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

酒精和丙型肝炎病毒导致的 Nanog 阳性癌症干细胞和肝癌发生

• 批准号: 8133144
• 负责人: Keigo Machida
• 金额: $ 36.61万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133144
• 关键词: Alcohol consumption; Alcohol-Related Hepatocellular Carcinoma; Alcohols; Anchorage-Independent Growth; Binding; Binding Sites; Biological Assay; Cell Line; Cells; Chronic; Comorbidity; Complementary DNA; DNA Methylation; Development; EMSA; Endotoxemia; Endotoxins; Enhancers; Epigenetic Process; Ethanol; Future; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Goals; Hepatic; Hepatitis C; Hepatitis C virus; Hepatocarcinogenesis; Hepatocyte; In Vitro; Incidence; Infection; Injection of therapeutic agent; Knowledge; Link; Liver; Liver neoplasms; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Messenger RNA; Modality; Modeling; Mus; Mutation; Nude Mice; Oncogenic; Outcome; Patients; Phenotype; Prevention; Primary carcinoma of the liver cells; Proteins; Regulation; Regulator Genes; Reporter; Research; Research Support; Risk; Role; Screening procedure; Signal Transduction; Site; Site-Directed Mutagenesis; Stem cells; Subfamily lentivirinae; Technology; Testing; Transcriptional Activation; Transgenic Mice; Translating; Transplantation; Validation; Wild Type Mouse; Work; alcoholism therapy; attenuation; base; cDNA Library; cancer stem cell; cell transformation; design; embryonic stem cell; feeding; genome wide association study; genome-wide; genome-wide analysis; histone modification; in vivo; mouse model; new therapeutic target; novel; novel therapeutics; oval cell; overexpression; prevent; progenitor; promoter; public health relevance; small hairpin RNA; stem; stemness; synergism; therapeutic target; toll-like receptor 4; tumor; tumor growth; tumorigenesis

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.

描述（由申请人提供）：令人信服的证据确定酒精和HCV感染之间的协同作用是发展出肝细胞癌（HCC）的风险。该应用寻求支持针对纳米阳性癌症干细胞在HCV和酒精引起的肝癌发生中作用的研究。我们发现，HCV NS5A蛋白在肝细胞中诱导了类似Toll样受体4（TLR4）的发现，饮酒和HCV之间的机理联系已揭示，这又介导了喂养酒精的小鼠的肝肿瘤的发展。通过酒精诱导的内毒素血症激活的TLR4信号传导诱导NS5A转基因（TG）小鼠的祖细胞/茎标记Nanog和肝肿瘤，但不诱导野生型（WT）或NS5A TG小鼠在TLR4中缺乏。 Nanog免疫染色与这些小鼠的肝肿瘤中的癌细胞标记CD133和CD49F共定位。体外启动子和mRNA分析表明NS5A转染细胞中Nanog的TLR4依赖性转录激活。用TLR4转导的p53 - / - 肝细胞的移植会在重复的LPS注射后在受体小鼠中产生肝肿瘤，并用Nanog ShRNA预防这种肿瘤形成。纳米+/CD133+/CD49F+细胞的移植，而不是从我们的小鼠模型的肝肿瘤中分离出的Nanog-/CD133-/CD49F+细胞，导致裸鼠的肿瘤生长，后者被重复注射LPS。慢病毒cDNA文库是从前癌干细胞建立的，而不是后一种细胞，在体外转化了椭圆形细胞系。这些发现支持以下假设：HCV/NS5A和酒精的协同肝脏肿瘤发生是由TLR4诱导的Nanog+癌症干细胞的致癌活性介导的。为了检验该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的：1）识别和验证纳米+癌干细胞中的致癌基因； 2）阐明了癌症干细胞中TLR4介导的纳米诱导的遗传和表观遗传机制。对于目标1，从癌症干细胞中建立的慢病毒cDNA文库将用于隔离候选致癌基因。基于ShRNA的敲低将用于在体外和体内验证这些致癌基因。将对癌症干细胞中的纳米结合进行全基因组结合进行CHIP-SEQ分析。对于AIM 2，我们将阐明TLR4通过启动子重复蛋白分析的转录机制，其具有缺失构建体和定向的诱变和芯片分析。并通过DNA甲基化分析和芯片分析确定表观遗传调节，以持续进行纳米诱导。总而言之，拟议的R01应用代表了针对Nanog+肝癌干细胞通过HCV NS5A和酒精产生的Nanog+肝癌干细胞的新机械研究。该应用的最终目标是最终将当前研究得出的基本知识转化为设计针对Nanog+癌症干细胞的新型治疗方式，用于HCV/酒精相关的HCC。 公共卫生相关性：丙型肝炎病毒（HCV）是肝癌最重要的原因之一，这是世界上第三大致命癌症。该提案的目的是了解HCV和酒精如何诱导肝癌，从而找到更好的预防和治疗。