**3. NF-κB signaling**

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

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

taken into account.

44 Current Topics in Lactation

It is clear that there are multiple mechanisms of regulation in early involution that synergise to ensure efficient induction of cell death, phagocytosis, suppression of inflammation, and remodeling of the architecture of the gland. Clarkson and Watson identified clusters of genes that are transcriptional targets of either NF-κB or STAT3, or indeed both, during early involution [23]. For example, among the NFκB targets, the TNF superfamily of death receptor (DR) ligands have been detected. These proteins induce apoptosis through binding to their receptor, which recruits caspase 8 (via FADD) and activates executioner caspases, finally leading to cell death [24]. Specifically, *Tnf*, *Tnfsf4*, *Tnfsf6*, *Tnfsf7*, *Tnfsf10,* and *Tnfsf12* are induced transiently at 12 h after weaning, and the proteins Fas ligand, TNF-α, TWEAK, and TRAIL are able to activate extrinsic apoptosis through their cognate receptors Fas, TNFR-1, TNFR-2, DR3, and DR4. The genes for the first two of these receptors (*Fas* and *Tnfrsf1a*) were also induced, and maximally coexpressed, within 24 h of weaning. NF-κB activity also correlated with the rapid activation of these TNF superfamily ligands [25].

Particularly, about TNF-α, our results have shown that this factor, through TNF-α receptor-2 (TNFR2) binding induces LIF expression mediated by ERK1/2 activation in nontumorigenic mouse mammary epithelial cells. In addition, the AP-1 has been implicated in this signaling cascade, since blocking the activity of this transcription factor resulted in a significant reduction of TNF-α induced LIF expression. Therefore, TNF-α may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation [26].

The NF-κB family of transcription factors primarily plays anti-apoptotic roles. DNA binding activity of this transcription factor is markedly upregulated within 3 h of forced involution and is suggested to promote survival of a subpopulation of mammary epithelial cells [25]. This hypothesis is consistent with the paradigm of NF-κB–mediated suppression of TNF-α cytotoxicity in TNF-α–responsive cells. NF-κB activity is mediated by a multiprotein signaling complex called the IκB kinase (IKK), which consists of two catalytic subunits: IKK1/α, IKK2/β and a regulatory subunit, NEMO (NF-kappa-B essential modulator). Activation of this complex leads to phosphorylation of the IκB proteins; phospho-IκB is rapidly ubiquitinated and degraded via the 26S-proteasome releasing NF-κB and unmasking its nuclear localization signal, allowing its activity as transcription regulator of many target genes [27]. NF-κB then inhibits the death signal by trans-activating genes that promote resistance to apoptosis. The effect of this negative feedback mediated by NF-κB is the modulation of apoptosis in response to the TNF-α death signal. However, deletion of the gene encoding IKK2 resulted in delayed apoptosis and remodeling, as well as blockade of caspase 3 activation in the postlactational mammary gland. This failure to induce cell death was associated with reduced expression of TNF and its receptor TNFR1, which are known NF-κB targets. In addition, the observed high levels of active AKT together with downregulation of TWEAK, another DR ligand, also contributed to retard the involution process in these genetically engineered mice [28]. These results suggest that NF-κB may provide either proapoptotic or antiapoptotic signals during involution, depending on the timing and cellular context in which this transcription factor is activated.
