Abstract LB-462: AGO2 PAR-CLIP suggests a shift in specific miRNA target spectra upon modulation of EWS-FLI1 in Ewing's sarcoma

Abstract

Abstract The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing's sarcoma family tumors (ESFT). As an aberrant ETS transcription factor it directly deregulates a multitude of genes related to proliferation control and differentiation. Recently, we reported on the EWS-FLI1 microRNA (miRNA) signature based on knockdown experiments in ESFT cell lines and on differential expression in primary tumors versus mesenchymal stem cells. In order to define the mRNA targets of EWS-FLI1 regulated miRNAs, we now performed pull-down experiments of AGO2 complexes after photoactivated ribonucleoside-enhanced cross-linking followed by NGS sequencing of associated mRNAs (PAR-CLIP). We obtained 92 Mio high quality reads of which 63 Mio were aligned to the reference genome. The genomic localization of these sequences demonstrated the specific enrichment of 3'UTRs (>17 fold). Among obtained reads, T to C conversions were highly enriched consistent with crosslinking via 4-thiouridine to AGO2 containing complexes. PAR-CLIP results indicate AGO2 binding next to miRNA seed sequences for 4695 and 5160 genes associated with 1876 and 1860 miRNAs in the presence and absence of EWS-FLI1, respectively. Strikingly, for individual miRNAs the mRNA target spectra drastically changed upon EWS-FLI1 knockdown. For instance, for the two top EWS-FLI1 signature miRNAs hsa-mir-145 and hsa-mir 424 a shift from genes involved in cell death and differentiation to protein activator activity, and from protein degradation and cell cycle control to protein kinases and protein phosphorylation was observed, respectively. Of note, a target shift was also observed for miRNAs that did not change in response to EWS-FLI1 knockdown. In addition for many specifc targets no correlation was observed between mRNA abundancy and the level of association with AGO2 as reflected by read counts. These data suggest that EWS-FLI1 perturbs post-translational gene regulation by changing access of the RISC complex to its target mRNAs. Supported by EC-FP7 grant 259348 “ASSET”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-462. doi:1538-7445.AM2012-LB-462