Effects of oestrogen and the antioestrogens, tamoxifen and LY117018, on four oestrogen-regulated RNAs in the EFM-19 breast cancer cell line

The EFM-19 cell line is a new breast cancer cell line whose proliferation has been reported to be stimulated by oestrogens and inhibited by the antioestrogen tamoxifen. Oestrogen receptor mRNA levels are higher in EFM-19 cells than in other oestrogen-responsive cell lines. The levels of four oestrogen-inducible RNAs [pNR-1, pNR-2, pNR-25 and pNR-100] were measured in EFM-19 cells. Oestradiol treatment increased the levels of the four regulated RNAs between 3-fold (pNR-100) and greater than 100-fold (pNR-2). The induction was half maximal between 1.5 x 10(-11) and 1.5 x 10(-10) M oestradiol. The effects of two antioestrogens, tamoxifen and LY117018, were measured on the expression of the oestrogen-regulated RNAs. Tamoxifen was a partial oestrogen agonist for the induction of the pNR-1 and pNR-25 RNAs but had very little effect on the pNR-2 and pNR-100 RNA levels. The pNR-2 RNA levels were less induced by tamoxifen in EFM-19 cells than in MCF-7 cells. LY117018 did not increase the levels of any RNA. The oestrogen-induced levels of the four RNAs were reduced by both antioestrogens to the RNA levels present in cells treated with the antioestrogens alone. LY117018 was at least 100-fold more potent than tamoxifen as an oestrogen antagonist.     

Uterine bioassay of tamoxifen,trioxifene and a new estrogen antagonist (LY117018) in rats and mice

Dose response uterotrophic and antiuterotrophic activity of antiestrogens was examined in immature rats, immature mice and adult ovariectomized mice. LY117018 was the most active antagonist and the least estrogenic, while tamoxifen induced the greatest uterine growth and the weakest antagonism. The reported estrogenic activity of tamoxifen in mice (1) was found to be related to maturity. All compounds caused uterotrophic changes in immature mice similar to those observed in immature rats. However, in adult mice tamoxifen was devoid of antagonism, and trioxifene was active only at a very high dose as both were extremely estrogenic in this model. LY117018 activity in adult mice was comparable to that observed in immature rats and mice. Results depict significant agonist and antagonist advantages of LY117018 over tamoxifen and trioxifene.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Biosynthesis of medium-chain fatty acids by mammary epithelial cells from virgin rats.

Epithelial cells were isolated from the undifferentiated mammary glands of mature virgin female rats, and their lipogenic characteristics were studied. These cells synthesized predominantly medium-chain fatty acids, albeit at a low rate. In contrast, whole tissue from mammary glands of virgin rats synthesized predominantly long-chain fatty acids at a relatively higher rate, indicating that the lipogenic activity is dominated by the adipocyte component of the gland. Enzyme assays revealed that thioesterase II, the enzyme which regulates production of medium-chain fatty acids by the fatty acid synthetase, was present at a high activity in the undifferentiated mammary epithelial cells of virgin rats. Immunohistochemical studies confirmed this observation and showed that the regulatory enzyme was present exclusively in the epithelial cells lining the alveolar and ductal elements of the undifferentiated gland. This study demonstrates that the potential to elaborate tissue-specific medium-chain fatty acids is already expressed in the undifferentiated tissue of virgin rats and is not acquired as a result of the differentiation associated with the lactogenic phase of development. In this species mammary epithelial cells apparently synthesize predominantly medium-chain fatty acids at all stages of development, and only the overall rate of synthesis is increased on induction of the fatty acid synthetase during lactogenesis.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

Effects of epidermal growth factor,estrogen, and progestin on DNA synthesis in mammary cells in vivo are determined by the developmental state of the gland

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are involved in the growth and development of the normal mammary gland. While studies have been carried out to investigate the in vivo effects of EGF in the immature mammary gland, nothing is known about the growth effects of EGF or its potential interactions with E and/or P in the adult mammary gland. The present studies were undertaken to investigate the effects of EGF, E, and P on mammary cell proliferation in immature, peripubertal vs. adult, sexually mature mice. We have found that EGF promotes epithelial and stromal cell proliferation in both the immature and adult mammary glands. In the immature gland, the end bud epithelium is most responsive to the proliferative effects of EGF and there is no apparent interaction between EGF, E, and/or P. In contrast, in the mature gland EGF adds to the proliferative effects of E+P in the ductal epithelium resulting in more extensive ductal sidebranching. Thus these results demonstrate that the developmental state of the mammary gland determines the nature and extent of the interactions between EGF, E, and P in growth and development. © 1993 Wiley-Liss, Inc.     

Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.     

Role of ovarian secretions in mammary gland development and function in ruminants

The mammary gland is a dynamic organ that undergoes cyclic developmental and regressive changes during the lifetime of a female mammal. Mammogenesis begins during embryonic life with the development of the first mammary gland rudiments and ductal system. After birth, during the pre-pubertal period, the ductal growth of the mammary parenchyma occurs through the fat pad. In most of the ruminant species allometric mammary parenchyma development begins with the onset of cyclic ovarian secretions activity. The two main hormones secreted during an ovarian cycle are estradiol and progesterone. These steroid hormones are derived from cholesterol and are synthesized by theca and granulosa cells in ovaries. During puberty, the mammary parenchyma develops in a compact, highly arborescent parenchymal mass surrounded by a dense connective matrix. Ductal elongation and lobulo-alveolar development are accomplished during growth and pregnancy to prepare for future milk production. At the end of lactation, the mammary gland undergoes involution, which corresponds to a regression of the secretory tissue, a reduction in the alveolar size and a loss of mammary epithelial cells (MECs). Ovarian steroids (estradiol and progesterone) appear to be key regulators of the different stages of mammogenesis and mammary function. Through this review, the role and the importance of ovarian steroids on mammary gland and on MECs is described.     

Profile of estrogen‐responsive genes in an estrogen‐specific mammary gland outgrowth model

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen‐responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen‐induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17β‐estradiol from two to 28 days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: (a) gene expression changes in our estrogen‐only model mimic those changes that occur in normal pubertal development in intact mice, (b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and (c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol‐responsive genes that are also co‐expressed with estrogen receptor α in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development. Mol. Reprod. Dev. 76: 733–750, 2009. Published 2009 Wiley‐Liss, Inc.     

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG^−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.     

Attenuation of mammary duct development by menhaden oil in BALB/c mice

The predominant polyunsaturated fatty acids of the n-6 family found in corn oil (CO) are crucial for normal mammary duct formation when fed to animals. However, as shown here, not all polyunsaturated fatty acids are equally effective in stimulating mammary gland development. The n-3 fatty acids in a 10% menhaden oil (MO) diet fed to mice effectively reduced both the diameter and the length of the growing mammary ducts. Previously, we demonstrated a similar reduction in duct growth by feeding a 10% fat diet high in those saturated fats found in hydrogenated cotton seed oil. The inhibited rate of duct maturation caused by hydrogenated cotton seed oil was reversed when the mice were allowed to mature on a diet containing n-6 fatty acids prior to feeding the saturated fat diet. The addition of 1% CO to a 9% hydrogenated cotton seed oil diet fed to immature mice was also sufficient to restore duct growth. Mice fed menhaden oil diets, on the other hand, continued to show impaired ductal growth well into adulthood. Examination of the ovaries from MO-fed mice as compared with CO-fed mice revealed significantly fewer corpora lutea. When exogenous progesterone was given to MO-fed mice, ductal growth was partially restored, but not to the extent seen in mice fed corn oil diets. Investigation of the fatty acid contents of livers of these mice revealed reduced amounts of arachidonate (20:4) in MO-fed mice when compared with CO-fed animals. The addition of 1% CO to the 9% MO diets did not alter the arachidonate content, indicating a block in the conversion of linoleate (18:2) to 20:4 by the n-3 fatty acids. Hence, dietary n-6 fatty acids are essential for normal mammary ductal development when fed prior to maturation. Although saturated rats are ineffective, n-3 fatty acids can partially substitute for the required n-6 fatty acids in both ductal and ovarian development.     

Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane

We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.     

Mouse mammary epithelial cells express the Na-K-Cl cotransporter,NKCC1: characterization,localization, and involvement in ductal development and morphogenesis

Despite the fact that physiological evidence points to the existence of a functional Na-K-Cl cotransporter in the mammary gland, the molecular identity of this transport process remains unknown. We now show that the Na-K-Cl cotransporter isoform, NKCC1, is expressed in mammary tissue. Developmental profiling revealed that the level of NKCC1 protein was significantly influenced by the stage of mammary gland development, and immunolocalization studies demonstrated that NKCC1 was present on the basolateral membrane of mammary epithelial cells. To examine whether functional NKCC1 is required for mammary epithelial cell development, we used NKCC1 -/- mice. We demonstrate that NKCC1 -/- mammary epithelium exhibited a significant delay in ductal outgrowth and an increase in branching morphogenesis during virgin development. These effects were autonomous to the epithelium as assessed by mammary gland transplantation. Although the absence of NKCC1 had no apparent effect on gross mammary epithelial cell morphology during lactation, pups born to NKCC1 -/- mice failed to thrive. Finally, analysis of NKCC1 protein in mouse models that exhibit defects in mammary gland development demonstrate that high levels of NKCC1 protein are indicative of ductal epithelial cells, and the presence of NKCC1 protein is characteristic of mammary epithelial cell identity.     

Wnt5a is required for proper mammary gland development and TGF-beta-mediated inhibition of ductal growth

Transforming growth factor-beta (TGF-beta) plays an essential role in growth and patterning of the mammary gland, and alterations in its signaling have been shown to illicit biphasic effects on tumor progression and metastasis. We demonstrate in mice that TGF-beta (Tgfbeta) regulates the expression of a non-canonical signaling member of the wingless-related protein family, Wnt5a. Loss of Wnt5a expression has been associated with poor prognosis in breast cancer patients; however, data are lacking with regard to a functional role for Wnt5a in mammary gland development. We show that Wnt5a is capable of inhibiting ductal extension and lateral branching in the mammary gland. Furthermore, Wnt5a(-/-) mammary tissue exhibits an accelerated developmental capacity compared with wild-type tissue, marked by larger terminal end buds, rapid ductal elongation, increased lateral branching and increased proliferation. Additionally, dominant-negative interference of TGF-beta signaling impacts not only the expression of Wnt5a, but also the phosphorylation of discoidin domain receptor 1 (Ddr1), a receptor for collagen and downstream target of Wnt5a implicated in cell adhesion/migration. Lastly, we show that Wnt5a is required for TGF-beta-mediated inhibition of ductal extension in vivo and branching in culture. This study is the first to show a requirement for Wnt5a in normal mammary development and its functional connection to TGF-beta.     

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.     

Hormonal defect in maspin heterozygous mice reveals a role of progesterone in pubertal ductal development

Progesterone and PR are mainly thought to affect tertiary ductal side branching and alveologenesis in late stage of mammary gland development. Here, we present evidence that they also play a role in early ductal development. This conclusion derived from our analysis of maspin heterozygous (Mp+/-) mice that showed defective ductal development at puberty. The defect was due to a reduced systemic level of progesterone. We show that treatment of Mp+/- mice with progesterone rescued the defect of ductal development. When both wild-type and Mp+/- mice were ovariectomized at 4 wk of age, treatment with progesterone alone can stimulate their ductal growth. In addition, treatment of wild-type mice with the progesterone inhibitor RU486 slowed ductal development in a dose-dependent manner. To confirm that progesterone receptor (PR) was required for progesterone action in ductal development at pubertal stage, we treated ovariectomized PR-deficient (PRKO) and wild-type mice with progesterone and examined ductal development at 7 wk of age. Whereas wild-type mammary glands displayed abundant ductal growth after progesterone treatment, there was a significant retardation of ductal growth in PRKO mice. Furthermore, we observed reduced ductal development in intact PRKO mice at 7 wk of age compared with that of wild-type mice. However, the defect was rescued at late stage of mammary development in PRKO mice. These data demonstrate that progesterone signaling, which is mediated by PR, plays an important role in early ductal development. In PRKO mice, a compensatory mechanism occurs that rescues the ductal defect at a late stage of mammary development.     

Cripto: a novel epidermal growth factor (EGF)-related peptide in mammary gland development and neoplasia

Growth and morphogenesis in the mammary gland depend on locally derived growth factors such as those in the epidermal growth factor (EGF) superfamily. Cripto-1 (CR-1, human; Cr-1, mouse)--also known as teratocarcinoma-derived growth factor-1--is a novel EGF-related protein that induces branching morphogenesis in mammary epithelial cells both in vitro and in vivo and inhibits the expression of various milk proteins. In the mouse, Cr-1 is expressed in the growing terminal end buds in the virgin mouse mammary gland and expression increases during pregnancy and lactation. Cr-1/CR-1 is overexpressed in mouse and human mammary tumors and inappropriate overexpression of Cr-1 in mouse mammary epithelial cells can lead to the clonal expansion of ductal hyperplasias. Taken together, this evidence suggests that Cr-1/CR-1 performs a role in normal mammary gland development and that it might contribute to the early stages of mouse mammary tumorigenesis and the pathobiology of human breast cancer.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.