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

研究项目3：酒精诱导的肝肿瘤始发干细胞的细胞命运决定。 项目摘要/摘要 酒精相关性肝细胞癌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。肝细胞癌治疗耐药的核心是启动肝肿瘤 我们成功地从肝癌动物模型和患者中分离出干细胞(TIC)，以了解他们的 自我更新机制。我们在抽动中发现了一种新的癌蛋白，TBC1D15，它促进了 TLR4-NANOG-AURKA-APKCζ通路对极性蛋白NUMBER磷酸化失活，导致 Numb的P53解离、P53泛素化和降解，以及TIC的自我更新。功能意义 肝脏肿瘤发病率和肿瘤相关NANOG的显著降低证实了这一机制 由肝细胞特异性表达非磷酸化Numer或肝细胞特异性缺陷引起的抽动 酒精喂养的丙型肝炎病毒NS5A转基因小鼠的TBC1D15。然而，TBC1D15的表达赋予了非转换 P53基因缺陷小鼠成肝细胞的自我更新活性。这表明TBC1D15具有未知的致癌作用 除促进P53基因丢失外，还可能有其他作用机制。我们发现TBC1D15与所有缺口异构体相互作用， 激活缺口途径，并以缺口依赖的方式诱导Nanog和TIC自我更新。我们的 数据表明，TBC1D15促进缺口激活并拮抗Numb介导的NICD失稳。 在一个-5kb的远端增强子和一个 近端启动子(-212/-151)，两者都是完全Nanog转录所必需的。我们还将TBC1D15-NICD 并鉴定了一种新型的小分子抑制剂，它可以阻断相互作用和 TbC1D15依赖的NICD-Hey1和Nanog启动子活性。我们假设TBC1D15促进 TABC1D15-NICD相互作用的抑制因子与Noch通路的强制性协同致癌作用 有治疗作用。我们将致力于实现以下目标： AIM-1。确定TBC1D15如何促进缺口途径：Aim-1.1：确定 TbC1D15-NICD稳定性中的缺口相互作用；Aim-1.2：确定TbC1D15是否激活缺口。 AIM-2。确定TBC1D15激活的Notch通路如何促进TIC的自我更新和肿瘤的发生： AIM-2.1：确定NICD如何支持Nanog转录；AIM-2.2：确定NICD在体内的重要性 TIC介导的肿瘤发生中可能的Nanog CSL/NICD位点；AIM-2.3。检验这部小说的疗效 患者来源的异种移植(PDX)肝癌模型中TBC1D15-NICD相互作用的抑制剂。 这项拟议工作的结果将为研究人乳头瘤的新致癌特性提供机制方面的见解。 TBC1D15和临床前基础，为未来使用这种拮抗相互作用的新型抑制剂进行临床试验奠定基础 在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.