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。 项目概要/摘要 酒精相关性肝细胞癌（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 治疗耐药的核心是肝脏肿瘤起始 我们成功地从 HCC 动物模型和患者中分离出干细胞样细胞 (TIC)，以了解它们的情况 自我更新机制。我们在 TIC 中发现了一种新型癌蛋白 TBC1D15，它有助于 TLR4-NANOG-AurkA-aPKCζ 通路对极性蛋白 NUMB 进行磷酸灭活，从而导致 NUMB 的 p53 解离、p53 泛素化和降解以及 TIC 自我更新。功能意义 肝肿瘤发生率和肿瘤相关 NANOG 的显着降低证实了这一机制 TIC 由非磷酸化 NUMB 的肝细胞特异性表达或 NUMB 的肝细胞特异性缺乏引起 酒精喂养的 HCV NS5A Tg 小鼠中的 TBC1D15。然而，TBC1D15 表达赋予非转化 p53 缺陷小鼠成肝细胞的自我更新活性。这表明 TBC1D15 具有未知的致癌性 除了促进p53损失之外的机制。我们发现 TBC1D15 与所有 NOTCH 亚型相互作用， 激活 NOTCH 通路，并以 NOTCH 依赖性方式诱导 Nanog 和 TIC 自我更新。我们的 数据表明 TBC1D15 促进 NOTCH 激活并拮抗 NUMB 介导的 NICD 不稳定。 Nanog 基因中两个假定的功能性 CSL/NICD 位点在 -5kb 远端增强子和 近端启动子 (-212/-151)，两者都是完整 Nanog 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小分子抑制剂，该抑制剂可阻断相互作用 TBC1D15 依赖的 NICD-Hey1 和 Nanog 启动子活性。我们假设 TBC1D15 促进 肿瘤发生是通过与 NOTCH 通路的强制性合作来实现的，因此 TBC1D15-NICD 相互作用的抑制剂是 治疗性的。我们将追求以下目标： 目标-1。确定 TBC1D15 如何促进 NOTCH 通路：Aim-1.1：确定 TBC1D15 的作用 TBC1D15-NOTCH 相互作用在 NICD 稳定中的作用； Aim-1.2：确定 TBC1D15 是否激活 NOTCH。 目标2。确定 TBC1D15 激活的 NOTCH 通路如何促进 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 介导的肿瘤发生中推定的 Nanog CSL/NICD 位点；目标-2.3。测试小说的治疗效果 患者来源的异种移植 (PDX) HCC 模型中 TBC1D15-NICD 相互作用的抑制剂。 拟议工作的结果将为新的致癌特性提供机制见解 TBC1D15 以及使用拮抗相互作用的新型抑制剂进行未来临床试验的临床前基础 TBC1D15 和 NOTCH 之间。

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.