Hormonal Regulation of the Immune Microenvironment in the Mammary Gland

It is well established that the development and homeostasis of the mammary gland are highly dependent upon the actions of ovarian hormones progesterone and estrogen, as well as the availability of prolactin for the pregnant and lactating gland. More recently it has become apparent that immune system cells and cytokines play essential roles in both mammary gland development as well as breast cancer. Here, we review hormonal effects on mammary gland biology during puberty, menstrual cycling, pregnancy, lactation and involution, and dissect how hormonal control of the immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, mast cells and lymphocytes. Collectively, these alterations may create an immunotolerant or inflammatory immune environment at specific developmental stages or phases of the menstrual cycle. Of particular interest for further research is investigation of the combinatorial actions of progesterone and estrogen during the luteal phase of the menstrual cycle and key developmental points where the immune system may play an active role both in mammary development as well as in the creation of an immunotolerant environment, thereby affecting breast cancer risk.     

Progesterone Signaling and Mammary Gland Morphogenesis

Progesterone was identified as a mammogenichormone several years ago but until now its precise rolein mammary development has remained obscure. Recentlywith the generation of several transgenic mouse models and development of reagents for analysisof progesterone receptor expression, the role ofprogesterone signaling in mammary development isbecoming more clear. The most significant observationsto emerge from these studies are (1) progesteronereceptors (PR)4 are present in a heterogeneous manner inthe epithelial cells and undetectable in the surroundingfat pad; (2) they are essential for lobuloalveolar and not for ductal morphogenesis; (3)progesterone signaling through progesterone receptors,leading to lobuloalveolar development, is initiated inthe epithelium and may occur through paracrinemechanisms; and (4) a regulated expression of the twoisoforms of progesterone receptor is critical formaintaining appropriate responsiveness to progesteroneand hence, epithelial cell replicative homeostasis.These studies also reveal that the consequences ofprogesterone signaling through progesterone receptor maydepend on the cell context, cell-cell andcell-extracellular matrix interactions, the dynamics ofPR turnover and the fate of PR positivecells.     

A Reappraisal of Progesterone Action in the Mammary Gland

The ovarian hormones estrogen and progesterone and their respective receptors are essential for maintenance of postnatal developmental plasticity of the mammary gland and play a key role in mammary tumorigenesis. Mouse models in which expression of the progesterone receptors was genetically ablated have recently become available. Studies of these models have demonstrated that progesterone is specifically required for pregnancy associated ductal proliferation and lobuloalveolar differentiation of the mammary epithelium, but not for immediate postpubertal ductal morphogenesis. Use of these mice in combination with mammary gland transplantation indicates that developmental regulation by progesterone appears to occur through a paracrine mechanism in which progesterone receptor (PR)³ positive cells represent a subset of non-proliferating epithelial cells that are capable of directing proliferation and/or differentiation of neighboring receptor negative cells. The hierarchical organization of these receptors in the epithelium and their segregation from proliferating cells is a conserved feature in rodent and human mammary tissue. The identification of paracrine mediators of the progesterone response is now an imminent goal as is the delineation of the individual contributions of the two PR isoforms using similar approaches.     

IGF-I, GH, and Sex Steroid Effects in Normal Mammary Gland Development

Although the pubertal surge of estrogen is the immediate stimulus to mammary development, the action of estrogen depends upon the presence of pituitary growth hormone and the ability of GH to stimulate production of IGF-I in the mammary gland. Growth hormone binds to its receptor in the mammary fat pad, after which production of IGF-I mRNA and IGF-I protein occurs. It is likely that IGF-I then works through paracrine means to stimulate formation of TEBs, which then form ducts by bifurcating or trifurcating and extending through the mammary fat pad. By the time pubertal development is complete a tree-like structure of branching ducts fills the rodent mammary fat pad. In addition to requiring IGF-I in order to act, estradiol also directly synergizes with IGF-I to enhance formation of TEBs and ductal morphogenesis. Together they increase IRS-1 phosphorylation and cell proliferation, and inhibit apoptosis. In fact, the entire process of ductal morphogenesis, in oophorectomized IGF-I^(−/−) knockout female mice, can occur as a result of the combined actions of estradiol and IGF-I. IGF-I also permits progesterone action in the mammary gland. Together they have been shown to stimulate a form of ductal morphogenesis, which is anatomically different from the kind induced by IGF-I and estradiol. Although both progesterone and estradiol synergize with IGF-I by increasing IGF-I action parameters, there must be other, as yet unknown mechanisms that account for the anatomical differences in the different forms of ductal morphogenesis observed (hyperplasia in response to IGF-I plus estradiol and single layered ducts in response to IGF-I plus progesterone). From the Bunnie Joan Sachs Laboratory, VA Medical Center, New York, NY, USA Supported in part by grants from DOD W81XWH-07-1-0488 and the Foundation for Growth and Endocrinology     

Hormonal regulation of mammary differentiation and milk secretion

The endocrine system coordinates development of the mammary gland with reproductive development and the demand of the offspring for milk. Three categories of hormones are involved. The levels of the reproductive hormones, estrogen, progesterone, placental lactogen, prolactin, and oxytocin, change during reproductive development or function and act directly on the mammary gland to bring about developmental changes or coordinate milk delivery to the offspring. Metabolic hormones, whose main role is to regulate metabolic responses to nutrient intake or stress, often have direct effects on the mammary gland as well. The important hormones in this regard are growth hormone, corticosteroids, thyroid hormone, and insulin. A third category of hormones has recently been recognized, mammary hormones. It currently includes growth hormone, prolactin, PTHrP, and leptin. Because a full-term pregnancy in early life is associated with a reduction in breast carcinogenesis, an understanding of the mechanisms by which these hormones bring about secretory differentiation may offer clues to the prevention of breast cancer.     

Use of PRKO Mice to Study the Role of Progesterone in Mammary Gland Development

To better understand the distinct physiological roles played by progesterone and estrogen receptors (PR amd ER)⁴ as well as to study directly PR function in an in vivo context, a novel mutant mouse strain, the PR knockout (PRKO) mouse, was generated carrying a germline loss of function mutation at the PR locus. Mouse mammary gland development has been examined in PRKO mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen and progesterone treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Stimulation of ductal development by the PR may, therefore, be mediated by an unknown secondary signaling molecule, possibly a growth factor. The continued stimulation of the stromal PR appears to be dependent on reciprocal signal(s) from the epithelium. Thus, the combination of gene knockout and reciprocal transplantation technologies has provided some new insights into the role of stromal-epithelial interactions and steroid hormones in mammary gland development.     

Establishing a framework for the functional mammary gland: from endocrinology to morphology

From its embryonic origins, the mammary gland in females undergoes a course of ductal development that supports the establishment of alveolar structures during pregnancy prior to the onset of lactogenesis. This development includes multiple stages of proliferation and morphogenesis that are largely directed by concurrent alterations in key hormones and growth factors across various reproductive states. Ductal elongation is directed by estrogen, growth hormone, insulin-like growth factor-I, and epidermal growth factor, whereas ductal branching and alveolar budding is influenced by additional factors such as progesterone, prolactin, and thyroid hormone. The response by the ductal epithelium to various hormones and growth factors is influenced by epithelial–stromal interactions that differ between species, possibly directing species-specific morphogenesis. Evolving technologies continue to provide the opportunity to further delineate the regulation of ductal development. Defining the hormonal control of ductal development should facilitate a better understanding of the mechanisms underlying mammary gland tumorigenesis.     

Mammary Gland Development and Tumorigenesis in Estrogen Receptor Knockout Mice

Estrogens are important for the development of the mammary gland and strongly associated with oncogenesis in this tissue. The biological effects of estrogens are mediated through the estrogen receptor (ER),³ a member of the nuclear receptor superfamily. The estrogen/ER signaling pathway plays a central role in mammary gland development, regulating the expression and activity of other growth factors and their receptors. The generation of the ER knockout (ERKO) mouse has made it possible to directly understand the contribution of ER in mammary development and has provided an unique opportunity to study estrogen action in carcinogenesis. A mammary oncogene (Wnt-1) was introduced into the ERKO background to determine if the absence of the ER would affect the development of tumors induced by oncogenic stimulation. The development, hyperplasia, and tumorigenesis in mammary glands from the ERKO/Wnt-1 mouse line are described. These studies provide the impetus to evaluate the effect of other oncogenes in mammary tumorigenesis in the absence of estrogen/ER signaling.     

Hyperplasia, reduced E-cadherin expression, and developmental arrest in mammary glands oxidatively stressed by loss of mitochondrial superoxide dismutase

To investigate the dysregulating effect of excess oxidative stress on mammary gland development, mammary anlage from newborn female mice with normal (+/+) or absent (null, -/-) manganese superoxide dismutase (SOD2) were excised and implanted under the renal capsule of normal host female nude mice with/without concurrent estrogen supplementation. After 30 days the transplanted glands were excised for wholemount, microscopic and immunohistochemical evaluation. In contrast to the normal growth and maturation of transplanted SOD2+/+ glands, SOD2-/- glands showed arrested development, reduced ductal outgrowth and branching, and absent lumen. These hypomorphic SOD2-/- ducts contained hyperplastic epithelium with increased Ki-67 labelling, loss of E-cadherin expression, and disorganized p63 and cytokeratin (K)-14 expressing basal and myoepithelial components. Estrogen treatment failed to upregulate progesterone receptor or normalize development. These findings suggest that excess oxidative stress from loss of SOD2 function can arrest mammary gland maturation and induce hyperplastic epithelium with early premalignant features.     

Steroid Receptors and Cell Cycle in Normal Mammary Epithelium

The ovarian steroids estrogen and progesterone (E(2) and P) are essential for normal mammary gland growth and development; however, the mechanisms by which they influence the proliferative activity of the mammary epithelium remain unclear. Mammary epithelial cells cells expressing the receptors for E(2) and P (ER and PR respectively) are separate from, although often adjacent to, those capable of proliferating, implying that the ovarian steroids act indirectly via paracrine or juxtacrine growth factors to stimulate entry into the cell cycle. A large number of candidate factors have been identified in a variety of different experimental systems, and it appears that transforming growth factor beta may play a role in preventing proliferation of steroid receptor-containing cells. Dysregulation of the strict inverse relationship between ERalpha expression and proliferation is detectable in premalignant human breast lesions, indicating that it might be essential to the tumorigenic process. Challenges for the future include determining which of the candidates identified as being mediators of the effects of E(2) are physiologically and clinically relevant as well as finding out how ERalpha-containing cells become proliferative during tumorigenesis. Answering these questions could greatly increase our understanding of the factors controlling mammary gland development and the processes leading to cancer formation.     

Parathyroid Hormone-Related Protein: A Developmental Regulatory Molecule Necessary for Mammary Gland Development

Parathyroid hormone-related protein (PTHrP) wasoriginally identified as the tumor factor responsiblefor a clinical syndrome known as humoral hypercalcemiaof malignancy. It is now appreciated that PTHrP3 is a developmental regulatory moleculeexpressed during the formation of a wide variety oforgans. Recently, our laboratory has demonstrated thatPTHrP is necessary for mammary gland development. Ourstudies have suggested that this molecule participatesin the regulation of epithelial-mesenchymal interactionsduring embryonic mammary development and perhaps alsoduring adolescent ductal morphogenesis. In addition, it has been suggested that PTHrP plays acritical role in the establishment of bone metastases inbreast cancer. In this article, we will discuss thecurrent knowledge of the mechanisms underlying PTHrPs actions during normal mammary development andin breast cancer.     

The Loss of Estrogen and Progesterone Receptor Gene Expression in Human Breast Cancer

Hormone responsiveness is a critical determinantof breast cancer progression and management, and theresponse to endocrine therapy is highly correlated withthe estrogen receptor (ER)³ and progesterone receptor (PR) status of tumor cells. Thus, keyareas of study in breast cancer are those mechanismsthat regulate ER and PR expression in normal andmalignant breast tissues. One-third of all breastcancers lack ER and PR; these conditions are associatedwith less differentiated tumors and poorer clinicaloutcome. In addition, approximately one-half ofER-positive tumors lack PR protein and patients withthis phenotype are less likely to respond tohormonal therapies than those whose tumors express bothreceptors. Since PR is induced by ER; its presence is amarker of a functional ER. In this review, we will discuss possible mechanisms for loss of ER andPR gene expression, especially structural changes withineach gene including deletions, polymorphisms ormethylation. Improved understanding of the pathways that lead to loss of ER and/or PR proteinsshould allow the development of better predictiveindicators as well as novel therapeutic approaches totarget these hormone-independent cancers.     

Progesterone Receptors in Mammary Gland Development and Tumorigenesis

The steroid hormone, progesterone (P), is a central coordinator of all aspects of female reproductive activity and plays a key role in pregnancy-associated mammary gland morphogenesis and mammary tumorigenesis. The effects of P on the mammary gland are mediated by two structurally and functionally distinct nuclear receptors PR-A and PR-B that arise from a single gene. Null mutation of both receptors in PR knockout (PRKO) mice has demonstrated a critical role for PRs in mediating pregnancy-associated mammary ductal branching and lobuloalveolar differentiation and in initiation of mammary tumors in response to carcinogen. Analysis of the molecular genetic pathways disrupted in PRKO mice has recently yielded important insights into the molecular mechanisms of regulation of mammary gland morphogenesis by PRs. In addition to its essential role in regulating proliferative and differentiative responses of the adult mammary gland during pregnancy, P plays a critical role in the protection against mammary tumorigenesis afforded by early parity. Thus, the effects of P on postnatal developmental plasticity of the mammary gland differ between young and adult glands. This review will summarize recent advances in our understanding of 1) the molecular mechanisms by which PRs mediate pregnancy-associated mammary gland morphogenesis, 2) the role of PRs in mediating tumorigenic responses of the adult mammary gland to carcinogen, and 3) the role of P in long-term protection of the juvenile mammary gland against tumorigenesis. In addition, we will summarize recent insights into the isoform selective contributions to some of these activities of PRs obtained from comparative analysis of P-dependent mammary gland development in PR isoform specific knockout mice lacking either the PR-A (PRAKO) or PR-B (PRBKO).     

Effects of gonadal hormones on thymus gland after bilateral ovariectomy and orchidectomy in rats

This study examined the histological changes that occurred in the thymus gland after gonadectomy and the administration of various sex steroids following gonadectomy. Male and female Wistar albino rats that were 6 weeks of age were used. The rats were subjected to bilaterally gonadectomy and then gonadal steroid hormones (testosterone, estrogen, and progesterone, 2.5 mg/kg) were given. Effects of gonadal steroid hormones on the thymus gland were microscopically examined. Thymic weight increased in all the groups after gonadectomy. Testosterone, estrogen, and estrogen + progesterone treatment decreased thymic weight after gonadectomy. Progesterone treatment also decreased weight, but there was no statistical significance. In the light microscopy, testosterone and estrogen treatment induced a loss of lymphoid elements from the thymic cortex, increased the number of phagocytic macrophages and mast cells, and enlarged blood vessels and connective tissue were observed in the thymic medulla. In the electron microscopic study it was observed that rough endoplasmic reticulum enlarged in the thymic lymphocytes. The same results were also found after estrogen + progesterone treatment. No histologically identifiable changes were observed in the thymus gland after progesterone treatment. This study demonstrated that the thymus gland undergoes involution after testosterone and estrogen treatment, but not progesterone, following gonadectomy.     

Hormone-induced protection against breast cancer

Reproductive history is a consistent risk factor for human breast cancer. Epidemiological studies have repeatedly demonstrated that early age of first full-term pregnancy is a strong protective factor against breast cancer and provides a physiologically operative model to achieve a practical mode of prevention. In rodents, the effects of full-term pregnancy can be mimicked by exposure to low doses of estrogen and progesterone or treatment with human chorionic gonadotropin. The cellular and molecular mechanisms that underlie hormone-induced refractoriness are largely unresolved. Several hypotheses have been proposed to explain the protective effects of hormones. These involve the induction of differentiation of the mammary gland to provide a less responsive cell population to carcinogens, a decrease in proliferative activity in the parous gland compared to the age-matched virgin, an altered hormonal environment mediated by a decrease in circulating growth hormone, and an alteration in cell fate mediated by specific molecular changes induced by estrogen and progesterone. The evidence for and against these hypotheses is discussed along with recent results on possible molecular alterations that may underlie the refractory state. One central question that is still unresolved is whether the refractoriness is intrinsic to the mammary epithelial cells and/or mediated by persistent alterations in the host environment.     

雌激素硫酸转移酶在肥大乳房乳腺组织中的表达及意义

目的 探讨雌激素硫酸转移酶(estrogen sulftransferase,EST)在肥大乳房乳腺组织中的表达及其意义.方法 采用免疫组织化学EnVision二步法,检测EST在32例肥大乳房和15例正常体积乳房乳腺组织中的表达,以及不同类型肥大乳房(19例腺性肥大乳房和13例脂性肥大乳房)乳腺组织中EST的表达状况.结果 32例肥大乳房和15例正常体积乳房乳腺组织中EST阳性表达率分别为34.4%(11/32)和93.3%(14/15),组间比较差异有统计学意义(P＜0.01).EST在19例腺性和13例脂性肥大乳房乳腺组织中的阳性表达率分别为10.5%(2/19)和69.2%(9/13),组间比较差异有统计学意义(P＜0.01).结论 EST的表达减少或缺失,对肥大乳房的形成,尤其与腺性肥大乳房的关系较为密切.

Abstract：

Objective To investigate the expression of estrogen sulfotransferase (EST) in the mammary gland of hypertrophic breast and its significance. Methods EST expression in the mammary gland was detected by EnVision two step method of immunohistochemistry in 15 cases with normal breasts and 32 cases with hypertrophic breasts, including 19 gland-associated cases and 13 fat-associated cases.Results The positive expression rate of EST in mammary gland was 34.4% (11/32 ) in hypertrophic group and 93.3% (14/15) in normal group, showing a significant difference between the two groups(P ＜0.01 ). The positive expression rate of EST was 10.5% (2/19) in gland-associated group and 69. 2% (9/13) in fat-associated group, showing a significant difference between the two groups (P ＜ 0.01 ).Conclusions Decrease or deletion of EST in the mammary gland may be related to the development of hypertrophic breast, especially gland-associated hypertrophic breast.