miR-21 Targets ASPP2 to Inhibit Apoptosis via CHOP-Mediated Signaling in Helicobacter pylori-Infected Gastric Cancer Cells.

Helicobacter pylori (H. pylori) infection affects cell survival pathways, including apoptosis and proliferation in host cells, and disruption of this balance is the key event in the development of H. pylori-induced gastric cancer (HPGC). H. pylori infection induces alterations in microRNAs expression that may be involved in GC development. Bioinformatic analysis showed that microRNA-21 (miR-21) is significantly upregulated in HPGC. Furthermore, quantitative proteomics and in silico prediction were employed to identify potential targets of miR-21. Following functional enrichment and clustered interaction network analyses, five candidates of miR-21 targets, PDCD4, ASPP2, DAXX, PIK3R1, and MAP3K1, were found across three functional clusters in association with cell death and survival, cellular movement, and cellular growth and proliferation. ASPP2 is inhibited by H. pylori-induced miR-21 overexpression. Moreover, ASPP2 levels are inversely correlated with miR-21 levels in HPGC tumor tissues. Thus, ASPP2 was identified as a miR-21 target in HPGC. Here, we observed that H. pylori-induced ASPP2 suppression enhances resistance to apoptosis in GC cells using apoptosis assays. Using protein interaction network and coimmunoprecipitation assay, we identified CHOP as a direct mediator of the ASPP2 proapoptotic activity in H. pylori-infected GC cells. Mechanistically, ASPP2 suppression promotes p300-mediated CHOP degradation, in turn inhibiting CHOP-mediated transcription of Noxa, Bak, and suppression of Bcl-2 to enact antiapoptosis in the GC cells after H. pylori infection. Clinicopathological analysis revealed correlations between decreased ASPP2 expression and higher HPGC risk and poor prognosis. In summary, the discovery of H. pylori-induced antiapoptosis via miR-21-mediated suppression of ASPP2/CHOP-mediated signaling provides a novel perspective for developing HPGC management and treatment.

S100A8, S100A9 and S100A8/A9 heterodimer as novel cachexigenic factors for pancreatic cancer-induced cachexia.

BACKGROUND: Cancer cachexia, occurring in ~ 80% pancreatic cancer (PC) patients overall, is a paraneoplastic syndrome mediated by cancer-induced systemic inflammation and characterized by weight loss and skeletal muscle wasting. Identifying clinically relevant PC-derived pro-inflammatory factors with cachexigenic potential may provide novel insights and therapeutic strategies. METHODS: Pro-inflammatory factors with cachexigenic potential in PC were identified by bioinformatic analysis. The abilities of selected candidate factors in inducing skeletal muscle atrophy were investigated. Expression levels of candidate factors in tumors and sera was compared between PC patients with and without cachexia. Associations between serum levels of the candidates and weight loss were assessed in PC patients. RESULTS: S100A8, S100A9, and S100A8/A9 were identified and shown to induce C2C12 myotube atrophy. Tumors of PC patients with cachexia had markedly elevated expression of S100A8 (P = 0.003) and S100A9 (P < 0.001). PC patients with cachexia had significantly higher serum levels of S100A8, S100A9 and S100A8/A9. Serum levels of these factors positively correlated with percentage of weight loss [correlation coefficient: S100A8: 0.33 (P < 0.001); S100A9: 0.30 (P < 0.001); S100A8/A9: 0.24 (P = 0.004)] and independently predicted the occurrence of cachexia [adjusted odds ratio (95% confidence interval) per 1ng/ml increase: S100A8 1.11 (1.02-1.21), P = 0.014; S100A9 1.10 (1.04-1.16), P = 0.001; per 1 µg/ml increase: S100A8/A9 1.04 (1.01-1.06), P = 0.009]. CONCLUSIONS: Atrophic effects of S100A8, S100A9, and S100A8/A9 indicated them as potential pathogenic factors of PC-induced cachexia. In addition, the correlation with the degree of weight loss and prediction of cachexia in PC patients implicated their potential utility in the diagnosis of PC-induced cachexia.

Extracellular domain of semaphorin 5A serves a tumor­suppressing role by activating interferon signaling pathways in lung adenocarcinoma cells.

Semaphorin 5A (SEMA5A), which was originally identified as an axon guidance molecule in the nervous system, has been subsequently identified as a prognostic biomarker for lung cancer in nonsmoking women. SEMA5A acts as a tumor suppressor by inhibiting the proliferation and migration of lung cancer cells. However, the regulatory mechanism of SEMA5A is not clear. Therefore, the purpose of the present study was to explore the roles of different domains of SEMA5A in its tumor­suppressive effects in lung adenocarcinoma cell lines. First, it was revealed that overexpression of full length SEMA5A or its extracellular domain significantly inhibited the proliferation and migration of both A549 and H1299 cells using MTT, colony formation and gap closure assays. Next, microarray analyses were performed to identify genes regulated by different domains of SEMA5A. Among the differentially expressed genes, the most significant function of these genes that were enriched was the 'Interferon Signaling' pathway according to Ingenuity Pathway Analysis. The activation of the 'Interferon Signaling' pathway was validated by reverse transcription­quantitative PCR and western blotting. In summary, the present study demonstrated that the extracellular domain of SEMA5A could upregulate genes in interferon signaling pathways, resulting in suppressive effects in lung adenocarcinoma cells.

Aberrant Upregulation of Indoleamine 2,3-Dioxygenase 1 Promotes Proliferation and Metastasis of Hepatocellular Carcinoma Cells via Coordinated Activation of AhR and ß-Catenin Signaling.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide. Chronic liver inflammation due to hepatitis virus infection and other major effectors is a major risk factor of HCC. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme enzyme highly expressed upon stimulation with proinflammatory cytokines such as interferon-γ (IFN-γ), is activated to modulate the tumor microenvironment and potentially crucial in the development of certain cancer types. Earlier studies have majorly reported an immunomodulatory function of IDO1. However, the specific role of IDO1 in cancer cells, particularly HCC, remains to be clarified. Analysis of The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) dataset in the current study revealed a significant correlation between IDO1 expression and HCC. We further established inducible IDO1-expressing cell models by coupling lentivirus-mediated knockdown and IFN-γ induction of IDO1 in normal and HCC cells. In functional assays, proliferation and motility-related functions of HCC cells were compromised upon suppression of IDO1, which may partially be rescued by its enzymatic product, kynurenine (KYN), while normal hepatocytes were not affected. Aryl hydrocarbon receptor (AhR), a reported endogenous KYN receptor, is suggested to participate in tumorigenesis. In mechanistic studies, IDO1 activation promoted both AhR and ß-catenin activity and nuclear translocation. Immunofluorescence staining and co-immunoprecipitation assays further disclosed interactions between AhR and ß-catenin. In addition, we identified a Src-PTEN-PI3K/Akt-GSK-3ß axis involved in ß-catenin stabilization and activation following IDO1-mediated AhR activation. IDO1-induced AhR and ß-catenin modulated the expression of proliferation- and EMT-related genes to facilitate growth and metastasis of HCC cells. Our collective findings provide a mechanistic basis for the design of more efficacious IDO1-targeted therapy for HCC.

Human pluripotent stem cell-derived DDX4 and KRT-8 positive cells participate in ovarian follicle-like structure formation.

Understanding the mechanisms of human pluripotent stem cells (hPSCs) specification, development and differentiation to gametes are useful for elucidating the causes of infertility and potential treatment. This study aims to examine whether hPSCs can be induced to DDX4 extracellularly expressing primordial germ cell-like cells (DDX4ec PGCLCs) and further into ovarian follicle stage in a combined in vitro and in vivo model. The transcriptional signatures show that these DDX4ec PGCLCs are characteristic of PGCs and express ovarian folliculogenesis markers. We also verify that keratin (KRT)-8 is highly expressed in the DDX4ec PGCLCs and plays a crucial role in germ cell migration. By co-culturing DDX4ec PGCLCs with human granulosa cells (GCs), these cells are further induced into ovarian follicle-like structures in a xenograft mice model. This approach can in the future design practical strategies for treating germ cell-associated issues of infertility.

Y-Box Binding Protein-1 Promotes Epithelial-Mesenchymal Transition in Sorafenib-Resistant Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.

Irisin, an exercise myokine, potently suppresses tumor proliferation, invasion, and growth in glioma.

Glioblastoma multiforme is the most common and aggressive glial tumor with poor prognosis. Importantly, effective treatment options for glioblastoma are unmet needs. Obesity and low physical activity have been linked with a high risk of cancer, and exercise is related to delayed cancer development and progression. Epidemiological studies have revealed a correlation between exercise and the survival rate of patients with glioblastoma. Nevertheless, the mechanisms by which exercise exerts its anticancer effects in glioblastoma remain unclear. Here, we found that irisin, an exercise-induced myokine, induced G2 /M cell cycle arrest and increased p21 levels in glioblastoma cells, leading to the inhibition of cell proliferation. In addition, irisin inhibited glioblastoma cell invasion by upregulating TFPI-2 and even reversed the aggressive tumor phenotype promoted by co-cultivation with cancer-associated adipocytes. Furthermore, irisin retarded xenograft glioblastoma tumor growth, and radiolabeled irisin demonstrated specific tumor-targeting capability in vivo. Therefore, this study identified one potential molecular mechanism by which exercise prevents cancer progression via irisin. Intriguingly, irisin has the potential to be developed as a molecular imaging and therapeutic anticancer agent.

Quantitative phosphoproteomic analysis identifies the potential therapeutic target EphA2 for overcoming sorafenib resistance in hepatocellular carcinoma cells.

Limited therapeutic options are available for advanced-stage hepatocellular carcinoma owing to its poor diagnosis. Drug resistance to sorafenib, the only available targeted agent, is commonly reported. The comprehensive elucidation of the mechanisms underlying sorafenib resistance may thus aid in the development of more efficacious therapeutic agents. To clarify the signaling changes contributing to resistance, we applied quantitative phosphoproteomics to analyze the differential phosphorylation changes between parental and sorafenib-resistant HuH-7 cells. Consequently, an average of ~1500 differential phosphoproteins were identified and quantified, among which 533 were significantly upregulated in resistant cells. Further bioinformatic integration via functional categorization annotation, pathway enrichment and interaction linkage analysis led to the discovery of alterations in pathways associated with cell adhesion and motility, cell survival and cell growth and the identification of a novel target, EphA2, in resistant HuH-7R cells. In vitro functional analysis indicated that the suppression of EphA2 function impairs cell proliferation and motility and, most importantly, overcomes sorafenib resistance. The attenuation of sorafenib resistance may be achieved prior to its development through the modulation of EphA2 and the subsequent inhibition of Akt activity. Binding analyses and in silico modeling revealed a ligand mimic lead compound, prazosin, that could abate the ligand-independent oncogenic activity of EphA2. Finally, data obtained from in vivo animal models verified that the simultaneous inhibition of EphA2 with sorafenib treatment can effectively overcome sorafenib resistance and extend the projected survival of resistant tumor-bearing mice. Thus our findings regarding the targeting of EphA2 may provide an effective approach for overcoming sorafenib resistance and may contribute to the management of advanced hepatocellular carcinoma.

Relationship between pancreatic cancer-associated diabetes and cachexia.

BACKGROUND: Pancreatic cancer-associated diabetes mellitus (PCDM) is a paraneoplastic phenomenon characterized by worsening hyperglycaemia and weight loss. Galectin-3 and S100A9, mediators of PCDM, have pro-inflammatory functions and might thereby induce systemic inflammation and cachexia. We aimed to examine whether PCDM directly mediates cachexia. METHODS: Consecutive pancreatic cancer (PC) patients with and without PCDM (n = 88 each) with complete information were included. Cachexia was defined as weight loss >5% within 6 months or weight loss >2% and body mass index <20 kg/m2 or sarcopenia. Skeletal muscle mass was measured with lumbar skeletal muscle index (SMI) using computed tomography images. Cachexia-related parameters (prevalence of cachexia, weight loss, and SMI) were compared between patients with and without PCDM. Relations between cachexia-related parameters and fasting blood glucose or serum levels of galectin-3 and S100A9 were analysed by Spearman correlation and logistic regression analyses. RESULTS: One hundred two (58.0%) patients had cachexia at diagnosis. No significant differences existed between patients with and without PCDM in prevalence of cachexia (64.8% vs. 51.1%, P = 0.093), percentage of weight loss (median 6.8 vs. 4.0, P = 0.085), and SMI (median 45.8 vs. 45.3 cm2 /m2 in men, P = 0.119; 34.9 vs. 36.3 cm2 /m2 in women, P = 0.418). In patients with cachexia, the percentage of weight loss and SMI were also similar between patients with and without PCDM. In patients with PCDM, fasting blood glucose was comparable between patients with and without cachexia (P = 0.458) and did not correlate with the percentage of weight loss (P = 0.085) or SMI (P = 0.797 in men and 0.679 in women). Serum S100A9 level correlated with fasting blood glucose (correlation coefficient 0.213, P = 0.047) but not with the percentage of weight loss (P = 0.977) or SMI (P = 0.247 in men and 0.458 in women). Serum galectin-3 level also did not correlate with the percentage of weight loss (P = 0.226) and SMI (P = 0.201 in men and 0.826 in women). Primary tumour size was associated with cachexia (adjusted odds ratio per 1 cm increase 1.28, 95% confidence interval 1.02-1.60, P = 0.034), whereas PCDM, fasting blood glucose, and levels of galectin-3 and S100A9 were not predictors of cachexia. CONCLUSIONS: Neither fasting blood glucose nor levels of galectin-3 and S100A9 were associated with cachexia-related parameters. Mediators of PCDM and hyperglycaemia do not directly mediate PC-induced cachexia.

Galectin-3 and S100A9: Novel Diabetogenic Factors Mediating Pancreatic Cancer-Associated Diabetes.

OBJECTIVE: Pancreatic cancer-associated diabetes (PCDM) is a paraneoplastic phenomenon accounting for 1% of new-onset diabetes. We aimed to identify the mediators of PCDM and evaluate their usefulness in distinguishing PCDM from type 2 diabetes. RESEARCH DESIGN AND METHODS: Secreted proteins of MIA PaCa-2 cells were identified by proteomics, and those with ≥10-fold overexpression in transcriptome analysis were assessed by bioinformatics and glucose uptake assay to identify candidate factors. Expression of factors was compared between tumors with and without PCDM by immunohistochemistry. Serum levels were measured in a training set including PC with and without PCDM, type 2 diabetes, pancreatitis, other pancreatic/peripancreatic tumors, and control subjects (n = 50 each). Cutoff values for differentiation between PCDM and type 2 diabetes from the training set were validated in a test set (n = 41 each). RESULTS: Galectin-3 and S100A9 were overexpressed in tumors with PCDM and dose-dependently suppressed insulin-stimulated glucose uptake in C2C12 myotubes. In the training set, serum galectin-3 and S100A9 levels were exclusively increased in patients with PCDM and distinguished PCDM from type 2 diabetes (area under the curve [AUC] galectin-3: 0.73 [95% CI 0.64-0.83]; S100A9: 0.79 [95% CI 0.70-0.87]). Similar results were observed in the test set (AUC galectin-3: 0.83 [95% CI 0.74-0.92]; S100A9: 0.77 [95% CI 0.67-0.87]), with sensitivity and specificity 72.1% and 86.1%, respectively, for galectin-3 and 69.8% and 58.1% for S100A9 in differentiating between PCDM and type 2 diabetes. CONCLUSIONS: Galectin-3 and S100A9 are overexpressed in PCDM tumors and mediate insulin resistance. Galectin-3 and S100A9 distinguish PCDM from type 2 diabetes in subjects with new-onset diabetes.

Different effects of long noncoding RNA NDRG1-OT1 fragments on NDRG1 transcription in breast cancer cells under hypoxia.

Hypoxia plays a crucial role in the aggressiveness of solid tumors by driving multiple signaling pathways. Recently, long non-coding RNA (lncRNA) has been reported to promote or inhibit tumor aggressiveness by regulating gene expression. Previous studies in our laboratory found that the lncRNA NDRG1-OT1 is significantly up-regulated under hypoxia and inhibits its target gene NDRG1 at both the mRNA and protein levels. At the protein level, NDRG1-OT1 increases NDRG1 degradation via ubiquitin-mediated proteolysis. However, the repressive mechanism of NDRG1 at the RNA level is still unknown. Therefore, the purpose of this study was to study how NDRG1-OT1 transcriptionally regulates its target gene NDRG1. Luciferase reporter assays showed that NDRG1-OT1 decreased NDRG1 promoter activities. Mass spectrometry, bioinformatics tools, genetic manipulation, and immunoblotting were used to identify the interacting proteins. Surprisingly, different fragments of NDRG1-OT1 had opposite effects on NDRG1. The first quarter fragment (1-149 nt) of NDRG1-OT1 had no effect on the NDRG1 promoter; the second quarter fragment (150-263 nt) repressed NDRG1 by increasing the binding affinity of HNRNPA1; the third quarter fragment (264-392 nt) improved NDRG1 promoter activity by recruiting HIF-1α; the fourth quarter fragment (393-508 nt) down-regulated NDRG1 promoter activity via down-regulation of KHSRP under hypoxia. In summary, we have found a novel mechanism by which different fragments of the same lncRNA can cause opposite effects within the same target gene.

High Serum Lipopolysaccharide-Binding Protein Level in Chronic Hepatitis C Viral Infection Is Reduced by Anti-Viral Treatments.

BACKGROUND: Lipopolysaccharide-binding protein (LBP) has been reported to associate with metabolic diseases, such as obesity, diabetes, and non-alcoholic fatty liver disease. Since chronic hepatitis C virus (HCV) infection is associated with metabolic derangements, the relationship between LBP and HCV deserves additional studies. This study aimed to determine the serum LBP level in subjects with or without HCV infection and investigate the change of its level after anti-viral treatments with or without interferon. METHODS AND FINDINGS: We recruited 120 non-HCV subjects, 42 and 17 HCV-infected subjects respectively treated with peginterferon α-2a/ribavirin and direct-acting antiviral drugs. Basic information, clinical data, serum LBP level and abdominal ultrasonography were collected. All the subjects provided written informed consent before being enrolled approved by the Research Ethics Committee of the National Taiwan University Hospital. Serum LBP level was significantly higher in HCV-infected subjects than non-HCV subjects (31.0 ± 8.8 versus 20.0 ± 6.4 µg/mL; p-value < 0.001). After multivariate analyses, LBP at baseline was independently associated with body mass index, hemoglobin A1c, alanine aminotransferase (ALT) and HCV infection. Moreover, the baseline LBP was only significantly positively associated with ALT and inversely with fatty liver in HCV-infected subjects. The LBP level significantly decreased at sustained virologic response (27.4 ± 6.6 versus 34.6 ± 7.3 µg/mL, p-value < 0.001; 15.9 ± 4.4 versus 22.2 ± 5.7 µg/mL, p-value = 0.001), regardless of interferon-based or -free therapy. CONCLUSIONS: LBP, an endotoxemia associated protein might be used as an inflammatory biomarker of both infectious and non-infectious origins in HCV-infected subjects.

Combination of OipA, BabA, and SabA as candidate biomarkers for predicting Helicobacter pylori-related gastric cancer.

Helicobacter pylori (H. pylori ) infection is a major cause of chronic gastritis and is highly related to duodenal ulcer (DU) and gastric cancer (GC). To identify H. pylori-related GC biomarkers with high seropositivity in GC patients, differences in levels of protein expression between H. pylori from GC and DU patients were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). In total, 99 proteins showed increased expression (>1.5-fold) in GC patients compared to DU patients, and 40 of these proteins were categorized by KEGG pathway. The four human disease-related adhesin identified, AlpA, OipA, BabA, and SabA, were potential GC-related antigens, with a higher seropositivity in GC patients (n = 76) than in non-GC patients (n = 100). Discrimination between GC and non-GC patients was improved using multiple antigens, with an odds ratio of 9.16 (95% CI, 2.99-28.07; p < 0.0001) for three antigens recognized. The optimized combination of OipA, BabA, and SabA gave a 77.3% correct prediction rate. A GC-related protein microarray was further developed using these antigens. The combination of OipA, BabA, and SabA showed significant improvement in the diagnostic accuracy and the protein microarray containing above antigens should provide a rapid and convenient diagnosis of H. pylori-associated GC.

Importance of the C-terminal histidine residues of Helicobacter pylori GroES for Toll-like receptor 4 binding and interleukin-8 cytokine production.

Helicobacter pylori infection is associated with the development of gastric and duodenal ulcers as well as gastric cancer. GroES of H. pylori (HpGroES) was previously identified as a gastric cancer-associated virulence factor. Our group showed that HpGroES induces interleukin-8 (IL-8) cytokine release via a Toll-like receptor 4 (TLR4)-dependent mechanism and domain B of the protein is crucial for interactions with TLR4. In the present study, we investigated the importance of the histidine residues in domain B. To this end, a series of point mutants were expressed in Escherichia coli, and the corresponding proteins purified. Interestingly, H96, H104 and H115 were not essential, whereas H100, H102, H108, H113 and H118 were crucial for IL-8 production and TLR4 interactions in KATO-III cells. These residues were involved in nickel binding. Four of five residues, H102, H108, H113 and H118 induced certain conformation changes in extended domain B structure, which is essential for interactions with TLR4 and consequent IL-8 production. We conclude that interactions of nickel ions with histidine residues in domain B help to maintain the conformation of the C-terminal region to conserve the integrity of the HpGroES structure and modulate IL-8 release.

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation.

UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1α signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy.

Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

Childhood nephrotic syndrome is mainly caused by minimal change disease which is named because only subtle ultrastructural alteration could be observed at electron microscopic level in the pathological kidney. Glomerular podocytes are presumed to be the target cells whose protein sieving capability is compromised by a yet unidentified permeability perturbing factor. In a cohort of children with non-hereditary idiopathic nephrotic syndrome, we found the complement fragment C5a was elevated in their sera during active disease. Administration of recombinant C5a induced profound proteinuria and minimal change nephrotic syndrome in mice. Purified glomerular endothelial cells, instead of podocytes, were demonstrated to be responsible for the proteinuric effect elicited by C5a. Further studies depicted a signaling pathway involving Rho/Rho-associated kinase/myosin activation leading to endothelial cell contraction and cell adhesion complex breakdown. Significantly, application of Rho-associated kinase inhibitor, Y27632, prevented the protein leaking effects observed in both C5a-treated purified endothelial cells and mice. Taken together, our study identifies a previously unknown mechanism underlying nephrotic syndrome and provides a new insight toward identifying Rho-associated kinase inhibition as an alternative therapeutic option for nephrotic syndrome.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Skp2-macroH2A1-CDK8 axis orchestrates G2/M transition and tumorigenesis.

Understanding the mechanism by which cell growth, migration, polyploidy, and tumorigenesis are regulated may provide important therapeutic strategies for cancer therapy. Here we identify the Skp2-macroH2A1 (mH2A1)-cyclin-dependent kinase 8 (CDK8) axis as a critical pathway for these processes, and deregulation of this pathway is associated with human breast cancer progression and patient survival outcome. We showed that mH2A1 is a new substrate of Skp2 SCF complex whose degradation by Skp2 promotes CDK8 gene and protein expression. Strikingly, breast tumour suppression on Skp2 deficiency can be rescued by mH2A1 knockdown or CDK8 restoration using mouse tumour models. We further show that CDK8 regulates p27 protein expression by facilitating Skp2-mediated p27 ubiquitination and degradation. Our study establishes a critical role of Skp2-mH2A1-CDK8 axis in breast cancer development and targeting this pathway offers a promising strategy for breast cancer therapy.

Biomarkers of early sepsis may be correlated with outcome.

BACKGROUND: Sepsis causes high mortality, and the mortality due to secondary infections is even higher. No studies to date have investigated the time from the primary infection to death due to a secondary infection; similarly, the factors that are significantly different in sepsis survivors relative to non-survivors or in severe sepsis patients who suffered a late death relative to those who recover have not been explored. We hypothesized that patients who survive sepsis have a weaker pro-inflammatory response than those who do not and that the mid-term survivors (which acquire secondary infections) would have a pronounced anti-inflammatory response (making them susceptible to infection); this hypothesis was verified in this study. METHODS: We examined 24 patients with severe sepsis; the patients were subdivided by outcome into early death (n=5), mid-term survival (survival through severe sepsis but death within six months or continued hospitalization for six months, n=6), and long-term survival (recovery and survival for more than six months, n=13) groups. The levels of CD3+, CD4+, CD8+, and CD19+ lymphocytes were analyzed by flow cytometry, and the plasma levels of carbonic anhydrase IX (CA IX), MCP-1, IL-6, IL-7, IL-8, and IL-10 were measured by ELISA on days 0, 1, 2, and 3. A statistical comparison of the variables in the groups was conducted using a mixed model. RESULTS: The plasma levels of MCP-1, IL-6, and IL-8 in early death and survivors were significantly different, and all had p values<0.01. The plasma levels of MCP-1, IL-6, and IL-8 were also significantly different in mid-term survivors and long-term survivors, with p values of <0.01, 0.04, and <0.01, respectively. CONCLUSIONS: Our data support the hypothesis that survivors have a weaker pro-inflammatory response than non-survivors, but the mid-term survivors did not have a more pronounced anti-inflammatory response. The levels of pro-inflammatory cytokines in the mid-term and long-term survivors were significantly different.