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

• 批准号: 8597101
• 负责人: Keigo Machida
• 金额: $ 19.87万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 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/8597101
• 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; Hepatocyte; Incidence; Lead; Liver; Liver neoplasms; MDM2 gene; Malignant neoplasm of liver; Mediating; Medical; Mus; Oncogene Proteins; Outcome; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Primary carcinoma of the liver cells; Property; Protein p53; Proteolysis; Research; Resistance; Role; Stem cells; TLR4 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

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.

长期饮酒会协同增加丙型肝炎患者肝细胞癌（HCC）的发病率，但其潜在机制仍知之甚少。 HCC 的一个关键事件是肿瘤起始细胞 (TIC) 的扩增失调，这些细胞是促进治疗耐药性 HCC 的干细胞样细胞。 p53 肿瘤抑制因子充当干细胞增殖的屏障，p53 或其稳定伙伴 NUMB 失活会导致 TIC 扩张。然而，关于 NUMB-p53 相互作用的控制以及酒精和 HCV 诱导的 HCC TIC 中 p53 失活的机制基础知之甚少。当 HCV NsSa 转基因 (Tg) 小鼠长期饮酒时，肝细胞中 TLR4 的异位诱导会产生 TIC 和肝肿瘤。我们证明了 TIC 的干性和肿瘤启动特性依赖于 TLR4-NANOG 通路和 p53 水平的降低。 NUMB-p53 复合物在 NANOG 介导的 NUMB 激酶 aPKCzeta 激活后分解，导致 MDM2 泛素连接酶依赖性 p53 蛋白水解。使用亲和纯化和串联质谱法，我们已将 TBC1D15 鉴定为一种 NUMB 相关癌蛋白，它还可以使 p53 脱离与 NUMB 的保护性关联，导致 p53 降解并解除对 TIC 传播的管制。 TBC1D15 的表达由 TLR4-NAN0G 通路诱导，并在酒精性 HCV 患者的 HCC 中升高。我们假设 TLR4-NAN0G 介导的 NUMB 磷酸化和 TBC1D15 上调协同触发 p53 降解，从而促进 HCV 和酒精引起的肝癌发生中 TIC 的自我更新。为了检验这一假设，我们将研究是否：1）NANOG 介导的上游 AurA 激酶的诱导和 aPKC 抑制剂 LGL 的抑制是 TIC 中 aPKCzeta 的激活和 p53 降解的原因； 2)新公开的aPKCzeta-NUMB和TBC1D15途径对于p53降解是相互需要的； 3) 这些途径是明显的，并且是从酒精性 HCV 患者的 HCC 中分离出的 TIC 自我更新所必需的； 4) NUMB的不可磷酸化突变体的肝细胞特异性表达或TbcldlS的肝细胞特异性消除抑制酒精喂养的HCV NsSaTg小鼠中的肿瘤发生。