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EWS/ETS proteins promote expression and regulate function of the homeodomain transcription factor BRN3A

Oncogene volume 29, pages 3134–3145 (2010)Cite this article

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Abstract

Ewing's sarcoma family tumors (ESFTs or EFTs) express neuronal markers, which indicates they may originate from cells at least partly committed to neuronal lineage. However, recent publications suggest EFT originates in mesenchymal stem cells, and EWS/ETS fusion proteins characteristic of EFT activate neuronal marker expression to confer a neural phenotype on EFT. Here we show that the neuronal marker BRN3A/POU4F1 is expressed abundantly at the protein level in primary EFT but not in rhabdomyosarcoma and neuroblastoma, and EFT cells exhibit high activity of the BRN3A proximal autoregulatory region. EWS/FLI-1 siRNA reduces BRN3A expression and promoter activity and EWS/ETS proteins are bound to the BRN3A locus, suggesting a direct function for EWS/ETS proteins in control of BRN3A expression. Differentiation-associated and autoregulatory activities of BRN3A are respectively impaired and altered in EFT cells, and EWS/FLI-1 siRNA can restore some BRN3A function. A potentially novel function for BRN3A in EFT cells is identified. These results extend the hypothesis that EWS/ETS proteins induce expression of neuronal markers such as BRN3A in EFT by showing that the function of those same markers may be restricted or controlled in an EWS/ETS-dependent manner.

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References

  • Arvand A, Denny CT . (2001). Biology of EWS/ETS fusions in Ewing's family tumors. Oncogene 20: 5747–5754.

    Article  CAS  Google Scholar 

  • Bailly RA, Bosselut R, Zucman J, Cormier F, Delattre O, Roussel M et al. (1994). Dna-binding and transcriptional activation properties of the Ews-Fli-1 fusion protein resulting from the T(11;22) translocation in Ewing sarcoma. Mol Cell Biol 14: 3230–3241.

    Article  CAS  Google Scholar 

  • Baird K, Davis S, Antonescu CR, Harper UL, Walker RL, Chen YD et al. (2005). Gene expression profiling of human sarcomas: insights into sarcoma biology. Cancer Res 65: 9226–9235.

    Article  CAS  Google Scholar 

  • Beauchamp E, Bulut G, Abaan O, Chen K, Merchant A, Matsui W et al. (2009). GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein. J Biol Chem 284: 9074–9082.

    Article  CAS  Google Scholar 

  • Bradford AP, Wasylyk C, Wasylyk B, GutierrezHartmann A . (1997). Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression. Mol Cell Biol 17: 1065–1074.

    Article  CAS  Google Scholar 

  • Collum RG, Fisher PE, Datta M, Mellis S, Thiele C, Huebner K et al. (1992). A novel Pou homeodomain gene specifically expressed in cells of the developing mammalian nervous-system. Nucleic Acids Res 20: 4919–4925.

    Article  CAS  Google Scholar 

  • Dauphinot L, De Oliveira C, Melot T, Sevenet N, Thomas V, Weissman BE et al. (2001). Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57(KIP2) and c-Myc expression. Oncogene 20: 3258–3265.

    Article  CAS  Google Scholar 

  • Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M et al. (1992). Gene fusion with an Ets Dna-binding domain caused by chromosome-translocation in human tumors. Nature 359: 162–165.

    Article  CAS  Google Scholar 

  • Eliazer S, Spencer J, Ye D, Olson E, Ilaria RL . (2003). Alteration of mesodermal cell differentiation by EWS/FLI-1, the oncogene implicated in Ewing's sarcoma. Mol Cell Biol 23: 482–492.

    Article  CAS  Google Scholar 

  • Eng SR, Gratwick K, Rhee JM, Fedtsova N, Gan L, Turner EE . (2001). Defects in sensory axon growth precede neuronal death in Brn3a-deficient mice. J Neurosci 21: 541–549.

    Article  CAS  Google Scholar 

  • Ensor E, Smith MD, Latchman DS . (2001). The BRN-3A transcription factor protects sensory but not sympathetic neurons from programmed cell death/apoptosis. J Biol Chem 276: 5204–5212.

    Article  CAS  Google Scholar 

  • Fedtsova NG, Turner EE . (1995). Brn-3.0 expression identifies early postmitotic CNS neurons and sensory neural precursors. Mech Dev 53: 291–304.

    Article  CAS  Google Scholar 

  • Frass B, Vassen L, Moroy T . (2002). Gene expression of the POU factor Brn-3a is regulated by two different promoters. Biochim Biophys Acta 1579: 207–213.

    Article  CAS  Google Scholar 

  • Fukuma M, Okita H, Hata J, Umezawa A . (2003). Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma. Oncogene 22: 1–9.

    Article  CAS  Google Scholar 

  • Gascoyne DM, Thomas GR, Latchman DS . (2004). The effects of Brn-3a on neuronal differentiation and apoptosis are differentially modulated by EWS and its oncogenic derivative EWS/Fli-1. Oncogene 23: 3830–3840.

    Article  CAS  Google Scholar 

  • Gonzalez I, Andreu EJ, Panizo A, Inoges S, Fontalba A, Fernandez-Luna JL et al. (2004). Imatinib inhibits proliferation of Ewing tumor cells mediated by the stem cell Factor/KIT receptor pathway, and sensitizes cells to vincristine and doxorubicin-induced apoptosis. Clin Cancer Res 10: 751–761.

    Article  CAS  Google Scholar 

  • Hahm KB, Cho KN, Lee C, Im YH, Chang J, Choi SG et al. (1999). Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein. Nat Genet 23: 222–227.

    Article  CAS  Google Scholar 

  • Hippenmeyer S, Vrieseling E, Sigrist M, Portmann T, Laengle C, Ladle DR et al. (2005). A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol 3: 878–890.

    Article  CAS  Google Scholar 

  • Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ . (2005a). Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res 65: 8984–8992.

    Article  CAS  Google Scholar 

  • Hu-Lieskovan S, Zhang JS, Wu LT, Shimada H, Schofield DE, Triche TJ . (2005b). EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing's family of tumors. Cancer Res 65: 4633–4644.

    Article  CAS  Google Scholar 

  • Jaishankar S, Zhang J, Roussel MF, Baker SJ . (1999). Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity. Oncogene 18: 5592–5597.

    Article  CAS  Google Scholar 

  • Kim S, Denny CT, Wisdom R . (2006). Cooperative DNA binding with AP-1 proteins is required for transformation by EWS-Ets fusion proteins. Mol Cell Biol 26: 2467–2478.

    Article  CAS  Google Scholar 

  • Landuzzi L, De Giovanni C, Nicoletti G, Rossi I, Ricci C, Astolfi A et al. (2000). The metastatic ability of Ewing's sarcoma cells is modulated by stem cell factor and by its receptor c-kit. Am J Pathol 157: 2123–2131.

    Article  CAS  Google Scholar 

  • Lanier J, Quina LA, Eng SR, Cox E, Turner EE . (2007). Brn3a target gene recognition in embryonic sensory neurons. Dev Biol 302: 703–716.

    Article  CAS  Google Scholar 

  • LeblondFrancillard M, Picon A, Bertagna X, deKeyzer Y . (1997). High expression of the POU factor Brn3a in aggressive neuroendocrine tumors. J Clin Endocrinol Metab 82: 89–94.

    CAS  Google Scholar 

  • Manara MC, Landuzzi L, Nanni P, Nicoletti G, Zambelli D, Lollini PL et al. (2007). Preclinical in vivo study of new insulin-like growth factor-1 receptor—specific inhibitor in Ewing's sarcoma. Clin Cancer Res 13: 1322–1330.

    Article  CAS  Google Scholar 

  • May WA, Arvand A, Thompson AD, Braun BS, Wright M, Denny CT . (1997). EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic. Nat Genet 17: 495–497.

    Article  CAS  Google Scholar 

  • May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O et al. (1993). Ewing sarcoma 11-22 translocation produces a chimeric transcription factor that requires the Dna-binding domain encoded by fli1 for transformation. Proc Natl Acad Sci USA 90: 5752–5756.

    Article  CAS  Google Scholar 

  • McEvilly RJ, Erkman L, Luo L, Sawchenko PE, Ryan AF, Rosenfeld MG . (1996). Requirement for Brn-3.0 in differentiation and survival of sensory and motor neurons. Nature 384: 574–577.

    Article  CAS  Google Scholar 

  • Nakatani F, Tanaka K, Sakimura R, Matsumoto Y, Matsunobu T, Li X et al. (2003). Identification of p21(WAF1/CIP1) as a direct target of EWS-Fli1 oncogenic fusion protein. J Biol Chem 278: 15105–15115.

    Article  CAS  Google Scholar 

  • Perez-Sanchez C, Budhram-Mahadeo VS, Latchman DS . (2002). Distinct promoter elements mediate the co-operative effect of Brn-3a and p53 on the p21 promoter and their antagonism on the Bax promoter. Nucleic Acids Res 30: 4872–4880.

    Article  CAS  Google Scholar 

  • Prieur A, Tirode F, Cohen P, Delattre O . (2004). EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3. Mol Cell Biol 24: 7275–7283.

    Article  CAS  Google Scholar 

  • Radig K, Schneider-Stock R, Rose I, Mittler U, Oda Y, Roessner A . (1998). p53 and ras mutations in Ewing's sarcoma. Pathol Res Pract 194: 157–162.

    Article  CAS  Google Scholar 

  • Riggi N, Cironi L, Provero P, Suva ML, Kaloulis K, Garcia-Echeverria C et al. (2005). Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res 65: 11459–11468.

    Article  CAS  Google Scholar 

  • Riggi N, Suva ML, Cironi L, Provero P, Tercier S, Joseph JM et al. (2008). EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal cells. Cancer Res 68: 2176–2185.

    Article  CAS  Google Scholar 

  • Rorie CJ, Thomas VD, Chen PC, Pierce HH, O'Bryan JP, Weissman BE . (2004). The Ews/Fli-1 fusion gene switches the differentiation program of neuroblastomas to ewing sarcoma/peripheral primitive neuroectodermal tumors. Cancer Res 64: 1266–1277.

    Article  CAS  Google Scholar 

  • Smith MD, Dawson SJ, Latchman DS . (1997). The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins. Mol Cell Biol 17: 345–354.

    Article  CAS  Google Scholar 

  • Smith R, Owen LA, Trem DJ, Wong JS, Jennifer SW, Golub TR et al. (2006). Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 9: 405–416.

    Article  CAS  Google Scholar 

  • Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y . (1997). EWS-FLi1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99: 239–247.

    Article  CAS  Google Scholar 

  • Theil T, Mcleanhunter S, Zornig M, Moroy T . (1993). Mouse Brn-3 family of pou transcription factors—a new aminoterminal domain is crucial for the oncogenic activity of Brn-3A. Nucleic Acids Res 21: 5921–5929.

    Article  CAS  Google Scholar 

  • Thiele CJ . (1990). Pediatric peripheral neuroectodermal tumors, oncogenes, and differentiation. Cancer Invest 8: 629–639.

    Article  CAS  Google Scholar 

  • Torchia EC, Jaishankar S, Baker SJ . (2003). Ewing tumor fusion proteins block the differentiation of pluripotent marrow stromal cells. Cancer Res 63: 3464–3468.

    CAS  PubMed  Google Scholar 

  • Trieu M, Ma A, Eng SR, Fedtsova N, Turner EE . (2003). Direct autoregulation and gene dosage compensation by POU-domain transcription factor Brn3a. Development 130: 111–121.

    Article  CAS  Google Scholar 

  • Trieu M, Rhee JM, Fedtsova N, Turner EE . (1999). Autoregulatory sequences are revealed by complex stability screening of the mouse brn-3.0 locus. J Neurosci 19: 6549–6558.

    Article  CAS  Google Scholar 

  • Welford SM, Hebert SP, Deneen B, Arvand A, Denny CT . (2001). DNA binding domain-independent pathways are involved in EWS/FLI1-mediated oncogenesis. J Biol Chem 276: 41977–41984.

    Article  CAS  Google Scholar 

  • Wood JN, Bevan SJ, Coote PR, Dunn PM, Harmar A, Hogan P et al. (1990). Novel cell-lines display properties of nociceptive sensory neurons. Proc R Soc Lond B Biol Sci 241: 187–194.

    Article  CAS  Google Scholar 

  • Xiang MQ, Gan L, Zhou LJ, Klein WH, Nathans J . (1996). Targeted deletion of the mouse POU domain gene Brn-3a causes a selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling. Proc Natl Acad Sci USA 93: 11950–11955.

    Article  CAS  Google Scholar 

  • Yamaguchi S, Yamazaki Y, Ishikawa Y, Noriyoshi KI, Mukai H, Nakamura T . (2005). EWSR1 is fused to POU5F1 in a bone tumor with translocation t(6;22)(p21;q12). Genes Chromosomes Cancer 43: 217–222.

    Article  CAS  Google Scholar 

  • Yeny CT, Eliazer S, Xiang LL, Richardson JA, Ilaria RL . (2005). Expression of the EWS/FLI-1 oncogene in murine primary bone-derived cells results in EWS/FLI-1-dependent, Ewing sarcoma-like tumors. Cancer Res 65: 8698–8705.

    Article  Google Scholar 

  • Zhang JS, Hu SW, Schofield DE, Sorensen PHB, Triche TJ . (2004). Selective usage of D-type cyclins by Ewing's tumors and rhabdomyosarcomas. Cancer Res 64: 6026–6034.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Sue Burchill (Leeds, UK) and Peter Houghton (Memphis, USA) for cell lines, Eric Turner (San Diego, USA) for α-Brn3a antiserum, Seong-Jin Kim (Bethesda, USA) for TGFβRII (−1670/+36), Sian Gibson and Dyanne Rampling for technical assistance and James Diss for PC-3 cells and useful discussions. Funding was provided by MRC and BBSRC (DMG and DSL), and CR-UK (JD).

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Authors and Affiliations

  1. Molecular Haematology and Cancer Biology Unit, University College London Institute of Child Health, London, UK

    D M Gascoyne, S Behjati & J Anderson

  2. Cancer Research UK Medical Oncology Laboratory, Barts and the London School of Medicine, London, UK

    J Dunne

  3. Department of Paediatric Pathology, Great Ormond Street Hospital, London, UK

    N J Sebire

  4. Birkbeck College, University of London, London, UK

    D S Latchman

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  1. D M Gascoyne
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Correspondence to D M Gascoyne.

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Gascoyne, D., Dunne, J., Behjati, S. et al. EWS/ETS proteins promote expression and regulate function of the homeodomain transcription factor BRN3A. Oncogene 29, 3134–3145 (2010). https://doi.org/10.1038/onc.2010.72

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