Mechanisms that promote hepatocellular carcinoma due to chronic ethanol exposure

摘要 肝细胞癌是世界上第五大常见癌症，每年新增病例超过60万例。 五年存活率不到9%，令人沮丧。大多数肝细胞癌患者都有慢性肝病 主要由丙型肝炎病毒/乙肝病毒感染、长期饮酒或接触黄曲霉毒素所致。尽管大多数患者 发展为肝细胞癌有肝硬变或肝炎，促进肝细胞癌的机制因病理不同而不同 并不是所有的情况都应该一视同仁。这项提议的目的是了解促进 由于长期接触酒精而导致的肝细胞癌，这是一种随着丙型肝炎病毒和乙肝病毒感染而上升的肝细胞癌 更好地预防和治疗。特别是，这项建议的重点是理解染色体8q24 在酒精诱导的肝细胞癌中观察到的更频繁的扩增。因为c-Myc驻留在这个放大的 我们认为，在肝细胞癌的晚期，染色体8q24被扩增，导致c-Myc水平升高。这个 高Myc促进Miz1转录抑制和上皮表型丧失及获得 间充质细胞特性。同时，高c-Myc激活ZEB1的表达，驱动细胞通过 上皮向间充质转化(EMT)进一步抑制上皮表型。随着细胞的转移和 在次级部位定植，随着细胞穿过间充质，部分上皮特征被重新获得 向上皮细胞转化(MET)。我们的假设是c-Myc、ZEB1和 Miz1和上皮间充质特征在酒精诱导的肝细胞癌患者中是独特的，并且可以 预测预后、耐药性和潜在的治疗策略。在目标1中，我们提出两个主要问题： 1)8q24扩增是否驱动酒精相关性肝细胞癌的EMT？2)转移灶是否会重新获得更多的 上皮表型？为了回答这些问题，我们将使用 石蜡包埋的人肝癌切片的免疫组织化学(IHC)染色(回答问题1)和 受影响的淋巴结和转移性病变(回答问题2)。染色将与癌症分期相关， 临床病理变量、饮酒和患者对治疗的反应。我们还将确认8Q24 FISH扩增分析c-Myc的表达。要直接确定时间关系， C-Myc、Miz1、ZEB1和上皮间充质表型，我们将进行靶向转录组 人良性和肝细胞癌组织的分析和形态分析。我们还将表演过度表达和 基因敲除研究旨在直接建立c-Myc、Miz1、ZEB1和上皮性VS之间的时间关系。 选定的细胞系和原代培养的人正常细胞和肝癌细胞的间充质表型。最后， 由于迫切需要开发有效的联合疗法来治疗肝癌的新策略，我们将 检测细胞对临床相关化合物的药物敏感性。我们已经得到了其他几个人的支持 为项目提供他们的专业知识。我们可以继续进入位于附近的成像设施 约翰霍普金斯医学院，是霍普金斯GI中心的成员，使我们能够获得许多服务 和资源。我们的合作者广博的专业知识，以及获取人体组织样本和高度... 终端资源加上我们在极化肝细胞生物学方面的丰富专业知识，使我们能够完美地 进行这些重要的机械和翻译研究。

SUMMARY Hepatocellular carcinoma (HCC) is the fifth most common cancer world-wide with over 600,000 new cases per year and a dismal five-year survival rate at less than 9%. Most HCC patients have chronic liver disease resulting mainly from HCV/HBV infection, chronic alcohol consumption or aflatoxin exposure. Although most patients that develop HCC have cirrhosis or hepatitis, the mechanisms promoting HCC differ between pathologies such that not all cases should be treated the same. This proposal is aimed at understanding the mechanisms that promote HCC due to chronic ethanol exposure, a form of HCC that is on the rise as HCV and HBV infections are being better prevented and treated. In particular, this proposal is focused on understanding chromosome 8q24 amplification that is observed more frequently in alcohol-induced HCC. Because c-Myc resides on this amplified region, we propose that at later stages of HCC, chromosome 8q24 is amplified leading to high c-Myc levels. The high Myc promotes Miz1 transcriptional repression and loss of the epithelial phenotype and acquisition of mesenchymal cell traits. Concomitantly, the high c-Myc activates Zeb1 expression driving cells through the epithelial to mesenchymal transition (EMT) further repressing the epithelial phenotype. As cells metastasize and colonize at secondary sites, epithelial traits are partially reacquired as cells progress through the mesenchymal to epithelial transition (MET). Our hypothesis is that the temporal expression patterns of c-Myc, Zeb1 and Miz1 and epithelial vs. mesenchymal traits are unique in patients with alcohol-induced HCC and can predict prognosis, drug resistance and potential treatment strategies. In Aim 1 we ask two major questions: 1) does 8q24 amplification drive EMT in alcohol-associated HCC? and 2) do metastatic lesions reacquire a more epithelial phenotype? To answer these questions, we will examine protein expression patterns using immunohistochemical (IHC) staining of paraffin-embedded human HCC resections (to answer question 1) and affected lymph nodes and metastatic lesions (to answer question 2). Staining will be correlated to cancer stage, clinicopathologic variables, alcohol consumption and patient responses to treatment. We will also confirm 8q24 amplification using FISH analysis of c-Myc expression. To directly determine the temporal relationship between c-Myc, Miz1, Zeb1 and the epithelial vs. mesenchymal phenotypes, we will perform targeted transcriptome analysis and morphological analysis of human benign and HCC tissue. We will also perform overexpression and knockdown studies to directly establish the temporal relationship between c-Myc, Miz1, Zeb1 and epithelial vs. mesenchymal phenotypes in selected cell lines and primary human normal and HCC cells in culture. Finally, due to the tremendous need to develop new strategies for effective combination therapies to treat HCC, we will examine drug sensitivity in cells to clinically relevant compounds. We have garnered the support of several others to provide their expertise to the project. We have continued access to the Imaging Facility located at nearby Johns Hopkins Medical School and are members of the Hopkins GI Center allowing us access to many services and resources. The expansive expertise of our collaborators and the access to human tissue samples and high- end resources coupled with our considerable expertise in polarized hepatic cell biology situate us perfectly to perform these important mechanistic and translational studies.