全基因组CRISPR/Cas9文库筛选洋橄榄叶素抑制自噬的靶基因

The survival rate of women with ovarian cancer has changed little since platinum-based treatment was introduced more than 30 years ago.Dysfunction of autophagy has been associated with a variety of human diseases，especially cancer. Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. We screened the North China Pharmaceutical Group Corporation’s pure compound library of microbial origin and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin eventually decreases cell viability, furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis. However, the mechanism(s) behind the elaiophylin-induced autophagy inhibition is poorly understood. In this study, we will perform a genome-wide screen using a CRISPR/Cas9 library to identify targets for elaiophylin-induced autophagy inhibition, verify its effectiveness, and explore the mechanism(s) of target(s) involving the elaiophylin-induced autophagy inhibition. This study will try to reveal the mechanism(s) behind the antitumor effect of elaiophylin, and provide directions for the development of new autophagy inhibitors.

卵巢癌是一类复杂的异质性肿瘤，尽管诊断和治疗不断进步，但患者5年生存率仍然不超过40%。细胞自噬在肿瘤的发生发展中起着重要作用，靶向自噬通路的抗肿瘤药物研发被视为一种很有潜力的新策略。申请者通过对华北制药集团有限公司（NCPC）微生物来源的纯化合物库进行系统筛选，发现洋橄榄叶素（elaiophylin）能够促进自噬体的累积，但是会在自噬过程的晚期阶段阻断自噬流，并最终抑制卵巢癌细胞的自噬。这种新型自噬抑制剂会最终导致细胞死亡，并在体内发挥单药抗肿瘤活性，但是其抑制自噬的具体作用靶点尚未阐明。因此，本项目拟通过CRISPR/Cas9文库筛选出elaiophylin抑制自噬的靶点并验证其有效性，阐明靶点参与elaiophylin抑制自噬的分子机制。本研究将进一步揭示elaiophylin抗肿瘤活性的具体机制，而且可为研发新的自噬抑制剂提供指导。

申请者通过对华北制药集团有限公司（NCPC）微生物来源的纯化合物库进行系统筛选，发现洋橄榄叶素（elaiophylin）能够促进自噬体的累积，但是会在自噬过程的晚期阶段阻断自噬流，并最终抑制卵巢癌细胞的自噬。但是其抑制自噬的具体作用靶点尚未阐明。寻找elaiophylin抑制自噬的具体靶点，将会进一步扩充elaiophylin的应用，而且可为研发新的自噬抑制剂提供指导。在本项目研究中，我们通过过CRISPR/Cas9文库筛选确定了一种磷脂酶C---PLCG2作为潜在的研究靶点。既往研究报道显示，细胞内钙离子信号与细胞自噬密切相关，钙离子信号可正向或负向调控细胞自噬过程，同时，PLCG2作为磷脂酶C（PLC）家族的一员，参与调控细胞内钙离子水平。本项目研究显示CRISPR/Cas9敲除PLCG2可减弱elaiophylin抑制自噬的效应。同时，我们通过流式细胞学的方法（FACS）显示elaiophylin可显著升高细胞内钙离子浓度。然而，敲除PLCG2可明显降低elaiophylin导致的细胞内钙离子浓度升高。总结来说，本项目通过CRISPR/Cas9文库筛选，确定了elaiophylin抑制自噬的可能靶点为PLCG2，并验证了其有效性，阐明了PLCG2可能通过调控细胞内钙离子浓度参与elaiophylin抑制自噬的过程。本研究进一步揭示了elaiophylin抑制自噬的具体机制，而且可为研发新的自噬抑制剂提供指导，具有一定的应用前景。