23 Physiologic and Pathological Cell Death
in the Mammary Gland
Armelle Melet and Roya Khosravi-Far
1. INTRODUCTION
Apoptosis is a regulated cell suicide program that functions to control mitosis during the development and maintenance of tissues. This type of cell death plays a key role in the extensive postnatal development of the breast. Dysregulation in apoptosis has been speculated to contribute to hyperplasia and to promote breast cancer and resistance to chemotherapy. Recent studies have highlighted that other modes of cell death additionally influence breast development, tumorigenesis, or response to chemotherapy. This chapter reviews the role and regulation of apoptosis in the normal and neoplastic breast. It also briefly summarizes the contribution of other types of cell death, such as autophagy, necrosis, or entosis.
2. APOPTOSIS IN THE NORMAL BREAST
2.1. Occurrence and role of apoptosis in the developing breast
The breast is a hormone-responsive organ that undergoes major functional and morphological changes postnatally, during puberty, and during pregnancy. Extensive studies in animal models have established that apoptosis plays a critical role in these physiologic processes. Apoptotic cell death occurs mainly in the breast epithelium, which develops gradually into hollow tree-like structures (ducts and terminal alveoli) surrounded by fatty, fibrous, and glandular connective tissues (the stroma).
At birth, the mammary gland consists of a rudimental ductal network protruding from the nipple into the stromal fat pad (Figure 23-1A). The ductal network expands and arborizes mostly at puberty. The ovarian hormone estrogen and the pituitary growth hormone stimulate
the growth and branching of highly proliferative bulbous structures at the ductal tips called the terminal end buds (TEBs) (Figure 23-1B). The TEBs are composed of two distinct cell types, the cap and body cells, which are the progenitors of the outer myoepithelium and the lumen epithelium, respectively. The luminal body cells undergo extensive detachment-induced apoptosis (anoikis) to hollow out the elongated part of the duct. When the expanding ductal branches reach the limits of the mammary fat pad, the TEBs differentiate and are permanently replaced by terminal end ducts or alveolar buds, with this alveolar differentiation starting as sexual maturity is reached.
Apoptosis not only occurs during ductal morphogenesis, but also takes place in mature females to maintain tissue homeostasis. During the menstrual/estrous cycle, the adult mammary gland responds to systemic hormonal changes by cycles of limited proliferation, differentiation, and apoptosis in a small subset of epithelial cells. Thereby, the mammary gland prepares for a possible pregnancy with a modest development of alveolar structures and regresses by apoptosis in the absence of pregnancy. The frequency of apoptosis fluctuates with steroid hormones levels, with a peak of apoptosis following a peak of proliferation close to the end of the menstrual cycle.
The final differentiation of the mammary gland only takes place during pregnancy and lactation. Pregnancy hormones (estrogen, progesterone, and prolactin) induce the shrinking of stromal adipocytes, additional ductal branching, and further growth and differentiation of the alveoli into milk-secretory lobules (the mammary acini). During lactation, the functional and morphogenetic development of the breast is fulfilled and apoptosis is inhibited. The milk produced in the lobuloalveolar structures can then be expelled thanks to the
PHYSIOLOGIC AND PATHOLOGICAL CELL DEATH IN THE MAMMARY GLAND
Figure 23-1. Apoptosis during the postnatal development of mammary gland. (A) Schematic overview of mammary gland development. Major events of cell proliferation, di erentiation, and apoptosis take place in the mammary epithelium during the postnatal development of the breast. During puberty, estrogen and growth hormone promote ductal outgrowth. The ends of the ducts form highly proliferative bulbous structures called terminal end buds (TEBs). These TEBs expand and hollow out by anoikis of luminal cells. In the mature female, the entire fat pad is filled with a treelike network of branching ducts. Cyclic hormonal changes stimulate a modest development of alveoli that are eliminated by apoptosis in the absence of pregnancy. Pregnancy hormones (progesterone, prolactin, and placental lactogens) induce the growth of alveoli that di erentiate into milk-secretory alveoli at the end of pregnancy. During lactation, luminal cells of mature alveoli secrete milk to feed the newborn. When lactation ceases, the majority of the secretory epithelium is removed by apoptosis during involution. The oval shapes depict the mammary fat pad (the stroma); the other labels are shown directly on the figure. (B) Schematic representation of the terminal end buds (TEBs). TEBs are highly proliferative bulbous structures that appear at the ductal tips at puberty. TEBs are composed of two types of stem cells: the outer cap cells and several layers of inner body cells. These two cell types are the progenitors of the ductal myoepithelium and the lumen epithelium, respectively. The central luminal body cells undergo detachment-induced apoptosis (anoikis) to clear the lumen of the elongating ducts during ductal morphogenesis. Labels are shown directly on the figure.
contractile myoepithelium and transported to the nipples through the luminal space of the ductal network. By clearing the lumen of the developing ducts and alveoli, apoptosis is thus essential to the breast’s ultimate function (i.e., milk production and secretion).
When lactation ends, the secretory epithelium undergoes massive apoptosis, and the gland remodels to a quiescent state in a two-phase involution process. The first apoptotic phase is triggered by milk stasis and is reversible by suckling up to 48 hours in mice. The second, irreversible phase is driven by the drop
in lactogenic hormones and involves proteolytic degradation of the basement membrane, further detachmentinduced apoptosis (anoikis), collapse of alveoli, and tissue remodeling. The involution process, which removes the majority of the secretory epithelium, constitutes the most dramatic occurrence of apoptosis in the normal breast.
Finally, after menopause, the aging mammary gland undergoes lobular involution through unknown mechanisms, probably combining epithelial apoptosis and senescence.
This overview of mammary development highlights the critical role of apoptosis in morphogenesis and homeostasis of the normal breast (Figure 23-1). Two major events of apoptosis take place in the epithelium of the mammary gland. First, during puberty and pregnancy, apoptosis contributes to shape the lumen of the developing ducts and alveoli. Second, during involution, post-lactational milk stasis triggers an extensive wave of apoptosis that removes the excess milk-secretory epithelium. Given its importance for the development and function of the breast, apoptosis is tightly regulated by both intracellular and extracellular factors.
2.2. Molecular regulation of apoptosis in the normal breast
The mammary gland is a complex organ with a highly organized cellular architecture composed of epithelial cells and stromal cells (fibroblasts, adipocytes, immune and inflammatory cells, endothelial cells) that communi-
cate with each other via an extracellular matrix (ECM) through adhesive connections and soluble secreted factors. Three-dimensional (3D) cell culture and in vivo animal models have been very useful to recapitulate the complexity of the 3D cellular organization and interactions in this organ. These models were used to identify the key components of apoptotic regulation in the mammary gland, those being derived from both the epithelial cells and their complex surrounding microenvironment. Apoptosis of mammary epithelial cells is thus regulated at three levels, by intracellular regulators (i.e., BCL-2),
mitochondrial pathway. Key molecules and pathways controlling apoptosis in the normal breast are described below.
2.2.1. Autocrine/paracrine regulation by growth factors, death ligands, and other cytokines
Mammary epithelial cells are exposed to a complex stromal microenvironment containing positive (epidermal growth factor [EGF], insulin-like growth factor [IGF]) or negative growth factors and cytokines (transforming growth factor [TGF] β, leukemia inhibitory factor [LIF], death ligands). The ratio between these
ARMELLE MELET AND ROYA KHOSRAVI-FAR
Figure 23-2. Death signaling pathways in mammary epithelial cells during involution. Apoptosis of mammary epithelial cells is controlled by systemic hormones, stromal growth factors and cytokines, and cell–cell and cell-matrix adhesion. Apoptotic death during involution is triggered by a combined gain of death signals and loss of survival signals. Main death pathways involved in involution are shown in bold. Transcription factors are symbolized by rectangles within the epithelial cell figure. Local milk stasis causes the stretching of alveoli, leading to the disruption of prosurvival cell-matrix and cell–cell adhesions. Along these lines, truncation of the β-catenin binding domain of E-cadherin was shown to precede epithelial apoptosis in early mammary involution. Milk stasis also induces the expression of several proapoptotic cytokines (LIF, TGFβ3, death ligands) that trigger apoptosis through the extrinsic death receptor pathway and the STAT3 pathway. Survival pathways such as the IGF and the PI3K/AKT signaling are inhibited, and proapoptotic members of the BCL-2 family such as BAD and BAX are upregulated to activate the mitochondrial intrinsic death pathway. (+) prosurvival signals which are inhibited during involution (stop signs) and (−) prodeath signals. DR, death receptor; TGFβR, transforming growth factor β receptor; LIFR, leukemia inhibitory factor receptor; IGF1R, insulin-like growth factor receptor type 1; EGFR, epidermal growth factor receptor; PrlR, prolactin receptor; ER, estrogen receptor; FRZ, frizzled receptor; sFRP4, secreted frizzled-related protein 4; GSK3, glycogen synthase kinase 4.
252
by local mammary factors (autocrine/ paracrine secreted factors, ECM, and cell adhesion proteins), and finally by systemic hormones that regulate the expression and activity of the latter.
Ovarian steroid hormones (estrogen, progesterone) and pituitary peptide hormones (prolactin) are potent inhibitors of apoptosis in hormoneresponsive tissues such as the breast. Decline in these systemic hormone levels is associated with apoptosis and regression of the mammary gland during involution. This hormonal control of apoptosis has been extensively described elsewhere. This review focuses on the local and intracellular regulation of apoptosis in the mammary gland, with major emphasis on the involution phase.
Involution has been used as a model to study apoptosis regulation in the mammary gland. At the onset of involution, milk accumulates locally within alveolar lumens, while systemically, levels of lactogenic hormones fall. Local milk accumulation (and not systemic hormones) appears to be the critical apoptotic inducer. Indeed, in a mouse model of lactation failure, the artificial addition of lactogenic hormones does not affect apoptosis, although it prevents the remodeling of the involuting gland. The first phase of involution is therefore initiated locally by mammary-derived factors. The precise initial trigger of apoptosis in the involuting breast is currently unknown,
but two hypotheses prevail. Apoptosis could be triggered by an accumulation of apoptosis-inducing factors in the milk and/or by a physical distortion of secretory epithelial cells generated by the engorgement. Subsequently, an interplay of different signaling pathways is activated to induce apoptosis in the mammary gland (Figure 23-2). Microarray analyses show that the two stages of involution are controlled by a temporal change in gene expression with progressive gain of death signals and loss of survival factors (Table 23-1). Apoptosis in the first phase involves both death receptor and mitochondrial pathways, whereas apoptosis/anoikis in the second phase is most likely mediated by the classic
PHYSIOLOGIC AND PATHOLOGICAL CELL DEATH IN THE MAMMARY GLAND |
253 |
Table 23-1. Transcription profiles of survival and death-related genes upregulated during the first 4 days of involution in the mouse mammary tissue
|
lnv1 |
lnv2 |
lnv3 |
lnv4 |
Transcription |
|
|
|
|
profiles |
|
|
|
|
Survival genes |
|
Nfkb2 |
|
Bcl-x |
|
|
|
|
Mcl1 |
Death genes |
Lifr |
Casp4 (Casp11) |
Bax |
lgfbp5 |
|
FasL (Tnfsf6) |
Casp12 |
Casp7 |
Casp1 |
|
Trail (Tnfsf10) |
p21 |
Fas (Tnfrsf6) |
Apaf1 |
|
Tweak (Tnfsf12) |
|
Tnfrsf1a |
Tgfb1 |
|
Tnf (Tnfa) |
|
Fadd |
|
|
p53 |
|
Stat3 |
|
|
|
|
Tgfb3 |
|
Note: Summary of microarray data (Clarkson et al. 2003 and 2004, Stein et al. 2004 and 2007). Four patterns of gene transcription are observed during involution (Inv1, Inv2, Inv3, Inv4). The dotted lines on the gene expression profiles highlight day 10 of lactation. The following time points correspond to 12-, 24-, 48-, 72-, and 96-hour involution. Inv1 corresponds to a rapid but transient upregulation 12 hours after weaning. Inv2 profiles show a peak at 12 hours, followed by a slow decrease in expression. Inv3 patterns exhibit a gene upregulation by 24/48 hours with prolonged expression. Inv4 corresponds to a delayed and progressive increase in transcription up to at least 4 days. Of note, the survival gene Akt1 is downregulated during the first 4 days of involution.
mitogen/survival and apoptotic factors regulates the epithelial cell fate through autocrine and paracrine pathways.
2.2.2. Death ligands and death receptor pathway
Microarray analyses of transcription during early involution indicate a rapid and transient increase in the mRNAs of several members of the tumor necrosis factor (TNF) superfamily of death ligands (Tnf, Trail, FasL, and tweak) and their receptors (TnfR1, Fas, Dr4). Increased nuclear factor kappa B (NF-κB) activity correlates with the rapid activation of these death ligands, suggesting that NF- κB could be the transcription factor for these death genes.
FAS protein is present in the mammary epithelium during normal breast development, absent during pregnancy and lactation, and returns after weaning. On the other hand, FAS-L protein is present during pregnancy, lactation, and weaning, but not in the virgin mouse. The overlapping expression of FAS and FAS-L during involution matches the occurrence of apoptosis in the mammary epithelium. Lack of FAS or FAS-L expression in transgenic mice prevents apoptosis of mammary epithe-
lial cells during the first 3 days of involution, suggesting that the FAS/FAS-L system may play an important role in early stages of involution.
These results demonstrate that autocrine death receptor signaling contributes to early apoptosis induction during mammary involution. Several death ligands act in concert during this physiologic process. However, they are not the exclusive death mediators because their absence only delays involution rather than preventing it. Regulators of the intrinsic pathway are upregulated at a later time point of the first involution stage, indicating a progressive shift in the cell death machinery from the extrinsic to the intrinsic pathway.
2.2.3. TGFβ3 proapoptotic pathway
TGFβ1, 2, and 3 are multifunctional cytokines that play critical roles in every phase of the mammary gland development. They are expressed by mammary epithelial cells as inactive precursors binding to the extracellular matrix and later activated by proteolytic cleavage. Among the three TGFβs, TGFβ3 seems to be the primary isoform regulating apoptosis in the mammary epithelium.
