Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Estrogen receptor beta growth-inhibitory effects are repressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells

Oncogene volume 32, pages 2390–2402 (2013)Cite this article

Subjects

Abstract

Two thirds of breast cancers express estrogen receptors (ER). ER alpha (ERα) mediates breast cancer cell proliferation, and expression of ERα is the standard choice to indicate adjuvant endocrine therapy. ERbeta (ERβ) inhibits growth in vitro; its effects in vivo have been incompletely investigated and its role in breast cancer and potential as alternative target in endocrine therapy needs further study. In this work, mammary epithelial (EpH4 and HC11) and breast cancer (MC4-L2) cells with endogenous ERα and ERβ expression and T47-D human breast cancer cells with recombinant ERβ (T47-DERβ) were used to explore effects exerted in vitro and in vivo by the ERβ agonists 2,3-bis (4–hydroxy–phenyl)-propionitrile (DPN) and 7-bromo-2-(4–hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY). In vivo, ERβ agonists induced mammary gland hyperplasia and MC4-L2 tumour growth to a similar extent as the ERα agonist 4,4′,4′′-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17β-estradiol (E2) and correlated with higher number of mitotic and lower number of apoptotic features. In vitro, in MC4-L2, EpH4 or HC11 cells incubated under basal conditions, ERβ agonists induced apoptosis measured as upregulation of p53 and apoptosis-inducible factor protein levels and increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. However, when extracellular signal-regulated kinase 1 and 2 (ERK ½) were activated by co-incubation with basement membrane extract or epidermal growth factor, induction of apoptosis by ERβ agonists was repressed and DPN induced proliferation in a similar way as E2 or PPT. In a context of active ERK ½, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) signalling was necessary to allow proliferation stimulated by ER agonists. Inhibition of MEK ½ with UO126 completely restored ERβ growth-inhibitory effects, whereas inhibition of PI3K by LY294002 inhibited ERβ-induced proliferation. These results show that the cellular context modulates ERβ growth-inhibitory effects and should be taken into consideration upon assessment of ERβ as target for endocrine treatment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

Abbreviations

Aif:

apoptosis-inducible factor

Akt:

RAC-alpha serine/threonine-protein kinase

BM:

basement membrane

BrdU:

bromodeoxyuridine

Casp 3:

caspase 3

DPN:

2,3-bis (4–hydroxy–phenyl)-propionitrile

E2:

17β-estradiol

erbB-2:

receptor tyrosine-protein kinase erbB-2

erbB-3:

receptor tyrosine-protein kinase erbB-3

EGF:

epidermal growth factor

ERK ½:

extracellular signal-regulated kinase 1 and 2

Mek 1:

ERK activator kinase 1

4OH-T:

4-hydroxytamoxifen

PI3K:

phosphatidylinositol-4,5-bisphosphate 3-kinase

PPT:

4,4′,4′′-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol

p-Ser:

phosphorylated serine

References

  1. Liu Y, Gao H, Marstrand TT, Strom A, Valen E, Sandelin A et al. The genome landscape of ERalpha- and ERbeta-binding DNA regions. Proc Natl Acad Sci USA 2008; 105: 2604–2609.

    Article  CAS  Google Scholar 

  2. Grober OM, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR et al. Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics 2011; 12: 36.

    Article  CAS  Google Scholar 

  3. Ciarloni L, Mallepell S, Brisken C . Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development. Proc Natl Acad Sci USA 2007; 104: 5455–5460.

    Article  CAS  Google Scholar 

  4. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K et al. Involvement of estrogen receptor beta in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 2002; 99: 15578–15583.

    Article  CAS  Google Scholar 

  5. Helguero LA, Faulds MH, Gustafsson JA, Haldosen LA . Estrogen receptors alfa (ERalpha) and beta (ERbeta) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11. Oncogene 2005; 24: 6605–6616.

    Article  CAS  Google Scholar 

  6. Helguero LA, Lindberg K, Gardmo C, Schwend T, Gustafsson JA, Haldosen LA . Different roles of estrogen receptors alpha and beta in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line. Cancer Res 2008; 68: 8695–8704.

    Article  CAS  Google Scholar 

  7. Su Y, Simmen RC . Soy isoflavone genistein upregulates epithelial adhesion molecule E-cadherin expression and attenuates beta-catenin signaling in mammary epithelial cells. Carcinogenesis 2009; 30: 331–339.

    Article  Google Scholar 

  8. Musgrove EA, Sutherland RL . Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 2009; 9: 631–643.

    Article  CAS  Google Scholar 

  9. Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM et al. Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression. J Biol Chem 2002; 277: 24353–24360.

    Article  CAS  Google Scholar 

  10. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA . Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 2006; 20: 534–543.

    Article  CAS  Google Scholar 

  11. Lazennec G, Bresson D, Lucas A, Chauveau C, Vignon F . ER beta inhibits proliferation and invasion of breast cancer cells. Endocrinology 2001; 142: 4120–4130.

    Article  CAS  Google Scholar 

  12. 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: 1566–1571.

    Article  Google Scholar 

  13. Lindberg K, Strom A, Lock JG, Gustafsson JA, Haldosen LA, Helguero LA . Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells. J Cell Physiol 2010; 222: 156–167.

    Article  CAS  Google Scholar 

  14. Lindberg K, Helguero LA, Omoto Y, Gustafsson JA, Haldosen LA . Estrogen receptor beta represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity. Breast Cancer Res 2011; 13: R43.

    Article  CAS  Google Scholar 

  15. Wu X, Subramaniam M, Grygo SB, Sun Z, Negron V, Lingle WL et al. Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen. Breast Cancer Res 2011; 13: R27.

    Article  CAS  Google Scholar 

  16. Green CA, Peter MB, Speirs V, Shaaban AM . The potential role of ER beta isoforms in the clinical management of breast cancer. Histopathology 2008; 53: 374–380.

    Article  CAS  Google Scholar 

  17. 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: 2537–2541.

    CAS  Google Scholar 

  18. Skliris GP, Munot K, Bell SM, Carder PJ, Lane S, Horgan K 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: 213–220.

    Article  CAS  Google Scholar 

  19. 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: 29–35.

    Article  CAS  Google Scholar 

  20. Honma N, Horii R, Iwase T, Saji S, Younes M, Takubo K et al. Clinical importance of estrogen receptor-beta evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol 2008; 26: 3727–3734.

    Article  Google Scholar 

  21. Shaaban AM, Green AR, Karthik S, Alizadeh Y, Hughes TA, Harkins L et al. Nuclear and cytoplasmic expression of ERbeta1, ERbeta2, and ERbeta5 identifies distinct prognostic outcome for breast cancer patients. Clin Cancer Res 2008; 14: 5228–5235.

    Article  CAS  Google Scholar 

  22. Skliris GP, Leygue E, Curtis-Snell L, Watson PH, Murphy LC . Expression of oestrogen receptor-beta in oestrogen receptor-alpha negative human breast tumours. Br J Cancer 2006; 95: 616–626.

    Article  CAS  Google Scholar 

  23. Jensen EV, Cheng G, Palmieri C, Saji S, Makela S, Van Noorden S et al. Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci USA 2001; 98: 15197–15202.

    Article  CAS  Google Scholar 

  24. Sanchez M, Picard N, Sauve K, Tremblay A . Challenging estrogen receptor beta with phosphorylation. Trends Endocrinol Metab 2010; 21: 104–110.

    Article  CAS  Google Scholar 

  25. Likhite VS, Stossi F, Kim K, Katzenellenbogen BS, Katzenellenbogen JA . Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity. Mol Endocrinol 2006; 20: 3120–3132.

    Article  CAS  Google Scholar 

  26. Soldati R, Wargon V, Cerliani JP, Giulianelli S, Vanzulli SI, Gorostiaga MA et al. Inhibition of mammary tumor growth by estrogens: is there a specific role for estrogen receptors alpha and beta? Breast Cancer Res Treat 2010; 123: 709–724.

    Article  CAS  Google Scholar 

  27. Rahal OM, Simmen RC . Paracrine-acting adiponectin promotes mammary epithelial differentiation and synergizes with genistein to enhance transcriptional response to estrogen receptor {beta} signaling. Endocrinology 2011; 152: 3409–3421.

    Article  CAS  Google Scholar 

  28. Lanari C, Luthy I, Lamb CA, Fabris V, Pagano E, Helguero LA et al. Five novel hormone-responsive cell lines derived from murine mammary ductal carcinomas: in vivo and in vitro effects of estrogens and progestins. Cancer Res 2001; 61: 293–302.

    CAS  Google Scholar 

  29. Merlo GR, Venesio T, Taverna D, Marte BM, Callahan R, Hynes NE . Growth suppression of normal mammary epithelial cells by wild-type p53. Oncogene 1994; 9: 443–453.

    CAS  Google Scholar 

  30. Haupt Y, Rowan S, Shaulian E, Vousden KH, Oren M . Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev 1995; 9: 2170–2183.

    Article  CAS  Google Scholar 

  31. Moll UM, Wolff S, Speidel D, Deppert W . Transcription-independent pro-apoptotic functions of p53. Curr Opin Cell Biol 2005; 17: 631–636.

    Article  CAS  Google Scholar 

  32. Aliaga A, Rousseau JA, Ouellette R, Cadorette J, van Lier JE, Lecomte R et al. Breast cancer models to study the expression of estrogen receptors with small animal PET imaging. Nucl Med Biol 2004; 31: 761–770.

    Article  CAS  Google Scholar 

  33. Frasor J, Barnett DH, Danes JM, Hess R, Parlow AF, Katzenellenbogen BS . Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus. Endocrinology 2003; 144: 3159–3166.

    Article  CAS  Google Scholar 

  34. Novaro V, Roskelley CD, Bissell MJ . Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells. J Cell Sci 2003; 116: 2975–2986.

    Article  CAS  Google Scholar 

  35. Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA . Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. Mol Endocrinol 1993; 7: 992–998.

    CAS  Google Scholar 

  36. Migliaccio A, Castoria G, Di Domenico M, Ciociola A, Lombardi M, De Falco A et al. Crosstalk between EGFR and extranuclear steroid receptors. Ann N Y Acad Sci 2006; 1089: 194–200.

    Article  CAS  Google Scholar 

  37. Williams C, Edvardsson K, Lewandowski SA, Strom A, Gustafsson JA . A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells. Oncogene 2008; 27: 1019–1032.

    Article  CAS  Google Scholar 

  38. Merlo GR, Basolo F, Fiore L, Duboc L, Hynes NE . p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin. J Cell Biol 1995; 128: 1185–1196.

    Article  CAS  Google Scholar 

  39. Merlo GR, Graus-Porta D, Cella N, Marte BM, Taverna D, Hynes NE . Growth differentiation and survival of HC11 mammary epithelial cells: diverse effects of receptor tyrosine kinase-activating peptide growth factors. Eur J Cell Biol 1996; 70: 97–105.

    CAS  Google Scholar 

  40. Le Romancer M, Poulard C, Cohen P, Sentis S, Renoir JM, Corbo L . Cracking the estrogen receptor's posttranslational code in breast tumors. Endocr Rev 2011; 32: 597–622.

    Article  CAS  Google Scholar 

  41. Shu XO, Zheng Y, Cai H, Gu K, Chen Z, Zheng W et al. Soy food intake and breast cancer survival. JAMA 2009; 302: 2437–2443.

    Article  Google Scholar 

  42. Nilsson S, Gustafsson JA . Estrogen receptors: therapies targeted to receptor subtypes. Clin Pharmacol Ther 2011; 89: 44–55.

    Article  CAS  Google Scholar 

  43. Novelli F, Milella M, Melucci E, Di Benedetto A, Sperduti I, Perrone-Donnorso R et al. A divergent role for estrogen receptor-beta in node-positive and node-negative breast cancer classified according to molecular subtypes: an observational prospective study. Breast Cancer Res 2008; 10: R74.

    Article  Google Scholar 

  44. Matthews J, Gustafsson JA . Estrogen signaling: a subtle balance between ER alpha and ER beta. Mol Interv 2003; 3: 281–292.

    Article  CAS  Google Scholar 

  45. Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA . Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 2002; 23: 1467–1474.

    Article  CAS  Google Scholar 

  46. Walker VR, Korach KS . Estrogen receptor knockout mice as a model for endocrine research. ILAR J 2004; 45: 455–461.

    Article  CAS  Google Scholar 

  47. Hamilton-Burke W, Coleman L, Cummings M, Green CA, Holliday DL, Horgan K et al. Phosphorylation of estrogen receptor beta at serine 105 is associated with good prognosis in breast cancer. Am J Pathol 2010; 177: 1079–1086.

    Article  CAS  Google Scholar 

  48. Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B . Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 1988; 7: 2089–2095.

    Article  CAS  Google Scholar 

  49. Reichmann E, Schwarz H, Deiner EM, Leitner I, Eilers M, Berger J et al. Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion. Cell 1992; 71: 1103–1116.

    Article  CAS  Google Scholar 

  50. Weitsman GE, Skliris G, Ung K, Peng B, Younes M, Watson PH, Murphy LC . Assessment of multiple different estrogen receptor-beta antibodies for their ability to immunoprecipitate under chromatin immunoprecipitation conditions. Breast Cancer Res Treat 2006; 100: 23–31.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr Lars-Arne Haldosén for enlightening discussions related to this work, Dr Anders Ström for T47-DERβ human breast cancer cells and Dr Margaret Warner for chicken anti-ERβ 503 and rabbit anti-ERβ2 antibodies. This work was supported by Federal funds through Programa Operacional Temático Factores de Competitividade (COMPETE) with co-participation from the European Community Fund (FEDER) and national funds through Fundação para a Ciência e Tecnología (FCT) under the project no. PTDC/SAU-ONC/112671/2009 (LAH); Project Ciência 2008 through FCT; Mass Spectrometry Unit at University of Aveiro and Swedish Cancer Fund.

Author information

Author notes

  1. L A Helguero: Previous address: Department of Biosciences and Nutrition, Karolinska Institutet, Sweden

Authors and Affiliations

  1. Department of Chemistry, Universidade de Aveiro, Campus Universitario de Santigo, Aveiro, Portugal

    C Z Cotrim, M L Doria & L A Helguero

  2. Intituto de Biologia y Medicina Experimental, Buenos Aires, Argentina

    V Fabris & C Lanari

  3. Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden

    K Lindberg & J-Å Gustafsson

  4. Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA

    J-Å Gustafsson

  5. School of Health Sciences, Universidade de Aveiro, Aveiro, Portugal

    F Amado

Authors
  1. C Z Cotrim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V Fabris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M L Doria
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K Lindberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J-Å Gustafsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. F Amado
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C Lanari
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L A Helguero
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L A Helguero.

Ethics declarations

Competing interests

Jan-Åke Gustafsson is consultant of KaroBio AB and BioNovo. All the other authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cotrim, C., Fabris, V., Doria, M. et al. Estrogen receptor beta growth-inhibitory effects are repressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells. Oncogene 32, 2390–2402 (2013). https://doi.org/10.1038/onc.2012.261

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.261

Keywords

This article is cited by

Search

Advanced search

Quick links