SAPK/JNK信号通路调控DNMT1表观遗传修饰RIP3/MLKL在大分割放疗诱导非小细胞肺癌程序性坏死中的作用及机制研究

Although hypofractionated radiotherapy (HFRT) in the treatment of early-stage non-small cell lung cancer (NSCLC) achieved a remarkable effect, there are still radiation resistances with unknown mechanism. Previously, we reported that HFRT induced the necroptosis of NSCLC cells through receptor-interacting protein kinases 3/mixed lineage kinase domain-like (RIP3/MLKL) pathways activation, and then affected the efficacy of HFRT. However, the intrinsic mechanisms were not clear. And recently, we found that the expression of RIP3/MLKL in NSCLC cells may be regulated by epigenetic modification of DNA methyltransferase 1 (DNMT1). And HFRT induced DNMT1 ubiquitination degradation and downregulation of its expression. By using the protein mass spectrum screening and identification methods, we identified that DNMT1 binded with stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and ubiquitination E3 enzyme S-phase kinase-associated protein 2 (Skp2). Inhibition of SAPK/JNK phosphorylation reversed DNMT1 degradation. Therefore, based on these preliminary data, in the current project, we will combine in vivo and in vitro experimental systems with data from clinical samples, in order to investigate the multi-dimensional mechanisms of the different expression of RIP3/MLKL in NSCLC cells and response to HFRT. This study will provide new insights and target for HFRT personalized therapy with great theoretical significance and applicable value.

大分割放射治疗（HFRT）早期NSCLC取得显著疗效，但HFRT抵抗是瓶颈。我们已证明：RIP3/MLKL介导NSCLC程序性坏死影响HFRT疗效，且HFRT上调RIP3/MLKL，但机制未知。我们深入研究发现：DNA甲基转移酶1（DNMT1）表观遗传修饰RIP3/MLKL启动子抑制其表达；HFRT诱导DNMT1泛素化降解下调其表达。蛋白质谱筛选鉴定出：DNMT1与应激活化蛋白激酶/c-Jun氨基末端激酶（SAPK/JNK）和泛素连接酶Skp2结合，抑制SAPK/JNK磷酸化逆转DNMT1降解。基于前期结果推测：HFRT激活SAPK/JNK导致DNMT1泛素化降解，降低RIP3/MLKL基因启动子甲基化而上调RIP3/MLKL，促进程序性坏死。本项目将对调控RIP3/MLKL在NSCLC细胞差异性表达的关键分子靶点系统解析，为完善HFRT疗效预测指标提供新思路、新靶点、具有潜在的应用前景。

大分割放射治疗（HFRT）早期NSCLC取得显著疗效，但HFRT抗拒是瓶颈。我们已证明：RIP3/MLKL介导NSCLC程序性坏死影响HFRT疗效，且HFRT上调RIP3/MLKL，但机制未知。我们深入研究发现：DNA甲基转移酶1（DNMT1）表观遗传修饰RIP3/MLKL启动子抑制其表达；HFRT诱导DNMT1泛素化降解下调其表达。蛋白质谱筛选鉴定出：DNMT1与应激活化蛋白激酶/c-Jun氨基末端激酶（SAPK/JNK）和泛素连接酶Skp2结合，抑制SAPK/JNK磷酸化逆转DNMT1降解。通过系统研究，初步确定HFRT激活SAPK/JNK导致DNMT1泛素化降解，降低RIP3/MLKL基因启动子甲基化而上调RIP3/MLKL，促进程序性坏死。本项目将对调控RIP3/MLKL在NSCLC细胞差异性表达的关键分子靶点系统解析，为完善HFRT疗效预测提供新思路、新靶点、具有潜在的应用前景。申请者正在扩大肿瘤细胞系及其移植瘤深入研究SAPK/JNK信号通路调控DNMT1表观遗传修饰RIP3/MLKL在大分割放疗诱导非小细胞肺癌程序性坏死中的作用及机制。