Stimulates the production of milk in the mammary glands following childbirth for female

The Glucocorticoid Receptor Gene and Mammary Glands

GCs are known to influence mammary gland function in vivo and to stimulate milk-protein gene expression in cell culture. Mice deprived of GR in their mammary epithelial cells were obtained using a Cre recombinase transgene expressed under the control of the whey acidic protein (WAP) gene promoter (GRWAPiCre). They produce and secrete milk and nurse their litters until weaning, suggesting that GCs do not act on the mammary gland via GR or that they act on other cells than those secreting WAP. The absence of GR reduces cell proliferation, retarding lobulo-alveolar development.

Introduction

The impact of the pituitary on mammary gland function was first discovered in 1928 by Stricker and Grüter when they injected pituitary extracts into ovariectomized rabbits and found that this induced mammary gland development and milk production (Stricker and Grueter, 1928; Riddle, 1940). This discovery was followed by additional analyses of pituitary extracts on the growth of the mammary gland and stimulation of lactation in the 1930s (Nelson, 1935, 1936; Trott et al., 2008). Soon after, several groups identified the pituitary hormone that controls lactation, which we now know as prolactin (Trott et al., 2008). Further supporting the importance of the pituitary in controlling development of the mammary gland, removal of the pituitary (hypophysectomy) abolished lactation in adult guinea pigs (Nelson, 1935). It is now widely accepted that the structure and function of the mammary gland is exquisitely controlled by neuronal and hormonal signaling generated from the hypothalamic/pituitary (HP) axis. Pituitary hormones that directly regulate mammary gland development, such as prolactin, oxytocin and growth hormone, and others that indirectly impinge upon the gland, are necessary for complete morphogenesis and the production and ejection of milk. Neuronal signals from the hypothalamus control synthesis and secretion of these hormones, which in turn relay various signals to the mammary gland. Specifically, prolactin (PRL), growth hormone (GH), and oxytocin (OXT) directly impact the mammary gland by binding to their cognate receptors in the mammary epithelium or mesenchyme. Other pituitary hormones, such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate mammary gland function indirectly by promoting the synthesis and secretion of the ovarian hormones, estrogen and progesterone (Table 1).

Table 1. Impact of pituitary hormones on mammary morphogenesis and lactation

The basic morphological stages of mammary gland development are consistent across mammals and much of what we know about the molecular mechanisms contributing to hormonal regulation of the gland comes from studying developmental processes in genetically engineered mice (Brisken and O’Malley, 2010). Mammary gland development can be divided into three stages: embryonic, pubertal, and pregnancy/lactation (Fig. 7). While the pubertal and pregnancy/lactation stages of development are highly dependent upon the hormonal milieu and input from the HP axis, embryonic mammary morphogenesis is largely independent of systemic hormonal cues. Rather, early formation and growth of the mammary gland depends on paracrine signaling originating in the mesenchyme surrounding the mammary epithelium.

Mammary morphogenesis initiates with the formation of bilateral ectodermal ridges in the ventral skin to form the milk or mammary lines (Macias and Hinck, 2012). These quickly resolve into the mammary placodes, of which there are five on each side of the mouse, by embryonic day 11.5 (E11.5). The placodes then invaginate to form the mammary buds and subsequently primary ductal mammary sprouts. By E18.5 of the mouse, a rudimentary ductal tree has formed that remains morphogenetically quiescent until the onset of puberty. At this stage and those that follow, mammary gland development becomes dependent on hormones synthesized and secreted by the HP axis and its target organs, in particular, the ovary. Hormone-induced elongation and branching of the ductal tree occurs until it extends to the edges of the mammary fat pad. Pregnancy and lactation are considered to be the final stages of mammary development and differentiation. At the onset of pregnancy, tertiary side branching of the ducts and lobuloalveologenesis begins and extends through parturition at which time milk production commences and lactation can occur. When the pups are weaned and lactation ceases, the mammary epithelium undergoes apoptosis and involutes until the gland returns to a near-virgin state both morphologically and molecularly. Remarkably, this process of epithelial expansion, lactational differentiation, and involution can occur repeatedly without exhaustion of the gland and each of these waves of differentiation and remodeling is controlled, in part, by the hormones synthesized and secreted by the hypothalamic/pituitary axis.

1 Introduction

Estrogens, especially estradiol (E2), are essential for mammary gland development and function. E2 has pleiotropic effects on a wide variety of physiological pathways, including the growth and differentiation of estrogen receptor (ER)-expressing epithelial cells. The effects of E2 are mainly mediated through the ER, which is a nuclear receptor that regulates the expression of numerous genes. Two isoforms of ER exist, ERα and ERβ, but ERα has higher expression in the mammary gland and appears to be the principal mediator of estrogen action in this tissue. In fact, studies in knockout mice have highlighted the predominant role of ERα in breast cells. Mature ERα knockout (ERαKO) mice exhibit a rudimentary structured mammary gland with no pubertal development, while knockout of ERβ does not have a significant effect on breast development and growth (Couse & Korach, 1999).

Similar to other nuclear receptors, the ER consists of functional and structural domains. The N-terminal region (domain A/B) includes the transactivation domain, AF1. The central region (domain C/D) contains the well-conserved zinc finger DNA-binding domain (DBD) and the nuclear localization signal. The C-terminal region (domain E/F) contains the ligand-binding domain (LBD), dimerization sites, the AF2 ligand-dependent transactivation domain, and cofactor interaction surfaces (Fig. 4.1). Classically, ligand binding induces dimerization and activation of the receptor, which then binds to estrogen-responsive elements (EREs) contained in the promoters of target genes. The conformational changes in the receptor induced by ligand binding allow the recruitment of transcriptional coactivators or corepressors, such as CBP/p300, SRC, and NCOA1, via its AF1 and AF2 domains. However, there is growing evidence suggesting that ERα actions are not just mediated by direct binding to DNA. In fact, the receptor can also interact with transcription factors, such as specific protein 1 (Sp1), activator protein 1 (Ap1), or nuclear factor-kappa B (NFκB), already bound to DNA. These interactions partially explain why ERα is capable of interacting with gene promoters that do not contain EREs, which represent one-third of E2-target genes (Stender et al., 2010). Recently developed technologies, such as chromatin immunoprecipitation followed by high-throughput sequencing, have allowed the identification of thousands of ERα recruitment sites across the genome, which defines its cistrome. Genome-wide studies of ERα binding have led to the conclusion that most ERα-mediated gene regulation is the result of long-range binding of the receptor to distal cis-regulatory elements (Carroll et al., 2005). In addition, ERα recruitment patterns are cell-type specific and can differ upon various stimuli. In fact, the ERα cistrome is controlled by the activity of the so-called pioneer factors, such as FOXA1 or GATA3, which are transcription factors capable of interacting with compacted chromatin, thereby modulating chromatin accessibility for other proteins. In addition, both E2 and ERα are able to mediate nongenomic actions that participate in estrogenic effects, which influence the physiology of many target cells and tissues. Indeed, an ER subpopulation is localized to the cytosol or the plasma membrane and is able to rapidly activate intracellular signaling pathways, including MAPK and PI3K, via interactions with adaptor proteins, such as MNAR, or with growth factor receptors, such as IGFR, epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2). These nongenomic effects modulate the activities of several transcription factors, including ERα itself and its cofactors (Levin & Pietras, 2008; Fig. 4.1). As discussed later in this chapter, these nongenomic actions and cross talk with growth factor receptors are speculated to be involved in resistance to endocrine therapies.

Regardless, binding of E2 to its receptor promotes the proliferation and survival of ER-expressing cells by stimulating the expression of antiapoptotic and promitotic genes (Boudot, Kerdivel, et al., 2011; Kerdivel, Boudot, & Pakdel, 2013). The mitogenic action of E2 also favors the generation of mutations during replication and, consequently, can participate in tumor transformation (Platet, Cathiard, Gleizes, & Garcia, 2004). More than 70% of mammary tumors are positive for ERα expression and respond to estrogen signals. Moreover, it has been reported that the ERα gene (ESR1) can be amplified in breast cancers, but the frequency of this event is still under debate (Burkhardt et al., 2010; Holst et al., 2008; Moelans et al., 2010). In addition, amplification of ESR1 is often correlated with high ERα expression levels and better responses to endocrine therapies (Holst et al., 2007). On the other hand, ERα also has a role in maintaining a well-differentiated epithelial phenotype that contributes to making ERα-positive tumors less aggressive than ERα-negative tumors. ERα-positive tumors are generally separated from adjacent tissues by a basal layer and grow locally under estrogen stimulation. As a consequence, these tumors are frequently less invasive and metastatic than ERα-negative cancers.

Despite its implications in breast cancer development and progression, expression of ERα is generally a good prognostic marker. Indeed, ERα-expressing tumors are mostly sensitive to endocrine therapies. Currently, most endocrine therapies are targeted at blocking the ER signaling pathway at different levels; one strategy consists of depriving the receptor of estrogen by inhibiting aromatase or by ovarian ablation, while another strategy directly targets the ER with either selective ER modulators, such as tamoxifen, or selective ER downregulators, such as fulvestrant (also known as Faslodex or ICI182,780). Endocrine therapies have been shown to be effective for patients with ER-positive tumors and are at least partially responsible for the constant decrease observed in breast cancer mortality over the last few decades (Early Breast Cancer Trialists’ Collaborative Group (EBCTCG), 2005; Siegel, Naishadham, & Jemal, 2012). In addition, endocrine therapies are selective and less toxic relative to other anticancer therapies. Unfortunately, patients treated with endocrine therapies will frequently relapse within 15 years and develop endocrine resistance.

Hormonal escape is generally associated with loss of the epithelial phenotype and acquisition of an invasive and migratory phenotype (Bandyopadhyay, Wang, Chin, & Sun, 2007). Tumor cells undergo an epithelial–mesenchymal transition (EMT), followed by local invasion of surrounding tissues. In a second time, cells can enter into the general circulation and migrate to form metastasis. Several components of growth factor pathways are generally overexpressed or overactivated in hormone-resistant breast cancer cells, explaining at least in part the E2-independent proliferation of these cells (Massarweh & Schiff, 2006). The progesterone receptor is also often downregulated in hormone-resistant tumors. One explanation for endocrine therapy resistance is the loss or downregulation of ER, as demonstrated by histology (Allred, Brown, & Medina, 2004). ERα expression, in normal or pathological breast epithelial cells, is finely regulated at numerous stages, including the gene, mRNA, and protein levels. Consequently, many molecular mechanisms may be implicated in the decrease or loss of ERα in mammary cancer. However, approximately 30% of ERα-positive breast cancer cells do not respond to first-line endocrine therapies, and a majority of relapsing tumors still expresses ERα (Gutierrez et al., 2005; Johnston et al., 1995). Thus, it is only logical to postulate the existence of mechanisms that enhance or fully bypass the classical estrogenic response and that result in antiestrogen resistance.

Modulation of estrogen signaling and ERα expression can occur through a variety of both direct and indirect mechanisms, including altered expression, breast cancer stem cells, and cross talk with growth factor signaling pathways. Mechanisms such as these, which can influence ERα activity and response to endocrine therapies during breast cancer tumorigenesis and progression, will be discussed in this chapter (Fig. 4.2).

Changes in relative and absolute concentrations of immunoglobulins during lactation

Table 103.3 indicates that the transitions from colostrum to mature milk are associated with changes in absolute and relative immunoglobulin concentrations suggesting changes in MG function. Among Group III mammals, the drop in concentration is also associated with a change in IgG: IgA ratios. For example, in swine it changes from 6 to 0.3, in horses from 9 to 0.5, but in cattle only from 10 to 6. Although IgA levels in human and rabbit lacteal secretions also drop precipitously, there is only a slight change in IgG: IgA ratios. The precipitous decline in IgG in the lacteal secretions of farm animals immediately after parturition is paralleled by an increase (a rebound) in serum IgG levels that had steadily declined during the last month of gestation (Guidry et al., 1980a; Fig. 103.4). This decline in serum IgG before parturition was shown to result from active transport of IgG (IgG1 in cattle) from blood into the colostrum-forming MG at the end of gestation (Dixon et al., 1961). Because in cattle the serum level of IgG2 does not change during the reproductive cycle (Guidry et al., 1980a), it suggests that active IgG transport in cattle is restricted to IgG1. As discussed later, transport of mouse IgG subclasses is inversely correlated with their affinity for FcRn on epithelial cells (Cianga et al., 1999). Should a similar phenomenon explains IgG transport in ruminants, it suggests that IgG2 is recognized and recycled back into serum while IgG1 is not, although it must be recognized by some receptor that facilitates transepithelial transport into lacteal secretions. The origin of lacteal immunoglobulins either as (1) products of active transport from serum or (2) local synthesis can be distinguished from serum transudates by normalizing the concentration of lacteal immunoglobulins (or immunoglobulins in any secretion) to those of albumin, because albumin appears in secretions totally by passive transudation (Guidry et al., 1980a; Butler et al., 1983; Fig. 103.5). Using this yardstick, relative occurrence values greater than 1.0 indicate that a particular immunoglobulin is either locally synthesized or actively transported from serum to the secretion in question. Indices of less than 1.0 indicate the immunoglobulin is derived by transudation from serum, which typically results from weakened tight junctions in the distended gland. Applied to Fig. 103.5, IgM and IgG in human milk and colostrum can be explained by transudation; this result is consistent with organ culture studies (Hochwald et al., 1964). In all three species, IgA is either actively transported from serum or locally synthesized, whereas after 2 days postpartum, IgG2 in cattle and total IgG in swine result from serum transudation. Before this time, IgG in swine and IgG1 in cattle are either actively transported from blood or locally synthesized. Thus, Figure 103.5 illustrates both species diversity and evolutionary conservation. Conservation is demonstrated by the predominance of

IgA in all three species when measured as relative occurrence; this is true even in cattle in which absolute IgG1 levels exceed IgA levels 10:1 (Table 103.3). This suggests a conserved function for lacteal IgA in all three species. Diversity is illustrated by differences in the relative occurrence of IgG and/or IgG subclasses among the three species. The situation for IgG in swine versus humans is easiest to comprehend in terms of the different pathways used for delivering IgG passively to the newborn (Fig. 103.1). After the transition from colostrum to mature milk, IgG levels precipitously drop so that IgA constitutes 80% to 95% of the immunoglobulin in the mature milk of humans and swine (Table 103.3). By contrast, IgG1 levels and the relative occurrence of IgG1 in lacteal secretions of cattle remain high throughout lactation (Fig. 103.5), suggesting that IgG1 in cattle has a different function than IgG1 in swine and humans. Another example of diversity concerns IgM. Although ascribed to serum transudation in humans, relative occurrence suggests that IgM in cattle and swine is actively transported and/or locally produced. The former notion is consistent with the decline in serum IgM before parturition in swine (Fig. 103.4), whereas the latter is supported by immunohistochemical studies showing plasma cells of all major bovine isotypes (IgG1, IgG2, IgM, and IgA) in the MG, particularly 14 days prepartum (Sordillo and Nickerson, 1988).

Because the gut of newborn piglets and calves is “closed” to the indiscriminate absorption of protein after 12 hours and the capacity of the gut of human infants, even at birth, to absorb intact proteins is very low, the high relative occurrence of IgA in the mature milk of all three species, of IgM in cattle and swine, and of IgG1 in cattle suggests that the presence of these immunoglobulins is either related to immune defense of the lactating MG or that they act within the gut lumen of the suckling neonate.

More modest changes in IgG: IgA ratios are also seen in rodents, whereas carnivores, also placed in Group II, show changes similar to those in swine (Fig. 103.5; Heddle and Rowley, 1975). In rats, in which enterocytes actively transport IgG for 3 weeks, IgG2a levels can exceed IgA levels 1 week after parturition and a ratio of 1:5 is maintained during suckling (McGhee et al., 1975).

Secretion rates also change during lactation. In humans, ∼100 ml/day are secreted on day one, 500 ml/day on day five, and more than 1 liter/day after 4 weeks (McClelland et al., 1978). Also, day five milk contains 40% to 50% casein, whereas in colostrum casein accounts for ∼10% of total proteins in humans. Changes in secretion rates translate into differences in output. The daily output after 4 weeks in women is less than 1 g IgA, in swine it is greater than 30 g IgA, and amounts to ∼3 g IgA and 6 g IgG1 in cattle (Guidry et al., 1980a; Beyer et al., 1986; Lawrence, 1994; Sinclair et al., 1996).

Normal Breast Development

Studies of breast cancer from the 1970s to the mid-1990s focused mainly on changes in breast cancer with little regard for normal tissue or development. A lack of knowledge of normal mammary gland development and function limited the understanding of tumor-specific changes. In 1998, the NCI-directed Breast Cancer Progress Review Group stated, “Our limited understanding of the biology and developmental genetics of the normal mammary gland is a barrier to progress.”1 This statement led to a major increase in such studies, with mouse models giving invaluable insights into the molecular biology of both normal mammary gland development and breast cancer.2 Extensive genetic and molecular analysis of mammary gland development in small and large animals has rapidly defined many of the intricate molecular networks, such as interactions between steroid hormone and growth factors, that are critical for all stages of development and function.3 Intriguingly, many of these same pathways have major roles in breast cancer development and progression and thus are major therapeutic targets.4 One of the greatest advances has been the recent identification and characterization of mouse mammary stem cells. Sorting cell populations using cell surface markers has shown that the myoepithelial cell layer contains adult mammary stem cells and that a single cell transplanted into the mouse can produce every epithelial cell of the mammary gland.5,6 Evidence that mammary stem cell number and function are regulated by hormones such as progesterone and RANK ligand is consistent with the major functions of hormones in mammary gland function and may have implications for human breast cancer development and treatment.7,8 Intriguingly, BRCA1 has also been found to regulate mammary stem cell number and function,9 and evidence suggests that BRCA1 cancers may arise from a blockade of progenitor cell development.10

Publisher Summary

The chapter studies the anatomy of the endocrine system and the non-neoplastic changes associated with mammary glands. A mammary gland is a sensitive indicator of the functional activity of the hypothalamic-pituitary-gonadal axis, and its histological assessment provides important information about the compounds that modulate this activity. In rodents, the assessment of drug-induced changes in the mammary glands is complicated by species differences in hormonal regulation of mammary gland development and function, perhaps compounded by the artificial physiological state of overfed laboratory animals. The development and function of the mammary gland is under the control of a constellation of hormones that includes the sex steroids estrogen and progesterone, prolactin and growth hormone, insulin, catecholamines, and ACTH. A mammary gland is composed of a system of alveolar buds or acini connected by a system of branching ducts to the main ducts that converge on the nipple. Duct and alveolar tissues are not static structures, but respond to hormonal changes of the estrous cycle, pregnancy, and lactation as well as those that accompany aging, changes in diet, and environmental conditions. The usual safety studies have shown that a mammary gland is a relatively uncommon site of spontaneous inflammation, although it can be induced by the injection or implantation of foreign materials.

Summary

Human GH, human prolactin, and human placental lactogen share an intricate evolutionary relationship that created a network of multiple related forms of these proteins likely arising from a common ancestral gene. All three hormones share structural homology in their protein sequence, and in their binding to their canonical receptors with significant crossreaction between each subtype. Both human GH and human prolactin are produced and secreted by pituitary acidophil cells under a complex regulatory system that involves hypothalamic and other neural peptides, as well as metabolic regulators of secretion, and neither has functions in the fetus and newborn that are typically seen in the adult. The formation of the pituitary is under a complex system of transcription and other factors, and mutations in these gene encoding these factors result in distinct patterns of hormone deficiencies that, in the case of human GH and human prolactin, become apparent only after birth. Their role in fetal growth and mammary gland function is marginal. Both appear quite early in gestation, with high concentrations at birth that decline postnatally, as the inhibitory effects of somatotropin release–inhibiting factor and l-dopa and maturation of their receptors and signal transduction cascades become established over a period of 3 to 6 months. For “normal” pituitary GH, the primary role in the fetus and newborn appears to be the metabolic actions of lipolysis, amino acid incorporation into muscle, and “antiinsulin” effects that protect the fetus/newborn from hypoglycemia and ensure an adequate nutrient supply; this role is further magnified by human placental lactogen, and by human GH variant, both secreted by the syncytiotrophoblast. Human placental lactogen circulates in the mother at concentrations 103-fold higher than those of human GH and ensures fuel supplies to the fetus even under conditions of maternal nutritional deficiencies such as fasting; human GH variant plays a similar role. The functions of fetal human prolactin are poorly defined, although roles in regulating amniotic fluid osmolality and lung maturation have been proposed.

This chapter has summarized the molecular development of the pituitary and the patterns of hormone deficiencies, which may arise as a result of mutations in the developmental cascade of genes and transcription factors that act to create the normal structure and function of this organ in the fetus and newborn. This knowledge has proven invaluable in recognizing patterns of hormone deficiencies in the newborn; future studies may unravel the physiologic significance of their roles in the fetus and newborn.

Diet-Induced Obesity Impairs Mammary Gland Development and Function

Obesity in dairy cows has long been known to impair milk production,491 and rodent studies show similar effects.443,492 When an HF diet was employed for 16 days prior to puberty in the mouse, one group found that the branching frequency and the width of mammary ducts were reduced along with the presence of abnormal myoepithelial cells in virgin mice.492 In another study, lobuloalveolar development of the mammary gland was impaired when HF feeding was begun after puberty.443 Morphologically, these authors reported abnormal side branching and alveolar development by day 14 of pregnancy. In addition they showed a significant difference in mammary gland weight due to the increased size of the fat pad. Decreased pup weight, depressed milk synthesis genes, and the retention of large CLDs in the epithelium by day one of lactation suggested an inherent impairment in milk synthesis.443 Together, these studies support the concept that diet-induced obesity can interfere with mammary gland function at different stages of development, but they fail to differentiate the effects of chronic HF feeding and obesity.

Unlike its effects on mammary gland development, the impact of HF feeding and obesity on milk production and composition has been relatively well characterized in rodents. The overall consensus is that diet-induced obesity impairs milk production early in lactation.443,490,493 Observations of delayed pup growth and sometimes even death479 within the first few days after parturition were an indication of decreased milk production and/or secretion. Additionally, milk composition is altered by HF feeding and obesity.22,488 As mentioned previously, milk lipid composition is reflective of dietary lipids. However, it has also been shown that HF feeding can increase milk lipid content480,490,493 as well as decrease milk protein.443,480 However, in one study HF feeding during lactation actually led to decreased milk fat production.445 Additionally, feeding conjugated linoleic acid or transfatty acids during lactation has resulted in suppressed milk fat production.22,486,494 Therefore, the type of dietary fat may prove to be a determining factor in how milk composition is affected.

The effects of high-fat feeding and obesity during lactation on neonatal growth have also been inconsistent441,443,444,495,496 with some studies reporting impaired neonatal growth linked to lactation defects443,444 and others reporting increased neonatal growth correlating with an obese phenotype.495,496 These inconsistencies suggest that the impact of HF feeding and obesity may vary with the stage of mammary development.

Synergy of Endocrine Hormone Action in Normal Mammary Physiology

Given that the normal mammary gland’s primary function is to lactate and produce milk, it is essential to appreciate the endocrinological influences and important processes of the female lifespan that influence mammary gland development and lead to lactation. Over the course of her lifetime, a female will undergo birth, puberty, possible pregnancy and lactation, and menopause, all of which are endocrinologically mediated through different patterns of steroid and cytokine hormone stimulation that synergize with downstream growth factors and receptors, to mediate onset and ending and influence mammary gland development and function (Fig. 3). During the embryonic stage of female life, a rudimentary mammary gland develops from ectoderm and mesoderm germ layers influenced by PTHrP action. Neonatal milk production can occur transiently at birth in both sexes, due to high levels of maternal hormones passing through the placenta to stimulate the neonatal mammary tissue. Mammary gland tissue is then generally quiescent until puberty. Release of gonadotropin releasing hormone (GNRH1) from the hypothalamus triggers the pituitary to secrete gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which then stimulate the ovary to cyclically produce estrogens and progesterone. Their cyclic production acts on mammary ductal tissue to stimulate development of terminal end buds that then travel through the mammary fat pad to lead to the development of a branched ductal tree in the mammary gland by the end of puberty (Paine and Lewis, 2017). Mammary epithelial cells experience cyclic rounds of growth stimulation and regression with each menstrual cycle dictated by waves of higher and lower levels of estrogens and progesterone. During pregnancy, a surge of estrogen and progesterone from the ovaries, corpus luteum and placenta stimulate the anterior pituitary to increase production of prolactin and this coupled with synthesis of placental lactogens from the placenta and local production of IL-4 and IL-13 results in milk production and secretion into the alveolar lumens. Milk becomes available to the offspring upon suckling of the nipple. Nipple sucking stimulates the pituitary gland to release the hormone oxytocin (OXT) stimulating contractile action that moves the milk from the alveolar lumens through the ducts to the nipple for ingestion by the offspring. When the offspring are weaned from the mother and the milk is no longer removed from the gland, involution is triggered. A series of changes in hormonal stimulation including immediate changes in patterns of STAT5/STAT3 activation occur, which is followed by resolution of hormone levels to cyclical pre-pregnancy patterns (Li et al., 1997). Menopause is reached when the ovarian follicle reserve is exhausted and cyclical production of estrogens and progesterone no longer occurs (Hale et al., 2013) and the mammary epithelium regresses. In humans breast density decreases as the proportion of fatty as compared to collagenous stromal tissue increases. Significantly, risk of breast cancer development actually increases with age, coincident with menopause and loss of cyclic hormonal stimulation.

Progesterone

Clinical and experimental studies confirm that progesterone plays a key role in uterine leiomyoma growth and development (see Fig. 131-1).55 Progesterone acts via PR, a member of the nuclear hormone superfamily of ligand-activated transcription factors.56,57 The two predominant PR isoforms, PR-A and PR-B, are transcribed from the same gene by two distinct promoters, resulting in differently sized proteins and different transcriptional activities.58-66 Selective ablation of PR isoforms in mice showed that PR-A is necessary for ovulation and modulates the antiproliferative effects of progesterone in the uterus, and PR-B is required for normal mammary gland development and function.67,68 Several studies have reported increased expression of both PR-A and PR-B in leiomyoma tissue compared with adjacent normal myometrial tissue, but the specific role of each PR isoform in uterine leiomyoma has yet to be fully elucidated.69-71

Clinical studies have shown that, in postmenopausal women, leiomyoma proliferation increases significantly with hormone replacement therapy with combined estrogen and progesterone, but not with estrogen replacement alone, supporting a key role for progesterone in leiomyoma growth.39 Additionally, proliferation markers and mitotic counts are highest in leiomyoma tissue during the luteal/secretory phase, when circulating progesterone is highest.39,72 Despite the belief that progesterone is the primary hormone driving the growth of uterine leiomyoma, its specific role in the pathogenesis of leiomyoma is not completely understood. In cultured leiomyoma cells, treatment with antiprogestins decreases proliferation by downregulating several growth factors and their receptors, including IGF-I, EGF, TGFβ3, VEGF-A, and VEGF-B.73-78 These treatments also induce apoptosis through activation of multiple differential apoptotic pathways, including the tumor necrosis factor-related apoptosis-inducing ligand-mediated signaling pathway and the endoplasmic reticulum stress-induced pathway,79,80 and by reducing antiapoptotic protein BCL2 and stimulating expression of cleaved caspase-3 and cleaved PARP.76 Furthermore, antiprogestins decrease extracellular matrix formation by increasing extracellular matrix metalloproteinase inducer, MMP-1, MMP-8, and membrane type 1-MMP protein contents, and decreasing TIMP-1, TIMP-2, type I, and type III collagen protein levels.81,82 Most recently, in an in vivo human leiomyoma xenograft model, Qiang et al83 demonstrated that estrogen plus progesterone, but not estrogen alone, induces extracellular matrix production via downregulation of miR-29b. Although grafts containing control vector-transduced cells gave rise to typical solid leiomyoma tumors, leiomyoma grafts with ectopic mir-29b expression failed to form solid tumors in the presence of estrogen plus progesterone,83 supporting the hypothesis that dysregulation of mir-29b plays an important role in tissue fibrosis and tumor formation.84-88

Genomic studies have also shed some light on progesterone’s role in leiomyoma pathogenesis. Gene expression of the tumor suppressor Kruppel-like transcription factor 11 (KLF11) is significantly lower in leiomyoma tissues compared with adjacent myometrial tissues, and the CpG island in the KLF11 gene promoter is hypermethylated in leiomyoma tissue.89 Therefore, dysregulation of KLF11 may be a key factor involved in uterine fibroids. Using a genome-wide approach, a PR-binding site was found 20.5 kb upstream of KLF11 gene promoter, and KLF11 knockdown markedly increased leiomyoma cell proliferation.89 In primary cultures of uterine leiomyoma cells, treatment with the antiprogestin mifepristone robustly regulates the protein and mRNA levels of KLF11 by enhancing recruitment of Sp1, RNA polymerase II, PR, and its coactivator SRC-2 to both the distal enhancer and basal promoter region of the KLF11 gene. It is unclear whether these activities can alter KLF11 methylation status. Additionally, using a ChIP-sequencing assay, another novel PR target gene perilipin 2 (PLIN2) was discovered in leiomyoma cells.90 PLIN2 normally plays an important role in lipid droplet formation and stabilization, but its loss is linked to the expression of fibrogenic genes in hepatic fibrosis.91-93 Moreover, in clear cell renal carcinoma, higher PLIN2 expression is associated with better cancer-specific survival and cancer-free survival.94 These findings strongly suggest that progesterone-mediated expression of KLF11 and PLIN2 may regulate both cell proliferation and extracellular matrix formation.

Four antiprogestins—mifepristone (RU 486), asoprisnil (J867), ulipristal acetate (CDB2914), and telapristone acetate (CDB4124)—have been shown to reduce leiomyoma size and to improve the quality of life in women with uterine leiomyoma.95-100 The newest antiprogestin to be studied, ulipristal acetate, has shown promising results. In a clinical trial comparing the efficacy and side-effect profile of ulipristal acetate to GnRH agonist, ulipristal acetate provided more prolonged tumor volume reduction after termination of treatment, although leuprolide acetate caused greater reduction in fibroid volume overall,16,98,99,101 and ulipristal acetate was better tolerated with a significantly lower incidence of hot flashes, diminutive effect on bone density, and less profound suppression of estradiol levels.101,102 The biggest concern about antiprogestin treatment of uterine leiomyoma is endometrial thickening from unopposed estrogen action in the endometrium. An NIH-sponsored study specifically evaluated the endometrial histologic changes following treatment with mifepristone, asoprisnil, or ulipristal acetate and reported little evidence of mitosis and atypical hyperplasia; however, there was asymmetry of stromal and epithelial growth and prominent cystic, dilated glands, designated as progesterone receptor modulator-associated endometrial change (PAEC).96,103,104 The long-term significance of the morphological changes is unclear.