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

慢性酒精摄入可协同增加丙型肝炎患者肝细胞癌（HCC）的发病率，然而，其潜在机制尚不清楚。HCC的一个关键事件是肿瘤起始细胞（tic）的增殖失控，这是一种干细胞样细胞，可促进治疗耐药的HCC。p53肿瘤抑制因子作为干细胞增殖的屏障，p53或其稳定伙伴NUMB失活导致tic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。慢性酒精对HCV NsSa转基因（Tg）小鼠肝细胞TLR4的异位诱导可产生tic和肝脏肿瘤。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复合体在nanog介导的aPKCzeta（一种NUMB激酶）激活后分解，导致p53的MDM2泛素连接酶依赖性蛋白水解。通过亲和纯化和串联质谱分析，我们发现TBC1D15是一种与NUMB相关的癌蛋白，也可以使p53脱离其与NUMB的保护性关联，导致p53降解和TIC增殖失控。TBC1D15的表达受TLR4-NAN0G途径诱导，在酒精性HCV患者HCC中表达升高。我们假设tlr4 - nan0g介导的NUMB磷酸化和TBC1D15上调协同触发p53降解，促进HCV和酒精引起的肝脏肿瘤发生中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激酶的诱导和aPKC抑制剂LGL的抑制是否与TICs中aPKCzeta的激活和p53的降解有关；2)新发现的aPKCzeta-NUMB和TBC1D15通路是p53降解的相互需要；3)这些途径对于酒精性HCV患者HCC分离的tic自我更新是明显的和必需的；4)在酒精喂养的HCV NsSaTg小鼠中，肝细胞特异性表达一种非磷酸化的NUMB突变体或肝细胞特异性消融TbcldlS可抑制肿瘤发生。

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.