Advertisement

Journal of Neuro-Oncology

, Volume 139, Issue 1, pp 33–42 | Cite as

Reduced hydroxymethylation characterizes medulloblastoma while TET and IDH genes are differentially expressed within molecular subgroups

  • Karina Bezerra Salomão
  • Gustavo Alencastro Veiga Cruzeiro
  • Ricardo Bonfim-Silva
  • Lenisa Geron
  • Fernando Ramalho
  • Fabiano Pinto Saggioro
  • Luciano Neder Serafini
  • Daniel Antunes Moreno
  • Rosane Gomes de Paula Queiroz
  • Simone dos Santos Aguiar
  • Izilda Cardinalli
  • José Andres Yunes
  • Silvia Regina Brandalise
  • Maria Sol Brassesco
  • Carlos Alberto Scrideli
  • Luiz Gonzaga Tone
Laboratory Investigation

Abstract

Introduction

Medulloblastoma (MB) is an embryonal tumour that originates from genetic deregulation of cerebellar developmental pathways and is classified into 4 molecular subgroups: SHH, WNT, group 3, and group 4. Hydroxymethylation levels progressively increases during cerebellum development suggesting a possibility of deregulation in MB pathogenesis. The aim of this study was to investigate global hydroxymethylation levels and changes in TET and IDH gene expression in MB samples compared to control cerebellum samples.

Methods

The methods utilized were qRT-PCR for gene expression, dot-blot and immunohistochemistry for global hydroxymethylation levels and sequencing for the investigation of IDH mutations.

Results

Our results show that global hydroxymethylation level was decreased in MB, and low 5hmC level was associated with the presence of metastasis. TET1 expression levels were decreased in the WNT subgroup, while TET3 expression levels were decreased in the SHH subgroup. Reduced TET3 expression levels were associated with the presence of events such as relapse and death. Higher expression of IDH1 was observed in MB group 3 samples, whereas no mutations were detected in exon 4 of IDH1 and IDH2.

Conclusion

These findings suggest that reduction of global hydroxymethylation levels, an epigenetic event, may be important for MB development and/or maintenance, representing a possible target in this tumour and indicating a possible interaction of TET and IDH genes with the developmental pathways specifically activated in the MB subgroups. These genes could be specific targets and markers for each subgroup.

Keywords

Medulloblastoma TET genes 5-Hydroxymethylcytosine IDH genes 

Notes

Funding

This work was funded by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Process Number 2013/15125-3 and 2014/20341-0). Fundação de Apoio ao Ensino, Pesquisa e Assistência (FAEPA) do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo is also acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This research was submitted to and approved by the HC/FMRP-USP Research Ethics Committee (CAAE n° 37206114.1.0000.5440).

Informed consent

All samples were obtained after receiving informed consent from all participants included in the study.

Supplementary material

11060_2018_2845_MOESM1_ESM.tif (10.9 mb)
Kaplan-Meier curves for event-free survival according to 5hmC levels in MB. A. Using dot-blot (n=11); B. Using IHC (n=30) (TIF 11158 KB)
11060_2018_2845_MOESM2_ESM.tif (24.2 mb)
Kaplan-Meier curves for event-free survival according to A. TET1 gene expression; B. TET2 gene expression; C. TET3 gene expression; D. IDH1 gene expression; E. IDH2 gene expression in MB samples (TIF 24757 KB)
11060_2018_2845_MOESM3_ESM.tif (7.6 mb)
Representative sequencing electropherograms of Isocitrate Dehydrogenas (IDH) 1 and IDH2 genes in control cerebellum and medulloblastoma samples, in a hotspot mutation (TIF 7812 KB)

References

  1. 1.
    Chan AW, Tarbell NJ, Black PM et al (2000) Adult medulloblastoma: prognostic factors and patterns of relapse. Neurosurgery 47:623–631PubMedGoogle Scholar
  2. 2.
    Crawford JR, Macdonald TJ, Packer RJ (2007) Medulloblastoma in childhood: new biological advances. Lancet Neurol 6:1073–1085CrossRefPubMedGoogle Scholar
  3. 3.
    Louis DN, Ohgaki H, Wiestler OD et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Packer RJ, Cogen P, Vezina G et al (1999) Medulloblastoma: clinical and biologic aspects. Neuro Oncol 1:232–250CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Smoll NR, Drummond KJ (2012) The incidence of medulloblastomas and primitive tumours in adults and children. J Clin Neurosci 19:1541–1544CrossRefPubMedGoogle Scholar
  6. 6.
    Mabbot DJ, Penkman L, Witol A et al (2008) Core neurocognitive functions in children treated for posterior fossa tumors. Neuropsychology 22:159–168CrossRefGoogle Scholar
  7. 7.
    Hatten M, Roussel MF (2011) Development and cancer of the cerebellum. Cell Press 34:134–142Google Scholar
  8. 8.
    Northcott PA, Jones D, Kool M et al (2012) Medulloblastomics: the end of the beginning. Nat Rev 12:818–834CrossRefGoogle Scholar
  9. 9.
    Taylor MD (2012) Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 123:465–447CrossRefPubMedGoogle Scholar
  10. 10.
    Kongkham PN, Northcott PA, Croul SE et al (2010) The SFRP family of WNT inhibitors function as novel tumor suppressor genes epigenetically silenced in medulloblastoma. Oncogene 29:3017–3024CrossRefPubMedGoogle Scholar
  11. 11.
    Pócza T, Krenács T, Turányi E et al (2016) High expression of DNA methyltransferase in primary human medulloblastoma. Folia Neuropathol 54(2):105–113CrossRefPubMedGoogle Scholar
  12. 12.
    Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Willians K, Christensen J, Helin K (2012) DNA methylation: TET proteins-guardians of CpG islands? EMBO Rep 1:28–35Google Scholar
  14. 14.
    Haffner MC, Chaux A, Meeker AK et al (2011) Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget 2:627–637CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kudo Y, Tateishi K, Yamamoto K et al (2012) Loss of 5-hydroxymethylcytosine is accompanied with malignant cellular transformation. Cancer Sci 103(4):670–676CrossRefPubMedGoogle Scholar
  16. 16.
    Lian CG, Xu Y, Ceol C et al (2012) Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 6:1135–1146CrossRefGoogle Scholar
  17. 17.
    Liu C, Liu L, Chen X et al (2013) Decrease of 5-hydroxymethylcytosine is associated with progression of hepatocellular carcinoma through downregulation of TET1. PLoS ONE 8(5):e62828CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yang H, Liu Y, Bai F et al (2013) Tumor development is associated with decreased of TET gene expression and 5-methylcytosine hydroxylation. Oncogene 32:663–669CrossRefPubMedGoogle Scholar
  19. 19.
    Frycz BA, Murawa D, Borejsza-Wysocki M et al (2014) Decreased expression of ten-eleven translocation 1 protein is associated with some clinicopathological features in gastric cancer. Biomed Pharmacother 68:209–212CrossRefPubMedGoogle Scholar
  20. 20.
    Dong Z-R, Zhang C, Cai J-B et al (2014) Role of 5-hydroxymethylcytosine level in diagnosis and prognosis prediction of intrahepatic cholangiocarcinoma. Tumor Biol 4:2763–2771Google Scholar
  21. 21.
    Feng J, Wang Q, Guanguei L et al (2015) TET1 mediated different transcriptional regulation in prostate cancer. Inter J Clin Exp Med 8:203–211Google Scholar
  22. 22.
    Murata A, Baba Y, Ishimoto T et al (2015) Tet family proteins and 5-hydroxymethylcytosine esophageal squamous cell carcinoma. Oncotarget 6:23372–23382CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Chen Z, Shi X, Guo L et al (2017) Decreased 5-hydroxymethylcytosine levels correlate with cancer progression and poor survival: a systematic review and meta-analysis. Oncotarget 8(1):1944–1952PubMedGoogle Scholar
  24. 24.
    Xu Y, Yang H, Liu Y et al (2011) Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarato-dependente dioxygenases. Cancer Cell 19:17–30CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Figueroa ME, Abdel-Wahab O, Lu C et al (2010) Leukemia IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 6:553–567.  https://doi.org/10.1016/j.ccr.2010.11.015 CrossRefGoogle Scholar
  26. 26.
    Yan H, Parsons W, Jin G et al (2010) IDH1 and IDH2 in gliomas. NIH Public Access 8:765–773Google Scholar
  27. 27.
    Wang T, Pan Q, Lin L et al (2012) Genome-wide DNA hydroxymethylation changes are associated with neurodevelopment genes in the developing human cerebellum. Hum Mol Genet 21:5500–5510CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefPubMedGoogle Scholar
  29. 29.
    Pearson K (1896) Mathematical contributions to the theory of evolution. III. Regression, heredity, and panmixia. Philos Trans R Soc Lond Ser A 187:253–318CrossRefGoogle Scholar
  30. 30.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300Google Scholar
  31. 31.
    Patnaik MM, Hanson C, Hodnefield JM et al (2012) Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients. Leukemia 26(1):101–105CrossRefPubMedGoogle Scholar
  32. 32.
    Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A (2012) Geneious Basic: anintegrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647–1649CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jin SJ, Jiang Y, Qiu R (2011) 5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations. Cancer Res 71(24):7360–7365CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Orr B, Haffner MC, Nelson WG et al (2012) Decreased 5-hydroxymethylcytosine is associated with neural progenitor phenotype in normal brain and shorter survival in malignant glioma. PLoS ONE 7:e41036CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kraus TFJ, Globisch D, Wagner M et al (2012) Low values of 5-hydroxymethylcytosine (5hmC), the “sixth base,” are associated with anaplasia in human brain tumors. Int J Cancer 7:1577–1590CrossRefGoogle Scholar
  36. 36.
    Tsai K-W, Li G-C, Chen C-H et al (2015) Reduction of global 5-hydroxymethycytosine is a poor factor in breast cancer patients, especially for an ER/PR-negative subtype. Breast Cancer Res Treat 153:219–234CrossRefPubMedGoogle Scholar
  37. 37.
    Song C-X, Yin S, Ma L et al (2017) 5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res 27(10):1231–1242CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Li W, Zhang X, Lu X et al (2017) 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res 27(10):1243–1257CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kafer GR, Li X, Horii T et al (2016). 5-Hydroxymethylcytosine marks sites of DNA damage and promotes genome stability. Cell Rep 14:1–10CrossRefGoogle Scholar
  40. 40.
    Tomkova M, McClellan M, Kriaucionis S et al (2016). 5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA. eLife.  https://doi.org/10.7554/eLife.17082 PubMedPubMedCentralGoogle Scholar
  41. 41.
    Zhou Z, Zhang H-S, Liu Y et al (2017). Loss of TET1 facilitates DLD1 colon cancer cell migration via H3K27me3-mediated down-regulation of E-cadherin. J Cell Physiol.  https://doi.org/10.1002/jcp.26012 Google Scholar
  42. 42.
    Neri F, Dettori D, Incarnato D et al (2014) TET1 is a tumour suppressor that inhibits colon cancer growth by repressing inhibitors of the WNT pathway. Oncogene 32:4168–4176.  https://doi.org/10.1038/onc.2014.356 Google Scholar
  43. 43.
    Kim R, Sheaffer KL, Choi I et al (2016) Epigenetic regulation of intestinal stem cells by Tet1-mediated DNA hydroxymethylation. Genes Dev 30(21):2433–2442CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Duan H, Yan Z, Chen W et al (2017) TET1 inhibits EMT of ovarian cancer cells through activating Wnt/β-catenin signaling inhibitors DKK1 and SFRP2. Gynecol Oncol 147(2):408–417CrossRefPubMedGoogle Scholar
  45. 45.
    Schwalbe EC, Lindsey JC, Nakjang S et al (2017) Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study. Lancet Oncol 18(7):958–971CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Cui Q, Yang S, Ye P et al (2016). Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis. Nat Commun 7:10637CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Perera A, Eisen D, Wagner M et al (2015). TET3 is recruited by REST for context-specific hydroxymethylation and induction of gene expression. Cell Rep 11:283–294CrossRefPubMedGoogle Scholar
  48. 48.
    Uribes-Lewis S, Stark R, Carrol T et al (2015) 5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer. Genome Biol 16:69–84CrossRefGoogle Scholar
  49. 49.
    Balss J, Meyer J, Mueller W et al (2008) Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 116:597–602.  https://doi.org/10.1007/s00401-008-0455-2 CrossRefPubMedGoogle Scholar
  50. 50.
    Calvert AE, Chalastanis A, Wu Y et al (2017) Cancer-associated IDH1 promotes growth and resistance to targeted therapies in the absence of mutation. Cell Rep 19(9):1858–1873CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Du J, Martin SM, Levine M et al (2013) Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin Cancer Res 16(2):509–520CrossRefGoogle Scholar
  52. 52.
    Chen J, Guo L, Zhang L et al (2013) Vitamin C modulates TET1 function during somatic cell reprogramming. Nat Genet 45(12):1504–1509CrossRefPubMedGoogle Scholar
  53. 53.
    Dickson KM, Gustafson CB, Young JI et al (2013) Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate. Biochem Biophys Res Commun 439(4):522–527CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Minor EA, Court BL, Young JI et al (2013) Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine. J Biol Chem 288(19):13669–13674CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Yin R, Mao S-Q, Zhao B et al (2013) Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc 135(28):10396–10403CrossRefPubMedGoogle Scholar
  56. 56.
    Blaschke K, Ebata KT, Karimi MM et al (2013) Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500(7461):222–226CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Cieslak J, Cullen JJ et al (2015) Treatment of pancreatic cancer with pharmacological ascorbate. Curr Pharm Biotechnol 16(9):759–770CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Gustafson CB, Yang C, Dickson KM et al (2015) Epigenetic reprogramming of melanoma cells by vitamin C treatment. Clin Epigenetics 7(1):51CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Karina Bezerra Salomão
    • 1
    • 2
    • 4
  • Gustavo Alencastro Veiga Cruzeiro
    • 1
    • 2
    • 4
  • Ricardo Bonfim-Silva
    • 2
    • 4
  • Lenisa Geron
    • 1
    • 2
    • 4
  • Fernando Ramalho
    • 3
    • 4
  • Fabiano Pinto Saggioro
    • 3
    • 4
  • Luciano Neder Serafini
    • 3
    • 4
  • Daniel Antunes Moreno
    • 2
    • 4
  • Rosane Gomes de Paula Queiroz
    • 1
    • 2
    • 4
  • Simone dos Santos Aguiar
    • 6
  • Izilda Cardinalli
    • 6
  • José Andres Yunes
    • 6
  • Silvia Regina Brandalise
    • 6
  • Maria Sol Brassesco
    • 4
    • 5
  • Carlos Alberto Scrideli
    • 1
    • 2
    • 4
  • Luiz Gonzaga Tone
    • 1
    • 2
    • 4
  1. 1.Department of PaediatricsUniversity of São PauloRibeirão PretoBrazil
  2. 2.Department of GeneticsUniversity of São PauloRibeirão PretoBrazil
  3. 3.Department of PathologyUniversity of São PauloRibeirão PretoBrazil
  4. 4.Ribeirão Preto School of MedicineUniversity of São PauloRibeirão PretoBrazil
  5. 5.Faculty of Philosophy, Sciences and Letters at Ribeirão PretoUniversity of São PauloRibeirão PretoBrazil
  6. 6.Boldrini Centre of ChildrenUniversity of Campinas-UNICAMPCampinasBrazil

Personalised recommendations