Tumor Biology

, Volume 35, Issue 9, pp 9069–9078 | Cite as

Epigenetic alteration of p16 and retinoic acid receptor beta genes in the development of epithelial ovarian carcinoma

  • Rahul Bhagat
  • Sandeep Sriram Kumar
  • Shilpa Vaderhobli
  • Chennagiri S. Premalata
  • Venkateshaiah Reddihalli Pallavi
  • Gawari Ramesh
  • Lakshmi Krishnamoorthy
Research Article


Silencing of tumor suppressor and tumor-related genes by promoter hypermethylation is one of the major events in ovarian carcinogenesis. In this study, we analyzed aberrant promoter methylation of p16 and RAR-β genes in 134 epithelial ovarian carcinomas (EOCs), 23 low malignant potential (LMP) tumors, 26 benign cystadenomas, and 15 normal ovarian tissues. Methylation was investigated by methylation-specific PCR (MSP), and the results were confirmed by bisulfite DNA sequencing. Relative gene expression of p16 and RAR-β was done using quantitative reverse transcriptase PCR (qRT-PCR) on 51 EOC cases, 9 LMP tumors, and 7 benign cystadenomas with 5 normal ovarian tissues. Aberrant methylation for p16 and RAR-β was present in 43 % (58/134) and 31 % (41/134) in carcinoma cases, 22 % (05/23) and 52 % (12/23) in LMP tumors, and 42 % (11/26) and 69 % (18/26) in benign cystadenomas. No methylation was observed in any of the normal ovarian tissues. The mRNA expression level of p16 and RAR-β was significantly downregulated in EOC and LMP tumors than the corresponding normal tissues whereas the expression level was normal in benign cystadenomas for p16 and slightly reduced for RAR-β. A significant correlation of p16 promoter methylation was observed with reduced gene expression in EOC. For RAR-β, no significant correlation was observed between promoter methylation and gene expression. Our results suggest that epigenetic alterations of p16 and RAR-β have an important role in ovarian carcinogenesis and that mechanism along with methylation plays a significant role in downregulation of RAR-β gene in ovarian cancer.


Ovarian carcinoma p16 RAR-β MSP Gene expression 



The research work is supported by Department of Biotechnology, India, Grant No BT/PR 13440/MED/30/267/2009. We are grateful to Dr V Shanmugam (Research Assistant, NIMHANS) for helping with the statistical analysis.

Conflict of interest


Ethical clearance

The study was approved by Institutional Ethics Committee and all samples were collected after obtaining written, informed consent from patients.

Supplementary material

13277_2014_2136_MOESM1_ESM.docx (13 kb)
Supplementary Table S1 (DOCX 13 kb)
13277_2014_2136_MOESM2_ESM.docx (12 kb)
Supplementary Table S2 (DOCX 11 kb)


  1. 1.
    Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 1998. CA Cancer J Clin. 2005;55:74–108.CrossRefPubMedGoogle Scholar
  2. 2.
    Three-year report of population based cancer registries 2009–11: National Cancer Registry Programme (ICMR), Bangalore 2013.Google Scholar
  3. 3.
    Hennessy BT, Coleman RL, Markman M. Ovarian cancer. Lancet. 2009;374:1371–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Wei SH, Balch C, Paik HH, Kim YS, Baldwin RL, Liyanarachchi S, et al. Prognostic DNA methylation biomarkers in ovarian cancer. Clin Cancer Res. 2006;12:2788–94.CrossRefPubMedGoogle Scholar
  5. 5.
    Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Warnecke PM, Bestor TH. Cytosine methylation and human cancer. Curr Opin Oncol. 2000;12:68–73.CrossRefPubMedGoogle Scholar
  7. 7.
    Baylin SB, Herman JG. DNA Hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 2000;16:168–74.CrossRefPubMedGoogle Scholar
  8. 8.
    Liggett Jr WH, Sidransky D. Role of the p16 tumor suppressor gene in cancer. J Clin Oncol. 1998;16:197–206.Google Scholar
  9. 9.
    Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994;264:436–40.CrossRefPubMedGoogle Scholar
  10. 10.
    Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase- 4 inhibitor gene in multiple human cancers. Nature. 1994;368:753–6.CrossRefPubMedGoogle Scholar
  11. 11.
    Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–7.CrossRefPubMedGoogle Scholar
  12. 12.
    Cobrinik D, Dowdy SF, Hinds PW, Mittnacht S, Weinberg RA. The retinoblastoma protein and the regulation of cell cycling. Trends Biochem Sci. 1992;17:312–5.CrossRefPubMedGoogle Scholar
  13. 13.
    Sherr CJ, Roberts JM. Cdk inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13:1501–12.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang HS, Postigo AA, Dean DC. Active transcriptional repression by the Rb-E2F complex mediates G1 arrest triggered by p16INK4a, TGFbeta, and contact inhibition. Cell. 1999;97:53–61.CrossRefPubMedGoogle Scholar
  15. 15.
    Rocco JW, Sidransky D. p16 (MTS-1/CDKN2/INK4a) in cancer progression. Exp Cell Res. 2001;264:42–55.CrossRefPubMedGoogle Scholar
  16. 16.
    Grummer MA, Thet LA, Zachman RD. Expression of retinoic acid receptor genes in fetal and newborn rat lung. Pediatr Pulmonol. 1994;17:234–8.CrossRefPubMedGoogle Scholar
  17. 17.
    Mendelsohn C, Lohnes D, Decrimo D, Lufkin T, LeMeur M, Chambon P, et al. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development. 1994;120:2749–71.PubMedGoogle Scholar
  18. 18.
    Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J. 1996;10:940–54.PubMedGoogle Scholar
  19. 19.
    Yang Q, Mori I, Shan L, Nakamura M, Nakamura Y, Utsunomiya H, et al. Biallelic inactivation of retinoic acid receptor 2 gene by epigenetic change in breast cancer. Am J Pathol. 2001;158:299–303.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Ivanova T, Petrenko A, Gritsko T, Vinokourova S, Eshilev E, Kobzeva V, et al. Methylation and silencing of the retinoic acid receptor-beta 2 gene in cervical cancer. BMC Cancer. 2002;2:4 (e1).PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Yang Q, Yoshimura G, Mori I, Sakurai T, Kakudo K. Chromosome 3p and breast cancer. J Hum Genet. 2002;47:453–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Nakayama T, Watanabe M, Yamanaka M, Hirokawa Y, Suzuki H, Ito H, et al. The role of epigenetic modifications in retinoic acid receptor Beta2 gene expression in human prostate cancers. Lab Investig. 2001;81:1049–57.CrossRefPubMedGoogle Scholar
  23. 23.
    Fackler MJ, McVeigh M, Evron E, Garrett E, Mehrotra J, Polyak K, et al. DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and twist in in situ and invasive lobular breast carcinoma. Int J Cancer. 2003;107:970–5.CrossRefPubMedGoogle Scholar
  24. 24.
    Jerónimo C, Henrique R, Hoque MO, Ribeiro FR, Oliveira J, Fonseca D, et al. Quantitative RARbeta2 hypermethylation: a promising prostate cancer marker. Clin Cancer Res. 2004;10:4010–4.CrossRefPubMedGoogle Scholar
  25. 25.
    Fischer JR, Ohnmacht U, Rieger N, Zemaitis M, Stoffregen C, Kostrzewa M, et al. Promoter methylation of RASSF1A, RARbeta and DAPK predict poor prognosis of patients with malignant mesothelioma. Lung Cancer. 2006;54:109–16.CrossRefPubMedGoogle Scholar
  26. 26.
    He M, Vanaja DK, Karnes RJ, Young CY. Epigenetic regulation of Myc on retinoic acid receptor beta and PDLIM4 in RWPE1 cells. Prostate. 2009;69:1643–50.CrossRefPubMedGoogle Scholar
  27. 27.
    Björklund P, Akerström G, Westin G. Accumulation of nonphosphorylated beta-catenin and c-myc in primary and uremic secondary hyperparathyroid tumors. J Clin Endocrinol Metab. 2007;92:338–44.CrossRefPubMedGoogle Scholar
  28. 28.
    Bhagat R, Shilpa V, Premalata CS, Ramesh G, Ramesh C, Pallavi VR, et al. Aberrant promoter methylation of the RASSF1A and APC genes in epithelial ovarian carcinoma development. Cell Oncol. 2012;35:473–9.CrossRefGoogle Scholar
  29. 29.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821–6.PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D, et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn. 2006;8:209–17.PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Parrish RR, Day JJ, Lubin FD. Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues. Curr Protoc Neurosci. 2012 July; CHAPTER: Unit 7.24. doi: 10.1002/0471142301.ns0724s60.
  32. 32.
    Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. New Engl J Med. 2003;349:2042–54.CrossRefPubMedGoogle Scholar
  33. 33.
    Milde-Langosch K, Ocon E, Becker G, Loning T. p16/MTS1 inactivation in ovarian carcinomas: high frequency of reduced protein expression associated with hypermethylation or mutation in endometrioid and mucinous tumors. Int J Cancer. 1998;79:61–5.CrossRefPubMedGoogle Scholar
  34. 34.
    McCluskey LL, Chen C, Delgadillo E, Felix JC, Muderspach LI, Debeau L. Differences in p16 gene methylation and expression in benign and malignant ovarian tumors. Gynecol Oncol. 1999;72:87–92.CrossRefPubMedGoogle Scholar
  35. 35.
    Wong YF, Chung TK, Cheung TH, Nobori T, Yu AL, Yu J, et al. Methylation of p16INK4A in primary gynecologic malignancy. Cancer Lett. 1999;136:231–5.CrossRefPubMedGoogle Scholar
  36. 36.
    Furlan D, Carnevali I, Marcomini B, Cerutti R, Dainese E, Capella C, et al. The high frequency of de novo promoter methylation in synchronous primary endometrial and ovarian carcinomas. Clin Cancer Res. 2006;12:3329–36.CrossRefPubMedGoogle Scholar
  37. 37.
    Li M, Huang ZJ, Dong WH, Li XY, Wang XY, He XH, et al. Disfigurement of p16INK4A gene expression in development of ovarian cancer and the mechanism. Zhonghua Fu Chan Ke Za Zhi. 2006;41:408–12.PubMedGoogle Scholar
  38. 38.
    Wiley A, Katsaros D, Chen H, Rigault de la Longrais IA, Beeghly A, Puopolo M, et al. Aberrant promoter methylation of multiple genes in malignant ovarian tumors and in ovarian tumors with low malignant potential. Cancer. 2006;107:299–308.CrossRefPubMedGoogle Scholar
  39. 39.
    Leal Rojas P, Anabalón Rodríguez L, García Muñoz P, Tapia Escalona O, Guzmán González P, Araya Orostica JC, et al. Promoter hypermethylation gene patterns in gynecological tumors. Med Clin (Barc). 2009;132:371–6.CrossRefGoogle Scholar
  40. 40.
    Makarla PB, Saboorian MH, Ashfaq R, Toyooka KO, Toyooka S, Minna JD, et al. Promoter hypermethylation profile of ovarian epithelial neoplasms. Clin Cancer Res. 2005;11:5365–9.CrossRefPubMedGoogle Scholar
  41. 41.
    Bammidi LS, Neerukonda GN, Murthy S, Kanapuram RD. p16 gene alterations in human ovarian cancer: comparison between tissue and blood samples. Int J Gynecol Cancer. 2012;22(4):553–60.CrossRefPubMedGoogle Scholar
  42. 42.
    Widschwendter M, Berger J, Hermann M, Müller HM, Amberger A, Zeschnigk M, et al. Methylation and silencing of retinoic acid receptor-beta 2 gene in breast cancer. J Natl Cancer Inst. 2000;92(10):826–32.CrossRefPubMedGoogle Scholar
  43. 43.
    Gao YP, Li M, Zhang YY, Wang H, He XH, Wang ZH. Relationship between RAR-beta gene expression defect and its methylation. Zhonghua Fu Chan Ke Za Zhi. 2007;42(7):472–6.PubMedGoogle Scholar
  44. 44.
    Tang D, Kryvenko ON, Mitrache N, Do KC, Jankowski M, Chitale DA, et al. Methylation of RAR-β gene increases prostate cancer risk in black Americans. J Urol. 2013;190(1):317–24.PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Chmelarova M, Krepinska E, Spacek J, Laco J, Nekvindova J, Palicka V. Methylation analysis of tumour suppressor genes in ovarian cancer using MS-MLPA. Folia Biol (Praha). 2012;58:246–50.Google Scholar
  46. 46.
    Ozdemir F, Altinisik J, Karateke A, Coksuer H, Buyru N. Methylation of tumor suppressor genes in ovarian cancer. Exp Ther Med. 2012;4(6):1092–6.PubMedCentralPubMedGoogle Scholar
  47. 47.
    Khodyrev DS, Loginov VI, Pronina IV, Kazubskaya TP, Garkavtsera RF, Braga EA. Methylation of promoter region of RAR-β2 gene in renal cell, breast and ovarian carcinomas. Russ J Genet. 2008;44(8):983–8.CrossRefGoogle Scholar
  48. 48.
    Katsaros D, Cho W, Singal R, Fracchioli S, Rigault De La Longrais IA, Arisio R, et al. Methylation of tumor suppressor gene p16 and prognosis of epithelial ovarian cancer. Gynecol Oncol. 2004;94:685–92.CrossRefPubMedGoogle Scholar
  49. 49.
    Leu YW, Rahmatpanah F, Shi H, Wei SH, Liu JC, Yan PS, et al. Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation. Cancer Res. 2003;63:6110–5.PubMedGoogle Scholar
  50. 50.
    Marchini S, Codegoni AM, Bonazzi C, Chiari S, Broggini M. Absence of deletions but frequent loss of expression of p16INK4 in human ovarian tumours. Br J Cancer. 1997;76:146–9.PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Fujita M, Enomoto T, Haba T, Nakashima R, Sasaki M, Yoshino K, et al. Alteration of p16 and p15 genes in common epithelial ovarian tumors. Int J Cancer. 1997;74:148–55.CrossRefPubMedGoogle Scholar
  52. 52.
    Niederacher D, Yan HY, An HX, Bender HG, Beckmann MW. CDKN2A gene inactivation in epithelial sporadic ovarian cancer. Br J Cancer. 1999;80:1920–6.PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Kaiser PC, Körner M, Kappeler A, Aebi S. Retinoid receptors in ovarian cancer: expression and prognosis. Ann Oncol. 2005;16(9):1477–87.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Rahul Bhagat
    • 1
  • Sandeep Sriram Kumar
    • 1
  • Shilpa Vaderhobli
    • 1
  • Chennagiri S. Premalata
    • 2
  • Venkateshaiah Reddihalli Pallavi
    • 3
  • Gawari Ramesh
    • 1
  • Lakshmi Krishnamoorthy
    • 4
  1. 1.Department of BiochemistryKidwai Memorial Institute of OncologyBangaloreIndia
  2. 2.Department of PathologyKidwai Memorial Institute of OncologyBangaloreIndia
  3. 3.Department of Gynaec OncologyKidwai Memorial Institute of OncologyBangaloreIndia
  4. 4.Department of Laboratory MedicineSri Shankara Cancer Hospital and Research CentreBangaloreIndia

Personalised recommendations