A phosphoproteomics screen in a set of isogenic lung fibroblast cell lines expressing different Akt isoforms, identified 606 Akt target proteins. Ingenuity pathway network function analysis of the data identified a host of network functions that appear to be differentially regulated by the three isoforms. One of these functions is RNA processing, which was represented by 25 proteins that operate at different stages of RNA biogenesis and are phosphorylated by at least one of the three isoforms. The main focus of this work is on one of these proteins (IWS1), which plays a critical role in the assembly of a transcriptional elongation complex on the C- terminal domain (CTD) of the large subunit of RNA polymerase II. Based on studies we recently published (Sanidas et al Mol Cell 53: 577, 2014), we now know that IWS1 is phosphorylated by Akt3 and Akt1, but not Akt2 at the conserved Ser720/Thr721 site. More important, IWS1 phosphorylation at this site is required for the recruitment of the histone H3 K36 trimethyltransferase SetD2 to the IWS1-dependent transcriptional elongation complex. The inhibition of IWS1 phosphorylation, prevents the recruitment of SetD2 and the trimethylation of histone H3 at K36 in the body of target genes during transcription. This abrogates the recruitment of the histone H3K36me3-interacting protein MRG15 and its binding partner polypyrimidine tract binding protein (PTB) and results in shifts in alternative splicing of target genes, one of which is FGFR-2. Alternative splicing gives rise to two FGFR-2 isoforms, IIIb and IIIc, which are expressed in epithelial and mesenchymal cells respectively and of which only the IIIc isoform encodes a receptor that is recognized by FGF-2. In cancer cells, the switch from IIIb to IIIc is associated with EMT and increased cell motility an invasiveness. The phosphorylation of IWS1 by Akt3 and Akt1 promotes a shift from the IIIb to the IIIc isoform and stimulates the proliferation, migration, invasiveness and oncogenic potential of human tumor cell lines. Some, although not all, of these effects of IWS1 phosphorylation can be fully explained by the alternative splicing of FGFR-2. The role of IWS1 phosphorylation in primary human non-small cell lung carcinomas (NSCLCs), was determined by analyzing the expression and phosphorylation of IWS1 in a set of 24 primary NSCLCs. Twenty one of the 24 NSCLCs were found to express IWS1. More important, the stoichiometry of IWS1 phosphorylation in these tumors was fond to correlate with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer. Our plan is to take advantage of the window to the poorly understood regulation of RNA splicing that is opened by these data, to explore the role of Akt in alternative splicing in normal and tumor cells.
Despite significant progress in cancer management in recent years, the management of lung cancer continues to be problematic. Therefore progress in this area represents progress toward the fulfillment of an unmet need in cancer management. Our preliminary data identified a novel and unexpected link between the PI-3K/Akt pathway and alternative RNA splicing, which is frequently activated in lung cancer. Activation of this link results in changes in the proliferation and invasiveness of lung cancer cells. The proposed studies will dissect the molecular mechanisms involved in linking the PI-3K/Akt pathway with RNA splicing and will explore the importance of these findings in lung cancer. The results of these studies will be of great value for the design of truly novel diagnostic and therapeutic strategies for lung and perhaps other human cancers.
| McNiel, Elizabeth A; Tsichlis, Philip N (2017) Analyses of publicly available genomics resources define FGF-2-expressing bladder carcinomas as EMT-prone, proliferative tumors with low mutation rates and high expression of CTLA-4, PD-1 and PD-L1. Signal Transduct Target Ther 2: |
| Voulgarelis, Michael; Tsichlis, Philip N (2016) Proline hydroxylation linked to Akt activation. Science 353:870-1 |
