CPNE1 Is a Useful Prognostic Marker and Is Associated with TNF Receptor-Associated Factor 2 (TRAF2) Expression in Prostate Cancer.

BACKGROUND CPNE1 plays a vital role in regulating cell differentiation. The clinical and biological values of CPNE1 in prostate cancer are still unclear. The aim of this study was to investigate the clinicopathological value of CPNE1 and the association of CPNE1 with TRAF2 expression in patients with prostate cancer. MATERIAL AND METHODS CPNE1 expression in prostate cancer was analyzed using Gene Expression Omnibus (GEO) databases. The Cancer Genome Atlas (TCGA) dataset was used to investigate the association of CPNE1 expression with TRAF2 expression in prostate cancer. The association of CPNE1 expression with recurrence-free survival in patients was also analyzed using the TCGA dataset. Immunohistochemistry assay was performed to examine CPNE1 expression in 65 normal prostate samples and 114 prostate cancer samples. The recurrence-free survival in patients was evaluated using Kaplan-Meier curves and log-rank test. In addition, multivariate and univariate analyses of prognostic factors were investigated by Cox regression. The effect of CPNE1 on TRAF2 expression was explored in human prostate cancer DU-145 cells. RESULTS Our results showed that expression level of CPNE1 is higher in prostate cancer than in normal prostate tissues (P=0.006). In the GSE35988 dataset, CPNE1 expression was found to be upregulated in castration-resistant prostate cancer compared with non-castration-resistant prostate cancer (P<0.001). Furthermore, we found that CPNE1 high expression was significantly related to tumor stage, Gleason score, and poorer biochemical recurrence-free survival in prostate cancer patients. Co-expression analysis of TCGA data showed that CPNE1 is significantly associated with TRAF2 expression. CPNE1 overexpression can upregulate TRAF2 expression in prostate cancer DU-145 cells as determined by Western blotting and immunofluorescence assays. CONCLUSIONS Overall, our findings suggest that CPNE1 is a valuable prognostic marker for evaluating recurrence-free survival and is positively related to TRAF2 expression in prostate cancer.

Prognostic Significance of TNFR-Associated Factor 1 and 2 (TRAF1 and TRAF2) in Glioblastoma.

BACKGROUND TNFR-associated factor 1 (TRAF1) and TRAF2 have been demonstrated to inhibit apoptosis and promote cell survival in glioblastoma (GBM) cells with experiments in vitro. However, their clinical and prognostic significance have not been elucidated. MATERIAL AND METHODS In our study, we for the first time investigated the expression of TRAF1 and TRAF2 in 105 GBM tissues. Furthermore, we evaluated their clinical significance, including their association with clinicopathologic factors and prognostic value. The association with clinicopathologic factors was assessed by chi-square test. The relation of TRAF1/2 expression to survival rate was assessed by Kaplan-Meier method and Cox-regression model. RESULTS We demonstrated that TRAF1 expression had no significant prognostic value for GBM. On the contrary, high expression of TRAF2 can predict poorer prognosis of GBM and was identified as an independent biomarker in GBM prognosis. CONCLUSIONS High expression of TRAF2 was identified as an independent biomarker in GBM prognosis, indicating TRAF2 as a novel drug target in GBM treatment.

TRAF2 is a Valuable Prognostic Biomarker in Patients with Prostate Cancer.

BACKGROUND TRAF2 exerts important functions in regulating the development and progression of cancer. The aim of this study is to investigate whether TRAF2 is a valuable prognostic biomarker and to determine if it regulates TRAIL-induced apoptosis in prostate cancer. MATERIAL AND METHODS Microarray gene expression data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to determine TRAF2 expression in prostate cancer. TRAF2 expression in prostate cancer was further investigated by immunohistochemistry assay. Kaplan-Meier curves and log-rank test were used to assess the recurrence-free rate. Cox regression was used to analyze prognostic factors. Effects of TRAF2 on regulating TRAIL-induced apoptosis in DU-145 cells were further investigated. RESULTS We found that TRAF2 was significantly upregulated in prostate cancer compared with normal prostate samples (P<0.001). In addition, compared with primary prostate cancer, TRAF2 was upregulated in metastatic prostate cancer (P=0.006). Furthermore, our results showed that high expression of TRAF2 was significantly associated with tumor stage of prostate cancer (P=0.035). TRAF2 high expression was associated with poorer recurrence-free survival in prostate cancer patients (P=0.013). TRAF2 was found to be a valuable independent prognostic factor for predicting recurrence-free survival (P=0.026). In addition, the present results indicate that TRAF2 affects TRAIL-induced apoptosis in prostate cancer DU-145 cells via regulating cleaved Caspase-8 and c-Flip expression. CONCLUSIONS TRAF2 could be a novel prognostic biomarker for predicting recurrence-free survival in patients with prostate cancer, which might be associated with the effects of TRAF2 in regulating TRAIL-induced apoptosis in prostate cancer cells via c-Flip/Caspase-8 signalling.

Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A.

Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-ß) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy(2J)/dy(2J) mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-ß protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-ß and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy(2J)/dy(2J) mouse model of MDC1A.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Suppressive role of miR-502-5p in breast cancer via downregulation of TRAF2.

TRAF2 promotes cancer cell survival, proliferation and metastasis through the NF-κB pathway by directly interacting with various TNF recepors. However, the molecular mechanism of TRAF2 dysregulation in breast cancer remains to be elucidated. In the present study, miR-502-5p was predicted as a potential regulator of TRAF2. miR-502-5p was significantly downregulated in breast cancer tissues when compared to the level in paired normal breast tissues. The breast cancer cell lines including MCF-7 and MDA-MB-231 expressed a lower level of miR-502-5p when compared to the level in the non-malignant breast epithelial cell line MCF-10A. In vitro, miR-502-5p enhanced early apoptosis and inhibited proliferation of breast cancer cells. Luciferase reporter assay results showed that miR-502-5p could bind to the 3'-untranslated region of the TRAF2 gene, thus, exerting an inhibitory effect on TRAF2. Furthermore, silencing of TRAF2 exhibited effects similar to those of exogenous miR­502-5p, while overexpression of TRAF2 partially abrogated miR-502-5p-mediated suppression in breast cancer cells. In conclusion, miR-502-5p may act as a tumor-suppressor gene by targeting oncogenic TRAF2 in breast cancer and, therefore, may be a potential diagnostic and anticancer therapeutic marker for breast cancer.

Activation-induced tumor necrosis factor receptor-associated factor 3 (Traf3) alternative splicing controls the noncanonical nuclear factor κB pathway and chemokine expression in human T cells.

The noncanonical nuclear factor κB (ncNFκB) pathway regulates the expression of chemokines required for secondary lymphoid organ formation and thus plays a pivotal role in adaptive immunity. Whereas ncNFκB signaling has been well described in stromal cells and B cells, its role and regulation in T cells remain largely unexplored. ncNFκB activity critically depends on the upstream NFκB-inducing kinase (NIK). NIK expression is negatively regulated by the full-length isoform of TNF receptor-associated factor 3 (Traf3) as formation of a NIK-Traf3-Traf2 complex targets NIK for degradation. Here we show that T cell-specific and activation-dependent alternative splicing generates a Traf3 isoform lacking exon 8 (Traf3DE8) that, in contrast to the full-length protein, activates ncNFκB signaling. Traf3DE8 disrupts the NIK-Traf3-Traf2 complex and allows accumulation of NIK to initiate ncNFκB signaling in activated T cells. ncNFκB activity results in expression of several chemokines, among them B cell chemoattractant (CxCL13), both in a model T cell line and in primary human CD4(+) T cells. Because CxCL13 plays an important role in B cell migration and activation, our data suggest an involvement and provide a mechanistic basis for Traf3 alternative splicing and ncNFκB activation in contributing to T cell-dependent adaptive immunity.

Intestinal protein expression profile identifies inflammatory bowel disease and predicts relapse.

To date, most studies have applied individual factors as indicators of disease classification and prognosis. The aim of this study is to determine whether clustering analysis of protein expression profiles in intestinal epithelia improves classification and prognosis in patients with inflammatory bowel disease (IBD). One hundred and twenty Crohn's disease (CD) patients, 117 ulcerative colitis (UC) patients and 120 cases of nonspecific colitis provided intestinal biopsy samples for tissue microarray (TMA). Both unsupervised and supervised analyses were used for evaluation of clustering and association with relapse. There was a significant concordance between cluster groups based on immunostaining data of TMA and clinical classification in distinguishing IBD from nonspecific colitis (kappa= 0.498, p<0.001). CD27, CD70, CD40, TRAF3, TRAF4 and TRAF2 presented similar immunostaining features, which were different from clusters of CD154, CD80 and TRAF5. Moreover, higher expression of TRAF2 was a predictor of relapse in patients with UC (p=0.006).Thus, protein expression profiles can distinguish IBD and nonspecific colitis, and combination analysis protein expression profiles show that TRAF2 can predict relapse of UC.

Gene expression of tumor necrosis factor receptor associated-factor (TRAF)-1 and TRAF-2 in inflammatory bowel disease.

OBJECTIVE: This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor (TRAF)-1 and TRAF-2 in patients with inflammatory bowel disease (IBD). METHODS: Immunostaining, western blot and real-time polymerase chain reaction (PCR) were used to detect the expression of TRAF-1 and TRAF-2 in colonic mucosa of IBD patients and control. Furthermore, serum protein levels of TRAF-1 and TRAF-2 were measured by ELISA and the receiver operating characteristic (ROC) curve was used to determine their diagnostic value. RESULTS: The expression of TRAF-1 and TRAF-2 was significantly higher in inflamed and non-inflamed tissues of IBD patients than those in control (P < 0.05). Moreover, inflamed tissues had higher TRAF-1 and TRAF-2 expression than non-inflamed tissues (P < 0.05). Both TRAF-1 and TRAF-2 were shown to have a fair to excellent value in the differentiation of control and IBD patients with the area under the ROC curve (AUROC) of 0.680-1.000 (P < 0.001). CONCLUSION: The activation of TRAF-1 and TRAF-2 may be early events in the pathogenesis of IBD and their functions are not quite the same.

TNFR-associated factor 2 deficiency in B lymphocytes predisposes to chronic lymphocytic leukemia/small lymphocytic lymphoma in mice.

We have previously shown that transgenic (tg) mice expressing in B lymphocytes both BCL-2 and a TNFR-associated factor 2 (TRAF2) mutant lacking the really interesting new gene and zinc finger domains (TRAF2DN) develop small lymphocytic lymphoma and chronic lymphocytic leukemia with high incidence (Zapata et al. 2004. Proc. Nat. Acad. Sci. USA 101: 16600-16605). Further analysis of the expression of TRAF2 and TRAF2DN in purified B cells demonstrated that expression of both endogenous TRAF2 and tg TRAF2DN was negligible in Traf2DN-tg B cells compared with wild-type mice. This was the result of proteasome-dependent degradation, and rendered TRAF2DN B cells as bona fide TRAF2-deficient B cells. Similar to B cells with targeted Traf2 deletion, Traf2DN-tg mice show expanded marginal zone B cell population and have constitutive p100 NF-κB2 processing. Also, TRAF3, X-linked inhibitor of apoptosis, and Bcl-X(L) expression levels were increased, whereas cellular inhibitors of apoptosis 1 and 2 levels were drastically reduced compared with those found in wild-type B cells. Moreover, consistent with previous results, we also show that TRAF2 was required for efficient JNK and ERK activation in response to CD40 engagement. However, TRAF2 was deleterious for BCR-mediated activation of these kinases. In contrast, TRAF2 deficiency had no effect on CD40-mediated p38 MAPK activation but significantly reduced BCR-mediated p38 activation. Finally, we further confirm that TRAF2 was required for CD40-mediated proliferation, but its absence relieved B cells of the need for B cell activating factor for survival. Altogether, our results suggest that TRAF2 deficiency cooperates with BCL-2 in promoting chronic lymphocytic leukemia/small lymphocytic lymphoma in mice, possibly by specifically enforcing marginal zone B cell accumulation, increasing X-linked inhibitor of apoptosis expression, and rendering B cells independent of B cell activating factor for survival.

A common haplotype of the TNF receptor 2 gene modulates endotoxin tolerance.

Endotoxin tolerance is characterized by the suppression of further TNF release upon recurrent exposure to LPS. This phenomenon is proposed to act as a homeostatic mechanism preventing uncontrolled cytokine release such as that observed in bacterial sepsis. The regulatory mechanisms and interindividual variation of endotoxin tolerance induction in man remain poorly characterized. In this paper, we describe a genetic association study of variation in endotoxin tolerance among healthy individuals. We identify a common promoter haplotype in TNFRSF1B (encoding TNFR2) to be strongly associated with reduced tolerance to LPS (p = 5.82 × 10(-6)). This identified haplotype is associated with increased expression of TNFR2 (p = 4.9 × 10(-5)), and we find basal expression of TNFR2, irrespective of genotype and unlike TNFR1, is associated with secondary TNF release (p < 0.0001). Functional studies demonstrate a positive-feedback loop via TNFR2 of LPS-induced TNF release, confirming this previously unrecognized role for TNFR2 in the modulation of LPS response.

EBV LMP2A affects LMP1-mediated NF-kappaB signaling and survival of lymphoma cells by regulating TRAF2 expression.

A mechanism used by Epstein-Barr virus (EBV) for in vitro transformation of B cells into lymphoblastoid cell lines (LCLs) is activation of the NF-kappaB pathway, which is largely mediated by the EBV latent membrane protein 1 (LMP1). LMP1 is coexpressed with LMP2A in many EBV-associated lymphoid malignancies. Since inhibition of NF-kappaB leads to apoptosis of EBV-infected LCLs and lymphoma cell lines, we sought to determine whether LMP1 alone, or in combination with other viral proteins, is responsible for initiating NF-kappaB activation in these cells, thereby playing a role in cell survival. We found that suppression of LMP1 by RNA interference results in inhibition of basal NF-kappaB and induction of apoptosis. Unexpectedly, knockdown of LMP2A also resulted in comparable decrease of NF-kappaB activity and apoptosis. We report that LMP2A protein controls the expression of TRAF2 mRNA, which in turn is necessary for signaling by LMP1. Our data contrast with previous studies showing that transfected LMP1 can signal in the absence of LMP2A or TRAF2, and demonstrate that both LMP2A and TRAF2 are required for survival in naturally infected lymphoma cells and LCLs. These results also support LMP1, LMP2A, and TRAF2 as potential therapeutic targets in a subset of EBV-associated lymphoid malignancies.

A20 zinc finger protein inhibits TNF-induced apoptosis and stress response early in the signaling cascades and independently of binding to TRAF2 or 14-3-3 proteins.

A20 zinc finger protein is a negative regulator of tumor necrosis factor (TNF)-induced signaling pathways leading to apoptosis, stress response and inflammation. A20 has been shown to bind to TNF-receptor-associated factor 2 (TRAF2) and 14-3-3 chaperone proteins. Our data indicate that the zinc finger domain of A20 is sufficient and that neither TRAF2 nor 14-3-3 binding is necessary for the inhibitory effects of A20. Mutations in the 14-3-3 binding site of A20 did, however, result in a partial cleavage of A20 protein suggesting that 14-3-3 chaperone proteins may stabilize A20. Furthermore, we show that A20 acts early in TNF-induced signaling cascades blocking both TNF-induced rapid activation of c-Jun N-terminal kinase and processing of the receptor-associated caspase-8. Taken together our data indicate that the zinc finger domain of A20 contains all necessary functional domains required for the inhibition of TNF signaling and that A20 may function at the level of the receptor signaling complex.