Katherine Hughes

Conditional deletion of Stat3 in mammary epithelium impairs the acute phase response and modulates immune cell numbers during post-lactational regression.

Mammary gland regression following weaning (involution) is associated with extensive cell death and the acquisition of an inflammatory signature. Characterizing 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 also influences 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

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Significance Infectious diseases are responsible for one-third of all mortality worldwide. Innate immunity is critical for mounting host defenses that eliminate pathogens. Salmonella is a global food-borne pathogen that infects and replicates within macrophages. How inflammasomes—multimeric protein complexes that provide innate immune protection—function to restrict bacterial burden in macrophages remains unknown. We show that actin polymerization is critical for NLRC4 inflammasome activation in response to Salmonella infection. NLRC4 activation in Salmonella-infected cells prevents further uptake of bacteria by inducing cellular stiffness and antimicrobial responses, which prevent bacterial dissemination in the host. These results demonstrate a critical link between innate immunity and the actin cytoskeleton in the cellular defense against Salmonella infection. Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Toll-like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection, but not for the initiation of bacterial clearance

Toll‐like receptor‐4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4‐dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF‐related adaptor molecule (TRAM)/TIR‐domain‐containing adaptor‐inducing interferon‐b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF−/− mice have a higher bacterial load in comparison with wild‐type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro‐inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4−/−, MyD88−/− and TRIF−/− mice in vitro. Pro‐inflammatory responses from Mal−/− macrophages were similar to those from wild‐type cells. The pro‐inflammatory responses of TRIF−/− macrophages were partially restored by the addition of interferon‐γ (IFN‐γ), and TRIF−/− mice produced markedly enhanced IFN‐γ levels, in comparison to wild‐type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4−/−, MyD88−/−, TRIF−/− and Mal−/− mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

The spectrum of STAT functions in mammary gland development.

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.

Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress

Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM–ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy.

Overexpression of the oncostatin-M receptor in cervical squamous cell carcinoma is associated with epithelial–mesenchymal transition and poor overall survival

Background:Copy-number gain of the oncostatin-M receptor (OSMR) occurs frequently in cervical squamous cell carcinoma (SCC) and is associated with adverse clinical outcome. We previously showed that OSMR overexpression renders cervical SCC cells more sensitive to the major ligand oncostatin-M (OSM), which increases migration and invasion in vitro. We hypothesised that a major contribution to this phenotype would come from epithelial–mesenchymal transition (EMT).Methods:We performed a comprehensive integrated study, involving in vitro cell line studies, in vivo animal models and numerous clinical samples from a variety of anatomical sites.Results:In independent sets of cervical, head/neck and lung SCC tissues, OSMR expression levels correlated with multiple EMT-associated phenotypic markers and transcription factors. OSM treatment of OSMR overexpressing cervical SCC cells produced consistent EMT changes and increased tumour sphere formation in suspension culture. In a mouse model, OSMR overexpressing SCC cells treated with OSM showed significant increases in lung colonisation. The biological effects of exogenous OSM were mirrored by highly significant adverse overall survival in cervical SCCs with OSMR overexpression (N=251).Conclusions:OSM:OSMR interactions are able to induce EMT, increased cancer stem cell-like properties and enhanced lung colonisation in SCC cells. These changes are likely to contribute to the highly significant adverse outcome associated with OSMR overexpression in cervical SCCs.

Estrogen Receptor and Signal Transducer and Activator of Transcription 3 Expression in Equine Mammary Tumors

Equine mammary tumors are uncommon, and relatively sparse histopathologic and molecular data exist. The present study describes the histopathologic features of 7 such tumors, which exhibited infiltrative growth, intermediate to high mitotic rates, and focally extensive necrosis. The tumors exhibited variably strong staining for vimentin and cytokeratin 14, as well as frequently weak cytoplasmic staining for pan-cytokeratin. E-cadherin expression was strong. Interestingly, a subgroup of the tumors exhibited strong nuclear staining for estrogen receptor α. Three of 7 tumors exhibited nuclear expression of the transcription factor STAT3, suggesting that STAT3 was transcriptionally active. Rare to absent nuclear STAT3 expression was observed in carcinomas exhibiting moderate to intense staining for cytokeratin 14. This investigation confirms previous investigators’ assertions that equine mammary tumors have a malignant phenotype. A subset of the equine mammary tumors exhibited estrogen receptor α expression, suggesting that these tumors may potentially have similar molecular characteristics to their feline and canine counterparts.

Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue.

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.

Breast cancer: the menacing face of Janus kinase

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 clinicians 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.

The role of Stat3 in mammary gland involution: cell death regulator and modulator of inflammation.

Abstract Mammary gland regression post-weaning (involution) is a highly regulated, complicated process in which the transcription factor Stat3 is a key player. Over the last decade, microarray analyses have had a profound impact on our understanding of this role. Studies using mammary epithelial cells in which Stat3 was activated in a ligand-independent manner have allowed direct transcriptional targets of Stat3 to be identified. Additionally, global gene expression changes during involution have been profiled by microarray analyses, which allowed characterization of clusters of genes with distinct expression profiles during the first 4 days of involution. Such expression profiling led to the observation that one of the most strikingly upregulated genes in the absence of Stat3 is the serpin Spi2a. This led to the discovery that mammary epithelial cell lysosomes undergo lysosomal membrane permeablisation and leak cathepsins during involution. Stat3 upregulates the expression of cathepsins B and L within 24 h of weaning and is thus the critical inducer of lysosomal-mediated cell death during this process. In addition to its pivotal role in the control of cell death during involution, microarray-based studies have demonstrated that the expression of acute phase and inflammatory genes is regulated by Stat3 and that mammary epithelial expression of this transcription factor modulates the phenotype of macrophages present in the gland during second phase remodelling. Thus, Stat3 signalling may have effects that are not cell-autonomous, in addition to its cell-autonomous role in involution.