Limits...
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.

Show MeSH

Related in: MedlinePlus

The CD24/Sca1 and CD49fHigh/CD24 flow-cytometry profiles of parous and age-matched virgin mice are similar. (A) Schematic illustration of the cell-isolation strategy and representative flow-cytometry pseudocolor plots of mammary cells from age-matched virgin control mice. After depletion of CD45+ white blood cells, luminal and basal mammary epithelial cells were separated on the basis of CD24 and Sca1 expression. Further separation of basal cells into myoepithelial and basal stem/progenitor cell subpopulations was based on the expression of CD24 and CD49f. The isolated mammary epithelial cell subpopulations included luminal Sca1+ (CD24+HighSca1+) cells, luminal Sca1- (CD24+HighSca1-) cells, basal CD49fHigh (CD24+LowSca1-CD49fHigh) or basal stem/progenitor cells, and basal myoepithelial (CD24+LowSca1-CD49fLow) cells. (B) Outline of the mouse mating, parturition, weaning, and involution protocol. (C) Representative flow-cytometry pseudocolor plots of mammary cells from parous mice. The gates applied were the same as those for age-matched virgin controls. (D) Bar graph showing the distribution of mammary epithelial cell subpopulations comparing cells from parous with age-matched virgin control mice. Data represent the mean ± SEM of seven cell-isolation experiments with a minimum of 10 mice per experiment. The proportion of luminal Sca1+ cells was reduced by approximately 50% in parous mice (P = 0.02 with a two-tailed unpaired Student t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672662&req=5

Figure 1: The CD24/Sca1 and CD49fHigh/CD24 flow-cytometry profiles of parous and age-matched virgin mice are similar. (A) Schematic illustration of the cell-isolation strategy and representative flow-cytometry pseudocolor plots of mammary cells from age-matched virgin control mice. After depletion of CD45+ white blood cells, luminal and basal mammary epithelial cells were separated on the basis of CD24 and Sca1 expression. Further separation of basal cells into myoepithelial and basal stem/progenitor cell subpopulations was based on the expression of CD24 and CD49f. The isolated mammary epithelial cell subpopulations included luminal Sca1+ (CD24+HighSca1+) cells, luminal Sca1- (CD24+HighSca1-) cells, basal CD49fHigh (CD24+LowSca1-CD49fHigh) or basal stem/progenitor cells, and basal myoepithelial (CD24+LowSca1-CD49fLow) cells. (B) Outline of the mouse mating, parturition, weaning, and involution protocol. (C) Representative flow-cytometry pseudocolor plots of mammary cells from parous mice. The gates applied were the same as those for age-matched virgin controls. (D) Bar graph showing the distribution of mammary epithelial cell subpopulations comparing cells from parous with age-matched virgin control mice. Data represent the mean ± SEM of seven cell-isolation experiments with a minimum of 10 mice per experiment. The proportion of luminal Sca1+ cells was reduced by approximately 50% in parous mice (P = 0.02 with a two-tailed unpaired Student t test).

Mentions: Cells were labeled as previously described [26] by using the antibodies PE-Cy7-CD45 (1:33), FITC-CD24 (1:40), PE-CD49f (1:40), and APC-Sca1 (1:40). Detailed antibody information is given later. DAPI (0.2%, Invitrogen, Zug, Switzerland) was added 10 minutes before cell sorting (1:250). FACS was carried out on a MoFlo cell sorter (Becton Dickinson, Basel, Switzerland). Cells were gated based on their forward- and side-scatter profiles (FS Area and SS Area). A time-of-flight approach (pulse width) was used to exclude doublets and higher-order cell clumps. Dead cells (DAPI bright) and immune cells (CD45+) were gated out (see Additional file 1). The gate for basal stem/progenitor cells was set at the top 5% of CD49f-expressing cells, as described [19,21] (see Figure 1). Routine examination of the sorted mammary epithelial cell subpopulations showed a degree of purification higher than 95%.


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)

The CD24/Sca1 and CD49fHigh/CD24 flow-cytometry profiles of parous and age-matched virgin mice are similar. (A) Schematic illustration of the cell-isolation strategy and representative flow-cytometry pseudocolor plots of mammary cells from age-matched virgin control mice. After depletion of CD45+ white blood cells, luminal and basal mammary epithelial cells were separated on the basis of CD24 and Sca1 expression. Further separation of basal cells into myoepithelial and basal stem/progenitor cell subpopulations was based on the expression of CD24 and CD49f. The isolated mammary epithelial cell subpopulations included luminal Sca1+ (CD24+HighSca1+) cells, luminal Sca1- (CD24+HighSca1-) cells, basal CD49fHigh (CD24+LowSca1-CD49fHigh) or basal stem/progenitor cells, and basal myoepithelial (CD24+LowSca1-CD49fLow) cells. (B) Outline of the mouse mating, parturition, weaning, and involution protocol. (C) Representative flow-cytometry pseudocolor plots of mammary cells from parous mice. The gates applied were the same as those for age-matched virgin controls. (D) Bar graph showing the distribution of mammary epithelial cell subpopulations comparing cells from parous with age-matched virgin control mice. Data represent the mean ± SEM of seven cell-isolation experiments with a minimum of 10 mice per experiment. The proportion of luminal Sca1+ cells was reduced by approximately 50% in parous mice (P = 0.02 with a two-tailed unpaired Student t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672662&req=5

Figure 1: The CD24/Sca1 and CD49fHigh/CD24 flow-cytometry profiles of parous and age-matched virgin mice are similar. (A) Schematic illustration of the cell-isolation strategy and representative flow-cytometry pseudocolor plots of mammary cells from age-matched virgin control mice. After depletion of CD45+ white blood cells, luminal and basal mammary epithelial cells were separated on the basis of CD24 and Sca1 expression. Further separation of basal cells into myoepithelial and basal stem/progenitor cell subpopulations was based on the expression of CD24 and CD49f. The isolated mammary epithelial cell subpopulations included luminal Sca1+ (CD24+HighSca1+) cells, luminal Sca1- (CD24+HighSca1-) cells, basal CD49fHigh (CD24+LowSca1-CD49fHigh) or basal stem/progenitor cells, and basal myoepithelial (CD24+LowSca1-CD49fLow) cells. (B) Outline of the mouse mating, parturition, weaning, and involution protocol. (C) Representative flow-cytometry pseudocolor plots of mammary cells from parous mice. The gates applied were the same as those for age-matched virgin controls. (D) Bar graph showing the distribution of mammary epithelial cell subpopulations comparing cells from parous with age-matched virgin control mice. Data represent the mean ± SEM of seven cell-isolation experiments with a minimum of 10 mice per experiment. The proportion of luminal Sca1+ cells was reduced by approximately 50% in parous mice (P = 0.02 with a two-tailed unpaired Student t test).
Mentions: Cells were labeled as previously described [26] by using the antibodies PE-Cy7-CD45 (1:33), FITC-CD24 (1:40), PE-CD49f (1:40), and APC-Sca1 (1:40). Detailed antibody information is given later. DAPI (0.2%, Invitrogen, Zug, Switzerland) was added 10 minutes before cell sorting (1:250). FACS was carried out on a MoFlo cell sorter (Becton Dickinson, Basel, Switzerland). Cells were gated based on their forward- and side-scatter profiles (FS Area and SS Area). A time-of-flight approach (pulse width) was used to exclude doublets and higher-order cell clumps. Dead cells (DAPI bright) and immune cells (CD45+) were gated out (see Additional file 1). The gate for basal stem/progenitor cells was set at the top 5% of CD49f-expressing cells, as described [19,21] (see Figure 1). Routine examination of the sorted mammary epithelial cell subpopulations showed a degree of purification higher than 95%.

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