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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.

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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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Parity reduces the progenitor potential of mammary epithelial cell subpopulations. (A) Representative images of individual wells with colonies formed by the specified cell subpopulations from age-matched virgin and parous mice. Scale bar, 4 mm. (B) Bar graph comparing the colony-forming capacities of myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells of age-matched virgin and parous mice. Data are from three independent experiments and represent the mean ± SEM of colonies per well; 18 wells were assessed per cell type. *P < 0.015, NS: not significant (P = 0.08), by using two-tailed unpaired Student t test. (C) Representative images for the immunophenotyping of 5-day-old colonies grown from myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells. The colonies were stained for luminal Krt18 (green) and basal Krt14 (red) expression. Hoechst 33342 (blue) was used to distinguish nuclei and to label feeder cells (negative control). Scale bar, 200 μm.
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Figure 5: Parity reduces the progenitor potential of mammary epithelial cell subpopulations. (A) Representative images of individual wells with colonies formed by the specified cell subpopulations from age-matched virgin and parous mice. Scale bar, 4 mm. (B) Bar graph comparing the colony-forming capacities of myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells of age-matched virgin and parous mice. Data are from three independent experiments and represent the mean ± SEM of colonies per well; 18 wells were assessed per cell type. *P < 0.015, NS: not significant (P = 0.08), by using two-tailed unpaired Student t test. (C) Representative images for the immunophenotyping of 5-day-old colonies grown from myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells. The colonies were stained for luminal Krt18 (green) and basal Krt14 (red) expression. Hoechst 33342 (blue) was used to distinguish nuclei and to label feeder cells (negative control). Scale bar, 200 μm.

Mentions: Because decreased Wnt signaling and increased Notch signaling have been shown to decrease in vitro and in vivo proliferation of basal stem/progenitor cells [43,44], we next assessed the in vitro colony-formation capacities of mammary epithelial cell subpopulations from parous and age-matched virgin mice (Figure 5A). In virgin control mice, luminal Sca1- cells had the highest colony-formation capacity, with an average of 107 colonies per well (Figure 5B). This strong clonogenic potential suggests a pronounced progenitor identity of luminal Sca1- cells and is consistent with previous observations in younger virgin mice [16,17]. A high colony-formation capacity was also observed for the basal stem/progenitor cells of virgin mice (63 colonies per well) (Figure 5B), which is consistent with the notion that CD49fHigh cells contain a high proportion of basal progenitor cells as well as putative mammary stem cells [45]. With the exception of luminal Sca1- cells, the colony-formation capacities of all epithelial cell subpopulations were lower in parous mice than in age-matched virgins (Figure 5B). Thereby, by far the most pronounced difference was observed in basal stem/progenitor cells (Figure 5B). A substantial decrease in the colony-formation capacity was also seen for the myoepithelial cell subpopulation, which also contains basal progenitor cells. Of note, basal stem/progenitor cells from parous mice did not die but remained as quiescent single cells or divided only once during 5 days of culture (Figure 5C).


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)

Parity reduces the progenitor potential of mammary epithelial cell subpopulations. (A) Representative images of individual wells with colonies formed by the specified cell subpopulations from age-matched virgin and parous mice. Scale bar, 4 mm. (B) Bar graph comparing the colony-forming capacities of myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells of age-matched virgin and parous mice. Data are from three independent experiments and represent the mean ± SEM of colonies per well; 18 wells were assessed per cell type. *P < 0.015, NS: not significant (P = 0.08), by using two-tailed unpaired Student t test. (C) Representative images for the immunophenotyping of 5-day-old colonies grown from myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells. The colonies were stained for luminal Krt18 (green) and basal Krt14 (red) expression. Hoechst 33342 (blue) was used to distinguish nuclei and to label feeder cells (negative control). Scale bar, 200 μm.
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Related In: Results  -  Collection

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Figure 5: Parity reduces the progenitor potential of mammary epithelial cell subpopulations. (A) Representative images of individual wells with colonies formed by the specified cell subpopulations from age-matched virgin and parous mice. Scale bar, 4 mm. (B) Bar graph comparing the colony-forming capacities of myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells of age-matched virgin and parous mice. Data are from three independent experiments and represent the mean ± SEM of colonies per well; 18 wells were assessed per cell type. *P < 0.015, NS: not significant (P = 0.08), by using two-tailed unpaired Student t test. (C) Representative images for the immunophenotyping of 5-day-old colonies grown from myoepithelial cells, basal stem/progenitor cells, luminal Sca1-, and luminal Sca1+ cells. The colonies were stained for luminal Krt18 (green) and basal Krt14 (red) expression. Hoechst 33342 (blue) was used to distinguish nuclei and to label feeder cells (negative control). Scale bar, 200 μm.
Mentions: Because decreased Wnt signaling and increased Notch signaling have been shown to decrease in vitro and in vivo proliferation of basal stem/progenitor cells [43,44], we next assessed the in vitro colony-formation capacities of mammary epithelial cell subpopulations from parous and age-matched virgin mice (Figure 5A). In virgin control mice, luminal Sca1- cells had the highest colony-formation capacity, with an average of 107 colonies per well (Figure 5B). This strong clonogenic potential suggests a pronounced progenitor identity of luminal Sca1- cells and is consistent with previous observations in younger virgin mice [16,17]. A high colony-formation capacity was also observed for the basal stem/progenitor cells of virgin mice (63 colonies per well) (Figure 5B), which is consistent with the notion that CD49fHigh cells contain a high proportion of basal progenitor cells as well as putative mammary stem cells [45]. With the exception of luminal Sca1- cells, the colony-formation capacities of all epithelial cell subpopulations were lower in parous mice than in age-matched virgins (Figure 5B). Thereby, by far the most pronounced difference was observed in basal stem/progenitor cells (Figure 5B). A substantial decrease in the colony-formation capacity was also seen for the myoepithelial cell subpopulation, which also contains basal progenitor cells. Of note, basal stem/progenitor cells from parous mice did not die but remained as quiescent single cells or divided only once during 5 days of culture (Figure 5C).

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