Expression and Association of Tumor Necrosis Factor Receptor Associated Factor 4 (TRAF4) in Esophageal Squamous Cell Carcinoma.

BACKGROUND At present, there is no effective targeted therapy for esophageal squamous cell carcinoma (ESCC), and it is urgent to find new targets for the treatment of ESCC. TRAF4 has been regarded as a cause of carcinogenesis due to overexpression in many cancer types and participation in multiple signaling pathways. However, there are few studies on TRAF4 in ESCC worldwide. Its expression in ESCC and whether it affects the prognosis of patients still remain unclear. MATERIAL AND METHODS We detected the expressions of TRAF4, ki-67, and p53 in 100 cases of ESCC and 80 cases of adjacent normal esophageal squamous epithelium tissues by immunohistochemical technique. We further explored the relationship between TRAF4 and ESCC and its prognosis through statistical analysis. RESULTS TRAF4 was highly expressed in ESCC tissues and was mainly expressed in the cytoplasm. Overexpression of TRAF4 in ESCC was also associated with high expression of ki-67 and p53 (P<0.05). We also found that patients with high expression of TRAF4 had significantly lower OS than in patients with low TRAF4 expression (P<0.05). Overexpression of TRAF4 was an independent risk factor affecting the prognosis of patients (P<0.05). CONCLUSIONS We found that TRAF4 was highly expressed in ESCC tissues and was mainly expressed in the cytoplasm of cancer cells. Overexpression of TRAF4 was an independent risk factor affecting the overall prognosis of patients. The results indicated that TRAF4 may become a new target for the treatment of ESCC in the future.

TRAF4 Regulates Migration, Invasion, and Epithelial-Mesenchymal Transition via PI3K/AKT Signaling in Hepatocellular Carcinoma.

Overexpression of the tumor necrosis factor receptor-associated factor 4 (TRAF4) has been detected in many cancer types and is considered to foster tumor progression. However, the role of TRAF4 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that TRAF4 was highly expressed in HCC cell lines and HCC tissues compared with normal liver cell lines and adjacent noncancerous tissues. TRAF4 overexpression in HCC tissues was correlated with tumor quantity and vascular invasion. In vitro studies showed that TRAF4 was associated with HCC cell migration and invasion. An in vivo study verified that TRAF4 overexpression facilitated metastasis in nude mice. In addition, overexpressed TRAF4 promoted the phosphorylation of Akt and induced Slug overexpression, leading to downregulated E-cadherin and upregulated vimentin, while silencing TRAF4 moderated the phosphorylation of Akt and repressed the expression of Slug, which resulted in upregulated E-cadherin and downregulated vimentin. These effects were inversed after pretreatment of the PI3K/Akt inhibitor LY294002 or overexpression of constitutively active Akt1. Our study demonstrated that TRAF4 was involved in promoting HCC cell migration and invasion. The process was induced by the EMT through activation of the PI3K/Akt signaling pathway.

TRAF4 promotes the growth and invasion of colon cancer through the Wnt/ß-catenin pathway.

The tumor necrosis factor receptor-associated factor 4 (TRAF4) has been linked to carcinogenesis. However, the role of TRAF4 in colon cancer is still unclear. Therefore, we investigated the role of TRAF4 in colon cancer and the underlying mechanism. In the present study, we found that TRAF4 was overexpressed in colon cancer tissues and cells, and small interfering RNA (siRNA)-mediated gene knockdown of TRAF4 significantly inhibited cell proliferation, invasion and tumorigenesis, both in vitro and in vivo, but induced apoptosis in colon cancer cells. Furthermore, siRNA-TRAF4 significantly inhibited the expression levels of ß-catenin, cyclinD1, and c-myc proteins in colon cancer cells. Taken together, these results suggest that TRAF4 promoted colon cancer cell growth and invasion by potentiating the Wnt/ß-catenin pathway, suggesting that TRAF4 may be a potential molecular target for colon cancer prevention and therapy.

Proliferative role of TRAF4 in breast cancer by upregulating PRMT5 nuclear expression.

In this study, we examined protein arginine methyltransferase 5 (PRMT5) and tumor necrosis factor receptor-associated 4 (TRAF4) expression in breast cancer to find the interaction mechanism between the two. We examined TRAF4 and PRMT5 expression by immunohistochemistry and found that their expression is positively correlated in breast cancer. Besides, PRMT5 expression was significantly associated with histological type and tumor size (p < 0.05). PRMT5 nuclear expression was significantly associated with HER2 expression (p < 0.05). PRMT5 and TRAF4 were both overexpressed in breast cancer tissues and cells, and we found that PRMT5 binds to the zinc finger structures in TRAF4 by coimmunoprecipitation and Western blotting. We also tested the potential regulatory effect between TRAF4 and PRMT5. TRAF4 upregulated PRMT5 expression, which occurred predominantly in the nucleus, on which TRAF4 promotion of cell proliferation in breast cancer is mainly dependent. PRMT5 may play an important role in activation of the NF-κB signaling pathway.

Tumor necrosis factor receptor associated factor-4: an adapter protein overexpressed in metastatic prostate cancer is regulated by microRNA-29a.

The tumor necrosis factor receptor (TNFR)-associated factor 4 (TRAF4) is a member of TRAF family proteins that act as major signal transducers of the TNF receptor and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF4 has been reported to be overexpressed in various human cancers. However, the exact mechanisms that regulate the expression of TRAF4 still remain elusive. The objective of the present study was to investigate the regulatory mechanism of TRAF4 expression in prostate cancer. We initially identified microRNA-29a (miR­29a) as a possible candidate to bind TRAF4 3' untranslated region (3'UTR) by the algorithm, TargetScan. The expression of TRAF4 mRNA and protein was inversely associated with miR-29a expression in prostate cancer cell lines (LNCaP, DU145 and PC3). TRAF4 expression was reduced by the introduction of mimic miR-29a in LNCaP cells. Luciferase activity from the construct harboring wild-type TRAF4 3'UTR was reduced by the mimic miR-29a and this reduction was diminished by introducing mutations at the predicted miR-29a binding site. On the other hand, TRAF4 was upregulated when transfected with the inhibitor of miR-29a in DU145 and PC3 cells. TRAF4 was significantly upregulated in patients with metastatic prostate cancer compared to those with localized prostate cancer. Furthermore, there was a significant inverse correlation between TRAF4 and miR-29a expression in tumor tissues from radical prostatectomy. Considered together, our results suggest that the tumor suppressor microRNA, miR-29a, is one of the regulators of TRAF4 expression in metastatic prostate cancer.

Oncogene amplification in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.

TRAF4 is potently induced by TAp63 isoforms and localised according to differentiation in SCCHN.

p63, a member of the p53 family, is overexpressed in squamous cell carcinoma of the head and neck (SCCHN) and some other tumors of epithelial origin. As a transcription factor, p63 can bind to p53-type response elements and there is some overlap between p53 family transcriptional targets. Tumor necrosis factor receptor associated factor 4 (TRAF4) is a p53 regulated gene which is overexpressed in many human carcinomas. We investigated the involvement of p63 in regulation of TRAF4 and the expression of the TRAF4 protein in SCCHN. Disrupting endogenous p63 expression resulted in downregulation of TRAF4 mRNA and protein in an SCCHN cell line. Endogenous p63 bound to the TRAF4 promoter in vivo and reporter assays showed that p63, p73 and p53 can all transactivate TRAF4, with TAp63 isoforms being the most potent activators. The level of TRAF4 activation by TAp63 was two-fold higher than by p53, and TRAF4 was ten-fold more responsive to TAp63 than another p63-target, IGFBP3. Nuclear expression of TRAF4 was seen in normal oral epithelium and highly/moderately differentiated SCCHN, whereas cytoplasmic expression of TRAF4 was seen in poorly differentiated SCCHN. These results indicate that TRAF4 is a common target of p53 family members and that localization of TRAF4 is associated with differentiation of SCCHN cells.

Identification and characterization of proteins interacting with Traf4, an enigmatic p53 target.

Tumor necrosis factor receptor associated factor 4 (Traf4) mRNA expression is upregulated in various breast tumors and tumor cell lines. We previously identified Traf4 as a p53 target gene, and showed overexpression of Traf4 inhibited colony formation. However, basal Traf4 expression in cell lines does not appear to be dependent entirely on p53. To address the putative function of Traf4, a yeast 2-hybrid screen and coimmunoprecipitation/mass spectrometry experiments were performed to identify Traf4-interacting proteins. A yeast 2-hybrid using full length Traf4 as bait yielded several candidate interacting proteins including beta-catenin, GRIM19, PSMC3, p62 and dynamin. Although all of these proteins are novel interactors for Traf4, PSMC3 and p62 have been previously demonstrated to interact with the related protein, Traf6. Traf4 appears to enhance beta-catenin related transcription as well as to provide some protection of beta-catenin protein levels from p53-mediated degradation although a direct interaction was not observed in mammalian cells. To obtain interacting proteins using a more physiologically relevant environment, we immunoprecipitated Flag-tagged Traf4 followed by mass spectrometry which identified other novel Traf4 interacting proteins including Eg5, PRMT5, and MYH-9.