Histone methyltransferase G9a in liver pathophysiology and carcinogenesis

描述（由申请人提供）： 长期目标是了解肝癌发生的分子机制，以便开发有效预防和治疗肝癌的新策略。表观遗传异常是肝细胞癌发生的主要标志。肝细胞中肿瘤抑制性磷酸酶和张力蛋白同源物（PTEN）的缺失导致进行性脂肪性肝炎和肝癌; PTEN和其他肿瘤抑制因子的表观遗传沉默与组蛋白H3在赖氨酸-9（H3K9me2）的二甲基化相关，这是基因沉默的关键抑制标记。组蛋白甲基转移酶G9a通过催化H3K9me2和促进DNA甲基化在胚胎发育中起关键作用。G9a通过一组在脂肪性肝炎和癌发生中重要的转录阻遏物/辅阻遏物介导基因阻遏。G9a基因的敲除导致端粒逆转录酶的显著下调和癌细胞的凋亡。G9a在人类肝癌中被诱导;然而，关于G9a在肝脏病理生理学和致癌作用中的体内重要性知之甚少，这主要是由于G9a缺失小鼠的胚胎致死性。本提案的目的是阐明G9a在肝脏中的病理生理学重要性，利用肝细胞特异性敲除（HKO）G9a和PTEN的小鼠新模型。我们的初步数据表明，在小鼠和人肝癌细胞，一个特定的G9a抑制剂诱导肿瘤抑制基因，抑制细胞增殖，并促进凋亡。相比之下，G9a-HKO小鼠表现正常，表明成年肝脏中G9a的丧失耐受良好。核心假设是G9a通过促进脂肪生成和抑制肿瘤抑制基因在脂肪性肝炎和肝癌发生中起关键作用，因此G9a的缺失将抑制脂肪肝和细胞增殖，而促进肝癌细胞的凋亡。这一核心假设将在两个具体目标中得到检验。目的1利用G9a-HKO小鼠2/3部分肝切除模型，研究G9a在肝再生中的作用。工作假设是G9a-HKO小鼠将具有相对正常的肝再生，尽管再生过程中有中度/轻微延迟。目的2：利用肝细胞特异性双敲除PTEN和G9a基因的小鼠，研究G9a在脂肪性肝炎和肝癌发生中的作用。工作假设是G9a的缺失将通过抑制脂肪生成和细胞增殖而增强细胞凋亡来防止PTEN无效诱导的肝硬化-脂肪性肝炎向肝硬化和肝癌的进展。这项研究的结果将提供关于G9a在正常肝功能，肝再生和肝癌发生中的重要性的关键新知识，这可能最终有助于确定G9a抑制剂作为预防和治疗肝癌的新型表观遗传药物，肝癌是一种致命的疾病，在美国发病率不断上升。

DESCRIPTION (provided by applicant): The long term goal is to understand the molecular mechanism of hepatocarcinogenesis so that novel strategies for the effective prevention and treatment of liver cancer can be developed. Epigenetic abnormity is a major hallmark in hepatocellular carcinogenesis. Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) in hepatocytes causes progressive steatohepatitis and liver cancer; the epigenetic silencing of PTEN and other tumor suppressors is associated with dimethylation of histone H3 at lysine-9 (H3K9me2), a key suppressive mark for gene silencing. The histone methyltransferase G9a plays a key role in embryonic development through catalyzing H3K9me2 and facilitating DNA methylation by DNA methyltransferases. G9a mediates gene repression by a group of transcriptional repressors/corepressors important in steatohepatitis and carcinogenesis. Knockdown of G9a causes dramatic down-regulation of telomere reverse transcriptase and apoptosis of cancer cells. G9a is induced in human liver cancer; however, little is known about the in vivo importance of G9a in liver pathophysiology and carcinogenesis, largely due to the embryonic lethality of G9a-null mice. The objective of this proposal is to elucidate the pathophysiological importance of G9a in liver utilizing novel models of mice with hepatocyte-specific knockout (HKO) of G9a and PTEN. Our preliminary data demonstrate that in mouse and human hepatoma cells, a specific G9a inhibitor induces tumor suppressor genes, inhibits cell proliferation, and promotes apoptosis. In contrast, G9a-HKO mice appear normal, suggesting that loss of G9a in adult liver is well-tolerated. The central hypothesis is that G9a plays a key role in steatohepatitis and liver carcinogenesis via promoting lipogenesis and inhibiting tumor suppressor genes, and thus loss of G9a will inhibit hepatosteatosis and cell proliferation, whereas promote the apoptosis of liver cancer cells. This central hypothesis will be tested in two specific aims. Aim 1 will determine the importance of G9a in liver regeneration using models of 2/3 partial hepatectomy in G9a-HKO mice. The working hypothesis is that G9a-HKO mice will have relatively normal liver regeneration despite a moderate/minor delay in the regenerative process. Aim 2 will determine the importance of G9a in steatohepatitis and liver carcinogenesis using mice with hepatocyte-specific double knockout of PTEN and G9a. The working hypothesis is that loss of G9a will prevent PTEN-null-induced progression of hepatosteatosis-steatohepatitis to cirrhosis and liver cancer via inhibiting lipogenesis and cell proliferation whereas enhancing apoptosis. Results from this study will provide crucial novel knowledge about the importance of G9a in normal liver function, liver regeneration, and liver carcinogenesis, which may ultimately help to identify G9a inhibitor as a novel epigenetic drug for the prevention and treatment of liver cancer, a deadly disease with increasing incidence in the USA.