aPKC-ι介导的胆管癌细胞极化对Treg细胞功能影响及侵袭转移的调控研究

Polarization motion is a core aspect of the invasion and metastasis in tumor initiation stage, meanwhile tumor immunity is an important factor affecting the invasion and metastasis of cancer cells. In the previous studies, the applicant firstly found that as a key polarization regulatory protein, aPKC-ι was overexpressed in cholangiocarcinoma and metastatic tissue, and preliminarily found that aPKC-ι expression levels were correlate with Treg cell infiltration and TGF-β1 and IL-2 levels. Furthermore, the expression levels of aPKC-ι were also associated with Snail1. Based on the above statements, we infer that cholangiocarcinoma cell polarization regulated by aPKC-ι/Snail1 signaling pathway affect Treg cell function, and mediates cancer cell invasion and metastasis. This project intends to establish the polarization movement experimental model of the invasion and metastasis of cholangiocarcinoma cells in vitro, studies the regulation of the polarization state of cholangiocarcinoma cells and its effects on Treg cell function when aPKC-ι overexpressed or inactived, investigates the molecular mechanisms of aPKC-ι regulating Snail1, and studies the effects on invasion and metastasis by interfering aPKC-ι/ Snail1 signaling pathway. The project is expected to clarify the molecular mechanisms on invasion and metastasis of cholangiocarcinoma cells from a new perspective and it suggests us a new therapeutic approach for the treatment of cholangiocarcinoma.

癌细胞极化运动是肿瘤侵袭转移起始阶段的核心环节，肿瘤免疫也是影响癌细胞侵袭转移的重要因素。申请者前期研究首次发现：胆管癌及其转移灶高表达aPKC-ι（调节细胞极化的关键蛋白）；并初步发现，aPKC-ι表达水平与Treg细胞浸润及免疫调节因子TGF-β1和IL-2水平增高相关；aPKC-ι表达水平也与Snail1表达相关。申请者据此推测：aPKC-ι/Snail1通路可通过调控胆管癌细胞极化影响Treg细胞功能，并介导癌细胞侵袭转移。本项目拟建立胆管癌细胞体外侵袭转移的极化运动实验模型，观察aPKC-ι高表达或失活对胆管癌细胞极化状态的调控作用，及对Treg细胞功能的影响；探讨aPKC-ι调控Snail1表达的分子机制；通过干扰aPKC-ι/Snail1通路，观察对胆管癌细胞侵袭转移的影响。本项目可望从新的角度阐明胆管癌细胞侵袭转移的分子机制，并为探索胆管癌治疗的新策略提供依据。

胆管癌是源于胆管上皮的高度恶性肿瘤，因其具有极强的侵袭和转移能力，导致胆管癌复发率和死亡率较高。癌细胞上皮-间质细胞转化（EMT）是肿瘤侵袭转移起始阶段的核心环节，肿瘤免疫也是影响癌细胞侵袭转移的重要因素。目前已知，在体内参与肿瘤EMT进程的网络通路及相关信号分子极其复杂，其中，非典型蛋白激酶C-ι（atypical Protein Kinase C-ι, aPKC-ι）在EMT进程中发挥了重要的调控作用。通过研究发现，aPKC-ι、Snail表达及浸润性免疫抑制细胞数量在胆管癌组织中显著增多，且与患者的预后不佳相关。体内外实验证实，aPKC-ι通过诱导Snail表达进而促进胆管癌EMT及免疫抑制，但免疫共沉淀实验表明aPKC-ι与Snail无直接结合的作用关系。为了进一步探索aPKC-ι调控Snail的分子机制，我们应用基于iTRAQ技术的定量磷酸化蛋白质组学分析，检测aPKC-ι的磷酸化底物，结合免疫共沉淀检测发现Sp1 Ser59位点是aPKC-ι的直接磷酸化底物。而Sp1及P-Sp1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。应用染色质免疫共沉淀技术发现Sp1 Ser59位点磷酸化通过促进Sp1与Snial启动子的结合进而诱导Snail表达。而Sp1 Ser59位点磷酸化也可以促进aPKC-ι/Snail诱导的胆管癌EMT及免疫抑制。上述研究表明，aPKC-ι/P-Sp1/Snail信号通路可诱导胆管癌EMT及免疫抑制，最终导致肿瘤侵袭转移。本实验揭示了胆管癌侵袭转移的新机制，提示我们aPKC-ι/P-Sp1/Snail可作为分子靶点，为胆管癌抗肿瘤治疗提供新的思路。