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

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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.

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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

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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.

Author information

Author notes

  1. Matthew J. Sikora

    Present address: Women’s Cancer Research Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Authors and Affiliations

  1. Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA

    Matthew J. Sikora & James M. Rae

  2. Molecular and Behavioral Neuroscience Institute, University of Michigan Medical Center, Ann Arbor, MI, USA

    Viktoriya Strumba

  3. Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA

    James M. Rae

  4. Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA

    Marc E. Lippman

  5. Department of Oncology, Georgetown University Medical Center, Washington, DC, USA

    Michael D. Johnson

  6. Division of Hematology/Oncology, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, 1500 East Medical Center Drive, 6310 UMCCC, Ann Arbor, MI, 48109-0612, USA

    James M. Rae

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Correspondence to James M. Rae.

Additional information

Microarray data submission: GEO Accession # GSE33287.

Electronic supplementary material

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Supplementary Figure 1. Steroid hormone concentrations used for long term selection. (EPS 977 kb)

10549_2012_2032_MOESM2_ESM.eps

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

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

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)

Supplementary Table 1. Antibodies used in experiments. (EPS 994 kb)

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Sikora, M.J., Strumba, V., Lippman, M.E. et al. Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation. Breast Cancer Res Treat 134, 1027–1039 (2012). https://doi.org/10.1007/s10549-012-2032-6

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