Functional Dissection of EtOH-med Self-renew of Liver Tumor-Int Cells via TLR4

通过 TLR4 进行 EtOH 诱导的肝肿瘤 Int 细胞自我更新的功能剖析

基本信息

批准号：
8991273
负责人：
Keigo Machida
金额：
$ 20.36万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8991273
关键词：
Ablation Affinity Chromatography Albumins Alcohol consumption Alcoholic Hepatitis Alcoholism Alcohols Attenuated Auras California Cell Proliferation Cells Chronic Cirrhosis Clinical Clinical Research Complex Dissection Event Hepatitis C Hepatitis C virus Hepatocarcinogenesis Hepatocyte Incidence Lead Liver Liver neoplasms MDM2 gene Malignant neoplasm of liver Mediating Medical Mus Oncogenes Outcome Pathway interactions Patients Phosphorylation Phosphotransferases Primary carcinoma of the liver cells Property Protein p53 Proteolysis Research Resistance Role Stem cells TLR4 gene TP53 gene Tamoxifen Testing Transgenic Mice Tumor Expansion Up-Regulation aurora-A kinase base clinically relevant design feeding functional restoration improved inhibitor/antagonist mutant novel therapeutics overexpression prevent self-renewal stem stemness tandem mass spectrometry tumor tumorigenesis ubiquitin ligase

项目摘要

Chronic alcohol intake synergistically increases the incidence of hepatocellular carcinoma (HCC) in hepatitis C patients, however, the underlying mechanisms remain poorly understood. A critical event in HCC is the deregulated expansion of tumor initiating cells (TICs), which are stem-like cells that promote treatment resistant HCC. The p53 tumor suppressor acts as a barrier against stem cell proliferation, and inactivation of p53 or its stabilizing partner NUMB leads to expansion of TICs. However, little is known about the control of the NUMB-p53 interaction and the mechanistic basis for p53 inactivation in TICs from HCC induced by alcohol and HCV. Ectopic induction of TLR4 in hepatocytes in HCV NsSa transgenic (Tg) mice generates TICs and liver tumors when the mice are chronically given alcohol. We demonstrated that stemness and tumor-initiating properties of TICs are dependent on TLR4-NANOG pathway and diminished levels of p53. The NUMB-p53 complex disintegrates upon NANOG-mediated activation of aPKCzeta, a NUMB kinase, leading to MDM2 ubiquitin ligase-dependent proteolysis of p53. Using affinity purification and tandem mass spectrometry, we have identified TBC1D15 as a NUMB-associated oncoprotein that can also disengage p53 from its protective association with NUMB, causing p53 degradation and deregulated TIC propagation. The expression of TBC1D15 is induced by the TLR4-NAN0G pathway and elevated in HCC in alcoholic HCV patients. We hypothesize TLR4-NAN0G-medlated NUMB phosphorylation and TBC1D15 upregulation coordinately trigger p53 degradation to promote self-renewal of TICs in liver oncogenesis caused by HCV and alcohol. To test this hypothesis, we will investigate whether: 1) NANOG-mediated induction of upstream AurA kinase and inhibition of the aPKC inhibitor LGL are responsible for activation of aPKCzeta and p53 degradation in TICs; 2) the newly disclosed aPKCzeta-NUMB and TBC1D15 pathways are mutually required for p53 degradation; 3) these pathways are evident and required for self-renewal of TICs isolated from HCC of alcoholic HCV patients; and 4) hepatocyte-specific expression of a non-phosphorylatable mutant of NUMB or hepatocyte-specific ablation of TbcldlS suppresses tumorigenesis in alcohol-fed HCV NsSaTg mice.

慢性酒精摄入量从丙型肝炎患者中协同增加肝细胞癌（HCC）的发生率，但是，基本机制仍然鲜为人知。 HCC中的一个关键事件是肿瘤启动细胞（TICS）的放松调节，它们是促进耐药性HCC的干细胞。 p53肿瘤抑制剂起着抵抗干细胞增殖的障碍，p53或其稳定伴侣麻木的灭活会导致抽动的扩张。然而，对于Numb-P53相互作用的控制以及p53失活的机理基础，来自酒精和HCV引起的HCC的机理基础知之甚少。 HCV NSSA转基因（TG）小鼠的肝细胞中TLR4的异位诱导会在小鼠长期服用酒精时会产生抽动和肝肿瘤。我们证明，抽动的干性和肿瘤发射特性取决于TLR4-NANOG途径和p53水平降低。 Numb-P53复合物在纳米介导的APKCZETA激活（一种麻木激酶）上分解，导致MDM2泛素连接酶依赖性p53的MDM2泛素蛋白水解。使用亲和力纯化和串联质谱法，我们已经将TBC1D15鉴定为一种麻木相关的癌蛋白，它也可以使p53与Numb的保护性关联，从而导致p53降解并导致p53降解。 TLR4-NAN0G途径诱导TBC1D15的表达，并在酒精HCV患者的HCC中升高。我们假设TLR4-NAN0G - 矿体麻木的磷酸化和TBC1D15上调协调触发p53降解，以促进由HCV和酒精引起的肝脏造成的肝脏造成的自我更新。为了检验这一假设，我们将研究：1）纳米介导的上游光环激酶的诱导和对APKC抑制剂LGL的抑制是造成APKCZETA的激活和TICS中P53降解的激活； 2）新披露的apkczeta-nibm和tbc1d15途径是p53降解所必需的； 3）这些途径是明显的，并且需要从酒精HCV患者的HCC中分离出的抽动自我更新；和4）肝细胞特异性表达TBCLDLS的麻木或肝细胞特异性消融的非磷酸化突变体抑制了酒精喂养的HCV NSSATG小鼠中的肿瘤。