J Mammary Gland Biol Neoplasia (2006) 11:205–211 DOI 10.1007/s10911-006-9030-4
Illuminating the Center: Mechanisms Regulating Lumen Formation and Maintenance in Mammary Morphogenesis Mauricio J. Reginato & Senthil K. Muthuswamy
Published online: 18 November 2006 # Springer Science + Business Media, Inc. 2006
Abstract The lumens present in ductal structures are required for transport of fluids and air. Studies in model organisms and cells in culture suggest that lumens can be generated by multiple mechanisms including apoptosis of centrally located cells, and re-modeling of epithelia. Several studies point to a role for apoptosis during lumen formation in the mammary ducts. However, a role for other mechanisms during lumen formation in the mammary ducts is largely unexplored. Understanding how lumens are formed and maintained free of cells is of clinical importance because filling of the luminal space is associated with cancer and inflammation. Thus, further investigation can lead to new diagnostic and therapeutic opportunities. Keywords Lumen . Mammary . Morphogenesis . Duct . Bim . ErbB2 Abbreviations ERα estrogen receptor α ECM extracellular matrix EGF epidermal growth factor HGF hepatocyte growth factor MEC mammary epithelial cell MMTV Mouse Mammary Tumor Virus M. J. Reginato (*) Department of Biochemistry and Molecular Biology, College of Medicine, Drexel University, 245 N. 15th Street, Philadelphia, PA 19102, USA e-mail:
[email protected] S. K. Muthuswamy (*) Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA e-mail:
[email protected]
TEB 3D TRAIL VAC WAP
terminal end bud three-dimensional Tumor necrosis factor Related Apoptosis Inducing Ligand vacuolar apical compartment whey acidic protein
Introduction The mammary gland is composed of branched ductal structures that have hollow luminal spaces lined with epithelial cells. The epithelial cells lining the ducts secrete milk into the luminal space, which then flows through the ductal network to the nipple. Other organs such as the vasculature and lung also require luminal space for fluid and air flow, respectively. Understanding the molecular mechanisms of lumen formation (lumenization/cavitation) and lumen maintenance is thus of significant biological importance. In this review, we will discuss mechanisms of lumenization delineated from studies using model organisms and cells in culture, and will identify questions relevant to mammary gland biology and breast cancer. Several growth factors and steroid hormones regulate the initiation and maturation of branched ductal structures with central lumens. For instance, epidermal growth factor (EGF) [1], amphiregulin [2], and hepatocyte growth factor (HGF) [3] can promote branching morphogenesis. In addition, hormones including estrogen [4, 5], progesterone [6], glucocorticoids [7, 8], and retinoids [9] have also been implicated in the development and maintenance of mammary epithelial structures. It is likely that growth factors and hormones work in concert with each other to promote ductal morphogenesis in vivo (for recent reviews see [10, 11]).
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Studies using mammary or kidney epithelial cells in culture, as well as model organisms such as Drosophila, suggest three potential mechanisms that contribute to lumen formation: (1) apoptosis of centrally located cells, (2) autophagy of centrally located cells, and (3) re-modeling of cells around a luminal space (Fig. 1). Although the mechanisms by which lumens are created in the mammary gland are not fully understood, initial studies demonstrate a role for apoptosis of centrally located cells [12]. Whether other mechanisms are also involved in lumen formation in mammary ducts has not been well investigated. Disruption of the luminal space is associated with several disease states including inflammation and cancer. Aberrant activation of growth factor receptors and nuclear hormone receptors can disrupt ductal architecture and result in filling of the luminal space. For example, overexpression of either the receptor tyrosine kinase ErbB2 or the estrogen receptor alpha (ERα) is sufficient to induce ductal abnormalities by promoting development of multilayered epithelium that fills the luminal space [13, 14]. In humans, premalignant breast cancer lesions, such as carcinoma in situ, are characterized by a completely or partially filled lumen [15]. Very little is known about the molecular mechanisms that drive luminal filling in these early breast cancer lesions. An understanding of such mechanisms can provide insights into molecular events that regulate early events in carcinoma.
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Morphogenesis of Mammary Ducts Development of the mammary gland is initiated during midgestation with the formation of mammary placodes that contain both epithelial and mesenchymal cell layers. At birth, a rudimentary ductal network is observed in the mammary fat pad. Although the ductal network continues to grow with increases in body weight, most of the expansion occurs at puberty. A hormone-dependent expansion is initiated by the proliferative structures at the ends of the ducts referred to as terminal end buds (TEB) (for a recent review see [16]). The TEB contains two distinct cell types: cap cells and body cells (Fig. 2). The highly proliferative cap cells are organized as a single layer at the leading edge of the TEB and they are in contact with a thin layer of basal lamina. The body cells make up the bulk of the TEB and are organized in multicellular layers. The TEB penetrates the mammary fat pad forming the primary ducts. The secondary and tertiary ducts sprout from primary ducts to form the characteristic ductal arborization observed in mature virgins. Pregnancy hormones induce lobuloalveolar differentiation that results in formation of secretory alveolar structures required for milk production. The milk produced in alveolar structures is transported to the nipple through the luminal space in the ductal network. Thus, the formation and maintenance of lumens is essential for normal function of mammary gland.
Development of Lumens: Role for Apoptosis
Figure 1 Lumen formation: role for apoptosis, autophagy, and epithelial re-modeling. It is possible that development of luminal space occurs by one or more of three processes: apoptosis of centrally located cells, autophagy of centrally located cells, and epithelial re-modeling.
Although the TEB has multiple cells layers, the primary ducts behind the TEB have a single layer of luminal epithelial cells surrounding an empty hollow lumen. The body cells that border the TEB and the luminal space possess high rates of apoptosis, which contrasts with the low apoptotic rates observed in cap cells at the distal end of the developing duct, suggesting that apoptosis of body cells may contribute to lumen formation [12]. Consistent with this possibility, transgenic mice expressing Bcl-2 under the control of the whey acidic protein (WAP) promoter reduces apoptosis and shows delayed lumen formation in the mammary gland, demonstrating a critical role for apoptosis during lumenization in vivo [12]. Studies using a three dimensional (3D) cell culture approach pioneered by Bissell and colleagues [17] provide further support for a role of apoptosis during lumenization. When grown in a 3D matrix, primary mouse mammary epithelial cells form lumen-containing structures that show apoptosis of centrally located cells. Inhibition of apoptosis using caspase inhibitors delays lumen formation, again suggesting a role for apoptosis during lumenization [18].
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Figure 2 TEB, side branches, and lumens. a The TEB penetrates the mammary fat pad to establish the ductal arborization characteristic of the adult virgin mammary gland. b The TEBs have a characteristic organization with myoepithelial cells, a thin layer of basal lamina (see broken line), a layer of cap cells, and several layers of body cells. Side branches also develop luminal space and establish a continuum with the lumen in the primary ducts. c Cross section view of a developing duct. A clear luminal space is observed in a cross section through the mature duct (I), whereas partial (II) or complete lack of lumen (III) is represented in the cross sections from the distal end of the TEB.
A direct proof for a role for apoptosis during lumen formation in human mammary epithelial cells in culture was provided by studies by Joan Brugge and colleagues. A nontumorigenic human mammary epithelial cell line, MCF10A, forms organized structures with hollow central lumens when grown in a 3D matrix [19, 20]. As observed in primary mouse MECs, MCF-10A cells also undergo apoptosis of centrally located cells during 3D morphogenesis. Furthermore, overexpression of antiapoptotic proteins, Bcl-2 or Bcl-xL, blocks apoptosis and delays formation of lumens during 3D morphogenesis of these cells [21]. Expression of Bcl-2 results in multilayered 3D structures in another human MEC, HB4 [22]. Thus, morphogenesis of lumens in mammary epithelial structures involves apoptosis of centrally localized cells. Regulation of Bcl-2 family members may play a significant role in apoptosis during lumen formation. Using the MCF-10A model of lumen formation, we demonstrated that the proapoptotic BH3-only protein Bim is highly induced at the protein level during morphogenesis [23]. Although other BH3-only proteins such as Bad and Bid are detected in MCF-10A cells, only Bim induction coincides with detection of apoptosis in centrally located cells. Moreover, downregulation of Bim blocks apoptosis of these centrally located cells and significantly delays lumen formation. Regulation of Bcl-2 family members also occurs during cell death associated with mammary gland involution [24–26]. Apoptosis during lumen formation of the mouse mammary gland is independent of tumor suppressor protein p53 status. Loss of p53 neither decreases the rate of apoptosis observed in TEBs nor affects lumen formation [12, 27]. In addition, loss of p53 does not alter apoptosis during
involution [27]. However, one study reports that involution is delayed in p53−/− mice and suggests that loss of p53 alters the initial collapse of alveolar structure, yet apoptosis during involution still occurs in these mice [28]. Consistent with this data, Bim regulation and induction of apoptosis is independent of p53 [29]. Thus, as observed for apoptosis induced by growth factor withdrawal in mammary epithelial cells [30], the programmed cell death observed during lumenization uses mechanisms that may be insensitive to p53 status. Lack of survival signals from ECM can trigger apoptosis. When this occurs, epithelial cells undergo a process referred to as anoikis [31]. This is consistent with processes observed during cavitation of mouse embryoid bodies when the inner cell mass undergoes apoptosis to create an embryo with columnar epithelial cells surrounding a hollow cavity [32]. Homozygous loss of a basement membrane component, laminin γ1, results in embryoid bodies that are unable to cavitate, suggesting that basement membrane directly regulates cell death of the inner cell mass [33]. Consistent with this possibility, detachment of mammary epithelial cells, including MCF-10A cells and primary mouse MECs, from matrix causes apoptosis, which correlates with induction of Bim [34]. Downregulation of Bim expression prevented anoikis in MCF-10A cells, demonstrating that Bim is a critical effector of cell death trigged by loss of integrin-ECM interactions [34]. Subsequent studies by a number of groups have shown a similar role for Bim in regulating anoikis in both mammary and other epithelial cell types [35–39]. In addition, it is likely that ECM signals regulate multiple pro-apoptotic Bcl-2 family members, including Bad [40] or Bax [41], though their role in lumen
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formation has not been investigated. Thus, it is likely that changes in integrin function regulate expression and function of pro-apoptotic proteins that play a central role during lumen formation in culture. Although signaling from β1 integrin plays a critical role for survival of mammary epithelial cells in culture [42, 43], it is not clear whether integrins regulate apoptosis associated with lumen formation during morphogenesis in vivo. Recent studies using conditional inactivation of β1 integrin alleles during morphogenesis of mouse mammary glands have come to conflicting conclusions as to the role of β1 integrins in development of alveolar structures with hollow lumens. In two studies, excision of β1 integrin using Cre recombinase expressed under WAP or lactoglobulin promoters resulted in defective alveoli with cells bulging into the luminal space [44, 45]. However, in another study where β1 integrin was excised using Mouse Mammary Tumor Virus (MMTV) promoter-driven expression of Cre recombinase, there were no defects observed in normal morphogenesis in vivo [46]. Interestingly, loss of β1 integrin did not increase apoptosis levels during lactation and involution of the mammary gland [44], suggesting that the in vivo environment may possess compensatory mechanisms that allow cell survival in the absence of β1 integrin. Further analysis is required to fully understand the role played by integrin-ECM interactions during lumen formation in vivo. In addition to cell–matrix interactions, cell–cell interactions may also play a role in regulating lumen formation. For example CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a cell–cell adhesion molecule, has been implicated in regulating lumen formation as it has been shown that inhibiting its function blocks lumen formation in MCF-10A cells [47]. Moreover, forced expression of a CEACAM1 isoform restores lumens in the mammary carcinoma cell line MCF-7, which normally does not express CEACAM1 or form lumens. Formation of new lumens in these cells is associated with increased apoptosis of centrally located cells and is partially inhibited by caspase inhibitors [48]. It will be of interest to test whether CEACAM1 regulates Bim expression in developing lumens in vitro. In addition, several questions remain. Is Bim involved in TEB apoptosis/lumen formation in vivo? What is the morphogenetic program regulating Bim induction in developing acini? Are other Bcl-2 family members, including additional BH3only proteins, involved in lumen formation of MECs?
Development of Lumens: Role for Autophagy In addition to the classic form of cell death, regulated by the catalytic activity of capases, lumen formation may also be promoted by another death process, autophagy. Autophagy is an evolutionarily conserved process triggered in
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response to nutrient deprivation. During this process, cytoplasmic contents, including organelles and proteins, are engulfed by double membranous vacuoles referred to as autophagosomes and targeted for degradation. This results in a situation whereby a cell can eat itself to death. Whether autophagy regulates lumen formation or maintenance in the mouse mammary gland remains to be established. Early ultrastructural analyses investigating the mechanisms by which cells die during post-lactational involution demonstrated the frequent presence of autophagosomes. These autophagic vesicles had cytoplasmic organelles such as mitochondria and were located in the middle of the cells. Furthermore, these vesicle were observed during the early stages (days 2–3) of involution but rarely observed in lactating epithelia or during later stages (4 days) of involution, suggesting a role for autophagy during early stages of mammary gland involution [49]. Additional evidence for autophagy is suggested from studies using the MCF-10A 3D organotypic culture system. Although expression of Bcl-2 or Bcl-xL can delay lumen formation in MCF-10A cells, it cannot prevent it, suggesting that alternate cell death/clearing mechanisms must exist. Ultrastructural analysis of MCF-10A cells dying in the presence of sustained Bcl-2 expression demonstrates the presence of autophagocytic vesicles, suggesting that autophagy regulates lumen formation in human mammary epithelia [21]. Inhibition of Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL), by expression of a dominantnegative receptor, blocked lumen formation in Bcl-2-expressing cells, identifying TRAIL as a potential regulator of autophagy in this system [50]. Further analysis is required to investigate the role played by autophagy during lumen formation in mammary glands in vivo.
Development of Lumens: Role for Epithelial Re-modeling In the mammary gland, in addition to lumen formation at the leading edge of primary ducts (TEB), lumen formation is required at side branches that emanate from primary ducts. The mechanisms regulating formation of lumens at side branches are less well characterized. Under this circumstance, when lumens form in thin cords or in branches with only 2–3 cells, apoptosis may not be required [51]. Instead, lumens may form by localized generation of apical membranes, as observed during HGF-induced tubulogenesis of a kidney epithelial cell line, MDCK, grown in 3D matrix [51]. The tubules are initiated by cells sending out extensions from the basal surface, coupled with cell proliferation and the formation of epithelial cords that are 2–3 cells thick with no apical surface and no luminal space. At this stage, cells regain the apical surface by a process initiated by the
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fusion of vesicles, referred to vacuolar apical compartments (VACs), to the membrane at the future site of lumen [51]. It is possible the mechanisms delineated from MDCK cells in 3D organotypic culture may be relevant to lumen formation during branching morphogenesis in the mammary gland in vivo. Additional analyses are required to provide further insight.
Control of Lumen Size In addition to formation of lumens, there ought to be mechanisms that determine the diameter of the luminal space. Very little, if anything, is known about the mechanisms determining the size of lumens. Factors that induce proliferation may not be the primary determinant because, despite the continuous presence of growth factors, normal mammary ducts and acini have defined sizes. For instance, the primary ducts in a mouse mammary gland can be about 20 μm in diameter, whereas they can expand to about 80 μm during lactation (J. Fata and M. Bissell, personal communication). Although it should be noted that these size estimates are derived from fixed tissue and may not reflect lumen size in live animals, it seems likely that lumens expand and contract based on milk production. It is possible that lumen size is modulated by extrinsic factors such as the presence of specific morphogens or by physical tension exerted by the matrix composition in the microenvironment. For instance, in a 3D organotypic culture model of lumen formation, the morphogen epimorphin/syntaxin2 cooperates with growth factor-induced proliferation to increase lumen size [52, 53]. In addition, increasing the tensile strength of the extracellular matrix can significantly disrupt lumen formation [54]. Additionally, there may be proliferation-independent mechanisms that control lumen size. In Drosophila trachea, the lumen increases up to 40-fold in size during development without any increase in proliferation [55]. The epithelial cells lining the luminal space increase the area of their apical surface, which results in a cell shape change to make larger lumens. Whether such cell shape changes regulate lumen size in mammary gland is unknown. The dynamic changes in hormones during pregnancy and during every estrous cycle have significant effects on the proliferative potential of epithelial cells lining the ducts. Yet lumens are maintained free of cells, and dividing daughter cells do not accumulate in the luminal space. How is this achieved? It is possible that the plane of cell division determines the position of the daughter cell. If luminal epithelia divide in a plane that is perpendicular to the apical-basal axis, both the mother and daughter cells will remain in the same plane and contribute to expansion of lumen diameter [56]. However, a switch in the plane of cell division such that the cells divides parallel to the apical-basal axis generates a
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daughter cell that can enter the lumen. This switch may not be deleterious at all times because the daughter cells may bud-off from the ducts and become involved in initiation of a new side branch. If the daughter cell is not cleared from the lumen, however, there may be deleterious consequences (see below). In summary, it is likely that there are multiple molecular mechanisms that regulate lumen size and maintenance during normal development and physiology of the mammary gland. These mechanisms await investigation.
Luminal Filling in Breast Cancer Early premalignant breast cancer lesions, such as hyperplastic lesions with atypia and carcinoma in situ, are characterized by a complete or partially filled lumen [15]. Very little is known about the molecular mechanisms that disrupt normal ductal organization of epithelial cells. Understanding the molecular mechanisms that disrupt normal epithelial organization may provide new insight into events that regulate initiation of cancer, and lead to the identification of both molecular markers and drug targets for premalignant disease. A number of oncogenes have been implicated in breast cancer; however, not all of them can induce lumen filling. For instance, activation of the oncogenic receptor tyrosine kinase ErbB2, a dominant oncogene in breast cancer, induces proliferation and decreases apoptosis in lumens, leading to a DCIS-like phenotype in 3D acini generated by growing MCF-10A cells in a matrix [20]. In contrast, expression of cyclin D1 induces continuous proliferation, but does not promote filling of the luminal space. Coexpression of Cyclin D1 and Bcl-2 results in proliferating 3D structures with lumens with filled cells [21]. Thus oncogenes or oncogene combinations that induce proliferation and protect apoptosis can promote filling of luminal space. Activation of both Ras/MAPK and NF-kB pathways are thought to promote survival of cells in the luminal space [23, 57]. For instance, ErbB2 protects luminal apoptosis by downregulating Bim expression in a Ras/MAPK dependent manner, whereas the activated form of MEK suppresses Bim expression and blocks apoptosis, resulting in lumen filling [23]. Oncogenes may promote lumen filling by promoting integrin-ECM interactions. Consistent with this possibility, the ECM protein laminin-5 protects against apoptosis by engaging the α6β4 integrins and activating NF-kB signaling [57]. In addition to ErbB2, several oncogenes such as IGF-IR [58] and CSF-1R [59] induce filling of luminal space. Further investigations are required to identify the mechanisms used by oncogenes to protect from luminal apoptosis. In summary, understanding the mechanisms that regulate development and maintenance of luminal space in ductal
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structures is not only important for normal physiology but also for disease states such as cancer. Although we are beginning to gain insights into the process of lumenization/ cavitation, further understanding is critically important because a filled luminal space is one of the hallmarks of cancer initiation. Given the paucity of prognostic molecular markers for early breast lesions, this line of investigation is bound to open doors for a new class of markers and possibly new drug targets. Acknowledgements We thank Dr. Danielle K. Carroll (Harvard Medical School) for critical reading of the manuscript. We thank Jim Duffy for assistance with art work. MJR was supported by funds from Drexel University College of Medicine. SKM was supported by CA098830, The V Foundation Scholar award, Rita Allen Scholar award, FACT, Glen Cove C.A.R.E.S, and LIBC Foundation.
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