Skip to main content

Advertisement

Log in

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

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

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.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1

Similar content being viewed by others

References

  1. Silverman S Jr (2001) Demographics and occurrence of oral and pharyngeal cancers: the outcomes, the trends, the challenge. J Am Dent Assoc 132:7–11

    Google Scholar 

  2. Stewart BW, Kleihues P (2003) World cancer report. International Agency for Research on Cancer, Lyon

    Google Scholar 

  3. Tsantoulis PK, Kastrinakis NG, Tourvas AD et al (2007) Advances in the biology of oral cancer. Oral Oncol 43:523–534

    Article  PubMed  CAS  Google Scholar 

  4. Parkin DM, Laara E, Muir CS (1988) Estimates of the worldwide frequency of sixteen major cancers in 1980. Int J Cancer 41:184–197

    Article  PubMed  CAS  Google Scholar 

  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):293–298

    Article  PubMed  Google Scholar 

  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 56:2488–2492

    PubMed  CAS  Google Scholar 

  7. Slaughter DP, Southwick HW, Smejkal W (1953) Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6:963–968

    Article  PubMed  CAS  Google Scholar 

  8. Spitz MR (1994) Epidemiology and risk factors for head and neck cancer. Semin Oncol 21:281–288

    PubMed  CAS  Google Scholar 

  9. Morse DE, Katz RV, Pendrys DG (1996) Smoking and drinking in relation to oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 5:769–777

    PubMed  CAS  Google Scholar 

  10. Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33

    Article  PubMed  CAS  Google Scholar 

  11. Ha PK, Benoit NE, Yochem R et al (2003) A transcriptional progression model for head and neck cancer. Clin Cancer Res 9(8):3058–3064

    PubMed  CAS  Google Scholar 

  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 40:145–153

    Article  PubMed  CAS  Google Scholar 

  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 10:3825–3830

    Article  PubMed  CAS  Google Scholar 

  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 39:2306–2309

    Article  PubMed  Google Scholar 

  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 61:939–942

    PubMed  CAS  Google Scholar 

  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):1093–1103

    Article  CAS  Google Scholar 

  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 69:227–234

    Article  PubMed  CAS  Google Scholar 

  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 30:416–422

    Article  PubMed  CAS  Google Scholar 

  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):9821–9826

    Article  PubMed  CAS  Google Scholar 

  20. Jerónimo C, Henrique R, Campos PF et al (2003) Endothelin B receptor gene hypermethylation in prostate adenocarcinoma. J Clin Pathol 56(1):52–55

    Article  PubMed  Google Scholar 

  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):479–484

    Article  PubMed  CAS  Google Scholar 

  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 97:6049–6054

    Article  PubMed  CAS  Google Scholar 

  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):95–101

    PubMed  CAS  Google Scholar 

  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):903–910

    Article  PubMed  CAS  Google Scholar 

  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):9347–9354

    Article  PubMed  CAS  Google Scholar 

  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 8:238

    Article  PubMed  Google Scholar 

  27. Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167

    Article  PubMed  CAS  Google Scholar 

  28. Sidransky D (2002) Emerging molecular markers of cancer. Nat Rev Cancer 2:210–219

    Article  PubMed  CAS  Google Scholar 

  29. Ha PK, Califano JA (2006) Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma. Lancet Oncol 7:77–82

    Article  PubMed  CAS  Google Scholar 

  30. Sharma S, Kelly TK, Jones PA (2010) Epigenetics in cancer. Carcinogenesis 31(1):27–36

    Article  PubMed  CAS  Google Scholar 

  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):831–835

    Article  PubMed  CAS  Google Scholar 

  32. Kwabi-Addo B, Chung W, Shen L et al (2007) Age-related DNA methylation changes in normal human prostate tissues. Clin Cancer Res 13:3796–3802

    Article  PubMed  CAS  Google Scholar 

  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:e1000602

    Article  PubMed  Google Scholar 

  34. Feil R (2006) Environmental and nutritional effects on the epigenetic regulation of genes. Mutat Res 600:46–57

    Article  PubMed  CAS  Google Scholar 

  35. Waterland RA (2006) Assessing the effects of high methionine intake on DNA methylation. J Nutr 136:1706S–1710S

    PubMed  CAS  Google Scholar 

  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):807–811

    Google Scholar 

  37. Bollati V, Baccarelli A, Hou L et al (2007) Changes in DNA methylation patterns in subjects exposed to lowdose benzene. Cancer Res 67:876–880

    Article  PubMed  CAS  Google Scholar 

  38. Miremadi A, Oestergaard MZ, Pharoah PD et al (2007) Cancer genetics of epigenetic genes. Hum Mol Genet 16(Spec No1):R28–R49

    Article  PubMed  CAS  Google Scholar 

  39. Nakajima T, Enomoto S, Ushijima T (2008) DNA methylation: a marker for carcinogen exposure and cancer risk. Environ Health Prev Med 13:8–15

    Article  PubMed  CAS  Google Scholar 

  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 132:735–743

    Article  PubMed  CAS  Google Scholar 

  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 55:2656–2659

    PubMed  CAS  Google Scholar 

  42. Scully C, Field JK, Tanzawa H (2000) Genetic aberrations in oral or head and neck squamous cell carcinoma: clinico-pathological applications. Oral Oncol 36:404–413

    Article  PubMed  CAS  Google Scholar 

  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 107:612–616

    Article  PubMed  Google Scholar 

  44. Franceschi S, Barzan L, Talamini R (1997) Screening for cancer of the head and neck: if not now, when? Oral Oncol 33(5):313–316

    Article  PubMed  CAS  Google Scholar 

  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):39–44

    Article  Google Scholar 

  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 94:561–568

    Article  PubMed  CAS  Google Scholar 

  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):2174–2179

    Article  PubMed  CAS  Google Scholar 

  48. Shen L, Kondo Y, Rosner GL et al (2005) MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 97:1330–1338

    Article  PubMed  CAS  Google Scholar 

  49. Li QL, Ito K, Sakakura C et al (2002) Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 109:113–124

    Article  PubMed  CAS  Google Scholar 

  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 84:479–484

    Article  PubMed  CAS  Google Scholar 

  51. Ku JL, Kang SB, Shin YK et al (2004) Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene 23:6736–6742

    Article  PubMed  CAS  Google Scholar 

  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 65:7743–7750

    Article  PubMed  CAS  Google Scholar 

  53. Kim WJ, Kim EJ, Jeong P et al (2005) RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors. Cancer Res 65:9347–9354

    Article  PubMed  CAS  Google Scholar 

  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 25:380–388

    Article  Google Scholar 

  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 14:817–822

    PubMed  CAS  Google Scholar 

  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):739–747

    Article  PubMed  CAS  Google Scholar 

  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):6208–6214

    Article  PubMed  CAS  Google Scholar 

  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 57:35–37

    PubMed  CAS  Google Scholar 

  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):2072–2076

    PubMed  CAS  Google Scholar 

  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):817–824

    PubMed  CAS  Google Scholar 

  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 12:165–169

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Iúri Drumond Louro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Freitas Cordeiro-Silva, M., Oliveira, Z.F.L., de Podestá, J.R.V. et al. Methylation analysis of cancer-related genes in non-neoplastic cells from patients with oral squamous cell carcinoma. Mol Biol Rep 38, 5435–5441 (2011). https://doi.org/10.1007/s11033-011-0698-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-0698-1

Keywords

Navigation