Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.     

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.     

Atbf1 Regulates Pubertal Mammary Gland Development Likely by Inhibiting the Pro-Proliferative Function of Estrogen-ER Signaling

ATBF1 is a candidate tumor suppressor that interacts with estrogen receptor (ER) to inhibit the function of estrogen-ER signaling in gene regulation and cell proliferation control in human breast cancer cells. We therefore tested whether Atbf1 and its interaction with ER modulate the development of pubertal mammary gland, where estrogen is the predominant steroid hormone. In an in vitro model of cell differentiation, i.e., MCF10A cells cultured in Matrigel, ATBF1 expression was significantly increased, and knockdown of ATBF1 inhibited acinus formation. During mouse mammary gland development, Atbf1 was expressed at varying levels at different stages, with higher levels during puberty, lower during pregnancy, and the highest during lactation. Knockout of Atbf1 at the onset of puberty enhanced ductal elongation and bifurcation and promoted cell proliferation in both ducts and terminal end buds of pubertal mammary glands. Enhanced cell proliferation primarily occurred in ER-positive cells and was accompanied by increased expression of ER target genes. Furthermore, inactivation of Atbf1 reduced the expression of basal cell markers (CK5, CK14 and CD44) but not luminal cell markers. These findings indicate that Atbf1 plays a role in the development of pubertal mammary gland likely by modulating the function of estrogen-ER signaling in luminal cells and by modulating gene expression in basal cells.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

Mammary gland morphogenesis is enhanced by exposure to flaxseed or its major lignan during suckling in rats

The exposure of rats to 10% flaxseed (FS) or an equivalent level of its major lignan, secoisolariciresinol diglucoside (SDG), during suckling enhances mammary gland differentiation, which protects against mammary carcinogenesis at adulthood. We determined whether this diet-induced mammary gland differentiation is mediated through the estrogenic pathway via epidermal growth factor receptor (EGFR) and estrogen receptor (ER) signaling. Rats were fed the AIN-93G basal diet (BD) from day 7 of pregnancy until delivery and then randomized to consume BD, FS, or SDG during lactation. After weaning, female offspring were fed BD throughout the experiment. At postnatal day (PND) 21 and the proestrus phase on PND 49-51, mammary glands of offspring were analyzed for morphology, cell proliferation, and expression of EGFR, epidermal growth factor (EGF), transforming growth factor-alpha, ER-alpha, and ER-beta. At PND 21, compared with the BD control, the number of terminal end buds (TEBs) and terminal ducts were increased by FS, whereas mammary epithelial cell proliferation was increased by both FS and SDG, suggesting that mammary morphogenesis was enhanced. Epithelial EGFR and stromal fibroblast EGF were increased by SDG, whereas epithelial ER-beta was decreased by FS. Conversely, at PND 49-51, a lower number of TEBs but a higher ratio of lobules to TEBs with decreased expression of EGFR or EGF was observed in both treatment groups. EGFR expression was positively associated with EGF expression and cell proliferation in TEB epithelium at PND 21. Urinary lignans of lactating dams were related to their offspring's indices of mammary gland development. In conclusion, exposure to FS or SDG during suckling enhanced mammary gland morphogenesis by modulation of EGFR and ER signaling, which led to more differentiated mammary glands at PND 49-51. The physiological outcomes of FS and SDG were similar, which suggests that SDG is partly responsible for the mammary gland differentiation effect.     

Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

Pannexin1 (Panx1) subunits oligomerize to form large-pore channels between the intracellular and extracellular milieu that have been shown to regulate proliferation, differentiation and cell death mechanisms. These key cellular responses are ultimately necessary for normal tissue development and function but the role of Panx1 in development, differentiation and function in many tissues remains unexplored, including that of the breast. Panx1 was identified to be expressed in the mammary gland through western blot and immunofluorescent analysis and is dynamically upregulated during pregnancy and lactation. In order to evaluate the role of Panx1 in the context of mammary gland development and function, Panx1^-/- mice were evaluated in comparison to wild-type mice in the mammary glands of virgin, lactating and involuting mice. Our results revealed that Panx1 ablation did not affect virgin or involuting mammary glands following histological and whole mount analysis. Panx1 was necessary for timely alveolar development during early lactation based on a decreased number of alveolar lumen following histological analysis and reduced proliferation following Ki67 immunofluorescent labelling. Importantly, the loss of Panx1 in lactating mammary glands did not overtly affect epithelial or secretory differentiation of the mammary gland suggesting that Panx1 is not critical in normal mammary gland function. In addition, PANX1 mRNA expression was correlated with negative clinical outcomes in patients with breast cancer using in silico arrays. Together, our results suggest that Panx1 is necessary for timely alveolar development following the transition from pregnancy to lactation, which may have implications extending to patients with breast cancer.     

The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK−/−). Mammary glands from RonTK−/− mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK−/− epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK−/− glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.     

Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism. J. Cell. Physiol. 174:251–260, 1998. © 1998 Wiley-Liss, Inc.     

Estrogen and estrogen plus progestin act directly on the mammary gland to increase proliferation in a postmenopausal mouse model

Hormone replacement therapy (HRT) with ovarian hormones is an important therapeutic modality for postmenopausal women. However, a negative side effect of HRT is an increased risk of breast cancer. Surgical induction of menopause by ovariectomy (OVX) in mice is an experimental model that may provide insights into the effects of hormone replacement therapy on the human breast. We have developed a mouse model of early and late postmenopausal states to investigate the effects of HRT on the normal mammary gland. The purpose of this study was to determine if HRT-induced proliferation was due to the direct action of the hormones on the mammary gland, or mediated systemically by hormones or growth factors produced elsewhere in the body. Estrogen (E) or E plus the synthetic progestin, R5020, were implanted directly into the mammary glands of early (1 week post OVX) and late (5 week post OVX) postmenopausal mice instead of administration by injection. We report that responses of early and late postmenopausal mice to implanted hormones were the same as those observed previously with systemically administered hormones. Implanted E conferred an enhanced proliferative response in the late postmenopausal gland characterized morphologically by enlarged duct ends. E+R5020 implants induced similar degrees of cell proliferation in both postmenopausal states but the morphological responses differed. Ductal sidebranching was observed in early postmenopausal mice, whereas duct end enlargement was observed in late postmenopausal mice. The differences in morphological response to E+R5020 in 5 week post OVX were associated with an inability of E to induce progesterone receptors (PR) in the late postmenopausal gland. The responses of the late postmenopausal glands to E and E+P were very similar to that observed previously in immature pubertal glands in ovary-intact mice. In pubertal mice, PR cannot be induced by E unless the mammary gland is pre-treated with EGF-containing implants. Similarly, herein pre-treatment of the late postmenopausal mammary gland with EGF-containing implants restored PR induction by E. Thus, EGF may determine the sensitivity of the mammary gland to E and E+P in late postmenopause and at puberty.     

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.