Review

How pregnancy at early age protects against breast cancer Fabienne Meier-Abt1,2 and Mohamed Bentires-Alj1 1

Mechanisms of Cancer, Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, CH-4058 Basel, Switzerland 2 Faculty of Science, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland

Pregnancy at an early age has a strong protective effect against breast cancer in humans and rodents. Postulated mechanisms underlying this phenomenon include alterations in the relative dynamics of hormone and growth factor-initiated cell fate-determining signaling pathways within the hierarchically organized mammary gland epithelium. Recent studies in epithelial cell subpopulations isolated from mouse and human mammary glands have shown that early pregnancy decreases the proportion of hormone receptor-positive cells and causes pronounced changes in gene expression as well as decreased proliferation in stem/progenitor cells. The changes include downregulation of Wnt and transforming growth factor b (TGFb) signaling. These new findings highlight the importance of cell–cell interactions within the mammary gland epithelium in modulating cancer risk and provide potential targets for breast cancer prevention strategies. Pregnancy and breast cancer Pregnancy is the most significant modifiable factor affecting the risk of breast cancer in women. Although a transient increase in breast cancer risk is observed immediately after parturition (see Glossary) in women over 25 years, the long-term consequences of pregnancy include a strong and life-long breast cancer protective effect [1,2]. Parity-induced tumor protection is more pronounced the earlier in life pregnancy occurs (Box 1). In the text, a pregnancy at an early age will be referred to as early pregnancy. Parity-induced protection against breast cancer is well established not only in humans but also in experimental rodent models. In rats and mice, carcinogen administration leads on average to 75% fewer incidences of mammary cancers in parous compared with virgin control animals [3]. A similar cancer protective effect in rodents results from hormonal mimicry of pregnancy, by treatment with estrogen and progesterone or human chorionic gonadotroCorresponding author: Bentires-Alj, M. ([email protected]). Keywords: pregnancy; breast development; mammary stem/progenitor cells; hormone receptors; Wnt signaling; TGFb signaling. 1471-4914/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2013.11.002

pin for at least 21 days [3]. Apart from carcinogen-induced mammary carcinogenesis, such hormone treatment was also shown to provide protection in two different genetically engineered mouse models of breast cancer: (i) the p53null mammary transplant model; and (ii) the MMTV-Her2/ Neu transgenic mouse model [4]. Because early pregnancy, or its hormonal mimicry, induces long-lasting protection against mammary cancers in rodents, the latter serve as a valid experimental system for the human paradigm [3]. Glossary Basal: position of a cell adjacent to the basement membrane (versus luminal). BRCA1/BRCA2: breast cancer 1, early onset/breast cancer 2, early onset. BRCA1/2 are tumor suppressor genes. Carriers with loss-of-function mutations have a markedly increased breast cancer risk. Early pregnancy: pregnancy at an early age. Hormonal mimicry of pregnancy: the act of artificially imitating the hormonal milieu of pregnancy by administering hormones. Hormone replacement therapy: a therapy in which patients receive hormones to supplement or substitute naturally occurring hormones. Involution: a process that removes the milk-producing epithelial cells at weaning. Luminal: position of a cell adjacent to the lumen (versus basal). Mammary epithelial cell surface markers: CD24, cluster of differentiation 24 or heat stable antigen CD24; SCA1, stem cell antigen1; CD29, b1-integrin; CD49f, a6-integrin. These markers allow the isolation of distinct mammary epithelial cell subpopulations by FACS. Mammary repopulating units (MRUs): cells capable of reconstituting a deepithelialized mouse mammary fat pad after transplantation, typically thought of as mammary cells with stem cell properties. MMTV-Her2/Neu transgenic mouse model: these transgenic mice express the activated rat Erbb2 (Her2/c-neu) oncogene under the direction of the mouse mammary tumor virus promoter. Multipotent: able to form both luminal and basal epithelial cells. Notch signaling: the Notch signaling pathway involves Notch receptors (Notch 1–4), their ligands Jagged1/2 and Delta-like1/3/4, the Notch intracellular domain (NICD), the transcription factor RBP-J, and the mastermind-like (MAML) family members of coactivators. Activation of distinct Notch signaling pathways can inhibit cell proliferation and induce cell differentiation. Nulliparous (adjective): synonymous with ‘not’ having gone through pregnancy. Parity (noun)/parous (adjective): synonymous with having gone through pregnancy. Parturition: birth or the process of bringing forth offspring. Terminal ductal lobuloalveolar units (TDLUs): the fundamental developmental units in mature mammary tissue which give rise to milk-producing structures during pregnancy. Terminal end buds (TEBs): the structures at the ends of developing ducts that lead the process of ductal growth during puberty. Triple-negative breast cancers: breast cancers which do not express the genes for ER, PR, and human epidermal growth factor receptor 2 (Her2/neu). Wnt signaling: int/Wingless signaling. Best characterized is the canonical Wnt signaling pathway that involves seven-pass-transmembrane-spanning frizzled (FZD) receptors, coreceptors of the low density lipoprotein (LDL)-receptorrelated proteins LRP5/6, b-catenin, glycogen synthase kinase-b (GSK-3b), axin, and adenomatous polyposis coli (APC). Activation of canonical Wnt signaling stimulates cell proliferation and increases carcinogenic potential.

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Box 1. Age at pregnancy and risk of breast cancer in humans An extensive body of epidemiological studies has established a strong and life-long breast cancer protective effect of early full-term pregnancy in humans [1,2]. This protective effect is at least 50% for a pregnancy occurring before the age of 20 years, meaning that women which have undergone an early pregnancy develop 50% fewer cancers compared with nulliparous women. Interestingly, pregnancy-induced breast cancer protection is negligible for first full-term pregnancies between the ages of 30 and 34 and reverses to an overall increased risk of mammary tumors for first pregnancies after the age of 35 years. Furthermore, before the protective effect of pregnancy becomes apparent, there is an initial increase in breast cancer risk immediately after parturition in women over 25 years. This transient and pregnancy-associated elevation in breast cancer risk is most pronounced in women older than 30 years and may account at least in part for the overall increase in breast cancer risk observed in women

older than 35 years at first full-term pregnancy [1,2,94,95] (Figure I). In addition to early pregnancy, multiple pregnancies and breastfeeding also decrease breast cancer risk, although to a relatively small degree; the time of breastfeeding is inversely associated with breast carcinoma, and each additional pregnancy increases protection against breast cancer a further 10–13% [92,96]. Epidemiological data for breast cancer subtypes shows that parity specifically protects against ER- and PR-positive (ER+/PR+) breast cancer, whereas neither parity nor age at first birth affects the risk of ER–/PR– breast cancers [97–99]. A less pronounced or even negligible protective effect of early pregnancy has been found for the BRCA1 and BRCA2 familial breast cancers [90,91]. The data are less definitive with respect to breastfeeding versus tumor subtype, but recent studies have reported a protective effect of breastfeeding against luminal [99] and triplenegative breast cancers [100].

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Figure I. Effect of pregnancy and age at first birth on breast cancer risk in humans. Schematic illustration demonstrating that: (i) early pregnancy decreases breast cancer risk in the long term; (ii) the breast cancer protective effect of pregnancy is greater the earlier the pregnancy has occurred; (iii) pregnancy leads to a transient increase in breast cancer risk following parturition; and (iv) pregnancy-associated increase in breast cancer risk becomes more pronounced with increasing age at first pregnancy. The figure represents a qualitative summary adapted from several epidemiological studies [1,2,94,95] and highlights the principal relationship between age at first pregnancy and breast cancer risk.

Despite the well-established breast cancer protective effect of an early full-term pregnancy in both humans and rodent species, the molecular and cellular mechanisms underlying this phenomenon remain unclear. The prevailing hypotheses include pregnancy-induced persistent alterations in serum hormone levels, growth factors, the stromal composition of the mammary gland and/or hormone responsiveness, and the cell fates of distinct mammary epithelial cell subpopulations [5,6]. Notably, all postulated mechanisms include hormone-induced alterations in the dynamics of intra- and intercellular signaling cascades within the mammary gland [3]. The present review summarizes recent progress in this field and highlights new findings on early pregnancy-induced alterations in hormone-dependent cell fate-signaling pathways in mammary epithelial progenitor and stem cells, which are considered to be potential cells of origin for breast carcinomas [7]. Mammary gland development The mammary gland is unique in that it develops largely postnatally. Before puberty, the mammary gland contains only a rudimentary ductal system embedded in specialized stroma, known in mice as the mammary fat pad. With the 144

onset of puberty (3 weeks in mice, 9–12 years in humans), a network of ducts begins to grow from specialized structures described as terminal end buds (TEBs) in mice, and is also observed in humans (Figure 1). The ductal system continues to grow after sexual maturity (5 weeks in mice, 11– 14 years in humans) and reaches its full dimensions at approximately 8 weeks of age in mice, and 18–24 years in humans [6,8]. The mature virgin mammary gland consists of an extensive ductal network and numerous budding structures, known as alveolar buds in mice and terminal ductal lobuloalveolar units (TDLUs) in humans [5]. During pregnancy, in a second stage of postnatal mammary gland development, ductal branches of alveolar buds or TDLUs expand and differentiate into milk-producing structures, the lobular alveoli, during lactation. When lactation ceases, the mammary gland regresses to a virgin-like state in a process called involution. This cycle of alveolar bud/TDLU expansion, differentiation into lobular alveoli, and subsequent involution is repeated in following pregnancies. Postnatal mammary gland development is under the control of hormonal, for example, estrogen, progesterone, growth hormone (GH), prolactin (PRL), and cell fatedetermining signaling pathways (Figure 1). During puber-

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Figure 1. Simplified schematic of mammary gland development. Postnatal mammary gland development occurs in several stages that are regulated by distinct hormonal and cell fate-determining signaling pathways. The estrogen–EGF–FGF and GH–IGF-1 signaling cascades are involved in pubertal ductal morphogenesis, whereas the progesterone–Wnt and progesterone–RANKL signaling cascades promote ductal side branching and alveolar morphogenesis in mature mammary glands. Prolactin–IGF-2 signaling leads to final lactogenic differentiation of the mammary gland during pregnancy, and local Stat3, FasL, TGFb, and macrophage signaling is important for mammary gland involution at the cessation of lactation. The effect of progesterone on side branching in the cycling nulliparous mouse is not depicted. Ducts are dark brown. Ductal branches are light brown. TEBs are light green. Alveolar structures and lobular alveoli are dark green. Abbreviations: TEB, terminal end bud; LN, lymph node; EGF(R), epidermal growth factor (receptor); FGF, fibroblast growth factor; GH, growth hormone; IGF-1/2, insulin-like growth factor-1/2; Wnt, int/Wingless; RANKL, receptor activator for nuclear factor k B ligand; Stat3, signal transducer and activator of transcription 3; FasL, Fas ligand; TGFb, transforming growth factor b.

ty, ovarian estrogens are the major mitogenic factors for ductal morphogenesis (elongation/bifurcation). Accordingly, ovariectomy in mice at 5 weeks of age leads to failure of mammary ductal network development [9,10]. This effect can be countered by implantation of 17-b-estradiol pellets into the mammary glands of mice [11]. Mechanistically, estrogen signals primarily via estrogen receptor a (ERa) on mammary epithelial cells, which, in a paracrine manner involving stromal cells, activate downstream signaling pathways, such as the epidermal growth factor (EGF)– fibroblastic growth factor (FGF) signaling cascade, and mediate ductal morphogenesis (Figures 1 and 2). In addition to estrogens, peripubertal ductal morphogenesis also depends on GH and its downstream effector insulin-like growth factor-1 (IGF-1) [10]. This is demonstrated by the failure of estrogen to rescue mammary development in hypophysectomized, ovariectomized rats, and by its ability to restore duct formation if GH or IGF-1 is coadministered [12,13]. In the cycling mouse and during pregnancy, ductal side branching and alveolar morphogenesis in mature mammary glands require progesterone signaling (Figure 1). In line with this, deletion of both progesterone receptor (PR) isoforms, PR-A and PR-B, leads to failure of tertiary side branching and lobuloalveolar development in adult and pregnant mice [14]. Selective knockout experiments indicate that PR-B is essential and sufficient for these effects [15], whereas reciprocal transplantation studies support the importance of epithelial PR expression for lobuloalveolar development and, possibly, of stromal PR for ductal growth [16,17]. Two mediators of

progesterone paracrine signaling have been defined in the mammary gland: the tumor necrosis factor (TNF) family member receptor activator for nuclear factor kB ligand (RANKL) and the int/Wingless (Wnt) ligand, Wnt4. Both RANKL and Wnt4 colocalize with PRpositive luminal epithelial cells adjacent to proliferating cells, and their expression in mice is regulated by progesterone [18,19]. Moreover, deletion of RANKL or Wnt4 results in impaired pregnancy-induced side branching and alveogenesis in mice [18,20], whereas their ectopic expression causes tertiary side branching in the absence of pregnancy [21,22]. Final lactogenic differentiation of the mammary gland is under the control of PRL [10] (Figure 1). Transplantation experiments of PRL receptor (PrlR)-deficient epithelium demonstrate that PRL signaling is required for alveolar development and lactogenesis during late pregnancy, but not for ductal outgrowth and side branching [23]. Mechanistically, PRL induces mammary lactogenic differentiation via the downstream activation of Janus kinase 2 (Jak2) and signal transducer and activator of transcription 5 (STAT5) [24,25], and possibly also by upregulating IGF-2 and cyclin D1 expression [26,27]. Involution of the mammary gland to its virgin-like stage after lactation is under the control of signal transducer and activator of transcription 3 (Stat3), Fas ligand (FasL), transforming growth factor b (TGFb) signaling, and macrophages [28,29]. The latter are essential for epithelial cell death during mammary gland involution in mice, possibly involving paracrine signaling between macrophages and mammary epithelial cells [29]. TGFb1 signaling limits 145

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Figure 2. Mammary gland structure, epithelial cell hierarchy, and hormone-dependent intercellular signaling pathways. (A) The mammary gland consists of luminal and basal cell compartments that are separated from the surrounding stromal tissue by a basement membrane. Differentiated milk-producing luminal cells face the ductal lumen and are closely associated with contractile basal myoepithelial cells that serve in milk ejection. The entire epithelium is surrounded by a basement membrane and by stromal cells. (B) In the adult gland, differentiated luminal and basal myoepithelial cells develop from corresponding progenitor cells. Differentiated luminal cells comprise estrogen- and progesterone receptor-positive cells that relay the effects of estrogen and progesterone to other mammary epithelial cell types [8,35]. Differentiated myoepithelial cells constitute the contractile units of the mammary gland active in milk ejection. Luminal and basal progenitor cells are precursors of differentiated luminal and basal myoepithelial cells. Several biomarkers have been identified that distinguish the different mammary epithelial cell subpopulations. In mice, keratin 18 (KRT18) labels luminal cells, whereas keratin 14 (KRT14) labels basal cells. Similarly, CD24 expression discriminates between luminal and basal cells. SCA1 expression characterizes luminal hormone-sensing cells, whereas CD49f expression is a property of basal stem/progenitor cells. (C) Estrogen binds to ERa in luminal epithelial cells and activates the expression of the EGF family member AREG. AREG is active in paracrine signaling, binding to EGFRs in stromal cells and inducing the stromal release of FGFs. Stromal FGFs are thought to stimulate basal stem cells to proliferate, ultimately leading to ductal growth [40]. (D) Progesterone binds to PRs in luminal epithelial cells and activates the expression and secretion of the Wnt ligand Wnt4. Progesterone-stimulated Wnt4 acts on basal mammary stem and/or progenitor cells, promoting their proliferation and leading to tertiary side branching and lobuloalveogenesis. Abbreviations: CD24, cluster of differentiation 24 or heat stable antigen CD24; SCA1, stem cell antigen1; CD49f, a6-integrin; KRT, keratin; ER, estrogen receptor; ERa, estrogen receptor alpha; PR, progesterone receptor; AREG, amphiregulin; EGF(R), epidermal growth factor (receptor); FGF(R), fibroblast growth factor (receptor); FZD, frizzled receptor; Wnt, int/Wingless.

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Review mammary epithelial cell expansion by inhibiting cell proliferation [30] using the non-canonical Wnt ligand Wnt5a as a mediator in mice [31]. Active TGFb is thereby confined to the luminal cell compartment, yet the inhibitory effect of TGFb1–Wnt5a on cell proliferation extends to basal stem and/or progenitor cells [32]. These findings exemplify the importance of the intense interplay within the hierarchically organized mammary epithelial cell subpopulations between different hormone and growth factorinitiated cell fate-determining signaling pathways. Cell hierarchy in the mammary gland epithelium The mammary epithelium in humans and mice is organized hierarchically (Figure 2), with distinct populations of differentiated luminal and basal (myoepithelial) epithelial cells, luminal and basal progenitors, and mammary stem cells [7,33]. Mammary stem cells are long-lived and the source of various cell types; they give rise to new mammary epithelial tissue during puberty and pregnancy [7]. Traditionally, mammary cells with stem cell properties have been defined functionally by their potential to reconstitute complete mammary epithelium when transplanted into epithelium-free mouse mammary fat pads [33]. Such cells, also known as mammary repopulating units (MRUs), are basally located and multipotent when transplanted into cleared fat pads [34–36]. In the context of the intact adult mammary gland, recent lineage tracing experiments have indicated the unipotent nature of basal mammary stem cells and the existence of additional unipotent luminal mammary stem cells. In these studies, multipotent mammary stem cells were observed in the embryonic and possibly in the pregnant gland [37,38]. Distinct mammary epithelial cell subpopulations can be isolated from both human breast tissue and mouse mammary glands by fluorescent-activated cell sorting (FACS) using specific cell surface markers [34–36,39]. In mice, expression of heat stable antigen CD24, stem cell antigen1 (SCA1) and b1-integrin (CD29) or a6-integrin (CD49f), allows the separation of SCA1+ luminal (CD24+High SCA1+), SCA1– luminal (CD24+High SCA1–), basal myoepithelial (CD24+Low SCA1– CD49fLow), and basal stem/ progenitor (CD24+Low SCA1– CD49fHigh) cells. SCA1+ luminal cells comprise ER-positive cells and display limited in vitro and in vivo growth potential, suggesting the presence of many differentiated cells [35]. Similarly, myoepithelial cells show little in vitro and in vivo growth capacity. They express a-smooth muscle actin (a-SMA) and are thought to represent basal differentiated cells [39]. By contrast, SCA1– luminal cells give rise to numerous large colonies in vitro but have limited in vivo outgrowth capability. By exhibiting the potential to grow in the presence of specific stimulants in vitro but not in the more restricted environment of an epithelium-free mammary fat pad in vivo, these cells show the classic phenotype of progenitor cells [36,39]. Finally, isolated CD49fHigh basal stem/progenitor cells are highly enriched in MRUs and show strong in vivo and moderate in vitro growth potential [34–36]. These functional characteristics indicate the strong enrichment of basal mammary cells with stem and progenitor cell properties in this cell subpopulation. The isolation of specific mammary epithelial cell subpopu-

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lations allows the study of cell subtype-specific properties and, thus, of intercellular communication in the mammary gland. Cell–cell interactions and intercellular signaling pathways are of crucial importance for the regulation of proliferation and the differentiation of specific mammary epithelial cell subtypes. In response to estrogen signaling in luminal hormone-sensing cells, the EGF family member amphiregulin (AREG) is secreted by luminal cells and binds to EGFR in stromal cells. This stimulates the release of FGFs by stromal cells, which act on FGF receptors of basal cells with stem and progenitor cell properties causing them to proliferate (Figure 2C) [40]. In response to progesterone signaling in luminal hormone-sensing cells, the Wnt ligand Wnt4 is secreted by luminal cells. Wnt4 in turn binds to frizzled receptors on basal cells with stem and progenitor cell properties, which stimulates canonical Wnt signaling and cell proliferation (Figure 2D) [41]. Whereas failure of such intercellular signaling cascades promoting cell proliferation results in developmental defects [18,42], overactivity of the same pathways is associated with carcinogenesis [43]. Breast carcinogenesis and cell fate-determining Wnt, Notch, and TGFb signaling pathways Of the main signaling pathways determining cell fate in the mammary gland, aberrantly increased Wnt signaling (Box 2) is an important factor in mammary oncogenesis [7]. Transgenes encoding components of the Wnt signaling pathway target undifferentiated mammary progenitor cells for carcinogenesis in mice [43,44] and render them resistant to radiation therapy in mice [45]. The connection between Wnt signaling and breast carcinogenesis is further underscored by the activation of the Wnt/b-catenin pathway following knockdown of the tumor suppressor gene phosphatase and tensin homolog (PTEN) in human breast cells [46], and by the downregulation of the secreted Wnt inhibitor secreted frizzled-related protein1 (Sfrp1) found in most invasive human breast carcinomas [47]. Unlike Wnt signaling, which has tumor-promoting effects in the mammary gland, the influence of Notch and TGFb signaling is equivocal; oncogenic as well as tumor-suppressing properties have been observed for both pathways. The reported results of altered Notch signaling in human breast tumor tissue and its prognostic value are ambiguous [48,49]. High Notch2 mRNA expression found to be associated with a good clinical outcome is in line with a tumor-suppressive effect of Notch signaling [50], and ectopic expression of the active intracellular domain of Notch2 has been demonstrated to reduce growth and enhance apoptosis of basal-like breast cancer cells [51]. Interestingly, recent evidence suggests that the tumorsuppressive activity of Notch signaling may be due in part to counteracting WNT/b-catenin signaling [52]. In support of an oncogenic activity, positive associations have been observed for Notch2 and HER2 expression in invasive human breast cancers [53]. Furthermore, transgenic mice expressing constitutively active intracellular domains of the paralogs Notch1 or Notch3 in mammary epithelium formed mammary tumors [54], and Notch1 inhibition resulted in mammary tumor regression in transgenic 147

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Box 2. Wnt and Notch signaling as cell fate-determining signaling pathways Wnt signaling Wingless related protein (Wnt) signaling is central to cell fate decisions and stem cell homeostasis in many species and organs. Wnt ligands initiate receptor-mediated signaling cascades, of which the Wnt/b-catenin-dependent or ‘canonical’ pathway is best characterized [101]. Canonical Wnt signaling involves the interaction of Wnt ligands with seven-pass-transmembrane-spanning FZD receptors and with coreceptors of the LDL-receptor-related protein family (LRP5/6). Binding of Wnt ligands to the receptor complex prevents destruction of b-catenin by a degradation complex containing GSK3b, axin, and APC. This leads to b-catenin accumulation in the nucleus and interaction with the nuclear lymphoid enhancer factor/T cell specific transcription factor (LEF/TCF) family of transcription factors, resulting in expression of the respective target genes [101]. The identity of the target genes and thus also the effects on cell fate are tissue- and cell type-specific [102]. Of the Wnt proteins expressed in the mammary gland, specific roles have been defined for Wnt4 and Wnt5a. Whereas Wnt5a appears to regulate pubertal ductal morphogenesis [31], Wnt4 acts downstream of progesterone, initiating tertiary side branching during adulthood and early to midpregnancy. Definitive evidence for a role of Wnt/b-catenin signaling in mammary epithelial cell fate decisions and self-renewal properties is provided by Wnt coreceptor LRP5/6 knockout studies [103,104], administration of exogenous Wnt proteins to mammary basal stem/progenitor cells [105], and lineage tracing experiments for Wnt-responsive mammary epithelial cells [37]. Notch signaling Similar to Wnt signaling, Notch signaling is involved in many cell fate decisions during development. The Notch gene encodes a transmembrane receptor with an intracellular domain that is cleaved off upon specific ligand binding. In mammals, four Notch receptors (Notch1–4) and five transmembrane ligands (Jagged1, Jagged2, Delta-like1, Delta-like3, and Delta-like4) have been identified and characterized. Upon cleavage, the Notch intracellular domain (NICD) translocates to the nucleus and binds the recombination signal binding protein for immunoglobulin k J region (RBP-J) transcription factor as well as mastermind-like (MAML) family members of coactivators, thus activating Notch target gene expression [106]. In the mammary gland, the Notch pathway regulates stem and progenitor cell activity and commits mammary stem cells to the luminal cell lineage in both humans and mice [107,108].

mouse models [55]. Complexity with respect to the effects of Notch signaling may arise from the existence of four Notch paralogs in mammals (Notch1, Notch2, Notch3, Notch4), which have redundant but also distinct functions [56]. For example, overexpression of the active intracellular domain of Notch4 increased proliferation of the same basal-like breast cancer cells, which were inhibited by the intracellular domain of Notch2 [51]. Together, the data suggest cell type- and paralog-specific effects of Notch signaling in carcinogenesis, but further studies are needed to define more precisely the effects of distinct Notch paralogs on specific mammary epithelial cell subtypes. Similar to Notch signaling, TGFb signaling also has apparently paradoxical effects in mammary carcinogenesis. TGFb influences tumor resistance and represses carcinogenesis by mediating cell cycle arrest in normal cells [57], by inhibiting telomerase activity [58] and by inducing apoptosis [59]. In line with a tumor-suppressing role of TGFb signaling, conditional deletion of TGFb receptor type II (TbRII) in mammary epithelial cells that express an oncogene has been shown to shorten tumor latency and 148

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result in a 5-fold increase in lung metastases in mice when compared with the same cells with intact TGFb signaling [60]. By contrast, TGFb promotes epithelial-to-mesenchymal transition (EMT) and induces metastatic and invasive properties in tumor cells resistant to its tumor-suppressing effects [61]. Hence, signaling pathways that are essential for normal mammary gland biology may also play crucial roles in mammary gland carcinogenesis and, thus, represent potential targets for breast cancer protection. Mechanisms for the protection against breast cancer afforded by early pregnancy Hypotheses about the breast cancer protective effect of early full-term pregnancy or its hormonal mimicry cite both cell non-autonomous and cell autonomous mechanisms. Most probably, the individual mechanisms are not mutually exclusive and the full protective effect results from a combination of several processes. As crucial parity-induced alterations resulting in a decreased propensity for breast carcinogenesis, cell nonautonomous mechanisms potentially involve persistent changes in circulating hormones and/or changes in the stromal composition of the mammary gland [3,62,63]. Owing to diurnal, cyclical, and age-dependent variation, studies of hormone levels require especially large cohorts in humans and/or efficient control measures in rodents. Whereas no clear or reproducible changes in estrogen and progesterone hormone levels are found after pregnancy, PRL levels appear to decrease at least transiently in parous women, a finding that has been reproduced in some but not all rodent studies [63–65]. Moreover, the GH–IGF1 axis appears to be persistently suppressed after pregnancy in rats [63]. Interestingly, diminished PRL and GH secretion induces regression of mammary tumors in rats [66], and virgin GH-deficient rats are refractory to mammary carcinogenesis [67], whereas increased levels of PRL and GH or IGF-1 have been associated with an elevated incidence of mammary carcinogenesis in several studies [68,69]. Such findings suggest a possible effect of PRL and GH in parity-induced breast cancer protection. Furthermore, long-lasting alterations in mammary stroma extracellular matrix (ECM) composition have been observed upon parity in rodent mammary glands [62]. Altered collagen organization in matrix of parous mice thereby appears to impede phenotypes associated with tumor growth and invasion [70]. With respect to cell autonomous processes, hypotheses about parity-induced breast cancer protection note changes in the differentiation state and hormone responsiveness of the mammary gland, or alterations in cell fates of specific mammary epithelial cell subpopulations [3,5,6]. In the case of differentiation, it has been postulated that terminal differentiation of the mammary gland induced by pregnancy or pregnancy hormones removes cancer-prone cells, thereby decreasing the susceptibility of the gland to cancer [71]. This hypothesis is supported by studies of genome-wide expression profiles in lobular breast tissues of women or entire mammary glands of rodents that show a clear increase in the expression of differentiation genes in tissues from parous individuals [72,73]. However, although an attractive hypothesis, differentiation per se cannot

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explain all observations, given that neither the PRL-like molecule placental lactogen nor the dopamine receptor antagonist perphenazine protect against mammary carcinogenesis, even though both cause the mammary gland to differentiate [3,74]. Regarding the responsiveness of the mammary gland to reproductive hormones, a decrease in the ability of the mammary gland to sense estrogen may underlie the protective effect of an early pregnancy [5]. This theory is consistent with the observation that increased cumulative dosage of reproductive hormones, for example, by early menarche, late menopause, or progestin-containing hormone replacement therapy, increases the risk of breast carcinogenesis [75]. By contrast, decreasing hormone exposure by oophorectomy reduces mammary cancer risk in dogs [76]. Interestingly, ER-positive estrogen-sensing cells are known to increase in number with age and cancerous progression [77,78], whereas PR-A-positive progesteronesensing cells have been reported to be relatively low in parous women [79], and the expression of both ERa and PR was shown to be decreased in women after pregnancy [80]. These findings underscore a possible direct relationship between mammary gland hormone responsiveness and parity-induced breast cancer protection. The most hotly debated theory regarding the protective effect of an early pregnancy on breast cancer proposes a parity-induced change in the fate of specific mammary epithelial cells. According to this hypothesis, the hormonal environment of pregnancy alters the developmental fate of a subpopulation of mammary epithelial cells by inducing persistent changes in signaling pathways, growth factors, and/or other regulatory molecules. These changes reduce the proliferation potential of the subpopulation and render it relatively resistant to carcinogenesis, whereas the capacity to form complete differentiated lobular structures during a subsequent pregnancy is maintained [3]. In line with this theory, mammary cells from hormone-treated animals exhibit a block in carcinogen-induced proliferation [81]. Furthermore, a novel mammary epithelial cell population, termed parity-identified mammary epithelial cells (PI-MECs), has been detected in differentiating cells during pregnancy [82] and was found to exhibit the features of

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stem and/or progenitor cells [83]. Because the longevity and self-renewing property of stem/progenitor cells make this population a prime target for transformation and carcinogenesis [84,85], a molecular switch in mammary cells with stem cell properties may explain the breast cancer protective effect of early pregnancy [6]. Notably, studies in mice have addressed the issue of stem cell numbers in parous compared with virgin mammary glands by transplanting total mammary (epithelial) cells but have produced conflicting results. One study with mice mated at 5 weeks observed a parity-induced reduction in mammary repopulating cells [86], whereas another study with mice mated at 9 weeks found no effect of pregnancy on the mammary stem cell population [87]. Such discrepancies might be explained by the different mating protocols used and/or by transplantation of unfractionated total mammary (epithelial) cells, in which the presence of other dominant epithelial cell types might mask or even counteract subtle alterations in critical stem cell properties. For these reasons, more recent studies have addressed the cell fate hypothesis more directly in mice and humans by examining distinct mammary epithelial cell subpopulations from parous and nulliparous individuals. One study found that early pregnancy changes the dynamics and cell fate of specific mammary epithelial cell subtypes by inducing cell subtype-specific alterations in gene expression profiles, proliferation capacities, and differentiation potentials [88]. Most important, early pregnancy induced a decrease in the Wnt/Notch signaling ratio in the basal mammary stem/progenitor cell subpopulation. This alteration of cell fate-determining signaling pathways was accompanied by a more differentiated phenotype and by a decrease in the in vitro and in vivo proliferation potentials. Furthermore, early pregnancy reduced the response of the mammary gland to progesterone signaling through a decrease in the proportion of estrogen- and progesterone receptor-positive luminal cells. This resulted in reduced expression of the progesterone target Wnt4 despite constant progesterone plasma levels (Figure 3). Thus, a decrease in Wnt4 expression may well explain parity-induced reduction in Wnt signaling and associated proliferation failure in basal stem/progenitor cells, especially because

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Figure 3. Cascade of early pregnancy-induced molecular and cellular alterations. Early pregnancy leads to a decrease in the proportion of progesterone receptor (PR) positive cells (red arrow), which reduces expression of the Wnt ligand Wnt4 (yellow arrow). This in turn decreases Wnt signaling in basal stem and/or progenitor cells (blue arrow), reducing their proliferation potential and inducing a differentiated phenotype [88]. Abbreviations: PR, progesterone receptor; Wnt, int/Wingless; FZD, frizzled receptor.

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Review recombinant Wnt4 rescues the parity-induced proliferation defects of this cell subpopulation. Hence, the findings suggest a direct link between reduced numbers of hormone receptor-positive luminal cells and decreased Wnt signaling, as well as reduced proliferation potential and increased differentiation phenotype of basal mammary stem and/or progenitor cells after pregnancy. Whether early pregnancy-induced reduction in the proportion of hormone receptor-positive luminal cells and decrease in Wnt signaling in mice are of life-long duration and not induced by late pregnancy is currently under investigation. A further study investigating the effect of parity on human mammary epithelial cell fate found a pregnancyinduced decrease in the number of hormone receptorpositive cells as well as in the expression of stem celland development-related genes in CD44+/CD24–/CD10– (hereafter called CD44+) breast progenitor cells. In addition, decreases in the expression of the cell cycle regulator p27 (encoded by CDKN1B), in the number of p27+ cells (most of which were ER+) and in proliferation were observed in mammary epithelial cells from parous women [89]. Thereby, the observed parity-induced gene expression changes in CD44+ progenitor cells included a decrease in quiescence-inducing TGFb signaling and a downregulation of developmental and protumorigenic pathways such as cytoskeleton remodeling, DNA methylation, and Wnt signaling. Notably, an overlap of TGFb signaling and p27 was observed in breast tissue. Using breast explant cultures, a pronounced decrease in the frequency of p27+ cells and increase in bromodeoxyuridine-positive (BrdU+) cells (indicating cell proliferation) was observed upon inhibition of the TGFb receptor, suggesting that TGFb acts via p27 in inducing quiescence of these progenitor cells. These latter observations reinforce the significance of alterations in the cell fate of specific mammary epithelial cells in the protective effect of pregnancy on breast cancer. Importantly, a decrease in Wnt4 expression was observed in mammary epithelial cells from both parous women [89] and parous mice [88], highlighting the possible involvement of decreased Wnt signaling in mediating the proliferation-inhibiting effects of early pregnancy. In addition, decreased TGFb activity and the subsequent decline in p27, which was thought to regulate the proliferation of hormone-responsive cells (p27+ cells) with proliferative potential, has been suggested as a further potential mechanism of the protective effect of pregnancy [89]. Conceivably, as in mice, human p27+/ER+ cells may act as E and P ‘sensor cells’ and stimulate proliferation of other breast subpopulations in a paracrine manner. In assessments of the frequency of p27+ cells in parous and nulliparous control women, and also in BRCA1/ BRCA2 mutation carriers, p27+ progenitor cells were identified as a potential measure of breast cancer risk [89]. Whether the decrease in p27+ cells with progenitor features and decreases in hormone-responsive and Wnt4secreting cells that lead to reduced Wnt signaling are interdependent or complementary events also warrants further investigation. Interestingly, the gene expression profiles of progenitor cells from parous BRCA1/BRCA2 mutation carriers resembled nulliparous more than parous 150

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non-carriers, suggesting that parity-related changes are much less apparent in these high risk women [89]. These results are in accordance with epidemiological studies showing that pregnancy leads to less pronounced or even zero protective effects in BRCA1/BRCA2 mutation carriers [90,91]. In conclusion, the observations confirm the importance of altered stem/progenitor cell-related signaling pathways as potential mechanisms of the protective effect of an early pregnancy on breast cancer. Concluding remarks and future perspectives Studies in mammary epithelial cell subpopulations isolated from mouse and human mammary gland tissue strongly support the idea that alterations in paracrine signaling pathways determining cell fates in stem and progenitor cells are a key factor in early pregnancy-induced protection against breast cancer development. In parous mice, a persistent decrease in hormone receptor-positive and Wnt4-secreting luminal cells was associated with a persistent reduction in protumorigenic Wnt signaling, decreased proliferation potential, and increased differentiation phenotype of basal mammary stem/progenitor cells [88]. In women, pregnancy leads to a reduction in the number of hormone-responsive cells and preferential downregulation of protumorigenic genes and pathways in a subset of progenitor cells isolated from normal human breast tissue [89]. The studies point towards the use of Wnt inhibitors to mimic the protective effect against breast cancer of early pregnancy, a finding consistent with the known potent antiproliferation and anticancer activity of Wnt inhibition [92,93]. In addition, the results highlight the possible importance of inhibitors of TGFb signaling in breast cancer prevention [89], although this needs to be weighed against the known cancer-promoting effects of diminished TGFb signaling due to TGFb receptor type II knockouts [60]. Even though the studies summarized here provide a deeper understanding of the involvement of specific cell fate-determining signaling pathways in distinct mammary epithelial cell subpopulations in early parity-induced protection against breast cancer in mice and humans, important basic questions (Box 3) remain to be answered before we can develop a thorough prevention strategy against the development of breast cancer in humans. Acknowledgments Box 3. Outstanding questions  Which specific mammary cell subpopulation is the target in the initiation of early pregnancy-induced breast cancer protection?  What is the significance of canonical Wnt signaling and its molecular and cellular consequences for parity-induced breast cancer risk in the long term?  Are other signaling pathways or cell–cell and cell–microenvironment interactions important for the breast cancer protective effect of early pregnancy?  What causes the decrease in mammary hormone-sensing luminal cells after early pregnancy?  Does pregnancy at a late age (late pregnancy) fail to induce similar cellular and molecular changes as pregnancy at an early age? And if so, does this explain the absence of parity-induced protection against breast cancer after late pregnancy?

Review Research in the laboratory of M.B-A. is supported by the Novartis Research Foundation, the European Research Council (ERC starting grant 243211-PTPsBDC), the Swiss Cancer League, and the Krebsliga Beider Basel. F.M-A. is supported by a fellowship of the Swiss National Science Foundation (SNSF). We apologize to our colleagues whose work could not be cited owing to space limitations.

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