World Congress on Breast Cancer
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1 World Congress on Breast Cancer How pregnancy at early age protects against breast cancer Fabienne Meier-Abt, MD PhD
2 Background: Early age pregnancy protects against breast cancer. MacMahon, B. et al. 1970, Lambe, M. et al. 1996
3 Background: Early age pregnancy protects against breast cancer. Rodents can be used as model system. In rodents: early age pregnancy >75% protective effect against mammary tumors Medina, D MacMahon, B. et al. 1970, Lambe, M. et al. 1996
4 Background: Hypothetical mechanisms: - change in the proliferation/differentiation potential of specific mammary epithelial cells Russo, J. et al Siwko, S.K. et al Britt, K.L. et al systemic changes in circulating hormones Thordarson, G. et al changes in the stromal composition of the mammary gland Schedin, P. et al. 2004
5 Research Question: What is the effect of an early pregnancy on the gene expression profile and on the proliferation/differentiation potential of the various mouse mammary epithelial cell subpopulations?
6 Mammary Cell Type Hierarchy:
7 Mammary Cell Type Hierarchy: Basal compartment Luminal compartment Milk Basement Stroma membrane
8 Mammary Cell Type Hierarchy: Basal compartment Luminal compartment Milk Basement Stroma membrane
9 Methodology: 1. Isolation of mammary epithelial cell subpopulations by FACS 2. Transcriptome and bioinformatic transcription factor activity analysis 3. Ingenuity IPA and GSEA analysis 4. Colony formation assay 5. Mammary gland reconstitution assay 6. Immunohistochemistry for progesterone receptor 7. Rescue experiments
10 Methods: Early age parturition protocol
11 Results I Transcriptome analysis in mammary epithelial cell subpopulations from parous and age-matched virgin control mice II Effects of early parity on the clonogenic and proliferation potential of basal stem/progenitor cells III Putative mechanism of early parity-induced biofunctional alterations in basal mammary stem/progenitor cells IV Duration and age dependency of early parity-induced changes
12 Results I: Early age pregnancy-induced changes in gene expression in murine mammary epithelial cell subpopulations.
13 Results I: Upregulation of differentiation genes (blue), decreased Wnt signaling (green) and increased Notch signaling (orange) in basal stem/progenitor cells from parous mice. Gene expression in basal stem/progenitor cells from parous mice as compared to virgin control mice
14 Results I: Upregulation of differentiation genes (blue), decreased Wnt signaling (green) and increased Notch signaling (orange) in basal stem/progenitor cells from parous mice. Wnt signaling: major cell fate determining pathway linked to cell proliferation and carcinogenesis in basal stem/progenitor cells in the mammary gland Notch signaling: major cell fate determining pathway linked to reduced proliferation in basal stem/progenitor cells in the mammary gland Gene expression in basal stem/progenitor cells from parous mice as compared to virgin control mice
15 Results I Transcriptome analysis in mammary epithelial cell subpopulations from parous and age-matched virgin control mice II Effects of early parity on the clonogenic and proliferation potential of basal stem/progenitor cells III Putative mechanism of early parity-induced biofunctional alterations in basal mammary stem/progenitor cells IV Duration and age dependency of early parity-induced changes
16 Results II: Early parity decreases the number of cells with colony formation capacity with the most prominent effect in basal stem/progenitor cells. * P <0.015
17 Results II: Early parity decreases the in vivo reconstitution efficiency but not the number of mammary repopulating units (MRUs) of basal stem/progenitor cells. fat pad 3-week old mammary gland
18 Results II: Early parity decreases the in vivo reconstitution efficiency but not the number of mammary repopulating units (MRUs) of basal stem/progenitor cells. fat pad 3-week old mammary gland Cleared fat pad
19 Results II: Early parity decreases the in vivo reconstitution efficiency but not the number of mammary repopulating units (MRUs) of basal stem/progenitor cells. fat pad Basal stem/progenitor cells 3-week old mammary gland Cleared fat pad Transplantation
20 Results II: Early parity decreases the in vivo reconstitution efficiency but not the number of mammary repopulating units (MRUs) of basal stem/progenitor cells. fat pad Basal stem/progenitor cells 8 wks 3-week old mammary gland Cleared fat pad Transplantation Reconstituted mammary gland
21 Results II: Early parity decreases the in vivo reconstitution efficiency but not the number of mammary repopulating units (MRUs) of basal stem/progenitor cells. fat pad Basal stem/progenitor cells 8 wks 3-week old mammary gland Cleared fat pad Transplantation Reconstituted mammary gland * P =0.0004
22 Results I Transcriptome analysis in mammary epithelial cell subpopulations from parous and age-matched virgin control mice II Effects of early parity on the clonogenic and proliferation potential of basal stem/progenitor cells III Putative mechanism of early parity-induced biofunctional alterations in basal mammary stem/progenitor cells IV Duration and age dependency of early parity-induced changes
23 Results III: Parity leads to a >3-fold decrease in Wnt4 expression in total cell suspensions Wnt4
24 Results III: Parity leads to a >3-fold decrease in Wnt4 expression in total cell suspensions & to a 3-fold decrease of PR positive (Wnt4-secreting) mammary epithelial cells. Wnt4 * P =3.70E-07
25 Mechanistic model of the effect of an early age pregnancy
26 Results I Transcriptome analysis in mammary epithelial cell subpopulations from parous and age-matched virgin control mice II Effects of early parity on the clonogenic and proliferation potential of basal stem/progenitor cells III Putative mechanism of early parity-induced biofunctional alterations in basal mammary stem/progenitor cells IV Duration and age dependency of early parity-induced changes
27 Summary: Early parity leads to the following changes in mammary cell subpopulations: 1. an induction of differentiation and a downregulation of the Wnt/Notch signaling ratio in basal stem/progenitor cells 2. a downregulation of potentially tumorigenic biofunctions in the basal stem/progenitor cell subpopulation 3. a decrease in the in vitro and in vivo proliferation potential of isolated basal stem/progenitor cells 4. a reduction in progesterone-responsive and Wnt4-secreting luminal cells These early age pregnancy-induced changes are of life-long duration, and NOT induced by late age pregnancy. This is fully consistent with the life-long breast cancer protective effect of early but not late age pregnancy in women.
28 Conclusions and Perspectives: Early age pregnancy induces life-long cellular and molecular changes in mammary glands of mice which potentially explain the breast cancer protective effect of early age pregnancy. The decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells has now been confirmed in humans but further validations in humans are warranted. The results provide deeper understanding of the role of parity and open the door to future studies assessing whether inhibitors of the Wnt pathway might be useful to mimic the early parity-induced protective effect against breast cancer.
29 Acknowledgements: M. Bentires-Alj, S. Gasser, C. Rochlitz, D. Schübeler, M. Smalley Current and former Bentires-Alj lab members Genomics Facility: Tim Roloff, Stéphane Thiry Bioinformatics: Michael Stadler, Dimosthenis Gaidatzis MARA: Piotr Balwierz, Erik van Nimwegen FACS: Hubertus Kohler Imaging: Laurent Gelman, Steven Bourke, Raphael Thierry Histology: Sandrine Bichet, Augustyn Bogucki Funding: Swiss National Science Foundation, Novartis Research Foundation, European Research Council, Swiss Cancer League, Krebsliga beider Basel
30 Thank you!
31 Results I: Wnt targets are reduced on the protein level Mean number of versican (brown) pixels per image: Virgins: 38,700 ± 6954 Parous: 127 ± 33 P=5.48E-07
32 Results I: Wnt targets are reduced on the protein level and nuclear β-catenin is decreased in basal mammary epithelial cells from parous mice. Mean number of versican (brown) pixels per image: Virgins: 38,700 ± 6954 Parous: 127 ± 33 P=5.48E-07
33 Results: Early age pregnancy-induced decreases in progesterone receptor positive cells and Wnt target versican and keratin 15 expression persist in mammary glands of postmenopausal (22 months old) mice. *P =1.53E-04 *P =0.004 *P =0.0049
34 Results IV: In contrast to early parity, late age pregnancy (24wks) has NO effect on PR positive cells and on Wnt target versican and keratin 15 expression. P =0.72 P =0.28 P =0.71
Meier-Abt et al. Breast Cancer Research 2013, 15:R36 (29 April 2013)
Parity induces differentiation and reduces Wnt/ Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium Meier-Abt et al. Meier-Abt et al.
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