Mouse Modeling Dissecting Macrophage-Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis.

Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor-associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL-4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro-tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.

Older Adults' Experiences of a Physical Activity and Sedentary Behaviour Intervention: A Nested Qualitative Study in the SITLESS Multi-Country Randomised Clinical Trial.

BACKGROUND: The SITLESS programme comprises exercise referral schemes and self-management strategies and has been evaluated in a trial in Denmark, Spain, Germany and Northern Ireland. The aim of this qualitative study was to understand the implementation and contextual aspects of the intervention in relation to the mechanisms of impact and to explore the perceived effects. METHODS: Qualitative methodologies were nested in the SITLESS trial including 71 individual interviews and 12 focus groups targeting intervention and control group participants from postintervention to 18-month follow-up in all intervention sites based on a semi-structured topic guide. RESULTS: Overarching themes were identified under the framework categories of context, implementation, mechanisms of impact and perceived effects. The findings highlight the perceived barriers and facilitators to older adults' engagement in exercise referral schemes. Social interaction and enjoyment through the group-based programmes are key components to promote adherence and encourage the maintenance of targeted behaviours through peer support and connectedness. Exit strategies and signposting to relevant classes and facilities enabled the maintenance of positive lifestyle behaviours. CONCLUSIONS: When designing and implementing interventions, key components enhancing social interaction, enjoyment and continuity should be in place in order to successfully promote sustained behaviour change.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

The Animal Lectin Galectin-8 Promotes Cytokine Expression and Metastatic Tumor Growth in Mice.

Secreted animal lectins of the galectin family are key players in cancer growth and metastasis. Here we show that galectin-8 (gal-8) induces the expression and secretion of cytokines and chemokines such as SDF-1 and MCP-1 in a number of cell types. This involves gal-8 binding to a uPAR/LRP1/integrin complex that activates JNK and the NFkB pathway. Cytokine and chemokine secretion, induced by gal-8, promotes migration of cancer cells toward cells treated with this lectin. Indeed, immune-competent gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for gal-8 transgenic animals. Accordingly, gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These results suggest the existence of a 'vicious cycle' whereby gal-8 secreted by the tumor microenvironment, promotes secretion of chemoattractants at the metastatic niche that promote further recruitment of tumor cells to that site. This study further implicate gal-8 in control of cancer progression and metastasis through its effects on the production of immunoregulatory cytokines.

HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2-IL-36γ Pathway.

The Nrf2 transcription factor is well known for its cytoprotective functions through regulation of genes involved in the detoxification of reactive oxygen species or toxic compounds. Therefore, activation of Nrf2 is a promising strategy for the protection of tissues from various types of insults and for cancer prevention. However, recent studies revealed a proinflammatory activity of activated Nrf2 and a stimulating effect on epithelial cell proliferation, but the underlying mechanisms of action and the responsible target genes are largely unknown. Using a combination of gene expression profiling, chromatin immunoprecipitation, and targeted proteomics via selected reaction monitoring, we show that the gene encoding the proinflammatory cytokine IL-36γ is a novel direct target of Nrf2 in keratinocytes and hepatocytes in vitro and in vivo. As a consequence, upregulation of IL-36γ expression occurred upon genetic or pharmacological activation of Nrf2 in the epidermis and in the normal and regenerating liver. Functional in vitro studies demonstrate that IL-36γ directly stimulates proliferation of keratinocytes. In particular, it induces expression of keratinocyte mitogens in fibroblasts, suggesting that the Nrf2-IL-36γ axis promotes keratinocyte proliferation through a double paracrine loop. These results provide mechanistic insight into Nrf2 action in the control of inflammation and cell proliferation through regulation of a proinflammatory cytokine with a key function in various inflammatory diseases.

NF-κB/RelA and Nrf2 cooperate to maintain hepatocyte integrity and to prevent development of hepatocellular adenoma.

BACKGROUND & AIMS: The liver is frequently challenged by toxins and reactive oxygen species. Therefore, hepatocytes require cytoprotective strategies to cope with these insults. Since the transcription factors Nrf2 and NF-κB regulate the cellular antioxidant defense system and important survival pathways, we determined their individual and overlapping functions in the liver. METHODS: We generated mice lacking Nrf2 and the NF-κB RelA/p65 subunit in hepatocytes and we analyzed their liver by using histopathology, immunohistochemistry, quantitative RT-PCR, Western blot and Oxyblot analysis. Human inflammatory hepatocellular adenomas (iHCA) were analyzed by immunohistochemistry. RESULTS: Loss of either Nrf2 or NF-κB/RelA had only a minor effect on liver homeostasis, but the double knockout mice spontaneously developed liver inflammation and fibrosis. Upon aging, more than one-third of the female double mutant mice developed tumors, which histologically resemble human iHCA, a tumor that predominantly occurs in women. The mouse tumors also recapitulated the immunohistochemical marker profile characteristic for human iHCA. Moreover, pNRF2 and NF-κB RelA/p65 was not detectable in the nuclei of iHCA tumor cells. The mouse phenotype was not due to a synergistic effect of both transcription factors on cytoprotective Nrf2 target genes. Rather, loss of Nrf2 or NF-κB/RelA altered the expression of different genes, and the combination of these alterations likely affects liver homeostasis in the double mutant mice. CONCLUSIONS: Our results provide genetic evidence for a functional cross-talk of Nrf2 and NF-κB/RelA in hepatocytes, which protects the liver from necrosis, inflammation and fibrosis. Furthermore, the double mutant mice represent a valuable animal model for iHCA.

Activation of the Nrf2-ARE pathway in hepatocytes protects against steatosis in nutritionally induced non-alcoholic steatohepatitis in mice.

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2- animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and ß-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2- and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model.

Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell-cycle control and apoptosis.

UNLABELLED: The nuclear factor erythroid-derived 2, like 2 (Nrf2) transcription factor is a key regulator of the antioxidant defense system, and pharmacological activation of Nrf2 is a promising strategy for prevention of toxin-induced liver damage. However, the consequences of Nrf2 activation on liver regeneration (LR) have not been determined. To address this question, we generated mice expressing a constitutively active Nrf2 (caNrf2) mutant in hepatocytes. Expression of the transgene did not affect liver homeostasis. Surprisingly, however, there was no beneficial effect of Nrf2 activation on CCl4 -induced liver injury and fibrosis. Most important, LR after partial hepatectomy was impaired in caNrf2-transgenic mice as a result of delayed hepatocyte proliferation and enhanced apoptosis of these cells after liver injury. Mechanistically, this involved up-regulation of the cyclin-dependent kinase inhibitor p15 and the proapoptotic protein Bcl2l11 (Bim). Using chromatin immunoprecipitation, we show that the p15 and Bcl2l11 genes are direct targets of Nrf2, which are activated under hyperproliferative conditions in the liver. CONCLUSION: Activated Nrf2 delays proliferation and induces apoptosis of hepatocytes in the regenerating liver. These negative effects of Nrf2 activation on LR should be considered when Nrf2-activating compounds are used for prevention of liver damage.

Fructose protects murine hepatocytes from tumor necrosis factor-induced apoptosis by modulating JNK signaling.

Fructose-induced hepatic ATP depletion prevents TNF-induced apoptosis, whereas it contrarily enhances CD95-induced hepatocyte apoptosis in vitro and in vivo. By contrast, transformed liver cells are not protected against TNF due to metabolic alterations, allowing selective tumor targeting. We analyzed the molecular mechanisms by which fructose modulates cytokine-induced apoptosis. A release of adenosine after fructose-induced ATP depletion, followed by a cAMP response, was demonstrated. Likewise, cAMP and adenosine mimicked per se the modulation by fructose of CD95- and TNF-induced apoptosis. The effects of fructose on cytokine-induced apoptosis were sensitive to inhibition of protein kinase A. Fructose prevented the pro-apoptotic, sustained phase of TNF-induced JNK signaling and thereby blocked bid-mediated activation of the intrinsic mitochondrial apoptosis pathway in a PKA-dependent manner. We explain the dichotomal effects of fructose on CD95- and TNF-induced cell death by the selective requirement of JNK signaling for the latter. These findings provide a mechanistic rationale for the protection of hepatocytes from TNF-induced cell death by pharmacological doses of fructose.

Regulation of liver regeneration by growth factors and cytokines.

The capability of the liver to fully regenerate after injury is a unique phenomenon essential for the maintenance of its important functions in the control of metabolism and xenobiotic detoxification. The regeneration process is histologically well described, but the genes that orchestrate liver regeneration have been only partially characterized. Of particular interest are cytokines and growth factors, which control different phases of liver regeneration. Historically, their potential functions in this process were addressed by analyzing their expression in the regenerating liver of rodents. Some of the predicted roles were confirmed using functional studies, including systemic delivery of recombinant growth factors, neutralizing antibodies or siRNAs prior to liver injury or during liver regeneration. In particular, the availability of genetically modified mice and their use in liver regeneration studies has unraveled novel and often unexpected functions of growth factors, cytokines and their downstream signalling targets in liver regeneration. This review summarizes the results obtained by functional studies that have addressed the roles and mechanisms of action of growth factors and cytokines in liver regeneration after acute injury to this organ.

The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis.

The liver is frequently exposed to insults, including toxic chemicals and alcohol, viral infection or metabolic overload. Although it can fully regenerate after acute injury, chronic liver damage causes liver fibrosis and cirrhosis, which can result in complete liver failure. In this study, we demonstrate that the NF-E2-related factor 2 (Nrf2) transcription factor protects the liver from acute and chronic toxin-mediated damage. Repair of the liver injury that occurs after a single treatment with the hepatotoxin carbon tetrachloride (CCl(4)) was severely delayed in Nrf2-deficient mice. The defect in repair was accompanied by an enhanced and prolonged inflammatory and profibrotic response. After long-term CCl(4) treatment, liver fibrosis was strongly aggravated in the Nrf2 knockout mice and inflammation was enhanced. We demonstrate that these abnormalities are at least in part due to the reduced expression of known and novel Nrf2 target genes in hepatocytes, which encode enzymes involved in the detoxification of CCl(4) and its metabolites. These results suggest that activation of Nrf2 may be a novel strategy to prevent or ameliorate toxin-induced liver injury and fibrosis.

Long-term survival benefit after adjuvant treatment of cutaneous melanoma with dacarbazine and low dose natural interferon alpha: A controlled, randomised multicentre trial.

In a prospective, controlled, randomised, multicentre study 252 patients with totally resected cutaneous melanoma (248 in stage II-III and 4 in stage IV) were either treated with two doses of dacarbazine (DTIC) followed by a 6-month treatment with 3 MU thrice weekly of highly purified natural interferon-alpha (n = 128; arm A) or received no adjuvant treatment (n = 124; arm B). Treatment was well tolerated. After a median follow-up of 8.5 years ITT analysis showed that the difference in survival was statistically significant with respect to melanoma-related deaths (HR = 0.65, CI = 0.46-0.97, p = 0.022) and close to significance with respect to overall survival (HR 0.71, CI 0.49-1.00, p = 0.052). The risk reduction of melanoma-associated death, calculated by Cox proportional hazards modelling, after adjusting for identified predictive variables, was almost 50% (p = 0.002). The overall efficacy of the treatment appeared to be mainly attributable to effects observed in patients with deep and/or metastasizing tumours (HR 0.60, CI 0.40-0.90, p = 0.013).

Perisynaptic Schwann cells of the vertebrate motor endplate bear modified cilia.

Perisynaptic Schwann cells (PSCs), descendants of the myelinating Schwann cells, cover the axon terminal of the vertebrate motor endplate of the skeletal muscle fiber. PSCs are assumed to support the function of the axon terminal. This function suggests a net material transport in the direction of the axon terminal. Morphologically it is to be expected that these cells have a cytoskeleton aligned to the axon terminal. Investigations clarifying this statement have not yet been undertaken. From previous investigations we know, however, that the PSCs have a microtubule-organizing center, which is a part of this cytoskeleton. The centrioles of the organizing center may also participate in the formation of a modified cilium structure whose function is unknown. In the present investigation, characteristic ultrastructural features of the modified cilium structure and its relationship to the Golgi apparatus and the axon terminal are presented. A function for the modified cilium structure is discussed.