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

研究项目#3：酒精诱导的肝肿瘤启动干细胞样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肿瘤启动 干细胞样细胞（TIC），我们成功地从HCC动物模型和患者中分离出来，以了解其 自我更新机制。我们已经在TIC中鉴定了一种新的癌蛋白，TBC 1D 15， 通过TLR 4-NANOG-AurkA-aPKC β途径使极性蛋白NUMB磷酸化失活，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 这一机制的有效性得到了肝肿瘤发病率和肿瘤相关NANOG+显著降低的证实。 通过肝细胞特异性表达非磷酸化NUMB或肝细胞特异性缺乏 酒精喂养的HCV NS 5A Tg小鼠中的TBC 1D 15。然而，TBC 1D 15表达赋予非转化的 p53缺陷小鼠肝母细胞自我更新活性。这表明TBC 1D 15具有未知的致癌性。 除促进p53丢失外，我们已经发现TBC 1D 15与所有NOTCH亚型相互作用， 激活NOTCH途径，并以NOTCH依赖性方式诱导Nanog和TIC自我更新。我们 数据表明TBC 1D 15促进NOTCH活化并拮抗NUM介导的NICD去稳定化。 在Nanog基因中的两个推定的功能性CSL/NICD位点被鉴定为在-5kb的远端增强子和一个远端增强子中。 近端启动子（-212/-151），两者都是完整Nanog转录所需的。我们还绘制了TBC 1D 15-NICD 相互作用结构域，并确定了一种新的小分子抑制剂， TBC 1D 15依赖性NICD-Hey 1和Nanog启动子活性。我们假设TBC 1D 15促进了 通过与NOTCH途径的强制性合作来促进肿瘤发生，因此TBC 1D 15-NICD相互作用的抑制剂是 有治疗作用的我们将努力实现以下目标： 目标一确定TBC 1D 15如何促进NOTCH途径：目标1.1：确定 TBC 1D 15-NOTCH在NICD稳定中的相互作用;目的-1.2：确定TBC 1D 15是否激活NOTCH。 目标二确定TBC 1D 15激活的NOTCH通路如何促进TIC自我更新和肿瘤发生： 目的-2.1：确定NICD如何支持Nanog转录;目的-2.2：确定NICD在体内的重要性， TIC介导的肿瘤发生中的推定Nanog CSL/NICD位点; Aim-2.3.测试新的治疗效果 患者来源的异种移植物（PDX）HCC模型中TBC 1D 15-NICD相互作用的抑制剂。 从拟议的工作结果将提供机制的见解，新的致癌特性， TBC 1D 15和使用拮抗相互作用的新型抑制剂的未来临床试验的临床前基础 TBC 1D 15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.