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感染之间存在发生肝细胞癌（HCC）风险的协同作用。本申请旨在支持研究nanog阳性癌症干细胞在HCV和酒精诱导的肝癌发生中的作用。最近，我们发现HCV NS5A蛋白在肝细胞中诱导toll样受体4 (TLR4)，进而介导酒精喂养小鼠的肝脏肿瘤发展，揭示了酒精与HCV之间的机制联系。TLR4信号被酒精诱导的内毒素血症激活，在NS5A转基因（Tg）小鼠中诱导祖细胞/干细胞标记物Nanog和肝脏肿瘤，而在野生型（wt）或缺乏TLR4的NS5A Tg小鼠中则无此作用。在这些小鼠的肝肿瘤中，Nanog免疫染色与肿瘤干细胞标志物CD133和CD49f共定位。体外启动子和mRNA分析表明，在转染ns5a的细胞中，Nanog的转录激活依赖tlr4。经TLR4转导的p53-/-肝母细胞移植后，反复注射LPS可使受体小鼠产生肝脏肿瘤，而Nanog shRNA可阻止这种肿瘤的形成。从我们的小鼠模型肝肿瘤中分离Nanog+/CD133+/CD49f+细胞，而不是Nanog-/CD133-/CD49f+细胞，移植在重复注射LPS的裸鼠中引起肿瘤生长。用前一种肿瘤干细胞而非后一种肿瘤干细胞建立慢病毒cDNA文库，在体外转化卵形细胞系。这些发现支持了HCV/NS5A和酒精协同肝癌发生的假设，即tlr4诱导的Nanog+癌干细胞的致癌活性是介导的。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+肿瘤干细胞中的致癌基因；2)阐明tlr4介导的Nanog诱导肿瘤干细胞的遗传和表观遗传机制。对于Aim 1，从癌症干细胞中建立的慢病毒cDNA文库将用于分离候选致癌基因；基于shrna的敲除将用于在体外和体内验证这些致癌基因；和ChIP-seq分析将用于肿瘤干细胞中Nanog结合的全基因组分析。在Aim 2中，我们将通过缺失构建子和定点突变的启动子报告子试验以及ChIP分析阐明TLR4诱导Nanog的转录机制；并通过DNA甲基化分析和组蛋白修饰的ChIP分析确定持续Nanog诱导的表观遗传调控。综上所述，R01的应用代表了HCV NS5A和酒精通过激活TLR4信号产生Nanog+肝癌干细胞的新机制研究。该应用的最终目标是最终将从当前研究中获得的基础知识转化为设计针对Nanog+癌症干细胞治疗HCV/酒精相关性HCC的新治疗模式。

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.