Involuting Mammary Gland Research Articles

Mucosal Immunity in the Female Murine Mammary Gland.

The mammary gland is not classically considered a mucosal organ, although it exhibits some features common to mucosal tissues. Notably, the mammary epithelium is contiguous with the external environment, is exposed to bacteria during lactation, and displays antimicrobial features. Nonetheless, immunological hallmarks predictive of mucosal function have not been demonstrated in the mammary gland, including immune tolerance to foreign Ags under homeostasis. This inquiry is important, as mucosal immunity in the mammary gland may assure infant and women's health during lactation. Further, such mucosal immune programs may protect mammary function at the expense of breast cancer promotion via decreased immune surveillance. In this study, using murine models, we evaluated mammary specific mucosal attributes focusing on two reproductive states at increased risk for foreign and self-antigen exposure: lactation and weaning-induced involution. We find a baseline mucosal program of RORγT+ CD4+ T cells that is elevated within lactating and involuting mammary glands and is extended during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, and immunosuppressive Foxp3+ CD4+ T cells. Further, we demonstrate suppression of Ag-dependent CD4+ T cell activation, data consistent with immune tolerance. We also find Ag-independent accumulation of memory RORγT+ Foxp3+ CD4+ T cells specifically within the involution mammary gland consistent with an active immune process. Overall, these data elucidate strong mucosal immune programs within lactating and involuting mammary glands. Our findings support the classification of the mammary gland as a temporal mucosal organ and open new avenues for exploration into breast pathologic conditions, including compromised lactation and breast cancer.

Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization.

After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and β-catenin signaling contribute to phagocytosis downstream of TGF-β3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.

A Mouse Mammary Gland Involution mRNA Signature Identifies Biological Pathways Potentially Associated with Breast Cancer Metastasis

Mouse mammary gland involution resembles a wound healing response with suppressed inflammation. Wound healing and inflammation are also associated with tumour development, and a 'wound-healing' gene expression signature can predict metastasis formation and survival. Recent studies have shown that an involuting mammary gland stroma can promote metastasis. It could therefore be hypothesised that gene expression signatures from an involuting mouse mammary gland may provide new insights into the physiological pathways that promote breast cancer progression. Indeed, using the HOPACH clustering method, the human orthologues of genes that were differentially regulated at day 3 of mammary gland involution and showed prolonged expression throughout the first 4 days of involution distinguished breast cancers in the NKI 295 breast cancer dataset with low and high metastatic activity. Most strikingly, genes associated with copper ion homeostasis and with HIF-1 promoter binding sites were the most over-represented, linking this signature to hypoxia. Further, six out of the ten mRNAs with strongest up-regulation in cancers with poor survival code for secreted factors, identifying potential candidates that may be involved in stromal/matrix-enhanced metastasis formation/breast cancer development. This method therefore identified biological processes that occur during mammary gland involution, which may be critical in promoting breast cancer metastasis that could form a basis for future investigation, and supports a role for copper in breast cancer development.

Suppression of the NF-κB cofactor Bcl3 inhibits mammary epithelial cell apoptosis and, in breast tumours, correlates with poor prognosis

Background Several transcription factors have been shown to play important roles in the regulation of apoptosis at the onset of murine mammary involution. These include LIF-activated STAT3, c/ebpdelta, Ap-1 and IKK/NF-κB-mediated regulation of death receptor ligands. A study of STAT3 and STAT5 transcriptional targets in mammary epithelial cells in vitro showed that both c/ebpdelta and c-fos (a component of Ap-1) were upregulated by STAT3, suggesting a degree of interdependence between these transcription factor pathways in mediating their apoptotic effects. Interestingly, while no NF-κB or IKK genes were significantly regulated by STATs, the NF-κB cofactor gene, Bcl3, was found to be a principal transcriptional target of STAT3. This factor plays a role in altering the transcriptional capacity of specific NF-κB subunits and has previously been described as an oncogene in B-cell lymphomas. In this study we set out to establish whether Bcl3 had a role in regulating the cell fate of mammary epithelial cells either in the normal mammary gland or in mammary/breast cancer. Methods Archived material representing a range of tumour grades and types was collected from breast cancer patients immediately after surgery (tumour tissues = 122, normal tissues = 32). The median follow-up of the patients was 120 months (range 12 to 156 months). QRT-PCR for Bcl3 was performed and this information was used to determine statistically significant correlations with the clinical data on breast pathology. MCF7, T47D and MDA-MB231 human breast cancer cell lines were subjected to Bcl3-specific siRNA knockdown and subsequently assessed for cell motility characteristics using ECIS technology. Bcl3-knockout mice were assessed histologically for alterations in apoptosis rate during the adult pregnancy cycle. Western blots, quantitative PCR and DNA binding assays were used to determine the activity of molecular markers of apoptosis in these animals. Bcl3-deficient animals were crossed with mmtv-neu (c-erbB2) mice to establish the role of Bcl3 in primary (neu-dependent) mammary tumour growth, and magnetic resonance imaging was performed on tumour-bearing animals, to establish metastasis rates in the presence/absence of Bcl3. Results An analysis of 122 human breast cancer tissues showed that Bcl3 gene expression was suppressed in a significant proportion of invasive tumours, which correlated with poor prognosis. This also correlated with a significant decrease in Bcl3 gene expression in human breast cancer cell lines exhibiting increased motility characteristics. The effects of siRNA-mediated knockdown of Bcl3 are ongoing. In the mouse mammary gland, Bcl3 expression was restricted to epithelial cells during the first 24 hours of involution. Bcl3 deficiency resulted in a transient delay in the appearance of apoptotic bodies in the early involuting mammary gland in Bcl3-/- mice, while pSTAT3 levels were unchanged compared with equivalent timepoints in control animals. The activities of initiator/executor caspases of both intrinsic and extrinsic pathways were significantly decreased in Bcl3-/- tissues at this time, which correlated with decreases in the expression of key regulators of intrinsic/extrinsic apoptosis. Results from the ongoing magnetic resonance imaging study of tumour incidence/progression in mmtv-neu/Bcl3-/- mice will be presented. Conclusion These observations suggest that Bcl3 promotes apoptosis in the mammary gland and provides preliminary evidence of cross-talk between STAT3 and NF-κB pathways, both of which have been implicated in breast cancer. Our current data on Bcl3 in primary breast tumours and breast cancer cell lines contrasts with other studies, to suggest that Bcl3 suppresses the metastatic progression of primary breast cancer and has a neutral role in breast cancer incidence or primary tumour growth.

Scaffold Succumbs to Caspase Cleavage

Activation of the ERK (extracellular signal-regulated kinase) members of the mitogen-activated protein (MAP) kinase family is associated with cell survival signaling. In cells stimulated to undergo apoptosis, such survival signaling is often disrupted. McKay and Morrison provide insight into molecular mechanisms by which ERK signaling may be shut down in apoptotic cells. The protein known as kinase suppressor of Ras 1 (KSR1) is a scaffold that contributes to ERK activation by bringing together the kinases that participate in a cascade of sequential activation to ultimately stimulate the phosphorylation and activation of ERKs. The authors noted that KSR1 has a characteristic caspase cleavage site that would be expected to effectively break up KSR1’s scaffolding function by separating the C-terminal domain that binds to MEK (MAP kinase or ERK kinase) from the N-terminal domain that binds ERK (a substrate for MEK) and a domain that mediates targeting of the whole complex to the plasma membrane. An epitope-tagged version of KSR1 expressed in KSR –/– mouse embryo fibroblasts was indeed cleaved in cells stimulated to undergo apoptosis by treatment with tumor necrosis factor α and cycloheximide. Although the N-terminal fragment of KSR1 appeared to be further degraded, immunoblotting showed that the C-terminal fragment of KSR1 accumulated in apoptotic cells. This fragment can potentially act as a dominant-negative inhibitor, and expression of this fragment in KSR1 –/– cells resulted in increased apoptotic signaling and reduced amounts of active, phosphorylated ERK. On the other hand, expression of a cleavage-resistant version of KSR1 in the knockout cells led to accumulation of more activated ERK and diminished apoptotic signaling. Cleavage of KSR1 was also observed in a natural model of apoptosis--the involuting mammary gland. The authors propose that caspase-dependent dismantling of KSR1 may provide a one-two punch to the cell’s survival signaling by removing a scaffold that facilitates ERK activation and, at the same time, generating an inhibitor of ERK activation. M. M. McKay, D. K. Morrison, Caspase-dependent cleavage disrupts the ERK cascade scaffolding function of KSR1. J. Biol. Chem. 282 , 26225-26234 (2007). [Abstract] [Full Text]

Gene expression profiling of early involuting mammary gland reveals novel genes potentially relevant to human breast cancer

Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying "novel" genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse. We then examined the relevance of candidate genes to human tumorigenesis and identified a number of genes not previously implicated or not well characterized in human breast cancer. The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. This pilot study demonstrates proof of principle that through the analysis of gene expression during mammary gland involution, it may be possible to identify "novel" genes relevant human breast cancer.

Weaning-induced expression of a milk-fat globule protein, MFG-E8, in mouse mammary glands, as demonstrated by the analyses of its mRNA, protein and phosphatidylserine-binding activity.

A milk membrane glycoprotein, MFG-E8 [milk fat globule-EGF (epidermal growth factor) factor 8], is expressed abundantly in lactating mammary glands in stage- and tissue-specific manners, and has been believed to be secreted in association with milk fat globules. In the present paper, we describe further up-regulation of MFG-E8 in involuting mammary glands, where the glands undergo a substantial increase in the rate of epithelial cell apoptosis, and a possible role of MFG-E8 in mediating recognition and engulfment of apoptotic cells through its specific binding to PS (phosphatidylserine). Immunoblotting and RNA blotting analyses revealed that both MFG-E8 protein and MFG-E8 mRNA were markedly increased in mammary tissue within 3 days of either natural or forced weaning (pup withdrawal) of lactating mice. Using immunohistochemical analysis of the mammary tissue cryosections, the MFG-E8 signal was detected around the epithelium of such involuting mammary glands, but was almost undetectable at early- and mid-lactation stages, although strong signals were obtained for milk fat globules stored in the alveolar lumen. Some signals double positive to a macrophage differentiation marker, CD68, and MFG-E8 were detected in the post-weaning mammary tissue, although such double-positive signals were much smaller in number than the MFG-E8 single-positive ones. Total MFG-E8 in milk was also increased in the post-weaning mammary glands and, furthermore, the free MFG-E8 content in the post-weaning milk, as measured by in vitro PS-binding and apoptotic HC11 cell-binding activities, was much higher than that of lactation. In addition, the post-weaning milk enhanced the binding of apoptotic HC11 cells to J774 macrophages. Sucrose density-gradient ultracentrifugation analyses revealed that such enhanced PS-binding activity of MFG-E8 was present in membrane vesicle fractions (density 1.05-1.13 g/ml), rather than milk fat globule fractions. The weaning-induced MFG-E8 might play an important role in the recognition and engulfment of apoptotic epithelial cells by the neighbouring phagocytic epithelial cells in involuting mammary glands.

Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland.

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.