Skip to main content

Advertisement

SpringerLink
Log in

MYCN concurrence with SAHA-induced cell death in human neuroblastoma cells

  • Original Paper
  • Published:
Cellular Oncology Aims and scope Submit manuscript

Abstract

Background

In the past, the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) has been shown to induce apoptosis in several human tumor types, including neuroblastomas. Amplification and over-expression of the MYCN oncogene is a diagnostic hallmark and a poor prognostic indicator in high-risk neuroblastomas. Here, we studied the relationship between MYCN amplification and over-expression and the anti-tumor effect of SAHA to assess whether this drug may serve as a treatment option for high-risk neuroblastomas.

Methods

Different human neuroblastoma cell lines, over-expressing or not over-expressing MYCN, were used in this study. Targeted knockdown and exogenous over-expression of MYCN were employed to examine correlations between MYCN expression levels and SAHA responses. After various time periods and concentration exposures to the drug, cell viability was measured by MTS assay, and variations in MYCN mRNA and protein levels were assessed by qPCR and Western blotting, respectively.

Results

We found that SAHA decreased cell viability in all cell lines tested through apoptosis induction, and that SAHA had a stronger effect on cell lines carrying an amplified MYCN gene. A decrease in MYCN mRNA and protein levels was observed in the SAHA treated cell lines. Subsequent silencing and exogenous over-expression of MYCN changed the proliferation rate of the cells, but did not have any significant impact on the effect of SAHA on the viability of the cells. We also found that SAHA blocked the expression of MYCN and, by doing so, reduced the effects mediated by this protein.

Conclusions

Our results suggest that SAHA may be used as a single-drug treatment option for neuroblastomas with an amplified MYCN gene, and as an adjuvant treatment option for all neuroblastomas.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. J.M. Maris, Recent advances in neuroblastoma. N. Eng. J. Med. 362, 2202–2211 (2010)

    Article  CAS  Google Scholar 

  2. A. Bandino, D. Geerts, J. Koster, A.S. Bachmann, Deoxyhypusine synthase (DHPS) inhibitor GC7 induces p21/Rb-mediated inhibition of tumor cell growth and DHPS expression correlates with poor prognosis in neuroblastoma patients. Cell. Oncol. 37, 387–398 (2014)

    Article  CAS  Google Scholar 

  3. R.A. Ross, J.L. Biedler, B.A. Spengler, A role for distinct cell types in determining malignancy in human neuroblastoma cell lines and tumors. Cancer Lett. 197, 35–39 (2003)

    Article  CAS  PubMed  Google Scholar 

  4. S.L. Cohn, A.D. Pearson, W.B. London, T. Monclair, P.F. Ambros, G.M. Brodeur, A. Faldum, B. Hero, T. Iehara, D. Machin, V. Mosseri, T. Simon, A. Garaventa, V. Castel, K.K. Matthay, The international neuroblastoma risk group (INRG) classification system: an INRG task force report. Int. J. Oncol. 27, 289–297 (2009)

    Google Scholar 

  5. L.J. Valentijn, J. Koster, F. Haneveld, R.A. Aissa, P. van Sluis, M.E. Broekmans, J.J. Molenaar, J. van Nes, R. Versteeg, Functional MYCN signature predicts outcome of neuroblastoma irrespective of MYCN amplification. Proc. Natl. Acad. Sci. U. S. A. 109, 19190–19195 (2012)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. T.J. Pugh, O. Morozova, E. Attiyeh, S. Asgharzadeh, J.S. Wei, D. Auclair, S.L. Carter, K. Cibulskis, M. Hanna, A. Kiezun, J. Kim, M.S. Lawrence, L. Lichenstein, A. McKenna, C.S. Pedamallu, A.H. Ramos, E. Shefler, A. Sivachenko, C. Sougnez, C. Stewart, A. Ally, I. Birol, R. Chiu, R.D. Corbett, M. Hirst, S.D. Jackman, B. Kamoh, A.H. Khodabakshi, M. Krzywinski, A. Lo, R.A. Moore, K.L. Mungall, J. Qian, A. Tam, N. Thiessen, Y. Zhao, K.A. Cole, M. Diamond, S.J. Diskin, Y.P. Mosse, A.C. Wood, L. Ji, R. Sposto, T. Badgett, W.B. London, Y. Moyer, J.M. Gastier-Foster, M.A. Smith, J.M. Guidry Auvil, D.S. Gerhard, M.D. Hogarty, S.J. Jones, E.S. Lander, S.B. Gabriel, G. Getz, R.C. Seeger, J. Khan, M.A. Marra, M. Meyerson, J.M. Maris, The genetic landscape of high-risk neuroblastoma. Nat. Genet. 45, 279–284 (2013)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. A. Canette, M. Gerrard, H. Rubie, V. Castel, A. Di Cataldo, C. Munzer, R. Ladenstein, B. Brichard, J.D. Bermúdez, J. Couturier, B. de Bernardi, A.J. Pearson, J. Michon, Poor survival for infants with MYCN amplified metastatic neuroblastoma despite intensified treatment: the international society of paediatric oncology european neuroblastoma experience. J. Clin. Oncol. 27, 1014–1019 (2009)

    Article  Google Scholar 

  8. S. Gherardi, E. Valli, D. Erriquez, G. Perini, MYCN-mediated transcriptional repression in neuroblastoma: the other side of the coin. Front. Oncol. 3, 1–8 (2013)

    Article  Google Scholar 

  9. P.J. Hurlin, N-myc functions in transcription and development. Birth Defects Res. 75, 340–352 (2005)

    Article  CAS  Google Scholar 

  10. M. Huang, W.A. Weiss, Neuroblastoma and MYCN. Cold Spring Harbour Perspect. Med. (2013). doi:10.1101/cshperspect.a014415

    Google Scholar 

  11. E. Bell, L. Chen, T. Liu, G.M. Marshall, J. Lunec, D.A. Tweddle, MYCN oncoprotein targets and their therapeutic potential. Cancer Lett. 293, 144–157 (2010)

    Article  CAS  PubMed  Google Scholar 

  12. T.P. Stricker, A. Morales, A. Chlenski, L. Guerrero, H.R. Salwen, Y. Gosiengfiao, E.J. Perlman, W. Furman, A. Bahrami, J.M. Shohet, P.E. Zage, M.J. Hicks, H. Shimada, R. Suganuma, J.R. Park, S. So, W.B. London, P. Pytel, K.H. Maclean, S.L. Cohn, Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounterTM system. Mol. Oncol. (2014). doi:10.1016/j.molonc.2014.01.01

    PubMed Central  PubMed  Google Scholar 

  13. S.R. Frank, T. Parisi, S. Taubert, P. Fernandez, M. Fuchs, H.M. Chan, D.M. Livingston, B. Amati, MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Rep. 4, 575–580 (2003)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. R. Francisco, A. Pérez-Perarnau, C. Cortés, J. Gil, A. Tauler, S. Ambrosio, Histone deacetylase inhibition induces apoptosis and autophagy in human neuroblastoma cells. Cancer Lett. 318, 42–52 (2012)

    Article  CAS  PubMed  Google Scholar 

  15. F.A. Duijkers, R.X. de Menezes, I.J. Goossens-Beumer, D.J. Stumpel, P. Admiraal, R. Pieters, J.P. Meijerink, M.M. van Noesel, Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines. Cell. Oncol. 36, 351–362 (2013)

    Article  CAS  Google Scholar 

  16. O. Witt, H.E. Deubzer, M. Lodrini, T. Milde, I. Oehme, Targeting histone deacetylases in neuroblastoma. Curr. Pharm. Des. 15, 436–447 (2009)

    Article  CAS  PubMed  Google Scholar 

  17. D.C. Coffey, M.C. Kutko, R.D. Glick, L.M. Butler, G. Heller, R.A. Rifkind, P.A. Marks, V.M. Richon, M.P. La Quaglia, The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid. Cancer Res. 61, 3591–3594 (2001)

    CAS  PubMed  Google Scholar 

  18. P.A. Marks, W.S. Xu, Histone deacetylase inhibitors: potential in cancer therapy. J. Cell. Biochem. 107, 600–608 (2009)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. M. Fouladi, J.R. Park, C.F. Stewart, R.J. Gilbertson, P. Schaiquevich, J. Sun, J.M. Reid, M.M. Ames, R. Speights, A.M. Ingle, J. Zwiebel, S.M. Blaney, P.C. Adamson, Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children’s oncology group phase I consortium report. J. Clin. Oncol. 28, 3623–3629 (2010)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. C. Conti, E. Leo, G.S. Elchler, O. Sordet, M.M. Martin, A. Fan, M.I. Aladjem, Y. Pommier, Inhibition of histone acetylase in cancer cells slows down replication forks, activates dormant origins and induces DNA damage. Cancer Res. 70, 4470–4480 (2010)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. O. Witt, T. Milde, H.E. Deubzer, I. Oehme, R. Witt, A. Kulozik, A. Eisenmenger, U. Abel, I. Karapanagiotou-Schenkel, Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid yumor, lymphoma or leukemia. Klin. Padiatr. 224, 398–403 (2012)

  22. N.K.V. Cheung, M.A. Dyer, Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat. Rev. Cancer 13, 397–411 (2013)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. S. Breit, M. Schwab, Suppression of MYC by high expression of NMYC in human neuroblastoma cells. J. Neurosci. Res. 24, 21–28 (1989)

    Article  CAS  PubMed  Google Scholar 

  24. Y. Nakamura, O. Toshinori, H. Niizuma, M. Ohira, T. Kamijo, A. Nakagawara, Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line. Biochem. Biophys. Res. Commun. 354, 892–898 (2007)

    Article  CAS  PubMed  Google Scholar 

  25. J. Carr, E. Bell, A.D.J. Pearson, U.R. Kees, H. Beris, J. Lunec, D.A. Tweddle, Increased frequency of aberrations in the p53/MDM2/p14/ARF pathway in neuroblastoma cell lines established at relapse. Cancer Res. 66, 2138–2145 (2006)

    Article  CAS  PubMed  Google Scholar 

  26. R. Kleinert, Immunohistochemical characterization of primitive neuroectodermal tumors and their possible relationship to the stepwise ontogenetic development of the central nervous system. 1.Ontogenetic studies. Acta Neuropathol. 82, 502–507 (1991)

    Article  CAS  PubMed  Google Scholar 

  27. R. Huang, N.K. Cheung, J. Vider, I.Y. Cheung, W.L. Gerald, S.K. Tickoo, E.C. Holland, R.G. Blasberg, MYCN and MYC regulate tumor proliferation and tumorigenesis directly through BMI1 in human neuroblastomas. FASEB J. 25, 4138–4149 (2011)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. C. Zhang, V. Richon, X. Ni, R. Talpur, M. Duvic, Selective induction of apoptosis by histone deacetylase inhibitor SAHA in cutaneous T-cell lymphoma cells: relevance to mechanism of therapeutic action. J. Invest. Dermatol. 125, 1045–1052 (2005)

    Article  CAS  PubMed  Google Scholar 

  29. A. Muhlethaler-Mottet, R. Meier, M. Flahaut, K.B. Bourloud, K. Nardou, J.M. Joseph, N. Gross, Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells. Mol. Cancer (2008). doi:10.1186/1476-4598-7-55

    PubMed Central  PubMed  Google Scholar 

  30. J. Ribas, X. Gomez-Arbones, J. Boix, Caspase 8/10 are not mediating apoptosis in neuroblastoma cells treated with CDK inhibitory drugs. Eur. J. Pharmacol. 524, 49–52 (2005)

    Article  CAS  PubMed  Google Scholar 

  31. W. Lutz, M. Stohr, J. Schurmann, A. Wenzel, A. Lohr, M. Schwab, Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. Oncogene 13, 803–812 (1996)

    CAS  PubMed  Google Scholar 

  32. M.Y. Chen, W.S. Liao, Z. Lu, W.G. Bornmann, V. Hennessey, M.N. Washington, G.L. Rosner, Y. Yu, A.A. Ahmed, R.C. Bast Jr., Decitabine and suberoylanilide hydroxamic acid (SAHA) inhibit growth of ovarian cancer cell lines and xenografts while inducing expression of imprinted tumor suppressor genes, apoptosis, G2/M arrest, and autophagy. Cancer 117, 4424–4438 (2011)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. A. Slack, J.M. Shohet, MDM2 as a critical effector of the MYCN oncogene in tumorigenesis. Cell Cycle 4, 857–860 (2005)

    Article  CAS  PubMed  Google Scholar 

  34. V.H. Cowling, M.D. Cole, Mechanisms of transcriptional activation by the Myc oncoproteins. Semin Cancer Biol 16, 242–252 (2006)

    Article  CAS  PubMed  Google Scholar 

  35. K. Zhang, F. Faiola, E. Martinez, Six lysine residues on c-Myc are direct substrates for acetylation by p300. Biochem. Biophys. Res. Commun. 336, 274–280 (2005)

    Article  CAS  PubMed  Google Scholar 

  36. J. Vervoots, J. Lüscher-Firzlaff, B. Lüscher, The ins and outs of MYC regulation by posttranslational mechanisms. J. Biol. Chem. 281, 34725–34729 (2006)

    Article  Google Scholar 

  37. F. Westermann, D. Muth, A. Bennet, T. Bauer, K.O. Henrich, A. Oberthuer, B. Brors, T. Beissbarth, J. Vandesompele, F. Pattyn, B. Hero, R. König, M. Fischer, M. Schwab, Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas. Genome Biol. 9, R150–R150.14 (2008)

    Article  PubMed Central  PubMed  Google Scholar 

  38. MYCN oncogene targets, http://mellfire.ugent.be/public/mycnot/, Accessed 21 November (2014)

  39. M. Alaminos, J. Mora, N.K.V. Cheung, A. Smith, J. Qin, L. Chen, W.L. Gerald, Genome-wide analysis of gene expression associated with MYCN in human neuroblastoma. Cancer Res. 63, 4538–4546 (2003)

    CAS  PubMed  Google Scholar 

  40. U. Galderisi, G. Di Bernardo, M. Cipollaro, G. Peluso, A. Cascino, R. Cotrufo, M.A. Melone, Differentiation and apoptosis of neuroblastoma cells: role of N-Myc gene product. J. Cell. Biochem. 73, 97–105 (1999)

    Article  CAS  PubMed  Google Scholar 

  41. R. Janardhanan, N.L. Banik, S.K. Ray, N-Myc down regulation induced differentiation, early cell cycle exit, and apoptosis in human malignant neuroblastoma cells having wild type or mutant p53. Biochem. Pharmacol. 78, 1105–1114 (2009)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. S. Fulda, W. Lutz, M. Schwab, K.M. Debatin, MycN sensitizes neuroblastoma cells for drug-induced apoptosis. Oncogene 18, 1479–1485 (1999)

    Article  CAS  PubMed  Google Scholar 

  43. R. Cotterman, V.X. Jin, S.R. Krig, J.M. Lemen, A. Wey, P.J. Farnham, P.S. Knoepfler, N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor. Cancer Res. 68, 9654–9662 (2008)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Megumi Iisasa for correcting the English language. This work was supported by Dirección General de Investigación Científica y Técnica, BFU2009-09933 to A.T. and BFU2012-38867 to S.C.K. and A.T., as well as by Red Temática de Investigación Cooperativa en Cáncer, RD12/0036/0049 to A.T. and S.C.K. and RD12/0036/0029 to S.A. Constanza Cortés was recipient of a fellowship from Universitat de Barcelona.

Competing interests

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, 08028, Barcelona, Catalunya, Spain

    Constanza Cortés & Albert Tauler

  2. Laboratory of Cancer Metabolism, IDIBELL, Hospital Duran i Reynals, L’Hospitalet de Llobregat, Catalunya, 08907, Spain

    Constanza Cortés, Sara C. Kozma & Albert Tauler

  3. Unitat de Bioquímica, Dep. Ciències Fisiològiques II, Facultat de Medicina, Campus Universitari de Bellvitge - IDIBELL, Universitat de Barcelona, L’Hospitalet de Llobregat, Catalunya, 08907, Spain

    Santiago Ambrosio

Authors
  1. Constanza Cortés
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sara C. Kozma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Albert Tauler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Santiago Ambrosio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Santiago Ambrosio.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cortés, C., Kozma, S.C., Tauler, A. et al. MYCN concurrence with SAHA-induced cell death in human neuroblastoma cells. Cell Oncol. 38, 341–352 (2015). https://doi.org/10.1007/s13402-015-0233-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13402-015-0233-9

Keywords

Search

Navigation