PUB090 Identification of a Novel Approach to Phosphorylate IRF3 and Its Engagement to Induce Apoptosis via MDA5/RIG-I Pathway in Non-Small Cell Lung Cancer

Abstract

Immunotherapy for lung cancer is increasingly being contemplated as the most effective manners to counteract this malignancy in the future. The RIG-I-like receptors (RLRs) melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene 1(RIG-I) are indispensable receptors for recognizing pathogen-associated molecular patterns (PAMPs), and interferon regulatory factor 3(IRF3) endows the potentiality of inciting innate immunity in cancers. However, the roles of IRF3 in innate immunity of lung cancer are deeply obscure. We collected fresh non-small cell lung cancer samples/adjacent tissues and tested IRF3, MDA5/RIG-I by quantitative PCR(q-PCR) and Western Blot. Then non-small cell lung cancer (NSCLC) cell lines A549,H1299 were transfected with Poly(I:C),investigating the innate immunity activity and apoptosis by q-PCR, Western Blot, ELISA, and flow cytometry. Small interfering RNA(siRNA) and pLent-shRNA experiments were employed to knockdown MDA5/RIG-I and IRF3 respectively. Simultaneously, we constructed recombinant plasmid to over express IRF3 in A549,H1299 cell lines. In this study, we found that the expression of MDA5/RIG-I,IRF3 in NSCLC tissues were significantly higher than the corresponding adjacent tissues. In vitro, innate immunity pathway was triggered predominantly when transfecting the NSCLC cell lines A549,H1299 with Poly(I:C) independent direct addition to culture medium, accompanying the phosphorylation of Ribosomal protein S6 kinase(S6K ) and IRF3. In contrast, treatment with TBK1 inhibitor BX795 sharply decreased p-S6K,as a consequence of shrinkage of p-IRF3;In line, when p-S6K was inhibited by mTOR inhibitor Rapamycin, the variance of p-S6K and p-IRF3 resembled the effects of BX795, leading to the impairment of innate immunity. Furthermore, knockdown of MDA5/RIG-I with siRNA can dampen the phosphorylation of S6K and IRF3 ,the degree of innate immunity and the apoptosis induced by Poly(I:C). In addition, downregulation of IRF3 hindered the innate immunity and the activation of caspase3, caspase8. Conversely, overexpression of IRF3 promoted the expression of TRAIL and further activated the apoptotic signaling pathway. These results suggested that p-S6K under the control of MDA5/RIG-I regulated the phosporylation of IRF3 and the apoptosis induced by Poly(I:C) in NSCLC was mediated by MDA5/RIG-I/IRF3 axis. Therefore, the involvement of IRF3 in innate immunity may be the promising therapy in NSCLC.