title: High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.
abstract: A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.
authors.affiliation: VTT Technical Research Centre of Finland, Turku, Finland.
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.affiliation: Institut Curie, Unité de génétique somatique, Paris, France; Genetics and Biology of Cancers Unit, Institut Curie, PSL Research University, Paris, France; INSERM U830, Institut Curie Research Center, Paris, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Surdez
authors.affiliation: Misvik Biology, Turku, Finland.
authors.forename: Juha K
authors.initials: JK
authors.lastname: Rantala
authors.affiliation: VTT Technical Research Centre of Finland, Turku, Finland.
authors.forename: Saija
authors.initials: S
authors.lastname: Haapa-Paananen
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Jozef
authors.initials: J
authors.lastname: Ban
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Kauer
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Eleni
authors.initials: E
authors.lastname: Tomazou
authors.affiliation: VTT Technical Research Centre of Finland, Turku, Finland.
authors.forename: Vidal
authors.initials: V
authors.lastname: Fey
authors.affiliation: Unidad de Tumores Sólidos Infantiles, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Alonso
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria; Department of Pediatrics, Medical University, Vienna, Austria.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Kovar
authors.affiliation: Institut Curie, Unité de génétique somatique, Paris, France; Genetics and Biology of Cancers Unit, Institut Curie, PSL Research University, Paris, France; INSERM U830, Institut Curie Research Center, Paris, France; Institut Curie Genomics of Excellence (ICGex) Platform, Institut Curie Research Center, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Delattre
authors.affiliation: VTT Technical Research Centre of Finland, Turku, Finland. Electronic address: Kristiina.iljin@vtt.fi.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Iljin
mesh_terms: D001859:Bone Neoplasms / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008868:Microtubule Proteins / Q000235:genetics* / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034802:RNA-Binding Protein EWS / Q000235:genetics* / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D000071678:WD40 Repeats
chemical_list: C106031:EWS-FLI fusion protein; C000595278:LRWD1 protein, human; D008868:Microtubule Proteins; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D004318:Doxycycline
keywords: DNA replication; EWS-FLI1; Ewing sarcoma; LRWD1; RNA interference; WD repeat