Targeting EZH2 in Prostate Cancer by Luteolin

描述（由申请人提供）： 本研究的总体目标是评估木犀草素在预防进展中的疗效 前列腺癌的研究，并揭示其抗癌作用的分子基础。组蛋白的翻译后修饰对于染色质结构和基因表达的调节至关重要。Zeste同源物增强子2（Enhancer of Zeste homolog 2，EZH 2）是多梳抑制复合物（polycomb repressive complex，PRC）-2的催化亚基，通过催化组蛋白H3在赖氨酸27（Lysine 27，H3 K27）上的三甲基化，参与染色质重塑和基因沉默。EZH 2介导的表观遗传基因沉默在前列腺癌的发生和发展中起重要作用。EZH 2表达增加促进上皮细胞的肿瘤转化和癌症进展。从机制上讲，EZH 2介导的H3 K27三甲基化导致前列腺癌中大量肿瘤抑制基因如CDKN 2A/p16、CDH 1/E-钙粘蛋白、失能同源物2相互作用蛋白（DAB 2 IP）和金属蛋白酶组织抑制剂（TIMP）-3沉默。功能测定已经证明EZH 2是真正的癌基因。我们的初步数据表明：i）前列腺癌进展期间EZH 2的进行性增加及其在癌症中与相应的正常/良性组织中的差异表达，ii）来自计算机模拟研究，植物黄酮毛地黄黄酮与EZH 2的活性位点结合，iii）毛地黄黄酮与EZH 2和H3 k27 me 3蛋白的相互作用-离体研究，iii）毛地黄黄酮抑制前列腺癌细胞中的EZH 2表达和H3 K27 me 3，iv）毛地黄黄酮改变转录组和miRNA表达，v）毛地黄黄酮降低前列腺癌细胞的增殖和侵袭性，和vi）毛地黄黄酮诱导肿瘤抑制基因的再表达。我们的工作假设是，木犀草素介导的EZH 2及其组蛋白-赖氨酸N-甲基转移酶活性的抑制不仅通过组蛋白甲基化的变化，而且通过肿瘤抑制基因启动子的表观遗传修饰和EZH 2调节miRNA水平的翻译后变化来抑制前列腺癌的进展。我们提出了四个互补的具体目标，以确定：（1）毛地黄黄酮对EZH 2和其他PRC 2复合蛋白的作用，（2）毛地黄黄酮逆转EZH 2介导的表观遗传基因沉默的能力，（3）毛地黄黄酮对影响前列腺癌细胞中EZH 2介导的变化的特异性miRNA的调节的作用，和（4）使用小鼠前列腺的转基因腺癌（TRAMP）模型，其重现人类疾病，木犀草素介导的EZH 2抑制和相关分子机制对相关体内情况的影响。前列腺癌在美国是高度流行的，并且是男性癌症相关死亡的第二大原因。通过完成这些研究获得的知识有可能为VA医疗保健系统提供独特的机会，为诊断为低级别、低体积前列腺癌的VA男性提供有效的治疗策略。这种新的策略是必要的，目前的现实是，组织学上相同的癌症在不同的患者可以表现出广泛的差异生物学行为，前列腺癌患者比一般人群更可能有显着的合并症。最后，我们将定义1）的作用 EZH 2介导的表观遗传重编程在驱动前列腺癌进展中的作用，因为EZH 2-H3 K27 me 3表观遗传途径似乎对于肿瘤抑制基因的抑制非常重要，以及2）毛地黄黄酮的有益作用，这将在进行主动监测或处于生化复发风险的前列腺癌患者的管理中建立新的范例。

DESCRIPTION (provided by applicant): The overall objective of this study is to assess the efficacy of luteolin in preventing progression of prostate cancer and to uncover the molecular basis of its anticancer effects. Post-translational modification of histones is critical for regulation of chromatin structure and gene expression. Enhancer of Zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex (PRC)-2, which is involved in chromatin remodeling and gene silencing through its catalysis of the trimethylation of histone H3 on lysine 27 (H3K27). EZH2- mediated epigenetic gene silencing plays an important role in prostate cancer initiation and progression. Increased EZH2 expression promotes neoplastic transformation of epithelial cells and cancer progression. Mechanistically, EZH2-mediated trimethylation of H3K27 results in silencing of a large number of tumor suppressor genes such as CDKN2A/p16, CDH1/E-cadherin, Disabled homolog 2-interacting protein (DAB2IP) and tissue inhibitor of metalloproteinases (TIMP)-3 in prostate cancer. Functional assays have demonstrated EZH2 to be a bonafide oncogene. Our preliminary data suggest that: i) progressive increase in EZH2 during prostate cancer progression and its differential expression in cancer vs. corresponding normal/benign tissue, ii) from in silico studies, the plant flavone luteolin binds to the active site of EZH2, iii) interactin of luteolin with EZH2 and H3k27me3 proteins-ex vivo studies, iii) luteolin inhibits EZH2 expression and H3K27me3 in prostate cancer cells, iv) luteolin alters transcriptome and miRNA expression, v) luteolin decreases proliferation and invasiveness of prostate cancer cells, and vi) luteolin induces re-expression of tumor suppressor genes. Our working hypothesis is that luteolin-mediated suppression of EZH2 and its histone-lysine N- methyltransferase activity inhibits progression of prostate cancer not only through changes in histone methylation, but also through epigenetic modifications in the promoters of tumor suppressor genes and post-translational changes in the levels of EZH2-regulatory miRNAs. We propose four complementary specific aims to determine: (1) the effects of luteolin on EZH2 and other PRC2 complex proteins, (2) the ability of luteolin to reverse EZH2-mediated epigenetic gene silencing, (3) the effects of luteolin on the regulation of specific miRNAs affecting EZH2-mediated changes in prostate cancer cells, and (4) the effects of luteolin-mediated EZH2 inhibition and associated molecular mechanisms on relevant in vivo situations using the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, which recapitulate human disease. Prostate cancer is highly prevalent and the second leading cause of cancer-related deaths in men in the United States. The knowledge generated by completion of these studies has the potential to provide the VA healthcare system with unique opportunities for effective treatment strategies for VA men diagnosed with low- grade, low-volume prostate cancer. Such novel strategies are necessitated by the current realities that histologically identical cancers in different patients cn exhibit widely variant biologic behavior, and prostate cancer patients are more likely to have significant comorbidities than the general population. In conclusion, we will define the 1) role of EZH2-mediated epigenetic reprogramming in driving prostate cancer progression, as the EZH2-H3K27me3 epigenetic pathway appears to be very important for repression of tumor suppressor genes, and 2) the beneficial effects of luteolin, which will set a new paradigm in the management of prostate cancer patients undergoing active surveillance or at risk for biochemical recurrence.