新颖USP7抑制剂抑制肿瘤干细胞活性和作用机制研究

The cancer-stem-cell (CSC) model indicates that anti-cancer therapies targeting the cancer stem cells would ultimately improve conventional cancer treatments like surgery, radiotherapy and chemotherapy. Accumulating evidences have revealed a critical role for Wnt signaling in maintaining stem-cell like properties of CSCs and its self-renewal, and thus suggesting that this signaling is a promising target for treating cancers through inhibiting CSCs. Given that Wnt signaling is stringently regulated by ubiquitin-proteasome system, our recent work revealed that USP7 played a positive role in Wnt pathway through deubiquitinating and stabilizing β-catenin and validated that USP7 could be a novel drug target for anti-cancer agents targeting Wnt signaling. Taking the advantage of the strong research capability on natural products of the state key laboratory of phytochemistry and the west plant resources, we screened the library of natural compounds and derivatives and discovered β-carboline molecule Z86 as a novel inhibitor of USP7 activity. Preliminary study suggested that Z86 antagonized Wnt signaling via destabilizing β-catenin and inhibited self-renewal of CSCs. In this project, with chemical biology and pharmacology techniques, the molecular mechanisms and the structure-activity relationship of the β-carbolines as USP7 inhibitors will be investigated. Further we will elucidate molecular mechanisms by which Z86 regulating Wnt signaling and perform comprehensive pharmacological investigation on the inhibitory effect of Z86 on cancer stem cells. Especially, we will explore the roles of USP7 in the maintenance of cancer stem cells, in order to provide theoretical basis for the application of USP7 inhibitors as valuable candidate antitumor agents targeting cancer stem cells.

靶向肿瘤干细胞的治疗策略是改善传统肿瘤治疗效果的有效途径。Wnt信号通路是肿瘤干细胞自我更新和干性维持的关键通路，泛素蛋白酶体系统在Wnt信号通路调控中发挥重要作用。我们前期研究发现泛素特异性蛋白酶USP7通过稳定β-catenin正向调控Wnt信号通路，为靶向该信号通路的抗肿瘤药物提供了新的作用靶点。依托国家重点实验室的天然产物研究优势，我们对天然产物及衍生物库进行了USP7抑制剂筛选，发现β-咔啉化合物Z86具有明显活性。初步的研究表明Z86能促进β-catenin降解，抑制Wnt信号通路，显著抑制肿瘤干细胞自我更新，极具研究价值和开发潜力。本项目拟利用化学生物学和药理学手段，解析β-咔啉化合物作为新颖USP7抑制剂的分子机制和构效关系，阐明Z86调控Wnt信号通路的分子机制，系统评价Z86抑制肿瘤干细胞的药效和药理，为USP7抑制剂在靶向肿瘤干细胞药物中的可能应用提供理论依据。

肿瘤干细胞与肿瘤的发生，侵袭，复发和治疗抗性密切相关，Wnt/β-catenin信号通路是调控肿瘤干细胞自我更新和干性维持的关键通路。泛素蛋白酶体系统在Wnt信号通路调控中发挥重要作用，基于泛素蛋白酶体的Wnt信号通路及肿瘤干细胞调控和新颖抑制剂的发现对于靶向肿瘤干细胞药物的开发具有重要意义。泛素特异性蛋白酶USP7能够通过调控包括Wnt/β-catenin在内的多个关键肿瘤相关信号通路，促进肿瘤的发生发展。本项目基于构建的USP7抑制剂高通量筛选模型，筛选获得了系列活性化合物，阐明了新颖活性分子β-咔啉类化合物Z86和天然倍半萜内酯类化合物小白菊内酯（Parthenolide, PTL）靶向USP7的作用机制，并评价了其体内外肿瘤及肿瘤干细胞抑制活性和药理药效。利用b-VME/Ub-PA探针标记、细胞热转变分析以及表面等离子共振技术等研究发现Z86可以有效抑制USP7及其他去泛素化酶的活性，促进β-catenin的泛素化降解，通过抑制wnt信号通路活性抑制乳腺肿瘤干细胞的干性以及基于乳腺癌干细胞的动物体内的肿瘤形成、转移及化疗药物紫杉醇治疗后的肿瘤复发。而PTL能够直接结合USP7，通过选择抑制USP7的活性促进β-catenin的泛素化和降解，抑制Wnt通路活性以及结直肠肿瘤及肿瘤干细胞细胞增殖。构效关系研究表明α-亚甲基-γ-丁内酯是倍半萜内酯类化合物抑制USP7的活性基团。本项目对于小白菊内酯和Z86靶向USP7抑制Wnt/β-catenin信号通路以及肿瘤干细胞的发现，不仅丰富了Z86和PTL的抗肿瘤作用机制，而且还为USP7抑制剂的发现和药物研究提供了新颖的骨架类型，为靶向肿瘤干细胞的药物研究和发现奠定了科学基础。.此外，我们系统探索了多种去泛素化酶在肿瘤干细胞调控中的作用和分子机制，发现USP47在调控结直肠癌干细胞的干性维持和自我更新中发挥重要作用，而PTL同样作为USP47的抑制剂具有靶向结直肠癌干细胞的肿瘤治疗潜力，表明USP47有望作为结直肠癌干细胞的治疗靶点