Abstract 4777: A phosphoproteomics analysis reveals Akt isoform-specific signals that link RNA splicing to non-small cell lung cancer
Authors: Sanidas Ioannis , Polytarchou Christos , Hatziapostolou Maria , Ezell Scott A. , Kottakis Filippos , Hu Lan , Guo Ailan , Xie Jianxin , Comb Michael J. , Iliopoulos Dimitrios , Tsichlis Philip N. 
Published: May 2015

Abstract
Akt isoforms exhibit different functional properties. To address the signaling differences between Akt1, Akt2 and Akt3 we examined the phosphoproteomes of a set of isogenic mouse cell lines that express different Akt isoforms. Based on this screen, we identified a total of 606 Akt phosphorylation targets, of which many are phosphorylated in an isoform specific manner. Bioinformatics analyses of these data revealed that Akt isoforms regulate differentially multiple cellular functions. One of these functions was RNA metabolism which was represented by 25 proteins phosphorylated by at least one of the Akt isoforms. One of these proteins was IWS1, which is involved in the assembly of RNA Pol II transcriptional elongation complex, and which was found to be phosphorylated at the conserved site Ser720/Thr721 by Akt3 and Akt1. Here we show that this phosphorylation event is required for the recruitment of the histone methyltransferase SETD2 to the complex and the trimethylation of histone H3 at K36 in the body of the transcribed genes. H3K36me3 provides a docking site for MRG15 and its binding partner, the splicing suppressor PTB, and regulates PTB-dependent alternative splicing. One of the targets is FGFR-2 whose alternative splicing gives rise to two isoforms, IIIb, which is expressed in epithelial cells and IIIc, which is expressed in mesenchymal cells, promotes EMT and is associated with more aggressive tumors. IWS1 phosphorylation by Akt3/Akt1 shifts splicing toward the IIIc isoform and promotes tumor growth and invasiveness both in culture and in animals.
Addressing the expression of FGFR-2 in a set of lung-derived normal and tumor samples revealed that whereas the overall expression was similar in both, there was a shift toward the IIIc isoform in the tumor samples. More important, the relative expression of the IIIc and IIIb isoforms in non-small-cell-lung-carcinomas (NSCLCs) correlated with the stoichiometry of IWS1 phosphorylation and the latter correlated with Akt phosphorylation and Akt3 expression. These findings combined, underpin the importance of this pathway in the pathogenesis of lung cancer. Overall, our data suggest that Akt isoform-dependent phosphorylation events are essential for RNA processing and provide novel insights into the role of Akt in carcinogenesis.
Citation Format: Ioannis Sanidas, Christos Polytarchou, Maria Hatziapostolou, Scott A. Ezell, Filippos Kottakis, Lan Hu, Ailan Guo, Jianxin Xie, Michael J. Comb, Dimitrios Iliopoulos, Philip N. Tsichlis. A phosphoproteomics analysis reveals Akt isoform-specific signals that link RNA splicing to non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2014-4777