Aberrant overexpression of miR-421 downregulates ATM and leads to a pronounced DSB repair defect and clinical hypersensitivity in SKX squamous cell carcinoma

Background: Cellular and clinical sensitivity to ionizing radiation (IR) is determined by DNA double-strand breaks (DSB) repair. Here, we investigate the molecular mechanism underlying the extreme response of a head and neck tumor case (SKX) to standard radiotherapy. Methods: Immunofluorescence (IF) was used for the assessment of DSB repair, Western blot and real-time PCR for protein and mRNA expression, respectively. Results: SKX cells exhibited a pronounced radiosensitivity associated with numerous residual gamma-H2AX foci after IR. This was not associated with lacking canonical repair proteins. SKX cells did not express any ATM protein. Accordingly, immunoblotting revealed no ATM kinase activity toward substrates such as p-SMC1, p-CHK2 and p-KAP1. Sequencing of all 66 exons of ATM showed no mutation. ATM mRNA level was moderately reduced, which could be reverted by 5'-Aza-C treatment but without restoring protein levels. Importantly, we demonstrated a post-transcriptional regulation in SKX cells via 6-fold enhanced levels of miR-421, which targets the 3'-UTR of ATM mRNA. Transfection of SKX cells with either anti-miR-421 inhibitor or a microRNA-insensitive ATM vector recovered ATM expression and abrogated the hyper-radiosensitivity. Conclusion: This is the first report describing microRNA-mediated down-regulation of ATM leading to clinically manifest tumor radiosensitivity. (c) 2012 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 106 (2013) 147-154