IAPs拮抗剂调控肿瘤细胞自噬介导坏死性凋亡的分子机理

Exploring the way of tumor cells death induced by anti-tumor drugs and its regulatory mechanism is critical for discovering long-term benefit of cancer treatment strategies. IAPs antagonists target both intrinsic and extrinsic apoptotic pathways, activate caspase, and promote apoptosis in specific subset of tumor cells. Our previous results demonstrated that, besides inducing apoptosis in cancer cells, IAPs antagonists could also induce autophagy and promote caspase-independent cell death. Whereas RIPK1 inhibitor Nec-1 or autophagy inhibitor 3-MA could block this effect. On consideration that IAPs participate in death receptor and caspase-independent necroptosis pathways, and autophagy protein p62 regulates the transition between apoptosis and necroptosis, we speculated that IAPs antagonists may regulate the expression of p62 and its interaction with necroptosome through inducing autophagy, and subsequently trigger necroptosis. However, how IAPs antagonists regulate this process remains unclear. In this project, we intended to systematically investigate the interaction between autophagy and necroptosis induced by IAPs antagonists and reveal the molecular mechanisms of this process. This study will provide new idea and strategy for the development of novel anti-tumor drugs and explore new ways for clinical cancer treatment.

探索抗肿瘤药物诱发的肿瘤细胞死亡方式及其调控机制对于发现长期获益的肿瘤治疗策略具有重要价值。IAPs拮抗剂靶向内外源细胞凋亡通路，激活caspase促进特定肿瘤细胞凋亡。课题组前期研究发现，除诱导肿瘤细胞凋亡，IAPs拮抗剂还能诱导自噬与caspase非依赖的细胞死亡，而RIPK1抑制剂Nec-1或自噬抑制剂3-MA能阻断这种效应。鉴于IAPs参与死亡受体和caspase非依赖坏死性凋亡通路，且自噬蛋白p62调控细胞凋亡和坏死性凋亡间的转换，我们推测IAPs拮抗剂可能通过诱导细胞自噬，调控p62表达水平及其与坏死性凋亡小体间的相互作用，继而促进坏死性凋亡的发生。本课题拟以进入临床的IAPs拮抗剂为工具药，系统研究IAPs拮抗剂诱导的细胞自噬与坏死性凋亡间的相互作用，以期揭示IAPs拮抗剂调控自噬介导坏死性凋亡的分子机理。阐明上述问题，将为抗肿瘤药物研发提供新思路，为肿瘤临床治疗探索新途径。

探索抗肿瘤药物诱发的肿瘤细胞死亡方式及其调控机制对于发现长期获益的肿瘤治疗策略具有重要价值。IAPs拮抗剂靶向内外源细胞凋亡通路，激活caspase促进特定肿瘤细胞凋亡。课题组前期研究发现，除诱导肿瘤细胞凋亡，IAPs拮抗剂还能诱导自噬与caspase非依赖的细胞死亡，而RIPK1抑制剂Nec-1或自噬抑制剂3-MA能阻断这种效应，然而其具体分子机制还不清楚。本研究以进入临床的IAPs拮抗剂GDC-0152、TL32711及我所合成的新型单体IAPs拮抗剂WX20120108为工具药，评价IAPs拮抗剂对肿瘤细胞自噬活性的影响，明确IAPs拮抗剂诱导肿瘤细胞死亡的方式，系统研究其调控肿瘤细胞自噬活性及诱导细胞死亡的分子机制，探索IAPs拮抗剂调控肿瘤细胞自噬与坏死性凋亡之间的相互作用。结果显示，IAPs拮抗剂能够增加LC3-II蛋白表达水平，促进细胞自噬标志分子LC3-II颗粒聚集，促进细胞自噬体形成及其与溶酶体融合，诱导肿瘤细胞自噬的发生。应用siRNA敲减靶IAPs分子，结果表明IAPs自噬诱导活性部分依赖于靶IAPs分子。自噬起始相关蛋白报告细胞系筛查及验证结果显示，WX20120108选择性激活Foxo3，促进其下游自噬起始相关靶基因Bnip3、Pik3c3、Atg4b和Atg5的表达。WX20120108能够促进细胞ROS释放，而catalase拮抗WX20120108诱导的Foxo3及LC3-II激活，表明WX20120108主要通过激活ROS-Foxo3通路诱导自噬的发生。评价IAPs拮抗剂诱导肿瘤细胞的死亡方式，结果显示除诱导肿瘤细胞发生凋亡，GDC-0152还能够促进磷酸化MLKL的激活，进而诱导细胞发生坏死性凋亡。HCA和Western blot结果显示，IAPs拮抗剂靶标依赖地激活NF-κB通路。GDC-0152诱导p62与RIPK1共定位及相互作用，而siRNA敲减p62后，GDC-0152诱导的坏死性凋亡被显著抑制，表明p62介导了GDC-0152诱导的坏死性凋亡的发生。上述研究结果，可能为抗肿瘤药物研发提供新思路，为肿瘤临床治疗探索新途径。