1. Estrogen and Progestin Stimulation of Breast Proliferation

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1 1. Estrogen and Progestin Stimulation of Breast Proliferation Heidi N. Hilton, BSc, PhD J. Dinny Graham, BSc, PhD Christine L. Clarke, BSc, PhD The ovarian steroid hormones estrogen and progesterone are critical in the growth and proliferation of the breast during normal development, yet ovarian activity has also been shown to be a major driver of breast cancer risk. Through both the menstrual cycle and pregnancy, healthy women are frequently exposed to changes in their hormonal milieu, during which cycling estrogen and progesterone levels induce the breast to undergo significant morphological changes. This influence, and the fact that exposure to exogenous hormones such as the use of progestins in hormone replacement therapy and oral contraception are associated with increased breast cancer risk, highlights the importance of understanding the molecular mechanisms of estrogen and progesterone signaling, both in the normal breast and in the development and progression of breast cancer. Importantly, due to the limited availability of normal human breast tissue, the vast majority of current knowledge of the mechanisms of these hormones has evolved from animal models and cell line studies, and recapitulation of these mechanisms in the normal human breast largely remains to be confirmed. Here the authors review the current thinking on ovarian hormone action in the human breast and discuss the implications these effects may have on breast cancer risk throughout a woman s lifetime. Hormonal Regulation and Receptor Distribution in Human Breast Development The development of the human breast requires both estrogen (E) and progesterone (P) acting via their cognate nuclear receptors. E binds to and activates the estrogen receptor, ER, which comprises 2 distinct isoforms (ER α and ER β ), which in turn regulate the expression of genes that coordinate E-mediated proliferation. The 2 ER isoforms are almost identical in Translational Endocrinology & Metabolism, Volume 3, Number 1,

2 their DNA-binding domain ( 95% homology) but differ in the ligandbinding domain (about 60% homology), and each isoform is encoded by independent genes ( 1 ). Similarly to E, P action is mediated by binding to the progesterone receptor (PR), which also comprises 2 isoforms (PRA and PRB). In contrast to ER however, the 2 PR isoforms are encoded by the same gene, and are identical in sequence except that the shorter form, PRA, lacks 164 amino acids at the N-terminus ( 2 ). Data derived from animal models and transient cotransfection studies in various cell lines suggest that these 2 PR isoforms are functionally distinct, yet both are required to mediate physiologically relevant P signaling ( 3 ). Hormonal Regulation of Breast Development The breast is comprised of 2 major tissue compartments the stroma, which consists of adipocytes, fibroblasts, blood vessels, inflammatory cells, and extracellular matrix and the epithelium, which is made up of a branching ductal-lobular system. These ducts and lobules are lined by a single layer of luminal epithelial cells associated with secretory activity, surrounded by a basal cell layer, which consists mostly of myoepithelial cells with contractile properties ( 4 ). The main postnatal phase of breast development occurs during puberty, where increased levels of E and P are accompanied by an increase in breast volume. Mouse model studies have revealed that this phase of elongation and branching of the ductal system is driven primarily by E signaling ( 5 ). This initial phase of rapid growth ceases once sexual maturity is reached, and in the adult breast only brief proliferative spurts occur throughout the menstrual cycle ( 6 ). In the menstrual cycle, proliferation is considered to be regulated by the sequential and combined action of E and P; the follicular phase of the menstrual cycle is associated with a peak of circulating E, and this is followed by the appearance of a prominent peak of serum P and a second peak of serum E in the luteal phase ( 7 ). This luteal phase of the cycle is characterized by increasing complexity and size of breast lobules, which reflect proliferative activity within the epithelial compartment when mitosis is highest ( 8 10 ). The second major phase of development occurs during pregnancy, where E and P coordinate a massive wave of proliferative and morphological changes to stimulate the growth of functional milkproducing structures (named terminal ductal lobuloalveolar units) in preparation for lactation ( 11 ). Menopause is characterized by the cessation of E and P production by the ovaries, during which the epithelial component of the breast is gradually substituted by stromal cells and fatty tissue ( 12 ) ( Figure 1-1 ). 18 Translational Endocrinology & Metabolism: Breast Cancer Update

3 E P ERα PR puberty follicular luteal pregnancy lactation menopause menstrual cycle FIG 1-1. Schematic diagram depicting the levels of E and P and their cognate receptors throughout puberty, the menstrual cycle, pregnancy, and postmenopause. E and P levels first rise during puberty. During the menstrual cycle, E levels peak during the follicular phase, followed by a second peak during the luteal phase, which is also characterized by a prominent peak in P levels. Pregnancy is associated with very high levels of both E and P, in contrast to the drop to very low levels following the onset of menopause. Unlike the extensive expression of PR, ER α is virtually undetectable during puberty, and the normal premenopausal breast expresses low basal levels of ER α and PR overall. Aside from some expression in the follicular phase, ER α is virtually absent in the luteal phase and during pregnancy, despite PR still being present during these developmental phases. Both ER α and PR levels increase during lactation. Proportions of ER+ and PR+ cells increase with age until menopause is reached, when expression plateaus. Distribution of ER and PR in the Breast Although there are limited data on ER and PR expression in the developing human breast, it appears that ER protein is undetectable in the breast at puberty, unlike PR, which displays extensive distribution ( 13 ). The distribution of ER and PR in the adult breast has been more extensively studied, and it is clear that the normal premenopausal breast expresses low basal levels of ER and PR overall ( 14 ), with ER displaying cyclical variation in expression levels throughout the menstrual cycle. Some positive nuclear ER α staining is detected in the follicular phase of the menstrual cycle, but it is virtually absent in the luteal phase and during pregnancy, despite PR still being present during these phases of development ( 13, 15 ). Both ER α expression and PR expression are restricted to a subset of luminal epithelial cells in the normal breast, with receptor positivity observed in approximately 20% to 30% of luminal cells ( 16, 17 ). Unlike the PR isoforms PRA and PRB, which in the normal breast are coexpressed in PR+ cells, ER α and ER β display some contrasting expression patterns. While ER α expression is restricted to the luminal epithelial Estrogen and Progestin Stimulation of Breast Proliferation 19

4 compartment, ER β is expressed in luminal and myoepithelial cells, stromal cells, and the endothelium of blood vessels ( 18 ). Moreover, while ER β is ubiquitously expressed during all developmental stages, ER α displays fluctuating expression, which increases during puberty, declines during pregnancy, increases during lactation, and decreases again in the postlactating gland ( 19 ). Variation in steroid hormone receptor expression has been suggested to be associated with age and reproductive history. The proportion of ER+ cells has been shown to significantly increase with age until menopause is reached, when expression plateaus ( 20 ). There is also increasing evidence that full-term pregnancy induces long-term gene expression changes in the breast ( 21 ), with recent data suggesting that parous individuals display reduced expression levels of ER α and PR, compared with nulliparous subjects ( 22, 23 ), a mechanism that may possibly contribute to the long-term protective effect of pregnancy on breast cancer risk. Exogenous hormones can also alter ER and PR expression in the breast. The oral contraceptive pill can decrease ER expression ( 24 ), and PR expression in the The cumulative exposure to the postmenopausal breast can be regulated cycling levels of E and P by hormone replacement therapy (HRT), throughout a woman s as evidenced by the increased numbers of reproductive life significantly PR+ cells in the normal breast of women influences the lifetime risk of taking either E only, or combined E+P developing breast cancer HRT formulations ( 25, 26 ). The upregulation of PR by E in the postmenopausal breast recapitulates the limited available evidence in normal breast tissues and primary cell models that PR is increased by E in the normal human breast in vivo ( 27 ). In contrast to PR, no changes in ER expression were observed with either HRT formulation ( 26 ). Ovarian Activity, ER and PR Expression, and Breast Cancer The cumulative exposure to the cycling levels of E and P throughout a woman s reproductive life, depicted in Figure 1-1, significantly influences the lifetime risk of developing breast cancer ( 17 ). For example, an early age at menarche, late age at menopause, and late age at first fullterm pregnancy are associated with an increase in breast cancer risk ( 28 ). Although a number of studies have suggested a positive association between plasma hormone levels and breast cancer risk in postmenopausal women ( ), it has been difficult to similarly demonstrate a direct 20 Translational Endocrinology & Metabolism: Breast Cancer Update

5 causal relationship in premenopausal women because of difficulties in their quantitation, which in turn are caused by large variations in endogenous hormone levels during the menstrual cycle. Despite this, the involvement of hormone action in breast cancer risk is supported by recent genome-wide association studies, which have revealed a number of single nucleotide polymorphisms in genes involved in hormone signaling pathways, which contribute to breast cancer development. Examples of such genes that affect the reproductive hormone-related pathways include fibroblast growth factor receptor ( FGFR2 ) and MAP3K1, both of which are involved in breast cancer susceptibility, and in which mutations are present primarily in steroid hormone receptor-positive tumors ( ). The mechanisms by which lifetime exposure to cycling levels of E and P may contribute to increased breast cancer risk remain unknown. Paradoxically, however, the presence of ER and PR in breast tumors indicates a favorable prognosis, and positive steroid receptor expression remains the most reliable predictive marker for positive endocrine responsiveness in Approximately 60% of breast breast cancer. Approximately 60% of breast tumors coexpress ER and PR tumors coexpress ER and PR, and these are and these are the patients who the patients who have the best prognosis, have the best prognosis while tumors that display only 1 steroid hormone receptor are less likely to respond to endocrine therapy, yet still respond more favorably to hormonal therapies than ER-/PR- tumors ( 36 ). Although uncommon for ER, the levels of PR frequently decrease dramatically following intervening endocrine therapy, and the resulting ER+/PRsecondary tumors display more aggressive characteristics, compared with tumors retaining PR expression ( 37 ). Reports from the Arimidex, Tamoxifen and Combination (ATAC) adjuvant trial have shown that twice as many patients with ER+/PR- tumors suffered recurrences, compared with those with ER+/PR+ tumors ( 38 ), providing further support that ER combined with PR expression is associated with a favorable prognosis. The intensity of hormone receptor expression also directly correlates with the degree of responsiveness to hormone-based therapies. Increased ER α expression and loss of the near complete dissociation between ER expression and proliferation occur at the very earliest stages of breast carcinogenesis ( 17 ). Furthermore, ER β is preferentially lost in some cancers, whereas its introduction slows the growth of breast cancer cells ( 39 ). Finally, the normal human breast coexpresses PRA and PRB at similar levels; however, this PR isoform balance is disrupted early in breast carcinogenesis, resulting in frequent predominance of Estrogen and Progestin Stimulation of Breast Proliferation 21

6 1 isoform and altered PR signaling ( 40, 41 ). Together, these observations imply that dysregulation of ER and PR expression contributes to breast tumorigenesis. Mechanisms of Hormonal Action on Breast Tissue The steroid hormone receptors are members of a large superfamily of ligand-activated nuclear transcription factors, and the mechanisms by which E and P interact with their receptors to regulate hormone-responsive target genes are similar, although complex. All members of the nuclear receptor family share a common protein structure and are made up of a central DNA-binding domain (DBD), a carboxyl-terminal ligand-binding domain (LBD), and varying numbers of activation function (AF) elements ( Figure 1-2A ) ( 42 ). The current understanding of the mechanisms by which these steroid hormones act has largely evolved from cell line studies, and whether these mechanisms accurately reflect what occurs in the normal human breast in vivo remains to be shown. Steroid receptor function is complex, requiring a succession of molecular interactions to induce transcription of target genes. In the classical mode complex, requiring a Steroid receptor function is of action derived from in vitro studies, newly succession of molecular transcribed cytoplasmic ER and PR are assembled in the absence of ligand in inactive transcription of target genes interactions to induce multiprotein chaperone complexes. The receptors continually shuttle between the nucleus and cytoplasm, mediated via nuclear localization and nuclear export sequences, and in the presence of ligand, this dynamic equilibrium is shifted toward the nuclear compartment, where most steroid receptors reside. Upon hormone binding and receptor activation, the inactive complexes undergo a conformational change, leading to dissociation of chaperones, dimerization, and binding to specific hormone response elements (HREs) located in the promoter regions of target genes ( Figure 1-2B ). DNA-bound receptors then recruit specific coactivators, corepressors, and general transcription factors, through protein-protein interactions with AF elements, resulting in modulation of transcription of those genes ( 43 ). The protein products of these genes then mediate the downstream signaling pathways of the steroid hormones. Steroid hormone action can also occur by a number of other mechanisms. Rather than binding DNA directly themselves, activated steroid hormone receptors can also be tethered to DNA by other transcription factors (such as AP1 and Sp1 ) in order to regulate gene promoters that lack 22 Translational Endocrinology & Metabolism: Breast Cancer Update

7 A 1 AF-1 DBD LBD/AF AF-1 DBD LBD/AF PRA 1 AF-3 AF-1 DBD LBD/AF PRB 165 AF-1 DBD LBD/AF B steroid hormone membrane HR RTK Src kinase activation cell membrane HR P P cellular functions nuclear membrane coactivator gene transcription P P HRE FIG 1-2. (A) Schematic diagrams of ER α, ER β, PRA, and PRB structures depicting structural domains and sizes of each isoform. (B) Basic mechanism of steroid hormone action via classical genomic and rapid nongenomic pathways. In the classical mode of action, steroid hormones activate their receptors, which dimerize and translocate to the nucleus to bind to HREs of target genes. Steroids can also bind membrane-associated hormone receptors (HRs), leading to the activation of signal transduction cascades to control gene transcription and/or cellular functions. HRs can also form complexes with receptor tyrosine kinases (RTKs) (eg, EGFR) and non-rtks (eg, Src), and stimulation of tyrosine phosphorylation results in a transient activation of various kinase signaling cascades. canonical HRE sequences, providing a possible mechanism for liganddependent regulation of promoters without HREs ( 44 ). In addition to genomic effects, ER and PR may regulate transcription via rapid nongenomic pathways, such as activation of ion channels, second messenger signaling cascades, and protein kinase cascades ( 45 ). The nuclear receptor activation of the intracellular tyrosine kinase c-src, or enzymes such as MAPK and Akt, results in phosphorylation of a range of cytoplasmic enzymes, transcription factors, and other targets, including the receptors themselves ( 46 ). The existence of extranuclear steroid receptors located in Estrogen and Progestin Stimulation of Breast Proliferation 23

8 the cytoplasm or at the plasma membrane is increasingly being recognized ( 47 ), although this is still an issue of some debate, as it is possible that a small pool of conventional nuclear receptors may occasionally be found localized in the cytoplasm or at the membrane. So although data continue to emerge supporting the role of membrane steroid receptor signaling, further investigation of the receptor species mediating transcription via rapid ER and PR may also regulate these effects is warranted. Indeed, nuclear nongenomic pathways ER and PR have been shown to form complexes with cytoplasmic signaling molecules, mediating nuclear translocation and activation of genomic targets ( 48, 49 ). Moreover, cross-talk between ER and growth factor signaling pathways has been extensively studied ( 50 ). Therefore, steroids can act both in the nucleus to directly stimulate gene transcription, and in the cytoplasm and potentially at the membrane to activate protein kinases, which in turn may stimulate ER or PR activity ( Figure 1-2B ). Estrogen Receptor ER possesses 2 activation function domains by which it stimulates transcription of target genes: AF1 in the N-terminal domain, and AF2 in the ligand-binding domain. The activity of AF1 is ligand-independent, but can be modulated by phosphorylation by the MAPK pathway in response to growth factors, whereas the activity of AF2 requires E binding ( 51, 52 ). Although ER α and ER β are highly homologous in their DNA-binding domains and act through the same general mechanism in target cells, in vitro studies have shown that these 2 isoforms have overlapping but distinct transcriptional properties, with ER β being less transcriptionally active ( 51 ). ER α, which is generally more highly expressed, can also have opposite actions to ER β, in particular at the promoters of some genes involved in proliferation, suggesting that the balance between ER α and ER β signaling is important in mediating the proliferative response to E ( 53 ). There are also differences in the mode of action of the 2 ER isoforms; for example, only ER α can stimulate genes via the Sp1 pathway ( 54 ), and each isoform is differentially activated by E at AP1 sites ( 55 ). ER activity can also be regulated by multiple post-translational modifications, such as phosphorylation, acetylation, ubiquitylation, and sumoylation. ER is phosphorylated on multiple sites by a wide range of kinases, a mechanism that can control subcellular localization, dimerization, DNA binding, and transcriptional activity of ER ( 56, 57 ). More recently, attention has been drawn to the detection of specific phosphorylated forms of ER α 24 Translational Endocrinology & Metabolism: Breast Cancer Update

9 in primary breast tumors, which may predict response to tamoxifen and clinical outcome, reviewed by Murphy and colleagues ( 58 ). Progesterone Receptor The 2 PR isoforms have the potential to concurrently exist as 3 distinct molecular species (PRB homodimers, PRA homodimers, and PRA-PRB heterodimers), which contributes to the complexity of PR action. In humans, most PR+ cells coexpress PRA and PRB at equivalent levels, suggesting that both proteins are required to mediate physiologically relevant P signaling ( 40, 41 ). There is also increasing evidence that PRA and PRB are functionally unique transcriptional regulators, capable of differentially regulating gene transcription within the same promoter context, and capable of recognizing entirely different promoters. While PRB acts mostly as a transcriptional activator, PRA can act as a transdominant inhibitor of PRB in situations where PRA has little or no transactivational activity. Moreover, PRA can regulate the transcriptional activity of other nuclear receptors, including ER, and the glucocorticoid, mineralocorticoid, and androgen receptors ( 59, 60 ). P-mediated signaling via membrane-located PRs is increasingly gaining acceptance, and membrane PRs have been identified in human breast cancer biopsies and breast cancer cell lines, which may contribute to rapid nongenomic P signaling, such as transactivation of epidermal growth factor receptor (EGFR) ( 47, 61, 62 ). However, the importance of these extranuclear receptors in mediating progestin action in the breast requires further investigation. Like ER, PR also undergoes high levels of post-translational modifications, and PR phosphorylation is critical in regulating a diverse array of activities, such as transport in and out of the nucleus, binding to DNA, interactions with other proteins, transcription, and PR stability ( 57, 63 ). Cross-talk Between Steroid Hormone and Growth Factor Pathways Steroid hormone action is complicated by the ability of steroid hormone signaling to influence the activity of many other signaling pathways, and the interaction between the steroid and growth factor pathways may be required for a full proliferative response to be elicited. It is important to note that data concerning these complex pathway interactions have been determined from in vitro experiments only, and whether these interactions occur in the normal human breast in vivo has yet to be shown. For example, in breast cancer cells, E and insulin-like growth factor-1 (IGF-1) Estrogen and Progestin Stimulation of Breast Proliferation 25

10 cooperate to promote cell cycle progression ( 64 ), and IGF-1 receptor inhibitors block E-mediated cell proliferation ( 65 ). E also activates both the Src/Ras/MAPK cascade and the PI3K/Akt pathway, via direct interaction of the ER with Src and the regulatory subunit of PI3K ( 49, 66 ). Similarly, P and synthetic progestins rapidly activate these 2 signaling cascades in the breast ( 67, 68 ), a mechanism required for steroid hormone-driven proliferation, as well as migration and invasion in breast cancer cells ( 69 ), potentially contributing to the tumorigenic properties of these hormones. The P-mediated activation of Src can occur either by direct binding by PR ( 70 ), or via an interaction between PR and ER ( 68 ). This cross-talk with ER is essential for P-induced proliferation of breast cancer cells ( 71, 72 ). Other examples of PR pathway interactions include the demonstration that P can promote proliferation of breast cancer cells via the MAPK cascade via cross-talk with epidermal growth factor (EGF) signaling ( 73 ), and that ErbB-2 can act as a coactivator of Stat3 in P-induced breast tumor growth ( 48 ). Thus, steroid hormone activity can depend not only on various factors such as relative levels of receptor expression, different isoforms, splice variants, subcellular localization sites, and receptor phosphorylation, but also on different mechanisms of action as well as cross-talk with other signaling pathways. Effects of Estrogen and Progesterone on Proliferation and Apoptosis The steroid hormones can elicit different effects dependent on tissue type and the developmental context, demonstrated by studies performed in animal models, cell lines, and various organs in humans. Animal models have provided important insights into the mechanisms of hormone action in the mammary gland, and the pivotal role of E and P function in the proliferation of the breast has been clearly illustrated by both the ER and PR knockout mouse models. The ER α knockout mouse does not undergo any mammary gland development and only possesses a rudimentary ductal structure ( 74 ), while the PR knockout mouse does have a complete mammary epithelial ductal network, but displays severely limited lobuloalveolar development ( 75 ). In humans, the steroid hormones drive proliferation throughout the developmental phases, with the first phase of florid proliferation occurring during puberty, after which the normal adult breast does not rapidly expand. In the adult breast, as mentioned earlier, proliferation is restricted to the pregnant gland, and to the luteal phase of the menstrual cycle when P levels are maximal, although both hormones are present ( 10, 24, 76 ). Notwithstanding the range of mechanisms through which steroid hormones regulate cellular function, it is widely 26 Translational Endocrinology & Metabolism: Breast Cancer Update

11 accepted, from functional studies performed in mouse models and costaining of hormone receptors and proliferation markers in the human breast, that receptor-positive cells do not proliferate in direct response to hormone signals, but rather these cells exert a paracrine influence on the surrounding receptor-negative cells, supported by the inverse relationship between steroid receptor expression and proliferation ( 16, 77 ). Interestingly however, during breast cancer progression, an increasing number of proliferating cells express steroid hormone receptors, suggesting that steroid receptor-positive cells undergo an early switch to autocrine signaling mechanisms, or that they acquire the ability to divide as part of the carcinogenic process ( 78 ). Details of the mechanisms that mediate breast cell proliferation in response to ovarian steroids in the human breast thus remain incompletely understood. Estrogen Action For many years, a wealth of experimental and clinical data has shown that E is critical in the proliferation and progression of breast cancer ( 76 ). Indeed, in the mouse, E is the major mitogenic stimulus during puberty, when it promotes the elongation of the mammary ducts and expansion of the epithelium throughout the fat pad. Mouse mammary epithelial transplantation experiments have revealed that amphiregulin, an EGFR ligand, is an essential mediator of E-induced proliferation during puberty ( 79 ). The proliferative action of E has also been shown to occur by modulation of cell cycle gene expression, such as c-myc, cyclin D1, cyclin E, p21, and p27 ( 80 ). However, the proliferative properties of E can sometimes go awry, contributing to breast tumor development. In many malignant breast cells, the ER signaling pathway contributes to promoting an imbalance in cell proliferation and apoptosis rates, with prosurvival and proliferation signals overwhelming prodeath and quiescence signals, reviewed in Tyson and colleagues ( 81 ). Consistent with a pro-proliferative role, there is direct in vitro evidence in breast cancer cells that E inhibits apoptosis by up-regulation of Bcl-2, an antiapoptotic proto-oncogene ( 82 ). Because of its role in proliferation and tumor growth in breast cancer, the ER signaling network has served as an attractive target for the development of therapeutic agents. These therapies, which include selective ER modulators (SERMs) such as tamoxifen, aromatase inhibitors and pure antiestrogens, act to inhibit the proliferative effect of E, and have underpinned the significant reduction in recurrence risk in pre- and postmenopausal patients with ER+ disease observed over the last 20 or so years ( 83 ). Estrogen and Progestin Stimulation of Breast Proliferation 27

12 Progesterone Action Following puberty, E elicits only modest proliferation, although is permissive for the strongly mitogenic effects of P during this stage ( 84 ). Interestingly, P can induce distinct tissue-specific responses. For example, P can exert proliferative or antiproliferative effects in breast cancer cell lines in a context-dependent manner ( ), and it counteracts the proliferative effects of E in the endometrium ( 88, 89 ). These context-dependent effects may translate to the paradoxical effects of P in tumorigenesis; for example, women exposed to progestin-containing HRT have a significantly increased breast cancer risk compared to women taking E alone formulations, or no HRT at all ( 90, 91 ). Also, P can reverse the antitumorigenic effects of the antiestrogen tamoxifen via a PR-mediated mechanism ( 92 ), and ablation of PR is associated with a protective effect against mammary tumorigenesis in mice ( 93 ). Conversely, P treatment of breast cancer cells in culture elicits a biphasic response, whereby there is initially a proliferative burst, followed by onset of differentiation and long-term inhibition of proliferation ( 80, 85 ). Concordant with the ability of progestins to stimulate proliferation and inhibit apoptosis in breast cancer cells ( 94, 95 ), in vitro and clinical studies have shown that antiprogestins can inhibit proliferation and promote apoptosis ( 96, 97 ). P is recognized as a major proliferative hormone in both the mouse mammary gland and the normal human breast, and it is required to promote the massive proliferation that occurs during early pregnancy. In the adult mouse mammary gland, P-mediated signals trigger the formation of side branches from the mammary ducts during estrus cycles and early pregnancy ( 84 ), and they have been reported to drive proliferation in 2 waves the first smaller wave by a cell-intrinsic cyclin D1-dependent mechanism and a second larger wave by a paracrine mechanism ( 98 ). But although P has been shown to signal through molecules such as Wnt4 ( 99 ) and RANKL ( 100 ) in a paracrine manner in rodent models, these pathways are yet to be convincingly shown to play a role in P signaling in the human breast. So, the mechanistic details by which E and P promote proliferation in the human in vivo remain poorly understood. Interaction of E and P with the Breast Cancer Susceptibility Genes BRCA1 and BRCA2 Disruption of BRCA1, a breast cancer susceptibility gene, impairs normal breast proliferation ( 101 ), and as there are several lines of evidence linking hormone action and BRCA1 signaling, the potential relationship 28 Translational Endocrinology & Metabolism: Breast Cancer Update

13 between BRCA1 and the reproductive hormone signaling networks is an interesting aspect of their role in proliferation and breast cancer progression. Germline mutations in BRCA1, encoding a tumor suppressor protein, greatly enhance the risk of breast and ovarian cancer ( 102 ). BRCA1 function (including roles in DNA repair, cell cycle checkpoint control, and transcriptional regulation) ( 103 ) displays striking tissue-specificity, thus implicating the role of ovarian hormones ( 104 ). Studies performed primarily in breast cancer cell lines and mouse models supporting this idea include the demonstration that E has been shown to regulate BRCA1 expression, while BRCA1 can regulate ER α signaling. Specifically, BRCA1 has been shown to directly bind ER α to inhibit E-dependent transactivation by ER α ( 105, 106 ) and to mediate ligand-independent transcriptional repression of ER α ( 107 ). BRCA1 has also been demonstrated to regulate PR signaling, potentially via a direct interaction with PR, resulting in inhibition of PR activity ( 108 ). Importantly, exogenous P stimulated proliferation in the mammary glands of Brca1-deficient mice ( 108 ) and RU486, a PR antagonist, prevented mammary tumorigenesis in Brca1/p53-deficient mice ( 109 ), providing in vivo evidence that PR signaling is involved in BRCA1-related tumorigenesis. In addition, normal epithelium adjacent to BRCA1 mutant tumors, or from prophylactic mastectomies in BRCA1 mutation carriers, displays dysregulated PR expression ( ). Combined E and P treatment in ovariectomized mice was shown to synergistically induce Brca1 expression in the mammary gland ( 114 ), while BRCA1 mrna and protein levels are regulated by both E and P in human breast cancer cells. This, however, may be more a consequence of the ovarian hormones regulating BRCA1 expression by modulating the proliferative status of the cells, rather than directly inducing BRCA1 ( 115 ). Thus overall, altered steroid hormone signaling may be a factor in the increased risk of cancer, or represent an early event in BRCA1-related breast tumorigenesis. Furthermore, wild-type BRCA1 may be important in restraining uncontrolled proliferation elicited by E and/or P signaling pathways. Effects of Combined Estrogen and Progesterone on Proliferation and Apoptosis The effects of combined E and P action on cell growth in the breast have been shown to be context-dependent. In the adult mouse and the macaque, combined E and P treatment is mitogenic in the mammary gland to a much greater extent than treatment with E alone ( 116, 117 ). This is also seen in the human breast, where E and P stimulate higher breast proliferation than E or P alone ( 26 ), although to a lesser degree than in animal Estrogen and Progestin Stimulation of Breast Proliferation 29

14 models. Exogenous hormone use can also have profound effects on proliferation in the breast. For example, the oral contraceptive pill has been shown to increase proliferation in the normal premenopausal breast ( 24, 118 ). The synergistic effect of combined E and P action ( 116 ) is consistent with the now well-established increase in breast cancer incidence in women receiving combined E and P HRT formulations, rather than E alone ( 90, 91, 119 ). The cellular basis for this increased risk is not known, but past and current trends in breast cancer incidence suggest that progestins in HRT are hastening the appearance of existing breast cancers, rather than initiating new tumors ( 120 ). The proliferation of epithelial cells stimulated by exogenous hormone use occurs early, and can be observed within the first 3 months of continuous use of combined E and P HRT formulations ( 121 ). As previously mentioned, the involvement of P signaling in breast cancer has been inferred from rodent models for some time, from demonstrations of the requirement of PR for robust tumorigenesis ( 93 ), and the ability of P to reverse the antitumorigenic effects of tamoxifen ( 92 ). More recently, it has been reported that combined E and P treatment increased mouse mammary stem cell numbers, although no difference was observed with E treatment alone ( 122, 123 ). In the human breast, the impact of P action on cell fate determination is supported by the observations that P can alter mammary progenitor cell numbers ( 27, 124 ) and can induce a luminal to myoepithelial shift in a subset of PR+ tumors ( 125 ). Similarly, E has been reported to promote mammary stem cell proliferation, although the data are conflicting as to whether this proliferation is stimulated via ER+ cells situated adjacent to or within the stem cell compartment ( 126, 127 ). Hormone-mediated increased proliferation of stem and progenitor cells, often considered to be targets for carcinogenic transformation ( 128 ), may therefore provide an expanded pool that is more susceptible to acquiring mutations. However, this is still a controversial area, as these data describing hormone-mediated expansion of the stem/progenitor pool seemingly conflict with the strong protective factor against breast cancer offered by hormone exposure during pregnancy at an early age, and studies that have observed no association between pregnancy and mammary stem cell numbers ( ). Thus, further studies are required to define the role of steroid hormone exposure in the regulation of stem and progenitor cells, and in driving increased breast cancer risk. As the P component of HRT has been implicated in increasing breast cancer risk to a much greater extent than E-only formulations ( 90, 91 ), focus has more recently moved to understanding the role of P and its 30 Translational Endocrinology & Metabolism: Breast Cancer Update

15 analogues in the etiology of breast tumorigenesis. The natural hormone P is only weakly bioavailable orally, necessitating the development of longerlived analogues in HRT formulations designed for daily oral use. A range of P analogues has been used in HRT formulations ( Table 1-1 ), all with diverse pharmacokinetic and biological profiles. Additionally, they all have been associated with increased breast cancer risk ( 91, 132 ). All these progestins have progestogenic activity in classical assays and bind with comparable kinetics to PR, and some also to other nuclear receptors, in cell line studies. These progestins also regulate the expression of genes that are broadly similar to those regulated by the natural hormone ( 133 ), although progestins with higher affinities for PR tend to regulate more genes than P itself. In vitro therefore, progestins are largely functionally similar. In vivo, however, progestins may have diverse functional profiles, due to factors that include differences in pharmacokinetics and metabolism; differences in the relative expression of the two PR isoforms, PRA and PRB; cross-talk of PR with other signaling pathways; and finally the capacity of progestins to signal via nuclear receptors other than PR. Therefore, mechanistic differences between P and its analogues in vivo will require continued investigation. TABLE 1-1. Effect of Common Components in HRT Formulations on Proliferation and Apoptosis Animal models Breast cancer cell lines Normal breast in vitro Normal breast in vivo Breast tumors in vivo MPA Proliferation Apoptosis Proliferation Apoptosis Proliferation Apoptosis References 26, 91, 117, Norethisterone Cyproterone Acetate Tibolone Estradiol Antitumoral Proliferation Antitumoral Proliferation Proliferation Proliferation Proliferation Apoptosis No effect Not tested Proliferation Apoptosis Proliferation Proliferation Density Proliferation Apoptosis Proliferation Proliferation Apoptosis Proliferation Proliferation Proliferation Proliferation No effect Proliferation , , 117, 163, 167 Estrogen and Progestin Stimulation of Breast Proliferation 31

16 Effects of Estrogen in Conjunction with Progestins on Breast Density Mammographic density, as measured by both percent dense area and absolute dense area, is a strong risk factor for breast cancer ( 134, 135 ). Density is a highly heritable trait ( 136 ) and has been established as a hormonally regulated biological feature. For example, pregnancy (in particular, early age at first birth), early age at menopause, and tamoxifen use ( ), are all factors that have been observed to decrease breast density. In contrast, women taking either combined E and P or E-only formulations displayed significantly increased epithelial cell density, compared with those on no HRT at all ( 26 ). Furthermore, combined E and P formulations induced greater epithelial cell proliferation and breast density in postmenopausal women on HRT, compared with those taking E alone ( 26, 140, 141 ). This is important as not only does higher mammographic density represent increased breast cancer risk, but it also affects the sensitivity and specificity of mammographic screening ( 142 ). The increase in epithelial proliferation mediated by E and P treatment can occur within a short timeframe of HRT use ( 143 ), but decreases following discontinuation of HRT treatment ( 140 ). Thus, because of the established association between mammographic density and breast cancer risk, it may be advantageous to use density measurements as part of the risk-benefit assessment when advising women to begin or continue with E and P HRT formulations or not. Although women with increased mammographic density are at 4 to 6 times greater risk of breast cancer than those with lower density ( 144, 145 ), the biological mechanism responsible for the association between higher breast density and breast cancer risk has not yet been established. One possible explanation is that mammographic density reflects the total amount of epithelium and stroma in the breast, relative to fat ( 146 ), and thus higher areas of density present an increased population of epithelial cells susceptible to oncogenic mutation. Because of the hormonal influences on breast density, studies have focused on the pathways that regulate steroid hormone signaling and/or proliferation. Mammographic density has been correlated with proliferative activity of breast cells ( 76 ), and some studies have implicated the IGF pathway in increased breast density in premenopausal women ( 147, 148 ). IGF-1 production is stimulated by E, and the close association between the IGF-1 and ER signaling pathways has a critical role in regulating cell proliferation and apoptosis in normal breast and in breast cancer ( 149, 150 ). This led to the suggestion that IGF-1 signaling may be involved in breast density and increasing breast cancer risk by stimulating 32 Translational Endocrinology & Metabolism: Breast Cancer Update

17 proliferation or inhibiting apoptosis in breast epithelial cells ( 15, 151 ). Indeed, several earlier epidemiological studies reported high circulating levels of IGF-I were consistently found to be positively associated with breast cancer risk in premenopausal women ( 151, 152 ), and interestingly, a positive correlation between the ratio of serum E and P levels and IGF-1 mrna levels in breast tissue has been reported ( 153 ). However, updated studies have suggested that the association of IGF-1 with both breast density and breast cancer risk is much weaker than initially believed ( ), and instead may be an indirect effect due to its regulation by E. No significant association between steroid hormone levels or steroid receptor expression and breast tissue density has been found, however ( 157, 158 ), nor is there any difference in breast density between individuals at high risk of breast cancer (ie, carriers of germline BRCA1 and BRCA2 mutations) and the normal population ( 159 ). The idea of large areas of nondense fat tissue having an independent role in the development of cancer, potentially explained by the fact that fat tissue is known to be an important source of estrogens in the breast ( 160 ), is interesting, although it remains controversial, according to recent studies ( 161, 162 ). Finally, candidate gene approaches have shown an association between ER polymorphisms and postmenopausal breast density, which possibly influence breast cancer risk ( 149 ). Further investigation to identify any role the steroid hormones may have in influencing breast density is required. Concluding Remarks The fundamental role of ovarian steroid hormone signaling in the proliferation of the breast emphasizes the need to define the details of their mechanism of action. Studies so far have shown that E and P act through a range of complex interactions and mechanisms to both directly and indirectly regulate proliferation and apoptosis. These effects are mediated by multiple modes of action including genomic and nongenomic signaling and cross-talk with growth factor pathways. These mechanisms are further complicated by the existence of multiple functionally distinct receptor isoforms and different post-translational modifications of the receptors. These details have emerged largely from animal models and cell line studies, but whether these modes of action similarly occur in the normal and malignant human breast in vivo remains to be conclusively proven. Growing experimental and clinical evidence supports the role of sustained exposure to E and P (through exogenous hormone use or through extended numbers of menstrual cycles in a woman s lifetime) in being a key driver of breast cancer risk. Furthermore, the interaction Estrogen and Progestin Stimulation of Breast Proliferation 33

18 between the ER, PR, and BRCA1 signaling pathways and the steroid hormone regulation of mammographic density may contribute to this hormone-associated increased risk. Despite the breast cancer risk associated with HRT use, it is still one of the most frequently administered pharmaceuticals, reflecting both the need for, and effectiveness of, currently available formulations. Understanding how steroid hormones and their synthetic analogues mediate proliferation in the breast and contribute to breast cancer risk will be valuable for the development of novel targeted cancer therapies and potential strategies that would allow continued development and administration of hormone formulations to women worldwide. References 1. Hall JM, McDonnell DP. The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology. 1999;140(12): Kastner P, Krust A, Turcotte B, et al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 1990;9(5): Scarpin KM, Graham JD, Mote PA, Clarke CL. Progesterone action in human tissues: regulation by progesterone receptor (PR) isoform expression, nuclear positioning and coregulator expression. Nucl Recept Signal. 2009;7:e Howard BA, Gusterson BA. Human breast development. J Mammary Gland Biol Neoplasia. 2000;5(2): Silberstein GB, Van Horn K, Shyamala G, Daniel CW. Essential role of endogenous estrogen in directly stimulating mammary growth demonstrated by implants containing pure antiestrogens. Endocrinology. 1994;134(1): Söderqvist G, Isaksson E, von Schoultz B, Carlström K, Tani E, Skoog L. Proliferation of breast epithelial cells in healthy women during the menstrual cycle. Am J Obstet Gynecol. 1997;176(1 Pt 1): Soules MR, McLachlan RI, Ek M, Dahl KD, Cohen NL, Bremner WJ. Luteal phase deficiency: Characterization of reproductive hormones over the menstrual cycle. J Clin Endocrinol Metab. 1989;69(4): Anderson TJ, Ferguson DJ, Raab GM. Cell turnover in the resting human breast: influence of parity, contraceptive pill, age and laterality. Br J Cancer. 1982; 46(3): Ferguson DJ, Anderson TJ. Morphological evaluation of cell turnover in relation to the menstrual cycle in the resting human breast. Br J Cancer. 1981;44(2): Longacre TA, Bartow SA. A correlative morphologic study of human breast and endometrium in the menstrual cycle. Am J Surg Pathol. 1986;10(6): Fendrick JL, Raafat AM, Haslam SZ. Mammary gland growth and development from the postnatal period to postmenopause: ovarian steroid receptor ontogeny and regulation in the mouse. J Mammary Gland Biol Neoplasia. 1998;3(1): Hutson SW, Cowen PN, Bird CC. Morphometric studies of age related changes in normal human breast and their significance for evolution of mammary cancer. J Clin Pathol. 1985;38(3): Translational Endocrinology & Metabolism: Breast Cancer Update

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