At weaning, TGFβ3 mRNA and protein are dramatically upregulated in the mouse alveolar and ductal epithelium, and this expression precedes the onset of apoptosis in the first phase of involution. Transplantation of neonatal mammary tissue from Tgfβ3 null mice into recipient hosts results in normal development and lactation but causes reduced apoptosis upon milk stasis compared with wild-type controls. Hormonal reconstitution and inhibition of suckling by teat sealing showed that TGFβ3 is induced locally by milk stasis and not by the levels of circulating hormones.

TGFβs signal through an hetero-tetrameric receptor complex composed of type I and type II TGF serine threonine kinase receptors (TGFβRI and TGFβRII), leading to the phosphorylation of SMADs. In the involuting mammary gland, TGFβ3 induces apoptosis through two classical signaling mediators: the transcription factors SMAD3 and SMAD4. Indeed, like in Tgfβ3 null mice, the mammary gland of Smad3 null transplants undergoes reduced alveolar apoptosis upon forced weaning. Moreover, directed expression of TGFβ3 in the alveolar epithelium of lactating mice leads to apoptosis of those cells with the concomitant nuclear localization of SMAD4. Mammary epithelial cells over-expressing TGFβ3 also show strong nuclear localization of phosphorylated STAT3 during early involution, suggesting that STAT3 may be a downstream target of TGFβ3 signaling.

According to several studies, TGFβ signaling contributes to the negative regulation of the downstream survival kinase AKT. Bailey and coworkers reported that porcine TGFβ inhibits AKT activity and triggers apoptosis of mouse mammary epithelial cells in culture and that prolactin prevents this TGFβ-induced apoptosis. The same authors also show that over-expression of a dominant-negative TGFβ type II receptor in the mouse mammary epithelium causes a hyperplastic prolactindependent alveolar development and a delayed involution with increased phosphorylated AKT levels. These results suggest that prolactin and TGFβs exert opposing effects on alveolar development through careful regulation of apoptosis by AKT.

Taken together, literature data provide evidence that TGFβ3 is one of the local autocrine factors that induces apoptosis of the mammary epithelium during the first phase of involution, possibly through downstream regulation of survival AKT kinase.

2.2.4. LIF-STAT3 proapoptotic signaling

STATs are transcription factors activated by phosphorylation that mediate signaling from cytokines and

growth factors. It is now known that LIF is the major paracrine cytokine that activates STAT3 in vivo in early involution. LIF receptor is upregulated at the onset of involution. Moreover, knockout of LIF in mice suppresses the phosphorylation of STAT3 after weaning and delays involution. TGFβ3 has also been identified as an additional upstream regulator of STAT3 activity during involution.

STAT3 is activated by phosphorylation at the onset of involution and is critical for the first apoptotic phase of this process. Stat3 null mammary glands indeed show a reduced epithelial apoptosis and a dramatic delay in involution upon forced weaning.

STAT3 regulates the transcription of a number of genes promoting apoptosis in mammary epithelial cells. Many of these genes are upregulated at the transition from lactation to involution. A number of targets have been identified in vitro in mammary epithelial cells. STAT3 targets include CCAAT/enhancer binding protein delta (C/Ebpδ), suppressor of cytokine signaling 3 (Socs 3), c-fos, Smad1, B-cell leukemia lymphoma 3 (Bcl3), and the regulatory subunits p55α/50α of phosphoinositide 3-kinase (PI3K).

Stat3 and Lif null mammary epitheliums express reduced levels of C/EBPδ, confirming C/EBPδ as a STAT3 target in vivo. C/EBPδ is a critical regulator of the proapoptotic genes Bak, Igfbp-5, p53, and Sgp2 during mammary gland involution. Moreover, C/Ebpδ disruption in the mouse epithelium delays the onset of involution and the expression of proapoptotic factors such as p53 and the IGF binding protein IGFBP-5.

Other important targets of STAT3 are the PI3K regulatory subunits. Their induction mediates a negative switch in PI3K survival signaling and a decrease in downstream AKT1 activation.

Overall, the LIF-STAT3 pathway emerges as a key regulator of apoptosis within the mammary gland, stimulating the expression of proapoptotic genes such as p53 or Bak while inhibiting two major survival pathways, the IGF and PI3K/AKT pathways.

2.2.5. IGF survival signaling

The IGF system is composed of three ligands (IGF-1, IGF-2, insulin), three receptors (IGF-1R, IGF-2R, and IR), and six IGF binding proteins (IGFBPs). IGFs are both systemic growth factors coming from the liver and autocrine/paracrine factors secreted locally by many tissues, including breast. They act as cellular mitogens and survival factors, protecting epithelial cells from apoptosis in a wide variety of conditions. IGF-1 and 2 exert their physiologic effects mostly through the binding

to the tyrosine kinase receptor IGF-1R. Ligand stimulation triggers the receptor autophosphorylation and the subsequent recruitment of adaptor proteins (IRS-1, SHC, 14–3–3). Phosphorylation of IRS-1 activates the PI3K/AKT survival pathway, which in turn phosphorylates BAD. The IGF-1R suppresses apoptosis primarily through the PI3K pathway, but two alternative routes, the mitogen-activated protein kinase (MAPK) pathway and the translocation of RAF to the mitochondria, also contribute to BAD phosphorylation. The bioavailability and function of IGFs are modulated by high-affinity associations with IGFBPs. IGFBPs sequester the IGFs and prevent them from binding to their receptors.

Components of the IGF system are differentially expressed in the developing mammary gland. IGF-1R is expressed only in the epithelium, whereas IGFs and IGFBPs are synthesized by stromal cells (i.e., fibroblasts and adipocytes) or epithelial cells, depending on the particular isoform and developmental stage. These data emphasize the importance of stromal–epithelial interactions for the regulation of IGF signaling in the mammary gland.

Substantial in vitro and in vivo evidence supports a role for IGF-1 in the regulation of mammary epithelial cell survival. Inhibition of IGF signaling triggers apoptosis, whereas extra IGFs suppress it. In vitro, exogenous IGF-1 suppresses apoptosis of primary mammary epithelial cells in culture by inducing AKT activation and FKHRL1 phosphorylation. IGF-1R over-expression induces proliferation and antiapoptotic signaling in a three-dimensional culture model of breast epithelial cells. Mammary gland models confirm the importance of IGFs as survival factors in vivo. Over-expression of IGF- 1 or IGF-2 in the mammary gland of transgenic mice results in reduced apoptosis and incomplete involution. Sustained activation of AKT is also detected in IGF-2 transgenic mice.

The survival function of IGFs are counteracted by IGFBP-5 during involution. Indeed, Marshman et al. report that IGFBP-5 protein is upregulated during the early apoptotic phase of involution and that IGFBP-5 is able to inhibit IGF-mediated survival signaling to cause apoptosis of primary mammary epithelial cells in culture. Loss-of-function and gain-of-function studies in mice confirm the proapoptotic role of IGFBP-5 during involution. Knockout of Igfbp-5 in the mouse mammary epithelium does not affect the mammary gland development but reduces apoptosis in early involution. On the other hand, directed expression of IGFBP-5 in the mammary gland of transgenic mice

induces premature cell death and impaired mammary development, together with increased expression of the proapoptotic molecule caspase-3 and decreased expression of prosurvival members of the BCL-2 family.

Current data thus support a role for the IGFs in apoptosis regulation in the mammary gland and highlight IGFBP-5 as a physiologic inhibitor of IGF survival signaling in this organ.

2.2.6. Regulation by adhesion

Mammary epithelial cells, like all normal adherent cells, are dependent on anchorage for survival. They are anchored to the ECM and to neighboring epithelial cells via transmembrane adhesion proteins such as integrins and cadherins. Integrins are well-known adhesion receptors existing as α-β heterodimers and linking the ECM with the cell interior (cytoskeleton and intracellular signaling molecules). The alpha subunit interacts with specific ligands from the ECM, whereas the beta subunit recruits intracellular signaling molecules such as focal adhesion kinase (FAK) or integrin-linked kinase (ILK). Integrins, which regulate cell architecture, are also known to cooperate with growth factor receptors to control cell survival. Anchorage survival signal is transduced intracellularly by β1 integrins to FAK and ILK, which in turn activate the survival kinases PI3K and AKT, respectively. Cadherins are the major cell–cell adhesion molecules of adherens junctions and desmosomes that bind to identical partners in neighboring cells. They are linked intracellularly to the actin cytoskeleton via β-catenins and α-catenins. Cytoplasmic β-catenin is degraded by the ubiquitin-proteasome pathway. When stabilized by WNT and non-WNT pathways, cytoplasmic β-catenins can translocate to the nucleus, where they act as transcriptions factors to mediate proliferation and survival signals.

In nontransformed epithelial cells, disruption of cellmatrix and cell–cell adhesion leads to detachmentinduced apoptosis, called anoikis. Artificial disruption of cell-matrix interactions by anti-β1 integrin antibodies or over-expression of the matrix metalloproteinase 3 (MMP-3) induces anoikis in mammary epithelial cells in culture. Conditional deletion of E-cadherin or α-catenin in mouse mammary glands causes dramatic apoptosis at parturition, which generates a mutant mammary gland with involution-like features. In physiologic conditions, shedding of epithelial cells into the ductal lumen is detected during ductal morphogenesis and as early as 12 hours after forced weaning during involution.

This effect precedes caspase-3 activation and involves the disruption of both cadherin and integrin signaling. Indeed, truncation of the β-catenin–binding domain of E-cadherin was shown to precede epithelial apoptosis in early involution. Moreover, sFRP4, a decoy receptor for WNT ligands, is upregulated at involution and hence inhibits WNT signaling, stabilizing β-catenin. Immunohistochemistry studies revealed that β1 integrin adopts an inactive conformation or is downregulated together with FAK in the first reversible phase of involution. MMPs that degrade the ECM are maintained in the inactive state by tissue inhibitors of MMPs in early involution, and their activities are then upregulated to enable matrix degradation and remodeling in the second involution phase. There is evidence for the degradation of ECM components laminin and fibronectin concomitantly with increased MMP activities during involution.

The adhesion system thus exerts a stringent control on epithelial cell fate by sensing the microenvironmental context. Proper adhesion activates survival signals that synergize with growth factor and cytokine signaling. Loss of integrin and cadherin adhesion and signaling has been involved in anoikis during both phases of the involution process. First, milk stasis causes an alveolar stretch, which probably alters cell adhesion and thereby triggers a first wave of anoikis. Then, upregulation of MMP activities in the second phase results in the degradation of the ECM and subsequent anoikis of remaining secretory alveoli.

2.2.7. PI3K/AKT pathway: molecular hub for survival signals

The PI3K/AKT pathway is known as a critical survival pathway in cells. PI3K activates the kinase AKT, which in turn phosphorylates and inactivates proapoptotic factors such as caspase-9, BAD, or FKHRL1.

In the mammary gland, levels of Akt mRNA increase slightly during pregnancy and more dramatically during lactation, dropping sharply as the gland begins to involute. The activated AKT protein levels are likewise decreased during the first stage of involution.

Shutting down this survival pathway is important for the course of involution, as demonstrated in vivo. Tissuespecific expression of AKT1 has been shown to delay involution in transgenic mice. By contrast, expression of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K, enhances apoptosis in the mouse mammary gland and conversely, conditional deletion of this gene delays involution.

As previously described in this chapter, multiple cell surface stimuli feed into the PI3K/AKT pathway within the mammary epithelium. These include:

Prolactin signals through JAK2 to FYN and CBL, which in turn activates the PI3K/AKT pathway. This pathway is downregulated during involution.

IGF signaling activates PI3K and is suppressed by IGFBP-5 during early involution.

Interaction of epithelial cell surface integrins with the extracellular matrix proteins provides additional survival signals to PI3K/AKT through FAK or ILK.

PI3K negative regulatory subunits are upregulated by STAT3 during involution and mediate a negative switch in PI3K signaling, facilitating the decrease of active AKT.

Loss of prolactin, sequestration of IGFs by IGFBPs, and disruption of epithelium-matrix interactions all reduce signaling to PI3K/AKT in the involuting breast. Moreover, STAT3 upregulates the expression of PI3K regulatory subunits, thereby mediating a negative switch in PI3K signaling. Overall, AKT1 emerges as the critical molecular nexus for survival/death signals in the mammary epithelium.

2.2.8. Downstream regulators of apoptosis: the BCL-2 family members

BCL-2 family members are important downstream regulators of the mitochondrial apoptotic pathway that can act either as death promoters (BIM, BAX, BAD, BAK, BCL-XS) or death inhibitors (BCL-2, BCL-W, BCL-XL). The relative ratios of these various proand antiapoptotic members determine the sensitivity or resistance of the cells to diverse apoptotic stimuli.

The mammary epithelium expresses a number of BCL-2 family members, including BIM, BAX, BAK, BAD, BCL-X, BCL-W, BFL-1, MCL-1, and BCL-2. Expression patterns of these BCL-2 proteins vary throughout mammary gland development. For instance, BCL-2 expression is dependent on estrogen and fluctuates with the cyclic changes of estrogen levels during the menstrual cycle. BCL-2 protein is expressed throughout pregnancy and lactation and drops at involution.

The functional roles of individual BCL-2 family members have been investigated using dominant gain-of- function and loss-of-function in mice (germline or tissue-specific loss of function). BIM, BAX, BCL-2, and BCL-X are expressed at the TEBs during pubertal development. Disruption of Bim in mice prevents the induction of apoptosis and clearing of the lumen in