Molecular Biology Reports

, Volume 38, Issue 8, pp 5435–5441 | Cite as

Methylation analysis of cancer-related genes in non-neoplastic cells from patients with oral squamous cell carcinoma

  • Melissa de Freitas Cordeiro-Silva
  • Zilda Fagundes Lima Oliveira
  • José Roberto Vasconcelos de Podestá
  • Sonia Alves Gouvea
  • Sandra Ventorin Von Zeidler
  • Iúri Drumond Louro


Early detection of Oral Squamous Cell Carcinoma (OSCC) is important to reduce mortality rates and to help provide successful cancer treatment. Hypermethylation of CpG islands is a common epigenetic mechanism that leads to gene silencing in tumors and could be a useful biomarker in OSCC. Abnormal DNA hypermethylation can occur very early in cancer development and may be induced by exposure to environmental carcinogens. We set out to investigate the methylation status of cancer-related genes in normal oral exfoliated cells from OSCC patients and healthy volunteers, as well as possible associations with alcohol/tobacco exposure or specific tumor characteristics. The methylation status of CDKN2A (cyclin-dependent kinase inhibitor 2A or p16), SFN (stratifin or 14-3-3 σ), EDNRB (endothelin receptor B) and RUNX3 (runt-related transcript factor-3) was evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in non-neoplastic oral epithelial cells from OSCC patients (n = 70) and cancer-free subjects (n = 41). Hypermethylation was observed in CDKN2A, EDNRB and SFN genes, whereas no methylation was found in the RUNX3 gene. CDKN2A hypermethylation occurred only in the OSCC group (5.7%) while SFN and EDNRB hypermethylation occurred in both groups. There was no association between hypermethylation and smoking, drinking habits or specific tumor characteristics.


Oral squamous cell carcinoma Methylation status Cancer-related genes Normal oral mucosa 



This study was supported by grants from FACITEC/ES and Fibria ®. We would like to thank PRPPG–UFES for donating methylSEQr Bisulfite Conversion Kits. MFCS was sponsored by a CAPES scholarship.


  1. 1.
    Silverman S Jr (2001) Demographics and occurrence of oral and pharyngeal cancers: the outcomes, the trends, the challenge. J Am Dent Assoc 132:7–11Google Scholar
  2. 2.
    Stewart BW, Kleihues P (2003) World cancer report. International Agency for Research on Cancer, LyonGoogle Scholar
  3. 3.
    Tsantoulis PK, Kastrinakis NG, Tourvas AD et al (2007) Advances in the biology of oral cancer. Oral Oncol 43:523–534PubMedCrossRefGoogle Scholar
  4. 4.
    Parkin DM, Laara E, Muir CS (1988) Estimates of the worldwide frequency of sixteen major cancers in 1980. Int J Cancer 41:184–197PubMedCrossRefGoogle Scholar
  5. 5.
    Durazzo MD, Araujo CEN, Brandão Neto JS et al (2005) Clinical and epidemiological features of oral cancer in a medical school teaching hospital from 1994 to 2002: increasing incidence in women, predominance of advanced local disease, and low incidence of neck metastases. Clinics 60(4):293–298PubMedCrossRefGoogle Scholar
  6. 6.
    Califano J, van der Riet P, Westra W et al (1996) Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res 56:2488–2492PubMedGoogle Scholar
  7. 7.
    Slaughter DP, Southwick HW, Smejkal W (1953) Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6:963–968PubMedCrossRefGoogle Scholar
  8. 8.
    Spitz MR (1994) Epidemiology and risk factors for head and neck cancer. Semin Oncol 21:281–288PubMedGoogle Scholar
  9. 9.
    Morse DE, Katz RV, Pendrys DG (1996) Smoking and drinking in relation to oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 5:769–777PubMedGoogle Scholar
  10. 10.
    Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33PubMedCrossRefGoogle Scholar
  11. 11.
    Ha PK, Benoit NE, Yochem R et al (2003) A transcriptional progression model for head and neck cancer. Clin Cancer Res 9(8):3058–3064PubMedGoogle Scholar
  12. 12.
    Kulkarni V, Saranath D (2004) Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues. Oral Oncol 40:145–153PubMedCrossRefGoogle Scholar
  13. 13.
    Maruya S, Issa JP, Weber RS et al (2004) Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications. Clin Cancer Res 10:3825–3830PubMedCrossRefGoogle Scholar
  14. 14.
    López M, Aguirre JM, Cuevas N et al (2003) Gene promoter hypermethylation in oral rinses of leukoplakia patients–a diagnostic and/or prognostic tool? Eur J Cancer 39:2306–2309PubMedCrossRefGoogle Scholar
  15. 15.
    Rosas SL, Koch W, Carvalho MGC et al (2001) Promoter hypermethylation patterns of p 16, 06_ Methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res 61:939–942PubMedGoogle Scholar
  16. 16.
    Pattani KM, Zhang Z, Demokan S et al (2010) Endothelin receptor type B gene promoter hypermethylation in salivary rinses is independently associated with risk of oral cavity cancer and premalignancy. Cancer Prev Res (Phila) 3(9):1093–1103CrossRefGoogle Scholar
  17. 17.
    Christensen BC, Houseman EA, Godleski JJ et al (2009) Epigenetic profiles distinguish pleural mesothelioma from normal pleura and predict lung asbestos burden and clinical outcome. Cancer Res 69:227–234PubMedCrossRefGoogle Scholar
  18. 18.
    Marsit CJ, Christensen BC, Houseman EA et al (2009) Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma. Carcinogenesis 30:416–422PubMedCrossRefGoogle Scholar
  19. 19.
    Herman JG, Graff JR, Myöhänen S et al (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93(18):9821–9826PubMedCrossRefGoogle Scholar
  20. 20.
    Jerónimo C, Henrique R, Campos PF et al (2003) Endothelin B receptor gene hypermethylation in prostate adenocarcinoma. J Clin Pathol 56(1):52–55PubMedCrossRefGoogle Scholar
  21. 21.
    Kim TY, Lee HJ, Hwang KS et al (2004) Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest 84(4):479–484PubMedCrossRefGoogle Scholar
  22. 22.
    Ferguson AT, Evron E, Umbricht CB et al (2000) High frequency of hypermethylation at the 14–3-3σ locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA 97:6049–6054PubMedCrossRefGoogle Scholar
  23. 23.
    Bender CM, Pao MM, Jones PA (1998) Inhibition of DNA methylation by 5-aza-2′-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res 58(1):95–101PubMedGoogle Scholar
  24. 24.
    Pao MM, Tsutsumi M, Liang G et al (2001) The endothelin receptor B (EDNRB) promoter displays heterogeneous, site specific methylation patterns in normal and tumor cells. Hum Mol Genet 10(9):903–910PubMedCrossRefGoogle Scholar
  25. 25.
    Kim WJ, Kim EJ, Jeong P et al (2005) RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res 65(20):9347–9354PubMedCrossRefGoogle Scholar
  26. 26.
    Negraes PD, Favaro FP, Camargo JL et al (2008) DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection. BMC Cancer 8:238PubMedCrossRefGoogle Scholar
  27. 27.
    Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167PubMedCrossRefGoogle Scholar
  28. 28.
    Sidransky D (2002) Emerging molecular markers of cancer. Nat Rev Cancer 2:210–219PubMedCrossRefGoogle Scholar
  29. 29.
    Ha PK, Califano JA (2006) Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma. Lancet Oncol 7:77–82PubMedCrossRefGoogle Scholar
  30. 30.
    Sharma S, Kelly TK, Jones PA (2010) Epigenetics in cancer. Carcinogenesis 31(1):27–36PubMedCrossRefGoogle Scholar
  31. 31.
    Bornman DM, Mathew S, Alsruhe J et al (2001) Methylation of the E-cadherin gene in bladder neoplasia and in normal urothelial epithelium from elderly individuals. Am J Pathol 159(3):831–835PubMedCrossRefGoogle Scholar
  32. 32.
    Kwabi-Addo B, Chung W, Shen L et al (2007) Age-related DNA methylation changes in normal human prostate tissues. Clin Cancer Res 13:3796–3802PubMedCrossRefGoogle Scholar
  33. 33.
    Christensen BC, Houseman EA, Marsit CJ et al (2009) Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS Genet 5:e1000602PubMedCrossRefGoogle Scholar
  34. 34.
    Feil R (2006) Environmental and nutritional effects on the epigenetic regulation of genes. Mutat Res 600:46–57PubMedCrossRefGoogle Scholar
  35. 35.
    Waterland RA (2006) Assessing the effects of high methionine intake on DNA methylation. J Nutr 136:1706S–1710SPubMedGoogle Scholar
  36. 36.
    von Zeidler SV, Miracca EC, Nagai MA et al (2004) Hypermethylation of the p16 gene in normal oral mucosa of smokers. Int J Mol Med 14(5):807–811Google Scholar
  37. 37.
    Bollati V, Baccarelli A, Hou L et al (2007) Changes in DNA methylation patterns in subjects exposed to lowdose benzene. Cancer Res 67:876–880PubMedCrossRefGoogle Scholar
  38. 38.
    Miremadi A, Oestergaard MZ, Pharoah PD et al (2007) Cancer genetics of epigenetic genes. Hum Mol Genet 16(Spec No1):R28–R49PubMedCrossRefGoogle Scholar
  39. 39.
    Nakajima T, Enomoto S, Ushijima T (2008) DNA methylation: a marker for carcinogen exposure and cancer risk. Environ Health Prev Med 13:8–15PubMedCrossRefGoogle Scholar
  40. 40.
    Kato K, Hara A, Kuno T et al (2006) Aberrant promoter hypermethylation of p16 and MGMT genes in oral squamous cell carcinomas and the surrounding normal mucosa. J Cancer Res Clin Oncol 132:735–743PubMedCrossRefGoogle Scholar
  41. 41.
    El Naggar AK, Hurr K, Batsakis JG et al (1995) Sequential loss of heterozygosity at microsatellite motifs in preinvasive and invasive head and neck squamous carcinoma. Cancer Res 55:2656–2659PubMedGoogle Scholar
  42. 42.
    Scully C, Field JK, Tanzawa H (2000) Genetic aberrations in oral or head and neck squamous cell carcinoma: clinico-pathological applications. Oral Oncol 36:404–413PubMedCrossRefGoogle Scholar
  43. 43.
    Zochbauer-Muller S, Lam S, Toyooka S et al (2003) Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers. Int J Cancer 107:612–616PubMedCrossRefGoogle Scholar
  44. 44.
    Franceschi S, Barzan L, Talamini R (1997) Screening for cancer of the head and neck: if not now, when? Oral Oncol 33(5):313–316PubMedCrossRefGoogle Scholar
  45. 45.
    Bhutani M, Pathak AK, Fan YH et al (2008) Oral epithelium as a surrogate tissue for assessing smoking-induced molecular alterations in the lungs. Cancer Prev Res (Phila) 1(1):39–44CrossRefGoogle Scholar
  46. 46.
    Shaw RJ, Liloglou T, Rogers SN et al (2006) Promoter methylation of P16, RARb, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 94:561–568PubMedCrossRefGoogle Scholar
  47. 47.
    Hall GL, Shaw RJ, Field EA et al (2008) p16 Promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 17(8):2174–2179PubMedCrossRefGoogle Scholar
  48. 48.
    Shen L, Kondo Y, Rosner GL et al (2005) MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 97:1330–1338PubMedCrossRefGoogle Scholar
  49. 49.
    Li QL, Ito K, Sakakura C et al (2002) Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 109:113–124PubMedCrossRefGoogle Scholar
  50. 50.
    Kim TY, Lee HJ, Hwang KS et al (2004) Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest 84:479–484PubMedCrossRefGoogle Scholar
  51. 51.
    Ku JL, Kang SB, Shin YK et al (2004) Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene 23:6736–6742PubMedCrossRefGoogle Scholar
  52. 52.
    Ito K, Liu Q, Salto-Tellez M et al (2005) RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization. Cancer Res 65:7743–7750PubMedCrossRefGoogle Scholar
  53. 53.
    Kim WJ, Kim EJ, Jeong P et al (2005) RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res 65:9347–9354PubMedCrossRefGoogle Scholar
  54. 54.
    Mor T, Nomoto S, Koshikawa K et al (2005) Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma. Liver Int 25:380–388CrossRefGoogle Scholar
  55. 55.
    Yanada M, Yaoi T, Shimada J et al (2005) Frequent hemizygous deletion at 1p36 and hypermethylation downregulate RUNX3 expression in human lung cancer cell lines. Oncol Rep 14:817–822PubMedGoogle Scholar
  56. 56.
    Gao F, Huang C, Lin M et al (2009) Frequent inactivation of RUNX3 by promoter hypermethylation and protein mislocalization in oral squamous cell carcinomas. J Cancer Res Clin Oncol 135(5):739–747PubMedCrossRefGoogle Scholar
  57. 57.
    Wolff EM, Liang G, Cortez CC et al (2008) RUNX3 methylation reveals that bladder tumors are older in patients with a history of smoking. Cancer Res 68(15):6208–6214PubMedCrossRefGoogle Scholar
  58. 58.
    Nelson JB, Lee WH, Nguyen SH et al (1997) Methylation of the 5′ CpG island of the endothelin B receptor gene is common in human prostate cancer. Cancer Res 57:35–37PubMedGoogle Scholar
  59. 59.
    Gasco M, Bell AK, Heath V et al (2002) Epigenetic inactivation of 14–3-3 δ in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity. Cancer Res 62(7):2072–2076PubMedGoogle Scholar
  60. 60.
    Bhawal UK, Tsukinoki K, Sasahira T et al (2007) Methylation and intratumoural heterogeneity of 14–3-3 sigma in oral cancer. Oncol Rep 18(4):817–824PubMedGoogle Scholar
  61. 61.
    Bhatia K, Siraj AK, Hussain A et al (2003) The tumor suppressor gene 14–3-3σ is commonly methylated in normal and malignant lymphoid cells. Cancer Epidemiol Biomarkers Prev 12:165–169PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Melissa de Freitas Cordeiro-Silva
    • 1
  • Zilda Fagundes Lima Oliveira
    • 2
  • José Roberto Vasconcelos de Podestá
    • 2
  • Sonia Alves Gouvea
    • 3
  • Sandra Ventorin Von Zeidler
    • 4
  • Iúri Drumond Louro
    • 1
  1. 1.Núcleo de Genética Humana e Molecular, Departamento de Ciências Biológicas, Centro de Ciências Humanas e NaturaisUniversidade Federal do Espírito SantoVitóriaBrazil
  2. 2.Programa de Prevenção e Detecção Precoce do Câncer Bucal, Setor de Cirurgia de Cabeça e PescoçoHospital Santa Rita de CássiaVitóriaBrazil
  3. 3.Departamento de Ciências Fisiológicas, Centro de Ciências da SaúdeUniversidade Federal do Espírito SantoVitóriaBrazil
  4. 4.Departamento de Patologia, Centro de Ciências da SaúdeUniversidade Federal do Espírito SantoVitóriaBrazil

Personalised recommendations