Abstract A43: Therapeutic importance of a long noncoding RNA 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: May 2015

Background: Ewing sarcoma is a highly invasive pediatric bone malignancy with a 20% 5-year survival rate for patients with metastatic disease. The EWS-FLI1 or equivalent fusion gene is detected in 95% of these cases, and functions as a transcription factor that regulates oncogenesis-related gene expression. Various oncoproteins including EWS-FLI1 have been studied at a molecular level to explore their therapeutic potential in order to improve patient outcomes. With a similar aim, we study non-coding genes in Ewing sarcoma that can be targeted using RNA therapeutics.

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®, and Ingenuity Pathway Analysis®. 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 results indicated that several non-coding transcripts had a strong association with Ewing sarcoma. Molecular characterization of one such transcript, AK057037 (also known as FEZF1-AS), identified it as a multi-exonic, polyadenylated , long non-coding RNA (lncRNA) with multiple expressed isoforms. Like many other Ewing sarcoma associated biomarkers, EWS-FLI1 directly regulated this lncRNA by binding to the GGAA microsatellite region in its promoter. Increased stable expression of this transcript led to a statistically significant increase in anchorage independence. RNAi mediated decrease in its expression reduced the invasiveness of Ewing sarcoma cell lines. In vivo studies using AK057037-overexpressing cells showed increased tumor cell engraftment and growth in the mice liver. Pathway analysis of expression profiling data on the AK057037-knockdown cells identified cellular adhesion and movement pathways. RNA-seq analysis showed increased transcriptional activity in the metastatic cell line when compared to the primary. RNA-IP 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. H3K27me3 marks correlated with increased repression of metastatic genes on knockdown of the transcript.

Conclusion: AK057037 is a lncRNA that is highly expressed in Ewing sarcoma. It is directly regulated by the chimeric oncogene, EWS-FLI1. The lncRNA functions as an oncogene and promotes metastasis in this tumor. Its association with the PRC2 complex leads to methylation changes in promoters of genes that are involved in cell migration. Finally, AK057037 is a functional lncRNA that influences Ewing sarcoma pathogenesis by its genetic and epigenetic interactions and has the potential to be developed into a therapeutic target to improve survival of patients with metastatic disease.

Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, William A. May, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. Therapeutic importance of a long noncoding RNA in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A43.