通过Clathrin/AP2途径介导内皮细胞Occludin自噬性降解以靶向开放胶质瘤血肿瘤屏障的机制研究

胶质母细胞瘤（GBM）中的血肿瘤屏障会阻碍多数化疗药物渗透并杀伤侵袭的肿瘤细胞，是造成GBM化疗患者预后不佳的重要原因。前期工作中，利用斑马鱼GBM模型高通量筛选小分子抑制剂库，发现自噬激动剂靶向开放血肿瘤屏障；通过转录组和生物信息学分析，发现肿瘤内皮细胞自噬增加导致紧密连接蛋白Occludin的自噬性降解来开放血肿瘤屏障；通过蛋白免疫共沉淀实验，发现在肿瘤内皮细胞中Occludin与衔接蛋白AP2结合增加，可能通过网格蛋白Clathrin介导的内吞转入自噬体内发生降解。在此基础上，本项目拟阐明GBM中自噬促进血肿瘤屏障开放的分子机制，明确Clathrin/AP2-Occludin途径在内皮细胞血肿瘤屏障开放中的生物学功能，评价自噬激动剂对GBM化疗效果的影响。本项目的实施进一步明确血管内皮细胞自噬靶向开放血肿瘤屏障的分子机制，为开发GBM潜在治疗药物以及探寻新型治疗策略提供理论依据。

The blood-tumor barrier in glioblastoma (GBM) prevent most chemotherapeutic drugs from penetrating and killing infiltrated tumor cells, which results in poor prognosis of GBM patients treated with chemotherapeutic drugs. Our preliminary work showed that autophagy agonists selectively disrupted the blood-tumor barrier (BTB) in GBM tumors through high-throughput drug screening in zebrafish GBM models. Transcriptome sequencing suggested that autophagy-mediated Occludin degradation might attribute to elevated levels of autophagy in tumor associated endothelial cells (ECs) and lead to breakdown of the blood-tumor barrier. The co-immunoprecipitation studies indicated the elevated binding of Occludin with adaptor protein AP2 in tumor associated ECs. The complex of Occludin and AP2 that might be endocytosed into autophagosome through Clathrin-mediated Occludin endocytosis was degraded by autophagy-mediated degradation. In this project, we will clarify the molecular mechanism of ECs autophagy in breakdown of blood-tumor barrier and the biological function of the Clathrin/AP2-Occludin pathway in selectively disrupting the blood-tumor barrier. In addition, we will evaluate the therapeutic effect of autophagy agonists combined with chemotherapy in GBM. Our study will provide new insight into the role of autophagy in cerebrovascular endothelial cells in breakdown of blood-tumor barrier and provide theoretical basis for development of potential therapeutic drugs or strategies for GBM.