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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

Abstract

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.

Keywords

Breast cancer Estrogen Androgen Aromatase inhibitor Endocrine resistance 

Abbreviations

3βAdiol

5α-Androstane-3β,17β-diol

LTED

Long-term estrogen deprivation

AI

Aromatase inhibitor

CCS

Charcoal-stripped calf serum

SERM

Selective estrogen receptor modulator

eSERM

Endogenous selective estrogen receptor modulator

IPA

Ingenuity pathway analysis

Notes

Acknowledgments

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)

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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

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