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. 1997 Jan 15;99(2):239–247. doi: 10.1172/JCI119152

EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells.

K Tanaka 1, T Iwakuma 1, K Harimaya 1, H Sato 1, Y Iwamoto 1
PMCID: PMC507791  PMID: 9005992

Abstract

The translocation t(11;22) is a common chromosomal abnormality detected both in Ewing's sarcoma and in primitive neuroectodermal tumor cells. The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated possible roles of the fusion gene products. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive polymerase chain reaction technique, we show here that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of Ewing's sarcoma and primitive neuroectodermal tumor cells. When the EWS-Fli1 expression is inhibited by antisense oligodeoxynucleotides against the fusion RNA, the growth of the tumor cells is significantly reduced both in vitro and in vivo. The data further indicate the growth inhibition of the cells by the antisense sequence might be mediated by G0/G1 block in the cell cycle progression. These results suggest that EWS-Fli1 may play an important role in the proliferation of the tumor cells, and the EWS-Fli1 fusion RNA could be used as a target to inhibit the growth of Ewing's sarcoma and primitive neuroectodermal tumor with the specific antisense oligonucleotide.

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Selected References

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  1. Bailly R. A., Bosselut R., Zucman J., Cormier F., Delattre O., Roussel M., Thomas G., Ghysdael J. 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. 1994 May;14(5):3230–3241. doi: 10.1128/mcb.14.5.3230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barr F. G., Galili N., Holick J., Biegel J. A., Rovera G., Emanuel B. S. Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993 Feb;3(2):113–117. doi: 10.1038/ng0293-113. [DOI] [PubMed] [Google Scholar]
  3. Braun B. S., Frieden R., Lessnick S. L., May W. A., Denny C. T. Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis. Mol Cell Biol. 1995 Aug;15(8):4623–4630. doi: 10.1128/mcb.15.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burdach S., Jürgens H., Peters C., Nürnberger W., Mauz-Körholz C., Körholz D., Paulussen M., Pape H., Dilloo D., Koscielniak E. Myeloablative radiochemotherapy and hematopoietic stem-cell rescue in poor-prognosis Ewing's sarcoma. J Clin Oncol. 1993 Aug;11(8):1482–1488. doi: 10.1200/JCO.1993.11.8.1482. [DOI] [PubMed] [Google Scholar]
  5. Cavazzana A. O., Miser J. S., Jefferson J., Triche T. J. Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol. 1987 Jun;127(3):507–518. [PMC free article] [PubMed] [Google Scholar]
  6. Chen G., Jaffrézou J. P., Fleming W. H., Durán G. E., Sikic B. I. Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection. Cancer Res. 1994 Sep 15;54(18):4980–4987. [PubMed] [Google Scholar]
  7. Clark J., Rocques P. J., Crew A. J., Gill S., Shipley J., Chan A. M., Gusterson B. A., Cooper C. S. Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet. 1994 Aug;7(4):502–508. doi: 10.1038/ng0894-502. [DOI] [PubMed] [Google Scholar]
  8. Crooke S. T. Progress toward oligonucleotide therapeutics: pharmacodynamic properties. FASEB J. 1993 Apr 1;7(6):533–539. doi: 10.1096/fasebj.7.6.7682523. [DOI] [PubMed] [Google Scholar]
  9. Crozat A., Aman P., Mandahl N., Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature. 1993 Jun 17;363(6430):640–644. doi: 10.1038/363640a0. [DOI] [PubMed] [Google Scholar]
  10. Delattre O., Zucman J., Melot T., Garau X. S., Zucker J. M., Lenoir G. M., Ambros P. F., Sheer D., Turc-Carel C., Triche T. J. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994 Aug 4;331(5):294–299. doi: 10.1056/NEJM199408043310503. [DOI] [PubMed] [Google Scholar]
  11. Delattre O., Zucman J., Plougastel B., Desmaze C., Melot T., Peter M., Kovar H., Joubert I., de Jong P., Rouleau G. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992 Sep 10;359(6391):162–165. doi: 10.1038/359162a0. [DOI] [PubMed] [Google Scholar]
  12. Frassica F. J., Frassica D. A., Pritchard D. J., Schomberg P. J., Wold L. E., Sim F. H. Ewing sarcoma of the pelvis. Clinicopathological features and treatment. J Bone Joint Surg Am. 1993 Oct;75(10):1457–1465. doi: 10.2106/00004623-199310000-00006. [DOI] [PubMed] [Google Scholar]
  13. Galili N., Davis R. J., Fredericks W. J., Mukhopadhyay S., Rauscher F. J., 3rd, Emanuel B. S., Rovera G., Barr F. G. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993 Nov;5(3):230–235. doi: 10.1038/ng1193-230. [DOI] [PubMed] [Google Scholar]
  14. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Giovannini M., Biegel J. A., Serra M., Wang J. Y., Wei Y. H., Nycum L., Emanuel B. S., Evans G. A. EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations. J Clin Invest. 1994 Aug;94(2):489–496. doi: 10.1172/JCI117360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herschman H. R. Primary response genes induced by growth factors and tumor promoters. Annu Rev Biochem. 1991;60:281–319. doi: 10.1146/annurev.bi.60.070191.001433. [DOI] [PubMed] [Google Scholar]
  18. Jeon I. S., Davis J. N., Braun B. S., Sublett J. E., Roussel M. F., Denny C. T., Shapiro D. N. A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene. 1995 Mar 16;10(6):1229–1234. [PubMed] [Google Scholar]
  19. Kovar H., Aryee D. N., Jug G., Henöckl C., Schemper M., Delattre O., Thomas G., Gadner H. EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. Cell Growth Differ. 1996 Apr;7(4):429–437. [PubMed] [Google Scholar]
  20. Kretzner L., Blackwood E. M., Eisenman R. N. Myc and Max proteins possess distinct transcriptional activities. Nature. 1992 Oct 1;359(6394):426–429. doi: 10.1038/359426a0. [DOI] [PubMed] [Google Scholar]
  21. Ladanyi M., Gerald W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res. 1994 Jun 1;54(11):2837–2840. [PubMed] [Google Scholar]
  22. Ladenstein R., Lasset C., Pinkerton R., Zucker J. M., Peters C., Burdach S., Pardo N., Dallorso S., Coze C., Dollorso G. Impact of megatherapy in children with high-risk Ewing's tumours in complete remission: a report from the EBMT Solid Tumour Registry. Bone Marrow Transplant. 1995 May;15(5):697–705. [PubMed] [Google Scholar]
  23. Langer S. J., Bortner D. M., Roussel M. F., Sherr C. J., Ostrowski M. C. Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol Cell Biol. 1992 Dec;12(12):5355–5362. doi: 10.1128/mcb.12.12.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mao X., Miesfeldt S., Yang H., Leiden J. M., Thompson C. B. The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities. J Biol Chem. 1994 Jul 8;269(27):18216–18222. [PubMed] [Google Scholar]
  25. May W. A., Gishizky M. L., Lessnick S. L., Lunsford L. B., Lewis B. C., Delattre O., Zucman J., Thomas G., Denny C. T. 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 U S A. 1993 Jun 15;90(12):5752–5756. doi: 10.1073/pnas.90.12.5752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. May W. A., Lessnick S. L., Braun B. S., Klemsz M., Lewis B. C., Lunsford L. B., Hromas R., Denny C. T. 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. 1993 Dec;13(12):7393–7398. doi: 10.1128/mcb.13.12.7393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maygarden S. J., Askin F. B., Siegal G. P., Gilula L. A., Schoppe J., Foulkes M., Kissane J. M., Nesbit M. Ewing sarcoma of bone in infants and toddlers. A clinicopathologic report from the Intergroup Ewing's Study. Cancer. 1993 Mar 15;71(6):2109–2118. doi: 10.1002/1097-0142(19930315)71:6<2109::aid-cncr2820710628>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  28. McKeon C., Thiele C. J., Ross R. A., Kwan M., Triche T. J., Miser J. S., Israel M. A. Indistinguishable patterns of protooncogene expression in two distinct but closely related tumors: Ewing's sarcoma and neuroepithelioma. Cancer Res. 1988 Aug 1;48(15):4307–4311. [PubMed] [Google Scholar]
  29. Munroe S. H. Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J. 1988 Aug;7(8):2523–2532. doi: 10.1002/j.1460-2075.1988.tb03100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakabeppu Y., Oda S., Sekiguchi M. Proliferative activation of quiescent Rat-1A cells by delta FosB. Mol Cell Biol. 1993 Jul;13(7):4157–4166. doi: 10.1128/mcb.13.7.4157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Noguera R., Triche T. J., Navarro S., Tsokos M., Llombart-Bosch A. Dynamic model of differentiation in Ewing's sarcoma cells. Comparative analysis of morphologic, immunocytochemical, and oncogene expression parameters. Lab Invest. 1992 Feb;66(2):143–151. [PubMed] [Google Scholar]
  32. Ohno T., Rao V. N., Reddy E. S. EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Res. 1993 Dec 15;53(24):5859–5863. [PubMed] [Google Scholar]
  33. Ouchida M., Ohno T., Fujimura Y., Rao V. N., Reddy E. S. Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene. 1995 Sep 21;11(6):1049–1054. [PubMed] [Google Scholar]
  34. Peten E. P., Striker L. J., Carome M. A., Elliott S. J., Yang C. W., Striker G. E. The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction. J Exp Med. 1992 Dec 1;176(6):1571–1576. doi: 10.1084/jem.176.6.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rabbitts T. H. Chromosomal translocations in human cancer. Nature. 1994 Nov 10;372(6502):143–149. doi: 10.1038/372143a0. [DOI] [PubMed] [Google Scholar]
  36. Rabbitts T. H., Forster A., Larson R., Nathan P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet. 1993 Jun;4(2):175–180. doi: 10.1038/ng0693-175. [DOI] [PubMed] [Google Scholar]
  37. Ransone L. J., Verma I. M. Nuclear proto-oncogenes fos and jun. Annu Rev Cell Biol. 1990;6:539–557. doi: 10.1146/annurev.cb.06.110190.002543. [DOI] [PubMed] [Google Scholar]
  38. Roussel M. F., Davis J. N., Cleveland J. L., Ghysdael J., Hiebert S. W. Dual control of myc expression through a single DNA binding site targeted by ets family proteins and E2F-1. Oncogene. 1994 Feb;9(2):405–415. [PubMed] [Google Scholar]
  39. Siebert P. D., Larrick J. W. Competitive PCR. Nature. 1992 Oct 8;359(6395):557–558. doi: 10.1038/359557a0. [DOI] [PubMed] [Google Scholar]
  40. Sorensen P. H., Lessnick S. L., Lopez-Terrada D., Liu X. F., Triche T. J., Denny C. T. A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet. 1994 Feb;6(2):146–151. doi: 10.1038/ng0294-146. [DOI] [PubMed] [Google Scholar]
  41. Sreekantaiah C., Ladanyi M., Rodriguez E., Chaganti R. S. Chromosomal aberrations in soft tissue tumors. Relevance to diagnosis, classification, and molecular mechanisms. Am J Pathol. 1994 Jun;144(6):1121–1134. [PMC free article] [PubMed] [Google Scholar]
  42. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  43. Tanaka K., Iwamoto Y., Ito Y., Ishibashi T., Nakabeppu Y., Sekiguchi M., Sugioka Y. Cyclic AMP-regulated synthesis of the tissue inhibitors of metalloproteinases suppresses the invasive potential of the human fibrosarcoma cell line HT1080. Cancer Res. 1995 Jul 1;55(13):2927–2935. [PubMed] [Google Scholar]
  44. Tanaka K., Iwamoto Y., Noguchi Y., Oda Y., Sugioka Y. The establishment and characterization of a peripheral neuroepithelioma cell line in soft tissue of extremity. Lab Invest. 1995 Feb;72(2):237–248. [PubMed] [Google Scholar]
  45. Thiele C. J., McKeon C., Triche T. J., Ross R. A., Reynolds C. P., Israel M. A. Differential protooncogene expression characterizes histopathologically indistinguishable tumors of the peripheral nervous system. J Clin Invest. 1987 Sep;80(3):804–811. doi: 10.1172/JCI113137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Walder R. Y., Walder J. A. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5011–5015. doi: 10.1073/pnas.85.14.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Whang-Peng J., Triche T. J., Knutsen T., Miser J., Douglass E. C., Israel M. A. Chromosome translocation in peripheral neuroepithelioma. N Engl J Med. 1984 Aug 30;311(9):584–585. doi: 10.1056/NEJM198408303110907. [DOI] [PubMed] [Google Scholar]
  49. Whang-Peng J., Triche T. J., Knutsen T., Miser J., Kao-Shan S., Tsai S., Israel M. A. Cytogenetic characterization of selected small round cell tumors of childhood. Cancer Genet Cytogenet. 1986 Apr 1;21(3):185–208. doi: 10.1016/0165-4608(86)90001-4. [DOI] [PubMed] [Google Scholar]
  50. Wilhelm S. M., Collier I. E., Marmer B. L., Eisen A. Z., Grant G. A., Goldberg G. I. SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J Biol Chem. 1989 Oct 15;264(29):17213–17221. [PubMed] [Google Scholar]
  51. Zhang L., Lemarchandel V., Romeo P. H., Ben-David Y., Greer P., Bernstein A. The Fli-1 proto-oncogene, involved in erythroleukemia and Ewing's sarcoma, encodes a transcriptional activator with DNA-binding specificities distinct from other Ets family members. Oncogene. 1993 Jun;8(6):1621–1630. [PubMed] [Google Scholar]
  52. Zucman J., Delattre O., Desmaze C., Epstein A. L., Stenman G., Speleman F., Fletchers C. D., Aurias A., Thomas G. EWS and ATF-1 gene fusion induced by t(12;22) translocation in malignant melanoma of soft parts. Nat Genet. 1993 Aug;4(4):341–345. doi: 10.1038/ng0893-341. [DOI] [PubMed] [Google Scholar]

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