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Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis
Michelle Marques Howarth, … , Paul Khavari, E. Alejandro Sweet-Cordero
Michelle Marques Howarth, … , Paul Khavari, E. Alejandro Sweet-Cordero
Published November 17, 2014
Citation Information: J Clin Invest. 2014;124(12):5275-5290. https://doi.org/10.1172/JCI72124.
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Research Article Oncology

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

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Abstract

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma–associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

Authors

Michelle Marques Howarth, David Simpson, Siu P. Ngok, Bethsaida Nieves, Ron Chen, Zurab Siprashvili, Dedeepya Vaka, Marcus R. Breese, Brian D. Crompton, Gabriela Alexe, Doug S. Hawkins, Damon Jacobson, Alayne L. Brunner, Robert West, Jaume Mora, Kimberly Stegmaier, Paul Khavari, E. Alejandro Sweet-Cordero

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Figure 6

EWSAT1 mediates repression of genes downstream of EWS-FLI1.

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EWSAT1 mediates repression of genes downstream of EWS-FLI1.
(A) Venn dia...
(A) Venn diagram of all genes regulated by EWS-FLI1 compared with all genes regulated by EWSAT1. (B) Venn diagram of all EWS-FLI1–repressed genes compared with all EWSAT1-repressed genes. (C) Venn diagram of all EWS-FLI1–upregulated genes compared with all EWSAT1-upregulated genes. P values and odds ratios (OR) for overlaps in A–C were calculated using Fisher’s exact test. (D) Heat map of genes differentially regulated by EWSAT1 in the indicated samples. (E) UCSC genome browser view of reads mapping to the EWSAT1 locus in shGFP, shEWS-FLI1, and shEWSAT1 samples. The predominant EWSAT1 isoform expressed in Ewing cells is indicated with an asterisk. (F) Cumulative distribution function of Pearson correlations for genes repressed by EWSAT1 and that for genes not repressed. Left shift (anticorrelation, indicating repression) of EWSAT1-repressed genes (red) is seen. Correlations calculated from a data set of Ewing sarcoma primary tumor RNAseq. Two-sided KS test was used to calculate P value.
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