TNF-alpha-induced Protein 3 Research Articles

Ladostigil Reduces the Adenoside Triphosphate/Lipopolysaccharide-Induced Secretion of Pro-Inflammatory Cytokines from Microglia and Modulate-Immune Regulators, TNFAIP3, and EGR1.

Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1β, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10-11 M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1β and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats.

In vivo vitamin D target genes interconnect key signaling pathways of innate immunity.

The vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), its nuclear receptor VDR (vitamin D receptor) and hundreds of their target genes are not only key regulators of calcium homeostasis, but also important modulators of the immune system. Innate immune cells like monocytes use VDR for efficient differentiation and are very responsive to vitamin D. So far, most information on the gene regulatory function of vitamin D and its physiological impact had been obtained from in vitro studies using supraphysiological doses of 1,25(OH)2D3. Therefore, medical experiments like the study VitDHiD (NCT03537027), where 25 healthy individuals were supplemented once with a vitamin D3 bolus (80,000 IU), provide important insight into the response to vitamin D under in vivo conditions. In this study, we inspected 452 in vivo vitamin D target genes from peripheral blood mononuclear cells (PBMCs) detected in VitDHiD and found 61 of them involved in eight major KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of innate immunity. Under in vivo conditions in healthy individuals vitamin D either silences five pathways of innate immunity, stabilizes two and increases one, so that acute inflammation is suppressed and the release of cytokines is kept under control. A ranking of the 61 target genes by inducibility, basal expression and multiple involvements in the pathways highlighted the genes NFKBIA (NFκB inhibitor alpha), NFKBIZ, FOSL2 (FOS like 2, AP1 transcription factor subunit), JDP2 (Jun dimerization protein 2), PIK3R1 (phosphoinositide-3-kinase regulatory subunit 1), CLEC7A (C-type lectin domain containing 7A), DUSP6 (dual specificity phosphatase 6), NCF2 (neutrophil cytosolic factor 2), PLCB1 (phospholipase C beta 1), PLCG2 and TNFAIP3 (TNF alpha induced protein 3). In conclusion, vitamin D's in vivo effect on innate immunity in healthy adults is mediated by the interconnection of the pathways of neutrophil extracellular trap formation, Toll-like receptor, chemokine and phagosome signaling, NOD-like receptor, C-type lectin receptor, apoptosis and interleukin 17 through a limited set of proteins encoded by key target genes.

MEG3 alleviates ankylosing spondylitis by suppressing osteogenic differentiation of mesenchymal stem cells through regulating microRNA-125a-5p-mediated TNFAIP3.

Osteoblasts are important regulators of bone formation, but their roles in ankylosing spondylitis (AS) remain unclear. This study aims to explore the role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) MEG3 in AS. Serum from AS patients as well as AS mesenchymal stem cells (ASMSCs) and healthy donors mesenchymal stem cells (HDMSCs) was collected. Accordingly, poorly expressed MEG3 and TNF alpha induced protein 3 (TNFAIP3) as well as overexpressed microRNA-125a-5p (miR-125a-5p) were noted in the serum of AS patients and in ASMSCs during the osteogenic induction process. Meanwhile, the interaction among MEG3, miR-125a-5p, and TNFAIP3 was determined and their effect on osteoblast activity was examined in vitro and in vivo. Overexpression of MEG3 and TNFAIP3 or inhibition of miR-125a-5p was found to inactivate the Wnt/β-catenin pathway, thus suppressing osteogenic differentiation of MSCs. MEG3 competitively bound to miR-125a-5p to increase TNFAIP3 expression, thereby inactivating the Wnt/β-catenin pathway and repressing the osteogenic differentiation of MSCs. In proteoglycan (PG)-induced AS mouse models, MEG3 also reduced osteogenic activity of MSCs to inhibit AS progression through the miR-125a-5p/TNFAIP3/Wnt/β-catenin axis. Therefore, up-regulation of MEG3 or depletion of miR-125a-5p holds potential of alleviating AS, which sheds light on a new therapeutic strategy for AS treatment.

Paired Box 5-Induced LINC00467 Upregulation Promotes the Progression of Laryngeal Squamous Cell Cancer by Triggering the MicroRNA-4735-3p/TNF Alpha-Induced Protein 3 Pathway.

LINC00467 was reported as an oncogenic gene in different types of human cancers. In this study, we investigated the molecular mechanisms of LINC00467 in the tumorigenesis of laryngeal squamous cell cancer (LSCC). RT-qPCR was utilized to detect the mRNA expression of genes, and western blot assay was used to determine the protein levels of TNF alpha-induced protein 3 (TNFAIP3). The cell viability was detected by CCK-8 assay. Transwell assays were conducted to determine the cell migration and invasion of LSCC cells, and the cell cycle was assessed by flow cytometry. The association between paired box 5 (PAX5), LINC00467, miR-4735-3p, and TNFAIP3 was verified using ChIP, RNA pull-down, or luciferase reporter assays. In our study, we found that LINC00467 was upregulated in LSCC, and knockdown of LINC00467 suppressed cell viability and metastasis of LSCC. Besides, LINC00467 transcription could be activated by PAX5 in LSCC. Furthermore, LINC00467 acted as competitive endogenous RNA (ceRNA) for miR-4735-3p to accelerate LSCC progression. In the meantime, TNFAIP3 was identified as a downstream gene of miR-4735-3p. Finally, TNFAIP3 overexpression could overturn the effects of miR-4735-3p mimic on LSCC cellular activities. In conclusion, our results demonstrated that PAX5-induced LINC00467 facilitated LSCC progression by inhibiting miR-4735-3p to increase TNFAIP3 expression.

The ubiquitin-editing enzyme TNFAIP3 exerts neuroprotective roles in epilepsy rats through repressing inflammation.

The ubiquitin-editing enzyme TNF alpha-induced protein 3 (TNFAIP3) emerges protective roles in neurological disorder, such as cerebral trauma. However, the molecular mechanisms of TNFAIP3 in epilepsy are not very clear. Hereon, the epileptic mouse models and BV2 microglial cellular models were established by kainic acid (KA) and lipopolysaccharide (LPS) respectively. We found that TNFAIP3 was highly expressed in the hippocampus of epileptic mice. Besides, TNFAIP3 overexpression relieved the spatial learning and memory, reduced the hot plate latency, as well as inhibited neuronal apoptosis in KA-treated mice. In vivo and in vitro experiments indicated that inflammation, a key characteristic of epilepsy, was inhibited by TNFAIP3 upregulation, as evidenced by the downregulated expression of pro-inflammatory cytokine interleukin (IL)-1β and inducible NO synthase (iNOS), along with the decreased levels of NLRP3 inflammasome, which could activate inflammation. Collectively, we infer that TNFAIP3 relieves neuronal injury in epilepsy by suppressing inflammation.

Circ0008399 Interaction with WTAP Promotes Assembly and Activity of the m6A Methyltransferase Complex and Promotes Cisplatin Resistance in Bladder Cancer

Cisplatin (CDDP)-based chemotherapy is the first-line treatment for muscle-invasive and metastatic bladder cancer, yet most patients rapidly develop resistance. N6-methyladenosine (m6A) methylation is a pervasive RNA modification, and its specific role and potential mechanism in the regulation of CDDP chemosensitivity in bladder cancer remain unclear. Furthermore, studies have not yet fully elucidated whether circular RNA (circRNA) can directly regulate m6A modification of mRNA. Here we report upregulation of a novel circRNA, hsa_circ_0008399 (circ0008399), by eukaryotic translation initiation factor 4A3 (EIF4A3) in bladder cancer tissues and cell lines. Functionally, circ0008399 inhibited apoptosis of bladder cancer cells. Mechanistically, circ0008399 bound Wilms' tumor 1-associating protein (WTAP) to promote formation of the WTAP/METTL3/METTL14 m6A methyltransferase complex. Circ0008399 increased expression of TNF alpha-induced protein 3 (TNFAIP3) by increasing its mRNA stability in an m6A-dependent manner. In patients with bladder cancer, high expression of circ0008399 and WTAP was associated with poor outcomes. Importantly, activation of the circ0008399/WTAP/TNFAIP3 pathway decreased bladder cancer chemosensitivity to CDDP, and targeting the circ0008399/WTAP/TNFAIP3 axis enhanced the CDDP efficacy. Collectively, these findings give novel insights into circRNA-mediated regulation of m6A modifications and provide potential therapeutic targets for bladder cancer. SIGNIFICANCE: A newly characterized circRNA circ0008399 binds WTAP to modulate expression of target RNA through m6A modification and reduce cisplatin sensitivity in bladder cancer, implicating the potential therapeutic value of targeting this axis.

Retrospective analyses of other iatrogenic immunodeficiency-associated lymphoproliferative disorders in patients with rheumatic diseases.

SummaryOther iatrogenic immunodeficiency‐associated lymphoproliferative disorders (OIIA‐LPDs) occur in patients receiving immunosuppressive drugs for autoimmune diseases; however, their clinicopathological and genetic features remain unknown. In the present study, we analysed 67 patients with OIIA‐LPDs, including 36 with diffuse large B‐cell lymphoma (DLBCL)‐type and 19 with Hodgkin lymphoma (HL)‐type. After discontinuation of immunosuppressive drugs, regression without relapse was achieved in 22 of 58 patients. Spontaneous regression was associated with Epstein–Barr virus positivity in DLBCL‐type (P = 0·013). The 2‐year overall survival and progression‐free survival (PFS) at a median follow‐up of 32·4 months were 92·7% and 72·1% respectively. Furthermore, a significant difference in the 2‐year PFS was seen between patients with DLBCL‐type and HL‐type OIIA‐LPDs (81·0% vs. 40·9% respectively, P = 0·021). In targeted sequencing of 47 genes in tumour‐derived DNA from 20 DLBCL‐type OIIA‐LPD samples, histone‐lysine N‐methyltransferase 2D (KMT2D; eight, 40%) and tumour necrosis factor receptor superfamily member 14 (TNFRSF14; six, 30%) were the most frequently mutated genes. TNF alpha‐induced protein 3 (TNFAIP3) mutations were present in four patients (20%) with DLBCL‐type OIIA‐LPD. Cases with DLBCL‐type OIIA‐LPD harbouring TNFAIP3 mutations had shorter PFS and required early initiation of first chemotherapy. There were no significant factors for spontaneous regression or response rates according to the presence of mutations. Overall, OIIA‐LPDs, especially DLBCL‐types, showed favourable prognoses.

Ethanol suppresses the pulmonary response to Streptococcus pneumoniae: Role of NOD2 and NF-kB signaling in alveolar macrophages exposed to muramyl dipeptide.

Abstract Alcohol use decreases host immune defense and increases susceptibility to pathogens such as Streptococcus pneumoniae. NOD2 is an intracellular pattern recognition receptor that binds muramyl dipeptide (MDP), a cell wall component of S. pneumoniae, and activates NF-kB signaling. To limit inflammation, NF-kB signaling is negatively regulated in part by TNFAIP3 (TNF Alpha Induced Protein 3), or A20. We hypothesize that ethanol impairs NOD2 signaling and A20 expression in alveolar macrophages, leading to excessive inflammation and reduced bacterial clearance after infection. For in vivo experiments, we gavaged mice once daily for 3 consecutive days with water or ethanol (blood alcohol concentration ~ 80 mg/dL at 30 min), and infected with 104 cfu S. pneumoniae. In parallel experiments, MH-S cells were treated with 25 mM ethanol and 10 μg/ml MDP. We found that ethanol treated animals, compared to vehicle treated, had impaired respiratory function (17% lowered respiration rate [p < 0.01] and 33% increased penh [p = 0.09]) and increased lung bacteria (log10 increase of 0.8 [p = 0.11]) at 24 hours. RNA sequencing of alveolar macrophages from vehicle- or ethanol-gavaged mice revealed that ethanol intake led to 25% less Nod2 and 60% less A20 (p < 0.05) in the absence of infection. These results were confirmed by qPCR in ethanol-treated MH-S cells (40% decrease in Nod2 and 50% reduction in A20 [p < 0.01]), along with 15% lower A20 protein levels, which was sustained after MDP stimulation (29% less A20 compared to vehicle-treated, stimulated cells). Future experiments aim to further investigate ethanol’s effect on A20 regulation of NF-kB signaling, and will provide insight into immune dysregulation seen in alcohol-consuming patients with pneumonia.

Identifying the p65-Dependent Effect of Sulforaphene on Esophageal Squamous Cell Carcinoma Progression via Bioinformatics Analysis.

Understanding the mechanism by which sulforaphene (SFE) affects esophageal squamous cell carcinoma (ESCC) contributes to the application of this isothiocyanate as a chemotherapeutic agent. Thus, we attempted to investigate SFE regulation of ESCC characteristics more deeply. We performed gene set enrichment analysis (GSEA) on microarray data of SFE-treated ESCC cells and found that differentially expressed genes are enriched in TNFα_Signaling_via_the_NFκB_Pathway. Coupled with the expression profile data from the GSE20347 and GSE75241 datasets, we narrowed the set to 8 genes, 4 of which (C-X-C motif chemokine ligand 10 (CXCL10), TNF alpha induced protein 3 (TNFAIP3), inhibin subunit beta A (INHBA), and plasminogen activator, urokinase (PLAU)) were verified as the targets of SFE. RNA-sequence (RNA-seq) data of 182 ESCC samples from The Cancer Genome Atlas (TCGA) were grouped into two phenotypes for GSEA according to the expression of CXCL10, TNFAIP3, INHBA, and PLAU. The enrichment results proved that they were all involved in the NFκB pathway. ChIP-seq analyses obtained from the Cistrome database indicated that NFκB-p65 is likely to control the transcription of CXCL10, TNFAIP3, INHBA, and PLAU, and considering TNFAIP3 and PLAU are the most significantly differentially expressed genes, we used chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) to verify the regulation of p65 on their expression. The results demonstrated that SFE suppresses ESCC progression by down-regulating TNFAIP3 and PLAU expression in a p65-dependent manner.

Abstract P3-05-18: The complex landscape of breast cancer immune evasion and its implications to personalized and combined immunotherapy

Abstract Cancer immunotherapy activates the patient's immune system to recognize and kill cancer cells. The efficacy of immunotherapy in human breast cancer (BRCA) is hindered by the poor understanding of the immune system's anti-tumor response and tumor evasive mechanisms, and the lack of optimized treatment regimens for each BRCA patient. There are 6 known evasion mechanisms (M) by which tumors can escape immune surveillance. M1: immunosuppression via interleukin 10 (IL-10) and tumor growth factor beta (TGF-β); M2: induction of tolerance by cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and its ligands (PD-L1/PD-L2), and interferon gamma (IFN-γ); M3: apoptosis resistance via the expression of anti-apoptotic molecules, namely baculoviral IAP repeat-containing protein 3 (BIRC3), TNF receptor associated factor 1 (TRAF1), and TNF alpha induced protein 3 (TNFAIP3); M4: counterattack via decoy death receptors; M5: impaired antigen presentation by decreasing beta 2 microglobulin (B2M) and human leukocyte antigens A and B expression (HLA-A and HLA-B); and M6: ignorance caused by the absence of a danger signal or an antigen. Yet, which mechanisms or combination of mechanisms BRCA tumors utilize is unknown. Moreover, the currently used biomarkers for the choice of immunotherapy are not ideal, as for example, not all PD-L1+ patients respond to anti-PD-L1 treatment, and certain PD-L1- patients respond. Thus, there is a need for more precise biomarkers. Using the sequential biclustering method on the RNA-sequenced data of BRCA from The Cancer Genome Atlas (TCGA), eighty-one percent of BRCA patients were grouped into 7 different clusters (CL) of immune evasion subtypes. We found that the majority of breast cancer tumors utilize multiple immune evasion mechanisms. Tumors may use from 1 up to 4 combinatorial mechanisms simultaneously. We also identified classifier genes that would help sort patients based on their evasion mechanisms, and guide the choice of immunotherapy. This is especially important since the immune evasion molecules may not be the ideal biomarkers for the best choice of immunotherapy. Moreover, we showed that triple negative breast cancer (TNBC) patients were significantly associated with CL2 (M5). Citation Format: Bou-Dargham MJ, Liu Y, Sang Q-XA, Zhang J. The complex landscape of breast cancer immune evasion and its implications to personalized and combined immunotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-18.

TNFAIP3 inhibits migration and invasion in nasopharyngeal carcinoma by suppressing epithelial mesenchymal transition.

TNF alpha induced protein 3 (TNFAIP3), a member of zinc finger protein family, is a gene whose expression level is promptly induced by the tumor necrosis factor. In this study, the clinical significance of TNFAIP3 was analyzed based on available samples in The Cancer Genome Atlas database. TNFAIP3 downregulation was associated with distant metastasis and worse patient prognosis. TNFAIP3-overexpressing and TNFAIP3-knockdown NPC cell line models were established through plasmid-mediated overexpression and small interfering RNA (siRNA), respectively. Cell migration and invasion capacities were evaluated by wound-healing and transwell assays. Functional studies indicated that TNFAIP3 knockdown promoted migration and invasion, whereas TNFAIP3 overexpression alleviated these functions. Western blot analysis was used to examine protein changes from TNFAIP3 overexpression and knockdown, in which TNFAIP3 promoted the protein expression of E-cadherin and suppressed vimentin expression. Our data suggested that TNFAIP3 inhibited migration and invasion by suppressing epithelial mesenchymal transition in NPC.

Mutation analysis of tumor necrosis factor alpha-induced protein 3 gene in Hodgkin lymphoma

AimsSurvival and proliferation of Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (CHL), are dependent on constitutive activation of nuclear factor kB (NF-κB). A20, encoded by TNF alpha-induced protein 3 (TNFAIP3), one of the inhibitors of NF-kB, was found to be inactivated by deletions and/or point mutations in CHL. MethodsTNFAIP3 mutations were examined in 37 patients with CHL by using PCR and direct sequencing. In addition, protein expression of A20 was evaluated by immunohistochemistry. Epstein–Barr virus (EBV) status of HL samples was determined by EBV EBER chromogenic in situ hybridization (ISH). ResultsWe identified 8 mutation positive cases in a collective of 37 investigated cases (22%). Mutations were most frequent in the nodular sclerosis subtype. Our results revealed the tendency that cases harboring A20 mutations were negative for A20 staining. None of A20 mutation-positive CHL cases showed EBV infection. ConclusionsOur study confirms the involvement of the TNFAIP3 tumor suppressor gene in CHL. A20 may represent a suppressor of human lymphoma and provide a critical molecular link between chronic inflammation and cancer. None of A20 mutation-positive CHL cases showed EBV infection. This fact suggests complementing functions of TNFAIP3 inactivation and EBV infection in CHL pathogenesis and may represent an interesting point of further investigations.

Upregulation of the C/EBP β LAP isoform could be due to decreased TNFAIP3/TNIP1 expression in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus

Objectives: We aimed to examine CCAAT/enhancer-binding protein β (C/EBP β), TNF-alpha-induced protein 3 (TNFAIP3), and TNFAIP3-interacting protein 1 (TNIP1) expression in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients to assess their relationship in SLE pathogenesis.Methods: C/EBP β, TNIP1, and TNFAIP3 expression was assessed in PBMCs from 20 SLE patients and 20 controls by western blotting. The correlation between C/EBP β/TNFAIP3/TNIP1 expression and SLE disease activity was determined by Spearman’s rank. C/EBP β, TNIP1, and TNFAIP3 levels in THP-1 cells, THP-1 cells transfected with plasmids encoding TNFAIP3 shRNA, and THP-1 cells infected with lentiviral vectors encoding TNIP1 shRNA were assessed by western blotting.Results: C/EBP β LAP isoform expression was increased and LIP/TNFAIP3/TNIP1 expression was decreased in SLE patients. LAP expression was positively correlated with SLE disease activity; TNFAIP3 and TNIP1 expression was negatively correlated with SLE disease activity. LAP expression was increased in SLE patients with proteinuria and elevated anti-dsDNA antibody, as well as in THP-1 cells transfected with plasmids encoding TNFAIP3 shRNA and THP-1 cells infected with lentiviral vectors encoding TNIP1 shRNA.Conclusions: C/EBP β/TNFAIP3/TNIP1 is associated with SLE activity. The upregulated expression of C/EBP β LAP could be caused by reduced TNFAIP3/TNIP1 expression.

Abstract 4033: A20 gene promoter methylation confers resistance to colorectal carcinoma cell lines

Abstract Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors and most neoplasms show hypermethylation of one or more genes. A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. Previously, we elucidated the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. Loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH were studied in a microarray cohort of CRC cases and cell lines for mutations in A20 gene and A20 promoter methylation were also screened. We further elucidated the mechanism of A20 inactivation in colorectal carcinoma cell lines in this extended study. Methylation status of A20 promoter in CRC cell lines correlated with the degree of A20 protein expression: unmethylated cell line SW948 showed the highest expression of A20 protein whereas LOVO, which was methylated, showed almost complete loss of A20 expression. CRC cell lines with high levels of A20 expression (SW948 and COLO320), intermediate levels (HCT-15 and DLD-1) and very low levels (LOVO-1) were immunoblotted for detection of A20 protein expression. Treatment of methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2′deoxy-cytidine resulted in partial demethylation and re-expression of A20 gene, suggesting the role of methylation in silencing the gene. In addition, demethylation of A20 gene by 5-aza-2′deoxy-cytidine also decreased cell viability by 15-20X in CRC cell lines, suggesting that A20 expression may play an important role in CRC tumorigenesis. In conclusion, A20 methylation may be a promising molecular marker to predict patient outcome in colorectal cancer, and may be used as therapeutic demethylating target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4033. doi:1538-7445.AM2012-4033

TNFAIP3 Maintains Intestinal Barrier Function and Supports Epithelial Cell Tight Junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3−/− mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3−/− mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

Clinico-pathological significance of TNF alpha-induced protein3 (TNFAIP3) in Middle Eastern colorectal carcinoma

A20, also known as TNF-alpha-induced protein3 (TNFAIP3), is a well-known negative regulator of the NF-kB activation pathway (Coornaert et al.2009; Stilo et al.2008) and functions as a tumor suppressor gene in lymphomas (Honma et al.2009; Kato et al.2009). Aberrant NF-kB regulation in colorectal carcinoma (CRC) is associated with poor prognosis and resistance to therapy (Horst et al.2009; Kojima et al.2004; Rakitina et al.2003; Scartozzi et al.2007). The current study deals with an interesting and novel topic about oncogenic role of inactivation of tumor suppressor gene A20 in colorectal cancer, the mechanism of A20 dysfunction, and its potential as a novel prognostic marker and therapeutic target. This is the first report of A20 alterations in colon cancer where we comprehensively investigated the genomic profile and epigenetic changes in A20 gene. A better understanding of NF-kB modulators could lead to development of effective therapeutic strategies to target activated NF-kB (Rakitina et al.2003). Mechanistically, A20 exerts its NF-kB inhibitory role in TNF signaling by the cooperative and dual-functioning actions of the deubiquitinating and ubiquitinating enzyme activities located at the N and C terminus, respectively, of the A20 protein (Coornaert et al.2009). The amino terminal of A20 protein containing the deubiquitinating enzyme targets removal of ubiquitin moieties from receptor-interacting protein (RIP1), an essential mediator of the proximal tumor necrosis factor receptor signaling complex in NF-kB activation (Wertz et al.2004). This is followed by polyubiquitination of RIP by the ubiquitin ligase located at the carboxy terminal of A20 protein, thereby targeting RIP for proteosomal degradation. Recent studies have shown that A20 functions as a tumor suppressor gene in several types of lymphoma wherein not only is A20 inactivated by promoter methylation (Chanudet et al.2009), but it is also subject to gene deletion and mutation. Although A20 has been reported as a novel tumor suppressor gene in NHLs, the role of A20 in colorectal carcinoma is not yet known. In this study, we screened for A20 gene copy number changes and A20 expression in a large tissue microarray cohort of 434 colorectal carcinomas and adenoma samples. We further investigated the incidence of A20 mutations and A20 promoter methylation and the relationship of A20 alterations with CRC progression and phenotype. The incidence of A20 mutations, A20 deletions, A20 promoter hyper-methylation was 2.5%, 15.3%, and 50.8%, respectively. We analyzed the entire coding region of A20 gene in 116 CRC samples and 14 CRC cell lines and found that only three CRC samples and two CRC cell lines (LOVO and HCT-15) were mutated. Most of the A20 mutations found in Hodgkin's and mediastinal lymphomas were nonsense or frameshift mutations that prevented production of full-length A20 protein (Chanudet et al.2009; Honma et al.2009; Schmitz et al.2009). The three A20 mutations found in our study were replacement mutations. Two of these three mutations in our study were localized in the zinc finger (ZF) domains at the C terminus of the protein. The A20 ZF domain functions as an E3 ubiquitin ligase, adding K48-linked polyubiquitin chains to RIP1 and targeting the protein for proteasomal degradation. Most of the mutations seen in the three CRC samples as well as the two cell lines were missense mutations (four out of five). A20 deletions were seen in 15.3% (65/424) of the CRC samples analyzed by FISH. Homozygous deletions, i.e., presence of two centromeric signals but no A20 signals, were seen in four of the 65 (3.1%) deleted cases. Although A20 mutations were not observed in the CRC with A20 deletions, six cases showed co-existence of A20 methylation and A20 deletions. Thus, A20 alterations are widely prevalent in colorectal carcinoma, and A20 promoter methylation leads to reduced expression of A20 protein. A20 promoter hyper-methylation was correlated with loss of protein expression and associated with histology subtype of adenocarcinomas differentiated tumors and CRC located on the left side. A20 protein expression was significantly higher in colorectal adenomas as compared to colorectal carcinomas. A20 expression identifies a subgroup of colorectal cancers which show a poor outcome across all subgroups, in stage III alone, stage II and III subgroup, and also stage III and IV subgroup. Reduced A20 expression was an independent prognostic marker in colorectal carcinoma. Together, our results establish A20 as a key tumor suppressor molecule in colorectal carcinogenesis and identify a subgroup of CRC patients with reduced A20 expression showing a poor overall survival. A20 promoter methylation is the key epigenetic mechanism leading to suppression of A20 expression. Interestingly, complete loss of A20 expression results from multiple hits like A20 deletion and or mutation with methylation in accordance with Knudson's two-hit hypothesis. Our findings indicate that A20 is a tumor suppressor in CRC that can be used as a potential prognostic biomarker.

Abstract 2104: Frequent inactivation of A20 and its prognostic significance in Middle Eastern colorectal carcinoma

Abstract A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway in hematolymphoid neoplasms. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. However, role of A20 gene in CRC is unknown. Therefore, we sought to elucidate the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. We investigated the genetic and epigenetic abnormalities of A20 gene in CRC. In a tissue microarray cohort of 434 CRC, we studied loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH. We also screened 116 random CRC and 14 CRC cell lines for mutations in A20 gene and A20 promoter methylation. Of the 116 CRC samples, A20 deletions were seen in 15.3% and incidence of A20 mutation was 2.5%; most of the mutations (4/5) were missense mutations. A20 promoter hyper-methylation was seen in 50.8% and was correlated with loss of protein expression (p=0.0079). A20 inactivation, defined as loss or reduced expression of protein was observed in 63% of CRC and A20 inactivation was an independent prognostic marker for poor survival in all CRC. In addition A20 expression retained its prognostic value in the following subgroups: Stage III; Stage II and III; and Stage III and IV. A20 was inactivated in majority of the CRC due to promoter methylation and can be targeted to modulate NF-κb expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2011-2104

Identification of Marker Genes That Discriminate between Different Myeloproliferative Disorders and Normal Individuals Including RUNX3 (AML2).

Myeloproliferative disorders (MPD) are clonal stem cell diseases, which are defined by excessive production of cells in one or more hematopoietic lineages. The molecular mechanisms underlying the development of agnogenic myeloid metaplasia (AMM), polycythemia vera (PV), and essential thrombocythemia (ET) are currently poorly understood. We performed microarray analysis on 26 granulocyte samples from AMM (4), ET (5), PV (6) and normal (11) individuals in order to identify genes that: 1) distinguish MPD from normal samples and 2) distinguish between these diseases. Our data revealed a group of genes that were differentially expressed in MPD compared to normal or were differentially expressed between the three different diseases. Cytokine signaling has often been reported in development or progression of these diseases. Several of these aberrantly expressed genes included those involved in TGF-beta signaling. RUNX3 (AML2), a transcription factor that is involved in the signaling cascade mediated by TGF-beta, was markedly overexpressed in MPD (AMM 4.9-fold; PV 8.1-fold; ET 9.5-fold) compared to normal. TIEG1 (TGF-beta-inducible early growth response 1) was upregulated in AMM (6.4-fold), PV (9.5-fold), ET (16.7-fold) compared to normal. Moreover, TNFAIP3 (TNF alpha-induced protein 3) was overexpressed in MPD (AMM 13.8-fold; PV 11.6-fold; ET 9.3-fold) compared to normal, which might also suggest a potential role of TNF-alpha signaling in the pathogenesis of MPD. We also found several genes that could discriminate each disease from each other. For example, ZNF292 (zinc finger protein 292) was overexpressed in PV and ET (7.1-fold and 2.9-fold, respectively), but AMM had similar expression levels to normals. CCNL2 (cyclin L2) was overexpressed in ET (2.4-fold), unchanged in PV and downregulated by 5-fold in AMM compared to normal. Expression levels of all of these genes were confirmed by real-time PCR, and immunohistochemistry staining of normal and MPD samples for RUNX3 was comparable to our array data. We hypothesize that RUNX3 might play a role in myelopoiesis. RUNX3 mRNA levels in HL-60 cells cultured with ATRA (100 and 1000 nM) markedly (16 to 20-fold) and maximally increased by day 3. Levels subsided to control levels by day 7, suggesting that RUNX3 may be initially involved in HL-60 differentiation but returns to normal levels as these cells matured or underwent terminal differentiation. In MPD, the high levels of RUNX3 in the aberrant neutrophils might indicate that these cells are blocked at approximately the equivalent timepoint (day 3 for HL-60). Their differentiation program has been initiated, but they cannot undergo the final stages of terminal maturation as reflected by their high RUNX3 levels. In summary, this study identified genes, whose expression levels may serve as diagnostic markers in MPD.