NDRG2调控胆管癌细胞铁死亡（Ferroptosis）抑制肿瘤生长及增敏化疗与靶向治疗的机制研究

Ferroptosis, as an iron-dependent form of cell death, was recently discovered and characterized by accumulation of lipid peroxide reactive oxygen species . Recent studies suggested that ferroptosis was associated with tumorigenesis and anti-cancer drug resistance. However, the role and mechanisms of ferroptosis in human cholangiocarcinoma (CCA) are not clear. Our previous study (Hepatology, 2016) indicated that NDRG2 was downregulated in CCA, and NDRG2 overexpression could inhibit tumor cell proliferation, induce cell senescence and cell cycle arrest, but had no significant effects on apoptosis, which indicates that NDRG2 may regulate other ways of cell death in CCA. We have also found that iron chelator Dp44mT could inhibit metastasis and upregulate NDRG2 expression in hepatocellular carcinoma, suggesting NDRG2 might play a role in the regulation of iron levels. We recently found that the tumor growth inhibition mediated by ferroptosis in CCA was significantly associated with NDRG2 and other ferroptosis regulators (such as Nrf2 and SOCS1). Based on this, we propose the hypothesis that NDRG2 inhibits tumor growth and increases the sensitivity of tumor cells to chemotherapy and targeted therapies via up regulating SCOS1/SLC7A11, down regulating Keap1/Nrf2, and regulating iron levels in human CCA. To verify this hypothesis, we will perform experiments using the methods of Western blot, PCR, lentivirus transfection, ROS assay and GSH assay etc. to investigate the role of NDRG2 in tumorigenesis and the sensitivity to chemotherapy and targeted therapies via ferroptosis in human CCA. The project will provide new therapeutic targets and interventions for human CCA from the perspective of NDRG2 and ferroptosis.

铁死亡是新发现的以细胞内脂质活性氧堆积为特征的的铁依赖性细胞死亡方式，研究表明其与肿瘤密切相关，然而其在胆管癌中的作用不清楚。我们前期发现抑癌基因NDRG2与LIF/miR-181c形成负反馈环路抑制胆管癌细胞增殖、诱导细胞衰老及细胞周期阻滞，但并不影响细胞凋亡（Hepatology, 2016），提示其可能通过诱导其它的细胞死亡方式起作用。我们预实验结果发现铁死亡介导的胆管癌生长抑制和化疗增敏与NDRG2及其它铁死亡调控因子（Nrf2、SOCS1等）有关。基于此，我们提出NDRG2可能通过上调SCOS1，同时抑制Nrf2以及影响铁水平促进铁死亡发生进而抑制胆管癌生长并增敏药物治疗的假设，并拟进一步通过免疫组化、PCR、慢病毒感染、铁水平、脂质活性氧水平检测等实验手段探讨NDRG2通过调控铁死亡对胆管癌生长及化疗与靶向治疗的影响，以期率先从铁死亡及NDRG2角度出发为胆管癌治疗寻找新策略。

胆管癌是恶性度极高的肿瘤，患者预后差，目前缺乏有效的治疗药物，寻找胆管癌肿瘤治疗新策略至关重要。研究表明诱导肿瘤细胞发生铁死亡可有效抑制肿瘤生长，目前已发现多种药物可以诱导铁死亡，然而SHR6390是否可以诱导铁死亡还未知。在本研究中，通过检测铁死亡的多种表型，我们发现Erastin和SHR6390可以分别诱导胆管癌细胞发生铁死亡，而GPX4的表达水平低者，对SHR6390更敏感。我们还发现与癌旁相比，GPX4在癌中高表达，且GPX4高表达的患者预后差。通过将SHR6390分别与铁死亡的诱导剂Erastin和RSL3联合，结果表明与单药相比，联合治疗可显著抑制胆管癌细胞的增殖。此外，在本项目资助下，我们还探究了铁死亡相关基因在结肠癌中的预后作用，发现铁死亡基因可协同年龄和肿瘤分期提高预测结肠癌患者预后的准确率；发现了一种新型的干扰素基因刺激因子（STING）异构体（pmSTING）并探讨其功能，发现pmSTING在小鼠免疫细胞的细胞表面表达且其C末端在胞外，其可直接感知细胞外cGAMP并激活TBK1/IRF3/IFN信号通路，该研究为cGAMP作为免疫递质的作用提供了深入的认识；我们还通过单细胞测序比较了胆系肿瘤中不同解剖亚型之间T细胞特征的异同点，发现胆系肿瘤中不同解剖亚型之间的T细胞数量和功能存在区别。以上研究为胆管癌和结肠癌的治疗和预后预测提供了新思路。