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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.

长期饮酒协同增加丙型肝炎患者肝细胞癌的发病率，然而，其潜在的机制仍不清楚。肝细胞癌的一个关键事件是肿瘤起始细胞(TIC)的非调控扩张，TIC是促进耐药肝细胞癌的干细胞样细胞。P53肿瘤抑制因子起到阻止干细胞增殖的屏障作用，而P53或其稳定伴侣数量的失活会导致TICs的扩张。然而，在酒精和丙型肝炎诱导的TICS中，对Numb-P53相互作用的控制和P53失活的机制还知之甚少。在长期饮酒的情况下，异位诱导丙型肝炎病毒NSSA转基因(TG)小鼠的肝细胞中的TLR4会产生抽搐和肝肿瘤。我们证明TICS的干性和肿瘤启动特性依赖于TLR4-NANOG通路和P53的降低水平。Numb-P53复合体在NANOG介导的一种Numb蛋白激酶激活时解体，导致MDM2泛素连接酶依赖的P53蛋白分解。利用亲和纯化和串联质谱仪，我们已经鉴定了TBC1D15是一种Numb相关的癌蛋白，它也可以使P53与Numb的保护性结合解离，导致P53的降解和TIC的失控增殖。在酒精性丙型肝炎患者肝细胞癌中，TLR4-NAN0G途径诱导了TBC1D15的表达，并使其表达上调。我们假设TLR4-NAN0G介导的Numb磷酸化和TBC1D15上调协同触发P53的降解，以促进丙型肝炎病毒和酒精引起的肝脏肿瘤中TICS的自我更新。为了验证这一假说，我们将调查：1)NANOG介导的上游Aura激酶的诱导和aPKC抑制剂LGL的抑制是否与抽动症中aPKcheeta的激活和P53的降解有关；2)新公布的aPKcheeta-Numb和TBC1D15通路是P53降解相互必需的；3)这些通路是明显的并且需要酒精性丙型肝炎患者肝细胞癌分离株的自我更新；以及4)Numb的非磷酸化突变体的肝细胞特异性表达或肝细胞特异性消融TbcldlS抑制了酒精喂养的丙型肝炎NsSaTg小鼠的肿瘤发生。