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Clinical Orthopaedics and Related Research®

, Volume 472, Issue 3, pp 874–882 | Cite as

Loss of SS18-SSX1 Inhibits Viability and Induces Apoptosis in Synovial Sarcoma

  • Emily E. Carmody Soni
  • Silke Schlottman
  • Hayriye V. Erkizan
  • Aykut Uren
  • Jeffrey A. Toretsky
Symposium: Musculoskeletal Tumor Society 2012 Symposium

Abstract

Background

Most synovial sarcomas contain a chromosomal translocation t(X;18), which results in the formation of an oncoprotein SS18-SSX critical to the viability of synovial sarcoma.

Questions/purposes

We (1) established and characterized three novel synovial sarcoma cell lines and asked (2) whether inhibition of SS18-SSX1 decreases cell viability in these cell lines; and (3) whether reduction in viability after SS18-SSX1 knockdown is caused by apoptosis. After identifying a specific posttranscriptional splice variant in our cell lines, we asked (4) whether this provides a survival benefit in synovial sarcoma.

Methods

Cells lines were characterized. SS18-SSX1 knockdown was achieved using a shRNA system. Cell viability was assessed by WST-1 analysis and apoptosis examined by caspase-3 activity.

Results

We confirmed the SS18-SSX1 translocation in all cell lines and identified a consistent splicing variant. We achieved successful knockdown of SS18-SSX1 and with this saw a significant reduction in cell viability. Decreased viability was a result of increased apoptosis. Reintroduction of the exon 8 sequence into cells reduced cell viability in all cell lines.

Conclusions

We confirmed the presence of the SS18-SSX1 translocation in our cell lines and its importance in the survival of synovial sarcoma. We have also demonstrated that reduction in cell viability is related to an increase in apoptosis. In addition, we have identified a potential mediator of SS18-SSX function in exon 8.

Clinical Relevance

SS18-SSX represents a tumor-specific target in synovial sarcoma. Exploitation of SS18-SSX and its protein partners will allow us to develop potent tumor-specific therapeutic agents.

Keywords

Splice Variant Synovial Sarcoma Protein Partner shRNA Construct Synovial Sarcoma Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We acknowledge Felasfa M. Wodajo MD, for providing tumor samples from which two of the cell lines were started.

References

  1. 1.
    Amary MF, Berisha F, Bernardi FC, Herbert A, James M, Reis-Filho JS, Fisher C, Nicholson AG, Tirabosco R, Diss TC, Flanagan AM. Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR, and dual color FISH as diagnostic tools for synovial sarcoma. Mod Pathol. 2004;20:482–496.CrossRefGoogle Scholar
  2. 2.
    Bartel F, Taylor AC, Taubert H, Harris LC. Novel mdm2 splice variants identified in pediatric rhabdomyosarcoma tumors and cell lines. Oncol Res. 2001;12:451–457.PubMedGoogle Scholar
  3. 3.
    Brett D, Whitehouse S, Antonson P, Shipley J, Cooper C, Goodwin G. The SYT protein involved in the t(X;18) synovial sarcoma translocation is a transcriptional activator localized in nuclear bodies. Hum Mol Genet. 1997;6:1559–1564.PubMedCrossRefGoogle Scholar
  4. 4.
    Brodin B, Haslam K, Yang K, Bartolazzi A, Xie Y, Starborg M, Lundeberg J, Larsson O. Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression. Gene. 2001;268:173–182.PubMedCrossRefGoogle Scholar
  5. 5.
    de Bruijn DR, Baats E, Zechner U, de Leeuw B, Balemans M, Olde Weghuis D, Hirning-Folz U, Geurts van Kessel AG. Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas. Oncogene. 1996;13:643–648.PubMedGoogle Scholar
  6. 6.
    Busto R, Schally AV, Braczkowski R, Plonowski A, Krupa M, Groot K, Armatis P, Varga JL. Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors. Regul Pept. 2002;108:47–53.PubMedCrossRefGoogle Scholar
  7. 7.
    Chang TY, Wu YH, Cheng CC, Wang HW. Differentially regulated splice variants and systems biology analysis of Kaposi’s sarcoma-associated herpesvirus-infected lymphatic endothelial cells. Nucleic Acids Res. 2011;39:6970–6985.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Clark J, Rocques PJ, Crew AJ, Gill S, Grimer R, Chand A, Shipley J, Gusterson BA, Cooper CS. 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;7:502–508.PubMedCrossRefGoogle Scholar
  9. 9.
    Crew AJ, Clark J, Fisher C, Gill S, Grimer R, Chand A, Shipley J, Gusterson BA, Cooper CS. Fusion of SYT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. EMBO J. 1995;14:2333–2340.PubMedGoogle Scholar
  10. 10.
    D’Arcy P, Maruwge W, Ryan BA, Brodin B. The oncoprotein SS18-SSX1 promotes p53 ubiquination and degradation by enhancing HDM2 stability. Mol Cancer Res. 2008;6:127–138.PubMedCrossRefGoogle Scholar
  11. 11.
    Dos Santos NR, de Bruijn DRH, Balemans M, Janssen B, Gartner F, Lopes JM, de Leeuw B, van Kessel AG. Nuclear localization of SYT, SSX, and the synovial sarcoma-associated SYT-SSX fusion proteins. Hum Mol Genet. 1997;6:1549–1558.PubMedCrossRefGoogle Scholar
  12. 12.
    Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, Abaan OD, Chou TH, Dakshanamurthy S, Brown ML, Uren A, Toretsky JA. A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing’s sarcoma. Nat Med. 2009;5:750–756.CrossRefGoogle Scholar
  13. 13.
    Ferrari A, Gronchi A, Casanova M, Meazza C, Gandola L, Collini P, Lozza L, Bertulli R, Olmi P, Casali PG. Synovial sarcoma: a retrospective analysis of 271 patients of all ages treated at a single institution. Cancer. 2004;101:627–634.PubMedCrossRefGoogle Scholar
  14. 14.
    Fligman I, Lonardo F, Jhanwar SC, Gerald W, Woodruff J, Ladanyi M. Molecular diagnosis of synovial sarcoma and characterization of a variant SYT-SSX2 fusion transcript. Am J Pathol. 1995;147:1592–1599.PubMedGoogle Scholar
  15. 15.
    Gaffney R, Chakerian A, O’Connell JX, Mathers J, Garner K, Joste N, Viswanatha DS. Novel fluorescent ligase detection reaction and flow cytometric analysis of SYT-SSX fusions in synovial sarcoma. Mol Diagn. 2003;5:127–135.CrossRefGoogle Scholar
  16. 16.
    Gortzak E, Azzarelli A, Buesa J, Bramwell VH, van Coevorden F, van Geel AN, Ezzat A, Santoro A, Oosterhuis JW, van Glabbeke M, Kirkpatrick A, Verweij J. A randomized phase II study on neoadjuvant chemotherapy for ‘high-risk’ adult soft tissue sarcoma. Eur J Cancer. 2001;37:1096–1103.PubMedCrossRefGoogle Scholar
  17. 17.
    Gure AO, Türeci O, Sahin U, Tsang S, Scanlan MJ, Jäger E, Knuth A, Pfreundschuh M, Old LJ, Chen YT. SSX: a multigene family with several members transcribed in normal testis and human cancer. Int J Cancer. 1997;72:965–971.PubMedCrossRefGoogle Scholar
  18. 18.
    Hahnel A, Wichmann H, Greither T, Kappler M, Würl P, Kotzsch M, Taubert H, Vordermark D, Bache M. Prognostic impact of mRNA levels of osteopontin splice variants in soft tissue sarcoma patients. BMC Cancer. 2012;12:131.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Hiraga H, Nojima T, Abe S, Sawa H, Yamashiro K, Yamawaki S, Kaneda K, Nagashima K. Diagnosis of synovial sarcoma with the reverse transcriptase-polymerase chain reaction: analyses of 84 soft tissue and bone tumors. Diagn Mol Pathol. 1998;7:102–110.PubMedCrossRefGoogle Scholar
  20. 20.
    Ishida M, Miyamoto M, Naitoh S, Tatsuda D, Hasegawa T, Nemoto T, Yokozeki H, Nishioka K, Matsukage A, Ohki M, Ohta T. The SYT-SSX fusion protein down-regulates the cell proliferation regulator COM1 in t(x;18) synovial sarcoma. Mol Cell Biol. 2007;27:1348–1355.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Italiano A, Penel N, Robin YM, Bui B, Le Cesne A, Piperno-Neumann S, Tubiana-Hulin M, Bompas E, Chevreau C, Isambert N, Leyvraz S, du Chaterlard PP, Thyss A, Coindre JM, Blay JY. Neo/adjuvant chemotherapy does not improve outcome in resected primary synovial sarcoma: a study of the French sarcoma group. Ann Oncol. 2009;20:425–430.PubMedCrossRefGoogle Scholar
  22. 22.
    Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics. Cancer J Clin. 2004;54:8–29.CrossRefGoogle Scholar
  23. 23.
    Kawai A, Woodruff J, Healey JH, Brennan MF, Antonescu CR, Ladanyi M. SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma. N Engl J Med. 1998;338:153–160.PubMedCrossRefGoogle Scholar
  24. 24.
    Lewis JJ, Antonescu CR, Leung DH, Blumberg D, Healey JH, Woodruff JM, Brennan MF. Synovial sarcoma: a multivariate analysis of prognostic factors in 112 patients with primary localized tumors of the extremity. J Clin Oncol. 2000;18:2087–2094.PubMedGoogle Scholar
  25. 25.
    McCarter MD, Jaques DP, Brennan MF. Randomized clinical trials in soft tissue sarcoma. Surg Oncol Clin N Am. 2002;11:11–22.PubMedCrossRefGoogle Scholar
  26. 26.
    Mullen JR, Zagars GK. Synovial sarcoma outcome following conservation surgery and radiotherapy. Radiother Oncol. 1994;33:23–30.PubMedCrossRefGoogle Scholar
  27. 27.
    Nilsson G, Skytting B, Xie Y, Brodin B, Perfekt R, Mandahl N, Lundeberg J, Uhlén M, Larsson O. The SYT-SSX1 variant of synovial sarcoma is associated with a high rate of tumor cell proliferation and poor clinical outcome. Cancer Res. 1999;59:3180–3184.PubMedGoogle Scholar
  28. 28.
    Otsuka S, Nishijo K, Nakayama T, Aoyama T, Ishibe T, Shibata KR, Shima Y, Nakamura T, Otsuka T, Toguchida J. A variant of the SYT-SSX2 fusion gene in a case of synovial sarcoma. Cancer Genet Cytogenet. 2006;167:82–88.PubMedCrossRefGoogle Scholar
  29. 29.
    Peng C, Guo W, Yang Y, Zhao H. Downregulation of SS18-SSX1 expression by small interfering RNA inhibits growth and induces apoptosis in human synovial sarcoma cell line HS-SY-II in vitro. Eur J Cancer Prev. 2008;17:392–398.PubMedCrossRefGoogle Scholar
  30. 30.
    Perani M, Ingram CJE, Cooper CS, Garrett MD, Goodwin GH. Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodeling complexes, while the QPGY repeat domain forms homo-oligomers. Oncogene. 2003;22:8156–8167.PubMedCrossRefGoogle Scholar
  31. 31.
    Ryan JR, Baker LH, Benjamin RS. The natural history of metastatic synovial sarcoma: experience of the southwest oncology group. Clin Orthop Relat Res. 1982;164:257–260.PubMedGoogle Scholar
  32. 32.
    Skytting B, Nilsson G, Brodin B, Xie Y, Lundeberg J, Uhlén M, Larsson O. A novel fusion gene, SYT-SSX4, in synovial sarcoma. J Natl Cancer Inst. 1999;91:974–975.PubMedCrossRefGoogle Scholar
  33. 33.
    Smith S, Reeves BR, Wong L, Fisher C. A consistent chromosome translocation in synovial sarcoma. Cancer Genet Cytogenet. 1987;26:179–180.PubMedCrossRefGoogle Scholar
  34. 34.
    Spillane AJ, A’Hern R, Judson IR, Fisher C, Thomas JM. Synovial sarcoma: a clinicopathologic, staging and prognostic assessment. J Clin Oncol. 2000;18:3794–3803.PubMedGoogle Scholar
  35. 35.
    Takenaka S, Naka N, Araki N, Hashimoto N, Ueda T, Yoshioka K, Yoshikawa H, Itoh K. Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo. Int J Oncol. 2010;36:823–831.PubMedGoogle Scholar
  36. 36.
    Tamborini E, Agus V, Mezzelani A, Riva C, Sozzi G, Azzarelli A, Pierotti MA, Pilotti S. Identification of a novel spliced variant of the SYT gene expressed in normal tissues and in synovial sarcoma. Br J Cancer. 2001;84:1087–1094.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Uversky VN. Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol. 2011;43:1090–1103.PubMedCrossRefGoogle Scholar

Copyright information

© The Association of Bone and Joint Surgeons® 2013

Authors and Affiliations

  • Emily E. Carmody Soni
    • 1
    • 2
  • Silke Schlottman
    • 2
  • Hayriye V. Erkizan
    • 2
  • Aykut Uren
    • 2
  • Jeffrey A. Toretsky
    • 2
  1. 1.MedStar Georgetown Orthopaedic InstituteWashingtonUSA
  2. 2.Department of OncologyLombardi Comprehensive Cancer InstituteWashingtonUSA

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