Targeting EZH2 in Prostate Cancer by Luteolin

描述(由申请人提供)： 这项研究的总体目标是评估木犀草素预防疾病进展的效果。 并揭示其抗癌作用的分子基础。组蛋白的翻译后修饰对染色质结构和基因表达的调节至关重要。Zust同源增强子2(EZH2)是多梳抑制复合体(PRC)-2的催化亚基，通过催化组蛋白H3在赖氨酸27(H3K27)上的三甲基化参与染色质重塑和基因沉默。EZH2介导的表观基因沉默在前列腺癌的发生发展中起重要作用。EZH2的表达增加促进了上皮细胞的肿瘤转化和癌症的进展。在机制上，EZH2介导的H3K27三甲基化导致前列腺癌中CDKN2A/p16、CDH1/E-钙粘素、禁用同源2相互作用蛋白(DAB2IP)和金属蛋白酶组织抑制物(TIMP)-3等大量抑癌基因的沉默。功能分析已经证明EZH2是一个真正的癌基因。我们的初步数据表明：i)在前列腺癌进展过程中EZH2的表达逐渐增加，并且其在癌症和相应的正常/良性组织中的差异表达；ii)从电子研究中，植物黄酮木犀草素与EZH2和H3K27me3蛋白的活性部位结合，iii)木犀草素与EZH2和H3K27me3蛋白的相互作用-体外研究，iii)木犀草素抑制前列腺癌EZH2和H3K27me3的表达，iv)木犀草素改变转录组和miRNA的表达，v)木犀草素降低前列腺癌细胞的增殖和侵袭力，vi)木犀草素诱导肿瘤抑制基因的重新表达。我们的工作假设是，木犀草素介导的抑制EZH2及其组蛋白赖氨酸N-甲基转移酶活性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翻译后EZH2调节miRNAs水平的变化来抑制前列腺癌的进展。我们提出了四个互补的特异性目标来确定：(1)木犀草素对EZH2和其他PRC2复合蛋白的影响，(2)木犀草素逆转EZH2介导的表观遗传基因沉默的能力，(3)木犀草素对影响EZH2介导的前列腺癌改变的特定miRNAs的调节作用，以及(4)木犀草素介导的EZH2抑制作用和相关的分子机制在转基因小鼠前列腺癌(TRAMP)模型上的相关体内情况下的影响。前列腺癌非常普遍，是美国男性癌症相关死亡的第二大原因。完成这些研究所产生的知识有可能为退伍军人保健系统提供独特的机会，为被诊断为低级别、低体积前列腺癌的退伍军人提供有效的治疗策略。这些新的策略是有必要的，因为目前的现实是，不同患者的组织学相同的癌症可能表现出很大的不同的生物学行为，并且前列腺癌患者比普通人群更有可能有显著的合并症。总而言之，我们将定义1)角色 EZH2-H3K27me3表观遗传途径对于抑制肿瘤抑制基因非常重要，以及2)木犀草素的有益作用，这将为正在接受积极监测或面临生化复发风险的前列腺癌患者的管理设定新的范式。

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.