Active RAS/MEK pathway downregulates expression of IFN-inducible genes by targeting IRF1: implications for understanding molecular mechanisms of viral oncolysis

Abstract

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. The Hirasawa lab has previously demonstrated that oncogenic Ras/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. The objective of my PhD project was to elucidate the mechanisms underlying how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, the IFN regulatory factor 1 (IRF1) binding site was identified to be responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild-type mouse embryonic fibroblasts (MEFs), but not in IRF1− / − MEFs. Furthermore, IRF1 expression was lower in RasV12 cells compared with vector control NIH3T3 cells, which was restored to equivalent levels by inhibition of MEK. Similarly, MEK inhibition restored IRF1 expression in human cancer cells. IRF1 re-expression in human cancer cells increased cellular resistance to infection by the oncolytic vesicular stomatitis virus strain. Together, these results indicate that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis. I further sought to determine how active Ras/MEK downregulates IRF1 expression. MEK inhibition restored IRF1 expression at the protein level prior to mRNA induction; however, it did not affect IRF1 protein stability. The expression of IRF1- targeting microRNA, activity of IRF1 5’ and 3’-UTRs, and polysome loading of IRF1 mRNA in response to MEK inhibition were analyzed; however, the translational regulation of IRF1 mRNA by Ras/MEK remained inconclusive. To determine whether Ras/MEK modulates post-translational modifications (PTMs) of IRF1, phosphorylation, ubiquitination, sumoylation, and acetylation of IRF1 were examined. MEK inhibition promoted ubiquitination and inhibited sumoylation of IRF1, indicating that active Ras/MEK alters PTM of IRF1 protein. Lastly, siRNA screens and overexpression experiments identified RSK3 and RSK4 to be the ERK downstream effectors involved in Ras/MEK-mediated IRF1 regulation.