Nrf2-ARE通路活化介导铁过载及抑制铁死亡参与卵巢癌顺铂耐药的机制研究

Ovarian cancer is the most lethal gynecological malignancies. Cisplatin resistance has been a critical problem that associated with poor prognosis. Recent studies have shown that iron excess is one of key features of carcinoma, and ferroptosis is associated with chemoresistance. In our previous studies, we have shown that the activation of Nrf2-ARE signaling pathway plays an important role in ovarian cancer cisplatin resistance ,further studies showed that there was crosstalk between Nrf2 and SLC40A1 (FPN1). In chemoresistant cells the iron was overloaded and which promoted cisplatin resistance. Disturbing iron metabolism, particularly iron deficiency will decrease cisplatin sensitivity. Whatsmore, ferroptosis is also found to be associated with ovarian cisplatin resistance. However, the underling mechanism of chemoresistance induced by cross talking between Nrf2-ARE signaling pathway and iron excess is still unclear. Here, we propose that in ovarian chemoresistant cells, the Nrf2-ARE signaling pathway is activated, resulting in iron excess, which in return decreases the over production of ROS and introduces ferroptosis resistance, thus acquires chemoresistant survival. In this study, we are going to explore this underling mechanism by using both in vitro- and in vivo- evaluating system, the potential clinical value by targeting ferroptosis-associated pathway in the treatment of cisplatin resistance in ovarian cancer will also be investigated.

卵巢癌是致死率最高的妇科恶性肿瘤之一，顺铂耐药是限制卵巢癌疗效的关键问题。近年来研究发现铁过载是肿瘤的重要特征之一，与之相应的铁死亡(ferroptosis)也与肿瘤化疗耐药相关。我们前期研究发现Nrf2-ARE通路活化参与了卵巢癌顺铂耐药；近期研究发现Nr2f可直接抑制膜铁转运蛋白SLC40A1(FPN1)的转录，引起卵巢癌细胞内铁过载，并促进顺铂耐药；而干扰铁代谢状态可以改变细胞对顺铂的敏感性；进一步研究发现卵巢癌耐药细胞中存在铁死亡抵抗。基于此，我们提出科学假设：卵巢癌耐药细胞中Nrf2-ARE通路活化后干扰铁代谢关键步骤促进铁过载、后者抑制活性氧（ROS）过量产生、诱导细胞抵抗铁死亡、进而获得耐药性生存。本课题拟围绕上述假设，利用铁代谢体内、外评价体系展开研究，解析上述机制，并探索靶向铁死亡通路的治疗在卵巢癌顺铂耐药中的潜在临床价值，对提高卵巢癌疗效具有潜在科学意义。

本项目组前期研究已发现Nrf2-ARE信号通路活化在卵巢癌顺铂耐药中起重要作用，与铁过载和铁死亡相关。本课题着眼于这一现象背后的相关机制研究。经一系列体内、体外实验研究发现，HO-1作为代表性的Nrf2下游ARE信号分子之一，Nrf2/HO-1信号通路在顺铂耐药卵巢癌细胞中异常激活，HO-1通过调控铁自噬选择性货物载体NCOA4改变细胞内铁自噬水平，调控不稳定铁池中Fe2+含量以及铁储存蛋白Ferritin之间的动态平衡，进而调节顺铂诱导的铁死亡，最终逆转卵巢癌细胞对顺铂的耐药性。该结论与本课题假说相符。随后，我们通过TCGA与GEO数据库展开生物信息学分析，进一步评价铁死亡相关基因在卵巢癌诊断、预后评估以及治疗中的转化意义。我们发现，由10个铁死亡相关基因（HIC1, ACSF2, MUC1等）组成的基因特征集对卵巢癌具有极高的诊断价值。同时，由3个铁死亡相关基因（HIC1、LPCAT3以及DUOX1）构成的预后基因特征集对卵巢癌具有较好的预后预测作用，基于此构建的风险评分模型将卵巢癌患者分为高、低风险组。GSEA分析表明，肿瘤微环境中M2巨噬细胞浸润等免疫反应及免疫相关通路在高风险组富集，而且低风险组患者对化疗及免疫治疗表现出更好的反应性。在卵巢癌中，这3个预后特征基因均显示出与Nrf2相关。此外，我们还发现顺铂在卵巢癌细胞中可诱导发生铁死亡，铁死亡诱导剂erastin 通过抑制xCT 诱导卵巢癌细胞发生的铁死亡，erastin 联合顺铂给药可提高卵巢癌细胞对顺铂的敏感性。而不同卵巢癌细胞系对铁死亡敏感性差异可能与KRAS 突变相关。体内实验显示，erastin 联合顺铂可抑制人卵巢癌荷瘤小鼠皮下移植瘤的增殖，但并不增加顺铂的毒副作用。本项目研究结果为临床克服卵巢癌铂耐药难题提供了靶向HO-1、改变铁代谢、调控铁死亡等潜在的临床治疗措施，为改善卵巢癌患者的预后提供了理论基础。