Golgi phosphoprotein 3 (GOLPH3) promotes hepatocellular carcinoma cell aggressiveness by activating the NF‐κB pathway

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, in which the NF‐κB pathway plays an important role and is constitutively activated. Better understanding of the molecular pathogenesis of HCC and the NF‐κB pathway are needed to improve patient outcomes. Herein, we identified an unappreciated protein involved in NF‐κB‐induced activation, Golgi phosphoprotein 3 (GOLPH3). The mRNA and protein expression levels of GOLPH3 were frequently up‐regulated in HCC and GOLPH3 expression correlated closely with clinical stage and survival in both the testing and validation cohorts. Ectopic over‐expression of GOLPH3 in PLC/PRF/5 (PLC) and Huh7 HCC cells protected against cisplatin‐induced apoptosis, promoted angiogenesis and proliferation and increased the aggressiveness of HCC cells in vitro and in vivo, whereas inhibition of GOLPH3 led to decreased aggressiveness. Through analysis of two published HCC patient profiles, GOLPH3 expression significantly correlated with NF‐κB signalling. Furthermore, we demonstrated that GOLPH3 promoted K63‐linked polyubiquitination of tumour necrosis factor receptor‐associated factor 2 (TRAF2), receptor interacting protein (RIP) and NF‐κB essential modulator (NEMO) and substantially sustained the activation of NF‐κB in HCC cells. Taken together, our findings provided evidence that GOLPH3 is a prognostic and/or potential therapeutic biomarker for HCC patients and plays an important role in activation of the NF‐κB pathway during HCC progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

TAK1 maintains the survival of immunoglobulin λ‐chain‐positive B cells

TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex?nuclear factor‐κB (NF‐κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B‐cell receptor (BCR) signaling, the physiological roles of TAK1 in B‐cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ‐positive B cells, but not for receptor editing. In contrast, NF‐κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF‐κB signaling in early B‐cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B‐cell development. TAK1‐deficient mice showed significant reduction of BM Igλ‐positive B‐cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl‐2 was reduced in TAK1‐deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over‐expression of exogenous Bcl‐2 enhanced the survival of TAK1‐deficient Igλ‐positive B cells. TAK1–IKK–NF‐κB signaling contributes to the survival of λ‐chain‐positive B cells through NF‐κB‐dependent anti‐apoptotic Bcl‐2 expression.     

Brain‐Derived Neurotrophic Factor Enhances Bcl‐xL Expression Through Protein Kinase Casein Kinase 2‐Activated and Nuclear Factor Kappa B‐Mediated Pathway in Rat Hippocampus

Brain‐derived neurotrophic factor (BDNF) was shown to produce its neuroprotective effect through extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidylinositol‐3 kinase (PI3‐K) signaling. But whether other pathways also mediate the neuroprotective effect of BDNF is less known. In this study, we found that direct administration of BDNF to rat hippocampal CA1 area dose‐dependently increased the mRNA and protein levels of Bcl‐xL. BDNF also increased protein kinase casein kinase II (CK2) activity and NF‐κB phosphorylation at Ser529 dose‐dependently. Further, transfection of the wild‐type CK2α DNA to CA1 neurons increased nuclear factor kappa B (NF‐κB) phosphorylation and Bcl‐xL mRNA expression, whereas transfection of CK2α156A, the catalytically inactive mutant of CK2α, decreased these measures. Moreover, transfection of CK2α small interfering RNA (siRNA) blocked the enhancing effect of BDNF on NF‐κB phosphorylation and Bcl‐xL expression. These results were further confirmed by treatment of 4,5,6,7‐tetrabromobenzotriazole (TBB), a specific CK2 inhibitor. Transfection of NF‐κBS529A, the dominant negative mutant of NF‐κB, prevented the enhancing effect of BDNF on Bcl‐xL expression. More importantly, BDNF activation of CK2 is not affected by co‐administration of the ERK1/2 inhibitor, PD98059, and the PI3‐K inhibitor, LY294002. These results demonstrate a novel BDNF signaling pathway and provide an alternative therapeutic strategy for the protective effect of BDNF on hippocampal neurons in vivo.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Down‐regulation of miR‐301a suppresses pro‐inflammatory cytokines in Toll‐like receptor‐triggered macrophages

In many types of tumours, especially pancreatic adenocarcinoma, miR‐301a is over‐expressed. This over‐expression results in negative regulation of the target gene of miR‐301a, the nuclear factor‐κB (NF‐κB) repressing factor (NKRF), increasing the activation of NF‐κB and production of NF‐κB‐responsive pro‐inflammatory cytokines such as interleukin‐8, interferon‐β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX‐2). However, in immune cells, mechanisms that regulate miR‐301a have not been reported. Similar to tumour cells, Toll‐like receptor (TLR) ‐activated macrophages produce NF‐κB‐responsive pro‐inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR‐301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR‐301a was decreased in TLR‐triggered macrophages. Through targeting NKRF, miR‐301a affected the activity of NF‐κB and the expression of pro‐inflammatory genes downstream of NF‐κB such as COX‐2, prostaglandin E₂ and interleukin‐6. In addition, when lipopolysaccharide‐treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR‐301a increased, whereas NKRF and pro‐inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR‐301a and the inhibitory effect of trichostatin A on lipopolysaccharide‐induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR‐301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune‐mediated inflammatory responses.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Vitamin K3 attenuates lipopolysaccharide‐induced acute lung injury through inhibition of nuclear factor‐κB activation

Vitamin K is a family of fat‐soluble compounds including phylloquinone (vitamin K₁), menaquinone (vitamin K₂) and menadione (vitamin K₃). Recently, it was reported that vitamin K, especially vitamins K₁ and K₂, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti‐inflammatory effects of vitamin K₃ in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K₃ inhibited the tumour necrosis factor (TNF)‐α‐evoked translocation of nuclear factor (NF)‐κB into the nucleus, although vitamins K₁ and K₂ did not. Vitamin K₃ also suppressed the lipopolysaccharide (LPS)‐induced nuclear translocation of NF‐κB and production of TNF‐α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K₃ before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K₃ also suppressed the LPS‐induced increase in the serum TNF‐α level and inhibited the LPS‐evoked nuclear translocation of NF‐κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K₃ may be an effective therapeutic strategy against acute lung injury including ARDS.     

Hepatocyte‐specific activation of NF‐κB does not aggravate chemical hepatocarcinogenesis in transgenic mice

The NF‐κB signalling pathway plays important roles in liver organogenesis and carcinogenesis. Mouse embryos deficient in IKKβ die in mid‐gestation, due to excessive apoptosis of hepatoblasts. Although activation of the NF‐κB signalling pathway has been demonstrated in human hepatocellular carcinoma, the role of NF‐κB is controversial. Here, we have generated transgenic mice in which a constitutively active form of IKKβ was expressed in a hepatocyte‐specific manner. Using electrophoretic mobility shift assay, we documented increased NF‐κB activities and up‐regulated levels of NF‐κB downstream target genes, Bcl‐xL and STAT5, in the transgenic mouse livers. These results confirmed that the NF‐κB pathway was activated in the livers of the transgenic mice. However, there was no significant difference in tumour formation between transgenic and wild‐type mice up to an age of 50 weeks. When we treated the transgenic mice with the chemical carcinogen diethylnitrosamine (DEN), we observed no significant differences in the incidence and size of liver tumours formed in these mice with and without DEN treatment at 35 weeks of age, suggesting that the activated NF‐κB pathway in the livers of the transgenic mice did not enhance hepatocarcinogenesis. Interestingly, some of the transient transgenic embryos (E12.5) had abnormal excessive accumulation of nucleated red blood cells in their developing livers. In summary, NF‐κB activation in hepatocytes did not significantly affect chemical hepatocarcinogenesis. In addition, the TTR/IKKCA transgenic mice may serve as a useful model for studying the role of NF‐κB activation in hepatocarcinogenesis as well as inflammatory and metabolic diseases. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cordycepin as a sensitizer to tumour necrosis factor (TNF)-α-induced apoptosis through eukaryotic translation initiation factor 2α (eIF2α)- and mammalian target of rapamycin complex 1 (mTORC1)-mediated inhibition of nuclear factor (NF)-κB

Cordycepin (3′‐deoxyadenosine) is one of the major bioactive substances produced by Cordyceps militaris, a traditional medicinal mushroom. Cordycepin possesses several biological activities, including both pro‐apoptotic and anti‐apoptotic properties. In the present report, we investigated an effect of cordycepin on the survival of cells exposed to tumour necrosis factor (TNF)‐α. We found that subtoxic doses of cordycepin increased susceptibility of cells to TNF‐α‐induced apoptosis. It was associated with suppression of nuclear factor‐κB (NF‐κB), a major prosurvival component involved in TNF‐α signalling. The adenosine transporter and A₃ adenosine receptor, but not A₁ and A₂ adenosine receptors, mediated both anti‐NF‐κB and pro‐apoptotic effects. We found that cordycepin had the potential to phosphorylate eukaryotic translation initiation factor 2α (eIF2α) and that activation of eIF2α mimicked the suppressive effect of cordycepin on the NF‐κB pathway. Furthermore, activation of eIF2α sensitized cells to TNF‐α‐induced apoptosis. To identify molecular events downstream of eIF2α, the role of mammalian target of rapamycin complex 1 (mTORC1) was examined. Selective activation of ₃eIF2α, as well as treatment with cordycepin, caused phosphorylation of mTORC1. Rapamycin, an inhibitor of mTORC1, significantly reversed the suppressive effects of eIF2α on NF‐κB. These results suggest that cordycepin sensitizes cells to TNF‐α‐induced apoptosis, at least in part, via induction of the eIF2α–mTORC1 pathway and consequent suppression of NF‐κB.     

Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter‐κB/p65 in hepatoma cells

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real‐time quantitative RT‐PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor‐κB (NF‐κB) mediated by siRNA targeting NF‐κB/p65 mRNA or PDTC (an inhibitor of NF‐κB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF‐κB binding site of the Capn4 promoter was mutant, suggesting that NF‐κB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF‐κB/p65 to promote migration of hepatoma cells. J. Med. Virol. 82:920–928, 2010. © 2010 Wiley‐Liss, Inc.     

NF‐κB2/p100 deficiency impairs immune responses to T‐cell‐independent type 2 antigens

Formation of the splenic marginal zone (MZ) depends on the alternative NF‐κB signaling pathway. Recently, we reported that unrestricted activation of this pathway in NF‐κB2/p100‐deficient (p100^?/?) knock‐in mice alters the phenotype of MZ stroma and B cells. Here, we show that lack of the p100 inhibitor resulted in an expansion of both MZ B and peritoneal B‐1 cells. However, these cells failed to generate proliferating blasts in response to T‐cell‐independent type 2 (TI‐2) Ags, correlating with dampened IgM and absent IgG3 responses. This phenotype was in part due to increased activity of the NF‐κB subunit RelB. Moreover, p100^?/?→B6 BM chimeras were more susceptible to infection by encapsulated Streptococcus pneumoniae bacteria, pathogens that induce TI‐2 responses. In contrast to the TI‐2 defect, p100 deficiency did not impair immune responses to the TI‐1 Ag LPS and p100^?/? MZ B cells showed normal Ag transportation into B‐cell follicles. Furthermore, p100^?/? MZ B and B‐1 cells failed to respond to TI‐2 Ags in the presence of WT accessory cells. Thus, NF‐κB2/p100 deficiency caused a predominant B‐cell‐intrinsic TI‐2 defect that could largely be attributed to impaired proliferation of plasmablasts. Importantly, p100 was also necessary for efficient defense against clinically relevant TI‐2 pathogens.     

The B‐cell‐activating factor signalling pathway is associated with Helicobacter pylori independence in gastric mucosa‐associated lymphoid tissue lymphoma without t(11;18)(q21;q21)

We previously reported that activation of the B‐cell‐activating factor (BAFF) pathway upregulates nuclear factor‐κB (NF‐κB) and induces BCL3 and BCL10 nuclear translocation in Helicobacter pylori (HP)‐independent gastric diffuse large B‐cell lymphoma (DLBCL) tumours with evidence of mucosa‐associated lymphoid tissue (MALT). However, the significance of BAFF expression in HP independence of gastric low‐grade MALT lymphomas without t(11;18)(q21;q21) remains unexplored. Sixty‐four patients who underwent successful HP eradication for localized HP‐positive gastric MALT lymphomas without t(11;18)(q21;q21) were studied. BAFF expression was significantly higher in the HP‐independent group than in the HP‐dependent group [22/26 (84.6%) versus 8/38 (21.1%); p < 0.001]. Similarly, BAFF receptor (BAFF‐R) expression (p = 0.004) and nuclear BCL3 (p = 0.004), BCL10 (p < 0.001), NF‐κB (p65) (p = 0.001) and NF‐κB (p52) (p = 0.005) expression were closely correlated with the HP independence of these tumours. Moreover, BAFF overexpression was significantly associated with BAFF‐R expression and nuclear BCL3, BCL10, NF‐κB (p65) and NF‐κB (p52) expression. These findings were further validated in an independent cohort, including 40 HP‐dependent cases and 18 HP‐independent cases of gastric MALT lymphoma without t(11;18)(q21;q21). The biological significance of BAFF signalling in t(11;18)(q21;q21)‐negative lymphoma cells was further studied in two types of lymphoma B cell: OCI‐Ly3 [non‐germinal centre B‐cell origin DLBCL without t(11;18)(q21;q21) cell line] and MA‐1 [t(14;18)(q32;q21)/IGH‐MALT1‐positive DLBCL cell line]. In both cell lines, we found that BAFF activated the canonical NF‐κB and AKT pathways, and induced the formation of BCL10–BCL3 complexes, which translocated to the nucleus. BCL10 and BCL3 nuclear translocation and NF‐κB (p65) transactivation were inhibited by either LY294002 or by silencing BCL3 or BCL10 with small interfering RNA. BAFF also activated non‐canonical NF‐κB pathways (p52) through tumour necrosis factor receptor‐associated factor 3 degradation, NF‐κB‐inducing kinase accumulation, inhibitor of κB kinase (IKK) α/β phosphorylation and NF‐κB p100 processing in both cell lines. Our data indicate that the autocrine BAFF signal transduction pathway contributes to HP independence in gastric MALT lymphomas without the t(11;18)(q21;q21) translocation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Evaluation of Histone 3 Lysine 27 Trimethylation (H3K27me3) and Enhancer of Zest 2 (EZH2) in Pediatric Glial and Glioneuronal Tumors Shows Decreased H3K27me3 in H3F3A K27M Mutant Glioblastomas

H3F3A mutations are seen in ～30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb‐group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance.