**5. Mammary involution and cancer**

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

**4. Gene expression regulation at the level of mRNA stability**

It has been demonstrated that the stability of many messenger RNAs (mRNAs) encoding oncoproteins, chemokines, cytokines, and other inflammatory mediators is controlled by AU-rich elements (AREs), sequences located within the 3'-UTR of many transcripts [29, 30]. AREdirected control of mRNA decay is mediated, in part, through interactions with specific AREbinding proteins (AUBPs). One such protein is tristetraprolin (TTP), which accelerates the decay of targeted transcripts [31]. During inflammation, TTP plays a relevant role destabilizing different mRNAs, participating in glucocorticoid-mediated anti-inflammatory activity [32–34] and inhibiting NF-κB signaling [35]. The relevance of TTP as a negative regulator of these processes has been demonstrated by the severe chronic inflammation displayed by multiple tissues in TTP-KO mice, which was mostly due to the dramatic increase of TNF-α levels [32].

Several reports indicate that TTP participates in the inhibition of tumor progression. It has been shown that TTP mRNA levels are significantly decreased in many tumor types, including breast cancer [36]. We have also reported that TTP expression is lower in all breast cancer types compared with normal mammary tissue, and high levels of this protein negatively correlate with cancer cell aggressiveness. Interestingly, we have also determined that in the mouse mammary gland, expression of this protein reaches the highest level during lactation, and can be induced in culture by treatment with lactogenic

activated.

46 Current Topics in Lactation

hormones [37].

Breast cancer is the most frequent malignancy diagnosed in association with pregnancy [38–40]. In addition, different studies have demonstrated an increase in breast cancer risk in the years immediately following giving birth [41–43]. Importantly, not only a rise in breast cancer incidence during the postpartum years has been observed, but also a higher risk for poor outcomes in women diagnosed during that timeframe [44]. However, it has been well established that pregnancy provides lifetime protection for women who are under 35 years at first birth [45–47]. Therefore, pregnancy would exert two opposite effects on breast cancer development: induces protection, which is associated with the differentiation of mammary epithelium, and increases risk, through alteration of tissue microenvironment.

Postpartum breast cancers have been referred to as type II Pregnancy-Associated Breast Cancer (PABC) to distinguish these cancers from those diagnosed during pregnancy [45]. A physiological window unique to type II PABC is mammary gland involution and, in an effort to distinguish why type II PABC patients have worse prognoses, the normal postpartum breast microenvironment has been investigated for potential tumor-enhancing attributes. These studies reveal that postpartum involution utilizes wound-healing programs for gland remodeling, including increases in matrix metalloproteinase activity, release of bioactive fragments of extracellular matrix (ECM) termed matricryptins, accumulation of fibrillar collagen, and influx of immune cells, which also generates tumor-promotional microenvironments [48–51]. *In vivo* experiments showed that tumors from xenografts of breast cancer cells exposed to the postpartum involution microenvironment have increased growth, invasion, and metastasis compared to those growing in nulliparous hosts. In addition, these implants showed augmented fibrillar collagen accumulation and high cyclooxygenase-2 (COX-2) expression [52]. Coincidently, in the postlactating mammary gland, inhibition of COX-2 reduced the collagen fibrillogenesis, as well as tumor development and cancer cell invasiveness [53]. Therefore, it has been proposed that women at high risk for postpartum breast cancer might benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum, which would reduce COX-2 expression and its consequences on the behavior of breast initiated cells.

Without doubt, immune response plays a primordial role in mouse mammary involution, since molecular profiling of that phase is consistent with acute phase, innate and adaptive immune responses [51–54]. Interestingly, after weaning, mammary epithelial cells themselves express transcripts traditionally associated with immune cells [55, 56] and acquire phagocytic capability [57]. Therefore, it has not been possible to completely determine which cell types and in what part are responsible for the observed immune-like gene signatures. Particularly, there is not much data about the participation of adaptive immune cells, but innate immune cell populations have been partially characterized. Specifically, it has been observed that granulocyte infiltration in the mouse gland on the first day of involution suggest the involvement of this cell type in early involution [58]. In addition, resident macrophages seem to be required for this phase, while infiltrating macrophages are important during the remodeling stage [52]. It was observed that on this last phase, macrophages express low iNOS, high arginase-1, and the mannose receptor, which is consistent with alternative activation or M2 polarization of these cells [59]. Importantly, this phenotype correlated with breast tumor promotion in patients [60] and murine mammary tumor progression [61].

The earlier mentioned STAT3 and NF-κB signaling pathways, as well as others involving transforming growth factor beta (TGF-β) and the retinoid acid receptors (RARs)/retinoid X receptors (RXRs), participate in mammary gland involution as well as in breast cancer development. After weaning, target genes of RARα/p300 and RelA/p65, which belong to the NF-κB protein family, are induced, and high activity of the proteins coded by these genes, e.g., *MMP9*, *Capn1*, and *Capn2*, has been detected in breast cancer cells. Calpains belong to a family of calcium-dependent intracellular cysteine proteases involved in a wide variety of physiological and pathological processes. These proteases are heterodimers, consisting of a small regulatory subunit, encoded by CAPN4 gene, common for both members, and a large catalytic subunit encoded by either CAPN1 or CAPN2. During mammary gland involution and cancer progression, these proteins are relevant for modifying the extracellular matrix, allowing tissue remodeling and/or cell invasion. In addition, calpains also cleave intracellular proteins located in the cell membrane, lysosomes, mitochondria, and nuclei, favoring cell death during involution and cell anchoring loss during tumor progression [62].
