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

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.

[1]  A. McKenna,et al.  The genomic landscape of pediatric Ewing sarcoma. , 2014, Cancer discovery.

[2]  S. Dhanasekaran,et al.  The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex , 2013, Nature Genetics.

[3]  M. Rosenfeld,et al.  LncRNA-Dependent Mechanisms of Androgen Receptor-regulated Gene Activation Programs , 2013, Nature.

[4]  Elizabeth T. Wiles,et al.  BCL11B Is Up-Regulated by EWS/FLI and Contributes to the Transformed Phenotype in Ewing Sarcoma , 2013, PloS one.

[5]  Yunlong Liu,et al.  NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets , 2013, Bioinform..

[6]  R. Bell,et al.  Mechanism and relevance of EWS/FLI-mediated transcriptional repression in Ewing sarcoma , 2012, Oncogene.

[7]  Howard Y. Chang,et al.  Identification of proteins binding coding and non-coding human RNAs using protein microarrays , 2012, BMC Genomics.

[8]  David G. Knowles,et al.  The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression , 2012, Genome research.

[9]  Nadav S. Bar,et al.  Landscape of transcription in human cells , 2012, Nature.

[10]  Andrew H. Beck,et al.  Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers , 2012, Genome Biology.

[11]  Nicolò Riggi,et al.  A TARBP2-dependent miRNA expression profile underlies cancer stem cell properties and provides candidate therapeutic reagents in Ewing sarcoma. , 2012, Cancer cell.

[12]  Howard Y. Chang,et al.  Genome regulation by long noncoding RNAs. , 2012, Annual review of biochemistry.

[13]  M. Kretz,et al.  Abstract LB-248: BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration. , 2013 .

[14]  J. Lieb,et al.  Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription. , 2012, Genome research.

[15]  T. Derrien,et al.  The Long Non-Coding RNAs: A New (P)layer in the “Dark Matter” , 2012, Front. Gene..

[16]  Howard Y. Chang,et al.  Molecular mechanisms of long noncoding RNAs. , 2011, Molecular cell.

[17]  Cole Trapnell,et al.  Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. , 2011, Genes & development.

[18]  John T. Wei,et al.  Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression , 2011, Nature Biotechnology.

[19]  Howard Y. Chang,et al.  A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression , 2011, Nature.

[20]  L. Maquat,et al.  lncRNAs transactivate Staufen1-mediated mRNA decay by duplexing with 3'UTRs via Alu elements , 2010, Nature.

[21]  B. Blencowe,et al.  The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. , 2010, Molecular cell.

[22]  Howard Y. Chang,et al.  Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes , 2010, Science.

[23]  J. Rinn,et al.  A Large Intergenic Noncoding RNA Induced by p53 Mediates Global Gene Repression in the p53 Response , 2010, Cell.

[24]  Nicolò Riggi,et al.  EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells. , 2010, Genes & development.

[25]  W. Huber,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets , 2011 .

[26]  Howard Y. Chang,et al.  Long noncoding RNA HOTAIR reprograms chromatin state to promote cancer metastasis , 2010, Nature.

[27]  Andrew H. Beck,et al.  3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples , 2010, PloS one.

[28]  S. Lessnick,et al.  EWS/FLI and its downstream target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's sarcoma. , 2009, Cancer research.

[29]  Davis J. McCarthy,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[30]  Wolfgang Wagner,et al.  Aging and Replicative Senescence Have Related Effects on Human Stem and Progenitor Cells , 2009, PloS one.

[31]  E. Álava,et al.  Stable interference of EWS–FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target , 2009, British Journal of Cancer.

[32]  Mirko Francesconi,et al.  Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[33]  P. Meltzer,et al.  A Molecular Function Map of Ewing's Sarcoma , 2009, PloS one.

[34]  John N. Hutchinson,et al.  An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. , 2009, Molecular cell.

[35]  E. Barillot,et al.  The Oncogenic EWS-FLI1 Protein Binds In Vivo GGAA Microsatellite Sequences with Potential Transcriptional Activation Function , 2009, PloS one.

[36]  J. Mattick,et al.  Long non-coding RNAs: insights into functions , 2009, Nature Reviews Genetics.

[37]  H. Kovar,et al.  EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation , 2009, Proceedings of the National Academy of Sciences.

[38]  J. Einasto.  Dark Matter , 2011, Brazilian Journal of Physics.

[39]  Jennifer A. Mitchell,et al.  The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin , 2008, Science.

[40]  Stephen C. Haroldsen,et al.  Microsatellites as EWS/FLI response elements in Ewing's sarcoma , 2008, Proceedings of the National Academy of Sciences.

[41]  Natalie K. Wolf,et al.  IGF1 Is a Common Target Gene of Ewing's Sarcoma Fusion Proteins in Mesenchymal Progenitor Cells , 2008, PloS one.

[42]  C. Glass,et al.  Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription , 2008, Nature.

[43]  Stephen L. Lessnick,et al.  EWS/FLI Mediates Transcriptional Repression via NKX2.2 during Oncogenic Transformation in Ewing's Sarcoma , 2008, PloS one.

[44]  P. Bianco,et al.  Mesenchymal stem cells: revisiting history, concepts, and assays. , 2008, Cell stem cell.

[45]  M. Suvà,et al.  EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells. , 2008, Cancer research.

[46]  B. Larson,et al.  Human Multipotent Stromal Cells Undergo Sharp Transition from Division to Development in Culture , 2008, Stem cells.

[47]  B. Sacchetti,et al.  Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment , 2007, Cell.

[48]  Nicolò Riggi,et al.  Sarcomas: genetics, signalling, and cellular origins. Part 1: The fellowship of TET , 2007, The Journal of pathology.

[49]  Yong Zhang,et al.  CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine , 2007, Nucleic Acids Res..

[50]  Howard Y. Chang,et al.  Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs , 2007, Cell.

[51]  T. Gingeras,et al.  Genome-wide transcription and the implications for genomic organization , 2007, Nature Reviews Genetics.

[52]  O. Delattre,et al.  Mesenchymal stem cell features of Ewing tumors. , 2007, Cancer cell.

[53]  E. Álava,et al.  EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model , 2007, Journal of Molecular Medicine.

[54]  S. Lessnick,et al.  Expression of EWS-ETS Fusions in NIH3T3 Cells Reveals Significant Differences to Ewing’s Sarcoma , 2006, Cell cycle.

[55]  S. Lessnick,et al.  NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma , 2006, Molecular Cancer Research.

[56]  J. Ban,et al.  Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells. , 2006, Cancer research.

[57]  T. Golub,et al.  Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. , 2006, Cancer cell.

[58]  Stephen P. Jackson,et al.  hnRNP K: An HDM2 Target and Transcriptional Coactivator of p53 in Response to DNA Damage , 2005, Cell.

[59]  Q. Lin,et al.  BCL11B functionally associates with the NuRD complex in T lymphocytes to repress targeted promoter , 2005, Oncogene.

[60]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[61]  F. Lecanda,et al.  Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter. , 2005, The Biochemical journal.

[62]  Jerzy Ostrowski,et al.  hnRNP K: One protein multiple processes , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[63]  C. Denny,et al.  Functional analysis of the EWS/ETS target gene uridine phosphorylase. , 2003, Cancer research.

[64]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.

[65]  Akihiro Umezawa,et al.  Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma , 2003, Oncogene.

[66]  Christine Brun,et al.  In silico prediction of protein-protein interactions in human macrophages , 2001, BMC Research Notes.

[67]  Jeannie T. Lee,et al.  Tsix, a gene antisense to Xist at the X-inactivation centre , 1999, Nature Genetics.

[68]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[69]  D. Benayahu,et al.  Single‐Colony Derived Strains of Human Marrow Stromal Fibroblasts Form Bone After Transplantation In Vivo , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[70]  C. Denny,et al.  EAT-2 is a novel SH2 domain containing protein that is up regulated by Ewing's sarcoma EWS/FLI1 fusion gene. , 1996, Oncogene.

[71]  H. Zinszner,et al.  A novel effector domain from the RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP. , 1994, Genes & development.

[72]  C. Denny,et al.  The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1 , 1993, Molecular and cellular biology.

[73]  H. Willard,et al.  Characterization of a murine gene expressed from the inactive X chromosome , 1991, Nature.

[74]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[75]  Claude-Alain H. Roten,et al.  Fast and accurate short read alignment with Burrows–Wheeler transform , 2009, Bioinform..

[76]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[77]  Robert M. Stephens,et al.  DAVID Bioinformatics Resources : expanded annotation database and novel algorithms to better extract biology from large gene lists , 2007 .