Breast Cancer Research and Treatment

, Volume 134, Issue 3, pp 1027–1039 | Cite as

Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation

  • Matthew J. Sikora
  • Viktoriya Strumba
  • Marc E. Lippman
  • Michael D. Johnson
  • James M. RaeEmail author
Preclinical Study


Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15–20 % of patients receiving adjuvant AIs will relapse within 5–10 years of treatment initiation. Long-term estrogen deprivation (LTED) of breast cancer cells in culture mimics AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We expanded established LTED models to account for incomplete suppression of estrogen synthesis during AI therapy. MCF-7 cells were grown in medium with charcoal-stripped serum supplemented with defined concentrations of 17β-estradiol (E2) or the estrogenic androgen metabolite 5α-androstane-3β,17β-diol (3βAdiol), an endogenous selective estrogen receptor modulator. Cells were selected in concentrations of E2 or 3βAdiol that induce 10 or 90 percent of maximal proliferation (EC10 and EC90, respectively), or estrogen deprived. Estrogen independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. Following >7 months of selection, estrogen independence developed in estrogen-deprived cells and EC10-selected cells. Functional analyses demonstrated that estrogen-deprived and EC10-selected cells developed estrogen independence via unique mechanisms, ERα-independent and dependent, respectively. Estrogen-independent proliferation in EC10-selected cells could be blocked by kinase inhibitors. However, these cells were resistant to kinase inhibition in the presence of low steroid concentrations. These data demonstrate that further understanding of the total estrogen environment in patients on AI therapy who experience recurrence is necessary to effectively treat endocrine-resistant disease.


Breast cancer Estrogen Androgen Aromatase inhibitor Endocrine resistance 





Long-term estrogen deprivation


Aromatase inhibitor


Charcoal-stripped calf serum


Selective estrogen receptor modulator


Endogenous selective estrogen receptor modulator


Ingenuity pathway analysis



This study was supported in part by The Breast Cancer Research Foundation grant N003173, 1R01 GM099143 and by T-32 GM007767 from the National Institute of General Medical Sciences, Bethesda, MD. We thank the University of Michigan DNA Sequencing Core Facility for technical assistance. We also thank Dr. Richard Santen for his helpful review of our manuscript.

Conflict of interest

The authors have no relevant conflicts of interest to declare.

Supplementary material

10549_2012_2032_MOESM1_ESM.eps (977 kb)
Supplementary Figure 1. Steroid hormone concentrations used for long term selection. (EPS 977 kb)
10549_2012_2032_MOESM2_ESM.eps (1.4 mb)
Supplementary Figure 2. MCF-7 selected cell line growth following E2 treatment. (A), MCF-7 selected cell lines were treated with increasing concentrations of E2 as indicated. Growth was assessed at 5 days after treatment. Data are normalized to baseline growth in estrogen-free conditions; Y-axis values represent growth above that baseline. Points represent the average of 6 replicates ± SEM. (B), estimated EC50 values for growth induction and 95 % confidence intervals for the EC50. (EPS 1404 kb)
10549_2012_2032_MOESM3_ESM.tif (11.4 mb)
Supplemental Figure 3. IPA analysis highlighting enriched an enriched gene network in Veh cells in estrogen-free conditions. Network contains up-regulated nodes for EGFR, PRKCA and other oncogenic driver genes. Fold change in expression in Veh cells versus other selected cell lines is given; genes with decreased expression are indicated in green and those with increased expression indicated in red. Gray indicates no change in expression, and white genes did not have data available. Solid and dashed lines indicate direct and indirect relationships, respectively. (TIFF 11633 kb)
10549_2012_2032_MOESM4_ESM.tif (11.1 mb)
Supplemental Figure 4. IPA analysis highlighting an enriched gene network in 1pE/50p3β cells in estrogen-free conditions. 1pE/50p3β maintain an active ESR1 signaling pathway in the absence of estrogen, consistent with phenotypes described further in text. Fold change in expression in 1pE/50p3β cells versus other selected cell lines is given; genes with decreased expression are indicated in green and those with increased expression indicated in red. Gray indicates no change in expression, and white genes did not have data available. Solid and dashed lines indicate direct and indirect relationships, respectively. (TIFF 11363 kb)
10549_2012_2032_MOESM5_ESM.eps (994 kb)
Supplementary Table 1. Antibodies used in experiments. (EPS 994 kb)


  1. 1.
    Cuzick J, Sestak I, Baum M, Buzdar A, Howell A, Dowsett M, Forbes JF (2010) Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 10-year analysis of the ATAC trial. Lancet Oncol 11(12):1135–1141. doi: 10.1016/S1470-2045(10)70257-6 PubMedCrossRefGoogle Scholar
  2. 2.
    Dowsett M, Cuzick J, Ingle J, Coates A, Forbes J, Bliss J, Buyse M, Baum M, Buzdar A, Colleoni M, Coombes C, Snowdon C, Gnant M, Jakesz R, Kaufmann M, Boccardo F, Godwin J, Davies C, Peto R (2010) Meta-analysis of breast cancer outcomes in adjuvant trials of aromatase inhibitors versus tamoxifen. J Clin Oncol 28(3):509–518. doi: 10.1200/JCO.2009.23.1274 PubMedCrossRefGoogle Scholar
  3. 3.
    Martin LA, Farmer I, Johnston SR, Ali S, Dowsett M (2005) Elevated ERK1/ERK2/estrogen receptor cross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation. Endocr Relat Cancer 12(Suppl 1):S75–S84. doi: 10.1677/erc.1.01023 PubMedCrossRefGoogle Scholar
  4. 4.
    Martin LA, Farmer I, Johnston SR, Ali S, Marshall C, Dowsett M (2003) Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation. J Biol Chem 278(33):30458–30468. doi: 10.1074/jbc.M305226200 PubMedCrossRefGoogle Scholar
  5. 5.
    Masamura S, Santner SJ, Heitjan DF, Santen RJ (1995) Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. J Clin Endocrinol Metab 80(10):2918–2925PubMedCrossRefGoogle Scholar
  6. 6.
    Santen RJ, Song RX, Masamura S, Yue W, Fan P, Sogon T, Hayashi S, Nakachi K, Eguchi H (2008) Adaptation to estradiol deprivation causes up-regulation of growth factor pathways and hypersensitivity to estradiol in breast cancer cells. Adv Exp Med Biol 630:19–34PubMedCrossRefGoogle Scholar
  7. 7.
    Miller TW, Hennessy BT, Gonzalez-Angulo AM, Fox EM, Mills GB, Chen H, Higham C, Garcia-Echeverria C, Shyr Y, Arteaga CL (2010) Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer. J Clin Invest 120(7):2406–2413. doi: 10.1172/JCI41680 PubMedCrossRefGoogle Scholar
  8. 8.
    Sanchez CG, Ma CX, Crowder RJ, Guintoli T, Phommaly C, Gao F, Lin L, Ellis MJ (2011) Preclinical modeling of combined phosphatidylinositol-3-kinase inhibition with endocrine therapy for estrogen receptor-positive breast cancer. Breast Cancer Res 13(2):R21. doi: 10.1186/bcr2833 PubMedCrossRefGoogle Scholar
  9. 9.
    Miller TW, Balko JM, Fox EM, Ghazoui Z, Dunbier A, Anderson H, Dowsett M, Jiang A, Smith RA, Maira S-M, Manning HC, González-Angulo AM, Mills GB, Higham C, Chanthaphaychith S, Kuba MG, Miller WR, Shyr Y, Arteaga CL (2011) ERa-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer. Cancer Discov 1(4):338–351. doi: 10.1158/ PubMedCrossRefGoogle Scholar
  10. 10.
    Long BJ, Jelovac D, Thiantanawat A, Brodie AM (2002) The effect of second-line antiestrogen therapy on breast tumor growth after first-line treatment with the aromatase inhibitor letrozole: long-term studies using the intratumoral aromatase postmenopausal breast cancer model. Clin Cancer Res 8(7):2378–2388PubMedGoogle Scholar
  11. 11.
    Sabnis G, Brodie A (2011) Adaptive changes results in activation of alternate signaling pathways and resistance to aromatase inhibitor resistance. Mol Cell Endocrinol. doi: 10.1016/j.mce.2010.09.005 PubMedGoogle Scholar
  12. 12.
    Sikora MJ, Cordero KE, Larios JM, Johnson MD, Lippman ME, Rae JM (2009) The androgen metabolite 5alpha-androstane-3beta,17beta-diol (3betaAdiol) induces breast cancer growth via estrogen receptor: implications for aromatase inhibitor resistance. Breast Cancer Res Treat 115(2):289–296. doi: 10.1007/s10549-008-0080-8 PubMedCrossRefGoogle Scholar
  13. 13.
    Rossi E, Morabito A, Di Rella F, Esposito G, Gravina A, Labonia V, Landi G, Nuzzo F, Pacilio C, De Maio E, Di Maio M, Piccirillo MC, De Feo G, D’Aiuto G, Botti G, Chiodini P, Gallo C, Perrone F, de Matteis A (2009) Endocrine effects of adjuvant letrozole compared with tamoxifen in hormone-responsive postmenopausal patients with early breast cancer: the HOBOE trial. J Clin Oncol 27(19):3192–3197. doi: 10.1200/JCO.2008.18.6213 PubMedCrossRefGoogle Scholar
  14. 14.
    Gallicchio L, Macdonald R, Wood B, Rushovich E, Helzlsouer KJ (2011) Androgens and musculoskeletal symptoms among breast cancer patients on aromatase inhibitor therapy. Breast Cancer Res Treat. doi: 10.1007/s10549-011-1611-2 Google Scholar
  15. 15.
    Haynes BP, Dowsett M, Miller WR, Dixon JM, Bhatnagar AS (2003) The pharmacology of letrozole. J Steroid Biochem Mol Biol 87(1):35–45PubMedCrossRefGoogle Scholar
  16. 16.
    Dixon JM, Renshaw L, Young O, Murray J, Macaskill EJ, McHugh M, Folkerd E, Cameron DA, A’Hern RP, Dowsett M (2008) Letrozole suppresses plasma estradiol and estrone sulphate more completely than anastrozole in postmenopausal women with breast cancer. J Clin Oncol 26(10):1671–1676. doi: 10.1200/JCO.2007.13.9279 PubMedCrossRefGoogle Scholar
  17. 17.
    Rae JM, Johnson MD, Scheys JO, Cordero KE, Larios JM, Lippman ME (2005) GREB 1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Res Treat 92(2):141–149. doi: 10.1007/s10549-005-1483-4 PubMedCrossRefGoogle Scholar
  18. 18.
    Allegra JC (1965) Lippman ME (1980) The effects of 17β estradiol and tamoxifen on the ZR-75-1 human breast cancer cell line in defined medium. Eur J Cancer 16(8):1007–1015Google Scholar
  19. 19.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25(4):402–408. doi: 10.1006/meth.2001.1262 PubMedCrossRefGoogle Scholar
  20. 20.
    Dunbier AK, Martin LA, Dowsett M (2011) New and translational perspectives of oestrogen deprivation in breast cancer. Mol Cell Endocrinol. doi: 10.1016/j.mce.2010.12.034 PubMedGoogle Scholar
  21. 21.
    Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H (2001) Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 276(13):9817–9824. doi: 10.1074/jbc PubMedCrossRefGoogle Scholar
  22. 22.
    Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu V, Taylor J, Epstein RJ, Fuller-Pace FV, Egly JM, Coombes RC, Ali S (2002) Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene 21(32):4921–4931. doi: 10.1038/sj.onc.1205420 PubMedCrossRefGoogle Scholar
  23. 23.
    Likhite VS, Stossi F, Kim K, Katzenellenbogen BS, Katzenellenbogen JA (2006) 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 20(12):3120–3132. doi: 10.1210/me.2006-0068 PubMedCrossRefGoogle Scholar
  24. 24.
    Burris HA III (2004) Dual kinase inhibition in the treatment of breast cancer: initial experience with the EGFR/ErbB-2 inhibitor lapatinib. Oncologist 9(Suppl 3):10–15PubMedCrossRefGoogle Scholar
  25. 25.
    Moy B, Goss PE (2006) Lapatinib: current status and future directions in breast cancer. Oncologist 11(10):1047–1057. doi: 10.1634/theoncologist.11-10-1047 PubMedCrossRefGoogle Scholar
  26. 26.
    Partridge AH, LaFountain A, Mayer E, Taylor BS, Winer E, Asnis-Alibozek A (2008) Adherence to initial adjuvant anastrozole therapy among women with early-stage breast cancer. J Clin Oncol 26(4):556–562. doi: 10.1200/JCO.2007.11.5451 PubMedCrossRefGoogle Scholar
  27. 27.
    Litzenburger BC, Creighton CJ, Tsimelzon A, Chan BT, Hilsenbeck SG, Wang T, Carboni JM, Gottardis MM, Huang F, Chang JC, Lewis MT, Rimawi MF, Lee AV (2011) High IGF-IR activity in triple-negative breast cancer cell lines and tumorgrafts correlates with sensitivity to Anti-IGF-IR therapy. Clin Cancer Res 17(8):2314–2327. doi: 10.1158/1078-0432.ccr-10-1903 PubMedCrossRefGoogle Scholar
  28. 28.
    Fox EM, Miller TW, Balko JM, Kuba MG, Sánchez V, Smith RA, Liu S, González-Angulo AM, Mills GB, Ye F, Shyr Y, Manning HC, Buck E, Arteaga CL (2011) A Kinome-wide screen identifies the insulin/IGF-I receptor pathway as a mechanism of escape from hormone dependence in breast cancer. Cancer Res 71(21):6773–6784. doi: 10.1158/0008-5472.can-11-1295 PubMedCrossRefGoogle Scholar
  29. 29.
    McAuliffe PF, Meric-Bernstam F, Mills GB, Gonzalez-Angulo AM (2010) Deciphering the role of PI3 K/Akt/mTOR pathway in breast cancer biology and pathogenesis. Clin Breast Cancer 10:S59–S65PubMedCrossRefGoogle Scholar
  30. 30.
    Sheri A, Martin L-A, Johnston S (2010) Targeting endocrine resistance: Is there a role for mTOR inhibition? Clin Breast Cancer 10:S79–S85PubMedCrossRefGoogle Scholar
  31. 31.
    Baselga J, Campone M, Piccart M, Burris HA, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI, Lebrun F, Beck JT, Ito Y, Yardley D, Deleu I, Perez A, Bachelot T, Vittori L, Xu Z, Mukhopadhyay P, Lebwohl D, Hortobagyi GN (2011) Everolimus in postmenopausal hormone-receptorâ positive advanced breast cancer. N Engl J Med 366(6):520–529. doi: 10.1056/NEJMoa1109653 PubMedCrossRefGoogle Scholar
  32. 32.
    Ishikawa T, Takano K, Yasufuku-Takano J, Fujita T, Igarashi T, Miura M, Hata K (2001) Estrogenic impurities in labware. Nat Biotechnol 19(9):812. doi: 10.1038/nbt0901-812 PubMedCrossRefGoogle Scholar
  33. 33.
    DuSell CD, Umetani M, Shaul PW, Mangelsdorf DJ, McDonnell DP (2008) 27-Hydroxycholesterol is an endogenous selective estrogen receptor modulator. Mol Endocrinol 22(1):65–77. doi: 10.1210/me.2007-0383 PubMedCrossRefGoogle Scholar
  34. 34.
    Dauvois S, Labrie F (1989) Androstenedione and androst-5-ene-3β,17β-diol stimulate DMBA-induced rat mammary tumors—role of aromatase. Breast Cancer Res Treat 13(1):61–69. doi: 10.1007/bf01806551 PubMedCrossRefGoogle Scholar
  35. 35.
    Stanway SJ, Palmieri C, Stanczyk FZ, Folkerd EJ, Dowsett M, Ward R, Coombes RC, Reed MJ, Purohit A (2011) Effect of tamoxifen or anastrozole on steroid sulfatase activity and serum androgen concentrations in postmenopausal women with breast cancer. Anticancer Res 31(4):1367–1372PubMedGoogle Scholar
  36. 36.
    Voigt KD, Bartsch W (1986) Intratissular androgens in benign prostatic hyperplasia and prostatic cancer. J Steroid Biochem 25(5B):749–757PubMedCrossRefGoogle Scholar
  37. 37.
    Santen RJ, Demers L, Ohorodnik S, Settlage J, Langecker P, Blanchett D, Goss PE, Wang S (2007) Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy. Steroids 72(8):666–671PubMedCrossRefGoogle Scholar
  38. 38.
    Ingle JN, Buzdar AU, Schaid DJ, Goetz MP, Batzler A, Robson ME, Northfelt DW, Olson JE, Perez EA, Desta Z, Weintraub RA, Williard CV, Flockhart DA, Weinshilboum RM (2010) Variation in anastrozole metabolism and pharmacodynamics in women with early breast cancer. Cancer Res 70(8):3278–3286. doi: 10.1158/0008-5472.can-09-3024 PubMedCrossRefGoogle Scholar
  39. 39.
    Johnston SR (2009) Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 9(Suppl 1):S28–S36. doi: 10.3816/CBC.2009.s.003 PubMedCrossRefGoogle Scholar
  40. 40.
    Di Leo A, Jerusalem G, Petruzelka L, Torres R, Bondarenko IN, Khasanov R, Verhoeven D, Pedrini JL, Smirnova I, Lichinitser MR, Pendergrass K, Garnett S, Lindemann JP, Sapunar F, Martin M (2010) Results of the CONFIRM phase III trial comparing fulvestrant 250 mg with fulvestrant 500 mg in postmenopausal women with estrogen receptor-positive advanced breast cancer. J Clin Oncol 28(30):4594–4600. doi: 10.1200/JCO.2010.28.8415 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Matthew J. Sikora
    • 1
    • 7
  • Viktoriya Strumba
    • 2
  • Marc E. Lippman
    • 4
  • Michael D. Johnson
    • 5
  • James M. Rae
    • 1
    • 3
    • 6
    Email author
  1. 1.Department of PharmacologyUniversity of Michigan Medical CenterAnn ArborUSA
  2. 2.Molecular and Behavioral Neuroscience InstituteUniversity of Michigan Medical CenterAnn ArborUSA
  3. 3.Department of Internal MedicineUniversity of Michigan Medical CenterAnn ArborUSA
  4. 4.Department of MedicineUniversity of Miami Miller School of MedicineMiamiUSA
  5. 5.Department of OncologyGeorgetown University Medical CenterWashingtonUSA
  6. 6.Division of Hematology/Oncology, Department of Internal MedicineUniversity of Michigan Comprehensive Cancer CenterAnn ArborUSA
  7. 7.Women’s Cancer Research CenterUniversity of Pittsburgh Cancer InstitutePittsburghUSA

Personalised recommendations