title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.
abstract: BACKGROUND
  The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells.
  
  
  METHODS
  U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice.
  
  
  RESULTS
  Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis.
  
  
  CONCLUSIONS
  Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. gerwin.heller@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. gerwin.heller@meduniwien.ac.at.
authors.forename: Gerwin
authors.initials: G
authors.lastname: Heller
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. apm.rommer@gmail.com.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. apm.rommer@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Rommer
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. katarina.steinleitner@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. katarina.steinleitner@meduniwien.ac.at.
authors.forename: Katarina
authors.initials: K
authors.lastname: Steinleitner
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. julia.etzler@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. julia.etzler@meduniwien.ac.at.
authors.forename: Julia
authors.initials: J
authors.lastname: Etzler
authors.affiliation: Biocenter, Division of Bioinformatics, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria. hubert.hackl@i-med.ac.at.
authors.forename: Hubert
authors.initials: H
authors.lastname: Hackl
authors.affiliation: Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. petra.heffeter@meduniwien.ac.at.|Department of Medicine I, Institute of Cancer Research, and Research Platform "Translational Cancer Therapy Research", Borschkegasse 8A, 1090, Vienna, Austria. petra.heffeter@meduniwien.ac.at.
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. erwin.tomasich@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. erwin.tomasich@meduniwien.ac.at.
authors.forename: Erwin
authors.initials: E
authors.lastname: Tomasich
authors.affiliation: Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. martin.filipits@meduniwien.ac.at.|Department of Medicine I, Institute of Cancer Research, and Research Platform "Translational Cancer Therapy Research", Borschkegasse 8A, 1090, Vienna, Austria. martin.filipits@meduniwien.ac.at.
authors.forename: Martin
authors.initials: M
authors.lastname: Filipits
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. birgit.steinmetz@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. birgit.steinmetz@meduniwien.ac.at.
authors.forename: Birgit
authors.initials: B
authors.lastname: Steinmetz
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. thais.topakian@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. thais.topakian@meduniwien.ac.at.
authors.forename: Thais
authors.initials: T
authors.lastname: Topakian
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. sim.kling@gmx.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. sim.kling@gmx.at.
authors.forename: Simone
authors.initials: S
authors.lastname: Klingenbrunner
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. barbara.ziegler@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. barbara.ziegler@meduniwien.ac.at.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ziegler
authors.affiliation: Core Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. andreas.spittler@meduniwien.ac.at.
authors.forename: Andreas
authors.initials: A
authors.lastname: Spittler
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. sabine.zoechbauer-mueller@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. sabine.zoechbauer-mueller@meduniwien.ac.at.
authors.forename: Sabine
authors.initials: S
authors.lastname: Zöchbauer-Müller
authors.affiliation: Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. walter.berger@meduniwien.ac.at.|Department of Medicine I, Institute of Cancer Research, and Research Platform "Translational Cancer Therapy Research", Borschkegasse 8A, 1090, Vienna, Austria. walter.berger@meduniwien.ac.at.
authors.forename: Walter
authors.initials: W
authors.lastname: Berger
authors.affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria. rotraud.wieser@meduniwien.ac.at.|Comprehensive Cancer Center of the Medical University of Vienna, Vienna, Austria. rotraud.wieser@meduniwien.ac.at.
authors.forename: Rotraud
authors.initials: R
authors.lastname: Wieser
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D055785:Gene Knockdown Techniques; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000074008:MDS1 and EVI1 Complex Locus Protein; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D020411:Oligonucleotide Array Sequence Analysis; D011519:Proto-Oncogenes / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; C089821:MS4A3 protein, human; D008565:Membrane Proteins; D014157:Transcription Factors