**2. Involvement of STAT3 signaling**

The studies referred above found that cell death correlated with the induction of proapoptotic genes as *bax*, decreased expression of milk proteins, dephosphorylation of STAT5a and 5b (main transcription factors that mediate prolactin triggered signaling), and activation of STAT3. Signal transducer and activator of transcription (STATs) are a family of latent transcription factors, which are activated in response to a variety of cytokines and growth factors. This family of signaling molecules has been implicated in cell growth, differentiation, survival, and apoptosis. STAT3 and STAT5 have reciprocal patterns of activation throughout a mammary developmental cycle, suggesting that STAT5 may be a survival factor and STAT3 a death factor for differentiated mammary epithelium. Chapman et al. [4], using the lox/Cre recombination system, showed a decrease in epithelial apoptosis and a dramatic delay of the involution process upon forced weaning in conditional KO mice, in which STAT3 was specifically deleted in the lactating mammary gland. In addition, early activation of STAT1 and induction of p53 and p21 expression was observed, which suggested a potential compensatory mechanism for induction of eventual involution in the STAT3 null mammary glands. These results demonstrated the importance of STAT factors in signaling the initiation of physiological apoptosis *in vivo* and highlighted the utility of the lox/Cre system for addressing the function of genes with an embryonic lethal phenotype, specifically in the mammary gland.

STAT3 is the most ubiquitous of the members of this family of proteins, is activated by many different cytokines and growth factors, and plays many roles in different physiological processes. In addition, this protein is, the first STAT family member found to be constitutively activated in a variety of neoplastic tissues. It was determined that STAT3 modulates the expression of various target genes involved in cell-cycle regulation, angiogenesis, and apoptosis inhibition. Because of this, silencing or inhibiting STAT3 reduces tumor cell proliferation and survival in both animal and human studies. However, in the involuting mammary gland, STAT3 signaling induces cell death [5].

are reduced. This reversible phase is followed after 72 h by a nonreversible phase where widespread apoptosis and tissue remodeling takes place. This later phase requires *systemic factors and local* proteases such as MMP-3/Stromelysin-1 and MMP11/Stromelysin-3, MMP2/Gelatinase A, ICE (interleukin-1 beta converting enzyme), and Urokinase-type plasminogen activator [2]. In 1997, Li et al. [3] defined the role of local factors as compared with systemic hormones during the first and second stages of involution. When milk release was disrupted in the presence of systemic lactogenic hormones, they demonstrated that local signals were sufficient to induce alveolar cell death. These authors demonstrated that a variety of procedures successfully triggered mammary involution, although none of them prevented the presence of circulating lactogenic hormones. For example, sealing of the teats, mammary gland transplants unable to release milk due to the absence of a teat connection or inactivation of the oxytocin gene efficiently induced mammary cell death. On the other hand, in these scenarios, systemic hormones were able to preserve lobular-alveolar structure, although they did not prevent apoptosis. This chapter reviews the discovery of the local factors involved in the process of involution as well as the finding of the mechanisms involved in their ability to induce cell death shortly after weaning. In addition, the generation of a pro-oncogenic microenvironment during mammary involution, which is able to facilitate breast cancer progression, is

The studies referred above found that cell death correlated with the induction of proapoptotic genes as *bax*, decreased expression of milk proteins, dephosphorylation of STAT5a and 5b (main transcription factors that mediate prolactin triggered signaling), and activation of STAT3. Signal transducer and activator of transcription (STATs) are a family of latent transcription factors, which are activated in response to a variety of cytokines and growth factors. This family of signaling molecules has been implicated in cell growth, differentiation, survival, and apoptosis. STAT3 and STAT5 have reciprocal patterns of activation throughout a mammary developmental cycle, suggesting that STAT5 may be a survival factor and STAT3 a death factor for differentiated mammary epithelium. Chapman et al. [4], using the lox/Cre recombination system, showed a decrease in epithelial apoptosis and a dramatic delay of the involution process upon forced weaning in conditional KO mice, in which STAT3 was specifically deleted in the lactating mammary gland. In addition, early activation of STAT1 and induction of p53 and p21 expression was observed, which suggested a potential compensatory mechanism for induction of eventual involution in the STAT3 null mammary glands. These results demonstrated the importance of STAT factors in signaling the initiation of physiological apoptosis *in vivo* and highlighted the utility of the lox/Cre system for addressing the function of genes with an embryonic lethal phenotype, specifically in the mammary gland.

STAT3 is the most ubiquitous of the members of this family of proteins, is activated by many different cytokines and growth factors, and plays many roles in different physiological processes. In addition, this protein is, the first STAT family member found to be constitutively activated in a variety of neoplastic tissues. It was determined that STAT3 modulates the

also discussed.

42 Current Topics in Lactation

**2. Involvement of STAT3 signaling**

By 2003, it had been shown that STAT3 is the main factor involved in the initiation of apoptosis of mammary cells after weaning, but the mechanism of its activation remained unclear. In 2002, based on the hypothesis that IL-6 is the activating cytokine for STAT3, Hennighausen's group showed that expression of IL-6 increases during early involution together with STAT3 and p44/42 MAPK activation. Besides, it was shown that IL-6 treatment activated STAT3 in the mammary gland of virgin and lactating mice. In addition, IL-6-, STAT3-, and Bax-null mice showed similar mammary phenotypes, that is a significant delay in postlactational involution. Nevertheless, it was demonstrated that STAT3 activation during involution was independent of the IL-6 levels in the mammary after weaning. In contrast, the increase of p44/42 MAPK (ERK1/2) phosphorylation at the onset of involution was dependent on the presence of this cytokine. This suggested that either IL-6 does not induce STAT3 *in vivo* or its absence is compensated for by other cytokines, such as leukemia-inhibitory factor (LIF) [6].

By that time, there was no evidence in the literature reporting LIF expression and/or activities in the normal mammary gland tissue. Therefore, LIF expression profile was analyzed during the successive stages of mammary gland development, function, and involution. The results demonstrated that LIF is expressed in the mammary gland at low levels in postpubertal, adult virgin, and pregnant mice. But, expression of this protein almost disappear during lactation to then show a significant increase a few hours after weaning, maintaining these high levels during the following days. We demonstrated that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. In addition, implantation of LIF containing pellets in lactating glands resulted in a significant increase of STAT3 phosphorylation and epithelium apoptosis. We then concluded that LIF-regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution and that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by STAT3 activation [7].

Shortly after our paper was published, Christine Watson's lab also demonstrated that LIF is the physiological activator of STAT3, as they report that pSTAT3 is absent and C/EBPdelta (a well-known STAT3 target) is not upregulated in involuting transplanted mammary glands of LIF double knock-out (LIF(−/−) mice). Similarly to what was observed in the STAT3-null glands, LIF(−/−) mammary glands exhibit delayed involution, reduced apoptosis, and elevated levels of p53 [8].

STAT3 activation and LIF expression have not been observed only in the involuting mammary gland. It was determined that autocrine/paracrine LIF present in conditioned medium from primary cultures of mouse mammary tumors was also able to induce activation of that transcription factor and to increase cell survival in mammary tumor cell lines. However, although LIF blocking antibody prevented STAT3 phosphorylation, inhibition of STAT3 increased cell survival. These results indicated that LIF is overexpressed in mouse mammary tumors, where it acts as the main STAT3 activator. Nevertheless, the data also suggested that the positive LIF effect on tumor cell survival was not dependent on STAT3 activation, which seemed to inhibit tumor cell viability as it does in involuting mammary epithelium [9]. Kritikou et al. showed that pERK1/2 is significantly reduced in LIF(−/−) glands during pregnancy [8], suggesting that at this stage, LIF mediates its effects through pERK1/2. Therefore, it is possible that LIF proliferative effects on mammary tumors depend on ERK ½ activation. In addition, although it has been reported that STAT3 acts a potent oncogene in different tumor types, it was also demonstrated that the biological role of this factor is modulated by the stage of tumor progression [10]. Similarly, it can be proposed that in well-to-moderately differentiated mammary tumors, STAT3 activation induces cell death as observed in nontumorigenic mammary cells after lactation. This activity might be altered in more aggressive or less differentiated tumors, as it has been shown that STAT3 constitutive activation is very common in basal breast cancer [11], which have worse prognosis than luminal tumors. However, our results imply that in the development of therapeutic strategies for blocking STAT3 in breast cancer cells, the strong dependence on the cellular context that this factor activity displays should be taken into account.

Mechanical stress is a relevant factor to induce adaptive responses in multiple cell types [12–16]. Importantly, the signaling pathways triggered by this stimulus in those different examples also play a relevant role during mammary gland involution. Therefore, it was proposed that upon weaning, milk accumulation may cause cell stretching that, in turn, would induce the initiation of the molecular cascades that lead to the remodeling process of the lactating gland. To address this issue, we designed a new practical device that allowed us to evaluate the effects of radial stretching on the HC11 nontumorigenic mammary epithelial cell line cultured on flexible silicone membranes. The results showed that, as previously observed in other cell types, mechanical stress induced ERK1/2 phosphorylation and c-Fos expression induction, as well as LIF secretion, STAT3 activation, and AKT phosphorylation inhibition. Therefore, mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells [17].

STAT3 is essential, but not sufficient for the onset of apoptosis during mammary involution, as expression of a constitutively active *Akt*, a downstream effector of the phosphoinositide-3-OH kinase (PI3K) pathway, provides an overriding survival signal after lactation [18]. However, AKT downregulation depends on STAT3 activation, since PI(3)K regulatory subunits p55α and p50α (each of them, when overexpressed, reduces levels of activated AKT) are induced by that transcription factor during mammary involution. In fact, it has been shown that STAT3 binds directly to the promoters of p55α and p50α subunits *in vivo* and in STAT3 KO mice, upregulation of p55α and p50α is abrogated, levels of activated AKT are sustained, and apoptosis is prevented [19]. In addition, it was shown that deletion of both p55α and p50α subunits reduced cell death as well as expression and activity of cathepsin L during mammary involution. This protease participates in lysosomal-mediated programmed cell death (LM-PCD), which is upregulated during normal involution by activated STAT3. Furthermore, involution is delayed in cathepsin L-deficient mice, suggesting that the p55α/ p50α subunits mediate cell death in part by elevating the level of cathepsin L. Surprisingly, it was found that during involution, p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also STAT3 targets. Therefore, these findings revealed that postlactational regression of the mammary gland is accomplished through a nonclassical, lysosomal-mediated pathway of cell death, in which PI3K regulatory subunits participate as main regulators [20]. In fact, it has been demonstrated that cell death of mammary epithelium after weaning does not depend on the activation of executioner caspases 3, 6, and 7, although it requires STAT3 for cathepsin B and L induction as well as for the downregulation of their endogenous inhibitor Spi2A [21].

Global gene expression changes during involution have been profiled by microarray analysis, which allowed characterization of clusters of genes with distinct expression profiles during the first 4 days of involution. Such expression profiling led to the observation that one of the most strikingly upregulated genes in the absence of STAT3 is the serpin Spi2a. Interestingly, during mammary involution, STAT3 not only regulates LM-PCD by inhibiting serpin Spi2a, inducing the expression of cathepsins B and L, and the regulatory subunits p55α/p50α, but also by the uptake of secreted MFGs that lead to the formation and fusion of large lysosomal-like vacuoles, which are toxic to epithelial cells. Upon re-entry, the MFG (mammary fat globules) triglycerides are metabolized to free fatty acids, including oleic acid, that can distort membranes and result in leakage of cathepsins from lysosomes. Therefore, STAT3 promotes a phenotypic switch from secretion to phagocytosis of MFGs, the latter function delivering triglyceride to vacuoles with the ensuing consequences of LMP and cell death [22].
