Abstract 5338: A repressive EWS-FLI1 subsignature is the result of FOXO1 suppression and nuclear exclusion

Abstract

Abstract The EWS-FLI1 chimeric protein, characteristic of Ewing′s sarcoma (ESFT), constitutes the prototype of an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of ETS binding motifs in EWS-FLI1 activated target promoters, while EWS-FLI1 repressed promoters showed most prominently enrichment of recognition motifs for forkhead box (FOX) proteins. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibition of FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, ectopic FOXO1 was found excluded from the nucleus as consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, a directly EWS-FLI1 activated kinase, augmented by chemical inhibition of PI3K, or by mutation of CDK2 phosphorylation sites. These results suggest an important role for CDK2 in FOXO1 repression by EWS-FLI1. To test the contribution of FOXO1 to the EWS-FLI1 repressive signature on the ESFT transcriptome, the overlap between genes activated by EWS-FLI1 knockdown and genes activated by ectopically expressed, nuclear directed FOXO1 was determined and compared to the effects of EWS-FLI1 knockdown under concomitant FOXO1 silencing. A significant overlap between FOXO1 activated and EWS-FLI1 repressed genes was identified (P<10e-20, hypergeometric test). Taken together, these data confirm our hypothesis that a repressive subsignature of EWS-FLI1 regulated genes is due to suppression of FOXO1. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability suggesting that FOXO1 plays an important role in ESFT oncogenesis. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2011-5338