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Androgen Receptor Signalling Promotes a Luminal Phenotype in Mammary Epithelial Cells

  • Gerard A. Tarulli
  • Geraldine Laven-Law
  • Mona Shehata
  • Kirsty A. Walters
  • Iza M. Denis
  • Md. Mostafizur Rahman
  • David J. Handelsman
  • Nicola R. Dean
  • Wayne D. Tilley
  • Theresa E. Hickey
Article

Abstract

Androgens influence mammary gland development but the specific role of the androgen receptor (AR) in mammary function is largely unknown. We identified cell subsets that express AR in vivo and determined the effect of AR activation and transgenic AR inhibition on sub-populations of the normal mouse mammary epithelium by flow cytometry and immunohistochemistry. Immunolocalisation of AR with markers of lineage identity was also performed in human breast tissues. AR activation in vivo significantly decreased the proportion of basal cells, and caused an accumulation of cells that expressed a basal cell marker but exhibited morphological features of luminal identity. Conversely, in AR null mice the proportion of basal mammary epithelial cells was significantly increased. Inhibition of AR increased basal but not luminal progenitor cell activity in vitro. A small population of AR-positive cells in a basal-to-luminal phenotype transition was also evident in human breast lobules. Collectively, these data support a role for AR in promoting a luminal phenotype in mammary epithelial cells.

Keywords

Androgens Mammary epithelium Androgen receptor 

Abbreviations

AR

Androgen receptor

DHT

Dihydrotestosterone

ECM

Extracellular matrix

ER

Estrogen receptor-alpha

FACS

Fluorescence-activated cell sorting

HF

Hydroxyflutamide

HS-MEC

Hormone-sensing mammary epithelial cell

MEC

Mammary epithelial cell

PR

Progesterone receptor

Notes

Acknowledgements

The authors wish to acknowledge the support of the University of Adelaide Laboratory Animal Services, University of Adelaide Microscopy for imaging support, Dr. Randall Grose from the South Australian Heath and Medical Research Institute for providing cell sorting services, Ms. Marie Pickering and Ms. Zoya Khityak for assistance with histological experiments, and Ms. Tamara Crittenden from Flinders Medical Centre for recruitment and consent of patients for primary breast tissue collection.

Funding

This work was supported by funding from the National Breast Cancer Foundation (ID PS-15-041 to GAT and WDT), the National Health and Medical Research Council of Australia (ID APP1130077 and ID 1084416 to WDT and TEH), and Cancer Australia (ID 1043497). IMD is supported by a National Breast Cancer Foundation Fellowship ID PF-17-006. The laboratory of WDT & TEH is supported by research funds from GTx Inc., the manufacturers and suppliers of GTx-024 used in this study.

Compliance with Ethical Standards

Conflict of Interests

The authors declare no competing or financial interests.

Independent Author Contributions

GAT: Devised concepts & hypotheses, performed experiments, interpreted data, devised conclusions, prepared manuscript. GLL: Performed experiments and analyzed data. MS: Performed single-cell RNA experiments and analyzed data. IMD: Assisted in performing mammary digestion and cell labelling. MMR: Assisted in performing mammary digestion and cell labelling. KAW & DJH: Design and development of AR null mice and supply of their mammary tissue. WDT: Reviewed data, manuscript preparation and editing. TEH: Experimental conceptual design, data revision, study supervision, manuscript editing.

Supplementary material

10911_2018_9406_Fig5_ESM.png (65 kb)
Supplemental Fig. S1

- Treatment with GTx-024 does not alter the proportion of alveolar compared to hormone-sensing MECs when assessed by flow cytometry. (PNG 64 kb)

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High resolution image. (EPS 266 kb)
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Supplemental Fig. S2

- A) AR-positive primary human breast cancer tissue labeled with secondary antibodies alone. B) AR+ prostate cancer sample labeled with anti-AR. Scale bar = 50 μm. (PNG 197 kb)

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Supplemental Fig. S3

- A-B) Confocal microscopy images of normal breast tissue immunolabeled for p63 (green) and AR (red), and nuclei labeled with DAPI (blue). Dashed box in “i” denotes magnified image in “ii”. Individual channels from magnified image in “ii”, found in panel “iii” (DAPI), “iv” (p63) and “v” (AR). Circles denote cells expressing p63 and AR. Scale bar = 25 μm. (PNG 208 kb)

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Supplemental Fig. S4

– A-C) Confocal microscopy images of normal breast tissue immunostained for AR (blue), ER (green) and PR (red). “i” = All channels overlaid, “ii-iv” = same confocal image in “i”, with pairs of channels segregated (AR & PR - “ii”, AR & ER - “iii”, PR & ER - “iv”). Cells that are positive for all three nuclear receptors are observed (arrowheads), as are cells that are only AR-positive (arrows). Scale bar = 25 μm. (PNG 218 kb)

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Supplemental Fig. S5

- Gating hierarchy for flow cytometry and cell sorting experiments. Debris and cell clumps were excluded by FSC vs. SSC (A), followed by exclusion of doublets using FSC-H vs. FSC-W followed by SSC-H vs. SSC-W (B). Dead/dying cells were excluded by gating on DAPI-negative cells (C), and lymphocytes excluded by gating on CD45-negative cells (D). The proportion of epithelial cells was determined by gating on CD24/CD49f-positive cells (E). CD24 and CD49f allows for the distinction between basal and luminal epithelial cells (F). Luminal subsets were segregated based on CD49b (G). (PNG 206 kb)

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Table S1.

(PNG 131 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Gerard A. Tarulli
    • 1
    • 2
  • Geraldine Laven-Law
    • 1
  • Mona Shehata
    • 3
  • Kirsty A. Walters
    • 4
  • Iza M. Denis
    • 1
  • Md. Mostafizur Rahman
    • 1
  • David J. Handelsman
    • 5
  • Nicola R. Dean
    • 6
  • Wayne D. Tilley
    • 1
  • Theresa E. Hickey
    • 1
  1. 1.Dame Roma Mitchell Cancer Research Laboratories, Faculty of Health & Medical SciencesThe University of AdelaideAdelaideAustralia
  2. 2.School of BioSciencesThe University of MelbourneParkvilleAustralia
  3. 3.Princess Margaret Cancer CentreUniversity Health NetworkTorontoCanada
  4. 4.Discipline of Obstetrics & Gynaecology, School of Women’s & Children’s HealthUniversity of New South WalesSydneyAustralia
  5. 5.Andrology Laboratory, ANZAC Research InstituteUniversity of SydneySydneyAustralia
  6. 6.Department of Plastic & Reconstructive SurgeryFlinders Medical Centre/Flinders UniversityBedford ParkAustralia

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