Research Project 3: Cell fate decision of liver tumor-initiating stem-like cells induced by alcohol

研究项目3：酒精诱导的肝肿瘤起始干细胞的细胞命运决定

• 批准号: 10322380
• 负责人: Keigo Machida
• 金额: $ 11.21万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322380
• 关键词: Alcohols; Animal Model; Auras; Binding; C-terminal; CRISPR/Cas technology; California; Cancer Etiology; Cells; Cessation of life; ChIP-seq; Characteristics; Chemotherapy and/or radiation; Chromatin; Cirrhosis; Clinical Trials; Data; Diet; Dissociation; Distal; Docking; Elements; Enhancers; Funding; Future; Genes; Genetic Transcription; Hepatitis C virus; Hepatocyte; Incidence; Length; Ligands; Liver neoplasms; Luciferases; Maps; Mediating; Modeling; Mus; Mutation; NOTCH1 gene; Oncogenic; Oncoproteins; Parents; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Polyubiquitination; Primary carcinoma of the liver cells; Property; Protein Isoforms; Proteins; Recurrence; Recycling; Reporter; Research; Research Project Grants; Resistance; Role; Site; Site-Directed Mutagenesis; TLR4 gene; TP53 gene; Testing; Therapeutic; Therapeutic Effect; Transactivation; Transplantation; Treatment Efficacy; Ubiquitination; Work; aurora kinase A; chromatin immunoprecipitation; daughter cell; in vivo; inhibitor; insight; knock-down; liver cancer model; mouse model; mutant; neoplastic cell; novel; numb protein; overexpression; patient derived xenograft model; pre-clinical; preservation; prevent; promoter; radiation resistance; screening; self-renewal; small molecule; small molecule inhibitor; small molecule libraries; stem cells; stem-like cell; therapeutic evaluation; therapy resistant; tumor; tumor growth; tumorigenesis; tumorigenic; western diet

Research Project #3: Cell fate decision of liver tumor-initiating stem-like cells induced by alcohol. PROJECT SUMMARY/ABSTRACT Alcohol-associated hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths globally, yet efficacious therapeutic options are still limited. Central to therapy-resistance of HCC is the liver tumor-initiating stem-like cell (TIC) which we successfully isolated from HCC animal models and patients to understand their self-renewal mechanisms. We have identified a novel oncoprotein in TICs, TBC1D15 which facilitates phosphor-inactivation of the polarity protein NUMB by the TLR4-NANOG-AurkA-aPKCζ pathway, leading to dissociation of p53 of NUMB, p53 ubiquitination and degradation, and TIC self-renewal. Functional significance of this mechanism is confirmed by marked reductions in liver tumor incidence and tumor-associated NANOG+ TICs by hepatocyte-specific expression of non-phosphorylatable NUMB or hepatocyte-specific deficiency of TBC1D15 in alcohol-fed HCV NS5A Tg mice. However, TBC1D15 expression endows non-transformed p53-deficient mouse hepatoblasts self-renewal activity. This suggests TBC1D15 has an unknown oncogenic mechanism besides promoting p53 loss. We have discovered TBC1D15 interacts with all NOTCH isoforms, activates the NOTCH pathway, and induces Nanog and TIC self-renewal in a NOTCH-dependent manner. Our data suggest TBC1D15 promotes NOTCH activation and antagonizes NUMB-mediated NICD destabilization. Two putative functional CSL/NICD sites in the Nanog gene were identified in a -5kb distal enhancer and a proximal promoter (-212/-151), both required for full Nanog transcription. We also mapped the TBC1D15-NICD interaction domain and identified a novel small molecule inhibitor which blocks the interaction and TBC1D15-dependent NICD-Hey1 and Nanog promoter activities. We hypothesize that TBC1D15 promotes oncogenesis by obligatory cooperation with the NOTCH pathway thus an inhibitor of TBC1D15-NICD interaction is therapeutic. We will pursue the following aims: Aim-1. Identify how TBC1D15 promotes the NOTCH pathway: Aim-1.1: Determine the role of TBC1D15-NOTCH interaction in NICD stabilization; Aim-1.2: Determine if TBC1D15 activates NOTCH. Aim-2. Identify how TBC1D15-activated NOTCH pathway contributes to TIC self-renewal and tumorigenesis: Aim-2.1: Determine how NICD supports Nanog transcription; Aim-2.2: Determine in vivo importance of the putative Nanog CSL/NICD sites in TIC-mediated tumorigenesis; Aim-2.3. Test the therapeutic efficacy of the novel inhibitor for the TBC1D15-NICD interaction in patient-derived xenograft (PDX) HCC model. Results from the proposed work will provide mechanistic insights into the novel oncogenic property of TBC1D15 and a pre-clinical basis for a future clinical trial using the novel inhibitor that antagonizes the interaction between TBC1D15 and NOTCH.

研究项目＃3：酒精诱导的肝脏肿瘤样干细胞的细胞命运决策。 项目摘要/摘要 酒精相关的肝细胞癌（HCC）是全球癌症死亡的主要原因之一， 然而，有效的治疗选择仍然有限。 HCC治疗抗性的核心是肝脏肿瘤发射 我们成功地从HCC动物模型和患者中分离出来的干细胞（TIC），以了解其 自我更新机制。我们已经确定了TICS中的一种新型癌蛋白TBC1D15，可促进 TLR4-Nanog-aurka-apkCζ途径对极性蛋白的磷光灭活，导致 p53，p53泛素化和降解以及tic自我更新的解离。功能意义 通过明显降低肝肿瘤的发病率和肿瘤相关的纳米+，证实了这种机制 通过肝细胞特异性表达非磷酸化麻木或肝细胞特异性缺陷 酒精喂养的HCV NS5A TG小鼠中的TBC1D15。但是，TBC1D15表达式赋予未转化的 p53缺乏小鼠肝类母细胞自我更新活性。这表明TBC1D15具有未知的致癌性 除了促进p53损失外，机制。我们发现TBC1D15与所有缺口同工型相互作用， 激活Notch途径，并以缺口依赖性方式诱导Nanog和Tic自我更新。我们的 数据表明TBC1D15促进了Notch激活，并拮抗麻木介导的NICD不稳定。 在-5kb盘中增强子和A中鉴定了Nanog基因中的两个推定功能CSL/NICD位点 近端启动子（-212/-151），都是全纳米转录所必需的。我们还映射了TBC1D15-NICD 相互作用域并鉴定出一种新型的小分子抑制剂，该抑制剂阻止了相互作用和 TBC1D15依赖性的NICD-HEY1和NANOG启动子活动。我们假设TBC1D15促进 通过与Notch途径进行强制性合作的发生，因此TBC1D15-NICD相互作用的抑制剂是 治疗性。我们将追求以下目标： AIM-1。确定TBC1D15如何促进Notch途径：AIM-1.1：确定 NICD稳定中的TBC1D15-NOTCH相互作用； AIM-1.2：确定TBC1D15是否激活Notch。 AIM-2。确定TBC1D15激活的Notch途径如何有助于TIC自我更新和肿瘤发生： AIM-2.1：确定NICD如何支持Nanog转录； AIM-2.2：确定体内重要性 TIC介导的肿瘤发生中的推定纳米CSL/NICD位点； AIM-2.3。测试小说的治疗效率 患者衍生的Xenographic（PDX）HCC模型中TBC1D15-NICD相互作用的抑制剂。 提议的工作的结果将提供对新型致癌特性的机械见解 TBC1D15和使用新型抑制剂进行相互作用的临床试验的临床前基础 在TBC1D15和Notch之间。