IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing_images_depository
Our previous studies have shown that IWS1 (Interacts with Spt6) is a phosphorylation target of Akt and regulates the alternative RNA splicing of FGFR-2, linking IWS1 with lung cancer tumorigenesis. To further address the role of IWS1 in alternative RNA splicing and lung cancer, we performed an RNA-seq study using lung adenocarcinoma cells in which IWS1 was knocked down or replaced by its phosphorylation site mutant. This identified the splicing factor U2 Associated-Factor 2 (U2AF2) as a target of IWS1 phosphorylation, by showing that the loss of phosphorylated IWS1 results in U2AF2 transcripts lacking exon 2. Exon 2 encodes part of the U2AF65 Serine-Rich (SR) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). Here, we show that the loss of phosphorylated IWS1 interferes with histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes on the U2AF2 gene in a cell-cycle specific manner. The latter results in the downregulation of cell cycle division associated 5 (CDCA5), a phosphorylation target of ERK, leading to impaired cell proliferation, G2/M phase arrest and impaired tumor growth in mouse xenografts models, an effect more pronounced in cells harboring EGFR mutations. Analysis of human lung adenocarcinoma samples reveals robust correlations between IWS1 phosphorylation, U2AF2 splicing pattern, and CDCA5/p-ERK levels, especially in EGFR mutated patients. More importantly, IWS1 phosphorylation is positively correlated with tumor stage and grade and defines poor prognosis in lung adenocarcinoma patients, harboring EGFR, and not KRAS, mutations. This work highlights the instrumental role of the AKT/p-IWS1 axis to alternative RNA splicing in governing cell cycle progression and tumorigenesis, and proposes this axis as a novel drug target and prognosis factor in lung adenocarcinoma, by concomitantly affecting the epigenetic regulation of RNA processing and oncogenic signals. These data accompany the main dataset of our paper : doi: 10.17632/y82yx8gfkj.3 In this dataset, we will upload all the images from cell transformation assays and IHC from mouse xenografts and human samples.