JCAD 通过Hippo信号通路促进非酒精性脂肪性肝炎向肝癌进展的机制

It is common that hepatocellular carcinoma (HCC) occurs in cirrhosis secondary to nonalcoholic steatohepatitis (NASH). HCC may also occur in NASH prior to cirrhosis. The molecular mechanisms of carcinogenesis originating from NASH are largely elusive. In our preliminary study, Affymetrix microarray analysis revealed that expression of junctional protein-associated with coronary artery disease (JCAD) was much higher in NASH-HCC than peri-cancerous tissue, and JCAD was found to stimulate proliferation and in vitro tumorigenecity and xenograft formation. Suppressing JCAD expression by an RNAi approach decreased hepatoma cell proliferation and xenograft formation. JCAD was found to interact with LATS2, a critical phosphokinase in Hippo signaling, and attenuated phosphorylation of YAP, resulting in activation of its downstream target genes. However, how JCAD and Hippo signaling are activated in a steatotic microenvironment is unknown. The present study aims to dissect the molecular interplays of JCAD with Hippo signaling and other molecules under an inflamed, steatotic and fibrotic microenvironment using approaches such as CRISPR-cas9 gene-editing, inducing HCC development in JCAD knock-out mice, as well as biopsied specimens from NASH patients and NASH-HCC resection. The undertaking of this proposal will enable to uncover molecular cascades of hepatic carcinogenesis in steatotic inflammation and metabolic abnormalities in the lines of JCAD and Hippo signaling activation, as well as downstream genes, such as cyclin D1, Gli-2, notch, CTGF, etc. Moreover, the correlation of JCAD with NASH progression to HCC may point to its biomarker potential for clinical translation.

非酒精性脂肪性肝炎（NASH）无肝硬化者可发生肝癌，但机制不明。肝癌与癌旁肝组织基因谱分析表明肝癌组织中冠状动脉疾病相关连接蛋白（JCAD）表达比癌旁肝组织（伴脂肪性肝炎）明显升高。JCAD可促进肝癌细胞生长，过表达或表达水平受抑制后，肝癌细胞体内外成瘤率及生长速度得到显著提升或降低。进一步研究发现JCAD结合到Hippo信号通路关键分子LATS2蛋白激酶的活性区，启动下游靶分子，促进肝癌的形成和生长。然而，NASH导致JCAD表达升高及启动Hippo通路的机制不明。为进一步探讨该蛋白与NASH-HCC的关系，本立项拟应用CRISPR-Cas9技术及JCAD基因敲除小鼠，从动物模型及临床标本着手，探究代谢紊乱及肝持续炎症如何诱导JCAD、启动Hippo通路和癌基因分子，促使受损肝细胞转变为肝癌细胞。本立项的实施将揭示慢性脂肪性炎症恶性转变的分子机制，同时JCAD有望预警NASH向肝癌发展。

随着非酒精性脂肪性肝炎（NASH）发病数不断攀升，非酒精性脂肪性肝炎相关肝细胞癌（NASH-HCC）发生率也不断升高，已经成为欧美肝癌发病率升高的主要原因。然而其发生机制远不如其它病因如病毒感染所致肝癌清楚。前期研究发现冠状动脉疾病相关连接蛋白（JCAD）与Hippo信号关键磷酸激酶LATS2相互作用，通过Hippo-YAP信号轴，促进肝细胞癌的发生进展（Cancer Res 2017）。在此基础上建立JCAD敲除小鼠，以肯定此连接蛋白在NASH-纤维化-HCC进展中的作用。与预期结果相反，两种完全由代谢因素诱发的NASH-纤维化-HCC模型准证实JCAD敲除反而导致肝细胞损伤、纤维化加重、HCC发生的数量、频率和体积明显高于野生型小鼠喂食高脂高热量饲料+高糖饮水（HFCD-HF/G）或胆碱缺乏高脂饲料（CD/HFD）所致的肝癌。肝组织电镜检查发现JCAD敲除小鼠肝细胞毛细胆管扩张，微绒毛消失，紧密连接蛋白水平下降，通透性增加。肝细胞内有胆汁积蓄。血总胆酸和多种胆酸组分升高，总胆红素及直接胆红素升高，而肝细胞胆酸经典和替代途径合成酶：细胞色素氧化酶亚型（CYP7a1、CYP7b1、CYP8b1、CYP27a1）及胆酸摄取受体（NTCP、OATP4）的基因表达水平下降，符合毛细胆管受损、胆汁反流而致血胆酸组分升高的推测。并且，肝肿瘤组织中pStat3、TAZ、pErk水平比肝组织明显升高，而-catenin的水平变化不大。因此，这些信号分子可能参与NASH-纤维化-HCC的恶变过程。目前，正进行进一步探究胆汁反流如何激活这些肿瘤基因信号通路，以揭示JCAD在NASH-异型变-恶变过程中的分子调控机制，为NASH-HCC的防治提供科学数据。

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