Tumor Biology

, Volume 33, Issue 2, pp 315–323 | Cite as

The epigenetic silencing of the estrogen receptor (ER) by hypermethylation of the ESR1 promoter is seen predominantly in triple-negative breast cancers in Indian women

  • Jyothi S. PrabhuEmail author
  • Kanu Wahi
  • Aruna Korlimarla
  • Marjorrie Correa
  • Suraj Manjunath
  • N. Raman
  • B. S. Srinath
  • T. S. Sridhar
Research Article


The proportion of estrogen receptor (ER)-negative and triple-negative (TN) breast cancer in Indian women is higher than that reported in the West, and this difference persists even after their migration to the West. The causes for this significant difference are not entirely clear. Hypermethylation of the ER promoter, an epigenetic alteration, is known to be one of the mechanisms by which the expression of ER is suppressed. Two thirds of breast cancer specimens from an Indian center tested, using the highly sensitive, methylation-specific polymerase chain reaction (MSP) technique, were reported positive. We have used a quantitative assay, the MethyLight, to better assess the extent of methylation in the ESR1 promoter region in 98 breast cancer tumor specimens from Indian women. In addition, the amount of ER transcripts was determined by quantitative reverse transcriptase polymerase chain reaction. Using the stringent cutoff of at least 4% of the target sequence being methylated, 27% of TN tumors were methylated. In addition they demonstrated the highest levels of methylation. In contrast less than 2% ER-positive tumors were hypermethylated. While the proportion of hypermethylated tumors are lower in this study than that estimated using MSP, our results support the notion of increased epigenetic deregulations in ER-negative tumors in general and TN tumors in particular. The development of this assay also permits a rational approach to the selection of patients for clinical trials examining the efficacy of demethylating agents in the treatment of ER-negative breast cancer.


Quantitative methylation Estrogen receptor promoter Triple-negative breast cancer Epigenetic FFPE 



This work was funded by the Nadathur Holdings, Bangalore and the Bagaria Education Trust, Bangalore. The patient enrolment, sample collection, and patient follow-up were coordinated by Ms. Annie Alexander of “Aadhara”, our patient support group. We would also like to acknowledge the excellent technical support provided by Mr. Raju and Ms. Anupama as well as the meticulous collation of clinical information by Ms. Rohini our clinical research associate. Dr Tinku Thomas of the Epidemiology and Biostatistics division of SJRI reviewed the statistical analysis.

Conflicts of interest



  1. 1.
    Desai SB, Moonim MT, Gill AK, Punia RS, Naresh KN, Chinoy RF. Hormone receptor status of breast cancer in India: a study of 798 tumours. Breast. 2000;9(5):267–70.PubMedCrossRefGoogle Scholar
  2. 2.
    Agarwal G, Pradeep PV, Aggarwal V, Yip CH, Cheung PSY. Spectrum of breast cancer in Asian women. World J Surg. 2007;31:1031–40.PubMedCrossRefGoogle Scholar
  3. 3.
    Murthy NS, Agarwal UK, Chaudhry K, Saxena S. A study on time trends in incidence of breast cancer Indian scenario. Eur J Cancer Care. 2007;16:185–6.CrossRefGoogle Scholar
  4. 4.
    National Cancer registry Programme. Consolidated report of the population based cancer registries 1990–1996. New Delhi: Indian Council of Medical Research; 2001.Google Scholar
  5. 5.
    Raina V, Taneja V, Gulati A, Deo SVS, Shukla NK, Vij U. Oestrogen receptor status in breast cancer. Indian Pract. 2000;53:405–7.Google Scholar
  6. 6.
    Shet T, Agrawal A, Nadkarni M, Palkar M, Havaldar R, Parmar V, Badwe R, Chinoy RF. Hormone receptors over the last 8 years in a cancer referral center in India: what was and what is? Indian J Pathol Microbiol. 2009;52(2):171–4.PubMedCrossRefGoogle Scholar
  7. 7.
    Ambroise M, Ghosh M, Mallikarjuna VS, Kurian A. Immunohistochemical profile of breast cancer patients at a tertiary care hospital in South India. Asian Pac J Cancer Prev. 2011;12(3):625–9.PubMedGoogle Scholar
  8. 8.
    Kakarala M, Rozek L, Cote M, Liyanage S, Brenner DE. Breast cancer histology and receptor status characterization in Asian Indian and Pakistani women in the U.S.—a SEER analysis. BMC Cancer. 2010;10:191.PubMedCrossRefGoogle Scholar
  9. 9.
    Moran MS, Gonsalves L, Goss DM, Ma S. Breast cancers in U.S. residing Indian-Pakistani versus non-Hispanic White women: comparative analysis of clinical-pathologic features, treatment, and survival. Breast Cancer Res Treat. 2011;128(2):543–51.PubMedCrossRefGoogle Scholar
  10. 10.
    Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer. 2007;109:1721–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Stead LA, Lash TL, Sobieraj JE, Chi DD, Westrup JL, Charlot M, Blanchard RA, Lee JC, King TC, Rosenberg CL. Triple-negative breast cancers are increased in black women regardless of age or body mass index. Breast Cancer Res. 2009;11:R18.PubMedCrossRefGoogle Scholar
  12. 12.
    Stark A, Kleer CG, Martin I, Awuah B, Nsiah-Asare A, Takyi V, Braman M, Quayson SE, Zarbo R, Wicha M, Newman L. African ancestry and higher prevalence of triple-negative breast cancer. Cancer. 2010;116:4926–32.PubMedCrossRefGoogle Scholar
  13. 13.
    Hicks DG, Short SM, Prescott NL, Tarr SM, Coleman KA, Yoder BJ, Crowe JP, Choueiri TK, Dawson AE, Budd GT, Tubbs RR, Casey G, Weil RJ. Breast cancers with brain metastases are more likely to be estrogen receptor negative, express the basal cytokeratin CK5⁄6, and overexpress HER2 or EGFR. Am J Surg Pathol. 2006;30:1097–104.PubMedGoogle Scholar
  14. 14.
    Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K, Hess KR, Stec J, Ayers M, Wagner P, Morandi P, Fan C, Rabiul I, Ross JS, Hortobagyi GN, Pusztai L, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res. 2005;11:5678–85.PubMedCrossRefGoogle Scholar
  15. 15.
    Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F, Ollila DW, Sartor CI, Graham ML, Perou CM. The triple negative paradox: primary tumour chemosensitivity of breast cancer subtypes. Clin Cancer Res. 2007;13:2329–34.PubMedCrossRefGoogle Scholar
  16. 16.
    Clark GM, McGuire W. Steroid receptors and other prognostic factors in primary breast cancer. Semin Oncol. 1988;15:20–5.PubMedGoogle Scholar
  17. 17.
    McGuire WL, Tandon AK, Allred DC, Chamness GC, Clark GM. How to use prognostic factors in axillary node-negative breast cancer patients. J Natl Cancer Inst. 1990;82:1006–15.PubMedCrossRefGoogle Scholar
  18. 18.
    Ottaviano YL, Issa JP, Par FF, Smith HS, Baylin SB, Davidson NE. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994;54:2552–5.PubMedGoogle Scholar
  19. 19.
    Yoshida T, Eguchi H, Nakachi K, Tanimoto K, Higashi Y, Suemasu K, Iino Y, Morishita Y, Hayashi S. Distinct mechanisms of loss of estrogen receptor α gene expression in human breast cancer: methylation of the gene and alteration of trans-acting factors. Carcinogenesis. 2000;21:2193–201.PubMedCrossRefGoogle Scholar
  20. 20.
    Lapidus RG, Ferguson AT, Ottaviano YL, Parl FF, Smith HS, Weitzman SA, Baylin SB, Issa JP, Davidson NE. Methylation of estrogen and progesterone receptor gene 5′ CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumours. Clin Cancer Res. 1996;2:805–10.PubMedGoogle Scholar
  21. 21.
    Yan L, Yang X, Davidson NE. Role of DNA methylation and histone acetylation in steroid receptor expression in breast cancer. J Mammary Gland Biol Neoplasia. 2001;6:183–92.PubMedCrossRefGoogle Scholar
  22. 22.
    Iwase H, Omoto Y, Iwata H, Toyama T, Hara Y, Ando Y, Ito Y, Fujii Y, Kobayashi S, et al. DNA methylation analysis at distal and proximal promoter regions of the oestrogen receptor gene in breast cancers. Br J Cancer. 1999;80:1982–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Mirza S, Sharma G, Prasad CP, Srivastava A, Gupta SD, Ralhan R. Promoter hypermethylation of TMS1, BRCA1, ERalpha and PRB in serum and tumour DNA of invasive ductal breast carcinoma patients. Life Sci. 2007;81:280–7.PubMedCrossRefGoogle Scholar
  24. 24.
    Mirza S, Sharma G, Prasad CP, Srivastava A, Gupta SD, Ralhan R. Clinical significance of Stratifin, Erα and PR promoter methylation in tumour and serum DNA in Indian breast cancer patients. Clin Biochem. 2010;43:380–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW. MethylLight: a high throughput assay to measure DNA methylation. Nucleic Acids Res. 2000;28:E32.PubMedCrossRefGoogle Scholar
  26. 26.
    Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA, Laird PW. Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res. 2001;61:3410–8.PubMedGoogle Scholar
  27. 27.
    Widschwendter M, Siegmund KD, Muller HM, Fiegl H, Marth C, Müller-Holzner E, Jones PA, Laird PW. Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res. 2004;64:3807–13.PubMedCrossRefGoogle Scholar
  28. 28.
    Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci. 1996;93(18):9821–6.PubMedCrossRefGoogle Scholar
  29. 29.
    Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol. 2004;164:35–42.PubMedCrossRefGoogle Scholar
  30. 30.
    Lapidus RG, Nass SJ, Butash KA, Parl FF, Weitzman SA, Graff JG, Herman JG, Davidson NE. Mapping of ER gene CpG island methylation-specific polymerase chain reaction. Cancer Res. 1998;58:2515–9.PubMedGoogle Scholar
  31. 31.
    Gaudet MM, Campan M, Figueroa JD, Yang XR, Lissowska J, Peplonska B, Brinton LA, Rimm DL, Laird PW, Garcia-Closas M, Sherman ME. DNA hypermethylation of ESR1 and PGR in breast cancer: pathologic and epidemiologic associations. Cancer Epidemiol Biomarkers Prev. 2009;18(11):3036–4.PubMedCrossRefGoogle Scholar
  32. 32.
    Zhao L, Wang L, Jin F, Ma W, Ren J, Wen X, He M, Sun M, Tang H, Wei M. Silencing of estrogen receptor alpha (ERalpha) gene by promoter hypermethylation is a frequent event in Chinese women with sporadic breast cancer. Breast Cancer Res Treat. 2009;117(2):253–9.PubMedCrossRefGoogle Scholar
  33. 33.
    Falette NS, Fuqua SA, Chamness GC, Cheah MS, Greene GL, McGuire WL. Estrogen receptor gene methylation in human breast tumours. Cancer Res. 1990;50:3974–8.PubMedGoogle Scholar
  34. 34.
    Bediaga NG, Acha-Sagredo A, Guerra I, Viguri A, Albaina C, Ruiz Diaz I, Rezola R, Alberdi MJ, Dopazo J, Montaner D, de Renobales M, Fernández AF, Field JK, Fraga MF, Liloglou T, de Pancorbo MM. DNA methylation epigenotypes in breast cancer molecular subtypes. Breast Cancer Res. 2010;12:R77.PubMedCrossRefGoogle Scholar
  35. 35.
    Holm K, Hegardt C, Staaf J, Vallon-Christersson J, Jonsson G, Olsson H, Borg A, Ringnér M, et al. Molecular subtypes of breast cancer are associated with characteristic DNA methylation patterns. Breast Cancer Res. 2010;12:R36.PubMedCrossRefGoogle Scholar
  36. 36.
    Veeck J, Ropero S, Setien F, Gonzalez-Suarez E, Osorio A, Benitez J, Herman JG, Esteller M. BRCA1 CpG island hypermethylation predicts sensitivity to poly(adenosine diphosphate)-ribose polymerase inhibitors. J Clin Oncol. 2010;28(29):e563–4.PubMedCrossRefGoogle Scholar
  37. 37.
    Singh AK, Pandey A, Tewari M, Shukla HS, Pandey HP. Epigenetic silencing of BRCA1 gene associated with demographic and pathologic factors in sporadic breast cancer: a study of an Indian population. Eur J Cancer Prev. 2011;20(6):478–83.PubMedCrossRefGoogle Scholar
  38. 38.
    Perou CM. Molecular stratification of triple negative breast cancers. Oncologist. 2010;15:39–48.PubMedCrossRefGoogle Scholar
  39. 39.
    Girault I, Tozlu S, Lidereau R, Bieche I. Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas. Clin Cancer Res. 2003;9:4415–22.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2012

Authors and Affiliations

  • Jyothi S. Prabhu
    • 1
    Email author
  • Kanu Wahi
    • 1
    • 5
  • Aruna Korlimarla
    • 1
  • Marjorrie Correa
    • 2
  • Suraj Manjunath
    • 3
  • N. Raman
    • 4
  • B. S. Srinath
    • 4
  • T. S. Sridhar
    • 1
  1. 1.Division of Molecular MedicineSt. John’s Research InstituteBangaloreIndia
  2. 2.Department of PathologySt. John’s Medical College and HospitalBangaloreIndia
  3. 3.Department of Surgical OncologySt. John’s Medical College and HospitalBangaloreIndia
  4. 4.Rangadore Memorial HospitalBangaloreIndia
  5. 5.Dept of Molecular GeneticsOhio State UniversityColumbusUSA

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