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Journal of Mammary Gland Biology and Neoplasia

, Volume 24, Issue 3, pp 257–270 | Cite as

BRCA1 Attenuates Progesterone Effects on Proliferation and NFκB Activation in Normal Human Mammary Epithelial Cells

  • H. N. Hilton
  • L. J. Patterson McDonald
  • N. Santucci
  • F. R. van der Bent
  • A. Silvestri
  • J. D. GrahamEmail author
  • C. L. Clarke
Article
  • 414 Downloads

Abstract

Germline mutations in the breast cancer susceptibility gene BRCA1, encoding a tumor suppressor protein, greatly enhance the risk of breast and ovarian cancer. This tissue-specificity implicates the role of ovarian hormones. Indeed, BRCA1 has been demonstrated to regulate the signalling axis of the hormone, progesterone, and its receptor, the progesterone receptor (PR), and progesterone action has been implicated in BRCA1-related tumorigenesis. BRCA1 also plays important roles in oxidative stress and activating nuclear factor kappaB (NFκB) signalling pathways. Like wildtype BRCA1 function, PR signalling has also been shown to inhibit NFκB activation. Although PR and BRCA1 networks are known to interact, their interaction at the level of NFκB activation in the human breast is not understood. This study investigates the effect of reduced BRCA1 expression on proliferation and NFκB activation in human breast cells, and the impact of progesterone on these effects. The major findings are that: 1) Reduced BRCA1 levels inhibit cell growth in normal human mammary cells and breast cancer cells; 2) Reduced BRCA1 levels stimulated inflammatory targets and NFκB activity in normal human mammary cells; 3) Wildtype BRCA1 inhibited the pro-proliferative effects of progesterone in normal mammary epithelial cells, and; 4) Progesterone attenuated BRCA1-mediated NFκB activation in normal human mammary cells. These data have important implications for our understanding of progesterone action in BRCA1 mutation carriers, and how inhibition of this action may potentially delay tumorigenesis or impart a more favourable prognosis.

Keywords

Progesterone BRCA1 Breast cancer NFκB Proliferation 

Notes

Acknowledgments

This work was made possible by funding from the National Breast Cancer Foundation (HNH; Career Development Fellowship; Grant ID: ECF-16-007), Cure Cancer Australia Foundation/Cancer Australia (HNH; Grant ID: APP1098796). CLC is a National Health and Medical Research Council Principal Research Fellow (Grant ID: APP1081334). HNH, CLC and JDG are supported by a project grant from The Cancer Council NSW (Grant ID: RG-15-17).

Supplementary material

10911_2019_9431_MOESM1_ESM.pptx (361 kb)
ESM 1 a Relative levels of BRCA1 transcripts in T47D cells following transduction of 2 individual shRNAs. b Relative levels of BRCA1 transcripts in T47D cells following transduction of a pool of 2 individual BRCA1 shRNAs, without (V) or with (P) progesterone treatment. c Representative immunofluorescent images of T47D breast cancer cells stained with α-BRCA1 (red), treated with control shRNA (i and ii) or BRCA1 shRNA (iii and iv). Scale bar represents 25 μm. d Reduction in BRCA1 transcripts following transduction of BRCA1shRNA in AB32 cells (n = 3). Error bars indicate standard error (PPTX 360 kb)
10911_2019_9431_MOESM2_ESM.pptx (48 kb)
ESM 2 Decreased average signal intensity on microarray of selected cyclins following knockdown of BRCA1 in AB32 cells. Error bars indicate standard error from triplicate samples. (PPTX 48 kb)
10911_2019_9431_MOESM3_ESM.pptx (49 kb)
ESM 3 Decreased average signal intensity on microarray of selected cyclins following knockdown of BRCA1 in T47D breast cancer cells. Error bars indicate standard error from triplicate samples (PPTX 49 kb)
10911_2019_9431_MOESM4_ESM.pptx (43 kb)
ESM 4 Average fold change in mRNA levels after BRCA1 knockdown, relative to control shRNA, in (a) AB32 cells (n = 3) and in (b) MCF-10A cells (n = 4), as determined by qPCR. Charts are plotted on a logarithmic scale. Error bars indicate standard error. c Relative levels of BRCA1 transcripts in MCF10A cells compared with AB32 cells (PPTX 43 kb)
10911_2019_9431_MOESM5_ESM.pptx (459 kb)
ESM 5 Induction of PR transcripts (a) and PR protein (b) following transduction of BRCA1shRNA in AB32 cells, and treatment with progesterone or progestins, as indicated. Error bars indicate standard error (n = 2). c Relative densitometric intensities of PR-A and PR-B bands (PPTX 459 kb)
10911_2019_9431_MOESM6_ESM.pptx (56 kb)
ESM 6 a Effect of progestins on cell proliferation in control shRNA cells. Cell growth is expressed as an increase in percentage of confluence. b Fold change in confluency of cells (± BRCA1 shRNA ± progestin treatment), relative to vehicle control shRNA cells at 120 h. c Effect of progestins on cell proliferation in BRCA1 shRNA cells. Charts represent mean ± standard error (n = 4) (PPTX 56 kb)
10911_2019_9431_MOESM7_ESM.pptx (72 kb)
ESM 7 Volcano plot analysis (integrating p values and log2 fold changes) for progesterone-regulated targets in AB32 cells treated with control-shRNA. Blue and yellow dots indicate probes that showed a fold-change ≤2 or ≥ 2, respectively (PPTX 72 kb)
10911_2019_9431_MOESM8_ESM.docx (14 kb)
ESM 8 (DOCX 13 kb)
10911_2019_9431_MOESM9_ESM.docx (13 kb)
ESM 9 (DOCX 12 kb)
10911_2019_9431_MOESM10_ESM.docx (14 kb)
ESM 10 (DOCX 14 kb)
10911_2019_9431_MOESM11_ESM.docx (14 kb)
ESM 11 (DOCX 13 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • H. N. Hilton
    • 1
  • L. J. Patterson McDonald
    • 1
  • N. Santucci
    • 1
  • F. R. van der Bent
    • 2
  • A. Silvestri
    • 1
  • J. D. Graham
    • 1
    Email author
  • C. L. Clarke
    • 1
  1. 1.Centre for Cancer Research, The Westmead Institute for Medical Research, Sydney Medical School – WestmeadThe University of SydneyWestmeadAustralia
  2. 2.Department of Medicine, Academic Medical CenterUniversity of GroningenGroningenThe Netherlands

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