Abstract LB261: The Akt-IWS1 signaling axis regulates microRNA activity

Abstract

Abstract MicroRNAs are small single-stranded non-coding RNAs that regulate gene expression post-transcriptionally, by targeting the 3‘-untranslated region (3’-UTR) of mRNAs. The regulation of microRNAs at the transcriptional level is well-studied, however, how their activity is regulated remains largely elusive. The family of Akt serine/threonine protein kinases, comprised of Akt1, Akt2 and Akt3, regulates important cell functions including metabolism, survival, proliferation, and migration. We have previously shown that IWS1 (Interacts with Spt6), a factor involved in mRNA splicing and nuclear export, is phosphorylated specifically by Akt1 and Akt3 at Ser720/Thr721. Here, RNA extracts from NCI-H522 lung cancer cells, transduced with shIWS1 and reconstituted with wild type (Ser720/Thr721-IWS1) or phosphorylation-deficient IWS1 (Ala720/Ala721-IWS1) were subjected to RNA and microRNA sequencing. Bioinformatic analysis suggested that microRNAs expressed at similar levels in the two cell types have a different impact on their mRNA targets. microRNA activity reporter assays and western blot analysis for microRNA targets in these cells revealed increased microRNA activity in Ala720/Ala721-IWS1-expressing cells. Our findings were verified in a second cell line, the non-transformed immortalized colonic epithelial cells NCM460. microRNA activity reporter assays, western blot analyses and cell growth assays showed that microRNA effects are enhanced in cells expressing Ala720/Ala721-IWS1. Immunoprecipitation and proximity ligation assays showed that IWS1 interacts with proteins known to be associated with RISC, and this interaction is enhanced by Akt-mediated IWS1 phosphorylation. To evaluate the dependence of IWS1/RISC interaction on IWS1 phosphorylation, we employed Akt1− / −Akt2− / −Akt3− / − immortalized mouse lung fibroblasts, transduced with myc-Akt1 or myc-Akt2 or the empty retroviral vector. Immunoprecipitation experiments in these cells, before and after treatment with IGF1, revealed that IWS1 interacts with the RISC components specifically in Akt1-expressing cells upon Akt1 activation by IGF1. Overall, our data demonstrate that IWS1 interacts with RISC in a conserved Akt1-dependent manner and regulates the activity of microRNAs with significant effects on cellular properties. Citation Format: Niki Christodoulou, Maria Hatziapostolou, Cristina Montiel-Duarte, Elisabetta Verderio Edwards, Philip N. Tsichlis, Christos Polytarchou. The Akt-IWS1 signaling axis regulates microRNA activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB261.