Abstract 1099: MiR-200c overexpression radiosensitizes lung cancer cells

Abstract

Abstract MiR-200 family are key regulators of the epithelial-to-mesenchymal transition (EMT) process and because this process is related to the development of tumor cell resistance and metastasis, these microRNAs represent potential biomarkers of cancer progression and therapy response. Nevertheless, their roles in resistance and sensitivity to radiotherapy are poorly understood. In the present work, we investigated the potential application of miR-200 family for overcoming radioresistence in non-small lung cancer cells (NSLC). First, we demonstrated that NSCLC cell lines that have gone through EMT lose the ability to express miR-200s. We established that miR-200s expression is lost in mesenchymal-like NSCLC cell lines by using real-time polymerase chain reaction analysis of two E-cadherin-positive cell lines H358 and H322 and two E-cadherin-negative cell lines A549 and H1299. We confirmed that restoring miR-200s in mesenchymal cells induces E-cadherin expression. E-cadherin expression was increased and ZEB1 and BIM expression were decreased after miR-200a overexpression in A549 cells. Moreover, we analyzed the effect of radiation treatment in an epithelial-like NSCLC cell line. H358 cells were treated with 4-Gys radiation doses over a 2-week period. Morphologic changes consistent with the development of mesenchymal-like characteristics were found and Western blotting demonstrated a 53% reduction in E-cadherin, suggesting that radiation drives epithelial cells into EMT. Next, we demonstrated that miR-200c replacement radiosensitizes mesenchymal-like cells. A549 cells with stable expression of miR-200c and cells transiently transfected with miR-200c mimics were exposed to ionizing radiation at 0, 2 and 4-Gys. Cell survival and DNA Double-strand breaks were monitored by clonogenic assay and the formation of γ-H2AX foci by immunofluorescence, respectively. We found that miR-200c treatment significantly sensitized A549 cells to radiation treatment. Moreover, miR-200c overexpression increased radiation-induced double-stranded DNA breaks relative to mock-transfected control cells. We also demonstrated that overexpression of miR-200c influenced two key DNA repair pathways, Rad-51 and Chk-1. To further verify the mechanism by which miR-200c promotes radiosensitization in lung cancer cells, we used reverse-phase protein arrays (RPPA) to identify EMT and DNA-repair proteins that are regulated by miR-200c. Comparison of several NSCLC lines that were or were not transfected with miR-200c revealed reductions in the expression of caveolin, AKT, phosphorylated AKT, phosphorylated SRC, and PI3K, BRCA1 and BRCA2, ATM, Ku80, XRCC1 and XIAP. Taken together, these results suggest that miR-200c can significantly impact the ability of lung cancer cells to respond to irradiation and may represent potential therapeutic target in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1099. doi:1538-7445.AM2012-1099