Abstract 1441: Role of a long non-coding RNA in oncogenesis of Ewing sarcoma

Abstract

Abstract BACKGROUND: Ewing sarcoma family of tumors (EFT) is the second most common primary malignant bone tumor in children and adolescents. It is a highly invasive disease with a 30% 5-year survival rate for patients with metastatic disease. EFT are defined by a chromosomal translocation that encodes for the fusion gene EWS-FLI1 in 90% of cases. This chimeric gene functions as a transcription factor and regulates tumorigenesis-related gene expression. Our hypothesis is that like protein-coding genes, the non-coding genome in EFT is also important for the biology of Ewing sarcoma. This study focuses on one such non-coding locus of the human genome and explores its role in EFT pathogenesis. METHODS: Expression profiles from a panel of 500 tissue samples including over 100 EFTs, other primary tumors, and normal tissues were generated using Affymetrix Human Exon 1.0 ST microarrays. Data were analyzed using institutional software, Genetrix. RNA-Seq data was generated on two EFT cell lines derived from a primary tumor and its metastatic derivative. Data was validated by polymerase chain reaction (PCR) on 33 cell lines including EFTs and other tumors. Functional studies on EFT cell lines were performed using various RNAi techniques and viral transductions. RESULTS: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFT from all other tumor types. One such transcript, AK057037, was the most highly expressed RNA (1.07 x 10−20) in EFT. RNA-Seq analysis and PCR studies revealed that this transcript is a multi-exonic, presumably, long non-coding RNA (lncRNA) with a maximum predicted open reading frame of 137 amino acids that had no homology, protein domains, or characteristic motifs. Validation studies confirmed that this lncRNA was exclusively expressed in EFT. EWS-FLI regulates expression of this transcript by directly binding to its promoter region. Furthermore, functional data based on knocking down and overexpressing the transcript showed that the lncRNA acts as an oncogene in EFT and enhances tumorigenicity by increasing the metastatic potential of the tumor cell lines. RNA-Seq results showed that the metastatic tumor-derived cell line had increased transcriptional activity of the lncRNA than the primary tumor-derived cell line. Chromatin immunoprecipitation assay detected binding of the lncRNA to the polycomb repressor complex 2 (PRC2) proteins. CONCLUSION: This study establishes lncRNA AK057037 as a promoter of EFT pathogenesis. Its association with the PRC2 complex suggests its role in chromatin modification. Targeting this lncRNA may help improve outcomes in children with metastatic Ewing disease. Using AK057037 as an example, we show that the non-coding genome is important in EFT. Further work on the non-coding transcriptome may reveal additional functional RNAs that contribute to the biology of this disease. 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 1441. doi:1538-7445.AM2012-1441