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The mammary gland provides a spectacular example of physiological cell death whereby the cells that produce milk during lactation are removed swiftly, efficiently, and without inducing inflammation upon the cessation of lactation. The milk-producing cells arise primarily during pregnancy and comprise the alveolar lineage that is specified by signalling pathways and factors that are activated in response to pregnancy hormones. There are at least two alveolar sub-lineages, one of which is marked by the presence of binucleate cells that are especially susceptible to programmed cell death during involution. This process of post-lactational regression, or involution, is carefully orchestrated and occurs in two phases, the first results in a rapid switch in cell fate with the secretory epithelial cells becoming phagocytes whereupon they destroy dead and dying cells from milk. This reversible phase is followed by the second phase that is marked by an influx of immune cells and a remodelling of the gland to replace the alveolar cells with re-differentiated adipocytes, resulting in a return to the pre-pregnant state in preparation for any subsequent pregnancies. The mouse mammary gland provides an excellent experimental tool with which to investigate lineage commitment and the mechanisms of programmed cell death that occur in a normal physiological process. Importantly, involution has highlighted a role for lysoptosis, a mechanism of cell death that is mediated by lysosomal cathepsins and their endogenous inhibitors, serpins. In this review, I discuss alveolar lineage commitment during pregnancy and the programmed cell death pathways that destroy these cells during involution.

Post-lactational mammary gland regression encompasses extensive programmed cell death and removal of milk-producing epithelial cells, breakdown of extracellular matrix components and redifferentiation of stromal adipocytes. This highly regulated involution process is associated with a transient increased risk of breast cancer in women. Using a syngeneic tumour model, we show that tumour growth is significantly altered depending on the stage of involution at which tumour cells are implanted. Tumour cells injected at day 3 involution grew faster than those in nulliparous mice, whereas tumours initiated at day 6 involution grew significantly slower. These differences in tumour progression correlate with distinct changes in innate immune cells, in particular among F4/80-expressing macrophages and among TCRδ+ unconventional T cells. Breast cancer post-pregnancy risk is exacerbated in older first-time mothers and, in our model, initial tumour growth is moderately faster in aged mice compared with young mice. Our results have implications for breast cancer risk and the use of anti-inflammatory therapeutics for postpartum breast cancers.

Mammary stem cells (MaSCs) have been defined by cell surface marker expression and their ability to repopulate a cleared fat pad, a capacity now known to result from reprogramming upon transplantation. Furthermore, lineage-tracing studies have provoked controversy as to whether MaSCs are unipotent or bi/multipotent. Various innovative experimental approaches, including single-cell RNA sequencing (scRNA-Seq), epigenetic analyses, deep tissue and live imaging, and advanced mouse models, have provided new and unexpected insights into stem and progenitor cells; thus, it is now timely to reappraise our concept of the MaSC hierarchy. Here, I highlight misconceptions, suggest definitions of stem and progenitor cells, and propose a way forward in our search for an understanding of MaSCs.

The mammary gland is a unique tissue and the defining feature of the class Mammalia. It is a late-evolving epidermal appendage that has the primary function of providing nutrition for the young, although recent studies have highlighted additional benefits of milk including the provision of passive immunity and a microbiome and, in humans, the psychosocial benefits of breastfeeding. In this Review, we outline the various stages of mammary gland development in the mouse, with a particular focus on lineage specification and the new insights that have been gained by the application of recent technological advances in imaging in both real-time and three-dimensions, and in single cell RNA sequencing. These studies have revealed the complexity of subpopulations of cells that contribute to the mammary stem and progenitor cell hierarchy and we suggest a new terminology to distinguish these cells.

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape.

The adult mammary gland undergoes dynamic changes during puberty and the postnatal developmental cycle. The mammary epithelium is composed of a bilayer of outer basal, or myoepithelial, cells and inner luminal cells, the latter lineage giving rise to the milk-producing alveolar cells during pregnancy. These luminal alveolar cells undergo Stat3-mediated programmed cell death following the cessation of lactation. It is established that immune cells in the microenvironment of the gland have a role to play both in the ductal outgrowth during puberty and in the removal of dead cells and remodelling of the stroma during the process of postlactational regression. However, most studies have focussed on the role of the stromal immune cell compartment or have quantified immune cell populations in tissue extracts. Our recent development of protocols for deep imaging of the mammary gland in three dimensions (3D) has enabled the architectural relationship between immune cells and the epithelium to be examined in detail, and we have discovered a surprisingly dynamic relationship between the basal epithelium and leucocytes. Furthermore, we have observed morphological changes in the myoepithelial cells, as involution progresses, which were not revealed by previous work in 2D tissue sections and whole tissue. This dynamic architecture suggests a role for myoepithelial cells in the orderly progression of involution. We conclude that deep imaging of mammary gland and other tissues is essential for analysing complex interactions between cellular compartments.

The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. 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 resulting in increased cytosolic activity. Surprisingly, we found that 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. Our findings reveal a novel role for these PI3K regulatory subunits as regulators of LM-PCD in vivo.

Involution of the mammary gland occurs at the end of every period of lactation and is an essential process to return the gland to a pre-pregnant state in readiness for the next pregnancy. Involution is a complex process of regulated alveolar cell death coupled with tissue remodeling and requires exquisite control of transcription and signaling. These processes can be investigated using a variety of molecular and morphological approaches.In this chapter we describe how to initiate involution and collect mammary glands, measure involution morphologically, and quantify lysosomal leakiness in mammary tissue and in cultured mammary epithelial cells. These procedures encompass a range of microscopy and molecular biology techniques.

The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium., The identity and origin of adult mammary stem cells has been much debated. Here, the authors use a stochastic genetic labelling approach, together with optical tissue clearing, to visualize clonal progeny and show that unipotent stem/progenitor cells contribute to adult mammary gland development.

Background High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated. Methods Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. Results Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue. Conclusions The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.

Isolation of mammary epithelial subpopulations, including stem and progenitor cells, has become a standard technique in recent years. However, a number of methods and approaches for this have developed and the relative benefits of the different approaches, and the reason for their development, have not always been clear. Here, three of the leading laboratories working on the separation of mammary cell subpopulations have summarised their methods, highlighted their differences and similarities and also discussed the reasoning behind the approaches they have taken. This article will assist workers establishing mammary cell separation protocols in their laboratories to make informed choices about the methods they should use.

Mammary gland regression following weaning (involution) is associated with extensive cell death and the acquisition of an inflammatory signature. Characterising the interplay between mammary epithelial cells, the re-emerging stroma, and immune cells has implications for the understanding of the pathogenesis of pregnancy associated breast cancer. Stat3 has a role in orchestrating cell death and involution, and we sought to determine whether expression of Stat3 by the mammary epithelium influences also the innate immune environment and inflammatory cell influx in the gland. We examined mice in which Stat3 is conditionally deleted only in the mammary epithelium. Distinct sets of genes associated with the acute phase response and innate immunity are markedly up-regulated during first phase involution in a Stat3-dependent manner. During second phase involution, chitinase 3-like 1, which has been associated with wound healing and chronic inflammatory conditions, is dramatically up-regulated by Stat3. Also at this time, the number of mammary macrophages and mast cells increases per unit area, and this increase is impaired in the absence of epithelial Stat3. Furthermore, expression of arginase-1 and Ym1, markers of alternatively activated macrophages, is significantly decreased in the absence of Stat3, whilst iNOS, a marker associated with classically activated macrophages, shows significantly increased expression in the Stat3 deleted glands. Thus, Stat3 is a key transcriptional regulator of genes associated with innate immunity and wound healing and influences mammary macrophage and mast cell numbers. The presence of epithelial Stat3 appears to polarize the macrophages and epithelial cells towards an alternatively activated phenotype, since in the absence of Stat3, the gland retains a phenotype associated with classically activated macrophages. These findings have relevance to the study of pregnancy associated breast cancer and the role of Stat3 signalling in recruitment of alternatively activated tumour-associated macrophages in breast cancer. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

It is well established that lysosomes play an active role during the execution of cell death1. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme2, 3. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions4, 5, 6, 7. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively1, 9, 10.

Prominin-1 (Prom1) is recognized as a stem cell marker in several tissues, including blood, neuroepithelium, and gut, and in human and mouse embryos and many cancers. Although Prom1 is routinely used as a marker for isolating stem cells, its biological function remains unclear. Here we use a knockout model to investigate the role of Prom1 in the mammary gland. We demonstrate that complete loss of Prom1 does not affect the regenerative capacity of the mammary epithelium. Surprisingly, we also show that in the absence of Prom1, mammary glands have reduced ductal branching, and an increased ratio of luminal to basal cells. The effects of Prom1 loss in the mammary gland are associated with decreased expression of prolactin receptor and matrix metalloproteinase-3. These experiments reveal a novel, functional role for Prom1 that is not related to stem cell activity, and demonstrate the importance of tissue-specific characterization of putative stem cell markers.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in tumors of different origin, but the molecular bases for STAT3 requirement are only partly understood. To evaluate the contribution of enhanced Stat3 activation in a controlled model system, we generated knock-in mice wherein a mutant constitutively active Stat3C allele replaces the endogenous wild-type allele. Stat3C could enhance the tumorigenic power of the rat Neu oncogene in mouse mammary tumor virus (MMTV)–Neu transgenic mice, triggering the production of earlier onset, more invasive mammary tumors. Tumor-derived cell lines displayed higher migration, invasion, and metastatic ability and showed disrupted distribution of cell-cell junction markers mediated by Stat3-dependent overexpression of the COOH terminal tensin-like (Cten) focal adhesion protein, which was also significantly upregulated in Stat3C mammary tumors. Importantly, the proinflammatory cytokine interleukin-6 could mediate Cten induction in MCF10 cells in an exquisitely Stat3-dependent way, showing that Cten upregulation is a feature of inflammation-activated Stat3. In light of the emerging pivotal role of Stat3 in connecting inflammation and cancer, our identification of Cten as a Stat3-dependent mediator of migration provides important new insights into the oncogenic role of Stat3, particularly in the breast. Cancer Res; 70(6); 2558–67

The tumor suppressor promyelocytic leukemia protein (PML) is possibly unique in that its tumor suppressive functions may be attributed to both the protein and the conspicuous nuclear bodies (PML-NBs) that PML builds. Untangling the role of either the protein or its domain in cell fate has been a decade long task which has just received new impetus from developmental biologists. PML appears to play a central role in regulating stem and progenitor cell fate in tissues as diverse as blood, brain and breast. Our studies have uncovered an inverse relationship between the activity of certain Stat transcription factors and PML in controlling normal mammary gland development and the regulation of lineage commitment. Genetic loss of Pml delays differentiation of the milk-producing alveolar cells and disrupts ductal development, defects which may result from a skewing of the progenitor population to favor estrogen receptor positive cells (ERalpha). This is of considerable interest as ERalpha cells are non-cycling in normal breast while promiscuous cell cycle entry is a feature of these cells in breast cancer. These data begin to show the cell types and tissues that are most sensitive to PML dose and provide new perspectives for the regulation of mammary gland development and tumorigenesis.

Cysteine cathepsins are proteolytic enzymes that reside in endolysosomal vesicles. Some are expressed constitutively while others are transcriptionally regulated. However, the expression and subcellular localization of cathepsins changes during cancer progression and cathepsins have been shown to be causally involved in various aspects of tumorigenesis including metastasis. The use of mouse models of breast cancer genetically ablated for cathepsin B has shown that both the growth of the primary tumor and the extend of lung metastasis is reduced by the loss of cathepsin B. The role of cathepsins in involution of the mammary gland has received little attention although it is clear that cathepsins are involved in tissue remodeling in the second phase of involution. We discuss here the roles of cathepsins and their endogenous inhibitors in breast tumorigenesis and post-lactational involution.

Mammary gland development occurs through distinctive stages throughout embryonic and pubertal development and reproductive life. At each stage,different signals are required to induce changes in both the epithelium and the surrounding mesenchyme/stroma. Recent studies have provided new insights into the origin, specification and fate of mammary stem and progenitor cells and into how the differentiated lineages that comprise the functional mammary gland are determined. The development of new tools and culture techniques has also enabled the factors that influence branching morphogenesis in the embryonic and pubertal gland to be identified. A surprising recent discovery has been that mammary epithelial cells commit to differentiated lineages using the same signalling pathways that regulate lineage determination in T helper cells.

Naïve T helper cells differentiate into Th1 and Th2 subsets, which have unique cytokine signatures, activators and transcriptional targets. The Th1/Th2 cytokine milieu is a key paradigm in lineage commitment, and IL-4(Il4), IL-13 (Il13) and Stat6 are important mediators of Th2 development. We show here, for the first time, that this paradigm applies also to mammary epithelial cells, which undergo a switch from Th1 to Th2 cytokine production upon the induction of differentiation. Thus, the Th1 cytokines IL-12 (Il12),interferon gamma (INFγ; also known as Ifng) and Tnfα are downregulated concomitantly with the upregulation of the Th2 cytokines IL-4,IL-13 and IL-5 (Il5) as epithelial cells commit to the luminal lineage. Moreover, we show that Th2 cytokines play a crucial role in mammary gland development in vivo, because differentiation and alveolar morphogenesis are reduced in both Stat6 and IL-4/IL-13 doubly deficient mice during pregnancy. This unexpected discovery demonstrates a role for immune cell cytokines in epithelial cell fate and function, and adds an unexpected tier of complexity to the previously held paradigm that steroid and peptide hormones are the primary regulators of mammary gland development.

The Nuclear Factor-kappaB (NF-kappaB) family of transcription factors are ubiquitously expressed and control a wide range of cellular responses, including apoptosis, proliferation, differentiation, inflammation and immunity. Here, we investigated the function of the NF-kappaB upstream regulator IkappaB kinase 2/beta (IKK2) in apoptosis regulation in the normal physiological setting of regressing mammary gland. Conditional deletion of the gene encoding IKK2 resulted, surprisingly, in delayed apoptosis and remodelling, and abrogation of caspase 3 cleavage. This failure to induce involution was associated with reduced expression, within 24 hours of involution, of the death receptor (DR) ligand TNF and its receptor TNFR1, which are known NF-kappaB targets. This was associated with elevated levels of active AKT and phosphorylated FOXO3a. Furthermore, we show that expression of TWEAK, another DR ligand, is dramatically downregulated, even in heterozygous IKK2 mammary glands. Unlike other DR ligands, the TWEAK promoter has six consensus FOXO-binding sites, further suggesting that it is differentially regulated. Interestingly, a cleaved form of TWEAK is upregulated during involution. This unexpected function of the IKK2/NF-kappaB pathway as a regulator of TWEAK expression and inducer of apoptosis has significant consequences for future therapeutic approaches for cancer and inflammatory diseases.

Prolactin and leukemia inhibitory factor (LIF) have different roles in the adult mammary gland, which are mediated in part by the signal transducers and activators of transcription (STAT)5 and STAT3. In vivo studies have shown that STAT5 contributes to prolactin-dependent lobuloalveolar development and lactation whereas STAT3 mediates LIF-dependent epithelial apoptosis during postlactational involution. To understand the molecular basis of these STAT-dependent pathways, we demonstrate the ligand-independent effects of STAT5 and STAT3 in mammary epithelial cells in vitro and also identify the genes regulated by these related transcription factors. Thus, using conditionally active STAT3- or STAT5a-GyraseB fusion proteins, we observed that enforced and specific dimerization of STAT3 induced apoptosis whereas STAT5 induced differentiation of mammary epithelial cells. Furthermore, STAT5 attenuated apoptosis mediated by LIF, the physiological inducer of STAT3. Microarray analysis of STAT3- and STAT5-induced genes using this system demonstrated a marked specificity, which reflected their different physiological effects in vitro and in vivo. STAT5-specific gene targets included the milk protein genes alpha-casein and kallikrein-8 and the survival factors prosaposin and Grb10. STAT3-specific genes included the apoptosis regulators CCAAT enhancer binding protein-delta, phosphatidylinositol 3-kinase-regulatory subunits, purine nucleoside phosphorylase, and c-fos. These data illustrate that specific activation of STAT3 and STAT5 alone is sufficient to induce and suppress apoptosis, respectively, and that these transcription factors elicit their actions by inducing distinct subsets of target genes in mammary epithelial cells.

STAT3 is the key mediator of apoptosis in mammary gland. We demonstrate here that LIF is the physiological activator of STAT3, because in involuting mammary glands of Lif -;/-; mice, pSTAT3 is absent and the STAT3 target, C/EBPδ, is not upregulated. Similar to Stat3 knockouts, Lif -;/-; mammary glands exhibit delayed involution, reduced apoptosis and elevated levels of p53. Significantly, Lif -;/-; glands display precocious development during pregnancy, when pSTAT3 is not normally detected. We show that pERK1/2 is significantly reduced in Lif -;/-; glands at this time, suggesting that at this stage LIF mediates its effects through pERK1/2. Inhibition of LIF-mediated ERK1/2 phosphorylation potentiates the proapoptotic effects of STAT3. LIF therefore signals alternately through ERK1/2, then STAT3, to regulate mammary growth and apoptosis.

Triple-negative breast cancer (TNBC) has poor prognostic outcome compared with other types of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. Here, we report that the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumours. Exogenous BCL11A overexpression promotes tumour formation, whereas its knockdown in TNBC cell lines suppresses their tumourigenic potential in xenograft models. In the DMBA-induced tumour model, Bcl11a deletion substantially decreases tumour formation, even in p53-null cells and inactivation of Bcl11a in established tumours causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Thus, BCL11A has an important role in TNBC and normal mammary epithelial cells. This study highlights the importance of further investigation of BCL11A in TNBC-targeted therapies., Triple-negative breast cancers (TNBCs) tend to have poor prognosis; however, the mechanisms underlying TNBC pathology are not well understood. Here the authors utilize epidemiologic data and animal models to demonstrate an important role for BCL11A in the genesis and propagation of TNBCs.

Involution is a process whereby the mammary gland undergoes extensive tissue remodelling involving exquisitely coordinated cell death, extracellular matrix degradation and adipose tissue regeneration following the weaning of offspring. These processes are mediated in part through Jak/Stat signalling pathways, which can be deregulated in breast cancer. Synthetic in vitro analogues of the breast could become important tools for studying tumorigenic processes, or as personalized drug discovery platforms and predictors of therapeutic response. Ideally, such models should support 3D neo-tissue formation, so as to recapitulate physiological organ function, and be compatible with high-throughput screening methodologies. We have combined cell lines of epithelial, stromal and immunological origin within engineered porous collagen/hyaluronic acid matrices, demonstrating 3D-specific molecular signatures. Furthermore seeded cells form mammary-like branched tissues, with lobuloalveolar structures that undergo inducible involution phenotypes reminiscent of the native gland under hormonal/cytokine regulation. We confirm that autophagy is mediated within differentiated mammary epithelial cells in a Stat-dependent manner at early time points following the removal of a prolactin stimulus (H/WD). In addition, epithelial cells express markers of an M2 macrophage lineage under H/WD, a process that is attenuated with the introduction of the monocyte/macrophage cell line RAW 264.7. Thus, such 3D models are suitable platforms for studying cell-cell interactions and cell death mechanisms in relation to cancer.

The transcription factor NF-kappaB is a key modulator of apoptosis in a variety of cell types, but to date this specific function of NF-kappaB has not been demonstrated in epithelia. Here, we describe the activation of NF-kappaB during post-lactational involution of the mouse mammary gland, a period of extensive apoptosis of luminal epithelial cells. Significantly, active NF-kappaB localized exclusively to nonapoptotic epithelial cells both in vivo and in the mammary epithelial cell line, KIM-2, transduced with an NF-kappaB-dependent green fluorescent protein reporter. Activation of NF-kappaB in vitro coincided with a decrease in the cytosolic repressor, IkappaBalpha. Furthermore, induction of NF-kappaB either by extracellular ligands or, more specifically, by inhibition of the IkappaB repressor with adenoviral constructs expressing antisense mRNA, resulted in enhanced survival of KIM-2 cells. Therefore, although coincident with induction of apoptosis both in vivo and in vitro, NF-kappaB appeared to exert a selective survival function in epithelial cells. This study highlights for the first time a role for NF-kappaB in modulating apoptosis in epithelium.

Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, Stat3 is specifically activated. To address the function of this signaling molecule in mammary epithelial apoptosis, we have generated a conditional knockout of Stat3 using the Cre-lox recombination system. Following weaning, a decrease in apoptosis and a dramatic delay of involution occurred in Stat3 null mammary tissue. Involution is normally associated with a significant increase in IGFBP-5 levels. This was observed in control glands, but not in the absence of Stat3. IGFBP-5 has been suggested to induce apoptosis by sequestering IGF-1 to casein micelles, thereby inhibiting its survival function. Our findings suggest that IGFBP-5 is a direct or indirect target for Stat3 and its upregulation is essential to normal involution. No marked differences were seen in the regulation of Stat5, Bcl-x(L), or Bax in the absence of Stat3. Precocious activation of Stat1 and increases in levels of p53 and p21 occurred and may act as compensatory mechanisms for the eventual initiation of involution observed in Stat3 null mammary glands. This is the first demonstration of the importance of a Stat factor in signaling the initiation of physiological apoptosis in vivo.

Janus, the two-faced Roman god of gates and doors, beginnings and endings, symbolizes important transitions. How appropriate therefore that Janus kinase 2 has been discovered to control the transition from normal mammary gland development to breast cancer in a recent manuscript from the Schreiber laboratory published in Cell Death and Differentiation.1 The lifetime risk of breast cancer for women in the western world is 1 in 8 and the incidence rate is rising toward epidemic levels with 1.4 million women diagnosed annually with this disease worldwide http://www.cancerresearchuk.org/cancer-info/cancerstats/world/breast-cancer-world/. Breast cancer is a heterogeneous disease with three main histological subtypes: estrogen receptor alpha/progesterone receptor (ERα/PR) positive, HER2 overexpressing, and triple negative tumors, which do not express ERα, PR or HER2. Six subtypes have been identified on the basis of gene expression signatures, each with a different prognosis.2 Patients with luminal subtype tumors, which are ERα positive and account for over 70% of breast cancers, have the best prognosis and are treated clinically with anti-estrogen therapies such as tamoxifen or aromatase inhibitors. However, despite an overall good prognosis for ERα-positive breast cancer, ∼25% of women develop resistance to anti-estrogen therapy, have recurrent tumors and succumb to metastatic disease.3 Furthermore, ERα+ breast cancer is itself heterogeneous. There is thus a pressing need to understand the origins of ERα+ tumors and the molecular mechanisms that give rise to their diversity and differential response to anti-estrogen therapy. Murine breast cancer models have been invaluable in delineation of both the genes and signaling pathways that regulate tumorigenesis. However, although ERα+ tumors are the most common type of breast cancer in women, there is a paucity of experimental mouse models of ERα+ tumors. Recently, the Schreiber laboratory described a new mouse model of ERα+ mammary carcinoma that arises in mice deficient for Stat1.4 Stat1 is a member of the Stat family of latent transcription factors, which bind to cytokine and growth factor receptors on their engagement by ligand, resulting in activation of receptor-associated JAK kinases that in turn tyrosine phosphorylate Stats, which dimerize, translocate to the nucleus and bind to promoters of target genes. Genetic ablation of Stat1 resulted in the spontaneous development of mammary tumors, with a long latency, that recapitulate many of the features and gene expression profiles of ERα+ luminal breast cancer. Thus, Stat1 is an unexpected tumor suppressor in mammary gland and this correlates with reduced expression levels of STAT1 in 45% of ERα+ human breast tumors, suggesting that Stat1−/− mice may be a useful model of ERα+ breast cancer. Conversely, other members of the Stat family of transcription factors, in particular Stat3 and Stat5, are potent oncogenes in mammary gland and appear to have reciprocal effects on target gene expression.5, 6, 7 During normal mammary gland development, Stat5 is essential for alveologenesis, the process by which differentiated milk-producing cells arise during pregnancy,8 whereas Stat3 is a critical mediator of cell death during post-lactational regression of the gland.9, 10 Stats 3 and 5 are generally thought of as having opposing functions and, despite recognizing a similar DNA-binding motif, bind distinct promoters.10 This Stat1−/− ERα+ tumor model has now been characterized in more detail and the authors show that control of Jak2 activity by the Stat1-SOCS1 axis is essential to maintain mammary gland homeostasis and that deletion of Stat1 and the concomitant loss of SOCS1, a negative regulator of Jak2, results in hyperactivation of Jak2 and unopposed signaling through the prolactin receptor (PrlR). This corroborates a previous study showing that loss of a single allele of SOCS1 can rescue lactation failure that occurs in PrlR+/− mice.11, 12 Interestingly, persistent PrlR signaling is a feature also of human ERα+ and ERα− breast cancer cells13 and elevated serum levels of prolactin (Prl) have been associated with increased risk of invasive ERα+ tumors and poor long-term survival.14 Furthermore, over 95% of human breast cancers overexpress PrlR and human breast cancer cells have been shown to upregulate local synthesis of Prl.15 The primary transcription factor downstream of PrlR in mammary gland is Stat5a.16 However, in Stat1−/− tumors, pJak2, pStat3 and pStat5a/b were all detected with pStat3 and pStat5 being observed in a proportion of the tumor cells raising the possibility that they are either co-activated in a subset of cells or that they are activated in two discrete populations. This distinction may be critically important to understanding the perturbation of signaling pathways in these tumors. Previous studies demonstrated that breast tumors exhibiting both activated STAT3 and STAT5 were more differentiated than tumors with just pSTAT3.5 As only Stat5 engages with the PrlR in normal mammary gland during pregnancy, when Stat3 is present at high levels but is not phosphorylated, this suggests that active Jak2 is bound also to receptors that engage Stat3 such as gp130/LIFR in Stat1-deficient mammary cells. It is well established that both Stat1 and Stat3 are activated downstream of the common gp130 receptor chain but in many contexts, gp130 is preferentially bound by Stat3, so in this instance it is interesting that deficiency of Stat1 has a profound effect on levels of pStat3.17 Using knockdown or inhibition of Jak2, the authors demonstrated that Jak2 is required for phosphorylation of Stat3 and Stat5 and that persistent activation of the PrlR-Jak2-Stat3/5a/5b axis provides a potent survival signal to tumor-derived cells that have presumably become addicted to Stat3 and/or Stat5a/5b, as observed in other human tumors and cell lines.18 As these studies utilized cell lines, the requirement for PrlR signaling in vivo was investigated by treating Stat1−/− tumor-bearing animals with a Jak2 inhibitor. This blocked, as anticipated, pStat3 and pStat5 and resulted in an increase in cleaved caspase 3-positive cells and a remarkable and prolonged inhibition of tumor growth for 7 months following inhibitor withdrawal. Furthermore, Jak2 drives tumor growth in both hormone-dependent and hormone-independent Stat1−/− tumors. This interesting and important work provides a useful model for further studies on the initiation and progression of ERα+ breast tumors (Figure 1). JAK2 inhibitors are currently undergoing clinical trials and these could be a valuable addition to the clinician's armory for tamoxifen-resistant breast cancers. Also, given the demonstration in this manuscript of the prophylactic efficacy of a JAK2 inhibitor, women at risk for breast cancer could receive anti-JAK2 therapy. In this context, it is notable that Jak2 has been shown to be required for initiation but not the maintenance of mammary tumors driven by Prl overexpression,19 as genetic ablation of Jak2 before, but not after, neoplastic transformation abolished tumorigenesis. The discrepancy between these two studies suggests that signaling through Jak2, downstream of receptors other than PrlR in the Stat1−/− mice, results in tumors that are dependent on Jak2. Figure 1 Summary of outcomes downstream of the prolactin receptor and JAK2 in breast cancer and in normal mammary gland. During pregnancy, engagement of the prolactin receptor by its ligand prolactin results in phosphorylation of Stat5, which regulates alveologenesis ... Further work is required to determine whether inhibition or ablation of either Stat5 or Stat3 alone is sufficient to inhibit tumor growth or whether it is the combination of both of these Stats that is required to generate ERα+ tumors. Given the competition between Stat5 and Stat3 for binding to specific promoters, and their overall distinct sets of transcriptional targets, it will be informative to investigate whether relative levels of these Stats dictate the clinical outcome. This ERα+ breast tumor model described by the Schreiber laboratory further endorses the view that there is an intricate interplay between Janus kinases and their corresponding Stats during mammary tumorigenesis. The irrefutable conclusion that can be drawn from this work is that perturbing the delicate balance between the activities of JAK2, STAT1, STAT3 and STAT5 leads to breast cancer development and targeting this pathway provides opportunities for therapeutic intervention.

Mammary gland regression at the cessation of lactation (involution) is an exquisitely orchestrated process of cell death and tissue remodelling in which Stat3 signalling has an essential role. The involution microenvironment of the mammary gland is considered to be pro-tumourigenic and a proportion of cases of pregnancy-associated breast cancer are suggested to originate in tandem with involution. However, the apparent paradox that STAT3 is required for cell death in normal mammary gland, but is associated with breast cancer cell survival, has not been resolved. Herein, we investigate Stat3-mediated regulation of expression of members of the calcium-activated chloride channel regulator (CLCA) family of proteins during involution and mammary carcinogenesis. Using the conditionally immortal mammary epithelial cell line KIM-2, together with mice exhibiting mammary epithelial cell-specific deletion of Stat3 during lactation, we demonstrate that expression of mCLCA1 and mCLCA2 is elevated in concert with activation of Stat3. By contrast, murine CLCA5 (mCLCA5), the murine orthologue of human CLCA2, is significantly upregulated at 24, 72 and 96 h of involution in Stat3 knockout mice, suggesting a reciprocal regulation of these proteins by Stat3 in vivo. Interestingly, orthotopic tumours arising from transplantation of 4T1 murine mammary tumour cells exhibit both phosphorylated Stat3 and mCLCA5 expression. However, we demonstrate that expression is highly compartmentalized to distinct subpopulations of cells, and that Stat3 retains a suppressive effect on mCLCA5 expression in 4T1 tumour cells. These findings enhance our understanding of the regulation of CLCA channel expression both in vitro and in vivo, and in particular, demonstrate that expression of mCLCA1 and mCLCA2 during involution is profoundly dependent upon Stat3, whereas the relationship between mCLCA5 and Stat3 activity is reciprocal and restricted to different subpopulations of cells., KH was funded by the Medical Research Council (MRC) (project grant number G0900980) and is a co-applicant on MRC project grant number MR/K011014/1. MB was funded by a Department of Pathology PhD studentship. SP was funded by the MRC (project grant number MR/K011014/1). Work in CJW’s laboratory is funded by the MRC, NC3Rs and the Wellcome Trust

Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors.

Evidence is emerging that the mouse mammary epithelium is arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells to differentiated luminal and myoepithelial cells. The use of fluorescence-activated cell sorting (FACS) in combination with quantitative functional clonal assays represents a powerful tool for studying the properties of mouse mammary stem and progenitor cells. This chapter outlines the experimental procedures for generating single viable cell suspensions of mouse mammary epithelial cells, immunostaining cells for flow cytometry, in vitro assays for the detection and enumeration of mouse mammary progenitor cells, and in vivo assays for the detection and enumeration of mouse mammary stem cells.

The functions of members of the large family of transcriptional repressors, the KRAB domain zinc finger proteins, are not well described. We have identified a new member of this family, Zfp157, as a downstream target of the transcription factor Stat6 in mammary gland. Using a gene-trap approach, we have generated mice harboring a Zfp157-LacZ reporter gene. We have characterized the expression of this reporter during mouse embryogenesis and show that it is expressed in the epiblast and subsequently in a number of embryonic tissues including brain, ovary, intestine, kidney, lung, mammary gland, and hair follicle. In the adult, Zfp157 continues to be expressed in a wide range of tissues while specific patterns of reporter gene expression are apparent in the mammary gland, primarily in the basal epithelial cells of ducts and in the sebaceous glands of hair follicles. These data lay the foundation for further work on the function of Zfp157.

The mammary gland is unusual in that most development occurs in the adult. At birth, the gland comprises a rudimentary branched structure that elongates and branches during puberty to fill the fat pad. Ducts are comprised of a polarized bilayered epithelium of luminal and basal cells, the latter being primarily differentiated myoepithelium. At the onset of pregnancy, the alveolar lineage arises from progenitors that are stimulated to proliferate and generate lobuloalveolar structures, comprised of luminal and basal cells, which subsequently differentiate to produce milk and are thus essential for survival of the offspring (Hennighausen and Robinson 2005). These distinct epithelial cell types arise from a common multipotent stem cell (Shackleton et al. 2006; Stingl et al. 2006). Stat5a is essential for mammary gland development during pregnancy and formation of lobuloalveolar structures, as germline deletion of stat5a resulted in impaired alveologenesis and lactation failure (Liu et al. 1997). This lactation defect can be rescued in subsequent pregnancies by elevated expression of Stat5b (Liu et al. 1998). Consequently, deletion of both stat5a/b genes in mammary epithelium severely impairs alveologenesis (Cui et al. 2004), and this can be rescued by transgenic expression of Stat5a in stat5a/b-null mammary cells during pregnancy (Yamaji et al. 2009). The latter study demonstrated that Stat5a is required for the generation and/or expansion of alveolar luminal progenitor cells from mammary stem cells (Yamaji et al. 2009). The Ets transcription factor Elf5 has been suggested to be a master regulator of alveologenesis, as conditional deletion of Elf5 in both luminal and basal cells of the mammary gland resulted in a complete failure to develop the secretory epithelium (Choi et al. 2009). Conversely, overexpression of Elf5 in virgin mammary glands using an inducible transgenic model resulted in precocious alveologenesis and milk secretion (Oakes et al. 2008). This resulted in a diminution in number of luminal progenitor cells that express CD61 (β3 integrin), while in Elf5-null glands, CD61+ luminal progenitor cells accumulated, suggesting a block in differentiation. The connection between Stat5a and Elf5, both downstream targets of prolactin signaling, is not clear. Elf5 has been shown to bind to the proximal stat5a gene promoter in late pregnancy, and levels of Stat5a are down-regulated in Elf5-deficient mammary epithelial cells (Choi et al. 2009). However, Stat5a/b-null luminal progenitor cells do not express Elf5, and there are multiple consensus Stat-binding sites in the distal region of the Elf5 gene promoter (Yamaji et al. 2009), suggesting that there could be a positive regulatory loop between Stat5 and Elf5. Gata-3, which is expressed in the luminal cells, is required to specify and maintain the differentiation status of luminal epithelial cells (Kouros-Mehr et al. 2006; Asselin-Labat et al. 2007). Conditional deletion of Gata-3 during gestation resulted in detachment of cells from the basement membrane, resulting in cell death and lactation failure (Kouros-Mehr et al. 2006). CD61+ luminal progenitors also accumulated in Gata-3-null mammary glands (Asselin-Labat et al. 2007). Stat6 and IL-4/IL-13 are upstream regulators of Gata-3, and these signaling molecules are required for efficient lobuloalveologenesis, as there was a considerable delay in proliferation and differentiation in the absence of Stat6 or IL-4/IL-13 (Khaled et al. 2007). A microarray analysis of 5-d-gestation (5dG) mammary tissue revealed that the most up-regulated transcript in stat6−/− tissue was a previously undescribed gene, zinc finger protein 157 (zfp157). In this study, we explore the function of Zfp157 in alveologenesis and reveal that this transcriptional regulator controls the balance of luminal populations and is a critical mediator of Gata-3 signaling.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

Site-directed mutagenesis of the three binding sites for the mammary factor MPBF in the β-lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon γ-activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor-induced transcription factors.

Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development, and this pathway is frequently hyper-activated in cancer, including tumours of the breast. Stats 3, 5 and 6 have important roles in the differentiation and survival of mammary alveolar cells, but somewhat paradoxically, both Stat3 and 5 can have oncogenic activity in the mammary gland. Constitutive activation of JAK2 could be anticipated to result in hyper-activation of Stats 1, 3, 5 and 6 with concomitant cell transformation, although the outcome is difficult to envisage, particularly since Stats 3 and 5 play opposing roles in normal mammary gland development. Here, we show that expression of a constitutively active JAK2 mutant, JAK2 V617F, leads to hyper-activation of Stat5 in mammary epithelial cells (MECs), and transgenic mice expressing JAK2 V617F specifically in the mammary gland exhibit accelerated alveologenesis during pregnancy and delayed post-lactational regression. Overexpressing JAK2 V617F in MECs in vitro results in elevated proliferation and resistance to cell death. Furthermore, constitutively active JAK2 enhances anchorage-independent cell growth in the presence of a co-operating oncogene and accelerates tumourigenesis in a xenograft model. Taken together, our results provide insights into signalling downstream of constitutively active JAK2 and could be important for understanding the molecular mechanisms of breast tumourigenesis.

The pro-oncogenic transcription factor STAT3 is constitutively activated in a wide variety of tumours that often become addicted to its activity, but no unifying view of a core function determining this widespread STAT3-dependence has yet emerged. We show here that constitutively active STAT3 acts as a master regulator of cell metabolism, inducing aerobic glycolysis and down-regulating mitochondrial activity both in primary fibroblasts and in STAT3-dependent tumour cell lines. As a result, cells are protected from apoptosis and senescence while becoming highly sensitive to glucose deprivation. We show that enhanced glycolysis is dependent on HIF-1α up-regulation, while reduced mitochondrial activity is HIF-1α-independent and likely caused by STAT3-mediated down-regulation of mitochondrial proteins. The induction of aerobic glycolysis is an important component of STAT3 pro-oncogenic activities, since inhibition of STAT3 tyrosine phosphorylation in the tumour cell lines down-regulates glycolysis prior to leading to growth arrest and cell death, both in vitro and in vivo. We propose that this novel, central metabolic role is at the core of the addiction for STAT3 shown by so many biologically different tumours.

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

The transcription factor Stat3 is essential for timely initiation of post-lactational regression and orchestrates the processes of cell death and tissue remodelling that occur during the first 6 days of involution in the mouse. Paradoxically, STAT3 is also frequently found to be constitutively active in breast cancer and tumors can become addicted to STAT3. This raises two interesting questions: 1) do the high levels of active Stat3 present in the mammary epithelium during involution promote tumor spread and 2) how do tumor cells escape the pro-apoptotic effects of Stat3? In order to address these questions, it is essential to understand the role of Stat3 in involution and the mechanisms by which Stat3 regulates both cell death and tissue remodelling. A number of studies have been undertaken using genetically modified mice and microarray analyses and two significant findings arose from these investigations. Firstly, post-lactational regression is associated with an acute phase and inflammatory response in addition to cell death and secondly, Stat3 alone is insufficient to induce involution in the absence of the NF-kappaB regulatory kinase IKKbeta. Both Stat3 and NF-kappaB have been shown to regulate the expression of genes involved in inflammatory signalling and the acute phase response. These findings suggest a role for the innate immune response in mammary epithelial cell fate during involution and highlight potential roles for this response in tissue remodelling-associated breast cancer metastasis.

The Stat family of transcription factors have diverse roles in mammary gland development. Genetic studies in mice have revealed an essential requirement for Stat5a in development of secretory alveolar cells during pregnancy while Stat6, which is normally associated with differentiation of T helper cells, is important in the commitment of luminal cells to this alveolar lineage. In contrast, Stat3 is specifically activated at the initiation of post-lactational regression when it has an essential function in the regulation of cell death and tissue remodelling. Stat1 and Stat4 have been shown to be regulated during a mammary developmental cycle although whether they have specific, non-redundant roles is not clear. Thus, the adult mammary gland is somewhat unusual in that it is a tissue where different Stats are sequentially activated to orchestrate the processes of functional differentiation, cell death and tissue remodelling.

BackgroundThe STAT (Signal Transducers and Activators of Transcription) transcription factor family mediates cellular responses to a wide range of cytokines. Activated STATs (particularly STAT3) are found in a range of cancers. Further, STAT3 has anti-apoptotic functions in a range of tumour cell lines. After observing a proteolytic cleavage in STAT3α close to a potential apoptotic caspase protease cleavage site we investigated whether STAT3α might be a caspase substrate.MethodsSTAT3α status was investigatedin vitroin several cell systems:- HM-1 murine embryonic stem (ES) cells following various interventions; IOUD2 murine ES cells following induction to differentiate along neural or adipocyte lineages; and in a number of breast cancer cell lines. STAT3α status was also analysedin vivoin wild type murine mammary glands undergoing controlled, forced involution.ResultsImmunoblotting for STAT3α in HM-1 ES cell extracts detected amino and carboxy terminal species of approximately 48 kDa and 43 kDa respectively – which could be diminished dose-dependently by cell treatment with the nitric oxide (NO) donor drug sodium nitroprusside (SNP). UV irradiation of HM-1 ES cells triggered the STAT3α cleavage (close to a potential caspase protease cleavage site). Interestingly, the pan-caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-FMK) and the JAK2 tyrosine kinase inhibitor AG490 both inhibited cleavage dose-dependently, and cleavage was significantly lower in a heterozygousJAK2knockout ES cell clone. STAT3α cleavage also occurredin vivoin normal murine mammary glands undergoing forced involution, coinciding with a pulse of phosphorylation of residue Y705 on full-length STAT3α. Cleavage also occurred during IOUD2 ES cell differentiation (most strikingly along the neural lineage) and in several human breast cancer cell lines, correlating strongly with Y705 phosphorylation.ConclusionThis study documents a proteolytic cleavage of STAT3α into 48 kDa amino and 43 kDa carboxyl terminal fragments in a range of cell types. STAT3α cleavage occurs close to a potential caspase site, and can be inhibited dose-dependently by SNP, AG490 and z-VAD-FMK. The cleavage seems to be caspase-dependent and requires the phosphorylation of STAT3α at the Y705 residue. This highly regulated STAT3α cleavage may play an important role in modulating STAT3 transcriptional activity.

Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution.

Epithelial-to-mesenchymal transition (EMT) is an essential embryogenic and developmental process, characterized by altered cellular morphology, loss of cell adhesion, and gain of migratory ability. Dysregulation of this process has been implicated in tumorigenesis, mediating the acquisition of migratory and invasive phenotypes by tumor cells. Mammary epithelial cells provide an excellent model in which to study the process, being derived from mammary gland tissue that utilizes EMT to facilitate branching morphogenesis through which the developing gland migrates into and invades the fat pad. Inappropriate EMT has been heavily implicated in the progression of ductal hyperplasia and mammary tumor metastasis. We examined the morphological and molecular changes of three murine mammary epithelial cell lines following EMT induction. EMT was induced in the EpH-4 and NMuMG cell lines by transforming growth factor (TGF)-beta1 but not by ethanol, while the KIM-2 cell line was partially resistant to TGF-beta1 but responded fully to ethanol. The response to EMT-inducing reagent was shown to be critically dependent on the time of treatment, with confluent cells failing to respond. Timelapse photography identified increased motility during wound healing in cells pre-treated with EMT-inducing reagent compared with untreated controls. Furthermore, EMT conferred resistance to UV-induced apoptosis. Our data indicate that evaluation of characteristics other than loss and gain of phenotypic markers may be of benefit when assessing EMT, and contribute to the evidence suggesting that inappropriate EMT facilitates the acquisition of resistance to apoptosis, a key characteristic required for tumor survival.

Physiological apoptosis is induced by a switch from survival to death signalling. Dysregulation of this process is frequently associated with cancer. A powerful model for this apoptotic switch is mammary gland involution, during which redundant milk-producing epithelial cells undergo apoptosis. Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of this switch but the mechanism has not yet been defined. Stat3-dependent cell death during involution can be blocked by activation of Akt/protein kinase B (PKB), a downstream effector of the phosphoinositide-3-OH kinase (PI(3)K) pathway. Here we show that expression of the PI(3)K regulatory subunits p55alpha and p50alpha is induced by Stat3 during involution. In the absence of Stat3 in vivo, upregulation of p55alpha and p50alpha is abrogated, levels of activated Akt are sustained and apoptosis is prevented. Chromatin immunoprecipitation assays show that Stat3 binds directly to the p55alpha and p50alpha promoters in vivo. Overexpression of either p55alpha or p50alpha reduces levels of activated Akt. We propose a novel mechanism in which Stat3 regulates apoptosis by inducing expression of distinct PI(3)K regulatory subunits to downregulate PI(3)K-Akt-mediated survival signalling.

Large amounts of calcium are transferred to offspring by milk. This demand results in negative calcium balance in lactating mothers and is associated with rapid bone loss. The mechanisms of bone loss during lactation are only partly understood. Several studies have suggested that parathyroid hormone-related protein (PTHrP) might be secreted into the circulation by the lactating mammary gland and regulate bone turnover during lactation. Because mammary development fails in the absence of PTHrP, conventional PTHrP knockout mice cannot be used to address this possibility. To examine this hypothesis, we therefore used mice carrying a beta-lactoglobulin promoter-driven Cre transgene, one null PTHrP allele, and one floxed PTHrP allele. Expression of Cre specifically in mammary epithelial cells during late pregnancy and lactation resulted in efficient deletion of the PTHrP gene; mammary gland PTHrP mRNA and milk PTHrP protein were almost completely absent. Removal of PTHrP from the lactating mammary glands resulted in reductions in levels of circulating PTHrP and 1,25-dihydroxy vitamin D and urinary cAMP. In addition, bone turnover was reduced and bone loss during lactation was attenuated. We conclude that during lactation mammary epithelial cells are a source of circulating PTHrP that promotes bone loss by increasing rates of bone resorption.

Translational recoding of mRNA through a –1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of –1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of –1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.

The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo - suppression of premature epithelial apoptosis during mammary gland involution.

We have examined the effects of inhibition of the 26S proteasome in a murine mammary cell line, KIM-2 cells using the peptide aldehyde inhibitor MG132. These studies have demonstrated a clear requirement for proteasome function in cell viability. Induction of apoptosis was observed following MG132 treatment in KIM-2 cells and this death was shown to be dependent on the cell actively traversing the cell cycle. KIM-2 cells were generated using a temperature sensitive T-antigen (Tag) and studies at the permissive temperature (33 degrees C) have shown that a Tag binding protein was essential for this apoptotic response. Studies in two additional cell lines, HC11, which is a mammary epithelial cell line carrying mutant p53 alleles and p53 null ES cells suggest that p53 is actively required for the apoptosis induced as a consequence of proteasome inhibition. These results suggest a pivotal role for the 26S proteasome degradation pathway in progression through the cell cycle in proliferating cells.