This study demonstrates that a history of early pregnancy changes the gene-expression profiles and functional properties of mammary epithelial cell subpopulations in a cell subtype-specific fashion. Most important, the following parity-induced alterations were observed in mice: (a) an induction of differentiation and downregulation of the Wnt/Notch signaling ratio in basal stem/progenitor cells; (b) a decrease in the in vitro
and in vivo
proliferation potential of isolated basal stem/progenitor cells; (c) a selective downregulation of potentially tumorigenic biofunctions in the basal stem/progenitor cell subpopulation; (d) a reduction in estrogen- and progesterone-responsive and Wnt4-secreting luminal cells; and (e) a rescue of the proliferation defect in basal stem/progenitor cells in vitro
by recombinant Wnt4. The finding of a decreased Wnt/Notch signaling ratio provides direct experimental evidence for the hypothesis that early pregnancy changes the "genomic signature" of mammary stem/progenitor cells [7
], causing their differentiation and reducing their proliferation potential. Furthermore, the data indicate a novel causal relation between parity-induced reduction in hormone-sensing and Wnt4-secreting luminal cells and altered biofunctions in basal stem/progenitor cells.
The basal CD49fHigh
cells, as isolated in this study, are a subfraction of basal epithelial cells [19
]. Basal CD49fHigh
cells have been demonstrated previously to be enriched in mammary repopulating units (MRUs) (known as mammary stem cells (MaSCs)) [16
] and to correspond to the Lin-
epithelial cell subpopulation isolated by an alternative method [18
]. However, the isolated basal CD49fHigh
epithelial cell subpopulation represents a heterogeneous cell fraction containing, in addition to MaSCs, basal progenitor cells and possibly mature myoepithelial cells [45
]. Progenitor cells can be characterized in vitro
by their colony-formation capacity [22
], whereas MaSCs have traditionally been defined by their in vivo
regenerative capacity [16
]. The observed effects of parity on the in vitro
and in vivo
proliferation capacity of the CD49fHigh
cell subpopulation (Table ) suggests that both basal progenitor cells and basal MaSCs are the target of an early pregnancy within the mammary epithelium. Although the dramatic decrease in the in vitro
proliferation capacity of the CD49fHigh
cell subpopulation (Figure ) indicates a predominant effect of an early pregnancy on basal progenitor cells, the additional reduction in large in vivo
outgrowths (≥ 10% of fat pad filled) and the prevalence of rudimentary outgrowths (≥ 3% of fat pad filled) after parity (Table ) suggest that isolated basal MaSCs are also affected by an early pregnancy. Because basal mammary stem and progenitor cells are closely related and likely to be interdependent in their proliferation potentials, our data do not permit a definite discrimination between basal stem and progenitor cells as primary targets of pregnancy. Therefore, we adhered to the combined term basal stem/progenitor cells throughout this study.
We found the p53-p21 pathway to be enriched to a similar extent in all mammary epithelial cell subpopulations (Additional file 7
], and hence, although parity-caused induction of the p53-p21 pathway may explain the relatively modest decrease in in vitro
colony-formation potential of the luminal Sca1+
cell subpopulation (Figure ), it may contribute but cannot account for the almost complete proliferation block in basal stem/progenitor cells. The most prominent parity-induced alterations in gene expression in basal stem/progenitor cells were downregulation of the Wnt-signaling pathway, upregulation of the Notch-signaling pathway, and upregulation of differentiation genes. Decreased Wnt signaling in basal stem/progenitor cells from parous mice was verified on the protein level by measuring versican and nuclear β-catenin expression (Figures and ). Wnt signaling has been shown to promote long-term expansion of cultured Lin-
cells and to provide a competitive advantage in mammary gland reconstitution assays [43
]. The latter is especially true for the expression of the classic Wnt target Lgr5
], which was found in our study to be downregulated in the basal stem/progenitor cell subpopulation after parity. Notably, Wnt signaling inhibition was demonstrated to have an antiproliferative effect in CD29High
]. Furthermore, Notch signaling was observed to reduce in vitro
and in vivo
proliferation of CD29High
cells while promoting their differentiation [44
]. Because the differentiation processes of stem and progenitor cells in many organs and in several model systems are dependent on the Wnt/Notch signaling ratio [50
], an overall reduction in Wnt/Notch signaling ratio would be expected to have a dramatic antiproliferation and prodifferentiation effect in mammary basal stem/progenitor cells. This is exactly what we observed in the CD49fHigh
cell subpopulation of parous mice. Thereby, the overall conclusion of our study is strengthened by the fact that all assays used (that is, transcriptome analysis, bioinformatics transcription factor, and gene-enrichment analyses, in vitro
colony-forming assay, in vivo
transplantation assay, and immunohistochemistry) pointed into the same direction. Thus, analysis of specific mammary epithelial cell subpopulations allowed the discovery of a decrease in the Wnt/Notch signaling ratio, which so far is the only plausible explanation for the observed differentiation burst and dramatic proliferation block experienced by basal stem/progenitor cells of parous mice.
As possible explanations for the parity-induced decrease in Wnt signaling, we found a marked increase in the activity of the Wnt repressor TCF3 in basal stem/progenitor cells and a more than threefold reduction in expression of the secreted Wnt ligand Wnt4
in total mammary cells from parous mice. The latter corresponded to a similar decrease in Wnt4-secreting [46
] and estrogen and progesterone receptor-positive luminal cells. Notably, a similar decrease in progesterone receptor α-positive cells after parity has also been observed in human breast epithelium [51
]. Hence, mechanistically, early parity decreases the hormone responsiveness of the mammary gland in mice by decreasing the number of estrogen/progesterone receptor-positive luminal cells. This reduces the paracrine signaling cascade mediated by Wnt4, inducing TCF3-dependent repression [52
] and/or primary downregulation [47
] of canonical Wnt signaling and secondary (reactive) upregulation of Notch signaling in basal stem/progenitor cells.
As a final consequence, proliferation is repressed and basal stem/progenitor cells differentiate. This mechanistic model is supported by the ability of recombinant Wnt4 to rescue the proliferation defect of basal stem/progenitor cells from parous mice in vitro (Figure ).
Our findings in specific mammary epithelial cell subpopulations are in part consistent with and in part contradictory to studies in entire breasts/mammary glands, total mammary cells, or total mammary epithelial cells. With respect to the transcriptome analysis, our studies in total mammary cells agree with previous reports in the entire mammary glands [9
]. However, in intact mammary glands or total mammary cells, the additional presence of stromal and dominant epithelial cell subtypes might mask the detection of key signaling-pathway changes. Indeed, our study demonstrates that isolation of specific mammary epithelial cell subpopulations is a prerequisite for the detection of a decrease in Wnt/Notch signaling ratio in basal stem/progenitor cells. A similar masking effect by stromal and dominant epithelial cell subtypes (for example, strong clonogenic luminal Sca1-
cells) might also explain why a previous study with a similar early-pregnancy protocol did not observe a parity-induced reduction in in vitro
proliferation of total mixed mammary cells [20
]. Controversial results have also been reported with respect to the effect of parity on the in vivo
mammary-repopulating capacity. Hence, although Britt et al.
] found no effects of late pregnancy (9 weeks) on mammary-repopulating units (MRUs) in total mammary epithelial cells, Siwko et al.
] (early-pregnancy protocol) observed a parity-induced reduction in the mammary-repopulating capacity of total mixed mammary cells. With a similar early-pregnancy protocol and the same cut-off for mammary gland outgrowth (≥ 10% of fat pad filled), our findings in isolated mammary basal stem/progenitor cells appear consistent with the observations of Siwko et al
. However, given that our studies were performed with isolated mammary basal stem/progenitor cell subpopulations, our findings are not directly comparable with and neither confirm nor contradict previous studies using total mammary (epithelial) cells [20
]. Furthermore, the fact that the number of smaller outgrowths (≥ 3% of fat pad filled) was unchanged after parity suggests that MRUs survive after pregnancy despite their reduced reconstitution efficiency. This conclusion is in line with the recent demonstration that Wnt-responsive mammary epithelial stem cells persist after parity in Axin2
reporter mice [15
It is intriguing to speculate that marked growth inhibitory effects and the downregulation of canonical Wnt signaling in basal stem/progenitor cells account, at least in part, for the cancer-protective effect of early pregnancy. Increases in canonical Wnt signaling have been linked repeatedly to oncogenesis [53
]. Moreover, downregulation of the Wnt inhibitory protein Sfrp1 and overexpression of the Wnt target versican have been associated with carcinogenesis [55
]. In the transcriptome analysis reported here, Sfrp1
was upregulated and versican downregulated, thus supporting a parity-induced anticarcinogenic effect. Moreover, Li et al.
] showed that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells. Hence, the contribution of decreased canonical Wnt signaling in basal stem/progenitor cells to the cancer-protective effect of early pregnancy may be in conjunction with other tumor-suppressing mechanisms, such as parity-induced induction of p53 [58
]. Thereby, the increase in the TCF3 repressor activity in basal stem/progenitor cells is expected to elevate the threshold further for the activation of tumorigenic Wnt signaling [52
]. In addition to decreased Wnt signaling in basal stem/progenitor cells, decreased ERα- and PR-positive cells could also be a mechanism for the breast cancer-protective effect of an early pregnancy. This is especially relevant, given the specific protective effect of pregnancy against ER/PR-positive tumors [60
Furthermore, mammary epithelial cell differentiation per se
has been suggested to exert a breast cancer-protective effect. This has been challenged, however, by the observation that differentiation-causing agents such as placental lactogen and perphenazine failed to protect against carcinogenesis in rodents [3
]. Also, the hypothesis of a potential breast cancer-protective effect of mammary stromal cells [62
] is not supported by our study, because stromal cells exhibited by far the fewest parity-induced gene-expression changes. However, our stromal cell subpopulation was not homogenous and devoid of immune cells, which may have masked some parity-induced alterations. In any case, the parity-induced downregulation of the Wnt/Notch signaling ratio in basal stem/progenitor cells represents a possible important mechanism for the breast cancer-protective effect of early pregnancy.