Medical Oncology

, 30:642 | Cite as

Potential clinical significance of ERβ ON promoter methylation in sporadic breast cancer

  • Ana Božović
  • Milan Markićević
  • Bogomir Dimitrijević
  • Snežana Jovanović Ćupić
  • Milena Krajnović
  • Silvana Lukić
  • Vesna Mandušić
Original Paper


The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERβ) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERβ gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERβ1 isoform, while ERβ1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERβ gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERβ1 protein levels were negatively correlated with ERα protein (ρ = −0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERβ1-mRNA and/or protein levels.


Metastases Estrogen receptor alpha Estrogen receptor beta Progesterone receptor Methylation Quantitative PCR 



This work was supported by the Ministry of Education and Science, Republic of Serbia, grants ON173049 (Mandusić, V., Božović, A., Dimitrijević, B., Jovanović-Ćupić, S., Krajnović, M.) and ON175068 (Markicevic, M. and Lukić, S.).

Conflict of interest

We declare that we have no conflict of interest.

Supplementary material

12032_2013_642_MOESM1_ESM.jpg (241 kb)
Online Resource 1. Melting curve profile analysis of PCR product amplified from samples of tumor DNA for: a) total reference, amplified using ERβ-EXT-F2 and ERβ-EXT-R primers; b) methylated target DNA, amplified using MSP primers: ERβ-M-F and ERβ-M-R (JPEG 241 kb)
12032_2013_642_MOESM2_ESM.jpg (148 kb)
Online Resource 2. Panels a), b) and c): The ERβ immunoblots of the same tumor sample probed with three different antibodies, after immunoprecipitation: a) Estrogen receptor beta antibody [9.88] (ab16813, Abcam, Cambridge, MA USA) showing two protein bands at MW~52 KDa and one at MW~76 KDa; b) Mouse antihuman estrogen receptor beta 1 (MCA1974S, AbD Serotec, Oxford, UK), an antibody that recognizes the C terminus of the ERβ1 isoform, showing one protein band at MW ~ 52 kDa; c) Novocastra lyophilized mouse monoclonal antibody estrogen receptor (beta) (NCL-ER-beta, Leica Biosystems Newcastle Ltd, United Kingdom), specific for ERβ1, showing one band at MW ~ 52 kDa.; d) Representative ERβ immunoblot of the tumor samples (lanes T1-T8), NC-negative control, MCF7-positive control, K calibrator (lane K), showing one protein band at MW~ 52 kDa corresponding to wt ERβ1 and some lower MW bands which represent different ERβ isoforms, as well as higher MW bands that are probably complexes of ERβ with other proteins; e) panel showing the corresponding GAPDH immunoblot of the samples listed above. Lane M has the Amersham High Range Rainbow Molecular Weight Marker (RPN756E, GE Healthcare, UK) (JPEG 147 kb)
12032_2013_642_MOESM3_ESM.jpg (441 kb)
Online Resource 3. Histogram showing the distribution pattern of the ERβ ON promoter methylation index in the analyzed samples (JPEG 441 kb)
12032_2013_642_MOESM4_ESM.jpg (196 kb)
Online Resource 4. Histogram showing the distribution pattern of ERβ1 protein levels in the analyzed samples (JPEG 195 kb)


  1. 1.
    Chang EC, Frasor J, Komm B, Katzenellenbogen BS. Impact of estrogen receptor beta on gene networks regulated by estrogen receptor alpha in breast cancer cells. Endocrinology. 2006;147(10):4831–42.PubMedCrossRefGoogle Scholar
  2. 2.
    Lazennec G, Bresson D, Lucas A, Chauveau C, Vignon F. ER beta inhibits proliferation and invasion of breast cancer cells. Endocrinology. 2001;142(9):4120–30.PubMedCrossRefGoogle Scholar
  3. 3.
    Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA. 2004;101(6):1566–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Hartman J, Lindberg K, Morani A, Inzunza J, Strom A, Gustafsson JA. Estrogen receptor beta inhibits angiogenesis and growth of T47D breast cancer xenografts. Cancer Res. 2006;66(23):11207–13.PubMedCrossRefGoogle Scholar
  5. 5.
    Hou YF, Yuan ST, Li HC, Wu J, Lu JS, Liu G, Lu LJ, Shen ZZ, Ding J, Shao ZM. ERbeta exerts multiple stimulative effects on human breast carcinoma cells. Oncogene. 2004;23(34):5799–806.PubMedCrossRefGoogle Scholar
  6. 6.
    Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H. Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res. 2001;61(6):2537–41.PubMedGoogle Scholar
  7. 7.
    Iwao K, Miyoshi Y, Ooka M, Ishikawa O, Ohigashi H, Kasugai T, Egawa C, Noguchi S. Quantitative analysis of estrogen receptor-alpha and -beta messenger RNA expression in human pancreatic cancers by real-time polymerase chain reaction. Cancer Lett. 2001;170(1):91–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Leygue E, Dotzlaw H, Watson PH, Murphy LC. Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis. Cancer Res. 1998;58(15):3197–201.PubMedGoogle Scholar
  9. 9.
    Shaw JA, Udokang K, Mosquera JM, Chauhan H, Jones JL, Walker RA. Oestrogen receptors alpha and beta differ in normal human breast and breast carcinomas. J Pathol. 2002;198(4):450–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Skliris GP, Munot K, Bell SM, Carder PJ, Lane S, Horgan K, Lansdown MR, Parkes AT, Hanby AM, Markham AF, et al. Reduced expression of oestrogen receptor beta in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model. J Pathol. 2003;201(2):213–20.PubMedCrossRefGoogle Scholar
  11. 11.
    Gustafsson JA. Estrogen signaling: a subtle balance between ERα and ERβ. Mol Interv. 2003;3(5):281–92.PubMedCrossRefGoogle Scholar
  12. 12.
    Speirs V. Oestrogen receptor beta in breast cancer: good, bad or still too early to tell? J Pathol. 2002;197(2):143–7.PubMedCrossRefGoogle Scholar
  13. 13.
    Vo AT, Millis RM. Epigenetics and breast cancers. Obstet Gynecol Int. 2012;2012:602720.PubMedGoogle Scholar
  14. 14.
    Hirata S, Shoda T, Kato J, Hoshi K. The multiple untranslated first exons system of the human estrogen receptor beta (ER beta) gene. J Steroid Biochem Mol Biol. 2001;78(1):33–40.PubMedCrossRefGoogle Scholar
  15. 15.
    Smith L, Coleman LJ, Cummings M, Satheesha S, Shaw SO, Speirs V, Hughes TA. Expression of oestrogen receptor beta isoforms is regulated by transcriptional and post-transcriptional mechanisms. Biochem J. 2010;429(2):283–90.PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao C, Lam EW, Sunters A, Enmark E, De Bella MT, Coombes RC, Gustafsson JA, Dahlman-Wright K. Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation. Oncogene. 2003;22(48):7600–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Xue Q, Lin Z, Cheng YH, Huang CC, Marsh E, Yin P, Milad MP, Confino E, Reierstad S, Innes J, et al. Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis. Biol Reprod. 2007;77(4):681–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Nojima D, Li LC, Dharia A, Perinchery G, Ribeiro-Filho L, Yen TS, Dahiya R. CpG hypermethylation of the promoter region inactivates the estrogen receptor-beta gene in patients with prostate carcinoma. Cancer. 2001;92(8):2076–83.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhang X, Leung YK, Ho SM. AP-2 regulates the transcription of estrogen receptor (ER)-beta by acting through a methylation hotspot of the 0 N promoter in prostate cancer cells. Oncogene. 2007;26(52):7346–54.PubMedCrossRefGoogle Scholar
  20. 20.
    Suzuki F, Akahira J, Miura I, Suzuki T, Ito K, Hayashi S, Sasano H, Yaegashi N. Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5’-untranslated region in human epithelial ovarian carcinoma. Cancer Sci. 2008;99(12):2365–72.PubMedCrossRefGoogle Scholar
  21. 21.
    Chang HG, Kim SJ, Chung KW, Noh DY, Kwon Y, Lee ES, Kang HS. Tamoxifen-resistant breast cancers show less frequent methylation of the estrogen receptor beta but not the estrogen receptor alpha gene. J Mol Med. 2005;83(2):132–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Rody A, Holtrich U, Solbach C, Kourtis K, von Minckwitz G, Engels K, Kissler S, Gatje R, Karn T, Kaufmann M. Methylation of estrogen receptor beta promoter correlates with loss of ER-beta expression in mammary carcinoma and is an early indication marker in premalignant lesions. Endocr Relat Cancer. 2005;12(4):903–16.PubMedCrossRefGoogle Scholar
  23. 23.
    Frederick M, Ausubel RB, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1999) Preparation of genomic DNA from mammalian tissue. Short Protoc Mol Biol, 4th edn. 2(2):2-9–2-10Google Scholar
  24. 24.
    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 USA. 1996;93(18):9821–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Grunau C, Clark SJ, Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 2001;29(13):E65–65.PubMedCrossRefGoogle Scholar
  26. 26.
    Lo PK, Watanabe H, Cheng PC, Teo WW, Liang X, Argani P, Lee JS, Sukumar S. MethySYBR, a novel quantitative PCR assay for the dual analysis of DNA methylation and CpG methylation density. J Mol Diagn. 2009;11(5):400–14.PubMedCrossRefGoogle Scholar
  27. 27.
    Zhu X, Leav I, Leung YK, Wu M, Liu Q, Gao Y, McNeal JE, Ho SM. Dynamic regulation of estrogen receptor-beta expression by DNA methylation during prostate cancer development and metastasis. Am J Pathol. 2004;164(6):2003–12.PubMedCrossRefGoogle Scholar
  28. 28.
    Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162(1):156–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Romain S, Laine Bidron C, Martin PM, Magdelenat H. Steroid receptor distribution in 47,892 breast cancers. A collaborative study of 7 European laboratories. The EORTC Receptor Study Group. Eur J Cancer. 1995;31A(3):411–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Poola I, Abraham J, Liu A. Estrogen receptor beta splice variant mRNAs are differentially altered during breast carcinogenesis. J Steroid Biochem Mol Biol. 2002;82(2–3):169–79.PubMedCrossRefGoogle Scholar
  31. 31.
    Esteva FJ, Hortobagyi GN. Prognostic molecular markers in early breast cancer. Breast Cancer Res. 2004;6(3):109–18.PubMedCrossRefGoogle Scholar
  32. 32.
    Rutherford T, Brown WD, Sapi E, Aschkenazi S, Munoz A, Mor G. Absence of estrogen receptor-beta expression in metastatic ovarian cancer. Obstet Gynecol. 2000;96(3):417–21.PubMedCrossRefGoogle Scholar
  33. 33.
    Bardin A, Hoffmann P, Boulle N, Katsaros D, Vignon F, Pujol P, Lazennec G. Involvement of estrogen receptor beta in ovarian carcinogenesis. Cancer Res. 2004;64(16):5861–9.PubMedCrossRefGoogle Scholar
  34. 34.
    Balfe P, McCann A, McGoldrick A, McAllister K, Kennedy M, Dervan P, Kerin MJ. Estrogen receptor alpha and beta profiling in human breast cancer. Eur J Surg Oncol. 2004;30(5):469–74.PubMedCrossRefGoogle Scholar
  35. 35.
    Smith L, Speirs V, Hughes TA. Estrogen receptor regulation: don’t forget translation. Breast Cancer Res Treat. 2010;121(1):251–2.PubMedCrossRefGoogle Scholar
  36. 36.
    Swedenborg E, Power KA, Cai W, Pongratz I, Ruegg J. Regulation of estrogen receptor beta activity and implications in health and disease. Cell Mol Life Sci. 2009;66(24):3873–94.PubMedCrossRefGoogle Scholar
  37. 37.
    O’Neill PA, Davies MP, Shaaban AM, Innes H, Torevell A, Sibson DR, Foster CS. Wild-type oestrogen receptor beta (ERbeta1) mRNA and protein expression in Tamoxifen-treated post-menopausal breast cancers. Br J Cancer. 2004;91(9):1694–702.PubMedGoogle Scholar
  38. 38.
    Vinayagam R, Sibson DR, Holcombe C, Aachi V, Davies MP. Association of oestrogen receptor beta 2 (ER beta 2/ER beta cx) with outcome of adjuvant endocrine treatment for primary breast cancer–a retrospective study. BMC Cancer. 2007;7:131.PubMedCrossRefGoogle Scholar
  39. 39.
    Skliris GP, Carder PJ, Lansdown MR, Speirs V. Immunohistochemical detection of ERbeta in breast cancer: towards more detailed receptor profiling? Br J Cancer. 2001;84(8):1095–8.PubMedCrossRefGoogle Scholar
  40. 40.
    Jarvinen TA, Pelto-Huikko M, Holli K, Isola J. Estrogen receptor beta is coexpressed with ERalpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. Am J Pathol. 2000;156(1):29–35.PubMedCrossRefGoogle Scholar
  41. 41.
    Fleming FJ, Hill AD, McDermott EW, O’Higgins NJ, Young LS. Differential recruitment of coregulator proteins steroid receptor coactivator-1 and silencing mediator for retinoid and thyroid receptors to the estrogen receptor-estrogen response element by beta-estradiol and 4-hydroxytamoxifen in human breast cancer. J Clin Endocrinol Metab. 2004;89(1):375–83.PubMedCrossRefGoogle Scholar
  42. 42.
    Sugiura H, Toyama T, Hara Y, Zhang Z, Kobayashi S, Fujii Y, Iwase H, Yamashita H. Expression of estrogen receptor beta wild-type and its variant ERbetacx/beta2 is correlated with better prognosis in breast cancer. Jpn J Clin Oncol. 2007;37(11):820–8.PubMedCrossRefGoogle Scholar
  43. 43.
    Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA. Low levels of estrogen receptor beta protein predict resistance to tamoxifen therapy in breast cancer. Clin Cancer Res. 2004;10(22):7490–9.PubMedCrossRefGoogle Scholar
  44. 44.
    Jarzabek K, Koda M, Kozlowski L, Mittre H, Sulkowski S, Kottler ML, Wolczynski S. Distinct mRNA, protein expression patterns and distribution of oestrogen receptors alpha and beta in human primary breast cancer: correlation with proliferation marker Ki-67 and clinicopathological factors. Eur J Cancer. 2005;41(18):2924–34.PubMedCrossRefGoogle Scholar
  45. 45.
    Nakopoulou L, Lazaris AC, Panayotopoulou EG, Giannopoulou I, Givalos N, Markaki S, Keramopoulos A. The favourable prognostic value of oestrogen receptor beta immunohistochemical expression in breast cancer. J Clin Pathol. 2004;57(5):523–8.PubMedCrossRefGoogle Scholar
  46. 46.
    Graff JR, Gabrielson E, Fujii H, Baylin SB, Herman JG. 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. 2000;275(4):2727–32.PubMedCrossRefGoogle Scholar
  47. 47.
    Omoto Y, Kobayashi S, Inoue S, Ogawa S, Toyama T, Yamashita H, Muramatsu M, Gustafsson JA, Iwase H. Evaluation of oestrogen receptor beta wild-type and variant protein expression, and relationship with clinicopathological factors in breast cancers. Eur J Cancer. 2002;38(3):380–6.PubMedCrossRefGoogle Scholar
  48. 48.
    Bozkurt KK, Kapucuoglu N. Investigation of immunohistochemical ERalpha, ERbeta and ERbetacx expressions in normal and neoplastic breast tissues. Pathol Res Pract. 2012;208(3):133–9.PubMedCrossRefGoogle Scholar
  49. 49.
    Honma N, Horii R, Iwase T, Saji S, Younes M, Takubo K, Matsuura M, Ito Y, Akiyama F, Sakamoto G. Clinical importance of estrogen receptor-beta evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol. 2008;26(22):3727–34.PubMedCrossRefGoogle Scholar
  50. 50.
    Shaaban AM, Green AR, Karthik S, Alizadeh Y, Hughes TA, Harkins L, Ellis IO, Robertson JF, Paish EC, Saunders PT, et al. Nuclear and cytoplasmic expression of ERbeta1, ERbeta2, and ERbeta5 identifies distinct prognostic outcome for breast cancer patients. Clin Cancer Res. 2008;14(16):5228–35.PubMedCrossRefGoogle Scholar
  51. 51.
    Silva JM, Dominguez G, Garcia JM, Gonzalez R, Villanueva MJ, Navarro F, Provencio M, San Martin S, Espana P, Bonilla F. Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer Res. 1999;59(13):3251–6.PubMedGoogle Scholar
  52. 52.
    Sanchez-Cespedes M, Esteller M, Wu L, Nawroz-Danish H, Yoo GH, Koch WM, Jen J, Herman JG, Sidransky D. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res. 2000;60(4):892–5.PubMedGoogle Scholar
  53. 53.
    Silva JM, Garcia JM, Dominguez G, Silva J, Miralles C, Cantos B, Coca S, Provencio M, Espana P, Bonilla F. Persistence of tumor DNA in plasma of breast cancer patients after mastectomy. Ann Surg Oncol. 2002;9(1):71–6.PubMedCrossRefGoogle Scholar
  54. 54.
    Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C, Widschwendter M. DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res. 2003;63(22):7641–5.PubMedGoogle Scholar
  55. 55.
    Widschwendter M, Apostolidou S, Raum E, Rothenbacher D, Fiegl H, Menon U, Stegmaier C, Jacobs IJ, Brenner H. Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study. PLoS ONE. 2008;3(7):e2656.PubMedCrossRefGoogle Scholar
  56. 56.
    Kurkjian C, Kummar S, Murgo AJ. DNA methylation: its role in cancer development and therapy. Curr Probl Cancer. 2008;32(5):187–235.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ana Božović
    • 1
    • 3
  • Milan Markićević
    • 2
  • Bogomir Dimitrijević
    • 1
  • Snežana Jovanović Ćupić
    • 1
  • Milena Krajnović
    • 1
  • Silvana Lukić
    • 2
  • Vesna Mandušić
    • 1
  1. 1.Vinča Institute of Nuclear SciencesUniversity of BelgradeBelgradeSerbia
  2. 2.Institute for Oncology and Radiology of SerbiaBelgradeSerbia
  3. 3.Department for Radiobiology and Molecular Genetics, Vinča Institute of Nuclear SciencesUniversity of BelgradeBelgradeSerbia

Personalised recommendations