以YAP/TAZ为靶点的恶性胶质瘤基因治疗新策略及分子机制研究

The transcriptional co-activators YAP/TAZ can upregulate the transcriptional level of multiple molecules involved in pathophysiological process of tumors, including cell proliferation, anti-apoptosis, migration, invasion, radiochemotherapy resistance and angiogenesis, by binding to the TEAD family members; moreover, YAP/TAZ present specific overexpression and nuclear localization in gliomas. Based on our previous study work and latest research progress of gliomas, we design a novel strategy of malignant glioma gene therapy by targeting YAP/TAZ. In this study, we try to use the expressed fusion molecules of TEAD binding domain located in YAP/TAZ amino terminals and protein transduction domain, to block the binding of YAP/TAZ to TEAD family members, then downregulate the expression of multiple tumor-associated molecules whose transcription depending on YAP/TAZ, further realize the inhibiting tumor effect by interfering multiple biological behavior of malignant gliomas, and evaluate systematically its treatment action, security and related molecular mechanism in vitro and in vivo. This strategy may open a new way of malignant glioma gene therapy, and its potential clinical application value may improve the therapeutic effect and elevate the control rate of malignant gliomas.

转录共激活因子YAP/TAZ能够依赖与TEAD家族成员结合的方式上调多种参与肿瘤细胞增殖、对抗凋亡、迁移、侵袭、放化疗抵抗以及血管形成等病理生理过程的分子转录水平，并且YAP/TAZ在胶质瘤中存在特异性高表达和细胞核定位。本项目根据以往的研究工作基础和胶质瘤的最新研究进展，设计了一种以YAP/TAZ为靶点的恶性胶质瘤基因治疗新策略，尝试使用表达YAP/TAZ氨基末端TEAD结合功能域及蛋白转导功能域的融合分子封闭YAP/TAZ与TEAD家族成员的结合，从而下调依赖YAP/TAZ转录的多个肿瘤相关分子的表达，以期通过同时干预恶性胶质瘤细胞的多种生物学行为实现抑瘤效应，并通过体外实验和动物实验系统探讨其对恶性胶质瘤的治疗作用、安全性和相关分子机制。该策略的建立将为恶性胶质瘤的基因治疗开启新思路，其潜在的临床应用前景将有助于改善恶性胶质瘤的临床疗效和提高肿瘤控制率。

YAPBD/TAZBD调节YAP/TAZ与TEAD蛋白互作，控制YAP/TAZ与TEAD复合物能否成功形成。本研究中我们以YAP/TAZ为靶点探讨了YAPBD、TAZBD在体内和体外对恶性胶质瘤细胞的影响。该研究通过使用pull down技术，观察到YAP/TAZ与TEAD在U87和U251细胞中能够互作，与空质粒载体组比较，YAP/TAZ过表达稳转细胞系中YAPBD/TAZBD过表达后，可以抑制YAP/TAZ和TEAD的结合，从而阻断YAP-TEAD、TAZ-TEAD复合物的形成，导致克隆形成率降低，细胞周期S期变短，细胞增殖减少，细胞周期调节蛋白Myc和Cylin D1表达降低，p21表达上调，抗凋亡蛋白BCL-2、BCL-XL及Survivin表达降低，促进肿瘤细胞的凋亡。荷瘤小鼠接种稳转YAPBD/TAZBD的U251细胞后，显著抑制了体内肿瘤的增长并促进瘤体凋亡。本研究首次表明YAPBD/TAZBD可抑制YAP/TAZ和TEAD的结合，进而阻断YAP-TEAD、TAZ-TEAD复合物的形成，在体外抑制胶质瘤细胞增殖，促进胶质瘤细胞发生凋亡，在体内显著抑制瘤体增殖，促进瘤体凋亡，对恶性胶质瘤具有高效的抑制和治疗作用。