Bcl-3 promotes multi-modal tumour cell migration via NF-κB1 mediated regulation of Cdc42.

A key challenge in the implementation of anti-metastatics as cancer therapies is the multi-modal nature of cell migration, which allows tumour cells to evade the targeted inhibition of specific cell motility pathways. The nuclear factor-kappaB (NF-κB) co-factor B-cell lymphoma 3 (Bcl-3) has been implicated in breast cancer cell migration and metastasis, yet it remains to be determined exactly which cell motility pathways are controlled by Bcl-3 and whether migrating tumour cells are able to evade Bcl-3 intervention. Addressing these questions and the mechanism underpinning Bcl-3's role in this process would help determine its potential as a therapeutic target. Here we identify Bcl-3 as an upstream regulator of the two principal forms of breast cancer cell motility, involving collective and single-cell migration. This was found to be mediated by the master regulator Cdc42 through binding of the NF-κB transcription factor p50 to the Cdc42 promoter. Notably, Bcl-3 depletion inhibited both stable and transitory motility phenotypes in breast cancer cells with no evidence of migratory adaptation. Overexpression of Bcl-3 enhanced migration and increased metastatic tumour burden of breast cancer cells in vivo, whereas overexpression of a mutant Bcl-3 protein, which is unable to bind p50, suppressed cell migration and metastatic tumour burden suggesting that disruption of Bcl-3/NF-κB complexes is sufficient to inhibit metastasis. These findings identify a novel role for Bcl-3 in intrinsic and adaptive multi-modal cell migration mediated by its direct regulation of the Rho GTPase Cdc42 and identify the upstream Bcl-3:p50 transcription complex as a potential therapeutic target for metastatic disease.

Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells.

The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.

Developmental programming mediated by complementary roles of imprinted Grb10 in mother and pup.

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk.

Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling.

The NF-κB co-factor Bcl3 is a proto-oncogene that promotes breast cancer proliferation, metastasis and therapeutic resistance, yet its role in breast cancer cell survival is unclear. Here, we sought to determine the effect of Bcl3 suppression alone on breast cancer cell viability, with a view to informing future studies that aim to target Bcl3 therapeutically. Bcl3 was suppressed by siRNA in breast cancer cell lines before changes in viability, proliferation, apoptosis and senescence were examined. Bcl3 suppression significantly reduced viability and was shown to induce apoptosis in all cell lines tested, while an additional p53-dependent senescence and senescence-associated secretory phenotype was also observed in those cells with functional p53. The role of the Bcl3/NF-κB axis in this senescence response was confirmed via siRNA of the non-canonical NF-κB subunit NFKB2/p52, which resulted in increased cellular senescence and the canonical subunit NFKB1/p50, which induced the senescence-associated secretory phenotype. An analysis of clinical data showed a correlation between reduced relapse-free survival in patients that expressed high levels of Bcl3 and carried a p53 mutation. Together, these data demonstrate a dual role for Bcl3/NF-κB in the maintenance of breast cancer cell viability and suggests that targeting Bcl3 may be more beneficial to patients with tumours that lack functional p53.

Development and Characterisation of a New Patient-Derived Xenograft Model of AR-Negative Metastatic Castration-Resistant Prostate Cancer.

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient's treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition.

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

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL.

INTRODUCTION: It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs, however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. METHODS: Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. RESULTS: Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. CONCLUSIONS: We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.

Evaluation of a fluorescent derivative of AMD3100 and its interaction with the CXCR4 chemokine receptor.

AMD3100 is a potent and selective antagonist of the CXCR4 receptor; it has been shown to block the route of entry of HIV into host T-cells. This compound and its analogues have since been found to act as haematopoietic stem cell mobilisation agents and, more recently, as anti-cancer agents. Here, we have examined a fluorescent derivative of AMD3100, L(1), which offered the potential to assess the behaviour of AMD3100 at the cell surface by using optical imaging modalities. The binuclear Zn(II) , Cu(II) and Ni(II) complexes of L(1) have also been investigated as these metals have been previously shown to enhance the binding properties of AMD3100. Furthermore, Zn(II) and Cu(II) are known to enhance and quench, respectively, the fluorescence of similar anthracenyl-based ligands. Whilst L(1) demonstrates an ability to inhibit the binding of the anti-CXCR4 monoclonal antibody 12G5 (IC(50) =0.25-0.9 µM), the incorporation of an anthracenyl moiety resulted in a significantly reduced affinity for CXCR4 compared to AMD3100 (IC(50) =10 nM). We observed no significant increase in fluorescence intensity following incubation with murine pre-B cells overexpressing CXCR4 compared to a control cell line. This limits the usefulness of L(1) as a fluorescent imaging probe. Interestingly, the Zn(II) complex, which carries an overall +4 charge, revealed marginally higher specificity and reduced toxicity in vitro compared to the free ligand, albeit with reduced affinity for CXCR4 (IC(50) =1.8-5 µM). We suggest that the incorporation of an anthracenyl group contributes to the lipophilic character of the free ligand, thereby resulting in transport across the plasma membrane. This effect is seemingly diminished when the ligand is complexed to charged metal ions.

Efficient Intravenous Tumor Targeting Using the αvß6 Integrin-Selective Precision Virotherapy Ad5NULL-A20.

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvß6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvß6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvß6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvß6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvß6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvß6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvß6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.

The Discovery of a Novel Antimetastatic Bcl3 Inhibitor.

The development of antimetastatic drugs is an urgent healthcare priority for patients with cancer, because metastasis is thought to account for around 90% of cancer deaths. Current antimetastatic treatment options are limited and often associated with poor long-term survival and systemic toxicities. Bcl3, a facilitator protein of the NF-κB family, is associated with poor prognosis in a range of tumor types. Bcl3 has been directly implicated in the metastasis of tumor cells, yet is well tolerated when constitutively deleted in murine models, making it a promising therapeutic target. Here, we describe the identification and characterization of the first small-molecule Bcl3 inhibitor, by using a virtual drug design and screening approach against a computational model of the Bcl3-NF-kB1(p50) protein-protein interaction. From selected virtual screening hits, one compound (JS6) showed potent intracellular Bcl3-inhibitory activity. JS6 treatment led to reductions in Bcl3-NF-kB1 binding, tumor colony formation, and cancer cell migration in vitro; and tumor stasis and antimetastatic activity in vivo, while being devoid of overt systemic toxicity. These results represent a successful application of in silico screening in the identification of protein-protein inhibitors for novel intracellular targets, and confirm Bcl3 as a potential antimetastatic target.

A dual role for oncostatin M signaling in the differentiation and death of mammary epithelial cells in vivo.

Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes beta-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed beta-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

BCL-3 promotes a cancer stem cell phenotype by enhancing ß-catenin signalling in colorectal tumour cells.

To decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with ß-catenin, it is perhaps surprising, given the importance of deregulated Wnt/ß-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of ß-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced ß-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes LGR5 and ASCL2 In contrast, the expression of canonical Wnt targets Myc and cyclin D1 remained unchanged. Furthermore, we show that BCL-3 increases the functional stem cell phenotype, as shown by colorectal spheroid and tumoursphere formation in 3D culture conditions. We propose that BCL-3 acts as a driver of the stem cell phenotype in CRC cells, potentially promoting tumour cell plasticity and therapeutic resistance. As recent reports highlight the limitations of directly targeting cancer stem cells (CSCs), we believe that identifying and targeting drivers of stem cell plasticity have significant potential as new therapeutic targets.This article has an associated First Person interview with the first author of the paper.

Identification of Pik3ca Mutation as a Genetic Driver of Prostate Cancer That Cooperates with Pten Loss to Accelerate Progression and Castration-Resistant Growth.

Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110α-dependent invasive prostate carcinoma in vivo Furthermore, we report that PIK3CA mutation and PTEN loss coexist in patients with prostate cancer and can cooperate in vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients.Significance: We show PIK3CA mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, PIK3CA mutation and PTEN loss coexist in prostate cancer and can cooperate in vivo to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. Cancer Discov; 8(6); 764-79. ©2018 AACR.See related commentary by Triscott and Rubin, p. 682This article is highlighted in the In This Issue feature, p. 663.

The genes induced by signal transducer and activators of transcription (STAT)3 and STAT5 in mammary epithelial cells define the roles of these STATs in mammary development.

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.

Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression.

INTRODUCTION: In order to gain a better understanding of the molecular processes that underlie apoptosis and tissue regression in mammary gland, we undertook a large-scale analysis of transcriptional changes during the mouse mammary pregnancy cycle, with emphasis on the transition from lactation to involution. METHOD: Affymetrix microarrays, representing 8618 genes, were used to compare mammary tissue from 12 time points (one virgin, three gestation, three lactation and five involution stages). Six animals were used for each time point. Common patterns of gene expression across all time points were identified and related to biological function. RESULTS: The majority of significantly induced genes in involution were also differentially regulated at earlier stages in the pregnancy cycle. This included a marked increase in inflammatory mediators during involution and at parturition, which correlated with leukaemia inhibitory factor-Stat3 (signal transducer and activator of signalling-3) signalling. Before involution, expected increases in cell proliferation, biosynthesis and metabolism-related genes were observed. During involution, the first 24 hours after weaning was characterized by a transient increase in expression of components of the death receptor pathways of apoptosis, inflammatory cytokines and acute phase response genes. After 24 hours, regulators of intrinsic apoptosis were induced in conjunction with markers of phagocyte activity, matrix proteases, suppressors of neutrophils and soluble components of specific and innate immunity. CONCLUSION: We provide a resource of mouse mammary gene expression data for download or online analysis. Here we highlight the sequential induction of distinct apoptosis pathways in involution and the stimulation of immunomodulatory signals, which probably suppress the potentially damaging effects of a cellular inflammatory response while maintaining an appropriate antimicrobial and phagocytic environment.

Bcl3 selectively promotes metastasis of ERBB2-driven mammary tumors.

Bcl3 is a putative proto-oncogene deregulated in hematopoietic and solid tumors. Studies in cell lines suggest that its oncogenic effects are mediated through the induction of proliferation and inhibition of cell death, yet its role in endogenous solid tumors has not been established. Here, we address the oncogenic effect of Bcl3 in vivo and describe how this Stat3-responsive oncogene promotes metastasis of ErbB2-positive mammary tumors without affecting primary tumor growth or normal mammary function. Deletion of the Bcl3 gene in ErbB2-positive (MMTV-Neu) mice resulted in a 75% reduction in metastatic tumor burden in the lungs with a 3.6-fold decrease in cell turnover index in these secondary lesions with no significant effect on primary mammary tumor growth, cyclin D1 levels, or caspase-3 activity. Direct inhibition of Bcl3 by siRNA in a transplantation model of an Erbb2-positive mammary tumor cell line confirmed the effect of Bcl3 in malignancy, suggesting that the effect of Bcl3 was intrinsic to the tumor cells. Bcl3 knockdown resulted in a 61% decrease in tumor cell motility and a concomitant increase in the cell migration inhibitors Nme1, Nme2, and Nme3, the GDP dissociation inhibitor Arhgdib, and the metalloprotease inhibitors Timp1 and Timp2. Independent knockdown of Nme1, Nme2, and Arhgdib partially rescued the Bcl3 motility phenotype. These results indicate for the first time a cell-autonomous disease-modifying role for Bcl3 in vivo, affecting metastatic disease progression rather than primary tumor growth.

Stat3 controls lysosomal-mediated cell death in vivo.

It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. 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.

Regulation of genes encoding proteolytic enzymes during mammary gland development.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

Microarray analysis of the involution switch.

Mammary epithelial cells (MEC) undergo a series of developmental decisions during a pregnancy cycle. The switches from proliferation to differentiation to secretion and then to cell death are precisely controlled. In order to identify critical changes associated with the transition from a secretory phenotype during lactation to dedifferentiation and cell death, we have undertaken a microarray analysis of mouse mammary gland development. We have focused on the involution switch and on the transcription profiles of genes that are targets of transcription factors known to influence involution and apoptosis. Our results show that both Stat3 and NF-kB target genes are induced by the involution switch while Stat5 target genes are distinct from Stat3 induced genes. Furthermore, a substantial number of genes that were specifically upregulated at the start of involution are regulators of inflammation and the acute phase response. These results provide a novel insight into the involution process and demonstrate the value of microarray analysis in defining molecular events associated with critical developmental transitions in mammary gland.

Gene structure of the human metabotropic glutamate receptor 5 and functional analysis of its multiple promoters in neuroblastoma and astroglioma cells.

The metabotropic glutamate receptor 5 (mGluR5) has a discrete tissue expression mainly limited to neural cells. Expression of mGluR5 is developmentally regulated and undergoes dramatic changes in association with neuropathological disorders. We report the complete genomic structure of the mGluR5 gene, which is composed of 11 exons and encompasses approximately 563 kbp. Three clusters of multiple transcription initiation sites located on three distinct exons (IA, IB, and II), which undergo alternative splicing, have been identified. The 5'-flanking regions of these exons were isolated and, using a luciferase reporter gene assay, shown to possess active promoter elements in SKN-MC neuroblastoma and U178-MG astroglioma cells. Promoter IA was characterized by a CpG island; promoter IB contained a TATA box, and promoter II possessed three active Oct-1-binding sites. Preferential luciferase activity was observed in SKN-MC concomitant with differential DNA binding activity to several responsive elements, including CREB, Oct-1, C/EBP, and Brn-2. Exposure to growth factors produced enhanced expression of promoters IB and II in astroglioma cells and activation of NF-kappa B. These results suggest that alternative 5'-splicing and usage of multiple promoters may contribute regulatory mechanisms for tissue- and context-specific expression of the mGluR5 gene.