Abstract
Abstract Introduction: Ewing sarcoma is the second most common primary malignant bone tumor in children and adolescents. Ewing sarcoma family of tumors (EFT) feature a characteristic chromosomal translocation t(11;22)(q24;q12), which encodes for the chimeric EWS-FLI1 oncoprotein. A minority of EFT have additional translocation variants partnering EWS with other members of the ETS family of transcription factors. This disease is highly invasive with a 30% 5-year survival rate for patients with metastatic disease. This study aimed to identify and validate candidate biomarker(s), both protein-coding and non-coding, that are unique to EFT and can potentially assist in objective diagnosis and therapeutic management. Methods: Using Affymetrix Human Exon 1.0 ST microarrays, genome-wide expression profiles were generated on a panel of 500 tissue samples including over 100 Ewing sarcomas, other primary tumors, normal tissues and stem cells. Whole transcriptome sequencing (RNASeq) was also performed on 2 cell lines. Microarray data was validated by RT-PCR. EFT cell lines were transfected with siRNA against EWS-FLI1 and subjected to retinoic acid (RA)-induced differentiation to monitor expression changes in EFT-specific transcripts. Cellular fractionation studies and northern blots were performed to characterize location and size of the candidate transcript, respectively. Results: Expression microarray profiles identified both genes and candidate non-coding RNAs (ncRNAs) that were strongly associated with EFT, including the candidate ncRNA AK057037. In silico modeling and RNASeq of this transcript revealed a large, intergenic, non-coding 2,641 base, 8-exon RNA (lincRNA) with a predicted open reading frame of 137 amino acids with no homology, protein domains, or characteristic motifs. RT-PCR data from 33 tumor cell lines validated over-expression of this unique and exclusive transcript in EFT. Alternative splicing of both exons and introns as well as extended forms of the transcript were also identified. Northern blot confirmed multiple variants of the transcript in addition to the known 2.6 kb region. Cellular fractionation indicated that the transcript was abundantly present in the nucleus. EWS-FLI1 knockdown in EFT cells reduced its expression. AK057037 was detected during early differentiation of human embryonic stem cells to neural crest stem cells (NCSC), putative cells of EFT origin. Furthermore, RA-induced differentiation of EFT cell lines decreased the transcript's expression, corroborating the hypothesis that AK057037 contributes to the primitive NCSC-like phenotype of EFT. Conclusions: AK057037 is highly and exclusively expressed in EFT, and its expression is regulated by EWS-FLI1. This multi-exon transcript with multiple splice variants has characteristic features of a lincRNA. Its strong association with EFT and expression in NCSC indicates that it may be a key component of Ewing sarcoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1182. doi:10.1158/1538-7445.AM2011-1182
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