Abstract B18: The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors

Abstract

Abstract Introduction: Ewing sarcoma family of tumors (EFT) is the second most common primary malignant bone tumor in children and adolescents. It features a characteristic chromosomal translocation; the most common being (t11;22)(q24;q12), which encodes for the chimeric EWS-FLI1 oncoprotein. The disease is highly invasive with a 30% 5-year survival rate for patients with metastatic disease. This study identifies and characterizes a non-coding locus that is unique to EFTs and mediates at least in part the role of the chimeric oncogene in EFT pathogenesis. This particular locus suggests that better understanding of the functional non-coding transcriptome of this developmental cancer can potentially assist in discovering biomarkers that improve the diagnosis and therapeutic management of the disease. 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. RNAseq data was generated on two EFT cell lines derived from a primary tumor and its metastatic derivative. Data was validated by 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. Additional characterization of the RNA was done by cellular fractionation studies and northern blots. Results: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFTs from all other tumor types. One such transcript, AK057037, was the most highly expressed RNA (1.07 × 10−20 in EFTs. In silico modeling and RNASeq analyses 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. Additionally, multiple variants of the transcript exist in both the nucleus and the cytoplasm. Validation studies confirmed that this lncRNA was exclusively expressed in EFTs. The EFT-specific oncoprotein, EWS-FLI1, regulates expression of this transcript by directly binding to its promoter region. Furthermore, functional data based on knocking down and overexpressing the transcript shows that AK057037 acts as an oncogene in EFT and enhances tumorigenicity by increasing the metastatic potential of the tumor cell lines. Conclusion: The study established that lncRNA AK057037 promotes EFT pathogenesis. Its exclusive presence in EFT suggests that expression of this lncRNA is tissue-specific and highly regulated. Targeting AK057037 in these tumors may help improve outcomes in children with metastatic disease. Further work on the non-coding transcriptome may reveal additional functional RNAs that contribute to the biology of this disease. Citation Format: Sheetal A. Mitra, Daniel H. Wai, William A. May, Jonathan D. Buckley, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B18.