雄激素受体-EZH2调控的表观遗传信号通路诱导肝细胞癌发生的研究

Histone epigenetic modification regulated by EZH2 is a hallmark of many cancers including hepatocellular carcinoma (HCC). While trimethylation of lysine 27 at histone H3 (H3K27me3) is well-known to cause tumor-suppressor gene silencing, accumulating evidence has illustrated that trimethylation of lysine 4 at histone H3 (H3K4me3) and the subsequent proto-oncogene activation allow the carcinogenic process to perpetuate. However, the molecular mechanisms and biological significance of H3K4me3 in HCC remain unclear. .Recent study showed that aberrant function of androgen receptor (AR) is one of the important factors to characterize male predominance of HCC. Our recent data unveil more important functional significance of AR activity in the epigenetic regulation in HCC. We found that over-expression of AR dramatically up-regulates expression of EZH2 by binding to the androgen-responsive element (ARE) in its promoter, causing cellular malignant transformation and tumor growth advantage. Colony formation assay in vitro and xenograft tumor model in vivo where AR-expressing hepatocytes confer enchanced cellular proliferation and tumorigenicity compared to non-expressing controls, pronounced AR expression are evident in concordant with EZH2 up-regulaton, and demonstrated AR depends on EZH2 expression to induce cell proliferation and tumorigenicity..Based on our preliminary findings and the existing evidence suggesting causal relationship between histone trimethylation-induced gene activation and carcinogenesis, we hypothesize that EZH2 up-regulation by AR in HCC exerts important function on H3K4me3 expression, which facilitates transcription of tumor-causing genes required for carcinogenic initiation and development. The present proposal hence aims to define the mechanistic basis of EZH2 up-regulation by AR and characterize the deregulated epigenome and transcriptome by integrated genome-wide analysis. This study will not only advance our understanding of the gender disparity in HCC, but may also provide a new paradigm for AR signaling in regulating cancer epigenome. Elucidating the detailed signaling pathway of AR-EZH2 will lead to the identification of novel drug targets for the development of more efficacious and specific therapies for HCC.

组蛋白表观遗传学修饰是诱发癌症的重要机制。EZH2是引发组蛋白表观遗传修饰基因异常表达的重要调控因子，但是其调节组蛋白4位赖氨酸三甲基化刺激原癌基因表达在癌症发生中的分子机制尚不清楚。近来研究表明，雄激素受体功能异常是男性高比率罹患肝癌的重要因素之一。体外细胞集落形成实验和体内小鼠异种肝癌模型显示，雄激素受体与EZH2呈明显正相关性，并且雄激素受体诱导细胞生长和肿瘤发生依赖EZH2的表达。由此，我们首次提出雄激素受体—EZH2信号通路诱导肝癌发生的表观遗传学机制：雄激素受体通过调控EZH2表达，引发组蛋白4位赖氨酸三甲基化从而激活原癌基因的转录。本项目拟用全基因组定位及体内外功能分析等方法，获得EZH2介导雄激素受体调控组蛋白4位赖氨酸三甲基化信号通路的可靠证据，并揭示此表观遗传学修饰信号通路在肝癌发生发展过程中的意义和作用，为肝癌治疗寻找新的有效靶点提供充分的科学依据。

肝癌是肿瘤死亡率排名第二的恶性疾病，全球每年发病率约为80万人。肝癌发生的机制复杂，其中基因突变和表观遗传学修饰在分子水平上具有重要意义。雄激素受体（AR）是调控多条促增殖信号通路的总集转录因子，在肝癌发生过程中具有重要功能。其中，通过改变AR活性而调控下游效应分子表达变化的表观遗传修饰，比编码AR基因发生突变，在肝癌患者样本中更具有普遍性。组蛋白甲基化是肿瘤发生的重要表观遗传学机制之一，人的zeste基因同源物2增强子（EZH2）能够催化组蛋白H3侧链赖氨酸发生三甲基化，从而诱导肝癌的发生。但是，AR信号通路与EZH2介导的表观遗传修饰在肝癌发生中是否存在相互作用以及相应的分子机制还不清楚。本研究采用体外细胞实验和体内小鼠成瘤实验，探索AR调控EZH2表达，进而影响组蛋白甲基化介导的下游效应分子的表达调控，诱导肝癌发生的机制。同时利用ChIP-seq基因组序列分析以及基因敲减和敲入的方法，结合集落形成实验和软琼脂集落形成实验，寻找和验证AR/EZH2调控的表观遗传信号通路在诱导肝癌发生中的关键靶点。我们发现AR能够上调EZH2表达，并且通过分别催化组蛋白H3上27位赖氨酸发生三甲基化（H3K27me3）和4位赖氨酸发生三甲基化（H3K4me3），展现出两种不同的表观遗传调控信号通路，诱导肝癌发生。相比于H3K27me3介导的肿瘤抑制因子调控途径，H3K4me3介导的促癌因子转录激活诱导肝癌发生的机制则鲜有研究。通过ChIP-seq分析，我们筛选得到泛素结合酶E2I（UBE2I）能够被AR/EZH2上调。利用基因表达和沉默方法，在体外细胞实验和体内小鼠模型中，证实UBE2I是EZH2直接调控的癌基因，能够诱导细胞恶变和肿瘤生成。因此，我们发现AR/EZH2/H3K4me3/UBE2I诱导肝癌发生的新的信号通路，阐明了AR调控EZH2表观遗传修饰的新机制，证实UBE2I是EZH2直接调控的靶点癌基因，为肝癌的治疗以及抗癌药物的开发提供新的策略。