Abstract 3158: Evidence for a role of EWS in alternative splicing in Ewing's sarcoma

Ewing's sarcoma family tumors (ESFT) are characterized in 85% of cases by the oncogenic fusion protein EWS-FLI1 resulting from a chromosomal translocation. While FLI1 is an ETS transcription factor, EWS belongs to the evolutionarily conserved FET family of RNA-binding proteins. So far, functional studies of oncogenic EWS fusion proteins have concentrated on their presumed role as transcriptional regulators. However, chimeric FET proteins are usually co-expressed with their intact counterparts and previous protein interaction studies have shown that EWS and EWS-FLI1 interact with each other. Therefore, it is possible that functional interference with normal EWS function accounts for part of the oncogenic activities of EWS-FLI1. However, the role of EWS in post-transcriptional and transcriptional gene regulation is poorly defined. Since FET proteins associate with a number of RNA processing factors, we investigated the influence of modulated EWS expression on genome wide splicing using Affymetrix HuEx-1.0stv2 arrays. For that purpose, we used two model systems: a unique ESFT cell line lacking endogenous EWS was studied upon stable restoration of EWS expression, and an ESFT cell line allowing for inducible RNAi-mediated EWS-FLI1 suppression was analyzed upon transient EWS knockdown. As a result, we found significant differences between cells with and without EWS expression in splicing patterns as well as alternative promoter usage for more than 50 genes. GO analysis of the top ranking genes is consistent with previously suggested functions of the FET protein family such as DNA repair, chromatin structure and RNA transcription. The alternative splicing patterns were verified by RT-qPCR of candidate genes which are currently tested for their functional relevance to ESFT. One of these candidate genes is structural maintainance of chromosome 5 (SMC5). As a core component of the SMC5-SMC6 complex it is involved in sister chromatid homologous recombination by recruiting the SMC1-SMC3 cohesin complex to DNA double-strand breaks during anaphase. Ongoing studies concentrate on the influence of EWS-FLI1 on EWS-dependent alternative RNA processing patterns in these model systems. This study is supported by grant P20665-B12 of the Austrian Science Fund FWF Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res