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Review

Mammary stem cells and parity-induced breast cancer protection- new insights Genevieve Dalla,b , Gail Risbridgerb , Kara Britta,b,c,* a

Metastasis Research Laboratory, Peter MacCallum Cancer Centre, 7 St Andrews Place, East Melbourne 3002, Australia Department of Anatomy and Developmental Biology, Monash University Clayton, Wellington Rd 3800, Australia c The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 January 2016 Received in revised form 9 February 2016 Accepted 18 February 2016 Available online xxx

Parity (childbearing) significantly decreases a woman’s risk of breast cancer and the protective effect is greater if the woman is younger and has more children. The mechanism/s of parity-induced protection are not known. Although several factors are postulated to play a role, we discuss how a reduction in the number of mammary stem cells (MaSCs) may lead to a reduction in breast cancer risk in parous women. Firstly we review the epidemiology linking childbearing to reduced breast cancer risk and discuss how additional births, a young age at first full term birth, and breastfeeding impact the protection. We then detail the mouse and human studies implicating MaSC in parity induced protection and the in-vivo work being performed in mice to directly investigate the effect of parity on MaSC. Finally we discuss the transplant and lineage tracing experiments assessing MaSC activity according to parity and the need to define if MaSC are indeed more carcinogen sensitive than mature mammary epithelial cells. Continuing and future studies attempting to define the parity induced mechanisms will aid in the development of preventative therapies. ã 2016 Elsevier Ltd. All rights reserved.

Keywords: Breast cancer Parity Mammary stem cells

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Parity protects against breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additional pregnancies are associated with/provide added protection against breast cancer 1.1. The protection offered by parity is long term and not immediately effective . . . . . . . . . . . . 1.2. The proposed mechanisms of parity-mediated protection against breast cancer . . . . . . . . . . . . . . . 2.1. Parity induces changes in circulating hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parity decreases the levels of estrogen receptor in the breast . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. 2.3. Parity increases differentiation in the mammary gland tissues . . . . . . . . . . . . . . . . . . . . . . . . Parity decreases mammary stem cell (MaSC) activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Development of the mammary gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Human and mouse experimental work on MaSC and parity induced protection . . . . . . . . . . . . . . . . Studies investigating carcinogen sensitivity of MaSC are limited . . . . . . . . . . . . . . . . . . . . . . 4.1. Mouse studies investigating MaSC activity with parity are conflicting . . . . . . . . . . . . . . . . . . 4.2. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Parity protects against breast cancer

* Corresponding author at: Metastasis Research Laboratory, Peter MacCallum Cancer Centre, 7 St Andrews Place, East Melbourne 3002, Australia. E-mail address: [email protected] (K. Britt).

Whilst treatments for breast cancer (BCa) continue to increase the length of time women can live with their disease, the aetiology of BCa and how to reduce its incidence is still unknown. In this

http://dx.doi.org/10.1016/j.jsbmb.2016.02.018 0960-0760/ ã 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: G. Dall, et al., Mammary stem cells and parity-induced breast cancer protection- new insights, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.02.018

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context, it is essential to consider BCa incidence trends in the population and attempt to define the factors that modulate BCa risk. Since the 1700s it has been known that nuns have an increased incidence of developing BCa compared to the general population. The underlying relationship to their lack of child-bearing was first investigated in epidemiological studies in the mid-1920s [38] and then confirmed in 1970 [46]. Parity, defined as “the condition of having given birth to an infant or infants” [72], was shown to decrease the risk of developing BCa by up to 50% [46]. The greatest protection conferred by parity occurs when the first full term birth takes place before the age of 20. The protective effects of parity do continue until the age of 35 (although at a decreasing rate), however after this age a first full-term birth leads to an increase in the risk of BCa. Numerous epidemiological studies following MacMahon’s landmark study have added support to his findings [39,2,56,82,25]. Meta-analyses then went on to show that parity only protected against hormone responsive BCa [3,43]. Hormone responsive, or estrogen receptor (ER) positive BCa makes up approximately 70% of all BCa [81]. It is not known why this is the case. Complicating matters further, the protection conferred by an early first full-term pregnancy is increased by additional childbearing, optimal intervals between births and breastfeeding. 1.1. Additional pregnancies are associated with/provide added protection against breast cancer Several groups have identified a trend between the number of births and reduced BCa risk [76,26,33]. Setting the relative risk of BCa following one full-term pregnancy to 1, [78] observed a relative risk of just 0.34 in women who had 4 or more full-term pregnancies. Similarly, [36] found that after the first full-term birth each additional birth reduced the risk of BCa by approximately 10%. The protection afforded by multiparity is affected by the age at the first birth. A study conducted in 2008 found that women who had 4 children with the first child born before 25 years of age had a lower risk of BCa than those women who also have 4 children but the first birth occurred after 25 years [42]. Whilst the number of children a woman has is important in determining her risk of BCa, so too is the spacing between the births. In 2009, a study showed that a 1–3 year interval between successive births significantly increased the number of cases of BCa compared to intervals of less than one year and greater than 3 years [34]. It is unknown why the risk of BCa is lower in women with a birth interval of less than 1 year and greater than 3 years. Prior to this the World Health Organisation had released a report detailing the outcomes of a meeting concerning birth spacing held in Geneva in 2005 where it was recommended that a 2–3 year space in between births be employed to reduce the risk of infant mortality [87]. These studies, largely performed in developing nations, have obvious benefits for the children, however the fact that mothers are increasing their own risk of disease is concerning. Considering the influence of age at first birth, number of births and the spacing between births it is not surprising that breastfeeding is also able to modulate risk of BCa in women [23]. The reduction in BCa risk found to be offered by breastfeeding is 4.3–4.5% for every 12 months of breast feeding [5,30], a reduction which is in addition to the reduced risk following each birth. Despite these findings and a recommendation of at least 6 months of breastfeeding from bodies such as the World Health organisation and the National Health and Medical Research Council of Australia, just 15% of Australian babies are exclusively breastfed up to 6 months of age, and only 1% of UK babies [47,1]. The protection conferred by breastfeeding is not limited to ER+ BCa [44,82] as is the case for parity itself. The mechanisms of breastfeeding-induced protection and why it protects against both ER+ and ER negative tumours are largely unknown.

1.2. The protection offered by parity is long term and not immediately effective As mentioned, a birth interval of 1–3 years leads to an increase in BCa risk that is believed to be attributed to the transient period of increased risk of developing BCa which follows for several years after giving birth. This was identified first in 1988 by Bruzzi and associates who observed an increased risk of BCa in women who’s years since last birth was less than three [10]. A 2002 case-control study in Sweden identified a 5 year period of increased incidence of BCa following the birth of the first child [41]. As with other protective effects, the age of the mother is important in determining the length of this increased transient risk. Women who had their first child at age 30 have been shown to only reach a lower risk than nulliparous women 15 years after the delivery of their child. They are at an increased risk for the 15 years immediately after delivery. This is compared to women who were 20 when they had their first child who had a lower risk than nulliparous women after just 2 years [36]. The period of increased risk is believed to be due to the complex tissue remodelling of the mammary gland which occurs during the period of post-lactational involution. Exhibiting a similar molecular profile to wound-healing [73,14], involution represents a time in the mammary gland of wide-spread apoptosis involving a large immune cell infiltrate and complex extracellular matrix (ECM) reorganization. Furthermore, ECM isolated from involuting mammary glands has been shown to induce a greater rate of metastasis of a tumorigenic human BCa cell line when compared to nulliparous ECM both in vitro and in vivo [48]. This transient period of increased risk must therefore be considered when designing and analysing parity studies to ensure correct modelling of the decreased risk period. 2. The proposed mechanisms of parity-mediated protection against breast cancer Pregnancy is currently postulated to confer protection against BCa through 4 mechanisms; changes in circulating hormone levels, a change in estrogen responsiveness, a reduction in stem cells, or as a result of permanent changes in differentiation status of the breast [8]. It is highly probable that each of these mechanisms may work in concert to mediate parity-associated protection, but for simplicity each will be discussed separately. In order to investigate the biological mechanisms of parityassociated protection against BCa, a model animal was chosen, circumventing the ethical issues surrounding human experiments. The rodent offered a good model for three main reasons. Firstly the similarities in cellular composition, and conservation of genes and pathways between human and mouse mammary cell subpopulations [40]. Secondly, the chemical carcinogen 7,12-dimethylbenz[a] anthracene (DMBA) can induce the development of estrogenresponsive mammary tumours in rodents and finally, that development of these tumours can be reduced by one round of pregnancy and weaning [11,65]. Thus all experimental evidence of the mechanisms of parity-associated protection reviewed below has thus far been demonstrated in the rodent model. 2.1. Parity induces changes in circulating hormones The prolonged exposure to elevated progesterone and estrogens during pregnancy has long been postulated to have permanent effects on the hormonal profile in parous women. The mediation of protection by progesterone and estrogen levels alone is supported by numerous observations of chemicallyinduced carcinogenesis being prevented in rodents by treatment with estradiol and progesterone at levels equivalent to those seen

Please cite this article in press as: G. Dall, et al., Mammary stem cells and parity-induced breast cancer protection- new insights, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.02.018

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in pregnancy [22,58,68]. Despite this, studies assessing serum levels of pregnancy hormones in parous women have failed to consistently show a permanent reduction in estrogens [19,52] which would be significant considering it’s known pro-proliferative effects [83]. A permanent reduction in estrogens in parous mice has also not been shown as the field is awaiting the development of hormone assays which have the appropriate level of sensitivity. 2.2. Parity decreases the levels of estrogen receptor in the breast The large body of epidemiological evidence correlating estrogen exposure and BCa [7,54,90] supports the notion that a decreased responsiveness of the breast to estrogens would be advantageous. Whether parity results in a decrease in estrogen responsiveness in the breast is currently unclear. Studies have shown lower levels of estrogen receptor alpha (ERa) expression in parous rodents compared to age matched controls [80,91,51] however, a direct link between a reduction in ER expression and the responsiveness of the mammary gland to estrogens has not been shown. 2.3. Parity increases differentiation in the mammary gland tissues Pregnancy is the first time the mammary gland undergoes terminal differentiation. This terminal differentiation is believed to permanently alter the morphology and gene expression profile of the parous mammary gland in such a way that prevents tumorigenic events. Morphological assessment of the mammary gland performed by the Russo group support this theory. They found that secretory lobules can be categorised within the mammary gland of humans based on the number of alveoli clusters within each lobule. These lobules are classified as 1, 2 or 3 in increasing order of complexity (and therefore maturity). Lobules 1 and 2 containing the least amount of clusters comprise the majority of lobule types within the nulliparous mammary gland whereas lobule types 3 are exclusively found within parous mammary glands [63]. The gene expression profile has also been confirmed to be more differentiated in the parous mammary gland compared to nulliparous counterparts. Wistar-furth rats treated with estradiol and progesterone were shown to have increased markers of differentiation, cell–cell contact and milk proteins compared to untreated controls [21]. Similarly D’Cruz et al. found an up regulation of milk proteins as well as regulators of cell growth in parous mice [17]. However, in these studies whole mammary glands were assessed, rather than isolating out, and performing analysis on, particular mammary epithelial sub-types and stroma. This means that any cell type specific differences between parous and nulliparous mammary glands would be masked. 2.4. Parity decreases mammary stem cell (MaSC) activity The similar self-renewing properties between stem cells and tumour cells have long provided justification for implicating stem cells in mediating cancer risk [59]. They are long-lived which may allow for the accumulation of mutations leading to tumorigenic transformation. They have a potentially limitless proliferative capacity and the ability to self-renew which are both hallmarks of cancer [24]. MaSC sensitivity to carcinogenesis is believed to explain the epidemiological observations of a higher incidence of BCa among women exposed to radiation at puberty [49,32] a time when stem cell numbers in the breast are believed to be at their peak. These observations were supported by rodent studies by the Russo group who have shown DMBA exposure in young rats leads to a greater incidence of tumour formation than in older rats [64]

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yet to date, no direct link between MaSC and sensitivity to carcinogenic insult has been made. As DMBA-induced carcinogenesis is reduced by pregnancy [66], it was postulated that pregnancy induces a proportion of the stem cells to differentiate, thus reducing the pool of transformation sensitive cells. The extensive tissue regeneration required after pregnancy is attributed to the presence of MaSC. It was thus postulated that parity induces the differentiation of stem cells into committed cells that are less susceptible to carcinogenic insult. In order to fully comprehend the link between differentiation of the mammary gland, stem cells, carcinogenesis and parity protection one must consider development of the mammary gland through embryogenesis, puberty, pregnancy and involution. 3. Development of the mammary gland Mammary gland development during embryogenesis is largely the same in mice and humans despite differences in timings due to the longer gestational period in humans. It is thought to begin with the formation of bilateral epidermal ridges, or ‘milk lines’ which in humans occurs during the fourth week of embryogenesis [28] and in rodents occurs at embryonic day 10 [86,60]. Shortly after this first step 1 pair (human) or 5 pairs (mouse) of placodes form (approximately 5–6weeks of embryogenesis in the human and embryonic day 11.5 in the mouse), corresponding to the sites of the future nipples [74]. Mammary epithelial buds forming at these sites elongate into the mammary sprout which functions to invade the underlying mesenchyme and associate with tissue fated to give rise to the mammary fat pad [60], a rich source of adipose and supportive cells into which the mammary gland grows. This occurs at the end of the first trimester in humans and embryonic day 11.5–12.5 in mice. Reciprocal signaling between the mammary sprout and primitive stroma within the mesenchyme are required to stimulate this invasion [20]. Up until this point, development of the mammary gland is largely equal in humans and rodents. Development of the mammary gland now diverges in mice and humans, as in the mouse further growth is inhibited until puberty [86]. In humans, the mammary parenchyma begins branching away from the nipples during the thirteenth to fifteenth week of gestation to form solid epithelial cord-like structures which by the thirty-second week become tubular ducts surrounded by loose periductal stroma [28]. At full-term, this stroma appears dense and alveolar structures with secretory function have formed at the distal tips of the developing glands. This secretory activity is thought to be a by-product of the placental and mammary hormones circulating within the fetus but once these decrease so do the majority of the alveolar structures. In both humans and rodents mammary gland development is hormone-independent until puberty, where the onset of estrogen release from the ovary, along with insulin growth factor and growth hormone stimulate branching morphogenesis [45]. This involves further invasion into the mammary fat pad and sidebranching in a cyclical manner to coincide with menstruation. ER expression in the mammary stroma is believed to be important for postpubertal growth. Complete loss of ER in both the epithelium and stroma leads to a rudimentary mammary gland however when ER knock-out epithelium is recombined with wild-type stroma the growth is restored [16]. In the rodent, puberty fosters the development of terminal end buds (TEBs) at the tips of growing ducts which are club-like structures able to further invade the mammary fat pad through extensive proliferation of the outer layer of cells surrounding the TEB known as ‘cap cells’. As these cap cells proliferate and move away from the leading edge of the TEB they differentiate into myoepithelial cells [88]. The inner layers of the TEB are made up of ‘body cells’ which eventually form a single layer of mature luminal

Please cite this article in press as: G. Dall, et al., Mammary stem cells and parity-induced breast cancer protection- new insights, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.02.018

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cells thus creating a lumen for future milk secretions after widespread apoptosis and selective differentiation [70]. The luminal cell layer also contains within it a cell type known as the basal cell due to its closer position to the myoepithelial cells than other luminal cells. This cell type is distinguished from the rest of the luminal cells by its small size and pale appearance [12] and is believed to be the stem cell compartment of the mammary gland [77,75]. Once the TEBs have reached the edge of the mammary fat pad they begin a process of regression where cap cells differentiate into myoepithelial cells and cells within the TEB reorganize into a single layer of lobular cells. These now mature distal tips, known as terminal ducts, of the gland are functionally similar to the terminal ductal lobular unit in humans [28,55]. The cellular composition of both the human and rodent pre-pregnant mammary gland are the same in that both comprise a bi-layer of cells made up of an outer layer of myoepithelial cells and an inner layer of luminal cells (containing basal cells), surrounded by interlobular stroma and adipose tissue. Moreover in both the rodent and human, complete differentiation of the mammary gland does not occur until pregnancy. 3.1. Pregnancy Pregnancy sees the development of a fully differentiated ductal tree capable of secreting milk. Release of progesterone initially from the ovary and later from the placenta induces widespread secondary and tertiary side-branching, and, along with prolactin, induces the formation of alveoli required for lactation. The interlobular adipose tissue loses it’s lipid content to make way for these alveoli structures, which resemble a bunch of grapes, rapidly expanding with milk secretions as well as the formation of a complex network of blood vessels [45]. At the cessation of weaning, a two-stage process of involution occurs to return the mammary gland back to its pre-pregnant state. The first and irreversible stage of involution is characterised by the initial steps of ECM decomposition and a small amount of apoptosis within the epithelial layer. The second stage is irreversible and involves rapid apoptosis of alveoli, immune cell influx and ECM reorganization as well as a return of the lipid component to adipocytes which was lost during pregnancy to allow space for ductal elongation. A schematic representation of the morphological changes during pre-pubertal and pubertal growth as well as pregnancy, lactation and involution is shown in Fig. 1 to illustrate the dramatic transformations that take place within the mammary gland during the reproductive cycle. The ability of the mammary gland to regress back to its prepregnant state following involution and then regenerate a

lactation-competent ductal system with successive pregnancies has long been thought to be attributed to stem cell activity. 4. Human and mouse experimental work on MaSC and parity induced protection The rationale for a reduction in MaSC as a mechanism of parity associated protection is based on two points: MaSCs are believed to be abundant in the young mammary gland (on which parity exerts its greatest protection), and the young mammary gland is particularly sensitive to carcinogenic insult. This notion first emerged from epidemiological observations of BCa incidence in women exposed to the atomic bombs during World War II. Women living in Hiroshima and Nagasaki who were exposed to the radiation between the ages of 10 and 19 had the highest absolute risk estimate of BCa (4.0) whilst women who were between the ages of 35 and 49 had the lowest (0.9) [49], a finding that has been supported by subsequent analysis of this unique population [79,37]. This suggested that breast tissue was most sensitive to carcinogenic insult during the pubertal growth phase of the mammary gland. In support of these findings, BCa risk in women exposed to x-rays during puberty is higher than those exposed at later ages [27,6] and exposure to the chemical carcinogen dichlorodiphenyltrichlorethane was shown to increase the risk of BCa 5-fold but only if women were exposed before 14 years of age [15]. Russo and colleagues have validated this epidemiological evidence by showing administration of DMBA to pubertal rats (55 days old) resulted in 100% incidence of carcinoma compared to only 63% in older virgin rats (180 days old) [64]. In the same year they also found 36 of 42 rats died shortly after administration of DMBA at 20 days of age [67]. The significance of this is that the TEBs, (which house the MaSC) are at their highest density at 21 days of age [62]. The high incidence of these estrogen responsive tumours developing was only decreased if the rats underwent one or more rounds of pregnancy Russo and Russo, 1978b,a or if the rats received an equivalent regiment of pregnancy hormones [58,22]. Despite these obvious links between stem cell number and carcinogen sensitivity, a functional assessment of carcinogenic insult to stem cells has not been performed. 4.1. Studies investigating carcinogen sensitivity of MaSC are limited To date, studies assessing DNA damage response in MaSCs have been limited and show conflicting results. Two studies reported an expansion in MaSC numbers following radiation exposure [31,53] whilst another showed a decrease [89]. These studies were also unable to find a difference in DNA damage levels, as indicated by

Fig. 1. Schematic of the morphology of the different stages of mammary gland development. Growth of the ductal tree begins at puberty with the formation of the TEBs. Once reaching adulthood major changes in mammary gland morphology are not observed unless pregnancy occurs where there is an increase in side-branching and formation of milk-producing alveoli. These alveoli are cleared after weaning (involution) before the mammary gland undergoes a repair period prior to returning to the resting parous stage.

Please cite this article in press as: G. Dall, et al., Mammary stem cells and parity-induced breast cancer protection- new insights, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.02.018

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gH2AX foci, between MaSC and more mature cell populations. The only study to date using DMBA as a carcinogen to assess DNA damage in mammary cells reported similar findings to the radiation studies mentioned above. They observed no difference in gH2AX foci between the MaSC-containing basal compartment compared to luminal cells, however basal cell numbers and in vivo repopulating activity were decreased [35]. Klos and colleagues did not specifically test the DNA damage levels and DNA damage response in the CD24 + CD49fhi MaSC-enriched population in comparison to the basal and luminal cells. Thus whether the decrease in stem cell activity was a reduction in MaSCs themselves or a decrease in surrounding supportive basal cells is unknown. 4.2. Mouse studies investigating MaSC activity with parity are conflicting There are four studies that have addressed whether parity leads to a decrease in MaSC number or activity. Collectively these studies show that early but not late pregnancy can reduce the number of MaSC [69,9,57,51], which supports the epidemiological findings of early pregnancy being the most protective reproductive factor against breast cancer. Two studies have assessed stem cell activity in animals experiencing a pregnancy as young adults (9-10weeks of age) and found no change in MaSC activity [9,57]. At this age, mice are only 3 weeks post sexual maturity, and so could still be considered quite young. To mimic the effects of older pregnancies in women, mice should be mated at ages closer to the decline of reproductive function. These studies used either non-fractionated mammary glands [57] or epithelial cell enriched fractions [9] rather than isolated MaSCs. Two additional studies have assessed MaSC activity in young mice (5–6weeks of age) and did show a decrease in MaSC activity [69,51]. The more recent study attributed the decrease in stem cell activity following pregnancy to a reduction in hormone-sensing luminal cells [51]. These ER+ PR+ luminal cells facilitate MaSC expansion by releasing the Wnt signaling ligand, Wnt4. Thus a reduction in their numbers means a reduction in the MaSC proliferation cue. The same group have since validated these findings showing the reductions in PR-positive luminal cells and Wnt signaling persists into aged-parous mice [50]. In contrast, mice mated at later ages do not show these same decreases in hormone-sensing cells or Wnt signaling, further supporting the epidemiological observations of an early and not late pregnancy providing the greatest protection against breast cancer [2,39,82]. Similarly, pregnancy has been shown to alter DNA methylation specifically within the CD61+ luminal progenitor subset [29], and importantly this involved a hypermethylation of MaSC-related genes. Downregulation of signaling pathways known to play a role in MaSC function have also been reported in breast tissue isolated from parous women [13]. Again this was attributed to a lasting effect on hormone-sensing cells within the luminal compartment. The aforementioned studies investigating MaSC function in parous mice used the mammary fat pad transplant assay as this is widely regarded as the gold-standard method to assess stem cell activity [71,18]. However, as this involves dissociating tissue from the parous or age-matched nulliparous hosts and transplanting into an artificially cleared donor fat pad many believe that this does not accurately represent normal physiological conditions [84,85]. To overcome this, lineage tracing studies have been performed, where markers for luminal cells such as keratin 8 (K8) or Elf5 and markers for MaSC-enriched basal cells such as keratin 5 (K5) or Axin2 control the expression of a traceable fluorescent protein [84,85,61]. Unexpectedly two studies were able to trace K5 and Axin2 positive basal cells through multiple rounds of pregnancy and whilst they did not quantitate their numbers, these cells (which contain the MaSCs) showed no obvious decline in

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regenerative behaviour with parity. They also did not report an obvious decline in luminal cell numbers, although this was not the focus of these studies. Collectively these data appear to contradict the mammary fat pad transplant assay results and the genetic profiling studies performed on mice and human tissue [51,29,13]. This highlights the fact that whilst lineage-tracing studies provide a more physiologically relevant setting for investigation, the results need to be interpreted with caution. Lineage tracing studies rely heavily on the expression of specific promoters which may be promiscuous or change expression patterns based on developmental and reproductive events. Furthermore, Tamoxifen was used to induce the expression of the promoters used in these studies [84,85] which may have impacted normal development in the estrogen dependent mammary gland [61,4]. Thus the cells that did not reduce their regenerative capacity through multiple rounds of pregnancy in the lineage tracing studies may not be the same cells that show reduced stem cell activity in more recent reports. The stark differences in results obtained from mating mice at different ages, differing MaSC isolation methods or techniques used to assess stem cell activity highlight the complexities of stem cell investigations in the mammary gland. Whilst the gene expression and fat pad assay data showing a specific reduction in luminal cells and a decrease in MaSC activity is compelling, it will need to be further validated in large scale assessments of human and mouse tissue. To add further weight to these data, it will also need to be shown by lineage tracing studies which do not use Tamoxifen. This is an exciting stage in mammary gland biology as we gain further understanding of the effect of pregnancy on complex cell-to-cell interactions and how these may contribute to parity-induced protection against breast cancer. 5. Conclusion Delineating a role for MaSCs in parity-induced protection against breast cancer remains a challenge for mammary gland biologists. The conflicting results may be rectified if someone was to test both early and late pregnancies using MaSC enriched populations as well as more mature progenitor populations. With recent advances in MaSC isolation markers and techniques, genetic screening and the increased access to biopsy specimens a thorough understanding of the mechanism at play may not be far off. Acknowledgments GD was supported by an Australian Postgradutate Scholarship. KB was supported by an NBCF Early Career Fellowship, an NHMRC New Investigator grant and a VCA early career seed grant. GRS was supported by an NHMRC fellowship. References [1] AIHW, Australian National Infant Feeding Survey: indicator results. Cat. no. PHE 156, Canberra, Australian Institute of Health and Welfare & Australasian Association of Cancer Registries, 2010. [2] G. Albrektsen, I. Heuch, S. Hansen, G. Kvale, Breast cancer risk by age at birth: time since birth and time intervals between births: exploring interaction effects, Br. J. Cancer 92 (2005) 167–175. [3] M. Althuis, J. Fergenbaum, M. Garcia-Closas, L. Brinton, M. Madigan, M. Sherman, Etiology of hormone receptor-defined breast cancer: a systematic review of the literature, Cancer Epidemiol. Biomark. Prev. 13 (2004) 1558– 1568. [4] M.L. Asselin-Labat, F. Vaillant, J.M. Sheridan, B. Pal, D. Wu, E.R. Simpson, H. Yasuda, G.K. Smyth, T.J. Martin, G.J. Lindeman, J.E. Visvader, Control of mammary stem cell function by steroid hormone signalling, Nature 465 (2010) 798–802. [5] V. Beral, Breast cancer and breastfeeding: collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries, including 50,302 women with breast cancer and 96,973 women without the disease by the Collaborative Group on Hormonal Factors in Breast Cancer, Lancet 360 (2002) 187–195.

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Please cite this article in press as: G. Dall, et al., Mammary stem cells and parity-induced breast cancer protection- new insights, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.02.018