Targeted Oncology

, Volume 10, Issue 4, pp 523–533 | Cite as

A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma

  • Livius Penter
  • Bert Maier
  • Ute Frede
  • Benjamin Hackner
  • Thomas Carell
  • Christian Hagemeier
  • Matthias Truss
Original Research

Abstract

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.

Keywords

Neuroblastoma F-box proteins DNA methyltransferase inhibitors RNA-interference Screening system Doxorubicin 

Supplementary material

11523_2014_354_MOESM1_ESM.pdf (78 kb)
Online Resource 1Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on cell cycle distribution. Cell cycle distribution measured by flow cytometry using propidium iodide fluorescence labeling: DNA content of individual cells is represented by pulse area (PE-A). Pulse width (PE-W) was used to exclude cell doublets. a) SK-N-AS and SH-EP cells (N-Myc single copy).b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 78 kb)
11523_2014_354_MOESM2_ESM.pdf (101 kb)
Online Resource 2Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on apoptosis quantified with flow cytometry using Annexin V (AV) and propidium iodide (PI) fluorescence labeling. Viable and dead cells are labeled AV-/PI- and AV+/PI+, while early and late apoptosis are characterized as AV+/PI- and AV+/PI+. a) SK-N-AS and SH-EP cells (N-Myc single copy). b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 101 kb)
11523_2014_354_MOESM3_ESM.pdf (14 kb)
Online Resource 3Overview of shRNAs against F-box proteins employed in the paper (PDF 14 kb)

References

  1. 1.
    Maris J (2010) Recent advances in neuroblastoma. N Engl J Med 362(23):2202–11PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Charlet J, Schnekenburger M, Brown KW, Diederich M (2012) DNA demethylation increases sensitivity of neuroblastoma cells to chemotherapeutic drugs. Biochem Pharmacol 83(7):858–65CrossRefPubMedGoogle Scholar
  3. 3.
    Chen ZC, Chen LJ, Cheng JT (2013) Doxorubicin-induced cardiac toxicity is mediated by lowering of peroxisome proliferator-activated receptor δ expression in rats. PPAR Res 2013:456042PubMedCentralPubMedGoogle Scholar
  4. 4.
    Takemura G, Fujiwara H (2007) Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis 49(5):330–52CrossRefPubMedGoogle Scholar
  5. 5.
    Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N et al (2006) Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 355(23):2408–17CrossRefPubMedGoogle Scholar
  6. 6.
    Cheung NK, Dyer MA (2013) Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer 13(6):397–411PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Esteller M (2008) Epigenetics in cancer. N Engl J Med 358(11):1148–59CrossRefPubMedGoogle Scholar
  8. 8.
    Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A et al (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10(3):223–32PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    George RE, Lahti JM, Adamson PC, Zhu K, Finkelstein D, Ingle AM et al (2010) Phase I study of decitabine with doxorubicin and cyclophosphamide in children with neuroblastoma and other solid tumors: a Children's Oncology Group study. Pediatr Blood Cancer 55(4):629–38PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Brueckner B, Garcia Boy R, Siedlecki P, Musch T, Kliem HC, Zielenkiewicz P et al (2005) Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. Cancer Res 65(14):6305–11CrossRefPubMedGoogle Scholar
  11. 11.
    Datta J, Ghoshal K, Denny WA, Gamage SA, Brooke DG, Phiasivongsa P et al (2009) A new class of quinoline-based DNA hypomethylating agents reactivates tumor suppressor genes by blocking DNA methyltransferase 1 activity and inducing its degradation. Cancer Res 69(10):4277–85PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Kuck D, Caulfield T, Lyko F, Medina-Franco JL (2010) Nanaomycin A selectively inhibits DNMT3B and reactivates silenced tumor suppressor genes in human cancer cells. Mol Cancer Ther 9(11):3015–23CrossRefPubMedGoogle Scholar
  13. 13.
    Wiebusch L, Hagemeier C (2010) p53- and p21-dependent premature APC/C-Cdh1 activation in G2 is part of the long-term response to genotoxic stress. Oncogene 29(24):3477–89CrossRefPubMedGoogle Scholar
  14. 14.
    Schiesser S, Pfaffeneder T, Sadeghian K, Hackner B, Steigenberger B, Schröder AS et al (2013) Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. J Am Chem Soc 135(39):14593–9CrossRefPubMedGoogle Scholar
  15. 15.
    Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–61CrossRefPubMedGoogle Scholar
  16. 16.
    Fulda S, Lutz W, Schwab M, Debatin K (1999) MycN sensitizes neuroblastoma cells for drug-induced apoptosis. Oncogene 18(7):1479–86CrossRefPubMedGoogle Scholar
  17. 17.
    Goldschneider D, Horvilleur E, Plassa LF, Guillaud-Bataille M, Million K, Wittmer-Dupret E et al (2006) Expression of C-terminal deleted p53 isoforms in neuroblastoma. Nucleic Acids Res 34(19):5603–12PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Spänkuch-Schmitt B, Bereiter-Hahn J, Kaufmann M, Strebhardt K (2002) Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. J Natl Cancer Inst 94(24):1863–77CrossRefPubMedGoogle Scholar
  19. 19.
    Spänkuch B, Heim S, Kurunci-Csacsko E, Lindenau C, Yuan J, Kaufmann M et al (2006) Down-regulation of Polo-like kinase 1 elevates drug sensitivity of breast cancer cells in vitro and in vivo. Cancer Res 66(11):5836–46CrossRefPubMedGoogle Scholar
  20. 20.
    Maier B, Wendt S, Vanselow J, Wallach T, Reischl S, Oehmke S et al (2009) A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes Dev 23(6):708–18PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Machida YJ, Dutta A (2007) The APC/C inhibitor, Emi1, is essential for prevention of rereplication. Genes Dev 21(2):184–94PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Shimizu N, Nakajima NI, Tsunematsu T, Ogawa I, Kawai H, Hirayama R, et al (2013)Selective enhancing effect of early mitotic inhibitor 1 depletion on the sensitivity of doxorubicin or X-ray treatment in human cancer cells. J Biol Chem. MayGoogle Scholar
  23. 23.
    Tang W, Li Y, Yu D, Thomas-Tikhonenko A, Spiegelman VS, Fuchs SY (2005) Targeting beta-transducin repeat-containing protein E3 ubiquitin ligase augments the effects of antitumor drugs on breast cancer cells. Cancer Res 65(5):1904–8CrossRefPubMedGoogle Scholar
  24. 24.
    Hegde V, McFarlane RJ, Taylor EM, Price C (1996) The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 251(4):483–92PubMedGoogle Scholar
  25. 25.
    García-Domínguez P, Dell'aversana C, Alvarez R, Altucci L, de Lera AR (2013) Synthetic approaches to DNMT inhibitor SGI-1027 and effects on the U937 leukemia cell line. Bioorg Med Chem Lett 23(6):1631–5CrossRefPubMedGoogle Scholar
  26. 26.
    Hagemann S, Kuck D, Stresemann, Carlo, Prinz F, Brueckner B, et al (2012) Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation. PLoS ONE; p. e36125.Google Scholar
  27. 27.
    Qiu YY, Mirkin BL, Dwivedi RS (2005) Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells. Cancer Detect Prev 29(5):456–63CrossRefPubMedGoogle Scholar
  28. 28.
    Qiu YY, Mirkin BL, Dwivedi RS (2002) Differential expression of DNA-methyltransferases in drug resistant murine neuroblastoma cells. Cancer Detect Prev 26(6):444–53CrossRefPubMedGoogle Scholar
  29. 29.
    Schlabach MR, Luo J, Solimini NL, Hu G, Xu Q, Li MZ et al (2008) Cancer proliferation gene discovery through functional genomics. Science 319(5863):620–4PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Gluschnaider U, Hidas G, Cojocaru G, Yutkin V, Ben-Neriah Y, Pikarsky E (2010) beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor. PLoS One 5(2):e9060PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Livius Penter
    • 1
  • Bert Maier
    • 2
  • Ute Frede
    • 1
  • Benjamin Hackner
    • 3
  • Thomas Carell
    • 3
  • Christian Hagemeier
    • 1
  • Matthias Truss
    • 1
  1. 1.Labor für Pädiatrische MolekularbiologieCharité - Universitätsmedizin BerlinBerlinGermany
  2. 2.Institut für Medizinische Immunologie, AG ChronobiologieCharité - Universitätsmedizin BerlinBerlinGermany
  3. 3.CIPSM, Fakultät für Chemie und PharmazieLudwig-Maximilians-UniversitätMünchenGermany

Personalised recommendations