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:

1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma

Abstract

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.

We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

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
Figure 6

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Abbas T, Sivaprasad U, Terai K, Amador V, Pagano M, Dutta A . (2008). PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex. Genes Dev 22: 2496–2506.

    Article  CAS  Google Scholar 

  • Aurias A, Rimbaut C, Buffe D, Zucker JM, Mazabraud A . (1984). Translocation involving chromosome 22 in Ewing's sarcoma. A cytogenetic study of four fresh tumors. Cancer Genet Cytogenet 12: 21–25.

    Article  CAS  Google Scholar 

  • Balcarkova J, Urbankova H, Scudla V, Holzerova M, Bacovsky J, Indrak K et al. (2009). Gain of chromosome arm 1q in patients in relapse and progression of multiple myeloma. Cancer Genet Cytogenet 192: 68–72.

    Article  CAS  Google Scholar 

  • Banks D, Wu M, Higa LA, Gavrilova N, Quan J, Ye T et al. (2006). L2DTL/CDT2 and PCNA interact with p53 and regulate p53 polyubiquitination and protein stability through MDM2 and CUL4A/DDB1 complexes. Cell Cycle 5: 1719–1729.

    Article  CAS  Google Scholar 

  • Brisset S, Schleiermacher G, Peter M, Mairal A, Oberlin O, Delattre O et al. (2001). CGH analysis of secondary genetic changes in Ewing tumors: correlation with metastatic disease in a series of 43 cases. Cancer Genet Cytogenet 130: 57–61.

    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 tumours. Nature 359: 162–165.

    Article  CAS  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  Google Scholar 

  • Friedrichs N, Kriegl L, Poremba C, Schaefer KL, Gabbert HE, Shimomura A et al. (2006). Pitfalls in the detection of t(11;22) translocation by fluorescence in situ hybridization and RT-PCR: a single-blinded study. Diagn Mol Pathol 15: 83–89.

    Article  CAS  Google Scholar 

  • Haas AL, Rose IA . (1982). The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 257: 10329–10337.

    CAS  PubMed  Google Scholar 

  • Haeusler J, Ranft A, Boelling T, Gosheger G, Braun-Munzinger G, Vieth V et al. (2010). The value of local treatment in patients with primary, disseminated, multifocal Ewing sarcoma (PDMES). Cancer 116: 443–450.

    Article  Google Scholar 

  • Hattinger CM, Rumpler S, Ambros IM, Strehl S, Lion T, Zoubek A et al. (1996). Demonstration of the translocation der(16)t(1;16)(q12;q11.2) in interphase nuclei of Ewing tumors. Genes Chromosomes Cancer 17: 141–150.

    Article  CAS  Google Scholar 

  • Hattinger CM, Potschger U, Tarkkanen M, Squire J, Zielenska M, Kiuru-Kuhlefelt S et al. (2002). Prognostic impact of chromosomal aberrations in Ewing tumours. Br J Cancer 86: 1763–1769.

    Article  CAS  Google Scholar 

  • Higa LA, Banks D, Wu M, Kobayashi R, Sun H, Zhang H . (2006a). L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5: 1675–1680.

    Article  CAS  Google Scholar 

  • Higa LA, Wu M, Ye T, Kobayashi R, Sun H, Zhang H . (2006b). CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation. Nat Cell Biol 8: 1277–1283.

    Article  CAS  Google Scholar 

  • Higa LA, Yang X, Zheng J, Banks D, Wu M, Ghosh P et al. (2006c). Involvement of CUL4 ubiquitin E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation. Cell Cycle 5: 71–77.

    Article  CAS  Google Scholar 

  • Hing S, Lu YJ, Summersgill B, King-Underwood L, Nicholson J, Grundy P et al. (2001). Gain of 1q is associated with adverse outcome in favorable histology Wilms’ tumors. Am J Pathol 158: 393–398.

    Article  CAS  Google Scholar 

  • Huang HY, Illei PB, Zhao Z, Mazumdar M, Huvos AG, Healey JH et al. (2005). Ewing sarcomas with p53 mutation or p16/p14ARF homozygous deletion: a highly lethal subset associated with poor chemoresponse. J Clin Oncol 23: 548–558.

    Article  CAS  Google Scholar 

  • Jin J, Arias EE, Chen J, Harper JW, Walter JC . (2006). A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 23: 709–721.

    Article  CAS  Google Scholar 

  • Kauer M, Ban J, Kofler R, Walker B, Davis S, Meltzer P et al. (2009). A molecular function map of Ewing's sarcoma. PLoS ONE 4: e5415.

    Article  Google Scholar 

  • Kim Y, Starostina NG, Kipreos ET . (2008). The CRL4Cdt2 ubiquitin ligase targets the degradation of p21Cip1 to control replication licensing. Genes Dev 22: 2507–2519.

    Article  CAS  Google Scholar 

  • Kjellman P, Lagercrantz S, Hoog A, Wallin G, Larsson C, Zedenius J . (2001). Gain of 1q and loss of 9q21.3-q32 are associated with a less favorable prognosis in papillary thyroid carcinoma. Genes Chromosomes Cancer 32: 43–49.

    Article  CAS  Google Scholar 

  • Knuutila S, Armengol G, Bjorkqvist AM, el-Rifai W, Larramendy ML, Monni O et al. (1998). Comparative genomic hybridization study on pooled DNAs from tumors of one clinical-pathological entity. Cancer Genet Cytogenet 100: 25–30.

    Article  CAS  Google Scholar 

  • Kullendorff CM, Mertens F, Donner M, Wiebe T, Akerman M, Mandahl N . (1999). Cytogenetic aberrations in Ewing sarcoma: are secondary changes associated with clinical outcome? Med Pediatr Oncol 32: 79–83.

    Article  CAS  Google Scholar 

  • Le Deley MC, Delattre O, Schaefer KL, Burchill SA, Koehler G, Hogendoorn PC et al. (2010). Impact of EWS-ETS fusion type on disease progression in Ewing's sarcoma/peripheral primitive neuroectodermal tumor: prospective results from the cooperative Euro-E.W.I.N.G. 99. Trial J Clin Oncol 28: 1982–1988.

    Article  CAS  Google Scholar 

  • Liu CL, Yu IS, Pan HW, Lin SW, Hsu HC . (2007). L2dtl is essential for cell survival and nuclear division in early mouse embryonic development. J Biol Chem 282: 1109–1118.

    Article  CAS  Google Scholar 

  • Lo KC, Ma C, Bundy BN, Pomeroy SL, Eberhart CG, Cowell JK . (2007). Gain of 1q is a potential univariate negative prognostic marker for survival in medulloblastoma. Clin Cancer Res 13: 7022–7028.

    Article  CAS  Google Scholar 

  • Nishitani H, Shiomi Y, Iida H, Michishita M, Takami T, Tsurimoto T . (2008). CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation. J Biol Chem 283: 29045–29052.

    Article  CAS  Google Scholar 

  • Ordoñez JL, Osuna D, Herrero D, de Alava E, Madoz-Gurpide J . (2009). Advances in Ewing's sarcoma research: where are we now and what lies ahead? Cancer Res 69: 7140–7150.

    Article  Google Scholar 

  • Ottaviano L, Schaefer KL, Gajewski M, Huckenbeck W, Baldus S, Rogel U et al. (2010). Molecular characterization of commonly used cell lines for bone tumor research: a trans-European EuroBoNet effort. Genes Chromosomes Cancer 49: 40–51.

    Article  CAS  Google Scholar 

  • Ozaki T, Paulussen M, Poremba C, Brinkschmidt C, Rerin J, Ahrens S et al. (2001). Genetic imbalances revealed by comparative genomic hybridization in Ewing tumors. Genes Chromosomes Cancer 32: 164–171.

    Article  CAS  Google Scholar 

  • Pezzolo A, Rossi E, Gimelli S, Parodi F, Negri F, Conte M et al. (2009). Presence of 1q gain and absence of 7p gain are new predictors of local or metatastic relapse in localized resectable neuroblastoma. Neuro Oncol 11: 192–200.

    Article  CAS  Google Scholar 

  • Ralph E, Boye E, Kearsey SE . (2006). DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1. EMBO Rep 7: 1134–1139.

    Article  CAS  Google Scholar 

  • Roberts P, Burchill SA, Brownhill S, Cullinane CJ, Johnston C, Griffiths MJ et al. (2008). Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children′s Cancer and Leukaemia Group. Genes Chromosomes Cancer 47: 207–220.

    Article  CAS  Google Scholar 

  • Sansam CL, Shepard JL, Lai K, Ianari A, Danielian PS, Amsterdam A et al. (2006). DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint. Genes Dev 20: 3117–3129.

    Article  CAS  Google Scholar 

  • Savola S, Klami A, Tripathi A, Niini T, Serra M, Picci P et al. (2009). Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors. BMC Cancer 9: 17.

    Article  Google Scholar 

  • Scotlandi K, Remondini D, Castellani G, Manara MC, Nardi F, Cantiani L et al. (2009). Overcoming resistance to conventional drugs in Ewing sarcoma and identification of molecular predictors of outcome. J Clin Oncol 27: 2209–2216.

    Article  CAS  Google Scholar 

  • Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S et al. (2009). An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458: 732–736.

    Article  CAS  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  Google Scholar 

  • Tarkkanen M, Kiuru-Kuhlefelt S, Blomqvist C, Armengol G, Bohling T, Ekfors T et al. (1999). Clinical correlations of genetic changes by comparative genomic hybridization in Ewing sarcoma and related tumors. Cancer Genet Cytogenet 114: 35–41.

    Article  CAS  Google Scholar 

  • Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM . (1984). Chromosome study of Ewing's sarcoma (ES) cell lines. Consistency of a reciprocal translocation t(11;22)(q24;q12). Cancer Genet Cytogenet 12: 1–19.

    Article  CAS  Google Scholar 

  • van de Wiel MA, Smeets SJ, Brakenhoff RH, Ylstra B . (2005). CGHMultiArray: exact P-values for multi-array comparative genomic hybridization data. Bioinformatics 21: 3193–3194.

    Article  CAS  Google Scholar 

  • van de Wiel MA, Kim KI, Vosse SJ, van Wieringen WN, Wilting SM, Ylstra B . (2007). CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics 23: 892–894.

    Article  CAS  Google Scholar 

  • van de Wiel MA, Wieringen WN . (2007). CGHregions: dimension reduction for array CGH data with minimal information loss. Cancer inform 3: 55–63.

    Article  Google Scholar 

  • van Doorninck JA, Ji L, Schaub B, Shimada H, Wing MR, Krailo MD et al. (2010). Current treatment protocols have eliminated the prognostic advantage of type 1 fusions in Ewing sarcoma: a report from the Children′s Oncology Group. J Clin Oncol 28: 1989–1994.

    Article  Google Scholar 

  • Whang-Peng J, Triche TJ, Knutsen T, Miser J, Douglass EC, Israel MA . (1984). Chromosome translocation in peripheral neuroepithelioma. N Engl J Med 311: 584–585.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Centro de Investigación del Cáncer-IBMCC, Heinrich-Heine-University Duesseldorf, Catholic University of Leuven, University of Valencia, Istituti Ortopedici Rizzoli, University of Helsinki, Leiden University Medical Center and University Children's Hospital Muenster are partners of the EuroBoNeT consortium, a network of excellence granted by the European Commission for studying the pathology and genetics of bone tumors. Daniel J Garcia-Domínguez is supported by a grant from the Maria Garcia-Estrada Foundation. Research in Enrique de Alava's lab is also supported by the Ministry of Science and Innovation of Spain-FEDER (PI081828, RD06/0020/0059). Trial center Muenster is supported by grants from Deutsche Krebshilfe, 50-2551 Jü3 und 50-2551-Jü4 and Federal Ministry of Education and Research Germany, BMBF (TranSaRNet) 01GM0869, Deutsches Zentrum für Luft- und Raumfahrt e.V. We thank Dr Hung-Wei Pan and Dr Hey-Chi Hsu (Department of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan) for the anti-CDT2 antibody and pBABE-CDT2 construction. Thanks are due to Cristina Teodosio and Martín Perez de Andrés for their kind help with the flow cytometer, to Andreas Ranft for helping with the clinical data, to Agustín Mayo Iscar for his help with statistics, and to Antonella Chiechi, Alicia Ginel and Alberto de Luis for their advice.

Author contributions:C Mackintosh made the experimental design, carried out most of the experiments, analyzed the data, wrote the R code, obtained the conclusions and wrote the manuscript; JL Ordóñez and DJ García manipulated the animals; V Sevillano replicated and amplified the shRNA construction collection and other retro and lentiviral vectors; A Llombart-Bosch performed and evaluated the Ki-67 IHC; K Szuhai carried out the COBRA-FISH analysis. M Debiec-Rychter, KL Schaefer, K Scotlandi, P Picci, U Dirksen, R Sciot, M Alberghini, F Sinnaeve, S Knuutila and PCW Hogendoorn supplied tumor samples, cell lines, clinical information, raw data from microarrays and revised the manuscript. E de Alava supervised the studies, coordinated the joint effort and revised the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to C Mackintosh or E de Álava.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mackintosh, C., Ordóñez, J., García-Domínguez, D. et al. 1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma. Oncogene 31, 1287–1298 (2012). https://doi.org/10.1038/onc.2011.317

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.317

Keywords

This article is cited by

Search

Quick links