15 The mammary gland ISABEL A. FORSYTH
INTRODUCTION Lactation represents an important, and for most species, an essential, part of the reproductive cycle. However, the adoption of breast milk substitutes to feed newborn babies has become increasingly widespread through the 20th century. Between 1946 and 1966, the proportion of American infants wholly breast fed on leaving hospital fell from 38 to 18% with similar declines evident in Europe, especially after the 1st month of age. Since the 1970s there has been a revival of interest and of the practice of breast feeding in North America, Europe and Australasia, but breast feeding remains undervalued worldwide. Its decline, especially in the urbanized Third World, has extremely serious implications for infant nutrition, infection and consequently mortality/morbidity. The alternative all too often is to feed inadequate quantities of formula feeds prepared with a contaminated water supply. There are major economic and energy costs of replacing breast milk with powdered milk and there are serious consequences for population growth of removing the now well-established contraceptive effects of a full lactation (see Jelliffe and Jelliffe, 1978). The proper management of lactation requires a good understanding of the physiology of mammary growth and function. Moreover, the mammary gland is an important site of malignant transformation, with breast cancer accounting for some 20% of all female cancer deaths in Europe and North America.
THE STAGES OF MAMMARY GLAND DEVELOPMENT The mammary gland consists of epithelial cells, which are ectodermal in origin (the parenchyma) and mesodermal connective tissue (the stroma). In the lactating gland, at least three types of epithelial cells can be recognized: non-secretory epithelial cells lining ducts, secretory epithelial cells in terminal ducts and in lobules of alveoli and myoepithelial cells which surround alveoli and small ducts within the basal lamina. A fatty stroma, although not necessarily the mammary fat pad specifically, is essential for normal mammary morphogenesis (see Daniel and Silberstein, 1987). Bailliere's Clinical Endocrinology and Metabolism-
vei.s, No.4, December 1991 ISBN 0--7020--1491-5
Copyright © 1991, by Baillierc Tindall All rights of reproduction in any form reserved
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c lob 1
lob3 Figure I. Representation of thc development of breast parenchyma in non-pregnant women. (A) In early adolescence: ducts grow, divide and form club-shaped terminal end buds. (B) After the first menstruation: some ducts terminate in terminal end buds, some in simple terminal ducts and the first lobules arc seen consisting of alveolar buds (AB). (C) With increasing age and after successive cycles: three types of lobules arc seen (lob I, 2 and 3) of increasing complexity, resulting from the sprouting of new alveolar buds. Maximum development is only attained in pregnancy, n, nipplc. Reproduced from Russo and Russo (1987).
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Excellent descriptions of human mammary gland development (Figure 1) are given by Salazar and Tobon (1974) and Russo and Russo (1987) . In brief, the mammary anlage are laid down early in fetal life, from week 4 in the 2.5 mm-long embryo. By birth, a branched tubular gland is established consisting of ducts and primitive ductolobular structures in which epithelial and myoepithelial cells can be already recognized. Transient secretory activity occurs at this time (witch's milk) , indicating competence to differentiate from a very early stage. Puberty marks the next important phase of mammary development. Ducts grow, divide and form club-shaped terminal end buds. Breast size is increased by fat deposition and connective tissue development. Through successive menstrual periods alveolar buds arise from terminal end buds and are organized as lobules of increasing complexity. Maximum development occurs in pregnancy with full lobuloalveolar growth and the attainment of secretory activity from mid-gestation. Morphological changes are minimal during lactation. Postlactational involution follows on weaning, although in parous women the breast epithelium remains more developed than if pregnancy had never occurred. After the menopause there is a reduction in epithelial structures. Comparative studies of mammary development in other species are reviewed by Cowie et al (1980). THE HORMONAL CONTROL OF MAMMARY DEVELOPMENT Studies in vivo in laboratory and farm species have defined hormones from the ovary (oestrogen and progesterone), adrenals (glucocorticoids) , pituitary (prolactin and growth hormone) , thyroid and, in pregnancy, the placenta (steroids, placental lactogen) as important controlling factors in mammary development. There is a clear association (at puberty, in the menstrual cycle and in pregnancy) between periods of mammary development and increases in oestrogen or oestrogen and progesterone in the circulation. However, in hypophysectomized animals, these steroids are without effect. Synergistic actions of steroid and peptide hormones are necessary to bring about mammary growth and function in vivo (see Cowie et aI, 1980; Neville and Neifert, 1983). Species may differ in minimal hormonal requirements, especially for maintenance of lactation (galactopoiesis). For example, prolactin suppression with dopamine agonists leads to rapid cessation of lactation in women, but has little effect in dairy cows in which milk yield is stimulated by growth hormone. The presence in subcellular fractions of the mammary gland of highaffinity binding sites for a large number of hormones suggests direct actions on the tissue to bring about biological effects, although the precise distribution of most receptors between different cell types is not well known. Hormones binding to intracellular receptors in epithelial cells primarily include oestrogen, progesterone, thyroid hormones and glucocorticoids. Peptide hormones binding to membrane-associated binding sites include prolactin and catecholamines and , in myoepithelial cells, oxytocin. Binding studies have previously failed to detect a growth hormone (GH) receptor in
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mammary tissue, but more recent work indicates that these may in fact be present (see Glimm et ai, 1990; Kleinberg et ai, 1990). In vitro systems such as mammary explants or mammary cell culture using collagen or extracellular matrix as substrata (Barcellos-Hoff and Bissell, 1989) allow demonstration of the direct effects of prolactin, glucocorticoids and thyroid hormones , in the presence of insulin, on functional differentiation and secretory activity, although other systemic factors may be involved (Hoeffter and Frawley, 1987). It has proved much more difficult to reconcile in vivo and in vitro findings on growth promotion. In mouse mammary explants, insulin is a sufficient and necessary stimulus to DNA synthesis, but supraphysiological concentrations are needed and there is no convincing evidence of a role for insulin as a major mammary mitogen in vivo (see Forsyth, 1989). The evidence that growth factors act as intermediaries in the hormonal stimulation of the normal mammary gland is reviewed here. Growth factors may act either in an endocrine manner, that is, being produced by distant tissues such as the liver (English et ai, 1990), or they may act locally in a paracrine or autocrine manner. Actions of nonsecreted, membrane-bound growth factors on adjacent cells (juxtacrine, see Anklesaria et ai, 1990) have also received recent attention. A requirement for successful experimentation in this field has been improved methods of culture which allow cells to be maintained, proliferate and differentiate in serum-free medium (see Barnes and Sato, 1980), in response to physiological concentrations of hormones and growth factors . Unphysiological conditions of medium and substratum are still in widespread use. Inconsistencies between different studies may, at least in part, result from differing culture conditions (see van der Burg et ai, 1988). Serum is complex and essentially uncontrolled and may, moreover, contain inhibitors. For example, Medrano et al (1990) found serum to inhibit expression of the oestradiol receptor gene in MCF-7 cells. Another almost universal media component , the pH indicator phenol red, is now known to contain a weak oestrogen which can mimic or mask the effects of oestradiol (Bindal et ai, 1988). NEOPLASTIC TRANSFORMATION IN THE MAMMARY GLAND The development of cancer is a multistage process in which external agents (environmental carcinogens and co-carcinogens, oncogenic viruses) and internal factors (cytoplasmic and nuclear oncogenes, hormones) are implicated. Clinically, about one-third of human mammary tumours show at least temporary regression after oestrogen stimulation is removed or blocked. Oestrogens may have direct effects on cell proliferation, but the concepts are developing that they mainly influence tumour growth through endocrine growth factors (estromedins; Sirbasku, 1978), through autocrine growth factors produced by and acting on epithelial cells and through mutual paracrine interactions between epithelial and adjacent stromal cells (Lippman and Dixon, 1989; Figure 2) . Breast cancer cell lines contain mRNA for, and secrete into conditioned media, a variety of growth factors or growth
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~EstrOmedins/ Secreted growth factors Autocrine (TGFa, TGFp. IGF-I, IGF-n, 52K, PDGF)
Breast Cancer Cell
Figure 2. Possible growth regulatory pathways in human breast cancer cells. Oestradiol (E z) may act directly and also via endocrine 'estrornedins'. It may also stimulate synthesis of stimulatory and inhibitory growth factors (IGFs. TGF·a. TGF-13. PDGF) and other proteins (such as the 52k procathepsin-D) which may act back on the cell and on neighbouring stromal cells, which may themselves produce growth factors. Receptors for some growth factors (insulin. IGFs, EGFrrGF-a) have been recognized on breast cancer and normal mammary cells. Reproduced from Osborne and Arteaga (1990).
factor-like activities with autocrine potential, which are oestrogen-regulated in some oestrogen receptor-positive cells. Lippman, Dixon and colleagues have further developed the hypothesis that the growth advantage of oestrogen receptor-negative tumours may relate to the escape of their growth factor production from regulation by oestrogens, although not all oestrogen dependence/independence may be explicable in this way (see Arteaga et ai, 1988a; Karey and Sirbasku, 1988; Osborne et ai, 1988). The testing of these important concepts has relied much on breast cancer cell lines and more work is needed on primary and metastatic breast tumours. Finally, the products of many oncogenes are being identified as growth factors or growth factor receptors (Gullick, 1990; see also Chapter 7), and, conversely, some hormones and growth factors can regulate the expression of proto-oncogenes (Franklyn and Sheppard, 1989). INSULIN·LIKE GROWTH FACTORS Receptors in the mammary gland Two types of membrane receptors for insulin-like growth factors (IGFs) have been recognized. The type 1 receptor has a 2-5-fold higher affinity for IGF-I than for IGF-II and a low affinity for insulin. The type 2 receptor has a high affinity for IGF-II, no significant affinity for insulin and, using
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recombinant molecules, little affinity for IGF-I (Barenton et ai, 1987). It also binds man nose 6-phosphate. Both types of receptors have been detected in normal mammary tissue from rabbits (Barenton et ai, 1987), sheep (Disenhaus et al, 1988) and cows (Dehoff et ai, 1988; Hadsell et ai, 1990). Breast cancer cell lines (see De Leon et ai, 1988) and human primary breast cancers (Foekens et al, 1989; Cullen et al, 1990) also contain type 1 and type 2 receptors. Using a ribonuclease protection assay, Cullen et al (1990) have shown expression of mRNA encoding type 1 and type 2 receptor proteins (and also the insulin receptor) in breast cancer and mammary cell lines, as well as in specimens of primary breast tumours. The cell lines were both positive and negative for the oestrogen receptor. In human mammary cancers, however, IGF-I receptors assessed by binding assays are positively correlated with concentrations of both oestradiol and progesterone receptors (Pekonen et ai, 1988; Peyrat et ai, 1988; Foekens et al, 1989). Binding of IGF to mammary tissue is affected by physiological state in sheep and cows. In both species the numbers of type 2 binding sites were always higher than the numbers of type 1 sites. Little change in affinity was observed, but lactating tissue contained more receptors of both types than mammary tissue from non-lactating cows (Dehoff et ai, 1988) or late pregnant sheep (Disenhaus et ai, 1988). In a more extensive study of dairy cows, Hadsell et al (1990) found a decline in IGF-I binding to mammary microsomes between day 150 pre-partum and term (approximately 250 days), a rise at parturition and then a slow decline to day 411 post-partum. IGF-II binding was more variable and showed no significant trends (Figure 3). Studies with a monoclonal antibody directed against the type 1 receptor suggest that, as in other tissues, this receptor mediates the mitogenic effects of both IGF-I and IGF-II in the MCF-7 breast cancer cell line (Rohlik et ai, 1987; Cullen et al, 1990). Mitogenic effects of insulin were not, however, blocked over a wide concentration range; an interaction was postulated between occupied insulin receptors and adjacent type 1 IGF receptors, leading to phosphorylation of the 13-subunit of the latter (Cullen et al, 1990). Although at very high concentrations insulin probably acts via the type 1 receptor, mitogenic actions of insulin via its own receptor may also occur; for example, effects on DNA synthesis occur at insulin concentrations too low to interact with the type 1 receptor in normal sheep mammary cells (Winder et al, 1989). The physiological relevance of the abundant type 2 receptors in mammary gland is unknown. The mannose 6-phosphate receptor transports proteins with mannose 6-phosphate moieties within the cell and IGF-II may associate with it for processing or degradation (Czech, 1989). Many breast tumours and cell lines produce an oestrogen-regulated 52 kDa protein, which is mitogenic for MCF-7 cells and able to degrade extracellular matrix, suggesting potential roles in mammary carcinogenesis. It has been identified as a pro cathepsin D-like protease (see Cavailles et al, 1989), which binds to the type 2 receptor via its man nose 6-phosphate moiety. It is taken up by this route by MCF-7 cells and processed (Capony et ai, 1987). Recently it has
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been shown to act on membrane-bound transforming growth factor a (TGF-a), also a mitogen for mammary cells, to release the free peptide (Henry and Shultz, cited by Cullen et aI, 1990). Procathepsin-D is regulated by growth factors (epidermal growth factor (EGF), IGF-I, basic fibroblast growth factor (FGF)) by a mechanism different from its transcriptional regulation by oestrogen (Cavailles et al, 1989). 10
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