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Growth Hormone and Insulin-Like Growth Factor-I in the Transition from Normal Mammary Development to Preneoplastic Mammary Lesions

Neuroendocrine Unit (D.L.K.), Department of Medicine, New York University School of Medicine and DVA Medical Center, New York, New York 10016; Department of Neurology and Neuroscience (T.L.W.), New Jersey Medical School, University of Medicine and Dentistry New Jersey, Newark, New Jersey 07103; Department of Oncology (P.A.F.), Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C. 20057; and Breast Center (A.V.L.), Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

Correspondence: Address all correspondence and requests for reprints to: David L. Kleinberg, M.D., Neuroendocrine Unit, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: david.kleinberg{at}med.nyu.edu.

	Abstract

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 
Adult female mammary development starts at puberty and is controlled by tightly regulated cross-talk between a group of hormones and growth factors. Although estrogen is the initial driving force and is joined by luteal phase progesterone, both of these hormones require GH-induced IGF-I in the mammary gland in order to act. The same group of hormones, when experimentally perturbed, can lead to development of hyperplastic lesions and increase the chances, or be precursors, of mammary carcinoma. For example, systemic administration of GH or IGF-I causes mammary hyperplasia, and overproduction of IGF-I in transgenic animals can cause the development of usual or atypical hyperplasias and sometimes carcinoma. Although studies have clearly demonstrated the transforming potential of both GH and IGF-I receptor in cell culture and in animals, debate remains as to whether their main role is actually instructive or permissive in progression to cancer in vivo. Genetic imprinting has been shown to occur in precursor lesions as early as atypical hyperplasia in women. Thus, the concept of progression from normal development to cancer through precursor lesions sensitive to hormones and growth factors discussed above is gaining support in humans as well as in animal models. Indeed, elevation of estrogen receptor, GH, IGF-I, and IGF-I receptor during progression suggests a role for these pathways in this process. New agents targeting the GH/IGF-I axis may provide a novel means to block formation and progression of precursor lesions to overt carcinoma. A novel somatostatin analog has recently been shown to prevent mammary development in rats via targeted IGF-I action inhibition at the mammary gland. Similarly, pegvisomant, a GH antagonist, and other IGF-I antagonists such as IGF binding proteins 1 and 5 also block mammary gland development. It is, therefore, possible that inhibition of IGF-I action, or perhaps GH, in the mammary gland may eventually play a role in breast cancer chemoprevention by preventing actions of both estrogen and progesterone, especially in women at extremely high risk for developing breast cancer such as BRCA gene 1 or 2 mutations.

I. Introduction

II. Mammary Development

A. Ductal morphogenesis during puberty

B. Lobular-alveolar development during pregnancy

C. Similarities and differences between human and mouse mammary development

III. Growth Hormone and IGF Control of Mammary Gland Development

A. Historical background

B. The role of the pituitary gland and GH in mammary gland development

C. Effects of IGF-I in pubertal mammary development

D. Interaction between IGF-I, estrogen, progesterone, and prolactin in mammary ductal morphogenesis

E. Role of IGF-I in lobular-alveolar development

F. Role of IGF binding proteins in GH and IGF-I action in the mammary gland

G. Interaction between IGF-I and other growth factor families in mammary gland development

IV. Growth Hormone and IGF Regulation of Mammary Gland Hyperplasia

A. Background

B. Circulating hormone levels and risk of mammary hyperplasia and cancer in women

C. Induction of mammary hyperplasia by exogenous hormone administration

D. Studies of mammary hyperplasia in genetically engineered mice

E. Results of decreasing the activity of the IGF signaling axis

F. Studies of mammary hyperplasia in the human breast

V. Molecular Mechanisms whereby Growth Hormone and IGF Promote Mammary Hyperplasia and Progression to Cancer

A. The GH and IGF signal transduction pathways

B. Transforming potential of GH and IGF signaling pathways

C. GH/IGF regulation of proliferation and apoptosis

D. Interaction with estrogen and progesterone receptor

E. GH/IGF regulation of other pathways in cancer progression

VI. Inhibition of Growth Hormone and IGF-I for the Prevention of Breast Cancer

A. Role of GH and IGF-I in pregnancy-mediated protection from breast cancer

B. Preclinical studies of inhibitors of GH and IGFs

C. Clinical studies of inhibitors of estrogen action to prevent breast cancer

D. Clinical studies based on GH or IGF-I inhibition for breast cancer prevention

	I. Introduction

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 
THE GH/IGF AXIS IS CENTRAL to many normal biological processes in animals including growth, development, metabolism, and longevity (Fig. 1). In humans, several clinical diseases involving loss of components of either the GH pathway or the IGF pathway result in decreased growth, and conversely aberrant overexpression can lead to hypertrophy and increased cancer risk. The observations in humans have been modeled in numerous animal systems. Both GH and IGF are critical regulators of metabolism via regulation of glucose homeostasis (1). Studies in worms originally identified a role for IGF pathway orthologs in longevity, and the same has been shown in Drosophila and rodents (2). Finally, IGFs are implicated in proliferation, survival, and differentiation of multiple cell types and development of some organs such as brain (3) and placenta (4).

View larger version (19K): [in this window] [in a new window]   FIG. 1. GH and IGF-I control of physiology. GH and IGF-I have been shown to regulate longevity, metabolism, development, and growth in several different lower organisms including worms, Drosophila, and rodents.

View larger version (63K): [in this window] [in a new window]   FIG. 2. A, Schematic of systemic endocrine (left) and local paracrine effects of GH on IGF-I production and action in mammary development. SRIF, Somatostatin. B, Effect of estradiol (E2) and IGF-I on mammary development in oophorectomized IGF-I(–/–) female mice after 0 d (upper left), 5 d (upper right), 14 d (lower left), and 28 d (lower right). Note the increased number of branching ducts at 14 d and the obvious hyperplasia that occurred at 28 d.

	II. Mammary Development

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

Because the hormones involved in the different phases of mammary development are distinct, mammary development has been divided into four phases as follows: 1) ductal development or morphogenesis as seen during puberty; 2) lobular-alveolar development as seen in pseudopregnant or pregnant rats; 3) lactation; and 4) involution. Because the focus of this review is on normal and abnormal ductal and lobular-alveolar development, only stages 1 and 2 will be addressed. The reader is referred to Refs. 13, 14, 15, 16, 17, 18 for a discussion of lactational development and Refs. 16, 19 and 20 for a discussion of involution.

A. Ductal morphogenesis during puberty
From birth to puberty, the murine mammary gland consists of a fat pad containing a small area of rudimentary ductal structures, also called "ductal anlagen" (21, 22). Further ductal development begins with the estrogen stimulation of puberty. Terminal end buds (TEBs), multilayered club-shaped structures with active cell division, travel through the fat pad leading the process of mammary development. TEBs undergo repeated bifurcation, or possibly trifurcation and extend into the substance of the mammary fat pad, leaving in their wake a network of branched ducts that fill the mammary fat pad (22, 23, 24, 25, 26). Programmed cell death behind the actively proliferating part of the TEB leads to luminal development (27). Some of the mechanisms of branching morphogenesis are still incompletely understood, but it is known that, in addition to IGF-I and estrogen (28), progesterone, fibroblast growth factor (FGF) family members, and TGF-β pathways all contribute in addition to other factors (5, 23, 26, 29, 30, 31). By the time the glandular tree reaches the limits of the mammary fat pad, TEB formation stops (23), and the gland is populated with a series of primary, secondary, and tertiary branched structures ending in structures smaller than TEBs called terminal duct ends. Thereafter, the mammary gland responds to each estrous cycle with the formation of alveolar buds and new tertiary branches that then regress if pregnancy does not occur (32, 33). True alveoli require the hormones of pregnancy to form (22) (see Section III. E). Overpopulation of the mammary fat pad by glandular elements is prevented by the action of TGF-β (34). This process ensures adequate space for formation of alveoli capable of producing milk after pregnancy.

B. Lobular-alveolar development during pregnancy
In the mouse, normal lobular-alveolar development occurs during pregnancy, and milk is produced in alveoli but not released until parturition. Both progesterone and estrogen can prevent release of milk during pregnancy so that active lactation only begins after parturition (35, 36). Lobular- alveolar development is mediated by the combination of estrogen, progesterone, IGF-I, placental lactogen and GH variant, and prolactin working in concert with peptide growth factors (37, 38). An essential role for the prolactin receptor in alveolar morphogenesis was demonstrated by systemic deletion of the Prlr gene in mice (39, 40). Loss of the Prlr results in an implantation defect; thus the function of this receptor in mammary epithelial growth was studied using epithelial transplantation strategies. Transplanted Prlr null epithelium showed normal ductal development but a lack of alveolar proliferation and differentiation (40). Subsequent studies on Jak2 epithelial knockout and Stat5 systemic or epithelial knockout mice demonstrated that Prlr activation requires these signaling molecules to promote alveolar development and milk secretion (41, 42, 43, 44). Stat5 directly activates expression of a number of genes important for milk secretion including beta-casein.

C. Similarities and differences between human and mouse mammary development
Although all female mammals undergo stages of pubertal ductal and pregnancy-related lobular-alveolar mammary gland development, there are species differences in structure and extent of development during the different stages. All mammary development starts with the formation of TEBs (21), in which DNA synthetic activity is very active and growth is very rapid compared with ducts. In mice, pubertal development produces terminal ductal ends that are simple undecorated ductal ends within the fat pad. In contrast, in humans ductal ends finish with a terminal ductal lobular unit (TDLU) composed of lobular structures surrounded by intralobular stroma and embedded within interlobular stroma (Fig. 3) (45). In humans, there is a significant increase in the DNA-labeling index during the luteal phase of the menstrual cycle when alveolar buds form. In rodents, ductal decorations appear and disappear during the estrous cycle (45). Normal lobular-alveolar development, which occurs in these as well as other species during pregnancy, is stimulated by the combination estrogen, progesterone, placental lactogens, GH variant, and prolactin, with IGF-I being permissive, and concludes with the generation of milk-producing alveolar cells (36). Abnormal lobular-type growth in the absence of pregnancy also can occur. It appears without the stimulation of pregnancy, is considered a preneoplastic lesion, and has been described in multiple species including mice, rats, dogs, primates, and humans. It has been termed hyperplastic alveolar nodules in mice and rats and atypical lobules, types A and B (ALA and ALB), in humans (46). These structures, originally described in the 1970s, are likely related to the hyperplastic enlarged lobular units (HELUs) more recently discussed by Allred (see discussion on hyperplasia in Section IV. A). Original descriptions of hyperplastic alveolar nodules in mice included the fact that they are able to develop into tumors when transplanted into gland-free mammary fat pads (47, 48).

View larger version (131K): [in this window] [in a new window]   FIG. 3. Comparison of the structure of mouse (A) and human (B) mammary ductal ends. The structure of mouse mammary ductal ends is less complex than that of humans. In the mouse, it is termed a terminal ductal end, the epithelial structure has only a few structural elements, the surrounding collagen is not very dense, and the epithelial/collagenous structure is embedded within a stroma primarily composed of fat (so-called fat pad). In comparison, the human mammary ductal end is termed a terminal ductal lobular unit (TDLU). The epithelial structures are more complex, and the epithelial cells are directly surrounded by a loose cellular intralobular stroma with this epithelial/collagenous structure, then embedded within a denser interlobular stroma. Images of mouse and human ductal ends were obtained by reflectance confocal microscopy. Epithelial structures appear as a brighter white due to enhanced reflectance from their cellular nuclei. [Reproduced with permission from M. T. Tilli et al.: J Biomed Opt 12:051901, 2007 (90 ). © SPIE.].

	III. Growth Hormone and IGF Control of Mammary Gland Development

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

B. The role of the pituitary gland and GH in mammary gland development
Although mammary gland ductal development is initiated by the estrogen surge during puberty, execution of the complex developmental program is dependent upon the interactions between multiple hormones and growth factors acting both systemically and on a local level. One of the key systemic factors is pituitary-derived GH. Employing advanced isolation techniques for the time (59), Lyons et al. (51) made major strides in identifying these hormones by testing the effects of various combinations of ovarian and pituitary hormones on restoring mammary development in hypophysectomized and oophorectomized female rodents, with or without adrenalectomy, or in hypophysectomized male rats. Male rats are responsive to the same hormone combinations as female animals. Lyons et al. found that GH was the factor capable of substituting for the pituitary gland (51) in promoting ductal development. Nandi (54, 60) noted that GH alone or GH together with estrogen had greater effects on ductal morphogenesis in hypophysectomized, oophorectomized C3H female mice than prolactin, although it was noted that even the purest preparations of GH or prolactin can be contaminated with one or the other (51), sometimes leading to confusing results. Kleinberg and colleagues (61, 62) later supported and extended the work of Lyons and Nandi and their colleagues. They showed that purer preparations of GH implanted locally into the mammary gland, whether lactogenic, nonlactogenic, wild type, recombinant, or mutant, caused significant ductal morphogenesis first in hypophysectomized, castrated male rats and later in hypophysectomized ovariectomized female rats, whereas prolactin and placental lactogen had no effect (61, 62). Estradiol, in the absence of GH, had little or no effect on ductal morphogenesis in these animals. However, it significantly enhanced the action of GH in pubertal mammary development when the hormones were administered together for 5-d periods (61, 63). To determine which lactogenic and somatogenic pituitary hormones were responsible for ductal morphogenesis, they compared the effects of naturally occurring and mutant hormones on various endpoints including the ability to bind to GH receptors, the ability to stimulate growth of NB2 cells (lactogenic activity) (64), and the ability to stimulate ductal morphogenesis. They found that only those hormones that bound to GH receptor were able to stimulate ductal morphogenesis, regardless of lactogenic activity (62, 61). The importance of GH in ductal development is underscored by experiments in nature. GH-deficient animal models including Ames, Snell, and Lit/Lit mice have impaired mammary development that can be rescued by treatment with GH if there is sufficient estrogen available (66) (D.L. Kleinberg, unpublished observations), but not by estrogen alone. Local expression of GH has been reported in mammary epithelium, particularly during puberty (67). However, the fact that estrogen alone does not take the place of GH plus estrogen in mammary development in hypophysectomized and ovariectomized rats (58) indicates that GH produced locally in mammary epithelium does not substitute for pituitary GH. Thus, the importance of locally produced GH in development is not established for ductal morphogenesis (67). It may, however, play other as yet unidentified roles in breast cancer and could be a target for GHRH through its receptor because both are found in mammary gland (68, 69, 70, 71).

The final known hormonal action of pituitary GH in mammary development is the stimulation of IGF-I mRNA and IGF-I itself (62, 63, 61, 72). IGF-I mRNA was increased in mammary glands by circulating GH. Estrogen enhanced that effect, but had no independent effect. Experimental evidence indicates that this process takes place largely in the mammary fat pad as opposed to the epithelial glandular tissue, a conclusion that was reached because GH stimulated as much IGF-I mRNA in mammary fat pad with or without epithelial structures (73). The source of IGF-I production in the fat pad has not been defined and may be from multiple sources including fat, fibroblast, immune, and/or endothelial cell types. GH also increases IGF-I mRNA in subscapular fat pads, suggesting the possibility that this action of GH on fat pads may be nonspecific (Fig. 2). Most of the subsequent action of GH on TEBs and ducts has been thought to be via a paracrine effect of GH-stimulated IGF-I. This concept is supported by data showing that stromal IGF-I enhances expression of cell cycle regulators during ductal development (37, 74). Members of the Wood laboratory (37, 72) has added a further dimension to the role of IGF-I in mammary development. They found that IGF-I is also produced in TEBs and that epithelial deletion of IGF-I results in a deficit in ductal branching. It is not known whether GH directly stimulates IGF-I expression in TEBs as was shown for IGF-I in the fat pad. A role for autocrine or paracrine GH in IGF-I production in TEBs has not been demonstrated (67).

C. Effects of IGF-I in pubertal mammary development
The preponderance of evidence indicates that IGF-I mediates all of the actions of GH in mammary gland ductal morphogenesis (66, 75). The fact that IGF-I can substitute for GH in mammary development in hypophysectomized and castrated male rats was shown in experiments in which either intact or aminoterminally shortened forms of IGF-I, whether administered locally or systemically, caused mammary development when given together with estrogen (76). Because the shortened form of IGF-I, which binds less well to IGF binding proteins (IGFBPs) than wild-type IGF-I (77), is far more potent than intact IGF-I in stimulating ductal morphogenesis, des(1–3) IGF-I was used in subsequent experiments instead of wild-type IGF-I to circumvent the effects of IGFBPs (see Section III.F). Although GH may have direct effects on metabolic actions or others, it has no effect on stimulating mammary development in animals incapable of making IGF-I (61). Thus, all of the actions of GH in mammary development are thought to be mediated by IGF-I. Although mammary development can be induced by either systemic or local administration of IGF-I (61, 62, 66), studies by Richards et al. (78) indicated that local production of IGF-I in the mammary stroma is likely more important than systemic IGF-I, at least for ductal development. Richards et al. found that pubertal mammary development is not impaired in mice with a liver-specific deletion of the igf1 gene in whose serum IGF-I was reduced by 75% (78). In another study, elevation of circulating IGF-I levels (in a transgenic mouse expressing the IGF-I gene under a liver-specific promoter) caused expected increases in body size and bone length (79) but had little or no effect on ductal morphogenesis (A. V. Lee, unpublished observations). It is possible that the levels of IGF-I produced in this animal model were not as high as levels reached when systemically administered IGF-I was found to cause mammary development (76).

IGF-I^(–/–) female mice, who like GH-deficient animals have deficient mammary development (66, 80), have been used to help determine whether IGF-I mediates the full effect or a partial effect of GH in mammary development. IGF-I, either alone, or together with estradiol stimulated ductal morphogenesis in these animals incapable of making IGF-I, but GH together with estradiol had no effect (66). This indicates that IGF-I mediates the entire effect of GH in pubertal mammary development. IGF-I actions are mediated through the IGF-IR in mammary epithelial cells. Bonnette and Hadsell (81) found that mammary epithelial cells lacking the IGF-IR had reduced ductal outgrowth and proliferation, particularly in the cap cells of the TEBs, when transplanted into wild-type fat pads. However, the ductal phenotype seen with epithelial loss of IGF-IR is less severe than that observed with a systemic deletion of IGF-I, even when the IGF-I null mice are given ovarian hormones (66). Taken together, these results suggest that IGF-I may have indirect effects on ductal morphogenesis through activating the IGF-IR in stroma in addition to its direct actions through binding the IGF-IR in epithelial cells.

D. Interaction between IGF-I, estrogen, progesterone, and prolactin in mammary ductal morphogenesis
Although IGF-I has an independent effect in stimulating TEB development, the addition of estradiol to IGF-I causes a synergistic increase in the number of TEBs formed (63). To assess the relative effects of IGF-I and estradiol, IGF-I^(–/–) female animals were oophorectomized and treated with IGF-I or estradiol or both for periods of 5 and 28 d. Estradiol alone had no effect on TEB development at 5 d, but after 28 d the area occupied by ducts was increased although there were no true TEBs formed. When IGF-I was given with the estrogen, the number of TEBs formed was almost double (82). Estradiol also enhances the effect of IGF-I, as evidenced by stimulation of insulin receptor substrate-1 (IRS-1) phosphorylation and cell division and inhibition of apoptosis, the known effects of IGF-I in the mammary gland (80, 83, 84, 85). In fact, continued administration of IGF-I and estradiol gradually increased ductal morphogenesis so that the mammary gland tree occupied 92% of the mammary fat pad after 28 d of treatment (66, 82). Thus, the entire process of ductal morphogenesis might be ascribed to the combined effects of IGF-I and estradiol, together with other factors including other growth factors and cell survival and cell proliferative pathways (5, 26, 86). Both the TEBs and ducts formed in response to IGF-I plus estradiol were hyperplastic (Fig. 2B)

Progesterone is not required for ductal morphogenesis because pubertal mammary development proceeds normally in PR^(–/–) mice (87). However, lobular-alveolar development in this animal model is distinctly abnormal. Recently, Ruan et al. (82) provided evidence that, like estradiol, progesterone had no independent effect on mammary development unless IGF-I was present. In the presence of IGF-I, progesterone was found to cause a form of ductal morphogenesis that was mediated by the progesterone receptor (PR) (Fig. 2C). The ductal tree was made up of thin ducts without ductal decorations, quite different from the ductal morphogenesis caused by estradiol and IGF-I (82). The fact that the PR mediates this effect was supported by the fact that RU486 prevented formation of TEBs and ducts that would have been induced by IGF-I together with progesterone (82). Although progesterone is able to stimulate ductal morphogenesis in the presence of IGF-I, there is no evidence that progesterone acts in this fashion in normal pubertal development. The mechanism by which progesterone acts is similar to that of estradiol. Progesterone enhances the action of IGF-I by stimulating phosphorylation of IRS-I and cell proliferation and inhibiting apoptosis and likely other postreceptor actions of IGF-I. However, it is likely that the specific downstream events that follow estrogen plus IGF-I stimulation are different than those induced by IGF-I plus progesterone because the type of ductal structures that are induced are not identical. Progesterone together with estrogen, and in the presence of IGF-I, produces secretory structures that decorate the ducts (88).

GHs that possess lactogenic activity have been shown to have a more profound effect on mammary development than nonlactogenic ones (61, 62). However, prolactin itself has no effect on mammary development when given together with estradiol. Therefore, it is possible but not proven that prolactin may have a role in ductal morphogenesis but, like estrogen and progesterone, requires IGF-I to have such an effect.

E. Role of IGF-I in lobular-alveolar development
IGF-I is required for lobular-alveolar development as it is for ductal morphogenesis (82). In mammary gland whole organ culture, lobular-alveolar development is induced in estrogen/progesterone primed glands by the combination of hydrocortisone, aldosterone, prolactin, and insulin (89). Similar studies demonstrated that the high levels of insulin required for epithelial growth in mammary gland whole organ culture could be replaced by IGF-I (72, 74).

True lobular-alveolar development occurs only in pregnancy or pseudopregnancy in response to rising and sustained levels of estrogen and progesterone (51, 54, 60, 91, 92). Experimental lobular-alveolar development in hypophysectomized, oophorectomized rodents requires IGF-I in order for estradiol and progesterone to act (82). Although the pituitary gland is essential for mammary development, lobular-alveolar mammary development can be rescued in hypophysectomized animals during pregnancy by placental GH variant and/or chorionic somatotropin (93). We are not aware of experiments to determine whether lobular-alveolar elements induced by this group of hormones would be maintained if IGF-I was withdrawn after the glandular development occurred. However, present data indicate a requirement for GH-induced IGF-I for lobular-alveolar development in addition to its role in pubertal development (68). IGF-I expression is maintained in mammary stroma throughout pubertal and pregnancy stages, and epithelial expression of IGF-I mRNA increases during the latter half of pregnancy (72, 74). Analysis of glands heterozygous for IGF-I revealed a decrease in alveolar budding after 5 d of pregnancy, supporting an essential role for IGF-I in early alveolar differentiation (37). In addition, the IGF-I heterozygous glands had delayed expression of milk proteins during alveolar differentiation. The induction of IGF-I in the epithelium during the latter half of pregnancy may reflect actions in epithelial cell survival because overexpression of IGF-I from a milk protein promoter causes delayed involution due to inhibition of apoptosis (83, 94).

Studies from Hovey et al. (95) and Brisken et al. (96) demonstrated that IGF-II is a direct target of prolactin signaling in mammary epithelial cells and that it mediates, in part, the effect of prolactin on alveolar differentiation. Studies by Brisken et al. (96) support the hypothesis that prolactin induction of IGF-II causes increased expression of cyclin D1. Thus, to determine which aspects of alveolar differentiation were mediated by IGF-II, a retrovirus was used to express IGF-II in Prlr null epithelial cells, and the cells were used in transplantation experiments. The results of this study suggested that IGF-II rescues a part of prl-mediated alveolar proliferation but not differentiation. The IGF-II null epithelium showed reduced alveolar formation in midpregnancy after transplantation but normal alveolar development by the end of pregnancy, suggesting that something else may compensate for the loss of IGF-II in these cells. The most likely candidate for this compensation is IGF-I, based on its similar ability to stimulate the IGF-IR and the demonstration that IGF-I expression increases in mammary epithelium from mid to late pregnancy (72).

F. Role of IGF binding proteins in GH and IGF-I action in the mammary gland
The six high-affinity IGFBPs, IGFBP-1 to -6, are present in developing mammary tissues of the mouse (74, 97, 98), where they are thought to mediate IGF-I availability and actions. IGFBP-1 protein is found in tissue extracts of pubertal glands but does not appear to be synthesized locally (97). In contrast, the other five IGFBPs are expressed locally during mammary development (97). IGFBP-2, -3, -4, and -5 are the most prominently expressed, and each has a distinct spatial and temporal pattern of expression. IGFBP-3 and IGFBP-5 are expressed in epithelium and isolated stromal cells during both ductal growth and alveolar differentiation. Both IGFBP-3 and IGFBP-5 are present in the TEBs; however, the cap cells express IGFBP-3, whereas the TEB body cells express IGFBP-5. In contrast, IGFBP-2 and IGFBP-4 are expressed predominantly in the stromal compartment of the developing gland, particularly in sites surrounding the growing ductal structures. The preponderance of IGFBP expression in and immediately surrounding the developing epithelial structures supports a role for these molecules in mediating epithelial-stromal interactions and likely regulating availability and localization of the IGF ligands. With respect to this hypothesis, several of the IGFBPs can bind the extracellular matrix (ECM) and thus are likely to provide a local pool of IGFs within the ECM. Expression of most of the IGFBPs decreases during lactation; IGFBP-2 and IGFBP-5 particularly are reexpressed during involution.

Although much of the function of the individual IGFBPs is still unknown in mammary development, the IGFBPs in mammary tissue likely have complex biological actions, including promoting or inhibiting IGF effects as well as IGF-independent actions. Similar to overexpression of the IGFs during lactation, transgenic overexpression of IGFBP-3 from a milk protein promoter results in delayed involution (75). In contrast, overexpression of IGFBP-5 accelerates involution (99), and a recent report demonstrated that loss of IGFBP-5 results in delayed involution (100). These data suggest that these two IGFBPs may have opposing effects; however, it is unclear whether these actions are through modulating IGF-mediated survival or whether they reflect IGF-independent actions such as has been demonstrated for IGFBP-5 (101). IGFBP-5 has been shown to decrease ductal morphogenesis experimentally, presumably by preventing IGF-I action (102). Although IGFBP-5 has the highest expression of any IGFBP in the mammary gland during developmental stages (97), glands from mice with a systemic deletion of IGFBP-5 appear to develop normally (100), possibly due to compensation by other IGFBPs. However, after ovariectomy and replacement with estrogen and progesterone to promote ductal development and alveolar budding, the IGFBP-5 null glands showed enhanced alveolar budding (100). There are clearly complex regulatory loops controlling levels of the IGFs and IGFBPs in response to hormones responsible for the phases of gland development. Decreased Prlr signaling in heterozygous Prlr glands results in increased levels of IGFBP-5 associated with increased cell death and ECM remodeling in early lactation (103). Epithelial cell death and ECM remodeling are normally characteristic of the involuting gland after cessation of lactation. Interestingly, GH treatment of the Prlr heterozygous animals restored alveolar development and reduced activity of IGFBP-5 on remodeling; however, GH treatment failed to restore milk production or rescue lactation defects (103). Kleinberg and colleagues (102) demonstrated that mammary expression of IGFBP-5 is induced by systemic treatment with a somatostatin analog and is associated with reduced growth of the gland. Consistent with the conclusion that the induction of IGFBP-5 was responsible for decreased growth of glandular epithelium in this study, direct administration of IGFBP-5 in the gland also resulted in decreased TEB formation and ductal growth (102). IGFBP-5 induction and decreased growth of gland structures also was observed with overexpression of phosphatase and tensin homolog (PTEN) (104), suggesting that IGFBP-5 may be a general negative regulator of gland growth and is induced to promote remodeling by a wide variety of factors.

G. Interaction between IGF-I and other growth factor families in mammary gland development
In addition to the IGF signaling axis, the major growth factors involved in mammary gland development are the FGF, epidermal growth factor (EGF), and TGF-β families. Although GH and IGF-I are central to mammary gland development, several studies have shown a role for other growth factors acting locally to integrate and coordinate the multiple signals required for this orchestrated process. Although not studied in detail, it is likely that IGF-I acts in concert with these other growth factors.

FGFs are critical for the embryonic initiation of mammary gland formation. In mice, five pairs of lens-like placodes form along a "milk line" around embryonic d 10 and develop into mammary buds. Systemic deletion of FGF-10 or FGF receptor 2 caused a loss of four out of five pairs of mammary placodes (105). Recent studies have shown that systemic gene deletion of IGF-IR or IRSs also disrupts mammary bud formation, with IGF-IR-null buds being small and IRS-null mice having small buds with a disrupted mesenchyme (106). These studies were interpreted to suggest that IGFs cross-talk with the rho signaling pathway to regulate mammary bud development. Overexpression of FGF receptor 1 or IGF-IR in cells in culture or in the mammary glands of transgenic mice produced strikingly similar results, with both receptors causing an epithelial-mesenchymal transition phenotype in vitro and excessive side branching and intraepithelial hyperplasia in vivo (7, 107, 108, 109, 110). Whether these similar phenotypes are due to the redundant nature of growth factor signaling or to a functional interaction between these pathways is currently unknown, and this is a fertile area of investigation given the recent implication of FGF receptors and IGF-IR in human breast cancer.

Postnatal ductal development can be enhanced with exogenous ligands of the EGF family; however, systemic gene deletion combined with mammary gland transplantation has revealed a specific requirement for amphiregulin (AREG) in ductal outgrowth and branching (111). Interestingly, the receptor for AREG [EGF receptor (EGFR)] is also required for branching; however, it is only required in stroma (112), whereas AREG is required in the epithelium (111, 113), indicating a paracrine mechanism of action. Importantly, recent studies have shown that estrogen induces AREG in mammary epithelium to drive mammary gland development via stromal EGFR (114). As mentioned in Section IV. F, estrogen receptor (ER) and AREG are dramatically increased in early hyperplastic lesions in the breast, possibly providing a mechanism for this early growth defect.

Although individual gene knockout models have shown critical roles for IGF-I and EGF family members in mammary gland development, the two growth factor families actually act in combination to regulate both mammary gland development and breast cancer progression. For example, mammary gland organ culture studies have shown that EGF and IGF-I act synergistically to regulate cell growth, with IGF-I being required for EGF-induced DNA replication (115). Of note, early studies using mouse fibroblasts that lacked IGF-IR also showed an absolute requirement of IGF-IR for EGF-induced proliferation (116). Subsequent studies have shown that EGFR and IGF-IR functionally interact and share many common downstream signaling intermediates (117, 118), leading to the notion of dual-targeting of these receptor systems in breast cancer (119).

Mammary gland development is regulated not only by positively acting growth factors, but also by growth inhibitors. The major growth inhibitor in the mammary gland is TGF-β1, which is able to inhibit both TEB growth and ductal branching. GH and IGF-I are able to block the growth suppressive effects of TGF-β1 in part by causing down-regulation of TGF-β1 in the mammary gland (120). IGFs can also rescue TGF-β1-induced apoptosis of mammary epithelial cells in culture (121).

Summary.
Ductal morphogenesis is begun by the pubertal spurt in estradiol. This stage of development, however, requires IGF-I whose production in the mammary fat pad is stimulated by GH of pituitary origin. Small ductal structures present from birth react to this hormone combination by forming TEBs, which in turn lead the process of ductal morphogenesis until the epithelial structures fill the mammary fat pad. Thereafter, the treelike structure remains quiescent until pregnancy, except for the ductal decorations that are caused by progesterone during the normal estrous or menstrual cycle. These hormones interact with other growth factors and IGFBPs in this process. When pregnancy occurs, lobular-alveolar development begins under the influence of estradiol, progesterone, prolactin, and placental factors. IGF-I is required for this phase of development. The alveoli become true glandular structures that fill the mammary gland and eventually produce milk. During pregnancy, although milk is produced within the alveoli, there is no or little milk flow due to the lactogenic inhibitory actions of estradiol and progesterone.

Clues suggesting that the hormones controlling normal mammary development might also be involved in abnormal development include the observations that GH can itself cause mammary hyperplasia, and IGF-I also causes hyperplasia in experimental mammary development in rats and in mice that are deficient in IGF-I.

	IV. Growth Hormone and IGF Regulation of Mammary Gland Hyperplasia

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 
A. Background
Mammary hyperplasia is a generic term that indicates that proliferative disease is present and that the number of mammary epithelial cells is abnormally increased. In the human breast, the more specific terms of ductal and lobular hyperplasia are used. These subtypes can be further subdivided into usual and atypical hyperplasia. Atypical ductal hyperplasia (ADH) or atypical lobular hyperplasia (ALH) demonstrates atypical tissue organization and/or structure and/or nuclear atypia. Hartmann et al. (122) and others have reported that women with proliferative lesions without atypia (Prlr., usual hyperplasia, sclerosing adenosis, and intraductal papilloma) have a higher relative risk of developing breast cancer over 15 yr (1.88) compared with the normal population (122, 123). Nonproliferative fibrocystic disease is associated with a somewhat lower relative risk compared with proliferative lesions (1.27). The relative risk for proliferative lesions rises considerably when they have histological atypia. For example, it is 4.24 for women with ADH. Elmore and Gigerenzer (124) translated these relative risks into actual numbers. They estimated that if you follow 100 women in the general population for 15 yr, five would be expected to develop breast cancer. A modest increase in relative risk such as in women with nonproliferative breast disease would increase that number from five to six. However, in patients with atypical hyperplasia, the number of women developing breast cancer would be expected to be 19.

In human hyperplasias, the proliferating cells largely fill the ductal structures. It has been suggested that a proliferating structure termed "hyperplastic enlarged lobular unit" (HELU) may be a precursor to breast cancer, perhaps even giving rise to ductal and/or lobular hyperplasias (125). Significantly in these structures, the proliferating mammary epithelial cells do not fill the ductal lumen but instead remain as a one- or two-cell layered structure that expands lobular dimensions by increasing the number of cells along the ductal lining. In rodent experimental systems, this latter type of hyperproliferative structure (e.g. HELU) is commonly observed, rather than the usual duct and lobular filling and atypical ductal and lobular hyperplasias (ADH and ALH, respectively) that indicate elevated cancer risk in humans.

The last several years have seen a revolution in the understanding of early breast lesions with the application of high throughput analysis of gene copy number and gene expression. Several of these studies support the notion that many of these early lesions show an imbalance of increased proliferation and decreased apoptosis (125, 126). In addition, many support the model of breast cancer progression via precursor lesions (127). For example, Ma et al. (128) performed microdissection and microarray analysis on normal breast and associated ADH, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) and found that the major change in gene expression occurred during normal to ADH, and that there was little further change during DCIS and IDC, thus supporting the notion that the heterogeneity of breast cancer is imprinted at the earliest stage of the disease. Supporting this, a recent comprehensive study of DCIS and IDC found that the major molecular subtypes of IDC (luminal, basal, and ErbB2) were present in DCIS as they were in IDC, again suggesting that these molecular subtypes are not the result of breast cancer progression and continued changes in gene transcriptomes, but that they represent major distinct groups of tumors that had a defined evolution from the earliest stages of the disease (129).

Three basic approaches to understanding the role of the IGF signaling axis in development of mammary hyperplasia in animal experimental systems have been used. The first is the use of exogenous IGF administered either as a single agent or in combination with other growth factors or hormones, including estrogen and progesterone; the second is through the use of inhibitors of the IGF signaling axis; and the third is through the use of genetically engineered mouse models in which overexpression and/or deletion of specific components of the IGF, GH, and/or estrogen signaling pathways have been made. These approaches are summarized in Table 1.

Numerous clinical studies also link circulating levels of the GH/IGF axis with risk of breast cancer. These include epidemiological studies indicating increased risk associated with polymorphisms of genes in the GH/IGF axis (132) and several studies that have linked circulating levels of IGF-I and risk of breast cancer (133). Initial studies reported an association between high circulating IGF-I increased risk for premenopausal breast cancer, and this was substantiated by three independent meta-analyses (133, 134, 135). Although some recent individual studies have failed to find an association between circulating IGF-I and risk of breast cancer (136, 137, 138), several of these recent studies have significant differences in design and reporting (139). Additionally, differences between studies may reflect different methods for measuring IGF-I, variability and reliability of single measurement of IGF-I (140), and lack of circulating IGF-I levels as a suitable biomarker of IGF activity, Prlr., blockade of IGF-I action via an IGF-IR blocking antibody is associated with elevated circulating levels of IGF-I (141).

The GH/IGF axis is correlated with numerous factors known to be major risk factors for breast cancer, including breast density, birth weight, parity, and height, and a case can be made that the GH/IGF axis is a modulator of these effects (132). A caveat of the GH/IGF-I axis being a major regulator of breast cancer risk is that patients with elevated GH/IGF-I due to acromegaly do not have an increased risk of breast cancer (142). This could indicate a difference in breast cancer formation between humans and rodents, or more likely the fact that women with acromegaly also often have impaired ovulatory function and therefore lower levels of estradiol and progesterone (143).

Prolactin plays a critical role in proliferation and differentiation of the mammary gland during pregnancy, and it may play a role in breast cancer development and progression (144). Several prospective studies have shown a correlation between high circulating prolactin and premenopausal breast cancer, although, similar to the data on circulating IGF-I, not all studies have shown an effect (145). Interestingly, some studies have suggested that women with prolactinomas may have an increased rate of breast cancer, although the hypogonadism and subsequent lowering of estrogen and androgen levels seen with this condition may blunt observations of increased risk (145). This may of course be similar to the hypothesis that acromegalics don’t have increased risk of breast cancer due to their impaired ovulatory function and highlights the imprecise nature of associating individual hormone levels with the risk of breast cancer. Before conclusions can be drawn regarding risk of breast cancer in women with acromegaly and prolactinomas, a consideration (and correction) for multiple hormones that may affect risk of breast cancer must be taken into account; this is highlighted by the increased risk of breast cancer by obesity being almost entirely accounted for by the associated elevated circulating levels of estrogen (131).

In contrast, clinical data indirectly suggest that GH and IGF-I deficiency may prevent breast cancer (146). In a survey of 222 patients with Laron dwarfism or isolated GH deficiency, none developed cancer, whereas 338 first- and second-degree relatives had a cancer incidence of 10–24% including breast cancer (147). Several studies have also suggested that taller individuals have a higher rate of breast cancer than shorter ones (146, 148).

C. Induction of mammary hyperplasia by exogenous hormone administration
Exogenous IGF administration rapidly activates the IGF receptor in a dose-dependent manner in mammary tissue, resulting in phosphorylation of Akt and ERK1/2 in the mammary gland (149). When relatively high concentrations of IGF-I are administered with follicular phase concentrations of estradiol, hyperplasia as defined by an increase in number and cellularity of TEBs results (82). When exogenous GH or IGF or the combination of the two is administered to aging female rhesus monkeys with measurable serum estrogen levels, increased mammary gland size and epithelial proliferation indices result (150). Increased levels of human GH have been linked to increased breast cancer risk (151). Because increased GH levels can increase IGF levels, it is difficult to unequivocally define specific roles in the development of hyperplasia. Moreover, in the mouse system, human GH is able to bind the mouse prolactin receptor and trigger development of mammary hyperplasia through the prolactin receptor-Stat5 signaling axis (152).

Exogenous estrogen alone does not typically result in development of hyperplasia in the absence of disease-associated mutations. This is because the growth-promoting ER is down-regulated by estrogen stimulation, resulting in no net growth (153, 154, 155). In contrast, in the presence of genetic predisposition [including BRCA1 mutation, transgenic c-erbB2 overexpression, or mouse mammary tumor virus (MMTV) infection in the mouse or the constellation of genetic changes in specific rat strains such as ACI and Noble], exogenous estrogen stimulation alone can trigger the development of mammary hyperplasia and adenocarcinoma (155, 156, 157, 158, 159). It is significant to note that loss of full-length Brca1 function is associated with disruption and overactivation of the IGF signaling axis (155, 160, 161, 162). In a similar vein, increased IGF-II expression levels have been found in the estrogen-induced mammary cancer cells of Noble rats (163).

In humans, exogenous estrogen alone does not appear to have a marked effect on breast cancer risk, but the combination of exogenous estrogen and progesterone is associated with increased risk (164). These two hormonal signaling pathways are linked together because estrogen stimulation of ER is a prime regulator of PR expression, but progesterone can also have an independent effect on ductal morphogenesis, with IGF-I being permissive (see Section III. D). Primate model studies provide mechanistic support for the human epidemiological observations demonstrating much more pronounced breast disease after combination treatment with estrogen and progesterone compared with estrogen alone (165). Like estrogen, exogenous progesterone alone does not appear to stimulate the development of breast cancer or premalignant hyperplasia in either rodents or humans, but it can be associated with the development of disease when genetic changes are present such as simultaneous loss of Brca1 and p53 function or when mice are treated with a chemical carcinogen (166, 167). Interestingly, when only Brca1 function is lost and p53 function is intact, exogenous progesterone results only in an exaggerated growth response resulting in increased mammary gland size with increased mammary epithelial cell proliferation but not cancer, similar to the phenotype as described above in IGF- and GH-treated rhesus monkeys (150, 168). In dogs, progestins have been reported to increase GH secretion from mammary hyperplasia, and interactions between the GH and IGF signaling axis and estrogen-progesterone hormonal signaling axis were suggested to be part of the mammary cancer development program (169, 170, 171). In one study, dogs but not monkeys demonstrated malignant mammary tumors after the combination of an estrogen with a progesterone, but not after the administration of either agent alone (172). Zoo cats are also reported to develop mammary cancers in response to exogenous progesterone treatment (173).

Exogenous IGF alone was found to be more potent than the combination of estrogen and progesterone in promoting chemically induced mammary carcinogenesis in rats and appears to activate estrogen signaling (174).

Interestingly, raising levels of IGF-II in the mammary gland by local injection into the mammary gland has been reported to reduce mammary epithelial cell proliferation and morphogenesis by increasing expression levels of PTEN (175). Prolactin is reported to increase IGF-II levels in mammary tissue and to mediate alveolar development in mammary gland organ culture (95).

In summary, exogenous IGF-I administration can trigger the development of mammary hyperplasia but appears to do so most potently in concert with estrogen signaling. Exogenous IGF-II can mediate alveolar development but has not been reported to induce hyperplasia.

D. Studies of mammary hyperplasia in genetically engineered mice
1. Direct.
Direct overexpression of both IGF-I and IGF-II in mammary epithelium have been reported to lead to the development of mammary cancers (5). Overexpression of IGF-I leads not only to hyperplastic changes but also an increase in mammary carcinoma. Likewise overexpression of des(1-3)hIGF-I using the mammary epithelial cell-targeted whey acidic protein enhancer/promoter results in mammary hyperplasia and mammary interepithelial neoplasia (6). Expression of ovine IGF-I using the relatively but not exclusively mammary epithelial cell-targeted MMTV long terminal repeat (MMTV-LTR) as the enhancer/promoter in transgenic mice induced the development of alveolar buds in virgin mice but was not reported to produce hyperplasia (176). Overexpression of bovine GH (bGH) and human GH (hGH) by the relatively systemic (but includes mammary gland as a target) metallothionein enhancer promoter in transgenic mice both cause mammary hyperplasia (177) (W. Ruan, A. Bartke, and D. L. Kleinberg, unpublished data), but carcinoma only occurs in response to hGH. That suggests that the lactogenic effect of hGH may be required for carcinoma to ultimately form. In rabbits, transgenic overexpression of wild-type IGF-I by the mammary epithelial cell-targeted bovine S1-casein enhancer promoter was presumed not to cause hyperplasia because its action was inhibited by a simultaneous increase in IGFBP-2 (178). Overexpression of IGF-II either diffusely targeted by H19 enhancer elements or targeted to the mammary gland by the beta-lactoglobulin enhancer promoter has resulted in the development of mammary cancers (8, 9). In contrast, in one model of MMTV-LTR, regulated transgenic IGF-II overexpression activation of PTEN expression apparently down-regulated the growth response, and no hyperplasia was found during ductal development (175); however, in this model the increased IGF-II decreased cell death after pup weaning, resulting in delayed involution (179).

In summary, whereas in many of the models direct IGF-I and IGF-II overexpression produced mammary hyperplasia, not all did. Phenotypic differences between the different models may in part be due to promoter-selected expression levels, mammary targeting vs. more general expression, and/or species differences related to either the transgene or transgenic host. In some models, induction of negative feedback mechanisms that can limit IGF-I action, such as IGFBPs and PTEN, may negate any proliferative phenotype.

In a like mode, direct IGF-IR overexpression by a doxycycline-inducible mammary epithelial cell-targeted MMTV-LTR enhancer promoter conditional system is also sufficient to induce mammary epithelial hyperplasia and tumor formation in vivo (10), as is overexpression of a MMTV-LTR mammary epithelial cell-directed constitutively active IGF-IR (7). The IGF-IIR is reported to act as a regulator of growth by lysosomal targeting and degradation of the growth-promoting IGF-II. In fact, nonmammary targeted overexpression of IGF-IIR under the regulation of its own enhancer promoter element reduces IGF-II driven mammary carcinogenesis (180).

2. Indirect.
Overexpression of hGH in transgenic mice, but not bGH, causes mammary hyperplasia (W. Ruan, A. Bartke, and D. L. Kleinberg, unpublished data). However, together with estradiol, high doses of bGH do cause mammary hyperplasia in rats (D. L. Kleinberg, unpublished data). In mice, Wennbo et al. (152) found that diffusely expressed mammary gland metallothionein enhancer promoter regulated hGH was able to bind the prolactin receptor and suggest that downstream gene activation through this receptor mediates the increase in mammary tumorigenesis. Targeted conditional loss of full-length Brca1 expression in mammary epithelial cells as well as conditional loss in other tissues is associated with an increase in the activity of the IGF signaling axis, coincident with the development of mammary hyperplasia and cancer (10, 160). Of note, in the clinical setting, Brca1 mutant tumors show elevation of both IGF-I (161) and IGF-IR (162). Overexpression of the p190-B Rho GTPase activating protein by a tetracycline-regulated MMTV-LTR conditional system during pregnancy results in the development of hyperplasia, and this has been linked to an increase in the activity of the IGF signaling axis (181). Increased proliferation of mammary epithelial cells without the development of hyperplasia follows overexpression of a diffusely targeted mammary gland N-terminally truncated version of amplified in breast cancer 1 (AIB1, also known as ACTR, SRC-3, RAC-3, TRAM-1, p/CIP) by a human cytomegalovirus immediate early gene 1 enhancer promoter element, and this is also associated with increased expression levels of IGF receptor (182). AIB1 transgene expression levels and possibly related changes in levels of mammary and serum IGF may mediate whether cell proliferation, hyperplasia, or frank mammary cancer develops in transgenic mice. In a model of MMTV-LTR mammary-targeted AIB1 overexpression, increased levels of mammary IGF and serum IGF were associated with mammary cancer development (183). Finally, Tip30 is a tumor suppressor that can down modulate expression of ER target genes. When Tip30 is deleted from the mouse genome, mice demonstrate increased expression levels of IGF-I as well as other genes in mammary epithelium and develop mammary hyperplasia (184). Although in many of the mouse models mentioned above, gene deletion or overexpression may have altered other pathways critical for mammary hyperplasia and cancer, the consistent link between circulating IGF-I and development of mammary hyperplasia and cancer warrants further direct investigation in these models.

E. Results of decreasing the activity of the IGF signaling axis
Reducing levels of serum IGF-I in mice through genetic engineering can delay both chemically induced and oncogene (SV40T-antigen) -driven mammary carcinogenesis. IGF signaling in mammary tissue is reduced by overexpression of tumor suppressor PTEN in the mammary gland, reportedly by increasing expression levels of IGFBP-5 (104). In mammary cancer-prone MMTV-regulated mammary epithelial cell-targeted Wnt-1 transgenic mice, PTEN overexpression significantly delays tumorigenesis, although a direct role for IGF-I in this effect has not been shown (185). Loss of the protein deacetylase SirT1 in the germline of genetically engineered mice reduces the activity of the IGF signaling pathway in the mammary gland, resulting in impaired ductal development. The phenotype can be reversed by exogenous estrogen stimulation, which increases local IGF-I production. Significantly, whereas mammary gland development may be impaired, the mice are able to reproduce normally (186). Loss of the gene AIB1/SRC3 in the germline of mice reduces serum IGF levels, which is associated with reduced sensitivity to chemical carcinogen induced, v-H-ras, and ErbB2-induced mammary carcinogenesis (185). Decreasing serum levels of IGF by a GH antagonist reduces susceptibility to dimethylbenzaanthracene-induced mammary carcinogenesis (187).

F. Studies of mammary hyperplasia in the human breast
Although there have been numerous studies that have examined the role of the GH/IGF axis in experimental systems of mammary hyperplasia and cancer (mainly rodent) and a number of studies have analyzed somatic growth in patients with IGF-IR mutations (188, 189, 190, 191, 192), there have been virtually no studies in mammary development in women with increased or reduced GH or IGF-I action, despite the existence of IGF-IR. In part this is due to the difficulty in obtaining specimens, as well as the small amount of tissue for study. A direct investigation of hGH mRNA levels in proliferative disorders of the breast (fibroadenoma and DCIS) found elevated levels in both epithelium and reactive stroma compared with adjacent normal breast (193). hGH protein levels measured in tissue extracts were also elevated. A comprehensive analysis of IGF ligands in several proliferative diseases of the human breast found slight elevations of both IGF-I and IGF-II in epithelium and a large increase in IGF-II in stroma (194). Although evidence implicates IGF-IIR as a tumor suppressor, with loss of heterozygosity and mutation in breast (195, 196), an immunohistochemistry study of IGF-IIR levels in benign breast disease, DCIS and IDC found elevated IGF-IIR during progression (197). These data argue against a role for IGF-IIR as a tumor suppressor in breast cancer.

A recent unbiased molecular determination of the commonest abnormalities in the human breast (HELU) indicates that this disease may be hyperproliferative via both ER and growth factor (EGFR and IGF-IR) signaling pathways (125). Thus, the comparison of gene transcriptomes between HELU and TDLU showed a dramatic elevation of ER, a 10-fold increase in amphiregulin (AREG—an activator of EGFR), and a concomitant 14-fold down-regulation of EGF (125). AREG has recently been shown to be a required paracrine factor for estrogen-induced mammary gland development (discussed in Section III. G).

Gene ontology analysis comparing TDLU and HELU identified "insulin receptor substrate binding" as a pathway that significantly discriminated TDLU from HELU. Indeed, IGF-IR was found to be 1.5-fold increased in HELU compared with TDLU, and insulin receptor was 3.5-fold down-regulated. Similar to the AREG/EGF switch, it is possible that the increased IGF-IR in the presence of decreased insulin receptor may allow a switch for the HELU to use the IGF-IR in proliferative signaling pathways compared with the metabolic pathways activated by insulin receptor. These data imply that the IGF system may be important in the hyperproliferation of early breast lesions and thus warrants further analysis of this pathway. More importantly, these data also suggest that inhibition of the IGF axis may potentially decrease proliferation in early breast lesions and thus be a potential prevention strategy.

Summary.
Animal studies have clearly shown that the disruption of the tight regulatory networks controlling mammary gland development can lead to mammary hyperplasia and cancer. This is apparent for the GH/IGF-I axis, where increased activity of the axis, via hormone administration or transgenic overexpression, results in the development of mammary hyperplasia and cancer. Not surprisingly, reduction of this axis, using pharmacological inhibitors or gene knockout studies, can delay cancer initiation and progression.

Although the evidence supporting a role for the GH/IGF-I axis in animal models of animal cancer is both strong and well established, evidence in humans is limited and controversial. Several epidemiological studies have shown that elevated circulating hormones (including estrogen, prolactin, and IGF-I) are associated with increased risk of developing breast cancer. With estrogen, this led to the notion of lowering estrogen levels or activity for the prevention of the disease, something that may eventually be tested for the GH/IGF-I axis. There have been few direct studies of GH/IGF-I activity in mammary hyperplastic tissue, in part due to the lack of appropriate tissue, and lack of measurable readouts of GH/IGF-I activity. As better markers of GH/IGF-I activity are discovered and techniques for measurement of signal transduction in small hyperplastic tissues advance, it is predicted that future studies will confirm a role for the GH/IGF-I axis in mammary hyperplasia and lead to the development of strategies for lowering activity of this axis, potentially in women at high risk of developing the disease.

	V. Molecular Mechanisms whereby Growth Hormone and IGF Promote Mammary Hyperplasia and Progression to Cancer

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 
A. The GH and IGF signal transduction pathways
The biochemistry of signaling pathways downstream of GH and IGF-I has become relatively well established; however, the exact mechanisms whereby GH and IGF-I regulate cell growth remain poorly understood, particularly in vivo. GH signals via GH receptor (GHR), a member of the cytokine receptor superfamily, by causing receptor dimerization (198). In contrast, IGF-I binds IGF-IR, a member of the tyrosine kinase receptor superfamily (199). GHR itself has no kinase activity but binds and activates a cytoplasmic kinase Jak2. Jak2 phosphorylates and activates STAT5, which translocates the nucleus, binds DNA, and alters gene transcription (with IGF-I being one of the most studied target genes). IGF-IR itself can phosphorylate adaptor proteins (Prlr., IRS-1 and shc), which then bind and activate downstream pathways such as ERK1/2 and phosphatidylinositol-3-kinase (PI3K). It should be noted however, that there are no linear signal transduction pathways, with most pathways showing some form of cross-talk. For example, GH has been shown to activate ERK1/2 (200), and IGFs can activate STATs (201). Several excellent reviews have highlighted the molecular pathways of GH and IGF-I important for mammary gland development and breast cancer (151, 202, 203, 204, 205).

B. Transforming potential of GH and IGF signaling pathways
The GH/IGF axis is central to many normal biological processes in animals, including growth, development, metabolism, and longevity (Fig. 1). Many of these same processes become deregulated in mammary hyperplasia and progression to cancer. Supporting a role of GH and IGF-IR in the process of cancer progression, GH and IGF-IR (and many intermediates of these pathways) are transforming oncogenes. Many of these studies were originally performed in mouse fibroblasts; however, recent studies have highlighted major differences in transformation between not only mouse and human cells, but also fibroblasts and epithelial cells (206). Thus, more recent studies have examined the role of the GH/IGF axis in transformation of human mammary epithelial cells. Overexpression of GH or IGF-IR in immortalized (but nontransformed) mammary epithelial cells (MCF-10A) causes complete transformation with growth of cells as xenografts in immunocompromised mice (109, 207). This result was surprising given that virtually all other described oncogenes (Prlr., ras, ErbB2, myc) when expressed alone are not able to transform MCF10A cells to grow in vivo. For both GH and IGF-IR, overexpression in breast cancer cells promotes a more aggressive transformed phenotype with increased cell spreading (208, 209), and survival (210, 211) can be reversed by blockade of GHR (210) or IGF-IR (109, 207). Although the majority of evidence implicates GH action via cell surface GHR, a small fraction of GHR can be found in the nucleus, and forced constitutive localization of GHR to the nucleus also causes transformation of pre-B cells (212).

C. GH/IGF regulation of proliferation and apoptosis (Fig. 4)
Although GH and IGF-IR can clearly act as transforming oncogenes, it is the ability of IGFs to modify many biological processes critical for transformation and maintenance of the transformed phenotype that has created such intense investigation into their permissive action in cancer progression. Probably the most studied is the ability of IGF-I to stimulate proliferation. As noted previously, early premalignant lesions in the breast show hyperproliferation that is associated with elevated IGF-IR levels, and IGF-I is one of the most potent mitogens for breast cancer cells (213). IGF-I is also a mitogen for normal mammary epithelial cells for both two dimensional cultures, in mammary gland whole organ culture and in developing glands in vivo (82). Moreover, IGF-I is required for EGF-related ligands to stimulate DNA synthesis in mammary organ cultures (115). IGFs act on many cyclins and cyclin-dependent kinases to overcome a restriction point in late G₁ and thus stimulate cell growth (86, 115). Studies in mammary epithelial cells also point to a role for IGFs in regulating the G₂/M phase of the cell cycle via induction of cyclin B (115).

View larger version (10K): [in this window] [in a new window]   FIG. 4. Schematic depicting the deregulation of proliferation and survival in mammary hyperplasia. Early progression to hyperplasia is associated with a dramatic increase in ER expression and elevation of GH and IGF-I/IGF-IR levels and IGF signaling.

D. Interaction with estrogen and progesterone receptor
As noted throughout this review, there is a unique interaction between steroid hormones (estrogen and progesterone) and the GH/IGF axis in mammary gland development and breast cancer progression. The exact molecular mechanisms of this interaction remain somewhat elusive in the rodent mammary gland due to the complexity of the in vivo system and the lack of tractable cell line models such as normal mammary epithelial cell expressing ER/PR. Accordingly, the majority of studies have been performed in ER/PR-positive breast cancer cell lines. In this situation, estrogen and IGF-I act in concert to stimulate breast cancer cell growth (215). This synergism involves a complex bidirectional feed-forward loop mediated in part by estrogen enhancing expression of IGF signaling intermediates such as IGF-IR and IRS. Both IGF-IR and IRS have been shown to be estrogen regulated in breast cancer cell lines and human tumors, and also in the normal mammary gland (85, 215, 216). Cui et al. (217) and others have also shown that progesterone regulates IGF-I signaling mainly via regulation of IRS-2. In contrast to estrogen regulation of IGF signaling, IGF-IR causes phosphorylation of ER and enhances its activity (218). A more recent and understudied area of interaction between ER and IGF signaling is the observance of direct interaction between ER and various IGF signaling intermediates including IGF-IR, IRS-1, shc, and PI3K (219, 220). Indeed, short-term activation of ER causes rapid activation of IGF-IR and its downstream intermediates IRS-1 and PI3K. These same intermediates also bind ER and may act in concert to amplify both estrogen and IGF-I signaling pathways.

E. GH/IGF regulation of other pathways in cancer progression (Fig. 5)
Although GH and IGF are critical regulators of mammary epithelial cell growth and survival, recent studies have shown a role for both of these signaling pathways in many other processes either implicated or thought to be important in cancer initiation and progression.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Schematic depicting the multiple molecular mechanisms whereby IGFs modulate tumorigenesis.

Loss of genomic stability is a critical feature of breast cancer progression, with large appearance of genomic instability occurring during the progression of ADH to DCIS (228). IGF signaling regulates the activity of many genes critical for genomic integrity, including telomerase. Short-term IGF-I activates telomerase potentially via phosphorylation mediated by Akt, whereas in the long-term IGF-I increased telomerase levels (229). IGF-IR also regulates DNA repair via activation of ATM and RAD51, and loss of IGF-IR sensitizes cancer cells to DNA-damaging agents (230). In stark contrast to this, two recent studies described mouse models with defects in nucleotide excision repair, which showed greatly reduced serum IGF-I and reduced life span (231, 232). The authors showed that DNA damage actually impaired IGF-I action, which the authors hypothesized may be an organismal mechanism to slow down growth and allow somatic preservation. Of note, p53 mutant mice, which show reduced life span, were found to have decreased IGF-I, reduced mammary gland development, and reduced transplantation, possibly due to a reduction in self-renewing mammary stem cells (233).

It is interesting to note that genomic stability is regulated by a number of tumor suppressor genes (such as p53, BRCA1, etc.), and many of these same tumor suppressor genes actually control expression of IGF-IR, including BRCA1, WT1, p53, ATM, and VHL (234). In all instances, loss of the tumor suppressor causes an elevation of IGF-IR levels that has been noted in human tumors and thus may contribute to tumor progression.

Increased metabolism is a feature of tumor cells that was first recognized by Warburg et al. (235) 70 yr ago and consists of increased glucose uptake and elevated glycolysis. Metabolic markers consistent with the Warburg hypothesis are seen in breast cancer and are associated with poor prognosis (236). Many molecular determinants of the increased metabolism have been elucidated, but given the importance of IGF-I in glucose uptake and glycolysis, it would not be surprising to find that these pathways play a major role in the Warburg effect. Indeed, loss of p53 and activation of Akt can mimic the Warburg effect, and as described in the two previous paragraphs, these are two genes that interact at numerous levels with the IGF signaling pathway (237). Despite this, there have been relatively few studies that have directly tested whether IGFs can enhance metabolism in tumorigenesis.

An important process during the transition from early premalignant breast lesions to developing cancer is the acquisition of cell migration and invasion. IGF signaling is intimately involved in both of these processes (238). IGF-IR can directly bind proteins that initiate migration such as RACK1 (239, 240), or it can alternatively interact with integrins and cell adhesion complexes (241). In addition, IGF-IR bidirectionally cross-talks with the rho signaling pathway (106, 181). IGF-I can induce matrix metalloproteinases to regulate invasion (242) and can stimulate invasion of many breast cancer cell lines. Supporting this, inhibition of IGF-IR can block invasion and metastasis (243, 244).

Summary.
The diverse biological actions of GH and IGF-I are becoming better understood due to a clearer understanding of the immediate signal transduction pathways after GH and IGF-I activation of their respective receptors. Intense investigation of IGF-IR signaling has shown classical regulation of proliferation and apoptosis via the well studied PI3K/Akt and Grb2/ERK1/2 pathways, but it has also shown novel regulation of many other signaling pathways controlling invasion, migration, DNA repair, and others. Although early work showed cooperation between IGF-I and many oncogenes, such as SV40T antigen, a recent area of investigation that has received much interest is the increased activity of the IGF-I axis after loss of tumor suppressor genes such as BRCA1. It is likely that a greater understanding of GH/IGF-I signaling will lead to the identification of new molecular targets to specifically block this axis for the prevention and treatment of cancer.

	VI. Inhibition of Growth Hormone and IGF-I for the Prevention of Breast Cancer

Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 
A. Role of GH and IGF-I in pregnancy-mediated protection from breast cancer
Numerous factors influence risk of breast cancer, and one of the strongest is reproductive history (245). Although many associations with reproductive history confer increased risk of developing breast cancer (Prlr., early menarche), an early first full-term pregnancy substantially lowers (up to 50%) a woman’s risk of getting breast cancer. Pregnancy protection (as it is termed) is one of the most effective natural protections against breast cancer in humans. The underlying principle of pregnancy protection is thought to pertain to persistent changes (due to pregnancy) in mammary epithelial differentiation and/or intracellular regulatory pathways (246). However, these changes alone are not sufficient to sustain the protective effect, with several studies pointing to a significant role for the mammary stroma and circulating hormones (247).

Recent evidence suggests that pregnancy protection is linked to alterations in key endocrine hormones responsible for stimulating mammary gland development. In rats, Thordarson et al. (248) showed that a single time point measurement of serum GH from 120-d-old parous Sprague-Dawley females was significantly lower than their age-matched virgin counterparts. Supporting this, an analysis of IGF-I levels in women from the Nurses Health study found that serum IGF-I levels were lower in parous compared with nulliparous women (249). These animal and human data strongly suggest that pregnancy-associated changes to the endocrine GH/IGF axis may confer protection from mammary tumorigenesis. Similarly, animal studies have shown large (but often nonsignificant) decreases in prolactin levels after pregnancy (248), and parity is associated with lower prolactin levels in humans (144, 250).

Alterations in circulating GH and IGF-I levels after pregnancy are important, given the wealth of literature suggesting that these hormones are critical in mammary tumorigenesis. For example, rodent studies using the Spontaneous Dwarf Rat, which has a mutation in the GH gene resulting in nondetectable circulating GH and subsequently low IGF-I, have shown that these animals are completely resistant to chemically induced mammary tumorigenesis (251), an action that was reversed with bGH treatment or IGF-I. A recent extensive study of hormone pulse profiles from two commonly used pregnancy protection rat models found that a single full-term pregnancy was sufficient to permanently lower basal levels of circulating GH in both rat strains but that this change was not associated with a change in pulse amplitude or frequency. Importantly, the changes in serum GH levels after pregnancy were associated with down-regulation of key GH signaling proteins in the mammary gland (A. V. Lee, unpublished data).

These studies suggest that by reducing circulating levels of GH and/or IGF-I, a full-term pregnancy reduces stimulatory inputs controlling mammary gland function, making it less susceptible to tumorigenesis. These results have important implications for the potential use of agents that lower GH and/or IGF-I for the prevention of breast cancer.

B. Preclinical studies of inhibitors of GH and IGFs
As this review points out, numerous lines of both basic and clinical evidence implicate the GH/IGF axis in the development and progression of breast cancer. This led many groups to test whether blockade of GH or IGF-I could block breast cancer progression and metastasis, and the preponderance of positive studies led to the development and recent testing of pharmaceutical compounds most of which target IGF-IR (252, 253, 254, 255, 256). Not surprisingly, researchers are already envisioning means by which targeting GH/IGF may prevent breast cancer.

C. Clinical studies of inhibitors of estrogen action to prevent breast cancer
Several strategies to prevent breast cancer in women at high risk of the disease have recently been shown to be effective. For example, risk factors such as BRCA mutations raise the likelihood of developing breast cancer to as high as 80% of patients during one’s lifetime. Those at highest risk often elect to have prophylactic bilateral mastectomies, which can drastically lower the development of breast cancer (257). Prophylactic oophorectomy has also been noted to reduce development of breast cancer by 50% in BRCA1 patients (258). Certainly, less drastic medical measures would be preferable to some surgical ones if they were as effective or more effective.

Estrogen action inhibition by tamoxifen has been reported to lower the cumulative rate of invasive breast cancer from 42.5 per 1000 women at increased risk to 24.8 per 1000 in the women taking tamoxifen, whereas the cumulative rate for developing noninvasive breast cancer fell from 15.8 per 1000 (placebo) to 10.2 per 1000 (tamoxifen) (259). Also, on the positive side, there was a reduction in osteoporotic fractures by 32%. Tamoxifen can also cause serious side effects, including endometrial carcinoma and thromboembolic events, in addition to sometimes intolerable menopausal symptoms (260). Other medications including selective ER modulators such as raloxifene (261) and aromatase inhibitors (262, 263) are possible choices for breast cancer prevention. All are associated with annoying menopausal symptoms. Thus, an alternative medical treatment for breast cancer prevention ideally would include a medication that prevents estrogen and progesterone action in the breast but does not require that estrogen be deficient.

D. Clinical studies based on GH or IGF-I inhibition for breast cancer prevention
The above review provides a rationale for such a treatment. In the first place, estrogen is inactive or almost inactive during development of the mammary gland unless IGF-I is available. Secondly, progesterone also requires IGF-I to act (82). Thus, inhibition of IGF-I would theoretically prevent mammary development and likely tumor formation without the need for reduction of estrogen. A number of compounds that inhibit IGF-I action have the potential of preventing estrogen- or progesterone-induced mammary development. These include pegvisomant (a competitive inhibitor of GH binding to its receptor) (264); IGFBP-1, which preferentially binds to IGF-I preventing it from binding to its own receptor (265); IGFBP-5, which acts similarly to IGFBP-1 (102); and SOM230, a somatostatin analog that lowers pituitary GH production but also has a direct inhibitory effect on IGF-I action in the mammary gland (102). Kleinberg and colleagues (102) have hypothesized that the effect of SOM230 is via stimulation of intramammary IGFBP-5, which in turn prevents mammary IGF-I action. Treatment of 21-d-old intact female rats with SOM230 completely prevented further mammary development over 7 d, whereas mammary development was unimpeded in controls. At d 21, control glands occupied 17% of the mammary fat pad. At 28 d of age, untreated animals had glands that had grown to occupy 30% of the fat pad. Treatment with SOM230 for 5 d resulted in only 14.8% of the fat pad being occupied by glands. SOM230 also prevented GH (supraphysiological), and estradiol induced mammary development in hypophysectomized, oophorectomized female rats (Fig. 6), but this local effect of SOM230 was overridden by the addition of IGF-I. This indicates that SOM230 prevents IGF-I action in the mammary gland. Consistent with the inhibition of IGF-I action, there was inhibition of phosphorylation of IRS-1 and cell division and stimulation of apoptosis. To determine whether SOM230 can be similarly effective in human beings, a 10-d proof of principle trial is underway in women with atypical hyperplastic lesions noted at core biopsy. The effect of the medication, given systemically, on the mammary tissue from excisional biopsies is being tested.

View larger version (95K): [in this window] [in a new window]   FIG. 6. A, Photomicrographs of whole mounts of mammary glands taken from female rats that were hypophysectomized and oophorectomized at 21 d of age and then treated with bGH + estradiol (E2) alone (left), bGH + E2 + SOM230 (center), or bGH + E2 + SOM230 + IGF-I (right). Although there was a system of ducts in the oophorectomized and hypophysectomized animals, there was no glandular development. TEBs were induced by bGH + E2 (left). Their formation was prevented by SOM230 (center) but restored by additional IGF-I (right). B, This set of photomicrographs of sections of mammary gland from hypophysectomized and oophorectomized rats treated with bGH and E2 shows the effect of SOM230 (bottom panel) on phosphorylation of IRS-1 (left), cell proliferation by Ki67 (center), and apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (right) on these glands. Brown staining (arrowheads) shows the effect. SOM230 reduced the IRS-1 phosphorylation and cell proliferation induced by bGH and E2 but increased apoptosis. [Reproduced with permission from W. Ruan et al.: Mol Endocrinol 20:426–436, 2006 (102 ) © The Endocrine Society.]

There is direct and indirect evidence that inhibition of the GH/IGF-I axis might prevent breast cancer. Pregnancies that occur at a young age protect women from developing breast cancer. GH is lower after early pregnancy in women and in animals. A relationship between the lower GH and protection from developing breast cancer has been suggested. Furthermore, GH- and IGF-I-deficient animals are protected from developing carcinogen-induced mammary cancer.

A reduction in IGF-I action is also potentially a viable alternative to employing antiestrogens, based on experimental evidence. The fact that IGF-I together with estrogen causes not only ductal morphogenesis but also hyperplasia, and that estrogen cannot do this in the absence of IGF-I, indirectly but importantly suggests a role for IGF-I inhibition. Also, women with Laron dwarfism do not develop breast cancer as frequently as their first- and second-degree relatives, indirectly suggesting that lower IGF-I may be the reason. It is the recent animal studies reporting that SOM230 reverses and prevents mammary development and hyperplasia that provide the best support for the concept that IGF-I inhibition might be important for prevention of forms of hyperplasia and hopefully breast cancer. A proof of principle study is under way to determine whether this drug will reduce cell division in preneoplastic breast lesions that put women at high risk for breast cancer and also increase apoptosis, as the drug does in rats. If this medication is found to do that in patients, as in rats, it would lead to further investigation on the role of targeted IGF-I inhibition to prevent breast cancer. A better understanding of the mechanism of such targeted therapy would lead to either choosing SOM230 as the drug of choice or developing other approaches. It has not yet been determined whether SOM230 causes IGF-I inhibition in organs other than mammary gland or whether it causes some degree of systemic IGF-I deficiency, but that should be an area of future interest. Finding a medication that can locally inhibit IGF-I action without causing long-term IGF-I deficiency might be particularly helpful in very high risk individuals, such as those with BRCA1 and BRCA 2 mutations.

	Footnotes

 
Disclosure Statement: T.L.W., P.A.F., and A.V.L. have nothing to declare. D.L.K. consults for and is in receipt of grants from Novartis Pharmaceutical Company, the makers of SOM230.

First Published Online December 15, 2008

Abbreviations: ADH, Atypical ductal hyperplasia; AIB1, amplified in breast cancer 1; ALH, atypical lobular hyperplasia; AREG, amphiregulin; bGH, bovine GH; DCIS, ductal carcinoma in situ; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FGF, fibroblast growth factor; GHR, GH receptor; HELU, hyperplastic enlarged lobular unit; hGH, human GH; IDC, invasive ductal carcinoma; IGFBP, IGF binding protein; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate-1; MMTV, mouse mammary tumor virus; MMTV-LTR, MMTV long terminal repeat; PI3K, phosphatidylinositol-3-kinase; PR, progesterone receptor; PTEN, phosphatase and tensin homolog; TEB, terminal end bud; TLDU, terminal ductal lobular unit.

Received for publication May 20, 2008. Accepted for publication November 18, 2008.

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Top Abstract I. Introduction II. Mammary Development III. Growth Hormone and... IV. Growth Hormone and... V. Molecular Mechanisms whereby... VI. Inhibition of Growth... References

 

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