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Cancer is a leading cause of death and a major health problem worldwide, particularly in more developed countries. There is, therefore, an urgent clinical need to develop more effective therapies to treat cancer and metastatic disease. In this Editorial, the content of The FEBS Journal's Special Issue on Cancer Therapeutics is outlined. The interesting collection of recent articles in this issue covers a wide repertoire of cancer therapeutic approaches. While some of the articles discuss broad-spectrum applications such as immunotherapy and oncolytic virus therapy, others focus on a particular type of cancer or a signalling pathway that has gone awry such as aberrant Ca2+ signalling, glycosylation or pre-mRNA processing. Finally, an article featured in this issue reviews our current understanding of how cancer cells can become dormant, often for decades, and which pathways reactivate these cells to cause relapse. I am sure there is something for everyone in this issue.

Christine J. Watson is Professor of Cell and Cancer Biology at the University of Cambridge. Christine obtained her Bachelor's (honors) degree in Biochemistry at the University of Glasgow in 1975 and, after a soujourn in Glauco Tocchini-Valentini's lab at the Institute of Cell Biology, Consiglio Nazionale delle Ricerche in Rome, she undertook a PhD in Molecular Genetics at Imperial College London. During her PhD, she looked at differences in gene expression between differentiated and undifferentiated embryonal carcinoma stem cells, inspiring an early interest in gene expression and cell fate determination. Between 1986 and 1992, Christine undertook three postdoctoral research positions that took her from London back to Scotland, where she was first introduced to mammary gland biology through her work with John Clark at the Roslin Institute in Edinburgh. During her time in the Clark lab, Christine identified a factor - later shown to be STAT5 - that binds to the promoter of the milk protein gene β-lactoglobulin. This prompted further work identifying the key role played by the STAT family of transcription factors in mammary gland development. Shortly afterwards, Christine became a group leader at the Roslin Institute and later relocated to the University of Edinburgh to collaborate with Andrew Wyllie. This led to her recruitment to the University of Cambridge in 1998, where she has remained to date. Over the last two decades, the Watson lab has focused on elucidating the mechanisms underlying lineage commitment of mammary stem and progenitor cells and the regulation of cell death in involuting mammary gland. In this interview, Christine discusses her research highlights and provides a glimpse into her personal interests, as she moves towards retirement.

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.

Funder: GlaxoSmithKline; funder-id: http://dx.doi.org/10.13039/100004330, Introduction: Delayed graft function (DGF) following renal transplantation is a manifestation of acute kidney injury (AKI) leading to poor long-term outcome. Current treatments have limited effectiveness in preventing DGF. Interleukin-18 (IL18), a biomarker of AKI, induces interferon-γ expression and immune activation. GSK1070806, an anti-IL18 monoclonal antibody, neutralizes activated (mature) IL18 released from damaged cells following inflammasome activation. This phase IIa, single-arm trial assessed the effect of a single dose of GSK1070806 on DGF occurrence post donation after circulatory death (DCD) kidney transplantation. Methods: The 3 mg/kg intravenous dose was selected based on prior studies and physiologically based pharmacokinetic (PBPK) modeling, indicating the high likelihood of a rapid and high level of IL18 target engagement when administered prior to kidney allograft reperfusion. Utilization of a Bayesian sequential design with a background standard-of-care DGF rate of 50% based on literature, and confirmed via extensive registry data analyses, enabled a statistical efficacy assessment with a minimal sample size. The primary endpoint was DGF frequency, defined as dialysis requirement ≤7 days post transplantation (except for hyperkalemia). Secondary endpoints included safety, pharmacokinetics and pharmacodynamic biomarkers. Results: GSK1070806 administration was associated with IL18-GSK1070806 complex detection and increased total serum IL18 levels due to IL18 half-life prolongation induced by GSK1070806 binding. Interferon-γ−induced chemokine levels declined or remained unchanged in most patients. Although the study was concluded prior to the Bayesian-defined stopping point, 4/7 enrolled patients (57%) had DGF, exceeding the 50% standard-of-care rate, and an additional two patients, although not reaching the protocol-defined DGF definition, demonstrated poor graft function. Six of seven patients experienced serious adverse events (SAEs), including two treatment-related SAEs. Conclusion: Overall, using a Bayesian design and extensive PBPK dose modeling with only a small sample size, it was deemed unlikely that GSK1070806 would be efficacious in preventing DGF in the enrolled DCD transplant population. Trial registration: NCT02723786.

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.

The signal transducer and activator of transcription (STAT) family of transcription factors have a spectrum of functions in mammary gland development. In some cases these roles parallel those of STATs in other organ systems, while in other instances the function of individual STATs in the mammary gland is specific to this tissue. In the immune system, STAT6 is associated with differentiation of T helper cells, while in the mammary gland, it has a fundamental role in the commitment of luminal epithelial cells to the alveolar lineage. STAT5A is required for the production of luminal progenitor cells from mammary stem cells and is essential for the differentiation of milk producing alveolar cells during pregnancy. By contrast, the initiation of regression following weaning heralds a dramatic and specific activation of STAT3, reflecting its pivotal role in the regulation of cell death and tissue remodeling during mammary involution. Although it has been demonstrated that STAT1 is regulated during a mammary developmental cycle, it is not yet determined whether it has a specific, non-redundant function. Thus, the mammary gland constitutes an unusual example of an adult organ in which different STATs are sequentially activated to orchestrate the processes of functional differentiation, cell death and tissue remodeling.

Sinus‐like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E‐cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14‐positive cells, supported by a thin layer of fibrous tissue. Multi‐segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3‐positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus‐like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus‐like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune 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.

Since seminal descriptions of signal transducer and activator of transcription 3 (STAT3) as a signal transducer and transcriptional regulator, which is most usually activated by phosphorylation of a specific tyrosine residue, a staggering wealth of research has delineated the key role of this transcription factor as a mediator of mammary gland postlactational regression (involution), and paradoxically, a pro-survival factor in breast cancer and some breast cancer cell lines. STAT3 is a critical regulator of lysosomal-mediated programmed cell death (LM-PCD) during mammary gland involution, where uptake of milk fat globules, and consequent high levels of free fatty acids, cause permeabilisation of lysosomal vesicle membranes, in turn leading to cathepsin protease leakage and cell death. A recent proteomic screen of STAT3-induced changes in lysosomal membrane protein components has highlighted wide-ranging effects of STAT3, which may coordinate LM-PCD via the stimulation of endocytosis, intracellular trafficking, and lysosome biogenesis. In parallel, STAT3 regulates the acute phase response during the first phase of involution, and it contributes to shaping the pro-tumourigenic ‘wound healing’ signature of the gland during the second phase of this process. STAT3 activation during involution is important across species, although some differences exist in the progression of involution in dairy cows. In breast cancer, a number of upstream regulators can lead to STAT3 activation and the effects of phosphorylation of STAT3 are equally wide-ranging. Recent studies have implicated microRNAs in some regulatory pathways. In this review, we will examine the multifaceted role of STAT3 in mammary gland involution and tumourigenesis, incorporating a review of these fundamental processes in tandem with a discussion of recent developments in this field.

The One Health concept promotes integrated evaluation of human, animal, and environmental health questions to expedite advances benefiting all species. A recognition of the multi-species impact of mastitis as a painful condition with welfare implications leads us to suggest that mastitis is an ideal target for a One Health approach. In this review, we will evaluate the role of the mammary microenvironment in mastitis in humans, ruminants and rabbits, where appropriate also drawing on studies utilising laboratory animal models. We will examine subclinical mastitis, clinical lactational mastitis, and involution-associated, or dry period, mastitis, highlighting important anatomical and immunological species differences. We will synthesise knowledge gained across different species, comparing and contrasting disease presentation. Subclinical mastitis (SCM) is characterised by elevated Na/K ratio, and increased milk IL-8 concentrations. SCM affecting the breastfeeding mother may result in modulation of infant mucosal immune system development, whilst in ruminants notable milk production losses may ensue. In the case of clinical lactational mastitis, we will focus on mastitis caused by Staphylococcus aureus and Escherichia coli. Understanding of the pathogenesis of involution-associated mastitis requires characterization of the structural and molecular changes occurring during involution and we will review these changes across species. We speculate that milk accumulation may act as a nidus for infection, and that the involution 'wound healing phenotype' may render the tissue susceptible to bacterial infection. We will discuss the impact of concurrent pregnancy and a 'parallel pregnancy and involution signature' during bovine mammary involution.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

The STAT family of transcription factors (signal transducers and activators of transcription) transduce signals from cytokine receptors to the nucleus, where STAT dimers bind to DNA and regulate transcription. STAT3 is the most ubiquitous of the STATs, being activated by a wide variety of cytokines and growth factors. STAT3 has many roles in physiological processes such as inflammatory signalling, aerobic glycolysis and immune suppression, and was also the first family member shown to be aberrantly activated in a wide range of both solid and liquid tumours. STAT3 promotes tumorigenesis by regulating the expression of various target genes, including cell-cycle regulators, angiogenic factors and anti-apoptosis genes. Paradoxically, in some circumstances, STAT3 signalling induces cell death. The best known example is the involuting mammary gland, where STAT3 is essential for induction of a lysosomal pathway of cell death. Nevertheless, direct silencing or inhibition of STAT3 diminishes tumour growth and survival in both animal and human studies. This suggests that abolishing STAT3 activity may be an effective cancer therapeutic strategy. However, despite this potential as a therapeutic target, and the extensive attempts by many laboratories and pharmaceutical companies to develop an effective STAT3 inhibitor for use in the clinic, no direct STAT3 inhibitor has been approved for clinical use. In this review, we focus on the role of STAT3 in tumorigenesis, and discuss its potential as a therapeutic target for cancer treatment.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Mammary gland involution involves a process that includes one of the most dramatic examples of cell death in an adult mammalian organism. We have previously shown that signal transducer and activator of transcription 3 (Stat3) regulates a lysosomal pathway of cell death in the first 48 h of involution and induces lysosome leakiness in mammary epithelial cells. Interestingly, Stat3 is associated also with the striking induction of autophagy that occurs concomitantly with cell death, presumably as a transient survival mechanism. The phosphatidylinositol 3-kinase regulatory subunits p55α and p50α are dramatically and specifically upregulated at the transcriptional level by Stat3 at the onset of involution. We show here that ablation of either Stat3 or p55α/p50α in vivo affects autophagy during involution. We used two different cell culture models (normal mammary epithelial cells and mouse embryonic fibroblasts) to further investigate the role of p55α/p50α in autophagy regulation. Our results demonstrate a direct role for p55α/p50α as inhibitors of autophagy mediated by p85α. Thus, Stat3 and its downstream targets p55α/p50α are key regulators of the balance between autophagy and cell death in vivo.

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.

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. genesis 51:179–186, 2013. © 2013 Wiley Periodicals, Inc.

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.

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.

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.

Evading programmed cell death is one of the hallmarks of cancer. Conversely, inducing cell death by pharmacological means is the basis of almost every non-invasive cancer therapy. Research over the past decade has greatly increased our understanding of non-apoptotic programmed cell death events, such as lysosomal-mediated cell death, necroptosis and cell death with autophagy. It is becoming clear that an intricate effector network connects many of these classical and non-classical death pathways. In this Review, we discuss converging and diverging features of these pathways, as well as attempts to exploit this newly gained knowledge pharmacologically to provide therapeutics for cancer.

Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.

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.

Mammary gland development occurs in three distinct stages during the lifetime of the female mammal: in embryonic, pubertal and reproductive life. At each of these developmental stages, different signalling molecules induce changes in both the epithelium and the surrounding stroma. However, it is during pregnancy that the most dramatic changes occur, resulting in a massive increase in the number of epithelial cells and in their function. Pregnancy initiates the development of a new epithelial lineage, the alveolar cells, which form the milk-producing lobuloalveolar structures. These cells become redundant at the end of lactation and are removed in an exquisitely controlled process of tissue remodelling coupled with extensive cell death. All of these events require not only steroid hormones but also sequential signalling by cytokines. A recent surprising discovery was that the signalling pathways and cytokines that regulate lineage determination in T helper cells are also involved in mammary gland development during pregnancy.

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.

The process of post-lactational regression, or involution, of the mammary gland is a complex event characterised by extensive death of the secretory epithelium coupled with remodelling of the extracellular matrix and adipogenesis to regenerate the fat pad. Associated with these events is an inflammatory cascade and acute phase response. The critical signalling pathways that regulated involution have been defined and a wide variety of genes have been shown to modulate the various processes involved, including cell death, phagocytosis, tissue remodelling and innate immune response.

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.

The mammary gland is a complex tissue comprised of a branching network of ducts embedded within an adipocyte-rich stroma. The ductal epithelium is a bi-layer of luminal and myoepithelial cells, the latter being in contact with a basement membrane. During pregnancy, tertiary branching occurs and lobuloalveolar structures, which produce milk during lactation, form in response to hormonal and cytokine signals. Postlactational regression is characterized by extensive cell death and tissue remodeling. These complex developmental events have been difficult to mimic in cell culture although many useful culture models exist. Recently, considerable advances in three-dimensional modelling of the mammary gland have been made with the use of collagen and other biomaterials for the study of branching morphogenesis and tumorigenesis, techniques which may enable rapid advances in our understanding of both basic biology and the study of cancer therapeutics.

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.

Breast cancer is a complex disease with many distinct subtypes being recognized on the basis of histological features and molecular signatures. It is difficult to predict how cancers will respond to therapy, which results in many women receiving unnecessary or inappropriate treatment. Advances in materials science and tissue engineering are leading the development of complex in vitro 3D breast tissue models that will increase our understanding of normal development and tumorigenic mechanisms. Ultimately, platforms that support primary tissue culture could readily be adapted to form high-throughput drug screening tools for personalized medicine. This review will summarize the control of mammary gland phenotype within in vitro 3D environments, in the context of a detailed analysis of mammary gland development and stem and progenitor cell controlled tumorigenesis.

Two members of the Stat family of transcription factors play a vital role in mouse mammary gland development. Stat5a was originally described as a regulator of milk protein gene expression and was subsequently shown to be essential for mammary development and lactogenesis. In contrast, Stat3 is an essential mediator of apoptosis and post-lactational regression. Other members of the Stat family may have specific, but as yet undemonstrated, functions in mammary development. However, since Stat1 activity is regulated during mammary development in a pattern different from Stats 3 and 5, this factor too may have a functional role. Although both Stat4 and Stat6 are expressed in mammary tissue, it seems unlikely that they will have a significant function as each of these Stats is activated in response to a limited number of cytokines. Given the essential regulatory roles of Stat signaling molecules in mammary development, it was not surprising to discover that constitutively activated Stat factors are a feature of human breast cancers. Sustained Stat activity has also been described in a variety of tumors including leukemias. The cause of this sustained activation is not clear but probably involves mutation of one of the many Stat regulatory proteins or dysregulation of other signaling pathways which modulate Stat activity. It is now important to understand the mechanism of constitutive Stat activity and to develop strategies which will abrogate aberrant Stat signaling in tumors in vivo.

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.

(2000). ‘Believe Me’: Acts of Witnessing in Aboriginal Women's Autobiographical Narratives. Journal of Australian Studies: Vol. 24, The Beautiful and the Damned, pp. 142-152.