有广泛的影响。

DESCRIPTION (provided by applicant): Epithelial ovarian cancer (EOC) remains the leading cause of death by gynecological malignancy in the United States. Thus, there is an urgent need for novel EOC therapeutics. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that silences gene expression by generating tri-methylation on lysine 27 residue of histone H3 (H3K27Me3). We recently published that EZH2 is frequently overexpressed in primary human EOCs. However, the mechanism underlying EZH2 upregulation in EOC is poorly understood. We have evidence to suggest that the CCAAT binding transcription factor NF-Y plays a critical role in regulating EZH2 expression in EOC cells. Significantly, we have showed that EZH2 expression regulates the proliferation, apoptosis and invasion of EOC cells. The objective of this proposal is to investigate the molecular basis whereby EZH2 promotes the malignant behaviors of EOC cells. We performed whole-genome chromatin immunoprecipitation (ChIP) followed by next generation sequencing analysis and gene expression profiling. Integrative analysis revealed a list of 60 direct EZH2 target genes whose expression was upregulated by EZH2 knockdown in EOC cells. To prioritize the identified genes for validation, we examined their expression in the Cancer Genome Atlas EOC database and discovered that 3 of the 60 genes are downregulated >2-fold with concurrent EZH2 upregulation in >75% of EOC cases. Interestingly, 3 prioritized genes are implicated in regulating cell growth, invasion, apoptosis and/or cancer stem cells. We validated the binding of EZH2/H3K27Me3 to their promoters by ChIP and confirmed their upregulation in EZH2 knockdown cells. Our central hypothesis is that EZH2 regulates malignant behaviors of EOC cells by epigenetically silencing the target genes we have identified. Our long-term goal is to target EZH2 for developing novel EOC therapeutics. Here we showed that two novel specific inhibitors of EZH2 methyltransferase activity suppress the proliferation and invasion of EOC cells. Three specific aims are proposed. Aim 1: To investigate the role of the CCAAT binding transcription factor NF-Y in regulating EZH2 expression in human EOC cells; Aim 2: To investigate the expression pattern and function of the validated EZH2 target genes in human ovarian tumors; and Aim 3: To investigate whether the effects observed in EOC cells treated with inhibitors of EZH2 methyltransferase activity are mediated by the validated EZH2 target genes. The research proposed is innovative because it will not only elucidate a novel molecular mechanism underlying EZH2 upregulation, but also provide new mechanistic insights into how EZH2 contributes to malignant behaviors of EOC. In addition, the proposed studies will reveal the mechanisms of action of two novel inhibitors of EZH2 methyltransferase. These studies are significant because it has potential to establish EZH2 as a novel target for developing EOC therapeutics. In addition, the mechanistic insights gained from the current studies have broad implications for many different types of cancers as well.