Abstract 1823: Influence of a long non-coding RNA on epigenetic, genetic and phenotypic changes in Ewing sarcoma.
Authors: Mitra Sheetal A. , Mitra Anirban P. , Buckley Jonathan D. , May William A. , Kapranov Philipp , Arceci Robert J. , Triche Timothy J. 
Published: August 2014

Background: Ewing sarcoma is a highly invasive pediatric bone malignancy with a 30% 5-year survival rate for patients with metastatic disease. The EWS-FLI1 fusion gene is detected in 90% of these cases, and functions as a transcription factor that regulates oncogenesis-related gene expression. The functions of EWS-FLI1 and other oncogenes have been explored in the past to detect potential therapeutic targets. With a similar aim, this study looks into the rapidly evolving non-coding genome of Ewing sarcoma. Methods: Total RNA from 500 tissues including over 100 primary Ewing, other cancers, normal tissues, and modified Ewing cell lines were run on Affymetrix Human Exon microarrays. RNA-seq data was generated on a pair of primary and metastatic Ewing cell lines. Data were analyzed using Genetrix®, Partek®, Ingenuity Pathway Analysis (IPA®) and iReport™. Functional studies on Ewing cell lines were performed using various RNAi techniques and viral transductions followed by quantitative PCR. Chromatin immunoprecipitation (ChIP) was performed to detect direct interactions of the RNA. Tail vein injection model was developed to study tumor metastasis model in mice. Results: Expression profiling showed that many unannotated transcripts were more significantly associated with Ewing sarcoma than coding genes. Molecular characterization of one such transcript, AK057037, identified it as a multi-exonic, multivariant, polyadenylated, long non-coding RNA (lncRNA). It was not expressed in other tumors or normal adult tissue and was poorly conserved across various vertebrates and lower organisms. The chimeric oncoprotein, EWS-FLI1, directly regulated AK057037 expression by binding to the GGAA microsatellite region in its promoter. Decrease in the lncRNA expression significantly reduced invasive capability of Ewing cells while an increase in expression increased anchorage independence. RNA-seq analysis showed increased transcriptional activity in the metastatic cell line. Pathway analysis on expression profiling data on the AK057037-knockdown cells detected involvement of cellular adhesion and movement pathways. In vivo studies using AK057037-overexpressing cells showed increased tumor cell engraftment and growth in the mice liver. ChIP studies showed that AK057037 was bound to Ezh2, a methyltransferase in the polycomb repressor complex 2 (PRC2). ChIP assays using histone antibodies showed changes in methylation pattern in promoter regions of genes affected by the lncRNA knockdown. Conclusion: AK057037, a lncRNA is significantly upregulated in Ewing sarcoma. Its direct regulation by EWS-FLI1 drives its function as an oncogene and promotes metastasis of the tumor cells, thus helping with the tumor pathogenesis. Its association with the PRC2 complex and perturbation of methylation status of its downstream effectors suggest chromatin modifying functions of AK057037.

Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, William A. May, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. Influence of a long non-coding RNA on epigenetic, genetic and phenotypic changes in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1823. doi:10.1158/1538-7445.AM2013-1823