EPHA2 Interacts with DNA-PK(cs) in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells

SIMPLE SUMMARY: Despite the introduction of targeted therapies against genomic drivers in non-small cell lung cancer (NSCLC), e.g., mutated epidermal growth factor receptor (EGFR) and EML4-ALK fusion or immune checkpoint blockade, many patients are receiving radiation therapy (RT). Thus, RT is important for the treatment of advanced NSCLC, where it is used alone or combined with targeted agents or chemotherapy. Unfortunately, a large fraction of the NSCLC cases show resistance to ionizing radiation (IR), calling for further understanding of the underlying mechanisms and novel ways to IR sensitization. Here, we demonstrate a novel function of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) pathway that controls cellular IR response. Moreover, we show that EphA2 can be targeted for IR sensitization of resistant NSCLC cells. Thus, we suggest that further understanding of this mechanism may allow for new RT sensitization approaches to treat LC. ABSTRACT: Ephrin (EFN)/Erythropoietin-producing human hepatocellular receptors (Eph) 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(cs). 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(cs) at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PK(cs) in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.