aPKC-ι/PDGF-BB双向调控癌细胞与血小板间cross-talk在肝内胆管癌侵袭转移中的作用及机制研究

Epithelial-Mesenchymal Transition is the core aspect of the invasion and metastasis in cholangiocarcinoma. In the previous studies, the applicant demonstrated that aPKC-ι could induce EMT in cholangiocarcinoma. Recently, the applicant found the intrahepatic cholangiocarcinoma cells with high level of aPKC-ι could promote platelet activation, and the expression of aPKC-ι in intrahepatic cholangiocarcinoma cells cocultured with platelet is increased. Based on the above research, it suggested that the intrahepatic cholangiocarcinoma cells had a cross-talk relationship with platelet through aPKC-ι signaling. For a further molecular mechanism, the applicant use the cytokine antibody microarray technology to find that the intrahepatic cholangiocarcinoma cells with high level of aPKC-ι could secret more PDGF-BB, and exogenous PDGF-BB could prompte platelet activation. But the PDGF-BB was mainly from activated platelets, and our research found the PDGF-BB could increase the expression of aPKC-ι and N-cadherin in intrahepatic cholangiocarcinoma cells. Therefore, the applicant propose a hypothesis that aPKC-ι/PDGF-BB regulated the cross-talk bi-directionally between the intrahepatic cholangiocarcinoma cells and platelet, then affect the process of EMT in tumor and the platelet activation. This project aim to elucidate the regulatory mechanism of invasion and metastasis in intrahepatic cholangiocarcinoma from a new perspective.

上皮-间质细胞转化（EMT）是胆管癌侵袭转移的核心环节，申请者前期研究表明aPKC-ι诱导胆管癌EMT。申请者近期发现，高表达aPKC-ι的肝内胆管癌细胞可促进血小板活化，而与血小板共培养后肝内胆管癌细胞aPKC-ι的表达显著上调。上述研究提示肝内胆管癌细胞可能通过aPKC-ι信号通路与血小板发生cross-talk。为了进一步明确分子机制，申请者利用细胞因子抗体芯片技术发现高表达aPKC-ι的肝内胆管癌细胞显著分泌PDGF-BB，且外源性PDGF-BB可诱导血小板活化。但PDGF-BB在体内主要来源于活化血小板，且申请者发现PDGF-BB可诱导肝内胆管癌细胞aPKC-ι、N-cadherin表达。申请者因此提出假说，aPKC-ι/PDGF-BB双向调控肝内胆管癌细胞与血小板间cross-talk,进而影响肿瘤EMT及血小板活化。本项目有望从新的角度阐明肝内胆管癌侵袭转移的调控机制。

肝内胆管癌是源于胆管上皮恶性肿瘤，恶性程度高，预后较差。肿瘤侵袭转移可以概括为一个复杂的多因素多步骤级联反应过程，而癌细胞与非肿瘤细胞之间的交联互作关系及其在调控癌变过程中发挥着重要作用。越来越多研究表明，进入到微血管系统的肿瘤细胞与血小板之间存在相互调控关系且已成为肿瘤恶性进展的关键因素。本项目通过研究发现，肝内胆管癌患者外周血血小板计数水平、血小板活化水平以及肿瘤微环境中血小板浸润水平都高于健康人，并可以作为肝内胆管癌患者预后因素。活化的血小板可以促进肝内胆管癌细胞发生EMT化，并促进胆管癌细胞的增殖、侵袭迁移等生物学功能。为进一步探索血小板调控肝内胆管细胞侵袭转移的生物学机制，我们应用蛋白质琥珀酰化修饰组学技术筛选发现TKT及其赖氨酸修饰K319位点是血小板促进胆管癌细胞进展的关键因素。进一步研究发现，TKT促进胆管癌细胞EMT、增殖和侵袭转移，而模拟高琥珀酰化修饰状态的TKT-K319E的胆管癌细胞表现出更强的恶性侵袭能力。此外，高琥珀酰化修饰状态下的TKT-K319E胆管癌细胞的TKT酶活性升高，并促进糖酵解进而影响胆管癌细胞的侵袭转移能力。而TKT-319ksuc表达在胆管癌组织中也显著增高，且与患者临床病理特征和预后不佳相关。此外，我们研究还发现高琥珀酰化修饰的的TKT-K319E胆管癌细胞可以促进血小板活化。综上所述，活化的血小板可以使胆管癌细胞发生琥珀酰化，而高琥珀酰化修饰但胆管癌细胞也可以促进血小板活化，二者交联互作形成cross-talk促进肝内胆管癌细胞侵袭转移。本实验揭示了血小板与癌细胞间交互促进肿瘤侵袭转移的新机制，提示我们TKT琥珀酰化可以作为治疗靶点，为肝内胆管癌治疗提供新策略。