Nanog-positive cancer stem cells in and liver oncogenesis by alcohol and HCV

描述（由申请人提供）：令人信服的证据表明，酒精和HCV感染对肝细胞癌（HCC）的风险具有协同作用。本申请旨在支持针对Nanog阳性癌症干细胞在HCV和酒精诱导的肝癌发生中的作用的研究。最近，我们发现HCV NS 5A蛋白诱导肝细胞中的Toll样受体4（TLR 4），进而介导喂食酒精的小鼠的肝脏肿瘤发展，从而揭示了酒精和HCV之间的机制联系。由酒精诱导的内毒素血症激活的TLR 4信号传导在NS 5A转基因（Tg）小鼠中诱导祖细胞/干细胞标志物Nanog和肝肿瘤，但在TLR 4缺陷的野生型（wt）或NS 5A Tg小鼠中不诱导。Nanog免疫染色与这些小鼠的肝肿瘤中的癌症干细胞标志物CD 133和CD 49 f共定位。体外启动子和mRNA分析证明了NS 5A转染细胞中Nanog的TLR 4依赖性转录激活。用TLR 4转导的p53-/-肝母细胞的移植在重复LPS注射后在受体小鼠中产生肝肿瘤，并且这种肿瘤形成用Nanog shRNA阻止。移植Nanog+/CD 133 +/CD 49 f+细胞，而不是从我们的小鼠模型的肝肿瘤中分离的Nanog-/CD 133-/CD 49 f+细胞，在给予重复LPS注射的裸鼠中引起肿瘤生长。从前者肿瘤干细胞而不是后者细胞建立的慢病毒cDNA文库，在体外转化卵圆细胞系。这些发现支持HCV/NS 5A和酒精的协同肝肿瘤发生是由Nanog+癌症干细胞的TLR 4诱导的致癌活性介导的假设。为了验证这一假设并了解模型中肿瘤发生的机制，我们将追求两个主要目标：1）鉴定和验证Nanog+癌症干细胞中的致癌基因; 2）阐明癌症干细胞中TLR 4介导的Nanog诱导的遗传和表观遗传机制。对于Aim 1，将使用从癌症干细胞建立的慢病毒cDNA文库分离候选致癌基因;将应用基于shRNA的敲减来在体外和体内验证这些致癌基因;并将进行ChIP-seq分析以进行癌症干细胞中Nanog结合的全基因组分析。对于目标2，我们将阐明TLR 4诱导Nanog的转录机制，通过启动子-报告基因测定与缺失构建体和定点突变，和ChIP分析;并确定持续Nanog诱导通过DNA甲基化分析和ChIP组蛋白修饰分析的表观遗传调控。综上所述，R 01申请代表了一种新的机制研究，其重点是通过HCV NS 5A和酒精激活TLR 4信号产生的Nanog+肝癌干细胞。本申请的最终目标是最终转化从当前研究中获得的基础知识，以设计针对HCV/酒精相关HCC的Nanog+癌症干细胞的新型治疗方式。 公共卫生相关性：丙型肝炎病毒（HCV）是导致肝癌的重要原因之一，肝癌是世界上第三大致死性癌症。该提案的目标是了解HCV和酒精如何诱发肝癌，以便找到更好的预防和治疗方法。

DESCRIPTION (provided by applicant): Compelling evidence identifies a synergism between alcohol and HCV infection for the risk of developing hepatocellular carcinoma (HCC). This application seeks support for research directed to the role of Nanog-positive cancer stem cells in hepatocarcinogenesis induced by HCV and alcohol. A mechanistic link between alcohol and HCV has recently been unveiled by our discovery that HCV NS5A protein induces Toll-like receptor 4 (TLR4) in hepatocytes, which in turn mediates liver tumor development in mice fed alcohol. TLR4 signaling activated by alcohol-induced endotoxemia, induces the progenitor/stem marker Nanog and liver tumors in NS5A transgenic (Tg) mice but not in wild type (wt) or NS5A Tg mice deficient in TLR4. Nanog immunostaining is co-localized with the cancer stem cell marker CD133 and CD49f in liver tumors of these mice. In vitro promoter and mRNA analyses demonstrate TLR4-dependent transcriptional activation of Nanog in NS5A-transfected cells. Transplantation of p53-/- hepatoblasts transduced with TLR4, produces liver tumors in recipient mice after repetitive LPS injection, and this tumor formation is prevented with Nanog shRNA. Transplantation of Nanog+/CD133+/CD49f+ cells but not Nanog-/CD133-/CD49f+ cells isolated from liver tumors of our mouse model, causes tumor growth in nude mice given repetitive LPS injection. Lentiviral cDNA library established from the former cancer stem cells but not the latter cells, transforms the oval cell line in vitro. These findings support the hypothesis that synergistic liver oncogenesis by HCV/NS5A and alcohol is mediated by TLR4-induced oncogenic activity of Nanog+ cancer stem cells. To test this hypothesis and understand the mechanisms of oncogenesis in the model, we will pursue two major aims: 1) to identify and validate oncogenic genes in the Nanog+ cancer stem cells; and 2) to elucidate genetic and epigenetic mechanisms of TLR4-mediated Nanog induction in the cancer stem cells. For the Aim 1, a lentiviral cDNA library established from the cancer stem cells will be used to isolate candidate oncogenic genes; shRNA-based knockdown will be applied to validate these oncogenic genes in vitro and in vivo; and ChIP-seq analysis will be performed for genome-wide analysis for Nanog binding in the cancer stem cells. For the Aim 2, we will elucidate the transcriptional mechanisms by which TLR4 induces Nanog via promoter-reporter assay with deletion constructs and site-directed mutagenesis, and ChIP analysis; and determine epigenetic regulation for sustained Nanog induction via DNA methylation analysis and ChIP analysis for histone modifications. In summary, the proposed R01 application represents novel mechanistic research focused on Nanog+ liver cancer stem cells generated via activated TLR4 signaling by HCV NS5A and alcohol. An ultimate goal of this application is to eventually translate basic knowledge derived from the current study to design novel therapeutic modalities targeted to Nanog+ cancer stem cells for HCV/alcohol-related HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is one of the most important causes of liver cancer, which is the third most deadly cancer in the world. The goal of this proposal is to understand how HCV and alcohol induces liver cancer so that better prevention and treatment can be found.