Estrogen receptor beta increases sensitivity to enzalutamide in androgen receptor-positive triple-negative breast cancer

  • Aristomenis Anestis
  • Panagiotis Sarantis
  • Stamatios Theocharis
  • Ilianna Zoi
  • Dimitrios Tryfonopoulos
  • Athanasios Korogiannos
  • Anna Koumarianou
  • Evangelia Xingi
  • Dimitra Thomaidou
  • Michalis Kontos
  • Athanasios G. PapavassiliouEmail author
  • Michalis V. KaramouzisEmail author
Original Article – Cancer Research



Androgen receptor (AR) is playing an important role in the progression of a subset of TNBC. We evaluated the impact of ERβ expression along with anti-AR drugs in AR-positive TNBC.


ERβ expression was examined in AR-positive TNBC cell line using MTT assay, scratch and Annexin V-FITC assay in the presence or absence of anti-androgens. Protein levels of involved molecules were assessed using Western blot. Receptors’ localization was detected by immunofluorescence and their physical association was examined using proximity ligation assay (PLA), which enables the visualization of interacting proteins in fixed cells and tissues.


Transient transfection of ERβ in MDA-MB 453 AR-positive TNBC cell line significantly inhibited cell proliferation, metastatic potential and induced apoptosis. ERβ expression reversed the aggravating role of AR in both indirect and direct ways. Indirectly, ERβ decreased AR activation through the inhibition of PI3K/AKT signaling pathway. Directly, ERβ formed heterodimers with AR in MDA-MB 453 cells and in human tissue samples impeding AR from forming homodimers. Enzalutamide is a more potent anti-androgen in AR + TNBC compared to bicalutamide. ERβ expression increased the sensitivity of MDA-MB 453 cells to anti-androgens and especially to enzalutamide. The administration of enzalutamide enhanced AR:ERβ heterodimers formation increasing the anti-tumor capacity of ERβ.


Collectively, our results provide evidence for a novel mechanism by which ERβ exerts oncosuppressive effect in AR-positive TBNC through direct and indirect interactions with AR. Moreover, ERβ expression may identify a new subset of TNBC that would respond more favorable to anti-androgens.


Triple-negative breast cancer Androgen receptor Estrogen receptor β (ERβEnzalutamide Bicalutamide 


Author contributions

AA, PS, ST, IZ, DT, AK, AK, DT, EX, and MK made substantial contributions to acquisition, analysis, and interpretation of data. AA, AGP, and MVK made substantial contributions in the conception, design, and interpretation of the data. AA, AGP, and MVK made substantial contributions in drafting the manuscript and revising it critically for important intellectual content.



Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

The protocol of this study was approved by the National and Kapodistrian University Ethics Committee following the principles of the Declaration of Helsinki.


  1. Akaza H, Hinotsu S, Usami M et al (2009) Combined androgen blockade with bicalutamide for advanced prostate cancer: long-term follow-up of a phase 3, double-blind, randomized study for survival. Cancer. PubMedGoogle Scholar
  2. Aleskandarany MA, Rakha EA, Ahmed MA et al (2011) Clinicopathologic and molecular significance of phospho-Akt expression in early invasive breast cancer. Breast Cancer Res Treat. Google Scholar
  3. Anestis A, Karamouzis MV, Dalagiorgou G, Papavassiliou AG (2015) Is androgen receptor targeting an emerging treatment strategy for triple negative breast cancer? Cancer Treat Rev 41:547CrossRefPubMedGoogle Scholar
  4. Cochrane DR, Bernales S, Jacobsen BM et al (2014) Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide. Breast Cancer Res. PubMedPubMedCentralGoogle Scholar
  5. Costa RLB, Han HS, Gradishar WJ (2018) Targeting the PI3K/AKT/mTOR pathway in triple-negative breast cancer: a review. Breast Cancer Res Treat. PubMedGoogle Scholar
  6. D’Amato NC, Gordon MA, Babbs B et al (2016) Cooperative dynamics of AR and ER activity in breast cancer. Mol Cancer Res. PubMedPubMedCentralGoogle Scholar
  7. Doane AS, Danso M, Lal P et al (2006) An estrogen receptor-negative breast cancer subset characterized by hormonically regulated transcriptional program and response to androgens. Oncogene. PubMedGoogle Scholar
  8. Farmer P, Bonnefoi H, Becette V et al (2005) Identification of molecular apocrine breast tumours by microarray analysis. Oncogene. PubMedGoogle Scholar
  9. Gao W, Bohl CE, Dalton JT (2005) Chemistry and structural biology of androgen receptor. Chem Rev 105:3352CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gucalp A, Tolaney S, Isakoff SJ et al (2013) Phase II trial of bicalutamide in patients with androgen receptor-positive, estrogen receptor-negative metastatic breast cancer. Clin Cancer Res. PubMedPubMedCentralGoogle Scholar
  11. Hall JM, Korach KS (2003) Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells. Mol Endocrinol. PubMedGoogle Scholar
  12. Hamilton N, Márquez-Garbán D, Mah V et al (2015) Biologic roles of estrogen receptor-β and insulin-like growth factor-2 in triple-negative breast cancer. Biomed Res Int. Google Scholar
  13. Hodges-Gallagher L, Valentine CD, Bader S El, Kushner PJ (2008) Estrogen receptor beta increases the efficacy of antiestrogens by effects on apoptosis and cell cycling in breast cancer cells. Breast Cancer Res Treat. PubMedGoogle Scholar
  14. Hu R, Dawood S, Holmes MD et al (2011) Androgen receptor expression and breast cancer survival in postmenopausal women. Clin Cancer Res. Google Scholar
  15. Joseph JD, Lu N, Qian J et al (2013) A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509. Cancer Discov. PubMedGoogle Scholar
  16. Karamouzis MV, Papavassiliou KA, Adamopoulos C, Papavassiliou AG (2016) Targeting androgen/estrogen receptors crosstalk in cancer. Trends Cancer 2:35CrossRefPubMedGoogle Scholar
  17. Kim SB, Dent R, Im SA et al (2017) Ipatasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer (LOTUS): a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Oncol. Google Scholar
  18. Lazennec G (2006) Estrogen receptor beta, a possible tumor suppressor involved in ovarian carcinogenesis. Cancer Lett 231:151CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lebert JM, Lester R, Powell E et al (2018) Advances in the systemic treatment of triple-negative breast cancer. Curr Oncol. PubMedPubMedCentralGoogle Scholar
  20. Lehmann BD, Bauer JA, Chen X et al (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Investig. PubMedGoogle Scholar
  21. Li Y, Liang Y, Sang Y et al (2018) MIR-770 suppresses the chemo-resistance and metastasis of triple negative breast cancer via direct targeting of STMN1 article. Cell Death Dis. Google Scholar
  22. Lindberg K, Helguero LA, Omoto Y et al (2011) Estrogen receptor β represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity. Breast Cancer Res. PubMedPubMedCentralGoogle Scholar
  23. Liu JC, Voisin V, Wang S et al (2014) Combined deletion of Pten and p53 in mammary epithelium accelerates triple-negative breast cancer with dependency on eEF2K. EMBO Mol Med. Google Scholar
  24. Loibl S, Muller BM, von Minckwitz G et al (2011) Androgen receptor expression in primary breast cancer and its predictive and prognostic value in patients treated with neoadjuvant chemotherapy. Breast Cancer Res Treat. Google Scholar
  25. Majumder A, Singh M, Tyagi SC (2017) Post-menopausal breast cancer: from estrogen to androgen receptor. Oncotarget. Google Scholar
  26. Murphy LC, Leygue E (2012) The role of estrogen receptor-β in breast cancer. Semin Reprod Med. PubMedGoogle Scholar
  27. Panet-Raymond V, Gottlieb B, Beitel LK et al (2000) Interactions between androgen and estrogen receptors and the effects on their transactivational properties. Mol Cell Endocrinol. PubMedGoogle Scholar
  28. Paruthiyil S, Parmar H, Kerekatte V et al (2004) Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res. PubMedGoogle Scholar
  29. Rahim B, O’Regan R (2017) AR signaling in breast cancer. Cancers (Basel) 9:E21CrossRefGoogle Scholar
  30. Reese JM, Bruinsma ES, Monroe DG et al (2017) ERβ inhibits cyclin dependent kinases 1 and 7 in triple negative breast cancer. Oncotarget. PubMedPubMedCentralGoogle Scholar
  31. Safarpour D, Pakneshan S, Tavassoli FA (2014) Androgen receptor (AR) expression in 400 breast carcinomas: is routine AR assessment justified? Am J Cancer Res 4:353PubMedPubMedCentralGoogle Scholar
  32. Smal I, Loog M, Niessen W, Meijering E (2010) Quantitative comparison of spot detection methods in fluorescence microscopy. IEEE Trans Med Imaging. PubMedGoogle Scholar
  33. Söderberg O, Gullberg M, Jarvius M et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. PubMedGoogle Scholar
  34. Tan W, Li Q, Chen K et al (2016) Estrogen receptor beta as a prognostic factor in breast cancer patients: a systematic review and meta-analysis. Oncotarget. Google Scholar
  35. Thomas C, Rajapaksa G, Nikolos F et al (2012) ERbeta1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR. Breast Cancer Res. PubMedGoogle Scholar
  36. Traina TA, Miller K, Yardley DA et al (2018) Enzalutamide for the treatment of androgen receptor-expressing triple-negative breast cancer. J Clin Oncol. Google Scholar
  37. Tran C, Ouk S, Clegg NJ et al (2009) Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. PubMedCentralGoogle Scholar
  38. Vladusic E, Guerra-Vladusic HaE FK, et al (2000) Expression and regulation of estrogen receptor beta in human breast tumors and cell lines. Oncol Rep 7:157PubMedGoogle Scholar
  39. Wang J, Zhang C, Chen K et al (2015) ERβ1 inversely correlates with PTEN/PI3K/AKT pathway and predicts a favorable prognosis in triple-negative breast cancer. Breast Cancer Res Treat. Google Scholar
  40. Wu W, Maneix L, Insunza J et al (2017) Estrogen receptor β, a regulator of androgen receptor signaling in the mouse ventral prostate. Proc Natl Acad Sci. Google Scholar
  41. Zhu A, Li Y, Song W et al (2016) Antiproliferative effect of androgen receptor inhibition in mesenchymal stem-like triple-negative breast cancer. Cell Physiol Biochem. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Aristomenis Anestis
    • 1
  • Panagiotis Sarantis
    • 1
  • Stamatios Theocharis
    • 2
  • Ilianna Zoi
    • 1
  • Dimitrios Tryfonopoulos
    • 3
  • Athanasios Korogiannos
    • 3
  • Anna Koumarianou
    • 4
  • Evangelia Xingi
    • 5
  • Dimitra Thomaidou
    • 5
  • Michalis Kontos
    • 6
  • Athanasios G. Papavassiliou
    • 1
    Email author
  • Michalis V. Karamouzis
    • 1
    • 7
    Email author
  1. 1.Molecular Oncology Unit, Department of Biological Chemistry, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  2. 2.Department of Pathology, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  3. 3.Second Oncology ClinicSaint Savvas Anti-Cancer HospitalAthensGreece
  4. 4.Hematology Oncology Unit, Fourth Department of Internal MedicineAttikon University Hospital, National and Kapodistrian University of AthensAthensGreece
  5. 5.Light Microscopy UnitHellenic Pasteur InstituteAthensGreece
  6. 6.First Department of Surgery, Laikon General Hospital, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  7. 7.First Department of Internal Medicine, Laikon General Hospital, Medical SchoolNational and Kapodistrian University of AthensAthensGreece

Personalised recommendations