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Progesterone stimulates progenitor cells in normal human breast and breast cancer cells

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Abstract

The epithelium of the human breast is made up of a branching ductal–lobular system, which is lined by a single layer of luminal cells surrounded by a contractile basal cell layer. The co-ordinated development of stem/progenitor cells into these luminal and basal cells is fundamentally important for breast morphogenesis. The ovarian steroid hormones, progesterone (P) and 17β-estradiol, are critical in driving this normal breast development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. We previously demonstrated that P treatment increases proliferation and augments the number of progenitor-like cells, and that the progesterone receptor (PR) is also expressed in the bipotent progenitor-enriched subfraction. Here we demonstrate that PR is expressed in a subset of CD10+ basal cells and that P stimulates this CD10+ cell compartment, which is enriched for bipotent progenitor activity. In addition, we have shown that P stimulates progenitor cells in human breast cancer cell lines and expands the cancer stem cell population via increasing the stem-like CD44+ population. As changes in cell type composition are one of the hallmark features of breast cancer progression, the demonstration that progenitor cells are stimulated by P in both normal breast and in breast cancer cells has critical implications in discerning the mechanisms of how P increases breast cancer risk.

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Abbreviations

ALDH:

Aldehyde dehydrogenase

CFC:

Colony forming cell

CK14:

Cytokeratin-14

CK18:

Cytokeratin-18

CSC:

Cancer stem cell

E:

17β-estradiol

ER:

Estrogen receptor

FACS:

Fluorescence-activated cell sorter

FITC:

Fluorescein isothiocyanate

HRT:

Hormone replacement therapy

IF:

Immunofluorescence

IPX:

Immunoperoxidase

MEC:

Mammary epithelial cell

miRNA:

MicroRNA

qPCR:

Quantitative PCR

P:

Progesterone

PE:

Phycoerythrin

PR:

Progesterone receptor

SD:

Standard deviation

SE:

Standard error

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Acknowledgments

We wish to thank Arno Therapeutics, Inc. for generously donating the onapristone. We wish to thank Dr **n Maggie Wang for assistance with flow cytometry, performed in the Flow Cytometry Centre at Westmead Millennium Institute which is supported by the National Health and Medical Research Council of Australia (NHMRC) and Cancer Institute New South Wales. This study was supported by an NHMRC project grant (1011496), Cure Cancer Australia Foundation and the National Breast Cancer Foundation. We gratefully acknowledge the advice and assistance of Dr. Karen Byth, Westmead Millennium Institute, with statistical analysis.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Westmead Institute for Cancer Research, Sydney Medical School, University of Sydney at Westmead Millennium Institute, Westmead, NSW, 2145, Australia

    Heidi N. Hilton, N. Santucci, A. Silvestri, S. Kantimm, J. D. Graham & C. L. Clarke

  2. Children’s Medical Research Institute, Westmead, NSW, 2145, Australia

    L. I. Huschtscha

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  1. Heidi N. Hilton
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  2. N. Santucci
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  3. A. Silvestri
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  4. S. Kantimm
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  5. L. I. Huschtscha
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  6. J. D. Graham
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  7. C. L. Clarke
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Correspondence to Heidi N. Hilton.

Electronic supplementary material

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10549_2013_2817_MOESM1_ESM.tif

Supp Fig. 1 a Live and single cell gating strategy for detection of MUC1+ and CD10+ cells. b Relative mRNA expression levels of (i) CK14 and (ii) CK18 in each subpopulation, normalised to TBP (chart represents mean + SD). (TIFF 1,336 kb)

10549_2013_2817_MOESM2_ESM.tif

Supp Fig. 2 a Day 10 of CFC assay performed using sorted CD10 + and MUC1 + cells b Percentage of CFUs yielded from 2,000 cells seeded c Proportions of different colony types yielded from CFC assays using CD10 + and MUC1 + sorted cells. c Fold change in proportion of MUC1 + cells with indicated hormone treatments (n = 3). d Fold change in proportion of (i) luminal, or (ii) basal acini with indicated hormone treatments. Data are representative of the mean + SE for 3 independent experiments using tissue from 2 individuals. (TIFF 873 kb)

10549_2013_2817_MOESM3_ESM.tif

Supp Fig. 3a Average proportion of CSCs in T47Ds following treatment with ORG (1nM) or MPA (1nM), ± onapristone (ONP; 100nM). Chart represents mean + SE (n = 3). b Relative mRNA levels of CD44 were normalised to TBP in T47D cells following 48 h treatment with P (100nM) ± onapristone (1uM) or progestins (1nM) ± onapristone (100nM). Graph represents mean + SE from 3 biological replicates. (TIFF 457 kb)

10549_2013_2817_MOESM4_ESM.tif

Supp Fig. 4 a (i) Representative image of IPX staining for PR in HCC1428 cells. Scale bar represents 50 μm (ii) Gating strategy for detection and collection of CD44hi and CD44- cell fractions. b Relative transcript numbers of PR and CD44 in each subfraction. Chart represents mean + SD (TIFF 1,028 kb)

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Hilton, H.N., Santucci, N., Silvestri, A. et al. Progesterone stimulates progenitor cells in normal human breast and breast cancer cells. Breast Cancer Res Treat 143, 423–433 (2014). https://doi.org/10.1007/s10549-013-2817-2

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