Functional analyses of TRAF6 gene in Argopecten scallops.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

TRAF4-Mediated LAMTOR1 Ubiquitination Promotes mTORC1 Activation and Inhibits the Inflammation-Induced Colorectal Cancer Progression.

Mechanistic target of rapamycin complex 1 (mTORC1) is a conserved serine/threonine kinase that integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activation requires tethering to lysosomes by the Ragulator-Rag complex. However, the dynamic regulation of the interaction between Ragulator and Rag guanosine triphosphatase (GTPase) remains unclear. In this study, that LAMTOR1, an essential component of Ragulator, is dynamically ubiquitinated depending on amino acid abundance is reported. It is found that the E3 ligase TRAF4 directly interacts with LAMTOR1 and catalyzes the K63-linked polyubiquitination of LAMTOR1 at K151. Ubiquitination of LAMTOR1 by TRAF4 promoted its binding to Rag GTPases and enhanced mTORC1 activation, K151R knock-in or TRAF4 knock-out blocks amino acid-induced mTORC1 activation and accelerates the development of inflammation-induced colon cancer. This study revealed that TRAF4-mediated LAMTOR1 ubiquitination is a regulatory mechanism for mTORC1 activation and provides a therapeutic target for diseases involving mTORC1 dysregulation.

TRAF4 regulates ubiquitination-modulated survivin turnover and confers radioresistance.

Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. Despite continuous improvement in treatment strategies, recurrence or persistence of cancer after radiotherapy is still inevitable, highlighting the need to identify therapeutic resistance factors and develop effective methods for NPC treatment. Herein, we found that TRAF4 is overexpressed in NPC cells and tissues. Knockdown TRAF4 significantly increased the radiosensitivity of NPC cells, possibly by inhibiting the Akt/Wee1/CDK1 axis, thereby suppressing survivin phosphorylation and promoting its degradation by FBXL7. TRAF4 is positively correlated with p-Akt and survivin in NPC tissues. High protein levels of TRAF4 were observed in acquired radioresistant NPC cells, and knockdown of TRAF4 overcomes radioresistant in vitro and the xenograft mouse model. Altogether, our study highlights the TRAF4-survivin axis as a potential therapeutic target for radiosensitization in NPC.

TRAF4 is crucial for ST2+ memory Th2 cell expansion in IL-33-driven airway inflammation.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4-cre Traf4fl/fl) mice and investigated the role of TRAF4 in memory Th2 cells expressing IL-33 receptor (ST2, suppression of tumorigenicity 2) (ST2+ mTh2 cells) in IL-33-mediated type 2 airway inflammation. We found that in vitro-polarized TRAF4-deficient (CD4-cre Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4-cre Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies.

TRAF4-mediated nonproteolytic ubiquitination of androgen receptor promotes castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) poses a major clinical challenge with the androgen receptor (AR) remaining to be a critical oncogenic player. Several lines of evidence indicate that AR induces a distinct transcriptional program after androgen deprivation in CRPCs. However, the mechanism triggering AR binding to a distinct set of genomic loci in CRPC and how it promotes CRPC development remain unclear. We demonstrate here that atypical ubiquitination of AR mediated by an E3 ubiquitin ligase TRAF4 plays an important role in this process. TRAF4 is highly expressed in CRPCs and promotes CRPC development. It mediates K27-linked ubiquitination at the C-terminal tail of AR and increases its association with the pioneer factor FOXA1. Consequently, AR binds to a distinct set of genomic loci enriched with FOXA1- and HOXB13-binding motifs to drive different transcriptional programs including an olfactory transduction pathway. Through the surprising upregulation of olfactory receptor gene transcription, TRAF4 increases intracellular cAMP levels and boosts E2F transcription factor activity to promote cell proliferation under androgen deprivation conditions. Altogether, these findings reveal a posttranslational mechanism driving AR-regulated transcriptional reprogramming to provide survival advantages for prostate cancer cells under castration conditions.

USP7 mediates TRAF4 deubiquitination to facilitate the malignant phenotype of ovarian cancer via the RSK4/PI3K/AKT axis.

Background: Ubiquitin-specific peptidase 7 (USP7) is upregulated in multiple human cancers, including ovarian cancer; however, its functional role in the latter remains largely unknown. Methods: We conducted quantitative real-time PCR to detect the expression of USP7, TRAF4, and RSK4 in ovarian cancer cell lines. In addition, Western blotting served to determine USP7, TRAF4, RSK4, PI3K, and AKT (protein kinase B,PKB) protein levels and USP7 expression in the tissues was detected by immunohistochemical staining. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate cell viability, transwell assays to evaluate cell migration and invasion, and co-immunoprecipitation to evaluate TRAF4 ubiquitination. Results: The results showed USP7 and TRAF4 upregulation, and RSK4 downregulation in ovarian cancer cell lines. Knocking down USP7 suppressed viability, migration, and invasion of ovarian cancer cells; TRAF4 knockdown and RSK4 overexpression had similar effects in ovarian cancer cells. TRAF4 is deubiquitinated and stabilized by USP7, whereas RSK4 is negatively regulated by TRAF4. A mouse xenograft model confirmed that knocking down USP7 suppressed ovarian tumor growth by regulating the TRAF4/RSK4/PI3K/AKT axis. Conclusion: Knocking down USP7 decreased the proliferation, migration, and invasion of ovarian cancer cells and suppressed ovarian tumor growth in mice. Mechanistically, USP7 increased TRAF4 ubiquitination, promoting its degradation and leading to RSK4 upregulation.

TRAF4 Silencing Induces Cell Apoptosis and Improves Retinoic Acid Sensitivity in Human Neuroblastoma.

Neuroblastoma (NB) is a pediatric malignancy that arises in the peripheral nervous system, and the prognosis in the high-risk group remains dismal, despite the breakthroughs in multidisciplinary treatments. The oral treatment with 13-cis-retinoic acid (RA) after high-dose chemotherapy and stem cell transplant has been proven to reduce the incidence of tumor relapse in children with high-risk neuroblastoma. However, many patients still have tumors relapsed following retinoid therapy, highlighting the need for the identification of resistant factors and the development of more effective treatments. Herein, we sought to investigate the potential oncogenic roles of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family in neuroblastoma and explore the correlation between TRAFs and retinoic acid sensitivity. We discovered that all TRAFs were efficiently expressed in neuroblastoma, but TRAF4, in particular, was found to be strongly expressed. The high expression of TRAF4 was associated with a poor prognosis in human neuroblastoma. The inhibition of TRAF4, rather than other TRAFs, improved retinoic acid sensitivity in two human neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells. Further in vitro studies indicated that TRAF4 suppression induced retinoic acid-induced cell apoptosis in neuroblastoma cells, probably by upregulating the expression of Caspase 9 and AP1 while downregulating Bcl-2, Survivin, and IRF-1. Notably, the improved anti-tumor effects from the combination of TRAF4 knockdown and retinoic acid were confirmed in vivo using the SK-N-AS human neuroblastoma xenograft model. In conclusion, the highly expressed TRAF4 might be implicated in developing resistance to retinoic acid treatment in neuroblastoma, and the combination therapy with retinoic acid and TRAF4 inhibition may offer significant therapeutic advantages in the treatment of relapsed neuroblastoma.

TRAF4-mediated ubiquitination-dependent activation of JNK/Bcl-xL drives radioresistance.

The E3 ligase TNF receptor-associated factor 4 (TRAF4) is upregulated and closely associated with tumorigenesis and the progression of multiple human malignancies. However, its effect on radiosensitivity in colorectal cancer (CRC) has not been elucidated. The present study found that TRAF4 was significantly increased in CRC clinical tumor samples. Depletion of TRAF4 impaired the malignant phenotype of CRC cells and sensitized irradiation-induced cell death. Irradiation activated the c-Jun N-terminal kinases (JNKs)/c-Jun signaling via increasing JNKs K63-linked ubiquitination and phosphorylation. Furthermore, c-Jun activation triggered the transcription of the antiapoptotic protein Bcl-xL, thus contributing to the radioresistance of CRC cells. TRAF4 was positively correlated with c-Jun and Bcl-xL, and blocking TRAF4 or inhibiting Bcl-xL with inhibitor markedly promoted ionizing radiation (IR)-induced intrinsic apoptosis and sensitized CRC cells to radiotherapy in vitro and in vivo. Our findings illustrate a potential mechanism of radioresistance, emphasizing the clinical value of targeting the TRAF4/Bcl-xL axis in CRC therapy.

SRC-3/TRAF4 facilitates ovarian cancer development by activating the PI3K/AKT signaling pathway.

OBJECTIVE: Ovarian cancer is the seventh most common cancer in women, and it causes many deaths in women worldwide. Patients with ovarian cancer have a poor prognosis and low survival rate. This study aimed to explore the role of the SRC-3/TRAF4/PI3K/AKT pathway in ovarian cancer development. METHODS: SRC-3 and TRAF4 expression in ovarian cancer cell lines were assessed using qRT-PCR and western-blotting. The expression of SRC-3 and TRAF4 in ovarian cancer cells was downregulated by transient transfection with sh-RNAs. An MTT assay was performed to evaluate cell proliferation. Cell migration and invasion were measured using a Transwell assay. Cell stemness was detected using a cell spheroidization assay and western blotting. The expression levels of stem cell factors and PI3K/AKT pathway proteins were determined by qRT-PCR and western blot analysis. RESULTS: SRC-3 and TRAF4 were upregulated in ovarian cancer cell lines. TRAF4 is a downstream factor of SRC-3, and the protein level of TRAF4 was regulated by SRC-3. SRC-3 knockdown reduced TRAF4 expression. Silencing SRC-3 or TRAF4 inhibited cell proliferation, migration, and invasion, as well as the expression of stem cell factors. Furthermore, sh-TRAF4 as well as treatment with LY294002, the PI3K/Akt inhibitor, inhibited the phosphorylation of Akt and PI3K, thus repressing the activation of PI3K/AKT signaling pathway in ovarian cancer cell lines. However, TRAF4 overexpression reversed the effect of SRC-3 silencing on cell proliferation, migration, invasion, and stemness. CONCLUSION: Our study demonstrated that SRC-3/TRAF4 promotes ovarian cancer cell growth, migration, invasion, and stemness by activating the PI3K/AKT pathway.

De Novo PACSIN1 Gene Variant Found in Childhood Lupus and a Role for PACSIN1/TRAF4 Complex in Toll-like Receptor 7 Activation.

OBJECTIVE: Increased Toll-like receptor 7 (TLR-7) signaling leading to the production of type I interferon (IFN) is an important contributor to human systemic lupus erythematosus (SLE). Protein kinase C and casein kinase substrate in neurons 1 (PACSIN1), a molecule that regulates synaptic vesicle recycling, has been linked to TLR-7/TLR-9-mediated type I IFN production in humans and mice, but the underlying mechanism is unknown. We undertook this study to explore the pathogenicity and underlying mechanism of a de novo PACSIN1 missense variant identified in a child with SLE. METHODS: PACSIN1 Q59K de novo and null variants were introduced into a human plasmacytoid dendritic cell line and into mice using CRISPR/Cas9 editing. The effects of the variants on TLR-7/TLR-9 signaling in human and mouse cells, as well as PACSIN1 messenger RNA and IFN signature in SLE patients, were assessed using real-time polymerase chain reaction and flow cytometry. Mechanisms were investigated using luciferase reporter assays, RNA interference, coimmunoprecipitation, and immunofluorescence. RESULTS: We established that PACSIN1 forms a trimolecular complex with tumor necrosis factor receptor-associated factor 4 (TRAF4) and TRAF6 that is important for the regulation of type I IFN. The Q59K mutation in PACSIN1 augments binding to neural Wiskott-Aldrich syndrome protein while it decreases binding to TRAF4, leading to unrestrained TRAF6-mediated activation of type I IFN. Intriguingly, PACSIN1 Q59K increased TLR-7 but not TLR-9 signaling in human cells, leading to elevated expression of IFNß and IFN-inducible genes. Untreated SLE patients had high PACSIN1 expression in peripheral blood cells that correlated positively with IFN-related genes. Introduction of the Pacsin1 Q59K mutation into mice caused increased surface TLR-7 and TRAIL expression in B cells. CONCLUSION: PACSIN1 Q59K increases IFNß activity through the impairment of TRAF4-mediated inhibition of TLR-7 signaling, possibly contributing to SLE risk.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

Stabilization of MCL-1 by E3 ligase TRAF4 confers radioresistance.

The E3 ligase TNF receptor-associated factor 4 (TRAF4) is frequently overexpressed and closely related to poor prognosis in human malignancies. However, its effect on carcinogenesis and radiosensitivity in oral squamous cell carcinoma (OSCC) remains unclear. The present study found that TRAF4 was significantly upregulated in primary and relapsed OSCC tumor tissues. Depletion of TRAF4 markedly improved the sensitivity of OSCC cells to irradiation (IR) treatment, showing that tumor cell proliferation, colony formation and xenograft tumor growth were reduced. Mechanistically, IR promoted the interaction between TRAF4 and Akt to induce Akt K63-mediated ubiquitination and activation. TRAF4 knockout inhibited the phosphorylation of Akt and upregulated GSK3ß activity, resulting in increased myeloid cell leukemia-1 (MCL-1) S159 phosphorylation, which disrupted the interaction of MCL-1 with Josephin domain containing 1 (JOSD1), and ultimately induced MCL-1 ubiquitination and degradation. Moreover, TRAF4 was positively correlated with MCL-1 in primary and in radiotherapy-treated, relapsed tumor tissues. An MCL-1 inhibitor overcame radioresistance in vitro and in vivo. Altogether, the present findings suggest that TRAF4 confers radioresistance in OSCC by stabilizing MCL-1 through Akt signaling, and that targeting TRAF4 may be a promising therapeutic strategy to overcome radioresistance in OSCC.

Grouper TRIM23 exerts antiviral activity against iridovirus and nodavirus.

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.

TRAF4 Promotes the Proliferation of Glioblastoma by Stabilizing SETDB1 to Activate the AKT Pathway.

The process of ubiquitination regulates the degradation, transport, interaction, and stabilization of substrate proteins, and is crucial for cell signal transduction and function. TNF receptor-associated factor 4, TRAF4, is a member of the TRAF family and is involved in the process of ubiquitination as an E3 ubiquitin protein ligase. Here, we found that TRAF4 expression correlates with glioma subtype and grade, and that TRAF4 is significantly overexpressed in glioblastoma and predicts poor prognosis. Knockdown of TRAF4 significantly inhibited the growth, proliferation, migration, and invasion of glioblastoma cells. Mechanistically, we found that TRAF4 only interacts with the Tudor domain of the AKT pathway activator SETDB1. TRAF4 mediates the atypical ubiquitination of SETDB1 to maintain its stability and function, thereby promoting the activation of the AKT pathway. Restoring SETDB1 expression in TRAF4 knockdown glioblastoma cells partially restored cell growth and proliferation. Collectively, our findings reveal a novel mechanism by which TRAF4 mediates AKT pathway activation, suggesting that TRAF4 may serve as a biomarker and promising therapeutic target for glioblastoma.

TRAF4 promotes the malignant progression of high-grade serous ovarian cancer by activating YAP pathway.

High-grade serous ovarian cancer (HGSOC) accounts for the majority of deaths caused by epithelial ovarian cancer. The specific molecular changes attributable to the pathogenesis of HGSOC are still largely unknown. TRAF4 has been identified to be up-regulated in certain cancers. However, the role and mechanism of TRAF4 in HGSOC remain unclear. In this study, we aim to explore the prognostic value and function of TRAF4 in HGSOC. Immunohistochemical staining and prognostic analysis were used to estimate the prognosis value of TRAF4 in HGSOC. Cell counting assays, colony formation assays, sphere formation assays and tumorigenic assays were used to explore the function of TRAF4 in ovarian cancer cells. Furthermore, RNA-seq, qPCR and western blotting were performed to investigate the molecular mechanism of TRAF4 in ovarian cancer cells. The results showed that TRAF4 was significantly higher expressed in ovarian cancer than normal ovarian epithelium. Moreover, high expression of TRAF4 was significantly associated with shorter overall survival and recurrence-free survival in HGSOC. Knockdown of TRAF4 significantly inhibited the proliferation and tumorigenicity of ovarian cancer cells, whereas overexpression of TRAF4 promoted the proliferation and tumorigenicity of ovarian cancer cells both in vitro and in vivo. Mechanistically, our study demonstrated that TRAF4 expression was positively correlated with the YAP pathway gene signatures, and the malignant progression induced by TRAF4 was inhibited after silencing YAP signaling by its selective inhibitor. In conclusion, our findings suggested that TRAF4 promoted the malignant progression of ovarian cancer cells by activating YAP pathway and might serve as a prognostic biomarker for HGSOC.

Novel Findings in Teleost TRAF4, a Protein Acts as an Enhancer in TRIF and TRAF6 Mediated Antiviral and Inflammatory Signaling.

Tumor necrosis factor receptor-associated factors (TRAFs) are important adaptor molecules that play important roles in host immune regulation and inflammatory responses. Compared to other members of TRAFs, the function of TRAF4 in vertebrate immunity remains unclear, especially in teleosts. In the present study, TRAF4 ortholog was cloned and identified in large yellow croaker (Larimichthys crocea), named as Lc-TRAF4. The open reading frame (ORF) of Lc-TRAF4 is 1,413 bp and encodes a protein of 470 amino acids (aa), which is consisted of a RING finger domain, two zinc finger domains, and a MATH domain. The genome organization of Lc-TRAF4 is conserved in teleosts, amphibians, birds, and mammals, with 7 exons and 6 introns. Quantitative real-time PCR analysis revealed that Lc-TRAF4 was broadly distributed in various organs/tissues of healthy large yellow croakers and could be significantly up-regulated in the gill, intestine, spleen, head kidney, and blood under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations. Notably, luciferase assays showed that overexpression of Lc-TRAF4 could significantly induce the activation of IRF3, IRF7, and type I IFN promoters, with the RING finger and zinc finger domains function importantly in such promoter activation. Confocal microscopy revealed that Lc-TRAF4 is located in the cytoplasm, whereas the deletion of the RING finger, zinc finger or MATH domain showed little effect on the subcellular localization of Lc-TRAF4. Interestingly, Lc-TRAF4 overexpression could significantly enhance Lc-TRIF and Lc-TRAF6 medicated IRF3 and IRF7 promoter activation. In addition, co-expression of Lc-TRAF4 with Lc-TRIF or Lc-TRAF6 could significantly induce the expression of antiviral and inflammation-related genes, including IRF3, IRF7, ISG15, ISG56, Mx, RSAD2, TNF-α, and IL-1ß compared to the only overexpression of Lc-TRAF4, Lc-TRIF or Lc-TRAF6. These results collectively imply that Lc-TRAF4 functions as an enhancer in Lc-TRIF and Lc-TRAF6 mediated antiviral and inflammatory signaling.

TRAF4 Maintains Deubiquitination of Caveolin-1 to Drive Glioblastoma Stemness and Temozolomide Resistance.

Glioblastoma (GBM) is the most common type of primary adult brain tumor. Glioma stem cell (GSC) residence and temozolomide (TMZ) resistance in GBM both contribute to poor patient outcome. TRAF4 is a scaffold protein with E3 ubiquitin ligase activity that has recently been discovered to promote invasion and metastasis in several malignancies, but the effects and functions of TRAF4 in GBM remain to be determined. Here, we report that TRAF4 is preferentially overexpressed in GSCs and is required for stem-like properties as well as TMZ sensitivity in GBM cells. TRAF4 specifically interacted with the N-terminal tail of Caveolin-1 (CAV1), an important contributor to the tumorigenicity of GBM cells. TRAF4 regulated CAV1 stability by preventing ZNRF1-mediated ubiquitination and facilitating USP7-mediated deubiquitination independently of its E3 ubiquitin ligase catalytic activity. TRAF4-mediated stabilization of CAV1 activated protumorigenic AKT/ERK1/2 signaling, and disruption of this axis resulted in defects in stemness maintenance. In addition, expression of TRAF4 and CAV1 was positively correlated and predicted poor prognosis in human GBM samples. Screening of common nervous system drugs identified risperidone interaction with TRAF4, and risperidone treatment resulted in the dissociation of TRAF4 and CAV1. Importantly, pharmacologic inhibition of TRAF4 with risperidone potently inhibited self-renewal, abrogated tumorigenicity, and reversed TMZ resistance in GBM. Overall, TRAF4-mediated stabilization of CAV1 promotes stemness and TMZ resistance in GBM, providing a therapeutic strategy that could improve patient outcomes. SIGNIFICANCE: The identification of a TRAF4/Caveolin-1 axis that plays a crucial role in malignant progression of glioblastoma provides new insights into the function of TRAF4 in ubiquitin signaling and suggests TRAF4 as a potential therapeutic target.

Overexpression of TRAF4 promotes lung cancer growth and EGFR-dependent phosphorylation of ERK5.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is overexpressed in a variety of carcinomas of different origins, but its role in tumorigenesis remains incompletely understood. Previous studies suggest that TRAF4 promotes epidermal growth factor receptor (EGFR) activation in non-small cell lung cancer (NSCLC). However, the downstream signaling pathway of TRAF4-mediated EGFR activation, as well as its effects on tumor cells, have not been fully elucidated. Here we report that TRAF4 overexpression is associated with increased activity of extracellular signal-regulated kinase 5 (ERK5) in NSCLC tissues. Activation of ERK5 was dependent on TRAF4-mediated EGFR activation, since inhibition of either TRAF4 or EGFR dramatically abolished phosphorylation of ERK5. Mechanistically, EGFR recruited mitogen-activated protein kinase kinase kinase 3 (MEKK3), an upstream kinase of ERK5, in a TRAF4-dependent manner. Thus, our data suggest that an EGFR-TRAF4-MEKK3-ERK5 axis promotes the proliferation of tumor cells, and this may be a potential target for therapeutic intervention of NSCLC.

TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor-associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells. IMPLICATIONS: Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

The Synergistic Cooperation between TGF-ß and Hypoxia in Cancer and Fibrosis.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine regulating homeostasis and immune responses in adult animals and humans. Aberrant and overactive TGF-ß signaling promotes cancer initiation and fibrosis through epithelial-mesenchymal transition (EMT), as well as the invasion and metastatic growth of cancer cells. TGF-ß is a key factor that is active during hypoxic conditions in cancer and is thereby capable of contributing to angiogenesis in various types of cancer. Another potent role of TGF-ß is suppressing immune responses in cancer patients. The strong tumor-promoting effects of TGF-ß and its profibrotic effects make it a focus for the development of novel therapeutic strategies against cancer and fibrosis as well as an attractive drug target in combination with immune regulatory checkpoint inhibitors. TGF-ß belongs to a family of cytokines that exert their function through signaling via serine/threonine kinase transmembrane receptors to intracellular Smad proteins via the canonical pathway and in combination with co-regulators such as the adaptor protein and E3 ubiquitin ligases TNF receptor-associated factor 4 (TRAF4) and TNF receptor-associated factor 6 (TRAF6) to promote non-canonical pathways. Finally, the outcome of gene transcription initiated by TGF-ß is context-dependent and controlled by signals exerted by other growth factors such as EGF and Wnt. Here, we discuss the synergistic cooperation between TGF-ß and hypoxia in development, fibrosis and cancer.