stromal communication in mammary gland development and carcinogenesis

Journal of Steroid Biochemistry & Molecular Biology 80 (2002) 213–230

Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis Walter Imagawa∗ , Vadim K. Pedchenko, Jennifer Helber, Hongzheng Zhang Department of Molecular and Integrative Physiology, Kansas Cancer Institute, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7417, USA

Abstract Epithelial/mesenchymal interactions begin during embryonic development of the mammary gland and continue throughout mammary gland development into adult life. Stromal and epithelial growth factors that may mediate interactions between these compartments of the mammary gland are reviewed. Since mammogenic hormones are the primary regulators of mammary gland development, special consideration is given to hormonal regulation of growth factors in order to explore the integration of hormones and growth factors in the regulation of mammary gland growth and neoplasia. Examination of hormonal regulation of the fibroblast growth factor (FGF)-7/FGFR2-IIIb receptor system in the mammary gland reveals that mammogenic hormones differentially regulate the synthesis of stromal growth factors and their epithelial receptors. These effects serve to optimize the action of estrogen and progesterone on mammary gland development and illustrate that the ratio of these two hormones is critical in regulating this growth factor axis. The role of stromal/epithelial mitogenic microenvironments in modulating the genotype and phenotype of preneoplastic and neoplastic lesions by chemical carcinogens is discussed. Finally, changes in growth factor expression during mammary tumor progression are described to illustrate the relative roles that stromally-derived and epithelial-derived growth factors may play during progression to hormone independent tumor growth. © 2002 Published by Elsevier Science Ltd. Keywords: Fibroblast growth factors; Stroma; Tumor progression

1. Introduction Numerous recent reviews have dealt with various aspects of epithelial/stromal interactions in the mammary gland [1,2] including hormonal regulation [3]. Functions of the extraparenchymal components of the fat pad that sustain epithelial or parenchymal development are many. One role is to support the formation of a basal lamina or extracellular matrix that mediates, in part, the proliferative and lactational responsiveness of the epithelium to hormones and the stabilization of form. Another role is to provide an environment permissive for morphogenesis by the provision of soluble growth regulatory factors mediating proliferation, ductal branching, alveolar differentiation, and vasculogenesis. Thus far, most of these factors are members of a peptide growth factor family but bioactive lipids may also play an important role. How the epithelial/stromal interaction is regulated by hormones is of major importance given that hormones initiate different phases of mammary gland development and influence the progression of breast cancer. Emerging from ∗

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new studies utilizing genetically manipulated mice is the realization that mammogenic hormones target the stroma as well as the parenchyma to co-ordinately regulate the growth and morphogenesis of the mammary gland. This review will focus on the physiological role of hormones in the regulation of epithelial/stromal growth factor networks and the impact of these interactions on carcinogenesis emphasizing primarily rodent systems.

2. Reciprocity between mammary epithelial and stromal: from fetal to adult life The mammary epithelium or parenchyma, from its initial appearance as an ectodermal bud to its full development into a milk-producing gland is in a dynamic and reciprocal relationship with its surrounding environment. Studies utilizing tissue recombination and mammary epithelial cell transplantation have clearly shown that the embryonic growth and morphogenesis of the mammary parenchyma is dependent upon the surrounding mesenchyme [4,5]. During embryogenesis a specific mesenchymal induction of overlying ectoderm creates the mammary rudiment. This mesenchyme also gives rise to the presumptive adipose tissue that will form the

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mammary fat pad. As the mesenchyme affects the development of the mammary bud, the incipient mammary epithelium later modifies the mesenchyme, both morphologically (dense mesenchyme surrounding the mammary cord) and by inducing the androgen sensitivity responsible for sexual differentiation of the mammary gland [5–7]. Thus, the nascent mammary gland and mesenchyme participate in a reciprocal regulatory relationship. This reciprocity continues into adult life. In the adult, only adipose tissue is permissive for full growth and morphogenesis [8] of the mammary parenchyma. The specificity of mammary adipose tissue for mammary epithelial development was illustrated by the ability of the mammary fat pad from adult mice (peripubertal or mature) to support the development of only fetal mammary tissue (with the exception of hair follicles) [9,10]. However, mammary epithelium can undergo limited mammary-like morphogenesis in non-mammary fat tissue [8] and tissue recombination studies have shown that the growth and morphogenesis of the adult glandular epithelium can be supported by certain non-mammary mesenchymes from estrogen-sensitive embryonic tissues [1]. It is also evident that the differentiation of stromal cells, adipocyte function, and the innervation and vascularization of the mammary adipose tissue are affected by the presence of the mammary epithelium [5,11,12].

3. The fat pad environment In a cogent editorial commentary, Neville et al. [13] summarize the compartments of the fat pad and discuss critical questions concerning possible roles of the fat pad in supporting the growth and morphogenesis of normal and tumor mammary epithelium. The fat pad is composed of adipose cells, preadipocytes, fibroblasts, a parenchyma-associated stroma that is distinct from the adipose stroma, cells from the immune system, and an associated vascular system. All of these fat pad elements are presumably regulated and direct the development of the parenchyma which is composed of different cell types (end bud, ductal, alveolar, myoepithelial, stem cells) whose number and appearance in the normal mammary gland depend upon the hormonal milieu or stage of physiological development. There are specific differences in the morphology of the fat pad. A comparison of ruminant, human, and mouse mammary fat pads among ruminants, human, and mice is provided by Hovey et al. [14] who also discuss possible roles of the fat pad in regulating mammary parenchymal development. There are two distinct morphological differences in the fat pad among rodent, human, and ruminant species: the degree of investment of the epithelial parenchyma by fibroblastic connective tissue and the proximity of epithelial cells to adipose cells. The periductal and interlobular connective tissue surrounding the epithelial parenchyma is thicker in ruminants and humans than in rodents. In rodents, ducts and alveolar structures are separated from the adipose cell compartment by a relatively thin layer of stroma (more so

in the rat) and growing epithelial structures (end buds, side branches) are in very close if not direct contact with adipose cells. In ruminants and humans, parenchymal growth takes place within a thick fibroblastic connective tissue far removed from the adipose cells. This morphological difference may have functional consequences since the connective tissue is the site of synthesis of growth factors and adipose cells can also be a potential source of lipids and non-lipid growth factors.

4. Hormonal regulation of mammary gland development 4.1. Hormones are the primary mammogens Mammogenic hormones are the primary regulators of mammary gland development while growth factors can be considered to be downstream co-effectors of hormone action [15–18]. It is difficult to penetrate beyond this conceptualization because of our limited understanding of the mechanisms governing the integration of hormone and growth factor regulation. This is highly relevant to mammary tumor progression because of the demonstrated transforming potential of growth factors in transfection or transgenic models, the elevated expression of growth factors or their receptors observed in breast cancer, and changes in hormonal regulation during breast cancer progression. Physiological studies in rodents have led to the identification of ovarian, pituitary, and placental hormones as primary regulators linking mammary gland development and reproduction. The emergence of breast cancer similarly has a hormonal component (oophorectomy dramatically reduces breast cancer incidence) indicating that an understanding of the hormonal regulation of breast development is essential to devise intervention strategies. There is agreement that hormones are required for breast development and are involved in tumor progression and hormone-dependent tumor growth but the relative roles of individual hormones in human breast development and tumorigenesis are controversial. This is primarily due to inadequacies in experimental systems used to study normal and tumor breast development. Estrogen has received the most attention but prolactin and progesterone are probably also involved [19,20] and recent evidence suggests the possibility that prolactin might be an autocrine factor in mammary cancer in humans and rodents [21–23]. The past decade and a half has produced a significant body of new information about growth factors many of which are found in the mammary gland and are capable of stimulating or inhibiting mammary epithelial cell proliferation. This has stimulated much speculation about the role of growth factors in mammary gland development for both normal mammary gland and mammary tumors. However, at this time our understanding of the physiological roles of growth factors and their specific roles in mammary gland development is far from complete. We need to define specific

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pathways delineating hormonal effects on the synthesis of growth factors and their receptors, the target cells for the growth factor, the biological effect of growth factors, synergistic or inhibitory interactions with hormones, and regulation during tumor progression. To answer these questions both in vivo systems and in vitro primary cell or organ culture models are needed in which hormonal responsiveness is preserved.

epithelium [39] and progesterone acts directly on mammary epithelium to stimulate ductal side branching [40] and alveolar development which may be regulated in a paracrine manner [24,41]. Compatible with the presence of their receptors, both progesterone and prolactin can directly stimulate proliferation of ductal epithelium in primary cell culture, particularly in a synergistic manner [30].

4.2. Hormonal regulation is combinatorial and compartmentalized

5. Growth factors that may mediate stromal/epithelial interactions

Mammary gland development represents a complex program of cell proliferation, cell differentiation, and morphogenesis that is regulated by multiple hormonal interactions [15,16]. Estrogen, progesterone, and prolactin or growth hormone, and placental lactogens co-ordinately regulate the physiological development of the mammary gland from puberty through lactation. Other hormones such as insulin, and glucocorticoids are required as well. Many studies using hormonal supplementation of endocrine-ablated animals, in vitro culture, and now mice deleted in genes for hormones and their receptors, have demonstrated that full ductal growth and lobuloalveolar development requires the combined action of ovarian hormones and prolactin [24]. These hormones affect different phases of mammary gland development and different compartments within the mammary gland. Overall findings indicate that estrogen can target the stromal and epithelial compartments of the mammary gland, progesterone can target the epithelium, and prolactin can target the epithelium and potentially the stroma. The specific stromal cell populations that are affected by estrogen or prolactin are unclear. Estrogen receptors are found in both stromal and epithelial cells within the mammary gland [25,26]. However, since most cells that proliferate in response to estrogen treatment in vivo do not contain estrogen receptors [27–29], and direct proliferative responses to estrogen are difficult to demonstrate in primary cell culture [30,31], estrogen most likely stimulates the proliferation of normal epithelium through indirect mechanisms. These mechanisms probably include the induction of local growth factors or their receptors. In support of this possibility, recent studies examining tissue recombinants from estrogen receptor knock-out and normal mice suggest that estrogen regulation of epithelial proliferation in the mammary gland is a paracrine event mediated by estrogen receptor positive stromal cells [32,33]. Estrogen can also indirectly affect mammary gland development by elevating prolactin and progesterone (P) levels and inducing progesterone receptors in mammary epithelium [30,34–36]. Prolactin can apparently affect ductal branching by an indirect mechanism [37] possibly involving the fat pad since prolactin receptors have been detected in adipose tissue [38]. In contrast, prolactin acts directly upon mammary epithelium to induce alveolar development [37]. Progesterone receptors are found only in mammary

There are multiple pathways, demonstrated and postulated, through which stromal/epithelial regulation can occur within the mammary gland. There can be one-way interactions in which synthesis of a factor is limited to stroma while it targets only the epithelium. The converse may be true for factors synthesized only in epithelium. More complicated interactions may involve the synthesis of a factor in stroma and epithelium with the capability to target both compartments as seen for members of the epidermal growth factor family. Several locally produced factors such as EGF, insulin-like growth factor I, and cytokines can be provided from the circulation and this contribution must be weighed in tandem with local synthesis. Table 1 emphasizes primarily stromal factors that are implicated in the regulation of parenchymal development or parenchymal factors that regulate the stroma. These factors are listed for their potential to mediate stromal/epithelial interactions (stromal to epithelial or vice versa). This table is somewhat artificial since it does not include known growth factors synthesized by mammary epithelium that are thought to act in an autocrine manner but may yet be shown to modify the stromal compartment. 5.1. Growth factors synthesized in stroma and targeting epithelium 5.1.1. Hepatocyte growth factor (HGF) and neuregulin (NDF) Hepatocyte growth factor (HGF) is a mitogen and candidate ductal morphogen for mammary epithelium [42–47]. Studies employing organ culture and the EpH4 mouse mammary cell line indicate that HGF can stimulate ductal branching or ductal morphogenesis [44,48]. Similar observations were obtained using (clone TAC-2) of normal murine mammary gland epithelial cells (NMuMG) embedded within collagen gels [43]. Neuregulin or NDF, a member of the epidermal growth factor (EGF) family of peptide growth factors, can stimulate alveolar-like but not duct-like morphogenesis of TAC-2 cells cultured on matrigel in vitro [48]. HGF and NDF are expressed in mammary stroma adjacent to ductal epithelium and their receptors, respectively, c-MET and ErbB3 or B4 (which associates with B2 after heterodimerization) are expressed in mammary epithelium in humans and mice [43–45]. Expression of HGF and

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Table 1 Epithelial or stromal factors implicated in epithelial–stromal interactions Factor

Site of synthesis/release

References

Cytokines Colony stimulating factor-1 Tumor necrosis factor-␣ (TNF-␣)

Epithelium Epithelium/stroma

[111,112] [114,115]

Epimorphin

Stroma

[97]

EGF family growth factors EGF Amphiregulin Transforming growth factor-␣ (TGF-␣) Neuregulin

Epithelium/stroma Epithelium/stroma Epithelium/stroma Stroma

[125] [128,181] [129,181] [44]

Fibroblast growth factors FGF-7 (KGF) FGF-10 FGF-2 FGF-1

Stroma Stroma Stroma/epithelium Epithelium

[80] [82,147] [82] [179]

Hepatocyte growth factor (HGF)

Stroma

[81]

Inhibin B and/or activin BB/AB

Stroma

[42–45,47,95]

Insulin-like growth factor-I (IGF-I)

Stroma/epithelium

[73]

Lipids Polyunsaturated fatty acids Phosphatidic/lysophosphatididic acids Prostaglandins Parathyroid hormone related protein (PTHrP)

Epithelium/stroma Epithelium/stroma Epithelium/stroma Epithelium

[61] [63,67,182] [14,16] [117]

Transforming growth factor-␤ (TGF-␤)

Epithelium

[183,184]

Wnt-2, 5a, 6

Stroma

[77,78]

c-MET decline during pregnancy and lactation which is possibly indicative of hormonal regulation. In vitro, prolactin has been observed to inhibit c-MET expression in TAC-2 cells [49]. In contrast we have observed a stimulation of c-MET expression by prolactin in primary mouse mammary epithelial cells cultured within collagen gels (unpublished observations). HGF is expressed in parenchyma-free fat pads indicating that its occurrence is not limited to periductal stroma although expression in mammary glands has been observed to be higher adjacent to epithelium. Neuregulin is also expressed only in stroma but primarily during pregnancy where it is hypothesized to be an alveolar morphogen [44]. The expression and activation of ErbB receptors and ligands has been examined in the mouse mammary gland and results show that NDF and its receptors are both present and activated in pregnancy [50] although expression is not necessarily confined to pregnancy (NDF and ErbB3). Direct evidence for hormonal regulation of HGF or neuregulin (NDF) is lacking. There is evidence that estrogen can stimulate HGF synthesis in the ovary [51] via cis-acting ERE elements. Other evidence indicates that estrogen may inhibit COUP-TF repression of the mouse HGF promoter [52]. We investigated the effect of estrogen on HGF expression in ovary-intact mature mice and found that subcutaneous estrogen injection (20 ␮g per day), for 7 days inhibited HGF expression in intact mammary glands by an average of 25% and in parenchyma-free fat pads by an average of 60%

(unpublished observations). There is evidence that the epithelium may secrete a factor that can inhibit HGF expression [42,45] pointing out the complex regulation that may exist. Perhaps, the most studied regulators of HGF secretion have been cytokines. In human skin fibroblasts, prostaglandins and interleukin-1 [53,54], and EGF and fibroblast growth factor (FGF)-2 [55] stimulate HGF gene expression. Interleukins-1 and -6, Tumor necrosis factor-␣ (TNF-␣), and progesterone have been reported to stimulate c-MET expression in ovarian carcinoma cell-lines and HGF in stroma [56,57]. Interleukin-1 and FGF-2 have also been shown to be secreted by carcinoma cell-lines (epidermoid, lung) leading to the induction of HGF secretion in stroma [58] which then may potentiate invasion. Transforming growth factor (TGF-␤) can inhibit HGF expression in lung fibroblasts [59] and may have a similar effect in mammary stroma since the expression of a dominant negative TFG-␤ receptor in stroma results in an increase in HGF expression [60]. 5.1.2. Insulin-like growth factor-I (IGF-I) Deletion of the insulin-like growth factor-I (IGF-I) receptor gene results in an arrest of ductal growth illustrating its importance in mammary gland development (reviewed in [61]). IGF-I and IGF-II are expressed in terminal end buds and in ductal and alveolar epithelium. IGF-II’s role in mammary gland development remains to be determined but because it is expressed only in epithelium and may have

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only an autocrine role it is not listed in Table 1. Its possible autocrine role is uncertain but may not involve proliferation since, in contrast to IGF-I, IGF-II was not capable of stimulating DNA synthesis in organ cultures of mouse mammary glands [62]. IGF-I may have paracrine as well as autocrine roles but the paracrine role is discussed here in the context of hormonal regulation. IGF-I is hypothesized to act as a paracrine regulator of mammary gland development [63] since IGF-I is expressed in mammary stroma where its synthesis is stimulated by GH in the rat mammary gland. The site of synthesis is in the adipose tissue stroma since expression is stimulated by GH in cleared fat pads [64], and the level of GH receptors is higher in stroma than epithelium [65]. The role of IGF-I in mammary gland development appears to be permissive and it is not the sole mediator of GH’s effect on mammary gland development. IGF-I is a partial mediator of GH action since maximum mammary gland development requires the presence of another co-stimulatory factor, such as estrogen which also synergizes with GH in stimulating IGF-I expression [66]. In organ culture of estrogen and progesterone primed mouse mammary glands, IGF-I supplementation is needed for ductal growth [67] but IGF-I alone cannot substitute for GH or prolactin [68]. These in vivo and organ culture observations agree with in vitro data indicating that IGF-I alone does not stimulate mammary epithelial cell proliferation but synergizes with other co-mitogens such as progesterone, prolactin, or EGF [69]. GH probably also stimulates development by IGF-I-independent pathways since IGF-I cannot replace GH in stimulating alveolar development in organ cultures of mouse mammary glands [68]. In addition, since IGF-I is also present in the circulation at high concentrations, the relative role of local as opposed to circulating growth factor levels needs to be considered. In breast cancer, there is a correlation between enhanced activation of the IGF-I receptor and tumor growth. Ovarian steroids, estrogen in particular, are associated with higher IGF-I receptor levels or may induce receptor synthesis [70]. The regulation of the synthesis of IGF binding proteins by hormones and their role as inhibitors or stimulators of breast cancer has been reviewed by Perks and Holly [71]. In cultured mouse mammary epithelial cells, placental lactogens and IGF-I and IGF-II induce the secretion of IGF binding proteins [72]which can act to bind and sequester the IGF. This effect raises the possibility that a loss of induction of binding proteins by increased levels of IGFs or loss of GH or prolactin responsiveness may lead to diminished feedback inhibition of IGF stimulation and the promotion of preneoplastic growth. 5.1.3. Inhibin B and activin B/AB Inhibin and activins are members of the transforming growth factor-␤ family of growth factors. Mice deleted in the ␤B-subunit of inhibin and activin show inhibited mammary ductal elongation and alveolar development [73]. This deficit was shown in transplantation experiments to result

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from a loss of stromal not epithelial inhibin and/or activin. For this reason, Table 1 lists this hormone as a stromal factor although it may be produced by epithelium [74]. Receptors for inhibin/activin have been detected in rat mammary gland and breast cancer cell-lines [75]. Human chorionic gonadotropin (HCG) can stimulate inhibin production in the rat mammary gland, promote lobular development, and inhibit tumorigenesis leading to the proposal of HCG as a hormonal intervention to reduce the risk of breast cancer [76]. 5.1.4. Wnt growth factors Also included in Table 1 are Wnt family members that are expressed only in stroma [77,78] and may target the epithelium as opposed to Wnt proteins involved in postulated autocrine regulation of parenchymal growth. 5.1.5. Fibroblast growth factors The role of members of the fibroblast growth factor (FGF) family in mammary gland development and breast cancer has been recently reviewed [79,80]. In general, FGFs affect proliferation, morphogenesis, and angiogenesis. The latter effect may be mediated by FGFs synthesized in the epithelium or stroma and acting in a paracrine manner on the stroma. Expression of FGFs 1, 2, 5, 6, 7, 8, 10 as well as all four FGF receptor isoforms have been detected in the mammary gland [80–82]. Listed in Table 1 are factors that are expressed principally in stroma and have been shown to target epithelium. FGF-2 can be expressed in myoepithelium (prinicpally in the human breast) and stroma. It can stimulate stromal angiogenesis, proliferation of mammary epithelium from virgin and pregnant mice, and inhibit milk protein synthesis [83,84]. Not listed in Table 1 are FGFs that are expressed in epithelial cells and may act as only autocrine growth factors although this designation may change as their roles are investigated further. FGF-1 is listed due to its possible role as a stimulator of angiogenic activity in the stroma. FGF-7 also known as keratinocyte growth factor (KGF) [80,82,85,86], and FGF-10 [82,87–91] are unique in being expressed only in stroma and targeting only the epithelium where their receptors are expressed. Studies using dominant negative forms of FGF receptors, FGFR2-IIIb and FGFR1-IIIc, indicate that FGFR2-IIIb which binds FGF-7 (KGF) with high affinity is needed for full alveolar developmet. FGF-1 and FGF-10 also bind to this receptor with high affinity [92] and may explain why KGF transgenic mice with a deletion of the KGF gene [93] undergo lactation. Hormonal regulation of KGF/FGFR2-IIIb is discussed below. FGF-10 is required for limb and lung development [87] since mice deleted in the FGF-10 gene are born with limited lung development and limb outgrowth [94]. FGF-10’s important role in epithelial branching morphogenesis during development suggests that it may have a similar function in the mammary gland. FGF-10 like FGF-7 is expressed in parenchyma-free fat pads indicating that expression of these growth factors occurs in the absence of epithelium

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and is thus not limited to periductal stroma. Expression of these growth factors and FGFR2-IIIb during mammary gland development is discussed below. Although FGF-10 targets the epithelium, it may also act through a paracrine mechanism on the stroma to stimulate the proliferation of preadipocytes [95]. FGF-10 expression in cultured fibroblasts is inhibited by TGF-␤ and TNF-␣ but unaffected by EGF and interleukin-1␤ [89]. Subcutaneous estrogen injection can inhibit FGF-10 expression while mammary gland elevating FGF-7 synthesis in peripubertal, intact mice (discussed below). 5.1.6. Epimorphin Epimorphin is synthesized in connective tissue and stroma of a variety of tissues including the mammary gland [96,97] and mediates epithelial morphogenesis. It can also be produced by mouse mammary epithelial cell-lines in vitro [97]. A single study [97] examining a mouse cell line finds that epimorphin may be required to act synergistically with other growth factors including HGF to stimulate branching morphogenesis. Regulation of epimorphin synthesis by hormones in the mammary gland has not been examined. Co-culture experiments with stromal fibroblasts and keratinocytes suggest that epithelium can upregulate epimorphin synthesis via a secreted factor [98]. 5.2. Growth factors synthesized in the epithelium and targeting epithelium and stroma 5.2.1. Transforming growth factor-β Three isoforms of transforming growth factor-␤ (␤1, ␤2, and ␤3) are expressed in the mammary gland. TGF-␤ is secreted as an inactive complex that is activated by proteases and its regulation involves a complex interaction of synthesis, secretion, and activation. TGF-␤ is present within epithelium and deposited into the periductal matrix. It specifically inhibits epithelial DNA synthesis (not affecting periductal stroma) by stimulating the local deposition of extracellular matrix [99]. There is also weak immunocytochemical staining for TGF-␤ in stroma and adipose tissue. Studies using transgenic animals indicate that TGF-␤ acts on the epithelium of alveolar cells in an autocrine manner to inhibit alveolar differentiation [100] but limits ductal growth and branching [18] via a paracrine stromal interaction [60]. Expression of a dominant negative TGF-␤ type II receptor in stromal cells stimulated ductal branching. This effect occurred concurrently with enhanced HGF expression [60] implying that TGF-␤ was inhibiting branching by inhibiting the synthesis of a ductal morphogen in stromal cells. TGF-␤ may have other effects on the stroma such as inhibition of preadipocytes differentiation into adipocytes [101–104] and may thus play a role in the formation of the periductal stromal tissue in addition to stimulating matrix deposition by stromal cells [105].

Hormonal regulation of TGF-␤ synthesis in normal mammary gland is unexpolored but may be regulated (either synthesis, activation, or degradation) by hormones that stimulate ductal branching. Loss of TGF-␤ over incipient ductal branches suggests that progesterone, which stimulates lateral branching [40], may be involved. TGF-␤ can inhibit the growth of breast cancer (sensitive to TGF-␤ autoregulation) in an estrogen-dependent manner; growth inhibitory antiestrogens stimulate TGF-␤ secretion [106]. It has been postulated that antiestrogens may stimulate TGF-␤ synthesis in stroma of early breast cancers to inhibit tumor growth [107]. However, in breast cancers resistant to TGF-␤ inhibition, TGF-␤ can promote tumorigenesis by probably a variety of mechanisms [108]. Interestingly, when stroma is irradiated the presence of active TGF-␤ in stroma increases dramatically possibly promoting preneoplastic or neoplastic progression [109]. 5.2.2. Cytokines Cytokines can be synthesized in mammary epithelium and may have autocrine effects. However, they are included in Table 1 since they can also target the stroma and/or be released from blood-derived cells within the stroma. Colony stimulating factor-1 (CSF-1) appears to positively affect lactation [110]. CSF-1 is expressed primarily in epithelium only at a high level during pregnancy and lactation. The receptor is also expressed in breast epithelium raising the possibility of autocrine regulation. However, CSF-1 may have a local effect on the surrounding stroma. Recent evidence suggests a role for eotaxin as well as CSF-1 in stimulating the recruitment of, respectively, eosinophils and macrophages around end buds during ductal growth which may affect end bud development and branching [111]. Prolactin and insulin up regulate CSF-1 in breast cells while glucocorticoids upregulate the CSF-1 receptor [112,113]. Tumor necrosis factor-␣ (TNF-␣) stimulates the growth of rat mammary epithelial cells in primary culture [114] and can inhibit casein synthesis by binding to different receptors. Expression of TNF-␣ in mammary epithelium peaks during pregnancy while expression of its receptors rises only during lactation suggesting possible hormonal regulation [115]. Since TNF-␣ can be released by macrophages, and epithelial-derived TNF-␣ can possibly stimulate adipogenesis its possible paracrine role is indicated in Table 1. TNF-␣ secreted by macrophages or adipocytes and interleukin-6 secreted by stromal cells and lymphocytes have been hypothesized to regulate estrogen biosynthesis within tumors [116]. 5.2.3. Parathyroid hormone related peptide (PTHrP) Parathyroid hormone related peptide (PTHrP) appears to be an epithelial morphogen that acts only upon the stroma. PTHrP is related to parathyroid hormone but is only produced locally in tissues (recently reviewed in [117]). Embryonic branching morphogenesis of the mammary bud is blocked in PTHrP knockout mice [118]. PTHrP is

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synthesized in the embryonic mammary bud, in end buds during postnatal ductal growth, and at a lower level in alveolar epithelium. (For a discussion of mesenchymal genes implicated in embryonic development see recent articles by Daniel and Smith [119] and Robinson et al. [120]). Its G-protein-coupled receptor is expressed in embryonic mesenchyme, and the fat pad and periductal stroma during ductal growth and pregnancy. Expression is highest in stromal cells surrounding end buds suggesting that PTHrP regulates end bud morphogenesis [117]. At present the only known hormonal interaction of PTHrP is mediation of androgen receptor induction in mammary mesenchyme surrounding the mammary stalk that is responsible for stalk regression in male mice [121]. An interesting aspect of PTHrP receptor signaling is the elevation of intracellular cAMP [121]. This effect on stromal target cells may be relevant to the regulation of FGF-7 or HGF synthesis since the expression of these growth factors is stimulated by cAMP or agents that raise intracellular cAMP levels. Thus, PTHrP is conceivably a regulator of the expression of morphoregulatory factors in the stroma which may mediate the stromal pathway of PTHrP regulation. 5.3. Growth factors synthesized in and targting epithelium and stroma [122] 5.3.1. EGF family members EGF family members and receptors in the mammary gland and breast cancer have been reviewed extensively [123–126]. There is much evidence indicating that estrogen or progesterone can stimulate TGF-␣, EGF and EGF receptor synthesis in mammary tumors or breast cancer [127]. This discussion will focus on hormonal regulation in normal mammary gland. NDF has already been discussed as a factor expressed only in stroma. Family members included here that may be synthesized or localized in both epithelium and stroma include EGF, TGF-␣ and amphiregulin. Cripto-1 is an interesting EGF family related peptide that is synthesized in mammary epithelium and may play an autocrine role in ductal growth [128]. EGF is expressed in epithelium primarily during lactation. Although EGF has been detected in stroma by immunocytochemistry, stromal synthesis appears to be insignificant. Analysis of EGF expression by RNAse protection assay shows that during lactation when EGF expression in the mouse mammary gland is highest (100-fold higher compared to mammary glands from virgin mice), there is no detectable expression in cleared fat pads in these animals (unpublished observations). TGF-␣ expression occurs in stroma and epithelium [129] and rises during pregnancy and lactation. Amphiregulin synthesis is primarily epithelial [122] but is detected in stroma by PCR amplification [130]. Amphiregulin was detected during all phases of mammary gland development [130]. Studies employing mice with deletions in one or more of the EGF, TGF-␣, and amphiregulin genes indicate that amphiregulin is required for ductal growth

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and all three factors contribute to alveolar development [122]. There is evidence supporting a role for estrogen in regulating EGF receptors. In an in vitro study using the NMuMG murine mammary epithelial cell line, estrogen was reported to synergize with EGF in stimulating cell proliferation by upregulating EGF receptors [131]. Haslam presented evidence that treatment of mature (but not pubertal) mice with estrogen and progesterone can cause an increase in iodinated EGF binding in epithelial not stromal cells [25]. A role for EGF in mediating estrogen induction of end bud formation and progesterone receptors has also been proposed [132]. Estrogen treatment can stimulate EGF receptor and ErbB2 phosphorylation in the stroma and epithelium during postpubertal ductal growth implying that estrogen makes ligand available for receptor activation [133]. Further examination of the relative importance of stromal versus epithelial receptors for ductal growth showed that stromal not epithelial EGF receptors were required for ductal growth but not alveolar development [133,134]. This result when considered together with the requirement for stromal estrogen receptors for ductal growth leads to the hypothesis that estrogen can act via stromal receptors to induce EGF family peptides that bind, in turn, to stromal EGF receptors. Stromal cells would then be stimulated to release factor(s) that modulate epithelial cell proliferation and morphogenesis. One would also then predict that in the normal environment of the mammary gland a stromal interaction can abrogate direct epithelial cell responsiveness to EGF ligands. A candidate EGF family member supporting ductal or alveolar growth is amphiregulin which may be an epithelial-derived growth factor that after an estrogen-dependent stromal response, is upregulated and binds to stromal EGF receptors. Hormonal regulation of amphiregulin synthesis has not been reported. One caveat concerning EGF is that it is also present in the circulation at physiologically relevant concentrations important for mammary development as shown by the inhibitory effect of sialoadenectomy on mammary growth [135,136]. This raises an alternative hypothesis, that circulating EGF may be sufficient to activate stromal EGF receptors causing the release of a factor that is a necessary co-factor (not directly locally upregulated by estrogen) for estrogen to promote ductal growth. In this view the major function of endogenous EGF synthesis (at least 10-fold higher during lactation than at any other physiological state) would be to provide a milk-borne factor that promotes neonatal development. The direct mitogenic response to EGF observed in primary mammary epithelial cell cultures could then be considered to be an inappropriate response that can become important in breast cancer where ligand and receptor can be overexpressed by the breast carcinoma cells. 5.3.2. Lipids The potential roles of lipids as factors stimulatory to mammary epithelial proliferation or mammary gland de-

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velopment have been reviewed previously [14,16]. Apart from direct effects of lipids on the proliferation of mammary epithelium, recent data showing that HGF [54] or FGF-7 [137] synthesis is stimulated by prostaglandins suggest that the release of these eicosanoids by epithelium or stromal cells can serve as a paracrine or autocrine signal regulating growth factor synthesis. Novel co-culture systems employing epithelium and adipocytes are now showing new effects of soluble factors released by adipocytes on mammary epithelial morphogenesis and differentiation [138]. Phospholipids (phosphatidic acid, lysophosphatidic acid) are mitogenic alone in constrast to prostaglandins and fatty acids which stimulate proliferation only in synergism with a mitogen. The in vivo roles of lipids have been difficult to resolve but new systems and marker genes may novel avenues for research. Recently, G-protein-coupled receptors for bioactive phospholipids (lysophosphatidic acid, sphingosine-1-phosphate) have been characterized (EDG family [139]) which activate a number of intracellular pathways leading to modulation of proliferation, movement, invasion, and metastasis. Initial studies examining the expression of several EDG family members show that lpA1, lpB3, and lpB1 receptors are expressed in mouse mammary gland (especially in glands from virgin animals) and in hormone-dependent and hormone-independent mammary tumors. Expression occurs at least in stroma since expression was detected in parenchyma-free fat pads (unpublished observations). Studies examining the regulation of these receptors, their distribution between epithelium and stroma, and their role in tumor progression and invasion may reveal novel roles for phospholipids in the mammary gland. Hormonal regulation of receptor and ligand synthesis in the mammary gland is unexplored.

6. Model system: hormonal regulation of KGF/KGFR in the mammary gland Our research has examined the regulation by mammogenic hormones of the unique stromal growth factor, keratinocyte growth factor (KGF, or FGF-7) that targets only associated epithelium in a variety of tissues such as prostate, lung, skin, kidney, and gut [86,140–142] and is expressed in breast cancer [143–145]. The important role that stroma/mesenchyme plays in mammary development and potentially in tumorigenesis makes KGF an attractive candidate stromal morphogen whose potential hormonal regulation would be of significance for hormonal regulation in the mammary gland. As a starting point, the pattern of expression of KGF (FGF-7), FGF-10 (another high affinity ligand for the KGFR), and KGFR (FGFR2-IIIb) during postnatal mammary gland development was examined. The steady-state levels of these mRNAs were quantitated by ribonuclease protection assays (RPA) of total mammary gland (gland#

4) RNA extracts [82]. KGFR expression in mammary parenchyma is maximum in the mature virgin animal, declines during pregnancy and lactation but rises rapidly after weaning. Our findings suggest that the increase in KGFR mRNA level before and after puberty is due to growth of the parenchyma which is complete at 8 weeks in the virgin mouse. During pregnancy, there is reduced expression in alveolar cells and a severe decline due to RNA dilution during lactational differentiation. KGF mRNA levels rapidly increase during the first 2 weeks of life then decline slightly by 10 weeks when ductal development is complete. This decline probably reflects RNA dilution by contribution from the fully grown parenchyma. KGF expression further declines during pregnancy and lactation (32-fold reduction) but rises rapidly during involution. In contrast to KGF, the level of FGF-10 mRNA in the mammary gland does not change during the first 2 weeks but peaks at 6 weeks of age [82]. It was drastically downregulated during pregnancy and lactation with 40-fold less expression at lactation compared to 6 week old virgin mice and rose at a slightly slower rate than KGF during involution. The decline in KGF and FGF-10 expression during pregnancy and lactation can be attributed to the changing ratio of epithelium to stroma since the hormonal milieu of pregnancy did not affect (inhibit) KGF mRNA levels in parenchyma-free fat pads [82]. It is becoming clear that mammogenic hormones differentially regulate KGF mitogenesis in the mammary gland. We have used an in vitro hormone-responsive system to test the hypothesis that mammogenic hormones and KGF can co-operate to stimulate mammary epithelial cell proliferation. Our data show that progesterone and prolactin but not estrogen can synergize (order of potency progesterone + prolactin > progesterone > prolactin) with KGF in stimulating the proliferation of normal mammary epithelium from virgin mice in vitro [146]. Progesterone also increased KGFR mRNA levels in vitro and in vivo which might be one mechanism involved in the synergistic effect. Prolactin had no effect on KGFR mRNA levels in vitro implying that its synergistic effect impacts a postreceptor KGFR signaling pathway. Prolactin’s in vivo effect remains to be investigated. This synergism between KGF and progeserone + prolactin is a compelling demonstration of how the mitogenic potency of hormones and KGF is modified by a co-operative interaction among these individual elements. While estrogen does not directly affect epithelial mitogenesis, it targets the stromal compartment by stimulating KGF synthesis. Estrogen, but not progesterone, is capable of elevating the level of KGF mRNA and protein in the stroma in vivo about 2-fold in whole mammary gland extracts from intact or ovariectomized mice injected with hormone [147]. This effect maximises after 7 days of estrogen treatment at 20 ␮g per day delivered subcutaneously in sesame oil. The relatively long time course of this response suggests that estrogen may be acting indirectly on the mammary gland. Progesterone does not appear to be involved

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since the response to estrogen is observed equally well in ovariectomized mice. FGF-10 is also a ligand for the KGFR but it is regulated differently by estrogen. Mature (10 weeks old) mice were injected subcutaneously with estradiol-17␤ (20 ␮g per day) for 7 days before removing the #3 mammary glands for RNA extraction and analysis of FGF-10 expression by RPA and quantitation of protected bands by densitometry. Values in arbitrary units (mean ± S.E.M., n = 6) for FGF-10 mRNA levels normalized to actin were for control and estrogen-treated animals, respectively, 1 ± 0.05 and 0.25 ± 0.03 representing 75% inhibition by estrogen treatment. Inhibition is observed at an estrogen dose of 2 ␮g and as early as 3 days after initiation of treatment. This inhibitory effect as well as the ability of FGF-10 to bind to FGFR1-IIIb receptors and different binding to and modulation of mitogenesis by heparin [92,148] leads to the prediction that these two growth factors will have differences in tissue localization and effects on mammary gland development. Interestingly, branching in embryonic lung may depend upon a co-operative interaction between KGF and FGF-10 [149] raising the possibility that co-ordinate regulation of these factors is involved in the regulation of branching morphogenesis in the mammary gland. Estrogen treatment in vivo affects KGFR expression in the epithelium as well but with different kinetics and dose-response compared to its effect on KGF. Subcutaneous estrogen injection in peripubertal or mature mice suppresses the level of KGFR mRNA in the mammary gland in vivo by ∼85% (Fig. 1). The effect is observed after 24 h and is near maximal at a estrogen dose of 2 ␮g per day. Estrogen’s

Fig. 1. Effect of 17␤-estradiol (E) on expression of KGFR mRNA in mammary glands of 5 and 11-week-old BALB/c mice. Starting from 4 or 10 weeks of age mice were injected daily with E (20 ␮g) or vehicle (sesame oil). After 7 days, expression of KGFR mRNA in mammary glands was analyzed by RNAse protection assay. Data were calculated as the ratio of KGFR to ␤-actin protected fragments in each sample and expressed in arbitrary units (au). Bars represent mean ± S.E.M. from three independent experiments (n = 9). Significant differences between vehicle control and E-treated animals were observed for each of age group (P < 0.001, by Student’s t-test or ANOVA).

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Fig. 2. Effect of co-administration of estrogen and progesterone on KGFR expression in mammary glands of 5-week-old BALB/c mice. Starting from 4 weeks mice were injected for 7 days with vehicle control, 17␤-estradiol (2 ␮g), progesterone (400 ␮g) or their combination. KGFR mRNA level was measured ribonuclease protection assay. The data are expressed as the percent change compared to vehicle control animals. Bars represent mean ± S.E.M. from three (n = 9) experiments. Significant differences between groups are indicated by different letters, i.e. a, b, c (P < 0.05, by Student–Newman–Keuls test).

ability to elevate KGF expression while inhibiting KGFR expression seems paradoxical but only occurs when estrogen is injected unopposed by other mammogenic hormones. Progesterone (400 ␮g per day) can elevate KGFR mRNA levels 2-fold in peripubertal mice in vivo only after 7 days of injections mostly as a consequence of the stimulation of ductal growth [185]. When estrogen (2 ␮g per day) and progesterone are co-injected we observe a maintenance of the overall level of KGFR mRNA in the mammary gland in the absence of any change in gland morphology (Fig. 2). Thus, progesterone can inhibit the ability of estrogen to downregulate the level of KGFR mRNA making it clear that synthesis of the KGFR is dependent upon the ratio of estrogen and progesterone. If estrogen and progesterone are administered in a sufficient dose for 7 days or more one observes the stimulation of lobuloalveolar development. Neither estrogen nor progesterone alone has this effect suggesting that their antagonism of KGFR expression exemplifies a co-operative interaction that preserves or enhances KGF/KGFR signaling. Thus, during pregnancy when estrogen and progesterone are elevated, the second phase of pregnancy-stimulated ductal proliferation is preserved which is necessary to support full alveolar development. This could explain why alveolar development, which is stimulated by estrogen in combination with progesterone, is blocked when KGFR function is attenuated by expression of dominant negative receptor [80]. Others have discussed a role for progesterone in the pregnancy phase of ductal growth [40]. This interaction between

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estrogen and progesterone may also have implications for preneoplastic progression and tumorigenesis. If estrogen and progesterone interactions can become unbalanced upon the loss of estrogen or progesterone receptor expression or alterations in their signaling pathways, estrogen or progesterone dominance could then shift the balance of growth control to an altered state of abnormal growth factor or growth factor receptor function. A possible local mediator of estrogen’s inhibitory effect on KGFR expression is suggested by the ability of EGF to reversibly inhibit KGFR expression in vitro (unpublished observations). There is an abundant literature describing the inductive effects of estrogen on EGF or TGF-␣ synthesis in breast cancer [123] and evidence has been presented hypothesizing that estrogen inducible stromal EGF plays a role in the acquisition of ovarian steroid hormone responsiveness during the peripubertal period [132]. Thus, it is possible that a stromal mediator of estrogen that affects KGFR expression is member of the EGF family of peptides whose expression is sensitive to estrogen. Amphiregulin may be the best candidate since recent data examining mice null for different combinations of EGF, TGF-␣, and amphiregulin genes show that only amphiregulin is required for ductal growth [122]. All are required for maximum alveolar development. To summarize, we have determined that mammogenic hormones differentially regulate KGF and KGFR synthesis and the mitogenic response to KGF (see Fig. 3). This regulation is dependent upon cell type and altered during tumor progression (discussed below) [150]. Among these hormones, only estrogen targets both the stromal and epithelial compartments to regulate the KGF/KGFR axis. Its mode of action appears to be both direct and indirect (local or systemic pathways). These findings indicate that among mammogenic hormones, estrogen is the key co-ordinator of

Fig. 3. Hormonal regulation of the KGF/KGFR axis in the mouse mammary gland. Estrogen treatment in vivo can elevate the steady-state mRNA and protein level of stromal KGF and inhibit the steady-state mRNA level of epithelial KGFR. The physiological pathways (local and/or systemic) are as yet undefined. Progesterone in vivo can counter regulate the inhibitory effect of E on KGFR expresion and, in vitro, inhibit KGFR mRNA turnover. Both progesterone and prolactin synergize with KGF in stimulating the proliferation of ductal epithelium in vitro. Not depicted is the positive effect of estrogen and prolactin on the level of progesterone receptors (PR), and progesterone secretion.

stromal/epithelial interactions acting via multiple direct and indirect mechanisms. 6.1. Epithelial/stromal microenvironments affect carcinogenesis Many studies examining growth factor synthesis, bioactivities, and employing growth factor transfection of cells or the generation of transgenic mice have demonstrated the potential of growth factors to induce preneoplasia and neoplasia in the mouse mammary gland and promote breast cancer growth in vivo [80,151–155]. Involvement of growth factors in tumor angiogenesis has been summarized [156,157]. The interactions between intracellular hormone and growth factor signaling pathways in breast cancer progression [158] and the roles of stroma in regulating breast cancer growth [159] have been reviewed recently. In this section, we examine how hormonal regulation of growth factors might be important for initiation and progression. The importance of tissue architecture in modulating epithelial growth, morphogenesis, and transformation has been well established. If normal cell/cell, cell/matrix, or epithelial/stromal relationships are disrupted then tissue morphology is altered. Resulting abnormal control of growth, differentiation, and invasion can foster the development and expression of inherent genetic changes leading to tumorogenesis [160–162]. An important concept is that the mammary parenchyma and associated stroma/adipose tissue contain structurally ordered microenvironments. These microenvironments may be composed of different set of mitogens, morphogens, and matrix molecules specifying and maintaining specific components of mammary epithelial architecture [162,163]. It is clear that the stroma can play a key organizing role in organizing these microenvironments. The powerful inductive effect of adult stroma or fetal mesenchyme on mammary gland growth, morphogenesis, and transformation has been demonstrated by Sakakura [9] and Cunha [1]. Fetal mesenchyme from mammary fat pad precursor tissue or salivary mesenchyme can induce abnormal proliferation and morphogenesis when implanted into mammary glands in vivo and promote carcinogen or mouse mammary tumor virus induction of neoplasia [164–166]. The transforming potential of these fetal mesenchymes was also demonstrated in primary co-culture with mouse mammary epithelium where proliferation and anchorage independent growth was reversibly stimulated [167]. Possible mediators of this mesenchymal/stromal effect have been discussed above (Table 1). It follows that hormones as primary regulators of growth factor networks can stimulate carcinogenesis via the regulation of stromally-derived growth factors or their receptors. Studies examining the initiation of preneoplasia or tumorigenesis by the chemical carcinogen N-methlyl-N-nitrosourea (MNU) have shown that this event may be modulated by microenvironments differing in their mitogenic compo-

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sition. In vitro transformation by MNU of mouse mammary epithelium in serum-free, primary culture showed that the mitogenic environment at the time of carcinogen exposure affects the morphology and tumorigenicity of the carcinogen-treated cells upon transplantation in vivo [168,169]. Cultured cells grown in the presence of EGF and exposed to MNU formed predominantly ductal hyperplasias and lobular carcinomas without evidence of a mutation in the 12 codon of c-Ki-ras after transplantation into cleared fat pads. If cells cultured in the presence of progesterone and prolactin were treated with carcinogen, lobular hyperplasias and adenocarcinomas with squamous metaplasia were observed after in vivo transplantation. Eighty percent of these tumors had c-Ki-ras mutations. These findings led to the hypothesis that the mitogenic environment (growth factors and/or mammogenic hormones) influenced carcinogen-induced initiation and the subsequent phenotype and genotype of the resulting preneoplastic and neoplastic lesions [17]. Thus, the genotypes and phenotypes which determine preneoplastic progression and tumorigenesis are profoundly affected by the hormonal and growth factor microenvironment at the time of carcinogen exposure. Nandi et al. further hypothesized that the emergence of hormone-dependent as opposed to hormone-dependent mammary tumors is determined by the mitogenic milieu, i.e. growth factor or hormone-stimulated cells give rise to, respectively, hormone-independent or hormone-dependent tumors [17]. In support of this concept, more recent work showed that MNU carcinogenesis in ovariectomized Lewis rats treated with EGF during the initiation phase resulted in a high percentage of hormone-independent (rather than

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hormone-dependent) mammary tumors with a more malignant phenotype than observed in ovary-intact animals [170]. If we consider hormones to be the primary regulators of mammary responsiveness, how may they affect carcinogenesis? Hormones may act directly and/or indirectly. The different phenotypes and genotypes observed in the above in vitro experiments most likely arose from direct effects of hormones and growth factors on mammary epithelial cells, although other changes affecting the phenotype occurring during in vivo growth cannot be ruled out. In vivo, hormones can act directly on epithelium or induce growth factors or their receptors in stroma and/or epithelium. We can hypothesize that the in vitro effect of EGF mimics an in vivo estrogen response mediated through the stroma as discussed earlier. Hormones and growth factors may also act in a co-operative interaction to facilitate transformation (Fig. 4). Progesterone in vivo could modify the cellular responsiveness to a growth factor as observed for the KGF/KGFR axis as well as act directly as a mitogen. It is not known if these two actions, alone or in synergism could induce different genotypes or phenotypes after carcinogen exposure. Examination of the protective effect of parity on carcinogen-induced mammary tumorigenesis also suggests a role for hormone/growth factor interactions. In the parous rat which is refractory to carcinogen-induced mammary tumorigenesis, the level of circulating GH is lower than in nulliparous rats [171]. Thus, a stromal target for GH may mediate susceptibility to breast carcinogenesis. In the same study, a lower level of EGF receptor and estrogen receptor was also

Fig. 4. Modulation of the mitogenic enviromnent dictates specific pathways for carcinogen-induction of neoplastic progression. Hormones (estrogen, progesterone, prolactin) can affect epithelial cell proliferation either directly, indirectly through the induction of epithelium-specific growth factors, or both effects may co-operate. Co-operative interations may include effects of growth factor-stimulated pathways that act independently of hormone pathways or modulation of hormone receptor activity or selectivity thus altering the hormone response. These different mitogenic stimuli modify carcinogen-induced initiation leading to the development of lesions differing in genotype and phenotype.

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noted in the mammary glands from parous rats suggesting decreased estrogen or EGF sensitivity of the stroma. These examples only highlight the potential pathways through which hormones by regulating both epithelial and stromal cell function can co-ordinately modulate carcinogenesis. In this light one can readily see how changes in hormone ratios, mutations resulting in growth factor or growth factor overexpression or loss can profoundly alter preneoplastic and neoplastic progression.

7. Epithelial versus stromal growth factors during tumor progression Mammary tumors can express growth factors which can support growth in vivo in a hormone-independent manner. It is also clear that hormones can stimulate the expression of growth factors in normal mammary tissue and hormone-dependent mammary tumors or breast cancer. What is not clear is how hormonal regulation of growth

Fig. 5. Expression of fibroblast growth factors and HGF during mammary tumor progression. RNAse protection assays were performed on total RNA extracted from mammary glands from virgin (v), midpregnant (MP), pregnancy-dependent mammary tumors (PDT), or ovarian-independent mammary tumors (OIT), all from DDD strain mice. Analysis of two separate samples from each group with the exception of MP are shown. (a) FGF-1 and FGF-4, (b) FGF-2 and FGF-3, (c) FGF-10, and (d) HGF. The position of the protected fragment for each growth factor and control (GAPDH or rat actin) are labeled.

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factor networks is altered during tumor progression. Can alterations in the regulation of stromal versus epithelial compartments by hormones have a role in the emergence of hormone-independent growth. We have examined the expression of FGFs and HGF in a mouse model of mammary tumor progression in order to view, within a single in vivo model system, the progressive changes in growth factor expression that may occur during progression from normal to hormone-dependent and hormone-independent growth. These results show that during tumor development and progression there is a progressive loss of stromal growth factor expression and the acquisition of the expression of growth factors in tumor epithelium that are not normally expressed. The DDD mouse is a mouse mammary tumor virus expressing strain in which a spontaneously arising, pregnancy-dependent tumor line (T-4) has been derived and maintained by serial passage in vivo [172]. Long-term implantation (i.e. chronic stimulation) of estrogen and progesterone in virgin hosts containing T-4 transplants accelerates the development of tumors with a shift towards ovarian-independence [173]. Using primary, serum-free collagen gel cell culture, the growth regulation of normal cells was compared with pregnancy-dependent and ovarianindependent tumors developed in this mouse strain [174]. In vitro, the growth of T-4 was stimulated by progesterone and prolactin while the ovarian-independent (OIT) tumor grew autonomously and did not respond to hormones. We observed similar levels of FGFR2-IIIb (KGFR) expression in T-4 and normal mammary epithelial cells but a reduction of more than 90% in OIT. However, FGFR2-IIIb mRNA was more stable in T-4 cells compared to normal and not affected by progesterone. KGF expression in tissues was lower in T-4 compared to normal mammary gland and not detectable in OIT [150]. When the effect of KGF on proliferation was examined, KGF could stimulate the proliferation of T-4 (not OIT) cells but synergism with progesterone or prolactin was not observed as seen in normal mammary epithelium [146]. Thus, in T-4, both KGF-stimulated mitogenesis and the regulation of FGFR2-IIIb expression show hormone independence while OIT has progressed to independence from any KGF influence. In Fig. 5 are shown RPA comparing the expression of FGF-1, 2, 3, 4, 10, and HGF among normal mammary glands from virgin (V) and midpregnant (MP) DDD mice and T-4 (or PDT) and OIT mammary tumors. Examination of panels b, c, and d reveals that the expression of stromal growth factors (FGF-2, FGF-10, HGF) is lost during progression. As previously shown, FGF-7 expression is also diminished in PDT and barely detectable in OIT [150]. Conversely, FGF-1, 3, and 4 which are expressed only in epithelial cells are upregulated. FGF-3 and -4 are not expressed in normal mammary epithelium and differ in their pattern of expression since FGF-4 is expressed only in OIT. These data imply that the loss of stromal growth factors is a relatively early event that may lead to the loss of the regu-

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lation ductal and alveolar morphogenesis, a key step during progression leading to the loss of normal tissue architecture. Stromal growth factor repression of malignancy in breast cancer has been suggested for FGF-2 [175] which may have an early role since it was detected in or around myoepithelial cells surrounding intraductal carcinomas but not in invasive tumors [176]. Loss of expression may be related to hormones if, e.g. increased or unopposed estrogen activity can down regulate the expression of a stromal growth factor leading to the selection of growth factor-independent phenotypes. Similarly, loss of the receptor (FGFR2-IIIb) for these ligands may be critical. In the prostate, studies indicate that FGFR2 limits while FGFR1 promotes tumorigenicity [177,178]. Expression of FGF-3 which binds to FGFR2-IIIb would appear to be futile for autocrine regulation but upregulation of FGF-1 and FGF-4 which bind to FGFR1 would compensate. Finally, the induction of the expression of growth factors such as FGF-1 and -4 expressed only in epithelial cells (FGF-4 is not detected breast cancer [179]) could promote metastasis and angiogenesis [180] as a later event in progression providing conditions permissive for autonomous, hormone-independent growth.

8. Conclusions Many growth factors have been found to be expressed in the mammary gland and good candidates for stromal morphogens regulating parenchymal morphogenesis and differentiation have been discovered. Novel epithelial factors such as PTHrP or epimorphin and new roles for cytokines that can also be synthesized by epithelium have added complexity to an already complex system. Comparison of Tables 1 and 2 shows that our understanding of the composition of and regulation by hormone/growth factor networks is sorely deficient. Clearly much work is needed to clarify and elucidate the hormonal integration and control of growth factor networks in vivo. It is apparent that hormones can target both epithelium and stroma and that a complex Table 2 Effect of hormones on the activity or expression of growth factors and their receptors in the mouse mammary gland or cultured mammary epithelial cells Growth factor/receptor

Hormone

Effect

FGF-7

Estrogen Glucocorticoids Estrogen Progesterone Estrogen Prolactin Estrogen/progesterone Estrogen Growth hormone Prolactin Glucocorticoids

Stimulation Inhibit Inhibition Stimulation Inhibition Stimulation Stimulation Stimulation Stimulation Stimulation Stimulation

FGFR2-IIIb HGF c-MET EGFR EGF IGF-I CSF-1 CSF-1R

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interplay among mammogenic targeting these compartments exists. This interplay strongly implicates imbalances in hormonal regulation occurring at any level (circulating or local hormone levels, receptor loss, altered matrix, altered co-regulation of intracellular signaling pathways) as promotional events leading to the disruption of normal control of proliferation and morphogenesis by growth factors. Unbalanced hormonal requlation may underly the mechanisms leading to the observed phenotypic and genotypic heterogeneity in mammary preneoplastic and neoplastic lesions.

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