Advertisement

Promoter DNA Methylation and mRNA Expression Level of p16 Gene in Oral Squamous Cell Carcinoma: Correlation with Clinicopathological Characteristics

  • Abdolamir Allameh
  • Abdolkarim Moazeni-Roodi
  • Iraj Harirchi
  • Mehrdad Ravanshad
  • Maziar Motiee-Langroudi
  • Ata Garajei
  • Azin Hamidavi
  • Seyed Alireza Mesbah-Namin
Original Article
  • 23 Downloads

Abstract

The aim of this study was to investigate the relationship between p16 methylation and its expression in oral squamous cell carcinoma (OSCC). Also the contribution of clinicopathological factors, HPV infection and smoking in p16 expression and promoter methylation has been investigated. In this study 67 consecutive OSCC patients and 59 normal individuals were enrolled. All patients were candidates for surgery of oral cavity and fresh tumor biopsies were collected and processed for DNA and RNA extraction. Normal gingival tissues were collected from individuals referred to dentistry clinic and considered as controls. All the cases and controls were checked for HPV infection and then promoter methylation and expression of p16 gene were determined using Methylation-specific PCR (MSP) and real-time PCR (QPCR), respectively. Methylation of p16 in tumors and normal tissues were 59.7 and 38.9%, respectively. Most of hypermethylated samples (>82%) were in high grades. P16 methylation was comparable in HPV+ and HPV- patients or smokers. P16 was overexpressed (~3 fold; p = 0.044) in HPV+ tumors, but it was significantly down-regulated in smoker patients (40% of all tumors). Comparison of P16 expression in OSCC tumors with different degrees of promoter methylation further suggest the relationship of methylation rate and down-regulation of P16 expression. The p16 methylation and expression was differentially affected in patients with HPV infection and the smoker cases. Regardless of the influence of environmental factors, it appears that P16 status is useful for classifying patients with OSCC and for influencing treatment strategies in accordance with this classification. Moreover, targeting the upregulation of p16 could be a promising therapeutic option.

Keywords

OSCC HPV Smoking P16 promoter methylation P16 gene expression Iran 

Notes

Acknowledgements

This study was financially supported by Tarbiat Modares University and Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, I.R. Iran. (Grant# 92-02-51-23399).

Compliance with Ethical Standards

Conflict of Interest

The authors have declared that no potential conflict of interest exists.

References

  1. 1.
    Rivera C (2015) Essentials of oral cancer. Int J Clin Exp Pathol 8:11884–11894PubMedPubMedCentralGoogle Scholar
  2. 2.
    Karsai S, Abel U, Roesch-Ely M, Affolter A, Hofele C, Joos S et al (2007) Comparison of p16(INK4a) expression with p53 alterations in head and neck cancer by tissue microarray analysis. J Pathol 211:314–322CrossRefGoogle Scholar
  3. 3.
    Al-Kaabi A, van Bockel LW, Pothen AJ, Willems SM (2014) p16INK4A and p14ARF gene promoter hypermethylation as prognostic biomarker in oral and oropharyngeal squamous cell carcinoma: a review. Dis Markers 2014:260549CrossRefGoogle Scholar
  4. 4.
    Petti S (2009) Lifestyle risk factors for oral cancer. Oral Oncol 45:340–350CrossRefGoogle Scholar
  5. 5.
    Laco J, Nekvindova J, Novakova V, Celakovsky P, Dolezalova H, Tucek L et al (2012) Biologic importance and prognostic significance of selected clinicopathological parameters in patients with oral and oropharyngeal squamous cell carcinoma, with emphasis on smoking, protein p16(INK4a) expression, and HPV status. Neoplasma 59:398–408CrossRefGoogle Scholar
  6. 6.
    Kreimer AR, Clifford GM, Boyle P, Franceschi S (2005) Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer Epidemiol Biomark Prev 14:467–475CrossRefGoogle Scholar
  7. 7.
    Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368:753–756CrossRefGoogle Scholar
  8. 8.
    Stone S, Jiang P, Dayananth P, Tavtigian SV, Katcher H, Parry D et al (1995) Complex structure and regulation of the P16 (MTS1) locus. Cancer Res 55:2988–2994PubMedGoogle Scholar
  9. 9.
    Serrano M, Hannon GJ, Beach D (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366:704–707CrossRefGoogle Scholar
  10. 10.
    Rocco JW, Sidransky D (2001) p16(MTS-1/CDKN2/INK4a) in cancer progression. Exp Cell Res 264:42–55CrossRefGoogle Scholar
  11. 11.
    Xu R, Wang F, Wu L, Wang J, Lu C (2013) A systematic review of hypermethylation of p16 gene in esophageal cancer. Cancer Biomark 13:215–226CrossRefGoogle Scholar
  12. 12.
    Jablonowski Z, Reszka E, Gromadzinska J, Wasowicz W, Sosnowski M (2011) Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer. Arch Med Sci 7:512–516CrossRefGoogle Scholar
  13. 13.
    Sharma G, Mirza S, Prasad CP, Srivastava A, Gupta SD, Ralhan R (2007) Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients. Life Sci 80:1873–1881CrossRefGoogle Scholar
  14. 14.
    Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D et al (2006) Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 94:561–568CrossRefGoogle Scholar
  15. 15.
    Khor GH, Froemming GR, Zain RB, Abraham MT, Omar E, Tan SK et al (2013) DNA methylation profiling revealed promoter hypermethylation-induced silencing of p16, DDAH2 and DUSP1 in primary oral squamous cell carcinoma. Int J Med Sci 10:1727–1739CrossRefGoogle Scholar
  16. 16.
    Demokan S, Chuang A, Suoglu Y, Ulusan M, Yalniz Z, Califano JA et al (2012) Promoter methylation and loss of p16(INK4a) gene expression in head and neck cancer. Head Neck 34:1470–1475CrossRefGoogle Scholar
  17. 17.
    Todd R, Hinds PW, Munger K, Rustgi AK, Opitz OG, Suliman Y et al (2002) Cell cycle dysregulation in oral cancer. Crit Rev Oral Biol Med 13:51–61CrossRefGoogle Scholar
  18. 18.
    Yuen PW, Man M, Lam KY, Kwong YL (2002) Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas. J Clin Pathol 55:58–60CrossRefGoogle Scholar
  19. 19.
    Ai L, Stephenson KK, Ling W, Zuo C, Mukunyadzi P, Suen JY et al (2003) The p16 (CDKN2a/INK4a) tumor-suppressor gene in head and neck squamous cell carcinoma: a promoter methylation and protein expression study in 100 cases. Mod Pathol 16:944–950CrossRefGoogle Scholar
  20. 20.
    Alves MK, Ferrasi AC, Lima VP, Ferreira MV, de Moura Campos Pardini MI, Rabenhorst SH (2011) Inactivation of COX-2, HMLH1 and CDKN2A gene by promoter methylation in gastric cancer: relationship with histological subtype, tumor location and helicobacter pylori genotype. Pathobiology 78:266–276CrossRefGoogle Scholar
  21. 21.
    Banzai C, Nishino K, Quan J, Yoshihara K, Sekine M, Yahata T et al (2014) Promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A genes in cervical carcinoma. Int J Clin Oncol 19:127–132CrossRefGoogle Scholar
  22. 22.
    Celebiler Cavusoglu A, Sevinc AI, Saydam S, Canda T, Baskan Z, Kilic Y et al (2010) Promoter methylation and expression changes of CDH1 and P16 genes in invasive breast cancer and adjacent normal breast tissue. Neoplasma 57:465–472CrossRefGoogle Scholar
  23. 23.
    Jitani AK, Raphael V, Mishra J, Shunyu NB, Khonglah Y, Medhi J (2015) Analysis of human papilloma virus 16/18 DNA and its correlation with p16 expression in Oral cavity squamous cell carcinoma in north-eastern India: a chromogenic in-situ hybridization based study. J Clin Diagn Res 9:EC04–EC07PubMedPubMedCentralGoogle Scholar
  24. 24.
    Salehinejad J, Sharifi N, Amirchaghmaghi M, Ghazi N, Shakeri MT, Ghazi A (2014) Immunohistochemical expression of p16 protein in oral squamous cell carcinoma and lichen planus. Ann Diagn Pathol 18:210–213CrossRefGoogle Scholar
  25. 25.
    Lingen MW, Kalmar JR, Karrison T, Speight PM (2008) Critical evaluation of diagnostic aids for the detection of oral cancer. Oral Oncol 44:10–22CrossRefGoogle Scholar
  26. 26.
    Kochl S, Niederstatter H (2005) Parson W. DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR. Methods Mol Biol 297:13–30PubMedGoogle Scholar
  27. 27.
    Sailasree R, Abhilash A, Sathyan KM, Nalinakumari KR, Thomas S, Kannan S (2008) Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma. Cancer Epidemiol Biomark Prev 17:414–420CrossRefGoogle Scholar
  28. 28.
    Cao J, Zhou J, Gao Y, Gu L, Meng H, Liu H et al (2009) Methylation of p16 CpG island associated with malignant progression of oral epithelial dysplasia: a prospective cohort study. Clin Cancer Res 15:5178–5183CrossRefGoogle Scholar
  29. 29.
    Ha PK, Califano JA (2006) Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma. Lancet Oncol 7:77–82CrossRefGoogle Scholar
  30. 30.
    Ishida E, Nakamura M, Ikuta M, Shimada K, Matsuyoshi S, Kirita T et al (2005) Promotor hypermethylation of p14ARF is a key alteration for progression of oral squamous cell carcinoma. Oral Oncol 41:614–622CrossRefGoogle Scholar
  31. 31.
    Papadimitrakopoulou VA, Izzo J, Mao L, Keck J, Hamilton D, Shin DM et al (2001) Cyclin D1 and p16 alterations in advanced premalignant lesions of the upper aerodigestive tract: role in response to chemoprevention and cancer development. Clin Cancer Res 7:3127–3134PubMedGoogle Scholar
  32. 32.
    Perez-Sayans M, Suarez-Penaranda JM, Padin-Iruegas ME, Gayoso-Diz P, Reis-De Almeida M, Barros-Angueira F et al (2015) The loss of p16 expression worsens the prognosis of OSCC. Appl Immunohistochem Mol Morphol 23:724–732CrossRefGoogle Scholar
  33. 33.
    Bhatia V, Goel MM, Makker A, Tewari S, Yadu A, Shilpi P et al (2014) Promoter region hypermethylation and mRNA expression of MGMT and p16 genes in tissue and blood samples of human premalignant oral lesions and oral squamous cell carcinoma. Biomed Res Int 2014:248419PubMedPubMedCentralGoogle Scholar
  34. 34.
    Schlecht NF, Ben-Dayan M, Anayannis N, Lleras RA, Thomas C, Wang Y et al (2015) Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma. Cancer Med 4:342–353CrossRefGoogle Scholar
  35. 35.
    Sargolzaei S, Farhadi S, Kazemi B, Bandehpour M, Kharazifard MJ (2014) The correlation between p16 expression and INK4a locus mutation with grades and stages in oral squamous cell carcinoma. Indian J Pathol Microbiol 57:24–30CrossRefGoogle Scholar
  36. 36.
    Dona MG, Spriano G, Pichi B, Rollo F, Laquintana V, Covello R et al (2015) Human papillomavirus infection and p16 overexpression in oropharyngeal squamous cell carcinoma: a case series from 2010 to 2014. Future Microbiol 10:1283–1291CrossRefGoogle Scholar
  37. 37.
    Talikka M, Sierro N, Ivanov NV, Chaudhary N, Peck MJ, Hoeng J, Coggins CRE (2012) ECR Peitsch MC genomic impact of cigarette smoke, with application to three smoking-related diseases. Crit Rev Toxicol 42(10):877–889CrossRefGoogle Scholar
  38. 38.
    Antonsson A, Neale RE, Boros S, Lampe G, Coman WB, Pryor DI et al (2015) Human papillomavirus status and p16(INK4A) expression in patients with mucosal squamous cell carcinoma of the head and neck in Queensland, Australia. Cancer Epidemiol 39:174–181CrossRefGoogle Scholar
  39. 39.
    Mai S, Welzel G, Ottstadt M, Lohr F, Severa S, Prigge ES et al (2015) Prognostic relevance of HPV infection and p16 overexpression in squamous cell anal Cancer. Int J Radiat Oncol Biol Phys 93:819–827CrossRefGoogle Scholar
  40. 40.
    Li J, Poi MJ, Tsai MD (2011) Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer. Biochemistry 50:5566–5582CrossRefGoogle Scholar
  41. 41.
    Konig F, Krekeler G, Honig JF, Cordon-Cardo C, Fischer G, Korabiowska M (2007) Relation between human papillomavirus positivity and p16 expression in head and neck carcinomas--a tissue microarray study. Anticancer Res 27:283–288PubMedGoogle Scholar
  42. 42.
    El-Naggar AK, Westra WH (2012) p16 expression as a surrogate marker for HPV-related oropharyngeal carcinoma: a guide for interpretative relevance and consistency. Head Neck 34:459–461CrossRefGoogle Scholar
  43. 43.
    Wang S, Deng Z, Seneviratne CJ, Cheung GS, Jin L, Zhao B et al (2015) Enterococcus faecalis promotes osteoclastogenesis and semaphorin 4D expression. Innate Immun 21:726–735CrossRefGoogle Scholar
  44. 44.
    Shaw RJ, Hobkirk AJ, Nikolaidis G, Woolgar JA, Triantafyllou A, Brown JS et al (2013) Molecular staging of surgical margins in oral squamous cell carcinoma using promoter methylation of p16(INK4A), cytoglobin, E-cadherin, and TMEFF2. Ann Surg Oncol 20:2796–2802CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2018

Authors and Affiliations

  • Abdolamir Allameh
    • 1
  • Abdolkarim Moazeni-Roodi
    • 1
    • 2
  • Iraj Harirchi
    • 3
  • Mehrdad Ravanshad
    • 4
  • Maziar Motiee-Langroudi
    • 3
  • Ata Garajei
    • 3
    • 5
  • Azin Hamidavi
    • 1
  • Seyed Alireza Mesbah-Namin
    • 1
  1. 1.Department of Clinical Biochemistry, Faculty of Medical SciencesTarbiat Modares UniversityTehranIslamic Republic of Iran
  2. 2.Department of Clinical BiochemistryIranshahr University of Medical SciencesIranshahrIran
  3. 3.Cancer Research Center, Cancer Institute of IranTehran University of Medical SciencesTehranIslamic Republic of Iran
  4. 4.Department of Medical Virology, Faculty of Medical SciencesTarbiat Modares UniversityTehranIslamic Republic of Iran
  5. 5.Department of Oral and Maxillofacial Surgery, School of Dentistry and Department of Head and Neck Surgical Oncology and Reconstructive Surgery, School of MedicineTehran University of Medical SciencesTehranIran

Personalised recommendations