Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Translational Therapeutics

Combination of a synthetic retinoid and a DNA demethylating agent induced differentiation of neuroblastoma through retinoic acid signal reprogramming

Abstract

Background

The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2’-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination.

Methods

Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment.

Results

Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes.

Conclusions

Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Effects of TBT on neuroblastoma differentiation and tumour growth.
Fig. 2: Effects of 5-aza-dC on tumour growth.
Fig. 3: Effects of 5-aza-dC on DNA methylation.
Fig. 4: Effects of the combination of 5-aza-dC and TBT on neuroblastoma differentiation.
Fig. 5: Effects of a combination of 5-aza-dC and TBT on tumour growth.

Data availability

The datasets used in this study are available at the Gene Expression Omnibus (GEO) database (accession no. GSE180645, GSE180660 and GSE180929, https://www.ncbi.nlm.nih.gov/geo/).

References

  1. 1.

    Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3:203–16.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Cheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer. 2013;13:397–411.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. 3.

    Abe M, Ohira M, Kaneda A, Yagi Y, Yamamoto S, Kitano Y, et al. CpG island methylator phenotype is a strong determinant of poor prognosis in neuroblastomas. Cancer Res. 2005;65:828–34.

    PubMed  CAS  Google Scholar 

  4. 4.

    Abe M, Westermann F, Nakagawara A, Takato T, Schwab M, Ushijima T. Marked and independent prognostic significance of the CpG island methylator phenotype in neuroblastomas. Cancer Lett. 2007;247:253–8.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Banelli B, Brigati C, Di Vinci A, Casciano I, Forlani A, Borzi L, et al. A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype. Lab Invest. 2012;92:458–65.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Banelli B, Merlo DF, Allemanni G, Forlani A, Romani M. Clinical potentials of methylator phenotype in stage 4 high-risk neuroblastoma: an open challenge. PLoS ONE. 2013;8:e63253.

    PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Kiss NB, Kogner P, Johnsen JI, Martinsson T, Larsson C, Geli J. Quantitative global and gene-specific promoter methylation in relation to biological properties of neuroblastomas. BMC Med Genet. 2012;13:83.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Abe M, Watanabe N, Mcdonell N, Takato T, Ohira M, Nakagawara A, et al. Identification of genes targeted by CpG island methylator phenotype in neuroblastomas, and their possible integrative involvement in poor prognosis. Oncology. 2008;74:50–60.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  9. 9.

    Wang C, Liu Z, Woo CW, Li Z, Wang L, Wei JS, et al. EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR. Cancer Res. 2012;72:315–24.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  10. 10.

    Bate-Eya LT, Gierman HJ, Ebus ME, Koster J, Caron HN, Versteeg R, et al. Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity. Eur J Cancer. 2017;75:63–72.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Li Z, Takenobu H, Setyawati AN, Akita N, Haruta M, Satoh S, et al. EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications. Oncogene. 2018;37:2714–27.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  12. 12.

    Qadeer ZA, Valle-Garcia D, Hasson D, Sun Z, Cook A, Nguyen C, et al. ATRX in-frame fusion neuroblastoma is sensitive to EZH2 inhibition via modulation of neuronal gene signatures. Cancer Cell. 2019;36:512–27.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Diesch J, Zwick A, Garz AK, Palau A, Buschbeck M, Gotze KS. A clinical-molecular update on azanucleoside-based therapy for the treatment of hematologic cancers. Clin Epigenetics. 2016;8:71.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Linnekamp JF, Butter R, Spijker R, Medema JP, Van Laarhoven HWM. Clinical and biological effects of demethylating agents on solid tumours—a systematic review. Cancer Treat Rev. 2017;54:10–23.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Tang XX, Robinson ME, Riceberg JS, Kim DY, Kung B, Titus TB, et al. Favorable neuroblastoma genes and molecular therapeutics of neuroblastoma. Clin Cancer Res. 2004;10:5837–44.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Westerlund I, Shi Y, Toskas K, Fell SM, Li S, Surova O, et al. Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2alpha to tumor suppression. Proc Natl Acad Sci USA. 2017;114:E6137–E6146.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. 17.

    Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med. 1999;341:1165–73.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Reynolds CP, Matthay KK, Villablanca JG, Maurer BJ. Retinoid therapy of high-risk neuroblastoma. Cancer Lett. 2003;197:185–92.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  19. 19.

    Ohnishi K. PML-RARalpha inhibitors (ATRA, tamibaroten, arsenic troxide) for acute promyelocytic leukemia. Int J Clin Oncol. 2007;12:313–7.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Shiohira H, Kitaoka A, Enjoji M, Uno T, Nakashima M. Am80 induces neuronal differentiation via increased tropomyosin-related kinase B expression in a human neuroblastoma SH-SY5Y cell line. Biomed Res. 2012;33:291–7.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  21. 21.

    Shiohira H, Kitaoka A, Shirasawa H, Enjoji M, Nakashima M. Am80 induces neuronal differentiation in a human neuroblastoma NH-12 cell line. Int J Mol Med. 2010;26:393–9.

    PubMed  CAS  PubMed Central  Google Scholar 

  22. 22.

    Nitani C, Hara J, Kawamoto H, Taguchi T, Kimura T, Yoshimura K, et al. Phase I study of tamibarotene monotherapy in pediatric and young adult patients with recurrent/refractory solid tumors. Cancer Chemother Pharmacol. 2021;88:99–107.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  23. 23.

    Kato H, Okamura K, Kurosawa Y, Kishikawa T, Hashimoto K. Characterization of DNA rearrangements of N-myc gene amplification in three neuroblastoma cell lines by pulsed-field gel electrophoresis. FEBS Lett. 1989;250:529–35.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  24. 24.

    Suzuki T, Hirota M, Iwabuchi M, Nomura N, Saito F. A new human neuroblasoma cell line: biological characteristics, cytogenetics and N-myc oncogene analysis. Tissue Cult Res Commun. 1990;8:6–15.

    Google Scholar 

  25. 25.

    Kang JH, Rychahou PG, Ishola TA, Qiao J, Evers BM, Chung DH. MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells. Biochem Biophys Res Commun. 2006;351:192–7.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. 26.

    Harenza JL, Diamond MA, Adams RN, Song MM, Davidson HL, Hart LS, et al. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines. Sci Data. 2017;4:170033.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Hattori N, Niwa T, Kimura K, Helin K, Ushijima T. Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells. Nucleic Acids Res. 2013;41:7231–9.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. 28.

    Yamashita S, Kishino T, Takahashi T, Shimazu T, Charvat H, Kakugawa Y, et al. Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues. Proc Natl Acad Sci USA. 2018;115:1328–33.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. 29.

    Ishihara H, Yamashita S, Liu YY, Hattori N, El-Omar O, Ikeda T, et al. Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease. Cancer Sci. 2020;111:4276–87.

  30. 30.

    Bibikova M, Barnes B, Tsan C, Ho V, Klotzle B, Le JM, et al. High density DNA methylation array with single CpG site resolution. Genomics. 2011;98:288–95.

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Iida N, Okuda Y, Ogasawara O, Yamashita S, Takeshima H, Ushijima T. MACON: a web tool for computing DNA methylation data obtained by the Illumina Infinium Human DNA methylation BeadArray. Epigenomics. 2018;10:249–58.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Moro H, Hattori N, Nakamura Y, Kimura K, Imai T, Maeda M, et al. Epigenetic priming sensitizes gastric cancer cells to irinotecan and cisplatin by restoring multiple pathways. Gastric Cancer. 2020;23:105–15.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Consortium M, Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, et al. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol. 2006;24:1151–61.

    Article  CAS  Google Scholar 

  34. 34.

    Fernandes ND, Sun Y, Price BD. Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells. J Biol Chem. 2007;282:16577–84.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Takenobu H, Shimozato O, Nakamura T, Ochiai H, Yamaguchi Y, Ohira M, et al. CD133 suppresses neuroblastoma cell differentiation via signal pathway modification. Oncogene. 2011;30:97–105.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Frumm SM, Fan ZP, Ross KN, Duvall JR, Gupta S, Verplank L, et al. Selective HDAC1/HDAC2 inhibitors induce neuroblastoma differentiation. Chem Biol. 2013;20:713–25.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  37. 37.

    Dedoni S, Marras L, Olianas MC, Ingianni A, Onali P. Downregulation of TrkB expression and signaling by valproic acid and other histone deacetylase inhibitors. J Pharmacol Exp Ther. 2019;370:490–503.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Chuang JC, Warner SL, Vollmer D, Vankayalapati H, Redkar S, Bearss DJ, et al. S110, a 5-Aza-2’-deoxycytidine-containing dinucleotide, is an effective DNA methylation inhibitor in vivo and can reduce tumor growth. Mol Cancer Ther. 2010;9:1443–50.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  39. 39.

    Henrich KO, Bender S, Saadati M, Dreidax D, Gartlgruber M, Shao C, et al. Integrative genome-scale analysis identifies epigenetic mechanisms of transcriptional deregulation in unfavorable neuroblastomas. Cancer Res. 2016;76:5523–37.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Isoherranen N, Zhong G. Biochemical and physiological importance of the CYP26 retinoic acid hydroxylases. Pharmacol Ther. 2019;204:107400.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. 41.

    Acosta S, Mayol G, Rodriguez E, Lavarino C, De Preter K, Kumps C, et al. Identification of tumoral glial precursor cells in neuroblastoma. Cancer Lett. 2011;312:73–81.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  42. 42.

    Hassan HM, Kolendowski B, Isovic M, Bose K, Dranse HJ, Sampaio AV, et al. Regulation of active DNA demethylation through RAR-mediated recruitment of a TET/TDG complex. Cell Rep. 2017;19:1685–97.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  43. 43.

    Hattori N, Sako M, Kimura K, Iida N, Takeshima H, Nakata Y, et al. Novel prodrugs of decitabine with greater metabolic stability and less toxicity. Clin Epigenetics. 2019;11:111.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  44. 44.

    Karahoca M, Momparler RL. Pharmacokinetic and pharmacodynamic analysis of 5-aza-2’-deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy. Clin Epigenetics. 2013;5:3.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. 45.

    Ponthan F, Johnsen JI, Klevenvall L, Castro J, Kogner P. The synthetic retinoid RO 13-6307 induces neuroblastoma differentiation in vitro and inhibits neuroblastoma tumour growth in vivo. Int J Cancer. 2003;104:418–24.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  46. 46.

    Dobrotkova V, Chlapek P, Mazanek P, Sterba J, Veselska R. Traffic lights for retinoids in oncology: molecular markers of retinoid resistance and sensitivity and their use in the management of cancer differentiation therapy. BMC Cancer. 2018;18:1059.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. 47.

    Jones PA, Ohtani H, Chakravarthy A, De Carvalho DD. Epigenetic therapy in immune-oncology. Nat Rev Cancer. 2019;19:151–61.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  48. 48.

    Mckeown MR, Corces MR, Eaton ML, Fiore C, Lee E, Lopez JT, et al. Superenhancer analysis defines novel epigenomic subtypes of Non-APL AML, including an RARalpha dependency targetable by SY-1425, a potent and selective RARalpha agonist. Cancer Discov. 2017;7:1136–53.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. H. Kawamoto for his critical discussion. We also thank Dr. T. Imai, Dr. N. Uchiya and Ms. Y. Shiotani, and the National Cancer Center Research Core Facility for their assistance in the analysis of xenografts.

Funding

The Core Facility was supported in part by National Cancer Center Research and Development Fund (2020-J-2). This study was supported by the AMED under Grant Number JP20ck0106421 to TU, NH, CN and JH, and JSPS KAKENHI Grant Number JP18H02704 to TU and NH.

Author information

Affiliations

Authors

Contributions

TU, NH, KA, CN and JH designed the study; NH, NM, AM, YN, KK, MW and HT performed most of the experiments; NH and TU wrote the manuscript; and all authors read and approved the manuscript.

Corresponding authors

Correspondence to Naoko Hattori or Toshikazu Ushijima.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All animal experiments were approved by the Committee of National Cancer Center and were conducted according to the institutional guidelines for animal care.

Consent for publication

Not applicable.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hattori, N., Asada, K., Miyajima, N. et al. Combination of a synthetic retinoid and a DNA demethylating agent induced differentiation of neuroblastoma through retinoic acid signal reprogramming. Br J Cancer (2021). https://doi.org/10.1038/s41416-021-01571-y

Download citation

Search

Quick links