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Study Objective: Amphiregulin is a member of the epidermal growth factor family. In breast tissue, amphiregulin is a mediator of estrogen and progesterone signaling. The objectives were to examine the relationship of amphiregulin levels during peripuberty with estrogen levels. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: The participants in this analysis were a subset from a longitudinal study of pubertal maturation, the Breast Cancer and the Environment Research Program. They were recruited between ages 3 and 7 years. Blood specimens were selected for hormone analysis between 24 months before and 6 months after breast development. Serum amphiregulin levels were analyzed using enzyme-linked immunosorbent assay., Results: Amphiregulin levels were measured in 188 girls; 8.5% had a maternal history of breast cancer, and 30.9% of samples were below the limit of detection. Amphiregulin levels were greatest at 18 months before the onset of breast development (P < .006), and the rise in estrone levels between -24 and -18 months was correlated with the increase in amphiregulin levels in the same time period (P = .0002). After adjustment for time relative to breast development, amphiregulin levels were associated with maternal breast cancer (P = .024). Tracking of amphiregulin levels was highly significant (P < .0001) within a given individual., Conclusion: Amphiregulin levels peaked at 18 months before the onset of breast development, were temporally related to the rise in serum estrone, and were significantly associated with maternal history of breast cancer. Elevated amphiregulin levels at puberty might be a predictor of increased breast cancer risk., (Copyright © 2017 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.)

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.

Introduction: Puberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined., Methods: Pubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL)., Results: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression., Conclusions: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women., (© The Author(s) 2010. This article is published with open access at Springerlink.com.)

Objective: Increased adolescent obesity rates in the United States are a significant public health concern. Obesity or increased adiposity during puberty in girls, an important period of breast development and a window of exposure sensitivity, may influence breast development and cancer risk. The purpose of this study was to investigate the impact of a high fat diet (HFD) on mammary gland development in obesity-susceptible C57BL/6 and obesity-resistant BALB/c mice., Design: Pubertal or adult C57BL/6 and BALB/c mice were fed an HFD or control diet (CD) from 3 to 7 weeks of age or from 10 to 14 weeks of age, respectively. The effects of HFD diet on body weight, adiposity, mammary gland development, and mammary gland response to estrogen were evaluated., Results: Pubertal C57BL/6 mice fed the HFD had a significant increase in body weight and adiposity, and this was accompanied by stunted mammary duct elongation and reduced mammary epithelial cell proliferation. Ovariectomy and estrogen (17-β-estradiol, E) treatment of pubertal HFD-fed C57BL/6 mice showed decreased mammary gland stimulation by E. Amphiregulin, a downstream mediator of pubertal E action, was reduced in mammary glands of HFD-fed C57BL/6 mice. Weight loss and reduced adiposity initiated by switching C57BL/6 mice from HFD to CD restored ductal elongation. Pubertal BALB/c mice fed the HFD did not exhibit a significant increase in body weight or adiposity; HFD caused increased mammary epithelial cell proliferation and had no effect on response to E. HFD had no effect on body weight or the mammary glands of adult mice., Conclusions: HFD during puberty had a profound strain-specific effect on murine mammary gland development. Obesity and increased adiposity were associated with reduced responsiveness to estrogen and stunted ductal growth. Importantly, the effect of diet and adiposity on the mammary gland was specific to the pubertal period of development.

Estrogen receptor positive (ER+), estrogen (E) responsive MCF-7 breast cancer cells cultured on the extracellular matrix (ECM) protein laminin (LM), exhibit significantly reduced E-induced proliferation compared with cells cultured on collagen I (Col I) that is not due to a loss of ER. Based on reported differences in integrin-activated pathways on Col I vs. LM, we investigated the potential role of Rac1/c-jun-N-terminal kinase (JNK) activation and downstream regulation of cyclin D1 by E on Col I vs.LM. E-induced proliferation was increased on LM in MCF-7 cells expressing constitutively active Rac1 (CA Rac1) and decreased in dominant negative Rac1-(DN Rac1) expressing cells on Col I. siRNA knockdown established the specificity and requirement for Rac1 activation for E-induced regulation of cyclin D1. More robust c-Jun activation occurred on Col I than on LM and E-induced proliferation was abolished after treatment with a JNK inhibitor. These results provide evidence that Rac1/JNK/c-Jun activation promotes E-induced proliferation on Col I and reduced Rac1/JNK/c-Jun activation on LM contributes significantly to reduced E-induced proliferation in MCF-7 cells on LM. These results identify a novel role for extracellular matrix (ECM)-integrin regulation of Rac1-JNK pathway in E-regulated proliferation in ER+ breast cancer cells. These findings suggest that tumor stromal environment, i.e., ECM composition, may contribute to loss of E regulation in ER+ breast cancers. Defining molecular markers for early identification of ER+ tumors that are ER+ but antiestrogen resistant would allow the design and use of alternative therapies to inhibit tumor growth and improve survival.

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

In normal mouse mammary gland, the mitogenic action of progesterone (P) is mediated by two P receptor (PR) isoforms, PRA and PRB. PRA is predominantly expressed in the adult virgin, and PRB is predominantly expressed during pregnancy. To investigate hormonal regulation of PR isoform expression and isoform-specific functions in vivo, adult ovariectomized BALB/c mice were treated for 3, 5, or 10 d with estrogen (E), P, or estrogen plus progesterone (E+P). Using an immunohistochemical approach with isoform-specific antibodies, we investigated hormonal regulation of PRA and PRB and their functional roles in proliferation and morphogenesis. Significant E-induced proliferation was only observed after 5 d at the distal tips of ducts; there was no sidebranching or alveologenesis. P induced proliferation that resulted in sidebranching and alveologenesis, but E+P treatment produced more proliferation sooner and more extensive sidebranching and alveologenesis. PRA levels were increased by E and decreased by P. Increased PRB levels were induced by treatment with P or E+P and coincided with the formation of alveoli. PRA was the predominant PR isoform expressed during sidebranching, and colocalization of PRA with 5-bromo-2'-deoxyuridine revealed that proliferation of PRA-positive and -negative cells was responsible for P-induced sidebranching. PRB was the predominant PR isoform expressed during alveologenesis, and colocalization of PRB with 5-bromo-2'-deoxyuridine showed that both PRB-positive and -negative cells proliferated during alveolar expansion. These results demonstrate different hormonal regulation of PRA and PRB levels in vivo and suggest that P can induce proliferation through either PRA or PRB via direct and paracrine mechanisms.

Menopausal hormone therapy, in particular estrogen plus progestin therapy, has been associated with increased breast cancer risk. In order to understand the basis for increased breast cancer risk, more information is needed about the effects of menopausal hormone therapies on the breast. In this review we describe studies carried out in a mouse model of early vs. late postmenopausal states. We investigated the effects of 1) estrogen alone, 2) combined continuous estrogen + progestin, 3) systemically vs. locally administered estrogen and progestin, and 4) the effect of pregnancy on the response to hormonal therapies. We analysed the effects on mammary gland morphology and proliferation. Estrogen therapy started in late postmenopause caused a greater proliferative response than when started in early postmenopause. In parous, late postmenopausal mice the greater proliferative response to estrogen was not observed. Overall, the greatest proliferative response was observed with combined continuous estrogen + progestin hormone therapy and did not differ significantly in early vs. late nulliparous or parous postmenopausal mice. Both estrogen and progestin were found to act directly on the mammary gland rather than through systemically mediated effects. The possible implications of these findings for menopausal hormone therapy in women and breast cancer risk are discussed.

Epithelial-stromal cell interactions are important for normal development and function of the mouse mammary gland. The steroid hormone estrogen is required for epithelial cell proliferation and ductal development in vivo. Recent studies of estrogen receptor alpha knockout mice indicate that estrogen-induced proliferation is dependent upon the presence of estrogen receptor in mammary stromal cells, but not in epithelial cells. The purpose of the present study was to identify the underlying mechanism of estrogen-dependent stroma-derived effects on mammary epithelium. We have developed a minimally supplemented serum-free medium, collagen gel primary mammary coculture system to address the issue of stroma-derived, estrogen-dependent effects on epithelial cell proliferation. Conditioned medium from mammary fibroblasts or coculture with mammary fibroblasts caused increased epithelial cell proliferation and produced tubular/ductal morphology. Hepatocyte growth factor (HGF) was identified as the mediator of this effect, as the proliferative activity in fibroblast-conditioned medium was completely abolished by neutralizing antibody to HGF, whereas neutralizing antibodies to either epidermal growth factor or IGF-I had no effect. Treatment of mammary fibroblasts with estrogen increased the production of HGF. From these results we conclude that estrogen may indirectly mediate mammary epithelial cell proliferation via the regulation of HGF in mammary stromal cells and that HGF plays a crucial role in estrogen-induced proliferation in vivo.

Extracellular matrix (ECM) proteins have been shown to regulate mammary epithelial cell proliferation, differentiation, and apoptosis in vitro. However, little is known about the hormonal regulation and functional role of ECM proteins and integrins during mammary gland development in vivo. We examined the temporal and spatial localization and hormone regulation of collagen I, collagen IV, laminin, and fibronectin. Among these ECM proteins only fibronectin changed appreciably. Fibronectin levels increased 3-fold between the onset of puberty and sexual maturity, remaining high during pregnancy and lactation. This increase occurred specifically in the epithelial basement membrane. Fibronectin was decreased 70% by ovariectomy and increased 1.5- and 2-fold by estrogen or estrogen plus progesterone treatment, respectively. The fibronectin-specific integrin, alpha(5)beta(1), was localized in myoepithelial cells; it increased 2.2-fold between puberty and sexual maturity and decreased in late pregnancy and lactation. The basal localization of alpha(5)beta(1) was notably increased in pubertal and adult virgin mice. alpha(5)beta(1) concentrations decreased 40-50% after ovariectomy in pubertal and adult mice, which was reversed by estrogen plus progesterone treatment in adult mice. The high basal expression of alpha(5)beta(1) during active proliferation and the low expression in nonproliferating and lactating glands indicate that fibronectin signaling may be required for hormone-dependent proliferation in the mammary gland.

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., (Copyright 2001 Wiley-Liss, Inc.)

Despite the critical importance of ovarian steroids in the treatment of breast cancer, little is known about the acquisition or loss of estrogen and progesterone responsiveness in either the normal or neoplastic mammary gland. This review focuses on the interactions among mammary stroma-derived extracellular matrix (ECM) proteins, integrins and ovarian hormone-dependent proliferation in normal and neoplastic mammary cells both in vivo and in vitro. In vitro studies show that fibronectin is required for progesterone-induced proliferation of normal mammary epithelial cells and that specific ECM proteins also regulate interactions between growth factors and ovarian hormones. Studies with human breast cancer cell lines have shown that laminin inhibits estrogen-induced proliferation and estrogen-response-element-mediated transcription in vitro and also inhibits estrogen-induced proliferation in vivo. Reciprocally, ovarian steroids regulate the expression of ECM proteins and their cellular receptors, integrins, during mammary gland development in vivo. The fibronectin-specific integrin, alpha5beta1 is regulated by ovarian steroids and its expression is positively correlated with developmental stages of peak proliferation. These studies suggest that the coordinated regulation of ovarian hormone responsiveness and ECM/integrin expression may be critical to normal mammary gland development and breast cancer growth and progression.

Breast tumors that lack estrogen responsiveness have a poor prognosis. Despite the critical importance to breast cancer treatment, little is known about the loss of estrogen responsiveness and the development of antiestrogen resistance. We have examined the regulation of estrogen-induced proliferation, estrogen regulation of progesterone receptor (PR) expression, and estrogen signaling pathways in estrogen receptor (ER) positive (MCF-7 and T47D) breast cancer cell lines by specific extracellular matrix proteins (ECM) under serum-free conditions. Estrogen, supplemented with submaximal concentrations of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), stimulated DNA synthesis of MCF-7 cells 7- to 10-fold and T47D cells 2-fold on collagen I or fibronectin. However, estrogen-induced proliferation was greatly reduced on laminin. In contrast, IGF-I or EGF, alone, stimulated proliferation of MCF-7 and T47D cells on all ECM. Thus, ER+ breast cancer cells were not refractory to mitogens when cultured on laminin. Similarly, estrogen induction of PR occurred on fibronectin or collagen I, but not on laminin. While ER content was similar on all ECM, estrogen stimulation of estrogen response element (ERE)-luciferase activity was significantly lower in MCF-7 cells cultured on laminin. Therefore, changes in ECM composition that occur in breast cancer may alter estrogen-responsiveness and the effectiveness of antiestrogen therapies in ER+ breast cancer cells.

Hormone replacement therapy (HRT) with estrogen alleviates menopausal symptoms and is effective in reducing osteoporosis and cardiovascular disease when taken in early postmenopause. Older, late postmenopausal women who never previously received HRT are also believed to benefit from estrogen treatment. On the other hand, increased lifetime exposure of the mammary gland to estrogen may increase the risk of breast cancer. The development of suitable experimental animal model systems can advance our understanding of the effects of estrogen and the timing of HRT on the postmenopausal breast. Toward this end, early and late postmenopausal states were induced in mice by short vs. long term ovariectomy (1 vs. 5 weeks), and the effects of 17beta-estradiol (E) on mammary gland morphology, cell proliferation, and progesterone receptor (PR) levels were investigated. We report that in late postmenopausal mice, E caused a pronounced enlargement of duct ends and 6.5- and 4-fold greater mitogenic responses in the duct end epithelium and adjacent stromal cells, respectively, compared with the response in early postmenopausal mice. Furthermore, after long term, daily treatment with E, steady state levels of proliferation remained 2-fold higher than those of similarly treated, early postmenopausal mice. E failed to increase mammary PR levels in late postmenopausal, but not in early postmenopausal mice. Stimulation of duct ends by E and lack of PR inducibility are characteristics of the immature pubertal mammary gland and indicate that the late postmenopausal mammary gland resembled the immature state. In contrast, minimal E-induced proliferation and increased PR inducibility, characteristics of the adult, sexually mature mammary gland, were retained in early postmenopausal mice. The lack of difference in the numbers of estrogen receptor-positive epithelial or stromal cells or in estrogen receptor cellular concentration after short vs. long term ovariectomy indicates that the observed greater efficacy of E is mediated at a step beyond receptor-ligand binding. This mouse model of experimentally induced early vs. late postmenopausal states should prove useful in better understanding alterations in hormone responsiveness and their implications for timing of HRT on the human breast.

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.

Postnatal mammary gland development is highly dependent on the ovarian steroids, estrogen and progesterone. However, evidence from both in vitro and in vivo studies indicates that steroid-induced development occurs indirectly, requiring stromal cooperation in epithelial proliferation and morphogenesis. Stromal cells appear to influence epithelial cell behavior by secretion of growth factors and/or by altering the composition of the extracellular matrix in which epithelial cells reside. This review will discuss the requirement for stromal tissue in modulating proliferative responses to ovarian hormones during postnatal development and the potential role of the EGF, IGF, HGF and FGF3 growth factor families. Additionally, the roles of extracellular matrix proteins, including fibronectin, collagens and laminin, will be summarized.

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.

Ovarian steroid hormones play a critical role in regulating mammary gland growth and development. The mammary gland sequentially acquires and cyclically exhibits proliferative responses to estrogen and/or progesterone from birth to postmenopause. The focus of this review is to present our current understanding of estrogen and progesterone receptor distribution in epithelial and stromal cells and their functions in relation to mammary gland development. Insights gained from the study of the normal mammary gland are relevant to our understanding of the conditions which may predispose women to the development of breast cancer as well as to alterations in hormonal regulation that occur in breast cancer.

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF-R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone-dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF-R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF-R levels were examined in immature, pubertal 5-week-old and sexually mature 10-week-old female mice. EGF-R were characterized as a single class of high affinity sites and EGF-R concentration was 2-fold higher in glands of 5-week-old mice. Ovariectomy had no significant effect on EGF-R concentration in either age group, and treatment with E and/or P had no effect on EGF-R levels in either epithelial or stromal cells in 5-week-old mice. In contrast, E+P treatment caused a 2-fold increase in receptor concentration in 10-week-old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of of EGF, E, and P.

Several lines of evidence indicate that there may be important stromal influences on epithelial behavior in a number of adult organs. The present studies were undertaken to examine the contribution of mammary gland stroma to mammary epithelial cell hormonal responsiveness in vivo. To accomplish this, mouse mammary epithelium from a developmental state that is not responsive to estrogen (E)-regulation of progesterone receptors (3-week-old) was surgically recombined with mammary stroma from a developmental state that is E-responsive (10-week-old) and vice versa. The recombinants were then tested in vivo for the ability of E to regulate epithelial progesterone receptors. The results demonstrate that when immature nonresponsive epithelium is transplanted to mature stroma of E-responsive mice, the epithelium prematurely acquires E responsiveness. When the converse experiment was carried out, mature E-responsive epithelium retained its responsiveness when transplanted to stroma of immature nonresponsive mice. The contribution of the host systemic milieu to changes observed upon transplantation was also assessed. To do this, mature stroma was transplanted to nonresponsive mice before transplantation with nonresponsive epithelium. Under these conditions, the mature stroma was still able to promote responsiveness. Analysis of mammary gland morphology of the surgical recombinants revealed no significant differences in epithelial structures or organization that could account for the observed differences in hormone responsiveness. From these results we conclude that the local environment of mammary stroma has the capacity to influence mammary epithelial cell behavior and can modulate epithelial hormonal responsiveness in vivo. The mechanisms underlying stromal influences on epithelial behavior remain to be elucidated.

Estrogen-dependent stimulation of progesterone receptor (PgR) concentration or cell proliferation of normal mammary epithelial cells in vitro has been shown to be associated with the presence of mammary fibroblasts. To investigate further the nature of fibroblast influence on epithelial cells, Percoll-purified epithelial cells from collagenase-dissociated mammary glands of mid-pregnant BALB/c mice were co-cultured with mammary fibroblasts that were either untreated, irradiated, or glutaraldehyde-killed or with fibroblast-conditioned medium. Epithelial cells were then assayed for either estrogen-dependent stimulation of PgR by measuring specific [3H]R5020 binding or for estrogen-dependent stimulation of DNA synthesis by [3H]thymidine autoradiography. The results demonstrate that stimulation of PgR does not require the presence of live fibroblasts; either glutaraldehyde-killed fibroblasts or conditioned medium was effective. Pretreatment of culture dishes with type I collagen was equally effective, indicating that fibroblasts may promote the PgR response via a substratum effect. In distinct contrast, estrogen-dependent stimulation of DNA synthesis occurred only when live fibroblasts were present in high numbers and/or in direct contact with epithelial cells. Furthermore, under these latter conditions, epithelial cells also promoted estrogen-dependent stimulation of fibroblast DNA synthesis. Differences in both epithelial and fibroblast cell morphologies were also observed under co-culture conditions, which suggested that cell-cell communication or another interactive phenomenon takes place and is bidirectional. Thus there appear to be at least two different mechanisms by which fibroblasts can influence two specific responses of epithelial cells to estrogen. The present results demonstrate that the specific nature of epithelial-stromal interactions can determine and modulate epithelial cell responses to estrogen and may reflect in vivo regulatory processes affecting normal and neoplastic mammary cells.

Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.

Progesterone is known to stimulate cell proliferation of normal and neoplastic rodent mammary tissues. Recently we have observed that while progesterone is highly effective in sexually mature mice, no significant effect of progesterone can be demonstrated in immature, pubertal mammary gland. In the case of the adult gland, the mitogenic effects of progesterone appear to be related to the presence of E-dependent progesterone receptors (PgR). By contrast, immature mammary glands lack E-dependent PgR. The purpose of the present studies was to study the ontogeny of E-dependent PgR and the factors that lead to the acquisition of responsiveness to progesterone. The results obtained demonstrate that E-dependent PgR are first detectable at 7 weeks of age. Analysis of changes occurring in the mammary gland around this time indicate that E-dependent PgR are not acquired until the pubertal mammary epithelial growth phase has occurred. Furthermore both the growth phase and acquisition of PgR are dependent upon the presence of the ovaries. Analysis of concentration and subcellular distribution of both PgR and estrogen receptors indicate that there are no differences between pubertal and adult mice that could account for the absence of this estrogenic response and lack of E-dependent PgR in immature mammary gland.