Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells

Abstract

The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT–PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Altincicek B, Tenbaum SP, Dressel U, Thormeyer D, Renkawitz R, Baniahmad A . (2000). Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita. J Biol Chem 275: 7662–7667.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM et al. (2000). Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet 25: 25–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bardoni B, Zanaria E, Guioli S, Floridia G, Worley KC, Tonini G et al. (1994). A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal. Nat Genet 7: 497–501.

    Article  CAS  PubMed  Google Scholar 

  • Carrillo J, Garcia-Aragoncillo E, Azorin D, Agra N, Sastre A, Gonzalez-Mediero I et al. (2007). Cholecystokinin down-regulation by RNA interference impairs Ewing tumor growth. Clin Cancer Res 13: 2429–2440.

    Article  CAS  PubMed  Google Scholar 

  • Crawford PA, Dorn C, Sadovsky Y, Milbrandt J . (1998). Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 18: 2949–2956.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Alava E, Gerald WL . (2000). Molecular biology of the Ewing's sarcoma/primitive neuroectodermal tumor family. J Clin Oncol 18: 204–213.

    Article  CAS  PubMed  Google Scholar 

  • Ferreira BI, Alonso J, Carrillo J, Acquadro F, Largo C, Suela J et al. (2008). Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma. Oncogene 27: 2084–2090.

    Article  CAS  PubMed  Google Scholar 

  • Guo W, Burris TP, Zhang YH, Huang BL, Mason J, Copeland KC et al. (1996). Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. J Clin Endocrinol Metab 81: 2481–2486.

    CAS  PubMed  Google Scholar 

  • Hahm KB, Cho K, 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  PubMed  Google Scholar 

  • Holter E, Kotaja N, Makela S, Strauss L, Kietz S, Janne OA et al. (2002). Inhibition of androgen receptor (AR) function by the reproductive orphan nuclear receptor DAX-1. Mol Endocrinol 16: 515–528.

    Article  CAS  PubMed  Google Scholar 

  • Hosack DA, Dennis Jr G, Sherman BT, Lane HC, Lempicki RA . (2003). Identifying biological themes within lists of genes with EASE. Genome Biol 4: R70.

    Article  PubMed  PubMed Central  Google Scholar 

  • Ito M, Yu R, Jameson JL . (1997). DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17: 1476–1483.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Janknecht R . (2005). EWS-ETS oncoproteins: the linchpins of Ewing tumors. Gene 363: 1–14.

    Article  CAS  PubMed  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  PubMed  PubMed Central  Google Scholar 

  • Kinsey M, Smith R, Lessnick SL . (2006). NR0B1 is required for the oncogenic phenotype mediated by EWS/FLI in Ewing's sarcoma. Mol Cancer Res 4: 851–859.

    Article  CAS  PubMed  Google Scholar 

  • Kovar H . (1998). Ewing's sarcoma and peripheral primitive neuroectodermal tumors after their genetic union. Curr Opin Oncol 10: 334–342.

    Article  CAS  PubMed  Google Scholar 

  • Kovar H . (2005). Context matters: the hen or egg problem in Ewing's sarcoma. Semin Cancer Biol 15: 189–196.

    Article  CAS  PubMed  Google Scholar 

  • Lalli E, Sassone-Corsi P . (2003). DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation. Mol Endocrinol 17: 1445–1453.

    Article  CAS  PubMed  Google Scholar 

  • Luckow B, Schutz G . (1987). CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res 15: 5490.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mendiola M, Carrillo J, Garcia E, Lalli E, Hernandez T, De Alava E et al. (2006). The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer 118: 1381–1389.

    Article  CAS  PubMed  Google Scholar 

  • Nakatani F, Tanaka K, Sakimura R, Matsumoto Y, Matsunobu T, Li X et al. (2003). Identification of p21WAF1/CIP1 as a direct target of EWS-Fli1 oncogenic fusion protein. J Biol Chem 278: 15105–15115.

    Article  CAS  PubMed  Google Scholar 

  • Nishimori H, Sasaki Y, Yoshida K, Irifune H, Zembutsu H, Tanaka T et al. (2002). The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors. Oncogene 21: 8302–8309.

    Article  CAS  PubMed  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  PubMed  PubMed Central  Google Scholar 

  • Reiner A, Yekutieli D, Benjamini Y . (2003). Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19: 368–375.

    Article  CAS  PubMed  Google Scholar 

  • Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N et al. (2003). TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34: 374–378.

    Article  CAS  PubMed  Google Scholar 

  • Song KH, Park YY, Park KC, Hong CY, Park JH, Shong M et al. (2004). The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation. Mol Endocrinol 18: 1929–1940.

    Article  CAS  PubMed  Google Scholar 

  • Staege MS, Hutter C, Neumann I, Foja S, Hattenhorst UE, Hansen G et al. (2004). DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets. Cancer Res 64: 8213–8221.

    Article  CAS  PubMed  Google Scholar 

  • Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102: 15545–15550.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suzuki T, Kasahara M, Yoshioka H, Umesono K, Morohashi K . (2002). LXXLL motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Endocr Res 28: 537.

    Article  PubMed  Google Scholar 

  • Tamai KT, Monaco L, Alastalo TP, Lalli E, Parvinen M, Sassone-Corsi P . (1996). Hormonal and developmental regulation of DAX-1 expression in Sertoli cells. Mol Endocrinol 10: 1561–1569.

    CAS  PubMed  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  PubMed  PubMed Central  Google Scholar 

  • Wasylyk B, Hahn SL, Giovane A . (1993). The Ets family of transcription factors. Eur J Biochem 211: 7–18.

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  • Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W et al. (1994). An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372: 635–641.

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Thomsen JS, Johansson L, Gustafsson JA, Treuter E . (2000). DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J Biol Chem 275: 39855–39859.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to B Bardoni for gift of the pDAX-1 PROM.CAT plasmid and CT Denny for gift of the pSRα EWS/FLI1 and the EWS/FLI1 mutants pSRα EWS/FLI1 Δ65 and pSRα EWS/FLI1 triple mutant used in this study. This study was funded by Ministerio de Educación y Ciencia grants SAF2006-07586 and SAF2007-62101; Fundación Inocente Inocente, Fundación Enriqueta Villavecchia and Fundación Científica Asociación Española Contra el Cáncer. E Lalli is supported by Fondation pour la Recherche Médicale, Association Recherche sur le Cancer and a CNRS-ATIP grant; E García-Aragoncillo and J Carrillo are predoctoral fellows from the Ministerio de Educación y Ciencia. N Agra is a predoctoral fellow from the Fundación General de la U.A.M; G Gómez-López is supported by a contract from de Fondo de Investigaciones Sanitarias; J Alonso has been supported by a Ramón y Cajal contract from the Ministerio de Educación y Ciencia.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J Alonso.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

García-Aragoncillo, E., Carrillo, J., Lalli, E. et al. DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells. Oncogene 27, 6034–6043 (2008). https://doi.org/10.1038/onc.2008.203

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2008.203

Keywords

This article is cited by

Search

Quick links