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: 8516909
• 负责人: Keigo Machida
• 金额: $ 34.05万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516909
• 关键词: Alcohol consumption; Alcohol-Related Hepatocellular Carcinoma; Alcohols; Anchorage-Independent Growth; Binding; Binding Sites; Biological Assay; Cell Line; Cells; ChIP-seq; 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; 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; screening; 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）：令人信服的证据表明，酒精和 HCV 感染之间存在协同作用，可降低患肝细胞癌 (HCC) 的风险。该申请寻求对 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的支持。最近，我们发现 HCV NS5A 蛋白诱导肝细胞中的 Toll 样受体 4 (TLR4)，进而介导喂食酒精的小鼠的肝肿瘤发展，从而揭示了酒精和 HCV 之间的机制联系。酒精诱导的内毒素血症激活的 TLR4 信号传导可在 NS5A 转基因 (Tg) 小鼠中诱导祖细胞/干标记 Nanog 和肝脏肿瘤，但在野生型 (wt) 或缺乏 TLR4 的 NS5A Tg 小鼠中则不会。 Nanog 免疫染色与这些小鼠肝脏肿瘤中的癌症干细胞标记物 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。移植用 TLR4 转导的 p53-/- 成肝细胞，在重复注射 LPS 后在受体小鼠中产生肝脏肿瘤，并且 Nanog shRNA 可以阻止这种肿瘤的形成。移植从我们的小鼠模型的肝肿瘤中分离的Nanog+/CD133+/CD49f+细胞而不是Nanog-/CD133-/CD49f+细胞，在重复注射LPS的裸鼠中引起肿瘤生长。从前者的癌症干细胞而非后者的细胞建立的慢病毒cDNA文库在体外转化了卵圆细胞系。这些发现支持这样的假设：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。为了检验这一假设并了解模型中的致癌机制，我们将追求两个主要目标：1）识别和验证 Nanog+ 癌症干细胞中的致癌基因； 2) 阐明癌症干细胞中 TLR4 介导的 Nanog 诱导的遗传和表观遗传机制。对于目标1，将利用从癌症干细胞建立的慢病毒cDNA文库来分离候选致癌基因；基于shRNA的敲除将用于在体外和体内验证这些致癌基因； ChIP-seq 分析将用于癌症干细胞中 Nanog 结合的全基因组分析。对于目标 2，我们将通过使用缺失构建体和定点诱变的启动子报告基因测定以及 ChIP 分析来阐明 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。总之，拟议的 R01 应用代表了新的机制研究，重点是通过 HCV NS5A 和酒精激活的 TLR4 信号产生的 Nanog+ 肝癌干细胞。该应用的最终目标是最终转化从当前研究中获得的基础知识，设计针对 Nanog+ 癌症干细胞的 HCV/酒精相关 HCC 的新型治疗方式。 公共卫生相关性：丙型肝炎病毒 (HCV) 是导致肝癌的最重要原因之一，肝癌是世界上第三大致命癌症。该提案的目标是了解丙型肝炎病毒和酒精如何诱发肝癌，以便找到更好的预防和治疗方法。