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Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium.

Meier-Abt F, Milani E, Roloff T, Brinkhaus H, Duss S, Meyer DS, Klebba I, Balwierz PJ, van Nimwegen E, Bentires-Alj M - Breast Cancer Res. (2013)

Bottom Line: Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3.This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo.As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Early pregnancy has a strong protective effect against breast cancer in humans and rodents, but the underlying mechanism is unknown. Because breast cancers are thought to arise from specific cell subpopulations of mammary epithelia, we studied the effect of parity on the transcriptome and the differentiation/proliferation potential of specific luminal and basal mammary cells in mice.

Methods: Mammary epithelial cell subpopulations (luminal Sca1-, luminal Sca1+, basal stem/progenitor, and basal myoepithelial cells) were isolated by flow cytometry from parous and age-matched virgin mice and examined by using a combination of unbiased genomics, bioinformatics, in vitro colony formation, and in vivo limiting dilution transplantation assays. Specific findings were further investigated with immunohistochemistry in entire glands of parous and age-matched virgin mice.

Results: Transcriptome analysis revealed an upregulation of differentiation genes and a marked decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells of parous mice. Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3. This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo. As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells. Because recombinant Wnt4 rescued the proliferation defect of basal stem/progenitor cells in vitro, reduced Wnt4 secretion appears to be causally related to parity-induced alterations of basal stem/progenitor cell properties in mice.

Conclusions: By revealing that parity induces differentiation and downregulates the Wnt/Notch signaling ratio and the in vitro and in vivo proliferation potential of basal stem/progenitor cells in mice, our study sheds light on the long-term consequences of an early pregnancy. Furthermore, it opens the door to future studies assessing whether inhibitors of the Wnt pathway may be used to mimic the parity-induced protective effect against breast cancer.

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Effect of Wnt4 on the proliferation capacity of basal stem/progenitor cells after early pregnancy in mice. (A) Recombinant Wnt4 rescues the parity-induced in vitro proliferation defect in basal mammary epithelial cells. Selected mammary epithelial cells from parous mice were cultured in the absence or presence of recombinant Wnt4. Three independent experiments were performed. Data represent the mean ± SEM of colonies per well, with six to nine wells assessed per cell type. *P ≤ 0.02 (two-tailed unpaired Student t test). (B) Mechanistic model illustrating the parity-induced decrease in hormone-sensing and Wnt4-secreting luminal cells on the Wnt/Notch signaling pathways and the proliferation/differentiation potential in basal stem/progenitor cells.
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Figure 7: Effect of Wnt4 on the proliferation capacity of basal stem/progenitor cells after early pregnancy in mice. (A) Recombinant Wnt4 rescues the parity-induced in vitro proliferation defect in basal mammary epithelial cells. Selected mammary epithelial cells from parous mice were cultured in the absence or presence of recombinant Wnt4. Three independent experiments were performed. Data represent the mean ± SEM of colonies per well, with six to nine wells assessed per cell type. *P ≤ 0.02 (two-tailed unpaired Student t test). (B) Mechanistic model illustrating the parity-induced decrease in hormone-sensing and Wnt4-secreting luminal cells on the Wnt/Notch signaling pathways and the proliferation/differentiation potential in basal stem/progenitor cells.

Mentions: Having observed most of the expected functional consequences of a decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells from parous mice, we finally examined the possible cause of parity-induced reduction in canonical Wnt signaling and proliferation capacity in basal stem/progenitor cells. Because parity induced a greater than threefold decrease in Wnt ligand Wnt4 gene expression (Figure 2A, B), and Wnt4 is known to be secreted in response to progesterone by hormone-sensing luminal cells [46], thus inducing canonical Wnt signaling in mammary stem/progenitor cells [47], a parity-induced decrease in estrogen/progesterone-sensitive luminal cells could explain the overall decrease in Wnt signaling in mammary stem cells. This hypothesis is supported by the reduction in the proportion of luminal Sca1+ cells isolated from parous mice (Figure 1D) and by the demonstration that luminal Sca1+ cells are hormone receptor positive (Additional file 6) [17]. Furthermore, immunohistochemical analysis of mammary gland sections for estrogen receptor alpha (ERα) and its target progesterone receptor (PR) showed a twofold decrease in ERα- and a threefold decrease in PR-positive cells in parous compared with age-matched virgin control mice (Figure 6A, B). These results were additionally verified by qPCR in total mammary cell suspensions (Figure 6C). Notably, expression of the luminal marker Krt8 was similar in cell suspensions from parous and age-matched virgin control mice, supporting the conclusion of a specific decrease in hormone receptor-positive cells rather than a general cell loss after pregnancy. Furthermore, parity-induced reduction in progesterone-stimulated Wnt4 expression was independent of blood progesterone, because average blood progesterone concentrations were similar in parous mice and age-matched virgin control mice in estrus (see Additional file 9). Finally, supplementation of the culture medium with recombinant Wnt4 stimulated in vitro proliferation capacity of basal myoepithelial and basal stem/progenitor cells from parous mice by +138% ± 22% and +140% ± 17%, respectively (Figure 7A). In contrast, no significant effects of recombinant Wnt4 on the colony-formation capacity of luminal Sca1- (+3.7% ± 3.0%) and luminal Sca1+ cells from parous mice (-5.1% ± 2.9%) were observed. These data strongly suggest a causal relation between reduced number of luminal progesterone receptor-positive/Wnt4-secreting cells and decreased Wnt/Notch signaling and proliferation potential of basal stem/progenitor cells after early pregnancy (Figure 7B).


Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium.

Meier-Abt F, Milani E, Roloff T, Brinkhaus H, Duss S, Meyer DS, Klebba I, Balwierz PJ, van Nimwegen E, Bentires-Alj M - Breast Cancer Res. (2013)

Effect of Wnt4 on the proliferation capacity of basal stem/progenitor cells after early pregnancy in mice. (A) Recombinant Wnt4 rescues the parity-induced in vitro proliferation defect in basal mammary epithelial cells. Selected mammary epithelial cells from parous mice were cultured in the absence or presence of recombinant Wnt4. Three independent experiments were performed. Data represent the mean ± SEM of colonies per well, with six to nine wells assessed per cell type. *P ≤ 0.02 (two-tailed unpaired Student t test). (B) Mechanistic model illustrating the parity-induced decrease in hormone-sensing and Wnt4-secreting luminal cells on the Wnt/Notch signaling pathways and the proliferation/differentiation potential in basal stem/progenitor cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Effect of Wnt4 on the proliferation capacity of basal stem/progenitor cells after early pregnancy in mice. (A) Recombinant Wnt4 rescues the parity-induced in vitro proliferation defect in basal mammary epithelial cells. Selected mammary epithelial cells from parous mice were cultured in the absence or presence of recombinant Wnt4. Three independent experiments were performed. Data represent the mean ± SEM of colonies per well, with six to nine wells assessed per cell type. *P ≤ 0.02 (two-tailed unpaired Student t test). (B) Mechanistic model illustrating the parity-induced decrease in hormone-sensing and Wnt4-secreting luminal cells on the Wnt/Notch signaling pathways and the proliferation/differentiation potential in basal stem/progenitor cells.
Mentions: Having observed most of the expected functional consequences of a decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells from parous mice, we finally examined the possible cause of parity-induced reduction in canonical Wnt signaling and proliferation capacity in basal stem/progenitor cells. Because parity induced a greater than threefold decrease in Wnt ligand Wnt4 gene expression (Figure 2A, B), and Wnt4 is known to be secreted in response to progesterone by hormone-sensing luminal cells [46], thus inducing canonical Wnt signaling in mammary stem/progenitor cells [47], a parity-induced decrease in estrogen/progesterone-sensitive luminal cells could explain the overall decrease in Wnt signaling in mammary stem cells. This hypothesis is supported by the reduction in the proportion of luminal Sca1+ cells isolated from parous mice (Figure 1D) and by the demonstration that luminal Sca1+ cells are hormone receptor positive (Additional file 6) [17]. Furthermore, immunohistochemical analysis of mammary gland sections for estrogen receptor alpha (ERα) and its target progesterone receptor (PR) showed a twofold decrease in ERα- and a threefold decrease in PR-positive cells in parous compared with age-matched virgin control mice (Figure 6A, B). These results were additionally verified by qPCR in total mammary cell suspensions (Figure 6C). Notably, expression of the luminal marker Krt8 was similar in cell suspensions from parous and age-matched virgin control mice, supporting the conclusion of a specific decrease in hormone receptor-positive cells rather than a general cell loss after pregnancy. Furthermore, parity-induced reduction in progesterone-stimulated Wnt4 expression was independent of blood progesterone, because average blood progesterone concentrations were similar in parous mice and age-matched virgin control mice in estrus (see Additional file 9). Finally, supplementation of the culture medium with recombinant Wnt4 stimulated in vitro proliferation capacity of basal myoepithelial and basal stem/progenitor cells from parous mice by +138% ± 22% and +140% ± 17%, respectively (Figure 7A). In contrast, no significant effects of recombinant Wnt4 on the colony-formation capacity of luminal Sca1- (+3.7% ± 3.0%) and luminal Sca1+ cells from parous mice (-5.1% ± 2.9%) were observed. These data strongly suggest a causal relation between reduced number of luminal progesterone receptor-positive/Wnt4-secreting cells and decreased Wnt/Notch signaling and proliferation potential of basal stem/progenitor cells after early pregnancy (Figure 7B).

Bottom Line: Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3.This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo.As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Early pregnancy has a strong protective effect against breast cancer in humans and rodents, but the underlying mechanism is unknown. Because breast cancers are thought to arise from specific cell subpopulations of mammary epithelia, we studied the effect of parity on the transcriptome and the differentiation/proliferation potential of specific luminal and basal mammary cells in mice.

Methods: Mammary epithelial cell subpopulations (luminal Sca1-, luminal Sca1+, basal stem/progenitor, and basal myoepithelial cells) were isolated by flow cytometry from parous and age-matched virgin mice and examined by using a combination of unbiased genomics, bioinformatics, in vitro colony formation, and in vivo limiting dilution transplantation assays. Specific findings were further investigated with immunohistochemistry in entire glands of parous and age-matched virgin mice.

Results: Transcriptome analysis revealed an upregulation of differentiation genes and a marked decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells of parous mice. Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3. This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo. As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells. Because recombinant Wnt4 rescued the proliferation defect of basal stem/progenitor cells in vitro, reduced Wnt4 secretion appears to be causally related to parity-induced alterations of basal stem/progenitor cell properties in mice.

Conclusions: By revealing that parity induces differentiation and downregulates the Wnt/Notch signaling ratio and the in vitro and in vivo proliferation potential of basal stem/progenitor cells in mice, our study sheds light on the long-term consequences of an early pregnancy. Furthermore, it opens the door to future studies assessing whether inhibitors of the Wnt pathway may be used to mimic the parity-induced protective effect against breast cancer.

Show MeSH
Related in: MedlinePlus