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GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

Abstract

Ewing's sarcoma is a malignant bone-associated tumor of children and young adults. Most cases of Ewing's sarcoma express the EWS/FLI fusion protein. EWS/FLI functions as an aberrant ETS-type transcription factor and serves as the master regulator of Ewing's sarcoma-transformed phenotype. We recently showed that EWS/FLI regulates one of its key targets, NR0B1, through a GGAA-microsatellite in its promoter. Whether other critical EWS/FLI targets are also regulated by GGAA-microsatellites was unknown. In this study, we combined transcriptional analysis, whole genome localization data, and RNA interference knockdown to identify glutathione S-transferase M4 (GSTM4) as a critical EWS/FLI target gene in Ewing's sarcoma. We found that EWS/FLI directly binds the GSTM4 promoter, and regulates GSTM4 expression through a GGAA-microsatellite in its promoter. Reduction of GSTM4 levels caused a loss of oncogenic transformation. Furthermore, reduction of GSTM4 resulted in an increased sensitivity of Ewing's sarcoma cells to chemotherapeutic agents, suggesting a role for this protein in drug resistance. Consistent with this hypothesis, patients with Ewing's sarcoma whose tumors had higher levels of GSTM4 expression had worse outcomes than those with lower expression levels. These data show that GSTM4 contributes to the cancerous behavior of Ewing's sarcoma and define a wider role for GGAA-microsatellites in EWS/FLI function than previously appreciated. These data also suggest a novel therapeutic resistance mechanism, in which the central oncogenic abnormality directly regulates a resistance gene.

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References

  • Andersen PK, Gill RD . (1982). Cox regression model for counting processes: A large sample study. Ann Stat 10: 1100–1120.

    Article  Google Scholar 

  • Camp RL, Neumeister V, Rimm DL . (2008). A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers. J Clin Oncol 26: 5630–5637.

    Article  Google Scholar 

  • Comstock KE, Johnson KJ, Rifenbery D, Henner WD . (1993). Isolation and analysis of the gene and cDNA for a human Mu class glutathione S-transferase, GSTM4. J Biol Chem 268: 16958–16965.

    CAS  PubMed  Google Scholar 

  • Comstock KE, Widersten M, Hao XY, Henner WD, Mannervik B . (1994). A comparison of the enzymatic and physicochemical properties of human glutathione transferase M4-4 and three other human Mu class enzymes. Arch Biochem Biophys 311: 487–495.

    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 

  • Dubois SG, Krailo MD, Lessnick SL, Smith R, Chen Z, Marina N et al. (2008). Phase II study of intermediate-dose cytarabine in patients with relapsed or refractory Ewing sarcoma: a report from the Children's Oncology Group. Pediatr Blood Cancer 52: 324–327.

    Article  Google Scholar 

  • Gangwal K, Lessnick SL . (2008). Microsatellites are EWS/FLI response elements: genomic ‘junk’ is EWS/FLI's treasure. Cell Cycle 7: 3127–3132.

    Article  CAS  Google Scholar 

  • Gangwal K, Sankar S, Hollenhorst PC, Kinsey M, Haroldsen SC, Shah AA et al. (2008). Microsatellites as EWS/FLI response elements in Ewing's sarcoma. Proc Natl Acad Sci USA 105: 10149–10154.

    Article  CAS  Google Scholar 

  • Grambsch PM, Therneau TM . (1994). Proportional hazards tests and diagnostics based on weighted residuals. Biometrika 81: 515–526.

    Article  Google Scholar 

  • Hancock JD, Lessnick SL . (2008). A transcriptional profiling meta-analysis reveals a core EWS-FLI gene expression signature. Cell Cycle 7: 250–256.

    Article  CAS  Google Scholar 

  • Hayes JD, Strange RC . (2000). Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 61: 154–166.

    Article  CAS  Google Scholar 

  • Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ . (2005). 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 

  • 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  Google Scholar 

  • Lessnick SL, Dacwag CS, Golub TR . (2002). The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts. Cancer Cell 1: 393–401.

    Article  CAS  Google Scholar 

  • Liloglou T, Walters M, Maloney P, Youngson J, Field JK . (2002). A T2517C polymorphism in the GSTM4 gene is associated with risk of developing lung cancer. Lung Cancer 37: 143–146.

    Article  Google Scholar 

  • Magwere T, Myatt SS, Burchill SA . (2008). Manipulation of oxidative stress to induce cell death in Ewing's sarcoma family of tumours. Eur J Cancer 44: 2276–2287.

    Article  CAS  Google Scholar 

  • May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O et al. (1993a). 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 

  • May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB et al. (1993b). The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 13: 7393–7398.

    Article  CAS  Google Scholar 

  • Myatt SS, Burchill SA . (2008). The sensitivity of the Ewing's sarcoma family of tumours to fenretinide-induced cell death is increased by EWS-Fli1-dependent modulation of p38(MAPK) activity. Oncogene 27: 985–996.

    Article  CAS  Google Scholar 

  • Owen LA, Kowalewski AA, Lessnick SL . (2008). EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma. PLoS ONE 3: e1965.

    Article  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 

  • Smith R, Owen LA, Trem DJ, Wong JS, Whangbo JS, 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 

  • Stegmaier K, Wong JS, Ross KN, Chow KT, Peck D, Wright RD et al. (2007). Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS Med 4: e122.

    Article  Google Scholar 

  • Tirado OM, Mateo-Lozano S, Villar J, Dettin LE, Llort A, Gallego S et al. (2006). Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells. Cancer Res 66: 9937–9947.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Andrea Bild and Matt Topham for critical reading of the manuscript; Nikko Ronquillo, Mohan Kaadige and Kym Zumbrennen for technical assistance; and Elizabeth Leibold and members of the Lessnick laboratory for helpful discussions. This work was supported by funds awarded to SLL from the Terri Anna Perine Sarcoma Fund, the Liddy Shriver Sarcoma Initiative, the Sunbeam Foundation and Huntsman Cancer Institute/Huntsman Cancer Foundation, and by a grant to WL from the Alex's Lemonade Stand Foundation. We also acknowledge the NIH support to the Huntsman Cancer Institute (P30 CA042014).

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Correspondence to S L Lessnick.

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Luo, W., Gangwal, K., Sankar, S. et al. GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance. Oncogene 28, 4126–4132 (2009). https://doi.org/10.1038/onc.2009.262

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Keywords

  • Ewing's sarcoma
  • EWS/FLI
  • GSTM4
  • transformation

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