Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

Genetic ablation of Tnfα demonstrates no detectable suppressive effect on inflammation-related mouse colon tumorigenesis

Colorectal cancer (CRC) is one of the most serious complications of inflammatory bowel disease. Tumor necrosis factor-α (Tnfα) is a major mediator of inflammation and there is increasing evidence that Tnfα/Tnf-receptor-1 (Tnfr1) signaling may act as an endogenous tumor promoter for colon carcinogenesis. In fact, a previous study revealed that mice lacking Tnfr1 develop significantly fewer colonic tumors in the inflammation-related CRC model. In addition, antibodies against Tnfα have been shown to inhibit the development of inflammation-related CRC. In the present study, Apc Min/+; Tnfα ?/? mice were treated with 2% dextran sodium sulfate (DSS) and the tumor development was compared with Apc Min/+; Tnfα +/+ control mice in order to investigate the role of Tnfα by itself in the inflammation-related CRC. Surprisingly, there were no detectable differences in either the severity of colonic inflammation or the expression of DSS-induced chemokines and cytokines (Ccl2, Cxcl1, Tnfβ, Il1β, Il6, and Cox-2) that relate to the colonic inflammation and tumorigenesis between these two groups. Furthermore, the genetic ablation of Tnfα did not suppress the colon tumorigenesis in comparison to the wild-type mice. Our observations suggest that intricate inflammatory responses promote the inflammation-related mouse colon tumorigenesis.     

Colonic inflammation accompanies an increase of β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of high-fat diet-fed mice

Consumption of an obesigenic/high-fat diet (HFD) is associated with a high colon cancer risk and may alter the gut microbiota. To test the hypothesis that long-term high-fat (HF) feeding accelerates inflammatory process and changes gut microbiome composition, C57BL/6 mice were fed HFD (45% energy) or a low-fat (LF) diet (10% energy) for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in body fat composition, inflammatory cell infiltration, inducible nitric oxide synthase protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, β-catenin expression was increased in colon (but not ileum). Consistent with gut inflammation phenotype, we also found that plasma leptin, interleukin 6 and tumor necrosis factor α concentrations were also elevated in mice fed the HFD, indicative of chronic inflammation. Fecal DNA was extracted and the V1–V3 hypervariable region of the microbial 16S rRNA gene was amplified using primers suitable for 454 pyrosequencing. Compared to the LF group, the HF group had high proportions of bacteria from the family Lachnospiraceae/Streptococcaceae, which is known to be involved in the development of metabolic disorders, diabetes and colon cancer. Taken together, our data demonstrate, for the first time, that long-term HF consumption not only increases inflammatory status but also accompanies an increase of colonic β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of C57BL/6 mice.     

Cox-2 expression, PGE2 and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE₂ production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1β, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE₂ and cytokine production.     

Dietary red raspberries attenuate dextran sulfate sodium-induced acute colitis

Persistent intestinal inflammation severely impairs intestinal integrity resulting in inflammatory bowel disease. Red raspberries (RB) are a rich source of bioactive compounds; their beneficial effect on the colitis protection was evaluated in the current study using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. Six-week-old mice were fed a standard rodent research diet supplemented with RB (0 or 5% w/w, n=20 each group) for 6 weeks. At the 4th week of dietary treatment, approximately half of mice in each dietary group (n=12 each group) were subjected to 2.5% DSS induction for 6 days, followed by 6 days of recovery, to induce colitis. RB supplementation decreased body weight loss (P≤.01), disease activity index (P≤.01), and colon shortening (P≤.05) in DSS-treated mice. In addition, RB supplementation protected the colonic structure (P≤.01), associated with suppressed NF-κB signaling and reduced expression of inflammatory interleukin (IL)-1β, IL-6, IL-17, cyclooxegenase-2, and tumor necrosis factor-α in DSS-treated mice. RB supplementation reduced neutrophil infiltration, monocyte chemoattractant protein-1 mRNA expression, and xanthine oxidase content, but enhanced catalase content in DSS-treated mice. Consistently, RB supplementation reduced pore forming tight junction protein claudin-2, increased barrier strengthening claudin-3, zonula occluden-1 protein content and mucin (MUC)-2 mRNA level, and activated AMP-activated protein kinase (AMPK) in DSS-treated mice. In conclusion, dietary RB protected against inflammation and colitis symptoms induced by DSS, providing a promising dietary approach for the management of colitis.     

Intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc(Min/+) mouse

The etiology of colon cancer is a complex phenomenon that involves both genetic and environmental factors. However, only about 20% have a familial basis with the largest fraction being attributed to environmental causes that can lead to chronic inflammation. While the link between inflammation and colon cancer is well established, the temporal sequence of the inflammatory response in relation to tumorigenesis has not been characterized. We examined the timing and magnitude of the intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc^Min/+ mouse. Apc^Min/+ mice and wildtype mice were sacrificed at one of 4 time-points: 8, 12, 16, and 20 weeks of age. Intestinal tissue was analyzed for polyp burden (sections 1, 4 and 5) and mRNA expression and protein concentration of MCP-1, IL-1β, IL-6 and TNF-α (sections 2 and 3). The results show that polyp burden was increased at 12, 16 and 20 weeks compared to 8 weeks (P < 0.05). Gene expression (mRNA) of MCP-1, IL-1β, IL-6 and TNF-α was increased in sections 2 and 3 starting at week 12 (P < 0.05), with further increases in MCP-1, IL-1β and IL-6 at 16 weeks (P < 0.05). Protein concentration for these cytokines followed a similar pattern in section 3. Similarly, circulating MCP-1 was increased at 12 weeks (P < 0.05) and then again at 20 weeks (P < 0.05). In general, overall polyp number and abundance of large polyps were significantly correlated with the inflammatory cytokine response providing further support for a relationship between polyp progression and these markers. These data confirm the association between intestinal cytokines and tumorigenesis in the Apc^Min/+ mouse and provide new information on the timing and magnitude of this response in relation to polyp development. These findings may lead to the development of inflammatory mediators as important biomarkers for colon cancer progression. Further, these data may be relevant in the design of future investigations of therapeutic interventions to effectively target inflammatory processes in rodent models.     

IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice

IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca²⁺/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2^-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2^-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2^-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.     

Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota

The gut microbiota has been proposed as an environmental factor that affects the development of metabolic and inflammatory diseases in mammals. Recent reports indicate that gut bacteria-derived lipopolysaccharide (LPS) can initiate obesity and insulin resistance in mice; however, the molecular interactions responsible for microbial regulation of host metabolism and mediators of inflammation have not been studied in detail. Hepatic serum amyloid A (SAA) proteins are markers and proposed mediators of inflammation that exhibit increased levels in serum of insulin-resistant mice. Adipose tissue-derived SAA3 displays monocyte chemotactic activity and may play a role in metabolic inflammation associated with obesity and insulin resistance. To investigate a potential mechanistic link between the intestinal microbiota and induction of proinflammatory host factors, we performed molecular analyses of germ-free, conventionally raised and genetically modified Myd88−/− mouse models. SAA3 expression was determined to be significantly augmented in adipose (9.9±1.9-fold; P<0.001) and colonic tissue (7.0±2.3-fold; P<0.05) by the presence of intestinal microbes. In the colon, we provided evidence that SAA3 is partially regulated through the Toll-like receptor (TLR)/MyD88/NF-kappaB signaling axis. We identified epithelial cells and macrophages as cellular sources of SAA3 in the colon and found that colonic epithelial expression of SAA3 may be part of an NF-kappaB-dependent response to LPS from gut bacteria. In vitro experiments showed that LPS treatments of both epithelial cells and macrophages induced SAA3 expression (27.1±2.5-fold vs. 1.6±0.1-fold, respectively). Our data suggest that LPS, and potentially other products of the indigenous gut microbiota, might elevate cytokine expression in tissues and thus exacerbate chronic low-grade inflammation observed in obesity.     

Adequacy of calcium and vitamin D reduces inflammation, β-catenin signaling,and dysbiotic Parasutterela bacteria in the colon of C57BL/6 mice fed a western-style diet

Adoption of an obesogenic diet low in calcium and vitamin D (CaD) leads to increased obesity, colonic inflammation, and cancer. However, the underlying mechanisms remain to be elucidated. We tested the hypothesis that CaD supplementation (from inadequacy to adequacy) may reduce colonic inflammation, oncogenic signaling, and dysbiosis in the colon of C57BL/6 mice fed a Western diet. Male C57/BL6 mice (4-weeks old) were assigned to 3 dietary groups for 36 weeks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). Compared to the AIN, mice receiving the NWD or NWD/CaD exhibited more than 0.2-fold increase in the levels of plasma leptin, tumor necrosis factor α (TNF-α) and body weight. The levels of plasma interleukin 6 (IL-6), inflammatory cell infiltration, and β-catenin/Ki67 protein (oncogenic signaling) were increased more than 0.8-fold in the NWD (but not NWD/CaD) group compared to the AIN group. Consistent with the inflammatory phenotype, colonic secondary bile acid (inflammatory bacterial metabolite) levels increased more than 0.4-fold in the NWD group compared to the NWD/CaD and AIN groups. Furthermore, the abundance of colonic Proteobacteria (e.g., Parasutterela), considered signatures of dysbiosis, was increased more than four-fold; and the α diversity of colonic bacterial species, indicative of health, was decreased by 30% in the NWD group compared to the AIN and NWD/CaD groups. Collectively, CaD adequacy reduces colonic inflammation, β-catenin oncogenic signaling, secondary bile acids, and bacterial dysbiosis in mice fed with a Western diet.     

Dietary inflammatory index and inflammatory gene interactions in relation to colorectal cancer risk in the Bellvitge colorectal cancer case–control study

Chronic inflammation is an important factor in colorectal carcinogenesis. However, evidence on the effect of pro-inflammatory and anti-inflammatory foods and nutrients is scarce. Moreover, there are few studies focusing on diet–gene interactions on inflammation and colorectal cancer (CRC). This study was designed to investigate the association between the novel dietary inflammatory index (DII) and CRC and its potential interaction with polymorphisms in inflammatory genes. Data from the Bellvitge Colorectal Cancer Study, a case–control study (424 cases with incident colorectal cancer and 401 hospital-based controls), were used. The DII score for each participant was obtained by multiplying intakes of dietary components from a validated dietary history questionnaire by literature-based dietary inflammatory weights that reflected the inflammatory potential of components. Data from four important single nucleotide polymorphisms located in genes thought to be important in inflammation-associated CRC: i.e., interleukin (IL)-4, IL-6, IL-8, and peroxisome proliferator-activated receptor-γ (PPARG) were analyzed. A direct association was observed between DII score and CRC risk (OR_{Q4 vs. Q1} 1.65, 95 % CI 1.05–2.60, and P trend 0.011). A stronger association was found with colon cancer risk (OR_{Q4 vs. Q1} 2.24, 95 % CI 1.33–3.77, and P trend 0.002) than rectal cancer risk (OR_{Q4 vs. Q1} 1.12, 95 % CI 0.61–2.06, and P trend 0.37). DII score was inversely correlated with SNP rs2243250 in IL-4 among controls, and an interaction was observed with CRC risk. Neither correlation nor interaction was detected for other inflammatory genes. Overall, high-DII diets are associated with increased risk of CRC, particularly for colon cancer, suggesting that dietary-mediated inflammation plays an important role in colorectal carcinogenesis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0447-x) contains supplementary material, which is available to authorized users.     

Worm Proteins of Schistosoma mansoni Reduce the Severity of Experimental Chronic Colitis in Mice by Suppressing Colonic Proinflammatory Immune Responses

Although helminthic therapy as a possible new option to treat inflammatory bowel disease is a well-established concept by now, the search for immunomodulatory helminth-derived compounds and their mechanisms of action is still ongoing. We investigated the therapeutic potential and the underlying immunological mechanisms of Schistosoma mansoni soluble worm proteins (SmSWP) in an adoptive T cell transfer mouse model of chronic colitis. Both a curative and a preventive treatment protocol were included in this study. The curative administration of SmSWP (started when colitis was established), resulted in a significant improvement of the clinical disease score, colonoscopy, macroscopic and microscopic inflammation score, colon length and myeloperoxidase activity. The therapeutic potential of the preventive SmSWP treatment (started before colitis was established), was less pronounced compared with the curative SmSWP treatment but still resulted in an improved clinical disease score, body weight loss, colon length and microscopic inflammation score. Both the curative and preventive SmSWP treatment downregulated the mRNA expression of the proinflammatory cytokines IFN-γ and IL-17A and upregulated the mRNA expression of the anti-inflammatory cytokine IL-4 in the colon at the end of the experiment. This colonic immunomodulatory effect of SmSWP could not be confirmed at the protein level. Moreover, the effect of SmSWP appeared to be a local colonic phenomenon, since the flow cytometric T cell characterization of the mesenteric lymph nodes and the cytokine measurements in the serum did not reveal any effect of SmSWP treatment. In conclusion, SmSWP treatment reduced the severity of colitis in the adoptive transfer mouse model via the suppression of proinflammatory cytokines and the induction of an anti-inflammatory response in the colon.     

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E–BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR = 0.49; 95% CI, 0.25–0.95; P = .036) and in tumor cells (HR = 0.37; 95% CI, 0.18–0.79; P = .010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells.     

R-Spondins Are Expressed by the Intestinal Stroma and are Differentially Regulated during Citrobacter rodentium- and DSS-Induced Colitis in Mice

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45^- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.     

Vitexin prevents colitis-associated carcinogenesis in mice through regulating macrophage polarization

BackgroundPatients with inflammatory bowel disease are at increased risks of developing ulcerative colitis-associated colorectal cancer (CAC). Vitexin can suppress the proliferation of colorectal carcinoma cells in vitro orin vivo. However, different from colorectal carcinoma, CAC is more consistent with the transformation from inflammation to cancer in clinical chronic IBD patients. Therefore, we aim to investigated that vitexin whether possess benefic effects on CAC mice.PurposeWe aimed to determine the beneficial effects of vitexin on CAC mice and reveal its underlying mechanism.MethodsThe mouse CAC model was induced by Azoxymethane and dextran sodium sulfate (AOM/DSS) and CAC mice were treated with vitexin. At the end of this study, inflammatory cytokines of IL-1β, IL-6, TNF-α, IL-10 as well as nitric oxide (NO) were detected by kits after long-term treatment of vitexin. Pathological changes and macrophage polarization were determined by H&E and immunofluorescence in adjacent noncancerous tissue and carcinomatous tissue respectively of CAC mice.ResultsOur results showed that oral administration of vitexin could significantly improve the clinical signs and symptoms of chronic colitis, relieve colon damage, regulate colonic inflammatory cytokines, as well as suppress tumor incidence and tumor burden. Interesting, vitexin caused a significant increase in serum level of NO and a higher content of NO in tumor tissue. In addition, vitexin significantly decreased M1 phenotype macrophages in the adjacent noncancerous tissue, while markedly up-regulated M1 macrophage polarization in the tumor tissue in the colon of CAC mice.ConclusionVitexin can attenuate chronic colitis-associated carcinogenesis induced by AOM/DSS in mice and its protective effects are partly associated with its alternations in macrophage polarization in the inflammatory and tumor microenvironment .     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.PIK3CA¹, the gene encoding the p110α catalytic subunit of phosphatidylinositide-3 kinase (PI3K), is one of the two most frequently mutated genes in breast cancer. Approximately 80% of these mutations occur in two hot spots in the helical domain (E545K, E542K) and in the catalytic domain (H1047R). PIK3CA activating mutations occur in ∼40% of luminal and HER2-enriched breast cancer subtypes and ∼10% of basal-like breast cancer (BLBC) (1). In this last tumor subtype, mutations in PIK3CA are the most frequent activating kinase mutation. Thus, understanding of how PIK3CA mutations operate in BLBC is important for identifying therapeutic targets in this subtype of the disease, which lacks approved targeted therapies.To elucidate mechanisms by which mutant PIK3CA transforms MECs, we used immortalized, nontumorigenic MCF10A cells, which exhibit basal-like gene expression. Although MCF10A cells require growth factors for proliferation (2), heterozygous knock-in of E545K or H1047R PIK3CA mutation allows growth factor-independent proliferation (3). These knock-in PIK3CA mutant MECs provide a robust model in which to study the impact of these mutations without the effects of random insertion and overexpression associated with ectopic gene transduction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of these cells identified 72 proteins concordantly altered by both PIK3CA mutations. A significant fraction of these were secreted proteins, cell surface receptors or ECM interacting molecules, suggesting PIK3CA mutations induce changes involving communication with the tumor microenvironment. This analysis identified a PI3K-induced amphiregulin (AREG)-EGFR-ERK signaling pathway that was required for growth of PIK3CA-mutant cells as well as adjacent PIK3CA-WT cells. In addition, these protein changes correlated with poor clinical outcome in BLBC. EGFR antagonists inhibited growth of PIK3CA mutant BLBC tumors, suggesting a potential therapeutic strategy for patients with this molecular subtype of breast cancer.     

Plasma Neutrophil Gelatinase-Associated Lipocalin Is Primarily Related to Inflammation during Sepsis: A Translational Approach

Acute kidney injury (AKI) during sepsis is common and underestimated. Plasma neutrophil gelatinase-associated lipocalin (plasma-NGAL) is discussed as new biomarker for AKI diagnosis, but during inflammation its function and diagnostic impact remain unclear. The association between plasma-NGAL and inflammatory markers in septic patients, but also in healthy controls and patients with chronic inflammation before and after either maximum exercise test or treatment with an anti-TNF therapy were investigated. In-vitro blood stimulations with IL-6, lipopolysaccharide, NGAL or its combinations were performed to investigate cause-effect-relationship. Plasma-NGAL levels were stronger associated with inflammation markers including IL-6 (Sepsis: r=0.785 P<0.001; chronic inflammation after anti-TNF: r=0.558 P<0.001), IL-8 (Sepsis: r=0.714 P<0.004; healthy controls after exercise r=0.786 P<0.028; chronic inflammation before anti-TNF: r=0.429 P<0.041) and IL-10 (healthy controls before exercise: r=0.791 P<0.028) than with kidney injury or function. Correlation to kidney injury or function was found only in septic patients (for creatinine: r= 0.906 P<0.001; for eGFR: r= -0.686 P=0.005) and in patients with rheumatic disease after anti-TNF therapy (for creatinine: r= 0.466 P<0.025). In stimulation assays with IL-6 and lipopolysaccharide plasma-NGAL was increased. Co-stimulation of lipopolysaccharide with plasma-NGAL decreased cellular injury (P<0.05) and in trend IL-10 levels (P=0.057). Septic mice demonstrated a significantly improved survival rate after NGAL treatment (P<0.01). Plasma-NGAL seams to be strongly involved in inflammation. For clinical relevance, it might not only be useful for AKI detection during severe inflammation - indeed it has to be interpreted carefully within this setting - but additionally might offer therapeutic potential.