Kaminskyy Vitaliy O, Hååg Petra, Novak Metka, Végvári Ákos, Arapi Vasiliki, Lewensohn Rolf, Viktorsson Kristina
Department of Oncology/Pathology, Karolinska Institutet, SE-17177 Stockholm, Sweden.
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, 1000 Ljubljana, Slovenia.
Cancers (Basel). 2021 Feb 28;13(5):1010. doi: 10.3390/cancers13051010.
Ephrin (EFN)/ Erythropoietin-producing human hepatocellular ) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PK. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PK at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PK in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.
此前有报道称，Ephrin（EFN）/促红细胞生成素产生性人肝细胞（Erythropoietin-producing human hepatocellular）信号传导可调节非小细胞肺癌（NSCLC）细胞的存活、死亡以及侵袭和迁移。在此，研究了Ephrin A型受体2（EphA2）在NSCLC细胞中对DNA损伤反应（DDR）信号传导和电离辐射（IR）细胞效应的作用。EphA2沉默导致IR致敏，伴有caspase-3激活增加、PARP-1裂解以及克隆形成存活率降低。在一组NSCLC细胞系中对EphA2表达进行分析，结果显示其与IR难治性表型相关。研究发现，EphA2在Ser897位点会因IR而发生瞬时快速磷酸化，这与核糖体蛋白S6激酶（RSK）的激活同时出现。通过细胞分级分离发现，IR可使核组分中总EphA2和pSer897 EphA2均出现瞬时增加。通过免疫沉淀和EphA2复合物的液相色谱-串联质谱（LC-MS/MS）分析，发现核定位的EphA2与DNA依赖性蛋白激酶（DNA-PK）形成复合物。IR后这种复合物的形成迅速增加，但在一小时内恢复到基础水平。在IR后的早期时间点，用小干扰RNA（siRNA）靶向EphA2或用EFNA1配体处理可部分降低DNA-PK在S2056位点的磷酸化。因此，我们报道EphA2在细胞核中与DNA-PK相互作用，提示EphA2受体在DDR信号传导和IR反应性中存在一种新机制。
