基于YAP/TAZ-TEAD三元复合物介导Hippo-YAP信号通路的冬凌草素抗肿瘤分子机制研究

Cancer is one of the top threats to people's health. Currently, the idea of hunting for new lead compounds from natural products on the basis of new targets is widely accepted in the world. Hippo-YAP pathway is a recently-found novel anti-tumor target, however, studies on natural lead compounds which can affect this pathway are scarce. Oridonin is one of the most important compounds isolated from Rabdosia rubescens (also known as Donglingcao) with anti-tumor activity. However, its mechanisms remain to be elucidated in depth and systematically. Further, its effect on the anti-tumor new target, Hippo-YAP pathway, is not clear. With regard to this, we carried out preliminary studies and found that oridonin could inhibit YAP's nuclear translocation to nucleus and disturb the YAP-TEAD interaction significantly. These findings suggest that it may exert its anti-tumor effect through interference with the YAP/TAZ-TEAD complex. Thus, we plan to utilize immunological techniques and knock-out mice to carry out systematic and deep studies at levels of in vivo animals, organs (tissues), cells and molecules and try to clarify its novel anti-tumor mechanisms from aspects of proteins’ nuclear localization, interaction between small molecules and proteins and genetic editing. This work has an important significance for the establishment of screening platform centering on Hippo-YAP pathway and the discovery of some new lead compounds. In addition, it will lay a strong foundation for new preventive and therapeutic strategies with drugs focusing on the Hippo-YAP pathway.

肿瘤是威胁人类健康的罪魁祸首，目前，以新靶点为线索，从天然产物中寻找药物先导物，正在全球形成共识。Hippo-YAP通路是近年发现的抗肿瘤新靶点，但天然先导物研究仍十分匮乏。冬凌草素是抗肿瘤中药冬凌草中的重要成分，其抗肿瘤作用引人注目，但其作用机制还缺乏深入而系统的研究，同时其对于肿瘤新靶点Hippo-YAP通路的作用尚不清楚。我们前期工作发现冬凌草甲素可抑制YAP核移位和YAP-TEAD的相互作用，提示其抗肿瘤机制可能与YAP/TAZ-TEAD复合物有关。据此，本项目拟从整体动物-器官(组织)-细胞-分子水平利用免疫学方法和基因敲除小鼠多层次开展系统深入研究，试图从蛋白细胞定位、小分子与蛋白相互作用、基因编辑等方面揭示其抗肿瘤的新分子机制。本研究对于建立以Hippo-YAP通路为核心的筛选平台及发现相关天然先导物具有重要意义，并将为形成以Hippo-YAP为核心的药物防治新策略奠定基础。

当今，癌症严重威胁人类健康，世界各国癌症发病率在不断上升，其死亡率已超过或接近心脑血管疾病，成为第一或第二位死亡原因。在跨入21世纪后，抗癌中药正日益受到全球医药界的关注，面向天然产物，寻找高效安全的抗癌药物，正在形成热潮。近年的研究表明，Hippo-YAP与肿瘤的发生、发展有密切相关。因此，ippo-YAP 通路已成为治疗肿瘤的新靶点，对它的干预已成为肿瘤治疗的新策略。本项目研究发现了包括冬凌草素在内的数种天然小分子化合物能够通过Hippo-YAP通路发挥抗肿瘤作用。研究发现：化合物能够抑制测试肿瘤细胞的增殖、迁移、侵袭和克隆形成，且抑制效果呈剂量依赖性；qRT-PCR结果表明YAP/TEAD及其下游靶基因CTGF、CYR61的mRNA水平均有所降低，Western blot结果显示其蛋白表达水平也降低，而p-YAP水平升高。同时，免疫荧光实验表明化合物使细胞核中的YAP减少，细胞质中YAP增加，p-YAP水平升高。YAP过表达或敲低可影响肿瘤细胞对化合物的敏感性；免疫共沉淀结果显示化合物影响了YAP与TEAD的结合，紫外吸收和荧光光谱提示化合物能够与YAP蛋白结合形成复合物，改变其分子构象；体内实验显示，肝脏特异性 Nf2 基因敲除小鼠发生肝肿大、肝硬化、腹水和黄疸，逐步发展为肝硬化、肝癌；化合物处理后能抑制胚胎期敲除鼠的胆管增生，还能使1-12月龄敲除鼠肝脏指数下降，血清 ALT、AST 和 TBIL 水平降低，胆管增生与胶原沉积减轻，肝脏细胞增殖减少、凋亡增加，肝组织 P-LATS1、P-YAP 蛋白表达水平升高，YAP 蛋白表达水平降低，Ctgf、Cyr61、Epcam 和 Opn 基因 mRNA 水平降低，肿瘤发生减少。以上结果提示，化合物可通过调节 Hippo-YAP信号通路发挥抗肿瘤作用，本研究为临床相关肿瘤的治疗提供了新的干预策略，并为研发以Hippo-YAP为靶点的新型抗肿瘤药物提供了先导物和实验基础。