208BrdU = bromodeoxyuridine; Col 1 = collagen type 1; ECM = extracellular matrix; EGF = epidermal growth factor; ER = estrogen receptor; FCM = fibroblast-conditioned medium; FN = fibronectin; HGF = hepatocyte growth factor; IGF-1 = insulin-like growth factor I; LM = laminin; PBS = phosphate-buffered saline; PR = progesterone receptor; R5020 = promegestone. Breast Cancer Research Vol 5 No 4 Haslam and Woodward IntroductionMammary gland growth and development are mediated through the complex interactions of steroid hormones, polypeptide hormones, growth stimulatory factors and growth inhibitory factors. Normal development and function of the mammary gland are also dependent upon complex interactions between epithelial cells and stromal cells [1,2]. Stromal cells can regulate the epithelium by the production of soluble growth stimulatory and/or inhibitory factors; and components of the extracellular matrix such as collagens, fibronectin and laminin can also act as signaling molecules for epithelial cells, via specific integrins on epithelial cells. Epithelial cells also secrete factors that influence proliferation and function of adjacent epithelial and stromal cells (Fig. 1).Although there have been numerous studies of signalling mediated by the extracellular matrix and integrin in normal mammary gland and breast cancer cell lines, none has addressed the role of stroma in mediating and modulating steroid hormone action. There is increasing evidence that a number of responses to estrogen and/or progesterone in the mammary gland may be mediated indirectly through paracrine effects. This review focuses on recent studies from our laboratory addressing interactions between epithelial cells and stromal cells and between steroid hormones and growth factors in the normal murine mammary gland and in human breast cancer cells. Steroid hormones and mammary gland developmentEstrogen and progesterone are required for proliferation and morphogenesis of the normal mammary gland. Estrogen drives ductal development during puberty, whereas estrogen + progesterone mediate the proliferative and morphological changes of ductal side-branching and alveologenesis that occur at sexual maturity and during pregnancy [1,2]. Progesterone is also mitogenic in the premenopausal and postmenopausal human breast [3]. The greater risk of breast cancer in postmenopausal women receiving combined estrogen plus progestin hormone replacement therapy than in those receiving AbstractMammary epithelial cells comprise the functional component of the normal gland and are the major target for carcinogenesis in mammary cancer. However, the stromal compartment of the normal gland and of tumors plays an important role in directing proliferative and functional changes in the epithelium. In vivo and in vitro studies of the murine mammary gland have provided insights into novel stromadependent mechanisms by which estrogen and progesterone action in the epithelium can be modulated by hepatocyte growth factor (HGF) and the extracellular matrix proteins, collagen typ...

The acute temporal effects of exogenous oestradiol (0.1 mg/kg per day), progesterone (0.25 mg/kg per day) or both together on the proliferative response of epithelial cells, fibroblasts, adipocytes and endothelial cells in the mammary tissue of prepubertal cross-bred heifers were determined. Mammary biopsies were taken immediately before, then 24, 48 and 96 h after the initiation of daily administration of hormones to three heifers per treatment group. Incorporation of [3H]thymidine into explants prepared from biopsies was evaluated after a 1-h incubation by measuring trichloroacetic acid (TCA)-insoluble radioactivity in explant homogenates as well as by quantitative histoautoradiography. Incorporation expressed as d.p.m./mg tissue or d.p.m./microgram DNA was increased (P < or = 0.05) approximately 11-fold by 96 h in oestradiol-treated heifers. Progesterone-treated animals were unresponsive and heifers treated with both hormones were intermediate in response compared with oestradiol-treated heifers. Autoradiographic data for ductal or terminal duct epithelial cells showed similar dramatic increases in labelling by 24 h with further increased (P < 0.01) labelling by 96 h (5.1% vs 0.1%) in heifers given oestradiol. As with incorporation, tissue from progesterone-treated heifers showed no time or treatment response compared with pretreatment biopsies and tissue from heifers given both showed intermediate responses, i.e. significantly increased labelling by 96 h compared with pretreatment (P < or = 0.05) but less labelling (P < 0.05) than oestradiol-treated heifers. A proliferative response of epithelial cells in oestradiol-treated heifers occurred prior to the response of fibroblasts adjacent to epithelial cells (< or = 50 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta (TGF-beta) has been shown to inhibit mammary morphogenesis, growth, and differentiation in murine studies. We have characterized TGF-beta receptors and their autoregulation, and the growth response to TGF-beta 1 and TGF-beta 2 in cultured bovine mammary epithelium (MAC-T) and fibroblasts. Affinity labelling studies revealed that fibroblast and epithelial cells contained type I, II, and III (betaglycan) receptors, with the type III receptor being the predominant binding component. On both fibroblasts and epithelial cells, TGF-beta 1 and TGF-beta 2 had equal binding affinities for the type I and II receptors, but TGF-beta 2 had a higher affinity for the type III receptor. Also, preincubation of MAC-T cells with 50 pM TGF-beta 1 or TGF-beta 2 markedly downregulated TGF-beta receptors. Proliferative response was measured using both total DNA and 3H-thymidine incorporation. Both TGF-beta isoforms were effective in inhibiting proliferation of MAC-T cells and fibroblasts. Inhibition of proliferation was not altered following immortalization of fibroblasts with SV-40 Large-T-antigen (LT), even when the cells acquired a transformed phenotype. Inhibition of proliferation was not a result of cytotoxicity, as TGF-beta at concentrations 1,000-fold higher than ED50 levels did not increase cell death. Moreover, the inhibition was reversible as shown by return of cellular proliferation to control levels following TGF-beta removal. Although growth inhibition was not transient as culture of MAC-T cells in TGF-beta resulted in sustained inhibition of proliferation for at least 144 h.

commentary review reports research article BM = basement membrane; ECM = extracellular matrix; EGF = epidermal growth factor; ER = estrogen receptor; ERE = estrogen response element; ERKO = estrogen receptor knockout; IGF-I = insulin-like growth factor-I; IGFBP = insulin-like growth factor binding protein; PR = progesterone receptor. AbstractDespite the critical importance of ovarian steroids in the treatment of breast cancer, little is known about the acquisition or loss of estrogen and progesterone responsiveness in either the normal or neoplastic mammary gland. This review focuses on the interactions among mammary stroma-derived extracellular matrix (ECM) proteins, integrins and ovarian hormone-dependent proliferation in normal and neoplastic mammary cells both in vivo and in vitro. In vitro studies show that fibronectin is required for progesterone-induced proliferation of normal mammary epithelial cells and that specific ECM proteins also regulate interactions between growth factors and ovarian hormones. Studies with human breast cancer cell lines have shown that laminin inhibits estrogen-induced proliferation and estrogenresponse-element-mediated transcription in vitro and also inhibits estrogen-induced proliferation in vivo. Reciprocally, ovarian steroids regulate the expression of ECM proteins and their cellular receptors, integrins, during mammary gland development in vivo. The fibronectin-specific integrin, α 5 β 1 is regulated by ovarian steroids and its expression is positively correlated with developmental stages of peak proliferation. These studies suggest that the coordinated regulation of ovarian hormone responsiveness and ECM/integrin expression may be critical to normal mammary gland development and breast cancer growth and progression.

The purpose of the present study was to investigate the role of extracellular matrix proteins (ECMs; collagens I and IV, fibronectin, and laminin) in modulating proliferative responses of normal mammary epithelial cells in serum-free culture to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I). As EGF and IGF-I can alter steroid responses, the interactions among growth factors, estrogen, and R5020 were also investigated. We report the novel finding that all ECMs tested, but not a nonspecific attachment factor, poly-L-lysine (PL), promoted a highly synergistic proliferative response to EGF plus IGF-I. EGF receptors were significantly increased with culture time on all ECMs, but not on PL. IGF receptor expression was significantly 2- to 4-fold higher on all ECMs compared with PL. EGF decreased IGF-binding protein-2 (IGFBP-2) and IGFBP-3 by more than 50% in the presence of IGF-I on PL or collagen I. These results indicate that ECM-specific IGF-I/EGF synergism occurs in response to ECM up-regulation of growth factor receptors and EGF down-regulation of inhibitory IGFBPs. Growth factors did not synergize with estrogen and/or R5020. Instead, estrogen plus R5020 decreased EGF-plus IGF-I-induced proliferation in an ECM-dependent manner. These studies demonstrate that proliferation of normal mammary epithelial cells involves complex interactions among steroids, growth factors, binding proteins, and ECMs.

Extracellular matrix (ECM) proteins have been shown to regulate mammary epithelial cell proliferation, differentiation, and apoptosis in vitro. However, little is known about the hormonal regulation and functional role of ECM proteins and integrins during mammary gland development in vivo. We examined the temporal and spatial localization and hormone regulation of collagen I, collagen IV, laminin, and fibronectin. Among these ECM proteins only fibronectin changed appreciably. Fibronectin levels increased 3-fold between the onset of puberty and sexual maturity, remaining high during pregnancy and lactation. This increase occurred specifically in the epithelial basement membrane. Fibronectin was decreased 70% by ovariectomy and increased 1.5- and 2-fold by estrogen or estrogen plus progesterone treatment, respectively. The fibronectin-specific integrin, alpha(5)beta(1), was localized in myoepithelial cells; it increased 2.2-fold between puberty and sexual maturity and decreased in late pregnancy and lactation. The basal localization of alpha(5)beta(1) was notably increased in pubertal and adult virgin mice. alpha(5)beta(1) concentrations decreased 40-50% after ovariectomy in pubertal and adult mice, which was reversed by estrogen plus progesterone treatment in adult mice. The high basal expression of alpha(5)beta(1) during active proliferation and the low expression in nonproliferating and lactating glands indicate that fibronectin signaling may be required for hormone-dependent proliferation in the mammary gland.

The purpose of the present study was to investigate the role of extracellular matrix proteins (ECMs; collagens I and IV, fibronectin, and laminin) in modulating proliferative responses of normal mammary epithelial cells in serum-free culture to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I). As EGF and IGF-I can alter steroid responses, the interactions among growth factors, estrogen, and R5020 were also investigated. We report the novel finding that all ECMs tested, but not a nonspecific attachment factor, poly-L-lysine (PL), promoted a highly synergistic proliferative response to EGF plus IGF-I. EGF receptors were significantly increased with culture time on all ECMs, but not on PL. IGF receptor expression was significantly 2- to 4-fold higher on all ECMs compared with PL. EGF decreased IGF-binding protein-2 (IGFBP-2) and IGFBP-3 by more than 50% in the presence of IGF-I on PL or collagen I. These results indicate that ECM-specific IGF-I/EGF synergism occurs in response to ECM up-regulation of growth factor receptors and EGF down-regulation of inhibitory IGFBPs. Growth factors did not synergize with estrogen and/or R5020. Instead, estrogen plus R5020 decreased EGF-plus IGF-I-induced proliferation in an ECM-dependent manner. These studies demonstrate that proliferation of normal mammary epithelial cells involves complex interactions among steroids, growth factors, binding proteins, and ECMs.

Retinoids are potent inhibitors of growth and tumor progression in many mammary carcinoma cell lines, though regulation of growth in nontumorigenic mammary epithelial cells by retinoids is less clear. Here, we have characterized the inhibition of MAC-T (a nontransformed bovine mammary epithelial cell line) cellular proliferation by retinoids and their role in regulating insulin-like growth factor binding proteins (IGFBPs). Retinoic acid (RA) (100 nM) was a potent inhibitor of MAC-T cell proliferation. Retinol was 10-100 times less effective. Neither retinoid could completely arrest growth at noncytotoxic concentrations. Retinoic acid inhibited cellular proliferation by 1 h (P < .05), but inhibition was fivefold greater by 24 h (P < .01). This second stage of growth inhibition (after 12 h) was dependent upon protein synthesis. However, RA-induced inhibition of cellular proliferation did not persist, with thymidine incorporation increasing toward control levels by 4 days in culture. Retinoic acid was less effective in inhibiting thymidine incorporation when cells were stimulated with insulin, des(1-3) IGF-I, or Long(R3) IGF-I when compared to cells stimulated with native IGF-I or serum. Inhibition of proliferation by RA was associated with increased levels of IGFBP-2 in conditioned media and in plasma membrane preparations. Treatment with insulin or des(1-3) IGF-I resulted in the appearance of IGFBP-3 in conditioned media and on the cell surface. However, RA significantly reduced IGFBP-3 levels in conditioned media and eliminated IGFBP-3 associated with the plasma membrane. Thus, RA is a potent but transient inhibitor of bovine mammary epithelial cell proliferation, and this growth inhibition is correlated with increased IGFBP-2 accumulation and inhibition of IGF-I stimulated IGFBP-3 protein secretion.