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Key Points A number of alternative cell death programmes, such as necroptosis, lysosomal-mediated programmed cell death (LM-PCD) and autophagy have been established alongside classical apoptosis. These are now known to act both as a backup to apoptosis and also as preferred death pathways in certain cell types. Necroptosis can be triggered by a RIP1- and RIP3-containing complex. This complex can form downstream of death receptors or in the cytosol following stress stimuli. It is tightly regulated by the initiator caspase 8, as well as inhibitors of apoptosis (IAPs) and FLICE-like inhibitory protein (FLIP). Hence, caspase inhibitors, as well as second mitochondria-derived activator of caspases (SMAC) mimetics, are strong sensitizers for necroptosis. LM-PCD, which occurs because of a loss of lysosomal integrity, is frequently seen in cancer cells. This is due to increased metabolism and protein turnover, as well as a reduction of important lysosomal structural proteins. Cancer cells are thus particularly responsive to drugs that target the lysosomes, and drugs ranging from lysosomotropic agents to monoclonal antibodies and microtubule-disrupting agents have all been shown to induce LM-PCD. Autophagy can have both tumour suppressive and tumour-promoting activities. Large-scale autophagy can eventually lead to cell death; however, it is not clear precisely how this type of death is induced. Inhibition and induction of autophagy have both proved to be beneficial under certain circumstances, and the decision as to whether inhibition or activation is preferable is still largely empirical. The alternative pathways of cell death prove to be intricately interconnected with many points of convergence, such as JUN N-terminal kinase (JNK), AMP-activated protein kinase (AMPK) or reactive oxygen species (ROS). Necroptosis has LM-PCD as a frequent downstream occurrence, and an impaired lysosomal compartment affects the ability of autophagosomes to mature. These converging and diverging features are increasingly better understood, leading to a number of targeted approaches aimed at these alternative pathways. 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. This Review discusses converging and diverging features of these pathways with a view to developing new therapeutics for cancer. 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.

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.

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 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.

We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo . However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo . We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death. Lysosomal membrane permeabilization releases cathepsins to promote cell death and mammary gland involution. Sargeant et al.  report that Stat3-driven phagocytic uptake of fatty acids in milk triglycerides permeabilizes lysosomes to induce cell death.

Post-lactational involution in the mammary gland is shown to be accomplished by a lysosome-mediated cell death pathway. This pathway is independent of the executioner caspases 3, 6 and 7, and instead relies on Stat3-mediated upregulation of cathepsins. It is well established that lysosomes play an active role during the execution of cell death 1 . A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme 2 , 3 . However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions 4 , 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 respectively 1 , 9 , 10 .

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

Gpr125 is an orphan G-protein coupled receptor, with homology to cell adhesion and axonal guidance factors, that is implicated in planar polarity and control of cell movements. By lineage tracing we demonstrate that Gpr125 is a highly specific marker of bipotent mammary stem cells in the embryo and of multiple long-lived unipotent basal mammary progenitors in perinatal and postnatal glands. Nipple-proximal Gpr125+ cells express a transcriptomic profile indicative of chemo-repulsion and cell movement, whereas Gpr125+ cells concentrated at invasive ductal tips display a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix. Gpr125 progenitors acquire bipotency in the context of transplantation and cancer and are greatly expanded and massed at the pushing margins of short latency MMTV-Wnt1 tumors. High Gpr125 expression identifies patients with particularly poor outcome within the basal breast cancer subtype highlighting its potential utility as a factor to stratify risk. Gpr125 has emerged as a specific marker of mammary stem cells and basal progenitors. Here they show that Gpr125 cells congregate at ductal tips during morphogenesis and amass at tumor margins, and that high Gpr125 predicts early tumor onset and poor outcome in basal breast cancer.

Background The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the mammary epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development. We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained. Results One third (n = 102) of all murine miRNAs analysed were detected during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show changes, we observed inverse patterns of miRNA and target expression. The data sets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research. Conclusion MicroRNAs were expressed in likely co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation provide new avenues for research into mammary gland biology and generate candidates for functional validation.