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

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

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 3

EWSAT1 is a lncRNA expressed in Ewing sarcoma.

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EWSAT1 is a lncRNA expressed in Ewing sarcoma. (A) Visualization of read...
(A) Visualization of reads mapped to the EWSAT1 locus in either control MPCs (pMPC UI) or EWS-FLI1–expressing MPCs (pMPC EWS-FLI1). Black arrow indicates accumulation of reads at the 3′ end of pMPCs expressing EWS-FLI1, whereas almost no reads are detected in the control. Tracks indicate pooled reads from either 3 controls or 3 pMPCs expressing EWS-FLI1. Predominant known isoforms are indicated below tracks. Asterisk indicates most abundant isoform, as determined by RT-PCR (see Results) (B) RT-PCR for EWSAT1 in uninfected pMPCs, EWS-FLI1–expressing pMPCs, and in 4 Ewing sarcoma cell lines. EWSAT1 expression is quantified relative to HPRT. Pound signs indicate expression below the threshold for detection. (C) cpm reads mapped for EWSAT1 in the RNAseq data set generated from a panel of primary human tumors and normal samples. DDLPS, dedifferentiated liposarcoma; EMC, extraskeletal myxoid chondrosarcoma; ESS, endometrial stromal sarcoma; ESFT, Ewing sarcoma family of tumors; GIST, gastrointestinal stromal tumor; HNSCC, head and neck; LMS, leiomyosarcoma; MLS, myxoid liposarcoma; NPC, nasopharyngeal carcinoma; PUC, papillary urothelial carcinoma; SS, synovial sarcoma. (D) RT-PCR for EWSAT1 in 4 Ewing cell lines and a primary patient-derived xenograft (PSS090) compared with the indicated non-EWS-FLI1–expressing cells. EWSAT1 expression is relative to HPRT and normalized to A673. (E) Northern blot detection of EWSAT1 in 3 Ewing cell lines. (F) Northern blot detection of EWSAT1 in A673 Ewing cells (control vs. knockdown of EWSAT1) and in osteosarcoma cell lines SAOS2, U20S, and KRIB. All lanes were run on the same gel but were noncontiguous.