钙粘蛋白相关家族分子2调节细胞接触抑制在肝癌中的作用机制研究

The Hippo pathway involved in cell contact inhibition plays a central role in tumorigenesis. However, its upstream pathway has remained elusive. In our previous studies, a membrane protein called cadherin-related family 2 (CDHR2), was detected downregulated in hepatocellular carcinoma. Ectopic expression of CDHR2 inhibited the proliferaton of cell in a manner of density-dependent, accompanying with cytoplasmic retention of YAP, a major downstream effector of the Hippo pathway, suggesting that CDHR2 may be associated with the Hippo pathway. In this study, we will further detect the expression and regulatory mechanism of CDHR2 gene in HCC and its clinical significance. And we will also unravel the role and mechanism of CDHR2 in contact inhibition through the Hippo pathway, as well as its effect in hepatocarcinogenesis. Finally, we will identify proteins interacting with CDHR2 and the crystal structure to clarify the structural basis for its function. Our study will not only elucidate the methanism of CDHR2 in contact inhibition, but also provide a new target for the early diagnosis, targeted cancer therapy and a clinical prognostic indicator of HCC or other types of tumors in the future.

Hippo信号通路调节细胞接触抑制在肿瘤发生中起着重要的作用，但其上游调节机制尚未完全阐明。我们在前期研究中发现钙粘蛋白相关家族分子2（CDHR2）在肝癌中表达下调，过表达CDHR2可以发生细胞密度依赖性的增殖抑制，伴随Hippo信号通路效应分子YAP的胞浆滞留，提示CDHR2可能与Hippo信号通路有关。在本课题研究中，我们将进一步明确肝癌中CDHR2的表达和调控机制在临床上的意义，阐释CDHR2通过Hippo信号通路发挥细胞接触抑制的作用和具体机制，及其在肝癌发生中的作用，确定其胞内外的相互作用分子，并通过蛋白质晶体结构解析确定CDHR2分子发挥作用的结构基础。我们的研究结果将有望阐明CDHR2分子发挥细胞接触抑制的具体机制，为今后肝癌等肿瘤的早期发现、靶向治疗和临床的预后判断等提供新靶点。

钙粘蛋白相关家族成员2（CDHR2）属于原钙粘蛋白家族，其在正常的肝、肾和结肠组织中含量丰富，但在这些相关组织引起的癌症中表达减弱。在本研究中，我们证实CDHR2在人肝癌（HCC）的癌旁组织中高度表达，但在HCC细胞系中通过半定量PCR和Western blot检测发现CDHR2在85.7%（6/7）中明显下调或沉默，而在79.1%（19/24）和80.2%（89/111）的肝癌组织中，CDHR2的mRNA和蛋白水平呈表达下调。通过甲基化特异性PCR检测发现，与癌旁组织相比，在肝癌细胞系和肝癌组织中CDHR2基因启动子中的CpG岛呈高度甲基化的状态，从而导致肝癌中CDHR2的转录抑制和表达沉默。此外，慢病毒介导的CDHR2过度表达实验发现其可延长肝癌细胞的倍增时间、降低体外克隆形成能力和体内致瘤性，具有抑制HCC细胞的生长和增殖的作用。进一步研究表明CDHR2过度表达可导致Akt去磷酸化，下调Akt下游靶点环氧化酶2（Cox2）的表达。激活型Akt（肉豆蔻酰化Akt）可逆转该作用，表明CDHR2-Akt-Cox2轴参与了抑制HCC生长。综上所述，我们的研究证实CDHR2是一种新的肝癌抑制因子，为肝癌的治疗提供了新的靶点。