JNK/P62信号通路在耐药绒癌中的保护作用及其机制研究

Gestational trophoblastic neoplasia (GTN) is one of the most highly curable forms of adult malignancy, largely due to the exceptional sensitivity of this tumor to chemotherapy. However, approximately 20% of patients with multidrug resistance will still fail treatment and die. This is emphasizes the necessity to find a new approach for the treatment of these patients with high risk. According to our previous investigation in the study of drug-resistance choriocarcinoma, we recognized that endoplasmic reticulum (ER) stress and autophagy might be associated with drug resistance of choriocarcinoma, which was not reported in the published literatures. We also found that the JNK/P62 signal pathway upregulated in drug resistance choriocarcinoma cell lines. We presumed that JNK/P62 signal pathway was related to drug resistance of choriocarcinoma. The mechanism of JNK/P62 pathway is involved in regulating endoplasmic reticulum stress induced apoptosis to autophagy switch. Therefore, the effect of autophagy will be investigated in drug-resistant choriocarcinoma by inhabitation of autophagy and apoptosis in our study. Then we would explore the mechanism of JNK/P62 signal pathway both in vitro and in vivo, which might play the protective role via attenuating ER stress-induced apoptosis and boosting autophagy switch. We think that we are presenting an attractive stand-alone anticancer strategy as well as an opportunity to enhance the efficacy of current chemotherapeutic agents in drug-resistant choriocarcinoma.

化疗是治疗恶性滋养细胞肿瘤有效的方法，然而对化疗耐药仍是目前恶性滋养细胞肿瘤临床治疗的主要瓶颈，约20%的患者最终因化疗耐药而死亡。本课题组前期工作中，我们发现绒癌耐药与内质网应激及自噬密切相关，但是绒癌耐药和内质网应激及自噬的关系国内外迄今无相关报道。预实验中我们观察到耐药绒癌JNK/P62信号通路上调，我们推测该通路可能参与耐药绒癌的发病机制，其机制涉及内质网应激介导的凋亡和自噬。为证实这个假说，该课题拟通过抑制自噬和凋亡的方法明确明确自噬在耐药绒癌中的作用，从细胞水平和动物水平进一步研究JNK/P62信号通路对内质网应激介导凋亡和自噬的调节作用和调控途径，探讨绒癌耐药的发生机制，为耐药绒癌的靶向治疗提供理论依据。

由于对化疗药物的高度敏感，妊娠滋养细胞肿瘤（GTN）已成为目前临床治愈率最高的实体瘤，然而全世界仍有约20%的恶性滋养细胞肿瘤患者死亡，耐药绒癌的治疗一直是GTN临床治疗的难题，是导致GTN患者死亡的重要原因。本课题组前期工作中发现，绒癌耐药与内质网应激及自噬密切相关，并观察到耐药绒癌细胞中ROS介导的JNK/P62通路的上调，遂开展了一系列JNK/P62信号通路对内质网应激介导凋亡和自噬的研究。. 研究结果显示：首先，化疗药物可以诱导耐药绒癌细胞产生保护性的自噬，药物诱导后可以在电镜下观察到自噬小体的产生，采用免疫印迹法可检测到细胞内自噬蛋白如LC3的表达水平升高。相反，抑制自噬或者沉默自噬相关基因可以增加耐药绒癌对药物的敏感性，诱导凋亡产生。其次，上述过程是由ERS介导凋亡向UPR介导自噬的转换（Switch）来实现的，结果表明甲氨喋呤药物诱导后JEG-3/MTXR耐药绒癌对ERS介导凋亡不敏感，导致caspase依赖凋亡的抵抗，而药物可以诱导耐药绒癌UPR介导自噬通路PERK-eIF2α-ATF4-LAMP3的激活，即实现了凋亡前信号向生存前信号的转换。再次，耐药绒癌细胞中观察到ROS介导的JNK/P62通路的激活，亲代和耐药绒癌细胞中均检测到了活性氧的变化，且耐药绒癌细胞中活性氧浓度较高。药物诱导下，耐药绒癌细胞中的P62 mRNA和蛋白水平均逐渐升高，而JNK的抑制剂可以抑制P62的转录和蛋白表达。ROS的抑制剂MnTMPyP可以抑制JNK的磷酸化，同时抑制P62的表达。故耐药绒癌细胞中JNK/P62通路的激活可由ROS介导。最后，P62有助于凋亡向自噬的转换，P62有助于降调GADD153的表达，参与ERS介导的凋亡抵抗。. 本项目对自噬、内质网应激、绒癌耐药三者的关系进行了深入的研究和讨论，系统阐述了ROS介导的JNK/P62通路通过诱导ERS介导的凋亡向UPR介导自噬的转换来参与绒癌细胞的耐药。目前，美国国立癌症研究所已经开始应用hydroxychloroquine（自噬抑制剂）治疗肿瘤的临床试验(http://clinicaltrials.gov)，我们的研究无疑为自噬及其相关通路作为潜在的绒癌治疗靶点提供了坚实的理论基础，具有重要的临床转化应用价值。