以酪氨酸磷酸酶为靶标的肝内胆管癌治疗策略及分子基础研究

Intrahepatic cholangiocarcinoma (ICC) is a treatment-refractory disease with a dismal outcome. Limited success in the clinical management and a persistent increase in the incidence world-wide have made ICC one of the most lethal and fastest growing malignancies. Understanding the driver molecular events of ICC and identifying new therapeutic targets are of great importance to achieve better patient outcome. In our previous publication, using whole exome sequencing, we have reported that pathways related to protein phosphorylation were the potential drivers in ICC, among which the protein tyrosine phosphatase (PTP) family was of crucial significance. However, due to the limited detection power and non-functional nature of exome sequencing, as well as the dense desmoplastic stroma of ICC, the full portrait of PTPs’ role in ICC remains elusive. In this study, we hypothesize that alterations in PTP family were the driver events in ICC and certain specific driver PTP gene(s) could be ideal target for ICC treatment. Using a large cohort of highly purified primary ICC cells, we will apply customized targeted sequencing to get detailed insights into the genetic profile of PTP family in ICC. Meanwhile, we aim to generate PTP knockout ICC cell lines by CRISPR/Cas-mediated genome editing to understand the phenotypic contribution of each PTP. Then, by combined analyses of the genetic profile and phenotypic screen data, the specific driver PTP(s) in ICC could be identified. We have previously constructed a large number of ICC patient derived cell lines (PDC) and patient derived xenografts (PDX). Herein, we will further test the effectiveness and feasibility of targeting the identified driver PTP(s) on PDCs in vitro and PDXs in vivo. Collectively, the findings of this study would provide insights into the optimum strategy for using targeted agents and thus providing the rationale for clinical trials in ICC.

肝内胆管癌（ICC）发病率持续上升、预后极差，化疗和靶向治疗几乎无药可用。鉴定ICC的驱动基因图谱，开发针对性的靶向治疗是当前的重点。借助外显子测序，申请人发现：“蛋白磷酸化”相关基因在ICC中起驱动性作用，而酪氨酸磷酸酶（PTP）家族更是其中的核心。由于外显子测序的灵敏度有限和非功能性、及ICC组织富含间质成分，制约了对PTP的深入研究。本项目以“PTP基因是ICC 的驱动基因和有效治疗靶点”为假设，以纯净的“原代癌细胞”为对象，利用高精度的平台—靶向捕获测序系统和CRISPR/Cas基因编辑技术，遵循“基因型和表型相结合”的原则，既精确灵敏的检测ICC中PTP变异图谱、又同时阐明其功能，以期鉴定出ICC中特定的“驱动性PTP”。接着，利用前期建立的“PDC和PDX模型（患者来源的ICC细胞系和移植瘤模型）”，阐明靶向该“驱动性PTP”的有效性，为ICC个体化靶向治疗提供借鉴和指导。

肝内胆管癌（Intrahepatic cholangiocarcinoma, ICC）是第二常见的肝脏恶性肿瘤，发病率持续上升、预后极差，仅10%患者可行根治性切除术，化疗和靶向治疗几乎无药可用。鉴定ICC的驱动基因图谱，开发针对性的靶向治疗是当前的重点。本项目借助临床样本、细胞实验、机制研究和动物模型，利用原代培养、外显子测序、药敏等研究技术，系统全面揭示了肝内胆管癌全基因组变异情况，研究了PTP家族中肝内胆管癌的表达谱，发现显著上调的30个（CDKN3、PTP4A1、PTPRM、SSH1等），显著下调的29个（包括PTPN5、EPM2A、CDC25A、DUSP1等）；成功鉴定了肝内胆管癌中多个功能性PTP 基因（如PTP4A1、SSH1、PTPN3、MAP3K4），并系统性的阐明其生物学功能、调控机制和临床意义；建立了一系列肝内胆管癌的PDC、PDO模型，用于后续治疗小分子的筛选和鉴定；系统揭示了肝内胆管癌的肿瘤内异质性、复杂的进化机制（分支进化、平行进化和克隆丢失等）及其对肿瘤进展和耐药的影响，提出了适应性治疗的临床策略。以第一或通讯作者共发表SCI论文14篇（其中5篇IF>10分，包括Cell Res、Gastroenterology、J Hepatol等知名杂志）、中文核心期刊3篇，申请获得国家发明专利2项（一种人肝内胆管癌细胞系及其用途, ZL 2015 1 0003158.9；用于研究人肝癌癌内异质性的细胞模型, ZL 2015 1 0003183.7），培养博士研究生4名。相关研究结果，项目负责人高强进一步申报并获得科研项目和人才计划6项，包括上海市教委曙光计划、上海市科委优秀学术带头人计划、上海市卫健委优秀学科带头人计划、上海市科委基础研究重点项目、国家自然科学基金重大研究计划和中美合作项目。项目执行期间，负责人高强入选教育部长江学者奖励计划特聘教授、教育部青年科学奖、药明康德生命化学研究奖、华夏医学科技一等奖（第九）和上海市科技进步一等奖等（第九）荣誉称号和奖励。

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