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

慢性酒精摄入协同增加丙型肝炎患者肝细胞癌（HCC）的发病率，然而，其潜在机制仍知之甚少。HCC中的一个关键事件是肿瘤起始细胞（TIC）的失控扩增，所述肿瘤起始细胞是促进治疗抗性HCC的干细胞样细胞。p53肿瘤抑制因子作为针对干细胞增殖的屏障，并且p53或其稳定性伴侣NUMB的失活导致TIC的扩增。然而，对于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。当长期给予小鼠酒精时，HCV NsSa转基因（Tg）小鼠肝细胞中TLR 4的异位诱导产生TIC和肝肿瘤。我们证明了TICs的干性和肿瘤起始特性依赖于TLR 4-NANOG通路和p53水平的降低。NUMB-p53复合物在NANOG介导的aPKCzeta（NUMB激酶）活化后解体，导致p53的MDM 2泛素连接酶依赖性蛋白水解。使用亲和纯化和串联质谱，我们已经确定TBC 1D 15作为一个NUMB相关的癌蛋白，也可以脱离p53从其保护协会与NUMB，导致p53降解和失调TIC传播。TBC 1D 15的表达由TLR 4-NAN 0 G通路诱导，并且在酒精性HCV患者的HCC中升高。我们假设TLR 4-NAN 0 G介导的NUMB磷酸化和TBC 1D 15上调协同触发p53降解，以促进HCV和酒精引起的肝肿瘤发生中TIC的自我更新。为了验证这一假设，我们将研究：1）NANOG介导的上游AurA激酶诱导和aPKC抑制剂LGL的抑制是否负责aPKCzeta的激活和TICs中p53的降解; 2）新公开的aPKCzeta-NUMB和TBC 1D 15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中分离的TIC自我更新所必需的;和4）NUMB的非磷酸化突变体的肝细胞特异性表达或TbcldlS的肝细胞特异性消融抑制酒精中的肿瘤发生。喂食HCV的NsSaTg小鼠。

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.