Differential roles of ATF-2 in survival and DNA repair contributing to radioresistance induced by autocrine soluble factors in A549 lung cancer cells

Abstract

Radioresistance is one of the obstacles to the effective radiotherapy for non-small cell lung cancer. Soluble factors in the tumour microenvironment are often implicated in radioresistance but the underpinning mechanism(s) remain largely elusive. We herein studied the wholesome effect of autocrine cytokines and growth factors in the form of self-conditioned medium (CM) on the radiosensitivity of A549 cells. A549 cells grown in CM exhibited radioresistance which was associated with increased survival and DNA repair. CM induced pro-survival pathways through increased intracellular cAMP and phosphorylation of JNK and p38. Downstream to JNK/p38 signalling, ATF-2 phosphorylated at Thr69/71 was accompanied with its increased transcriptional activity in CM treated cells. Pre-treatment with cAMP inhibitor and silencing of ATF-2 abrogated the CM-induced survival. Interestingly, in cells treated with CM followed by radiation, ATF-2 was found to be switched over from transcription factor to DNA damage response protein. In CM treated cells, after γ-radiation p-ATF-2Thr69/71 and subsequently the transcriptional activity of ATF-2 were declined with simultaneous rise in p-ATF-2Ser490/498. Immunoprecipitation/immunoblotting and inhibitor studies showed that phosphorylation of ATF-2 at Ser490/498 was mediated by ATM. Moreover, p-ATF-2Ser490/498 was found to be co-localised with γ-H2AX in DNA repair foci in CM-treated cells. The DNA repair activity of ATF-2 was assisted with higher activity MRN complex in cells grown in CM. Our study revealed that, autocrine soluble factors regulate dual but differential role of ATF-2 as a transcription factor or DNA repair protein, which collectively culminate in radioresistance of A549 cells.