Constitutive Activation Of Nuclear factor-κB Research Articles

Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-κB activation: common genetic etiology with Blau syndrome

AbstractEarly-onset sarcoidosis (EOS) and inheritable Blau syndrome (BS) share characteristic clinical features of juvenile-onset systemic granulomatosis syndrome that mainly affects skin, joints, and eyes. However, no direct evidence has been shown for the possible common origin of these 2 diseases. Recent discovery of CARD15 mutations in BS families encouraged us to investigate similar CARD15 mutations in EOS patients. Among 10 EOS cases retrospectively collected in Japan, heterozygous missense mutations were found in 9 cases; 4 showed a 1000C>T (R334W in amino acid change) that has been reported in BS, 4 showed novel 1487A>T (H496L), 1538T>C (M513T), 1813A>C (T605P), and 2010C>A (N670K), and 1 case showed double 1146C>G (D382E)/1834G>A (A612T) mutations on different alleles. All 6 of these variants of CARD15 showed increased basal nuclear factor (NF)–κB activity. These findings indicate that the majority of EOS and BS cases share the common genetic etiology of CARD15 mutations that cause constitutive NF-κB activation.

Ablation of miR-146b in mice causes hematopoietic malignancy

AbstractExcessive and constitutive activation of nuclear factor-κB (NF-κB) leads to abnormal cell proliferation and differentiation, leading to the development of malignant tumors, including lymphoma. MicroRNA 146a (miR-146a) and miR-146b, both of which carry an identical seed sequence, have been shown to contribute to inflammatory diseases and tumors by suppressing the expression of key molecules required for NF-κB activation. However, the functional and physiological differences between miR-146a and miR-146b in disease onset have not been fully elucidated. In this study, we generated miR-146b–knockout (KO) and miR-146a–KO mice by genome editing and found that both strains developed hematopoietic malignancies such as B-cell lymphoma and acute myeloid leukemia during aging. However, the B-cell lymphomas observed in miR-146a– and miR-146b–KO mice were histologically different in their morphology, and the malignancy rate is lower in miR-146b mice than miR-146a mice. Upon mitogenic stimulation, the expression of miR-146a and miR-146b was increased, but miR-146b expression was lower than that of miR-146a. Using a previously developed screening system for microRNA targets, we observed that miR-146a and miR-146b could target the same mRNAs, including TRAF6, and inhibit subsequent NF-κB activity. Consistent with these findings, both miR-146a– and miR-146b–KO B cells showed a high proliferative capacity. Taken together, sustained NF-κB activation in miR-146b KO mice could lead to the development of hematopoietic malignancy with aging.

Influence of autocrine growth hormone on NF-κB activation leading to epithelial-mesenchymal transition of mammary carcinoma.

Progression of breast cancers often depends on hormones among which human growth hormone is prominently involved in breast cancer progression. Earlier studies have reported constitutive activation of nuclear factor-κB, a key regulator of growth hormone receptor-mediated signaling pathway in breast carcinoma, but the precise molecular mechanisms are still elusive. In this study, we investigated the effect of human growth hormone on nuclear factor-κB activation and epithelial-mesenchymal transition in breast carcinoma. Our results explored that autocrine production of human growth hormone enhances cellular proliferation by the activation of nuclear factor-κB (65 kDa) and downregulation of E-cadherin expression. Furthermore, enhanced nuclear factor-κB expression significantly increases cell proliferation and diminishes apoptosis in MCF-7 cell line. Increased expression of nuclear factor-κB significantly enhances mammary carcinoma cell migration and invasion stimulated by autocrine human growth hormone, which results in epithelial-mesenchymal transition of MCF-7 cells. In conclusion, our study revealed the influence of human growth hormone on nuclear factor-κB activity and epithelial-mesenchymal transition in mammary carcinoma. Our findings will help to understand molecular role of "growth hormone-nuclear factor-κB axis" in mammary carcinogenesis which may facilitate the discovery of suitable pathway inhibitors for disease treatment.

Recombinant fibromodulin and decorin effects on NF-κB and TGFβ1 in the 4T1 breast cancer cell line.

Constitutive activation of nuclear factor-κB (NF-κB) stimulates cell proliferation and metastasis, and inhibits apoptosis in breast cancer. Transforming growth factor-β (TGF-β) signaling pathway is deregulated in breast cancer progression and metastasis. The aim of the present study was to investigate the inhibitory effects of the two small leucine rich proteoglycans fibromodulin (Fmod) and decorin (Dcn), overexpressed using adenovirus gene transfer, on NF-κB-activity and TGF-β1-expression in the highly metastatic 4T1 breast cancer cell line. The results demonstrate that Fmod and Dcn overexpression is associated with NF-κB and TGF-β1 downregulation, and that Fmod promotes this effect more effectively compared with Dcn.

Pevonedistat Sensitizes Diffuse Large B-Cell Lymphoma Cells to Death Receptor Ligand-Mediated Apoptosis

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in the U.S. DLBCL can be subdivided into the activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subgroups on the basis of gene expression profiling results. ABC-DLBCL is characterized by the constitutive activation of nuclear factor-κB (NFκB) transcription factor and high expression level of its target genes, including pro-survival BCL2 family members, cell cycles proteins, and chemokines. We and others have demonstrated that pevonedistat (MLN4924), an inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in DLBCL cell lines and primary chronic lymphocytic leukemia (CLL) cells. Pevonedistat has been shown to sensitize solid tumor cell lines to extrinsic apoptosis. Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists. By contrast, GCB-DLBCL and primary CLL cells are not sensitive to this combination.Methods We employed a panel of ABC- (OCI-LY3, U2932) and GCB-DLBCL (SUDHL6, SUDHL10, VAL) cell lines. CLL B-cells were obtained from patients in the hematology clinics at the Knight Cancer Institute and were subjected to in vitro co-cultures with CD40L-expressing stroma which we have previously shown to activate NFκB and thereby mimic the lymph node microenvironment. We used the Fas agonists Apo-1-L, CH-11 and SuperFasLigand as well as KillerTRAIL. Pevonedistat was provided by Millennium Pharmaceuticals Inc., a subsidiary of Takeda Pharmaceutical Company Limited.Results Either Fas or TRAIL agonists, as well as 0.5 µM pevonedistat alone induced modest apoptosis of ABC-DLBCL cells (<10%) upon a 24 hour incubation. Meanwhile, pre-treatment of OCI-LY3 cells with 0.5 µM pevonedistat overnight resulted in apoptosis of 72.6±6.2% cells following exposure to 100 ng/mL KillerTrail and 79.6±7.5% - 10 ng/mL SuperFasLigand (p<0.0001 compared with either agent alone). Similar findings were observed in U2932 cells. By contrast, pevonedistat had a minimal effect on death receptor ligand-mediated apoptosis in the GCB-DLBCL cell lines.Using immunoprecipitation, we confirmed that Fas engagement led to rapid assembly of the death-inducing signaling complex in OCI-LY3 cells (within 2 hours). This was accompanied by activation of caspase-8, as manifested by generation of its active forms (p41/43). Pre-treatment with pevonedistat promoted sustained and prolonged Fas-mediated cleavage of caspase-8 (for 6 hours, as opposed to transient cleavage following isolated Fas stimulation), accompanied by caspase-mediated PARP processing. We found that pevonedistat treatment downmodulated the transcript and protein levels of cFLIP in OCI-LY3 cells, an antagonistic caspase-8 homolog, whose expression is highly dependent on NFκB transcriptional activity. This is consistent with earlier findings where abrogation of NF-κB transcriptional activity was the dominant event preceding pevodinestat-induced apoptosis in ABC-DLBCL cells. By contrast, in GCB-DLBCL cells, targeting neddylation resulted in accumulation of Cdt1, DNA re-replication and cell cycle arrest. Thus, preferential sensitivity of ABC-DLBCL to death receptor ligands in combination with pevonedistat may be dependent on the effects of the latter on NFκB signaling and cFLIP.We previously reported that, like in ABC-DLBCL, pevonedistat abrogates NFκB in CD40-stimulated primary CLL cells. We found that CD40 upregulated Fas and TRAIL receptors, as well as cFLIP, in CLL cells. While treatment with pevonedistat downregulated cFLIP in CLL cells, it did not sensitize CLL cells to Fas or TRAIL; caspase-8 cleavage and Bid processing were not induced. Conversely, neutralizing antibodies against TNFα and TRAIL-R1/2 did not modulate the pro-apoptotic activity of pevonedistat in CLL. Hence, distinct from observations in normal monocytes, pevonedistat did not enhance death receptor-mediated apoptosis, consistent with previous reports indicating that such signaling is dysfunctional in CLL cells.Conclusions Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists by a mechanism which includes downmodulation of cFLIP. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies. DisclosuresDanes:3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment. Spurgeon:Gilead sciences: Honoraria, Research Funding; Bristol Meyers Squibb: Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria; Janssen: Research Funding; Genentech: Honoraria. Berger:3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment.

Romo1 and the NF-κB pathway are involved in oxidative stress-induced tumor cell invasion.

Reactive oxygen species (ROS) are important contributors to tumor cell invasion. ROS enhanced by reactive oxygen species modulator 1 (Romo1) expression has been reported to increase invasive potential and constitutive activation of nuclear factor-κB (NF-κB) in hepatocellular carcinoma (HCC). Therefore, we investigated whether constitutive NF-κB activation due to Romo1 expression is associated with breast cancer tumor cell invasion. In this study, we show that oxidative stress-induced invasion is mediated by Romo1 expression. The Romo1-induced increase of invasive activity was blocked by an inhibitor of κB kinase (IKK). These results demonstrate that tumor cell invasion in response to oxidative stress is associated with Romo1 expression and the NF-κB signaling pathway. Romo1 is therefore a promising therapeutic target for diseases characterized by NF-κB deregulation.

Hypermethylation of the tumor suppressor gene PRDM1/Blimp-1 supports a pathogenetic role in EBV-positive Burkitt lymphoma.

PRDM1/Blimp-1 is a tumor suppressor gene in the activated B-cell subtype of diffuse large B-cell lymphomas. Its inactivation contributes to pathogenesis in this setting by impairing terminal B-cell differentiation induced by constitutive nuclear factor-κB activation. The role of PRDM1 in Burkitt lymphoma (BL) lymphomagenesis is not known. Here we identified hypermethylation of the promoter region and exon 1 of PRDM1 in all six Epstein–Barr virus (EBV)-positive BL cell lines and 12 of 23 (52%) primary EBV-positive BL or BL-related cases examined, but in none of the EBV-negative BL cell lines or primary tumors that we assessed, implying a tumor suppressor role for PRDM1 specifically in EBV-associated BL. A direct induction of PRDM1 hypermethylation by EBV is unlikely, as PRDM1 hypermethylation was not observed in EBV-immortalized B lymphoblastoid cell lines. Treatment of EBV-positive BL cells with 5′ azacytidine resulted in PRDM1 induction associated with PRDM1 demethylation, consistent with transcriptional silencing of PRDM1 as a result of DNA methylation. Overexpression of PRDM1 in EBV-positive BL cell lines resulted in cell cycle arrest. Our results expand the spectrum of lymphoid malignancies in which PRDM1 may have a tumor suppressor role and identify an epigenetic event that likely contributes to the pathogenesis of BL.

Identification of a NFKBIA polymorphism associated with lower NFKBIA protein levels and poor survival outcomes in patients with glioblastoma multiforme.

The aberrant constitutive activation of nuclear factor-κB (NF-κB) has been observed in glioblastomas, while NF-κB inhibitor alpha (NFKBIA) inhibits the NF-κB signaling pathway under several physiological processes. However, the contribution of NFKBIA to glioblastomas is poorly understood. In the present study, using gene sequencing, we identified rs1957106 as a novel single nucleotide polymorphism (SNP) in NFKBIA in glioblastoma and found that it was more frequently present in glioblastoma patients. In addition, we examined the association between different genotypes of the rs1957106 SNP of NFKBIA and the gene copy number, mRNA level and protein expression of NFKBIA. The SNP rs1957106 CT and TT genotypes were found to be associated with lower NFKBIA protein levels and a poor prognosis of pateints with glioblastoma. Hence, by identifying rs1957106 as a novel SNP in NFKBIA in glioblastoma patients, we provide a new platform for further investigating the function of NFKBIA in the pathobiology of glioblastoma.

The cellular state determines the effect of melatonin on the survival of mixed cerebellar cell culture.

The constitutive activation of nuclear factor-κB (NF-κB), a key transcription factor involved in neuroinflammation, is essential for the survival of neurons in situ and of cerebellar granule cells in culture. Melatonin is known to inhibit the activation of NF-κB and has a cytoprotective function. In this study, we evaluated whether the cytoprotective effect of melatonin depends on the state of activation of a mixed cerebellar culture that is composed predominantly of granule cells; we tested the effect of melatonin on cultured rat cerebellar cells stimulated or not with lipopolysaccharide (LPS). The addition of melatonin (0.1 nM–1 µM) reduced the survival of naïve cells while inhibiting LPS-induced cell death. Melatonin (100 nM) transiently (15 min) inhibited the nuclear translocation of both NF-κB dimers (p50/p50, p50/RelA) and, after 60 min, increased the activation of p50/RelA. Melatonin-induced p50/RelA activity in naïve cells resulted in the transcription of inducible nitric oxide synthase (iNOS) and the production of NO. Otherwise, in cultures treated with LPS, melatonin blocked the LPS-induced activation of p50/RelA and the reduction in p50/p50 levels and inhibited iNOS expression and NO synthesis. Therefore, melatonin in vehicle-treated cells induces cell death, while it protects against LPS-induced cytotoxicity. In summary, we confirmed that melatonin is a neuroprotective drug when cerebellar cells are challenged; however, melatonin can also lead to cell death when the normal balance of the NF-κB pathway is disturbed. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response of melatonin.

Nuclear factor-κB activation in primary lymphoma of bone

Primary lymphoma of bone is a rare type of extranodal diffuse large B-cell lymphoma with a relatively favourable outcome. Recent scientific interest has focused on elucidating the role of nuclear factor-κB pathway in lymphomagenesis and its potential significance as a therapeutic target. In nodal B-cell non-Hodgkin lymphomas, constitutive activation of nuclear factor-κB appears to be involved in tumour cell survival, notably in the non-germinal centre type of diffuse large B-cell lymphoma. We investigated nuclear factor-κB activation via the classical and alternative pathway in primary lymphoma of bone, through immunohistochemical staining for nuclear factor-κB family members on tumour tissues of 50 patients. Nine cases (18 %) showed nuclear staining for p50, and one case showed nuclear co-expression of p52. None of the cases showed nuclear staining for c-Rel. The nuclear staining of p50 suggests that in a minority of primary lymphomas of bone nuclear factor-κB is constitutively activated via the classical pathway. In contrast to other extranodal types of diffuse large B-cell lymphoma, there was a lack of nuclear co-expression of p65, which might suggest activation of a different pathway. Activation of nuclear factor-κB through the alternative pathway does not appear to be significantly involved, as only one case showed significant nuclear expression for p52. Finally, nuclear expression of p50 was neither preferentially detected in non-germinal centre type or germinal centre type primary lymphoma of bone, nor related to poor prognosis. Therefore, in contrast to nodal diffuse large B-cell lymphoma, the nuclear factor-κB pathway does not appear to be an attractive therapeutic target in primary lymphoma of bone.

Involvement of Akt/NF-κB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo

BackgroundGlioblastoma is the most common and most aggressive form of malignant glioma and is very difficult to treat. Controlling tumour cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. Since constitutive activation of nuclear factor-κB (NF-κB) is necessary for tumour progression, NF-κB may be an important pharmacological target for this disease. Our study aimed to evaluate the antitumour effects of parthenolide, a NF-κB inhibitor, in two human glioblastoma cell lines (U87MG and U373) and in glioblastoma xenografts. Furthermore, we aimed to investigate the molecular mechanisms underlying these effects.MethodsThe anti-invasive and anti-angiogenic effects of parthenolide were analysed using in vitro invasion and angiogenesis assays. Parthenolide-induced growth inhibition of glioblastoma cells in vitro was determined using the MTT (methyl thiazolyl tetrazolium) assay. In addition, the effect of parthenolide on orthotropic implantation in vivo was evaluated using an intracerebral human glioblastoma xenograft model.ResultsWe found that parthenolide suppresses proliferation, invasion, and tumour- induced angiogenesis of glioblastoma cells. Molecular studies demonstrated that parthenolide suppresses gene and protein expression of angiogenic factors. Furthermore, parthenolide reduced Akt phosphorylation and activated mitochondrial signalling, suggesting that the antitumour function of parthenolide may be mediated not only by the inhibition of NF-κB but also by the inhibition of Akt signalling and the activation of apoptotic proteins. Parthenolide suppressed neovascularity and tumour growth in glioblastoma xenografts.ConclusionThe present study identified parthenolide as a new therapeutic agent for glioblastomas.

IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas

To clarify the relationships between marginal zone lymphomas (MZLs) and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas (WM/LPLs), immunoglobulin heavy chain variable gene (IGHV) features were analyzed and the occurrence of MYD88 L265P mutations was identified in a series of 123 patients: 53 MZLs from the spleen (SMZLs), 11 from lymph nodes (NMZLs), 28 mucosa-associated lymphatic tissue (MALT) lymphomas and 31 WM/LPLs. SMZLs were characterized by overrepresentation of IGHV1-2 gene rearrangements with a canonical motif, without selection pressure and with long CDR3 segments. NMZLs had increased frequencies of IGHV3 genes. The IGHV gene was unmutated in most cases, often with long CDR3 segments. MALT lymphomas were usually associated with a mutated IGHV gene, but with the absence of selection pressure. WM/LPLs were associated with an IGHV3-23 overrepresentation and high IGHV mutation rate, with features of selection pressure and short CDR3 segments. MYD88 L265P mutations were almost restricted exclusively to WM/LPL patients. Taken all diagnoses together, all patients with MYD88 L265P mutations had an immunoglobulin M peak and almost all patients except one had bone marrow infiltration. These results demonstrate that the history of antigen exposure of the four entities studied was different and MYD88 L265P was specifically associated with WM/LPLs. WM/LPL may thus be functionally associated with constitutive nuclear factor-κB activation.

Anti-neoplastic effects of fucoxanthin and its deacetylated product, fucoxanthinol, on Burkitt’s and Hodgkin’s lymphoma cells

Fucoxanthin (FX) is a natural carotenoid with reported antitumorigenic activity. This study explored the effects of FX and its deacetylated product, fucoxanthinol (FXOH), on B-cell malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma and Epstein-Barr virus-immortalized B cells. Both FX and FXOH reduced the viability of these malignant B cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during G1 phase and caspase-dependent apoptosis. FXOH was approximately twice more potent than FX in these activities. In contrast, normal peripheral blood mononuclear cells were resistant to FX and FXOH. Strong and constitutive activation of nuclear factor-κB (NF-κB) is a common characteristic of many B-cell malignancies, and FXOH suppressed constitutive NF-κB activity. NF-κB inhibition was accompanied by downregulation of NF-κB-dependent anti-apoptotic and cell cycle regulator gene products, including Bcl-2, cIAP-2, XIAP, cyclin D1 and cyclin D2. The results indicated that FX and FXOH are potentially useful therapeutic agents in B-cell malignancies characterized by aberrant regulation of NF-κB.

Biological aspects of angiogenesis in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by the aberrant expansion of malignant plasma cells within the bone marrow (BM). One of the hallmarks of this disease is the close interaction between myeloma cells and neighboring cells within the BM. Angiogenesis, through the activation of endothelial cells, plays an essential role in MM biology. In the current review, we describe the angiogenesis process in MM by identifying the interacting cells, the pro- and anti-angiogenic cytokines modulated, and the extracellular matrix degrading proteases liable to participate in the pathophysiology. Finally, we highlight the impact of hypoxia (through hypoxia-inducible factor-1) and constitutive activation of nuclear factor-κB in this tumor-induced neo-vascularization.

Abstract B171: Involvement of Akt/NF-κ B pathway in antitumor effect of parthenolide on glioblastoma cells.

Abstract Background: Glioblastoma is the most common and aggressive form of malignant glioma and is very difficult to treat. Controlling glioblastoma cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. Constitutive activation of Nuclear factor-κB (NF-κB) is crucial for tumor progression. Inhibition of NF-κB may be an important pharmacological target for the control of tumor progression. Our study aimed to evaluate the antitumour effects of parthenolide, NF-κB inhibitor, on two human glioma cell lines (U87MG, U373) and the molecular mechanisms underlying these effects. Methods: The anti-invasive effect of parthenolide was analysed by an in vitro invasion assay. An in vitro angiogenesis assay was also performed. In vitro growth inhibition of glioma cells by parthenolide was determined by the MTT assay. The effect of parthenolide on an orthotropic implantation model using athymic mice was also evaluated. Results: Parthenolide suppressed the proliferation of glioblastoma cells. Parthenolide also suppressed both the invasion of glioblastoma cells and tumor-induced angiogenesis. Molecular-based studies demonstrated that parthenolide suppressed gene and protein expression of angiogenic factors. Furthermore, parthenolide reduced the phosphorylation of Akt and activated mitochondrial cascade, suggesting that the antitumor effect of parthenolide may be mediated by the inhibition of the Akt signal and the activation of apoptotic proteins. An in vivo intracerebral human glioblastoma xenograft mouse model demonstrated that parthenolide suppressed neovascularity and tumor growth. Conclusions: The results of the present study suggest that parthenolide may be a new therapeutic agent for glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B171.

Inhibition of nuclear factor-κB activity by small interfering RNA in esophageal squamous cell carcinoma cell lines

Chemotherapy with 5-fluorouracil (5-FU) is commonly used in combination therapy for esophageal squamous cell carcinoma (ESCC), but its efficacy is limited in certain patients. Recent studies suggest that constitutive activation of nuclear factor-κB (NF-κB) has a critical role in tumorigenesis and is associated with poor prognosis and resistance to chemoradiation therapy in many types of human cancers. In the present study, we evaluated the effect of small interfering RNA targeting NF-κB (NF-κB siRNA) combined with 5-FU on the proliferation of two cell lines of cultured ESSCs. Immunofluorescence and immunoblot analyses revealed that the NF-κB protein was localized mostly in the cytoplasm of ESCCs. When cultured ESCCs were exposed to tumor necrosis factor-α, NF-κB was transferred to the nucleus and activated. ESCCs with activated NF-κB had poor sensitivity to 5-FU. When cells were transfected with NF-κB siRNA, the levels of NF-κB protein were significantly decreased in the cytoplasm and the nucleus. Transcriptional activity of NF-κB was significantly suppressed in cells treated with 5-FU and NF-κB siRNA compared to cells treated with 5-FU alone. 5-FU consistently suppressed proliferation of ESCCs in a dose-dependent manner, and this effect was significantly enhanced when combined with NF-κB siRNA. These results suggest that combination therapy of 5-FU with NF-κB siRNA may provide a new therapeutic option for ESCC.

B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice

AbstractThe ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose–dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.

A novel NF-κB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors

Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-κB (NF-κB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-κB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-κB, resulting in GC resistance. Taken together, interference of NF-κB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies.

Association of Constitutive Nuclear Factor-κB Activation With Aggressive Aspects and Poor Prognosis in Cervical Cancer

Although nuclear factor-kappaB (NF-kappaB) is generally believed to be involved in carcinogenesis, the relationship between NF-kappaB activation and progression of cervical cancer in clinical settings has not been reported. In this study, we investigated the association of NF-kappaB activation with aggressive aspects and prognosis in cervical cancer. Nuclear factor-kappaB subunits p65 and p50 were detected in 159 paraffin tissues including normal cervical, precancerous (squamous intraepithelial lesions), and cervical carcinoma tissues by immunohistochemistry. Nuclear factor-kappaB nuclear translocation and DNA-binding activity in precancerous or carcinoma tissues were examined by Western blot and electrophoretic mobility shift assay, respectively. A gradual NF-kappaB activation from normal cervical epithelial cells to precancerous and carcinoma cells was detected by immunohistochemistry (nuclear expression of p65 and p50, P < 0.001), Western blot (NF-kappaB nuclear translocation), and electrophoretic mobility shift assay (enhanced DNA-binding activity). In 79 cancer tissues, increased nuclear p65, an active NF-kappaB form, was correlated with poor tumor grade, lymphatic metastasis, interstitial invasion, and larger tumor size (P < 0.05). Similarly, increased nuclear p50 was correlated with poor tumor grade, interstitial invasion, and larger tumor size (P < 0.05). Moreover, increased nuclear p65 was associated with lower survival rate in patients with cancer (P < 0.05). Constitutive NF-kappaB activation is correlated to tumor progression, aggressive behaviors, and poor prognosis in cervical cancer, suggesting that NF-kappaB is a tumor promoter, a prognostic indicator, and a possible therapeutic target for this malignant disease.

TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas

AbstractThe constitutive activation of nuclear factor-κB (NF-κB) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-κB pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus–infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-κB activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-κB pathways may be advantageous for treatment.