芫花提取物PFP2降解IAP遏制耐药肿瘤细胞免疫逃逸的功能机制研究

Platinum resistance to lung cancer is the key cause of treatment failure and endangering patients' lives. To reveal the mechanism of cisplatin resistance and explore coping strategies is imminent. We have previously screened Daphne genkwa extract PFP2 with the functions of strengthening the body resistance to eliminate pathogenic factors and reversing drug resistance, it can degrade tumor cells themselves "disguised" protein IAP by the endosomal-lysosomal pathway and enhance "phagocytosis" function of macrophages. However, the mechanism remains unclear. Protein profile analysis showed that USP2, IAP and RagA exist "interaction" in tumor cell endosome. Further evidence suggested that PFP2 inhibits RagA de-ubiquitination, the de-ubiquitination enzyme of RagA is USP2. Molecular docking analysis showed that PFP2 may bind to USP2. The project proposes a hypothesis that PFP2 leads to the inactivation of USP2 de-ubiquitination, which subsequently activates the endosome-lysosomal pathway to degrade IAP and eventually suppress the anti-phagocytosis signal. Multiple techniques including protein binding experiments and deletion mutation will be applied to decipher the impact of PFP2 on ubiquitination modification involved in degradation of IAP, elucidate mechanisms of PFP2 to combat the immune evasion of drug-resistant tumor cells, and investigate the reversal effect of PFP2 in combination with platinum on drug resistance. The project is expected to provide new ideas and targets for revealing the mechanism of drug resistance and strategies for reversing drug resistance.

肺癌铂类耐药是造成治疗失败和危及患者生命的关键原因，揭示顺铂耐药的机制并探索应对策略迫在眉睫。我们前期筛选出具有扶正去邪、逆转肿瘤耐药功效的芫花提取物PFP2，其可能通过内体-溶酶体途径降解癌细胞自身“伪装”蛋白IAP，增强巨噬细胞“吞噬”功能，但机制不清；蛋白质谱分析肿瘤细胞内体USP2、IAP和RagA存在“互作”，进一步证据表明PFP2抑制RagA去泛素化，RagA的去泛素化酶是USP2；分子对接分析PFP2可与USP2存在结合。本项目提出“PFP2导致USP2去泛素化功能失活，继而激活内体-溶酶体途径降解IAP，最终遏制抗吞噬信号”的科学假说。拟采用蛋白质结合实验、基因定点突变等技术明确PFP2影响泛素化修饰参与降解IAP，阐明PFP2对抗耐药肿瘤细胞免疫逃逸的作用机制，探讨PFP2联合铂类对肺癌耐药的逆转效应，结果可望为揭示肿瘤耐药机制及逆转铂类耐药的治疗策略提供新思路和靶点。

肺癌铂类耐药是造成治疗失败和危及患者生命的关键原因，揭示顺铂耐药的机制并探索应对策略迫在眉睫。本项目以中医药理论为指导，结合现代分子生物学技术，挖掘出具有抗肿瘤活性及遏制免疫逃逸功效的芫花提取物Pimelea factor P2（PFP2），旨在揭示PFP2通过内体-溶酶体途径阻断“抗吞噬”信号逆转肺癌顺铂耐药的机制。研究发现，PFP2通过内体-溶酶体途径降解癌细胞自身“伪装”蛋白IAP（CD47），初步阐明了PFP2抑制RagA介导的IAP（CD47）稳定表达调控机制，证实PFP2结合USP2抑制RagA去泛素化，增强巨噬细胞的“吞噬”效应；探讨PFP2联合铂类药物在肺癌耐药治疗中的逆转效应及临床学意义；证实HMMR介导IAP（CD47）表达并抑制巨噬细胞的体内吞噬功能；证实SIRPγ通过YAP信号介导细胞因子分泌维持IAP（CD47）表达。本研究初步明确了顺铂耐药与肿瘤细胞免疫的关系，为揭示肺癌顺铂耐药发生演进的新机制及探索中药联合的肺癌免疫治疗策略提供新思路、新靶点。