Journal of Molecular Medicine

, Volume 83, Issue 2, pp 132–139 | Cite as

Tamoxifen-resistant breast cancers show less frequent methylation of the estrogen receptor β but not the estrogen receptor α gene

  • Ho Gun Chang
  • Sun Jung Kim
  • Ki-Wook Chung
  • Dong-Young Noh
  • Youngmee Kwon
  • Eun Sook Lee
  • Han-Sung KangEmail author
Original Article


No reports have examined the association between tamoxifen resistance and the methylation of the estrogen receptor (ER) α and β genes. Therefore we investigated the methylation patterns of the ER genes in the tamoxifen-resistant tumors. We used bisulfite genomic sequencing and reverse transcriptase PCR to determine the methylation patterns and mRNA expression of the two ER genes from control (n=68) and tamoxifen-resistant tissues (n=34) chosen by an age-matched sampling method. Bisulfite genomic sequencing allowed us to reveal the methylation of the ERα gene in 15 of the control tumors (22.1%) and in 11 tumors of the resistant group (32.4%). The methylation of ERβ was observed in 40 control tumors (58.8%) and in 8 recurrent tumors (23.5%). The methylation rate of the ERβ but not the ERα in the control group was significantly higher than in its counterpart (ERα, P=0.261; ERβ, P=0.001). Among the methylated tumors mean methylation density of ERα and ERβ in the resistant cases was significantly elevated (ERα, P=0.014; ERβ, P<0.001). Furthermore, the expression rate of ERβ mRNA was higher among the tumor in the resistant group than in the control with marginal significance (77.8% vs. 38.1%, P=0.109). Additionally, in the cancers from the resistant cases, the cells showed a higher percentage of positive staining for Ki67 than those from the control group (P=0.001). Our study indicates that there is an inverse relationship between the methylation rate of the ERβ gene and tamoxifen resistance. The tamoxifen-resistant tumors showed more dense methylation of the ERβ gene than control tumors. Although the number of case samples was limited, our results support the hypothesis that hypermethylation of the ERβ gene negatively affects the development of tamoxifen resistance.


Estrogen receptor α Estrogen receptor β Methylation Tamoxifen resistance 



Cytosine guanine dinucleotide


Estrogen receptor


Progesterone receptor


Reverse transcriptase



This work was supported by the National Cancer Center Grant.


  1. 1.
    Lohrisch C, Piccart MJ (2001) Standard medical treatment for early breast cancer. Eur J Cancer 37:S45–S58CrossRefPubMedGoogle Scholar
  2. 2.
    MacGregor JI, Jordan VC (1998) Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 50:151–196PubMedGoogle Scholar
  3. 3.
    Osborne CK, Coronado E, Allred DC, Wiebe V, DeGregorio, M (1991) Acquired tamoxifen resistance: correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Natl Cancer Inst 83:1477–1482PubMedGoogle Scholar
  4. 4.
    Takimoto GS, Graham JD, Jackson TA, Tung L, Powell RL, Horwitz LD, Horwitz KB (1999) Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors. J Steroid Biochem Mol Biol 69:45–50CrossRefPubMedGoogle Scholar
  5. 5.
    Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P (1995) Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494PubMedGoogle Scholar
  6. 6.
    Cariou S, Donovan J, Flanagan W, Milic A, Bhattacharya N, Slingerland J (2000) Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl Acad Sci USA 97:9042–9046CrossRefPubMedGoogle Scholar
  7. 7.
    Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology 138:863–870CrossRefPubMedGoogle Scholar
  8. 8.
    Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21:163–167CrossRefPubMedGoogle Scholar
  9. 9.
    Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE (1994) Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res 54:2552–2555PubMedGoogle Scholar
  10. 10.
    Issa J-P, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB (1994) Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 7:536–540CrossRefPubMedGoogle Scholar
  11. 11.
    Zhao C, Lam EW, Sunters A, Enmark E, De Bella MT, Coombes RC, Gustafsson JA, Dahlman-Wright K (2003) Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation. Oncogene 22:7600–7606CrossRefPubMedGoogle Scholar
  12. 12.
    Zhuang Z, Bertheau P, Emmert-Buck MR, Liotta, LA, Gnarra J, Linehan WM, Lubensky IA (1995) A microdissection technique for archival DNA analysis of specific cell populations in lesions <1 mm in size. Am J Pathol 146:620–625PubMedGoogle Scholar
  13. 13.
    Kang JH, Kim SJ, Noh DY, Park IA, Choe KJ, Yoo OJ, Kang HS (2001) Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma. Lab Invest 81:573–579PubMedGoogle Scholar
  14. 14.
    Li LC, Chui R, Nakajima K, Oh BR, Au HC, Dahiya R (2000) Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression. Cancer Res 2000:702–706Google Scholar
  15. 15.
    Li LC, Yeh CC, Nojima D, Dahiya R (2000) Cloning and characterization of human estrogen receptor beta promoter. Biochem Biophys Res Commun 275:682–689CrossRefPubMedGoogle Scholar
  16. 16.
    Geisler S, Lonning PE, Aas T, Johnsen H, Fluge O, Haugen DF, Lillehaug JR, Akslen LA, Borresen-Dale AL (2001) Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer. Cancer Res 61:2505–2512PubMedGoogle Scholar
  17. 17.
    Nishihara E, Nagayama Y, Inoue S, Hiroi H, Muramatsu M, Yamashita S, Koji T (2000) Ontogenetic changes in the expression of estrogen receptor alpha and beta in rat pituitary gland detected by immunohistochemistry. Endocrinology 141:615–620CrossRefPubMedGoogle Scholar
  18. 18.
    Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T (1997) Expression of estrogen receptor beta in rat bone. Endocrinology 138:4509–4512CrossRefPubMedGoogle Scholar
  19. 19.
    Mosselman S, Pohlman J, Dijkema R (1996) ERβ: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53CrossRefPubMedGoogle Scholar
  20. 20.
    Enmark E, Pelto-Huikko M, Grandien K, Fried G, Lagerkrantz S, Lagerkranzt J, Nordenskjöld M, Gustafsson JÅ (1997) Human estrogen receptor β: gene structure, chromosomal localisation and expression pattern. J Clin Endocrinol Metab 82:4258–4265CrossRefPubMedGoogle Scholar
  21. 21.
    Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustaffson JA, Kushner PJ, Scanlan TS (1997) Differential ligand activation of estrogen receptors ERα and ERβ at AP-1 sites. Science 277:1508–1510CrossRefPubMedGoogle Scholar
  22. 22.
    Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V (1997) Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 11:353–365CrossRefPubMedGoogle Scholar
  23. 23.
    Speirs V, Malone C, Walton DS, Kerin MJ, Atkin SL (1999) Increased expression of estrogen receptor beta mRNA in tamoxifen-resistant breast cancer patients. Cancer Res 59:5421–5424PubMedGoogle Scholar
  24. 24.
    Jensen EV, Cheng G, Palmieri C, Saji S, Makela S, Van Noorden S, Wahlstrom T, Warner M, Coombes RC, Gustafsson JA (2001) Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci USA 98:15197–15202CrossRefPubMedGoogle Scholar
  25. 25.
    Johnston SR, Haynes BP, Smith IE, Jarman M, Sacks NP, Ebbs SR, Dowsett M (1993) Acquired tamoxifen resistance in human breast cancer and reduced intra-tumoral drug concentration. Lancet 342:1521–1522PubMedGoogle Scholar
  26. 26.
    Johnston SR, Saccani-Jotti G, Smith IE, Salter J, Newby J, Coppen M, Ebbs SR, Dowsett M (1995) Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res 55:3331–3338PubMedGoogle Scholar
  27. 27.
    Robertson JF (1996) Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer 73:5–12PubMedGoogle Scholar
  28. 28.
    Widschwendter M, Jones PA (2002) DNA methylation and breast carcinogenesis. Oncogene 21:5462–5482CrossRefPubMedGoogle Scholar
  29. 29.
    Graff JR, Gabrielson E, Fujii H, Baylin SB, Herman JG (2000) Methylation patterns of the E-cadherin 5’ CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression. J Biol Chem 275:2727–2732CrossRefPubMedGoogle Scholar
  30. 30.
    Cameron EE, Baylin SB, Herman JG (1999) p15 (INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing. Blood 94:2445–2451PubMedGoogle Scholar
  31. 31.
    Lehmann U, Langer F, Feist H, Glockner S, Hasemeier B, Kreipe H (2002) Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol 160:605–612PubMedGoogle Scholar
  32. 32.
    Knoop AS, Bentzen SM, Nielsen MM, Rasmussen BB, Rose C (2001) Value of epidermal growth factor receptor, HER2, p53, and steroid receptors in predicting the efficacy of tamoxifen in high-risk postmenopausal breast cancer patients. J Clin Oncol 19:3376–3384PubMedGoogle Scholar
  33. 33.
    Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson JA (2000) Estrogen receptors α and β in the rodent mammary gland. Proc Natl Acad Sci USA 97:337–342CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Ho Gun Chang
    • 1
  • Sun Jung Kim
    • 1
  • Ki-Wook Chung
    • 2
  • Dong-Young Noh
    • 2
  • Youngmee Kwon
    • 3
  • Eun Sook Lee
    • 3
  • Han-Sung Kang
    • 3
    Email author
  1. 1.Department of BiologyDongguk UniversitySeoulKorea
  2. 2.Department of Surgery, College of MedicineSeoul National UniversitySeoulKorea
  3. 3.Research Institute and HospitalNational Cancer CenterGyeonggi doKorea

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