Abstract 1949: Positive feedback regulation between EWS-FLI1 and miR-145 in Ewing's sarcoma

Abstract

Abstract The EWS-FLI1 chimeric protein is a potent tumor specific transcriptional regulator and the molecular cause of Ewing's sarcoma family of tumors (ESFT). We had previously hypothesized that, since EWS-FLI1 is the major driver of aberrant gene expression in ESFT, there should be an inverse correlation between the changes in gene expression observed in ESFT cell lines in response to RNAi-mediated EWS-FLI1 knockdown and the corresponding gene expression in primary tumors relative to the tissue of origin. We found that mesenchymal progenitor cells (MPC) fit this assumption best and established an ESFT specific EWS-FLI1 transcriptional signature on the mRNA level. Following the same approach, we now studied the EWS-FLI1 dependent regulation of microRNAs (miRNAs) in ESFT. Genome-wide miRNA analysis was performed after shRNA-mediated stable (5 cell lines) and inducible (1 cell line) EWS-FLI1 knockdown, as well as in 6 primary ESFT and 6 MPC samples using the stem-loop reverse transcription quantitative PCR platform. Among miRNAs consistently affected by EWS-FLI1 silencing and inversely expressed in primary tumors compared to MPCs was hsa-miR-145 which we found upregulated upon EWS-FLI1 silencing. Since several target sites exist for this miRNA within the FLI1 3′UTR, we asked if hsa-miR-145 is capable of modulating EWS-FLI1 expression in ESFT cells. Upon ectopic expression of pri-miR-145 in wildtype p53 ESFT cells, strong modulation of EWS-FLI1 protein levels, but not RNA levels, was observed. In p53 mutant cell lines this effect was less pronounced consistent with a recently reported role of p53 in miR-145 maturation. Preliminary studies suggest that hsa-miR-145 is indirectly regulated by EWS-FLI1 by a mechanism that may involve the NOTCH signaling pathway. Our results identify a positive feed-back loop between EWS-FLI1 and hsa-miR-145 expression in ESFT. Supported by the 6th framework program of the European Commission, (STREP “E.E.T. Pipeline“ contract LSHC-CT-2006-037260) and 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 2010;70(8 Suppl):Abstract nr 1949.