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1.  TWEAK Promotes Peritoneal Inflammation 
PLoS ONE  2014;9(3):e90399.
Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r = 0.491, p = 0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD.
PMCID: PMC3944020  PMID: 24599047
2.  TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells 
Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-κB (NF-κB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-κB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast – and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.
PMCID: PMC1779446  PMID: 16945157
3.  Functional Expression of TWEAK and the Receptor Fn14 in Human Malignant Ovarian Tumors: Possible Implication for Ovarian Tumor Intervention 
PLoS ONE  2013;8(3):e57436.
The aim of this current study was to investigate the expression of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in human malignant ovarian tumors, and test TWEAK’s potential role on tumor progression in cell models in-vitro. Using immunohistochemistry (IHC), we found that TWEAK and its receptor Fn14 were expressed in human malignant ovarian tumors, but not in normal ovarian tissues or in borderline/benign epithelial ovarian tumors. High levels of TWEAK expression was detected in the majority of malignant tumors (36 out of 41, 87.80%). Similarly, 35 out of 41 (85.37%) malignant ovarian tumors were Fn14 positive. In these malignant ovarian tumors, however, TWEAK/Fn14 expression was not corrected with patients’ clinical subtype/stages or pathological features. In vitro, we demonstrated that TWEAK only inhibited ovarian cancer HO-8910PM cell proliferation in combination with tumor necrosis factor-α (TNF-α), whereas either TWEAK or TNF-α alone didn’t affect HO-8910PM cell growth. TWEAK promoted TNF-α production in cultured THP-1 macrophages. Meanwhile, conditioned media from TWEAK-activated macrophages inhibited cultured HO-8910PM cell proliferation and invasion. Further, TWEAK increased monocyte chemoattractant protein-1 (MCP-1) production in cultured HO-8910PM cells to possibly recruit macrophages. Our results suggest that TWEAK/Fn14, by activating macrophages, could be ovarian tumor suppressors. The unique expression of TWEAK/Fn14 in malignant tumors indicates that it might be detected as a malignant ovarian tumor marker.
PMCID: PMC3587594  PMID: 23469193
4.  Expression of TweakR in breast cancer and preclinical activity of enavatuzumab, a humanized anti-TweakR mAb 
The receptor for the cytokine TWEAK (TweakR) is a cell surface member of the tumor necrosis factor receptor superfamily with diverse biological roles. TNFRSF family members are appealing therapeutic targets in oncology due to their aberrant expression and function in tumor cells. The goal of the current study was to examine the potential of TweakR as a therapeutic target in breast cancer.
Expression of TweakR in primary breast cancer tissues and metastases was characterized using immunohistochemistry. To determine the functional relevance of TweakR, breast cancer cell lines were treated in vitro and in vivo with enavatuzumab, a humanized mAb against TweakR.
Overexpression of TweakR was observed in infiltrating tumors compared to normal adjacent breast tissues, and strong staining of TweakR was observed in all subtypes of invasive ductal breast cancer. In addition, a positive correlation of TweakR and HER2 expression and co-localization were observed, irrespective of ER status. TweakR expression was also observed in bone metastasis samples from primary breast cancer but rarely in benign tumors. Enavatuzumab inhibited the in vitro growth of TweakR-expressing breast cancer cell lines, and this activity was augmented by cross-linking the mAb. In addition, enavatuzumab significantly inhibited the in vivo growth of multiple breast cancer xenograft models including a model of metastasis.
TweakR is highly expressed in all subtypes of invasive ductal breast cancer, and enavatuzumab administration exhibited a dose-dependent inhibition of primary tumor growth and lung metastasis and enhanced the antitumor activity of several chemotherapy agents currently used to treat breast cancer. These data provide the rationale to evaluate enavatuzumab as a potential therapy for the treatment of breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1007/s00432-012-1332-x) contains supplementary material, which is available to authorized users.
PMCID: PMC3549414  PMID: 23073510
TweakR; Antibody; Breast cancer; Fn14
5.  High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis 
Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.
This study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.
The mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.
The results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.
PMCID: PMC4060194  PMID: 23800379
6.  TWEAK and its receptor Fn14 in the synovium of patients with rheumatoid arthritis compared to psoriatic arthritis and its response to tumour necrosis factor blockade 
Annals of the Rheumatic Diseases  2009;69(1):301-304.
To investigate the expression of tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor inducible 14 (Fn14) in the inflamed synovium of patients with arthritis, as TWEAK blockade has been observed to have a beneficial effect in an animal model of rheumatoid arthritis (RA).
Synovial tissue (ST) biopsies were obtained from 6 early, methotrexate-naive patients with RA as well as 13 patients with RA and 16 patients with psoriatic arthritis (PsA) who were matched for treatment and disease duration. Serial ST samples were obtained from a separate cohort of 13 patients with RA before and after infliximab treatment. TWEAK and Fn14 expression was evaluated by immunohistochemistry and digital image analysis.
TWEAK and Fn14 were clearly expressed in ST of patients with RA and PsA. TWEAK expression was significantly higher in RA (sub)lining samples compared to PsA (p = 0.005 and p = 0.014, respectively), but Fn14 expression was comparable. Double immunofluorescence showed TWEAK and Fn14 expression on fibroblast-like synoviocytes and macrophages, but not T cells. Of interest, persistent TWEAK and Fn14 expression was found after anti-TNF therapy.
TWEAK and Fn14 are abundantly expressed in the inflamed synovium of patients with RA and PsA. This raises the possibility that blocking TWEAK/Fn14 signalling could be of therapeutic benefit in inflammatory arthritis.
PMCID: PMC2789939  PMID: 19147618
7.  Urinary TWEAK as a biomarker of lupus nephritis: a multicenter cohort study 
Arthritis Research & Therapy  2009;11(5):R143.
TNF-like weak inducer of apoptosis (TWEAK) has been implicated as a mediator of chronic inflammatory processes via prolonged activation of the NF-κB pathway in several tissues, including the kidney. Evidence for the importance of TWEAK in the pathogenesis of lupus nephritis (LN) has been recently introduced. Thus, TWEAK levels may serve as an indication of LN presence and activity.
Multicenter cohorts of systemic lupus erythematosus (SLE) patients and controls were recruited for cross-sectional and longitudinal analysis of urinary TWEAK (uTWEAK) and/or serum TWEAK (sTWEAK) levels as potential biomarkers of LN. The performance of TWEAK as a biomarker for nephritis was compared with routinely used laboratory tests in lupus patients, including anti-double stranded DNA antibodies and levels of C3 and C4.
uTWEAK levels were significantly higher in LN patients than in non-LN SLE patients and other disease control groups (P = 0.039). Furthermore, uTWEAK was better at distinguishing between LN and non-LN SLE patients than anti-DNA antibodies and complement levels, while high uTWEAK levels predicted LN in SLE patients with an odds ratio of 7.36 (95% confidence interval = 2.25 to 24.07; P = 0.001). uTWEAK levels peaked during LN flares, and were significantly higher during the flare than at 4 and 6 months prior to or following the flare event. A linear mixed-effects model showed a significant association between uTWEAK levels in SLE patients and their disease activity over time (P = 0.008). sTWEAK levels, however, were not found to correlate with the presence of LN or the degree of nephritis activity.
High uTWEAK levels are indicative of LN, as opposed to non-LN SLE and other healthy and disease control populations, and reflect renal disease activity in longitudinal follow-up. Thus, our study further supports a role for TWEAK in the pathogenesis of LN, and provides strong evidence for uTWEAK as a candidate clinical biomarker for LN.
PMCID: PMC2787265  PMID: 19785730
8.  Fibroblast growth factor inducible 14 (Fn14) is expressed in the lower genital tract and may play a role in amplifying inflammation during infection 
TNF-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) cytokine superfamily which regulates a number of cellular responses, including inflammation and proliferation. TWEAK is primarily secreted by phagocytic cells and its receptor, fibroblast growth factor-inducible 14 (Fn14), is expressed on non-lymphoid cells, including epithelial, endothelial and mesenchymal cells. The TWEAK/Fn14 pathway is highly conserved from an evolutionary standpoint, and has been shown to play a role in tissue regeneration and inflammation in the liver, kidney, lung and skeletal muscle. We hypothesized that TWEAK/Fn14 might have a physiological role in regulating infection-induced inflammation in the lower female genital tract. To test this hypothesis, we examined expression of the receptor Fn14 in relevant cells and tissue. Receptor function was tested by treating cells with recombinant TWEAK, with and without other known pro-inflammatory stimuli. Flow cytometric analysis of vaginal and cervical epithelial cells revealed that Fn14 was highly expressed at the cell surface. We also detected both Fn14 and TWEAK in whole cervical tissue by RT-PCR. Treatment of vaginal and cervical epithelial cells with recombinant TWEAK led to a weak induction of the chemokine IL-8. However, TWEAK potentiated the effects of IL-1ß, the TLR2 ligand Pam3CysSK4, and live Neisseria gonorrhoeae in a synergistic manner. These data reveal a novel pathway for regulation of microbial-induced inflammation in the female reproductive tract and suggest that interference with the TWEAK/Fn14 pathway might be an approach to abrogate excessive infection-induced inflammation caused by sexually transmitted pathogens.
PMCID: PMC2814934  PMID: 19963275
Innate Immunity; Epithelial Cells; Cytokine Receptors
9.  TWEAK: A New Player in Obesity and Diabetes 
Obesity and type 2 diabetes (T2D) are associated with chronic low-grade inflammation. Mounting evidence suggests the involvement of an inflammatory switch in adipose tissue, both in mature adipocytes and immune-competent cells from the stromal vascular compartment, in the progression of obesity and insulin resistance. Several inflammatory cytokines secreted by obese adipose tissue, including TNFα and IL-6 have been described as hallmark molecules involved in this process, impairing insulin signaling in insulin-responsive organs. An increasing number of new molecules affecting the local and systemic inflammatory imbalance in obesity and T2D have been identified. In this complex condition, some molecules may exhibit opposing actions, depending on the cell type and on systemic or local influences. Tumor necrosis factor weak inducer of apoptosis (TWEAK), a cytokine of the tumor necrosis (TNF) superfamily, is gaining attention as an important player in chronic inflammatory diseases. TWEAK can exist as a full-length membrane-associated (mTWEAK) form and as a soluble (sTWEAK) form and, by acting through its cognate receptor Fn14, can control many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. Notably, sTWEAK has been proposed as a biomarker of cardiovascular diseases. Here, we will review the recent findings relating to TWEAK and its receptor within the context of obesity and the associated disorder T2D.
PMCID: PMC3874549  PMID: 24416031
TWEAK; obesity; type 2 diabetes; adipose tissue; TNFα; insulin resistance; inflammation
10.  Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis 
The aim of this study was to investigate the expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in patients with polymyositis (PM) and dermatomyositis (DM), and their relation to clinical manifestations.
Serum levels of TWEAK were detected in 98 PM/DM patients and 37 healthy controls by using the ELISA method. Total RNA isolated from fresh-frozen muscle tissue samples of 36 PM/DM patients and 10 healthy controls were used for analyzing the mRNA levels of TWEAK and Fn14 by quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence staining of TWEAK and Fn14 was conducted on muscle biopsy specimens from 23 PM/DM patients and seven healthy controls.
Serum levels of TWEAK were significantly decreased in the PM/DM patients compared to those in the healthy controls (P < 0.001), and serum TWEAK levels negatively correlated with serum CD163 levels in PM/DM patients (r = -0.49, P < 0.001). The expression of Fn14 mRNA was significantly increased in the muscle tissue of PM/DM patients than in the muscle tissue of healthy controls (P < 0.01), whereas the expression of TWEAK mRNA in PM/DM patients was not statistically different from that of the healthy controls (P > 0.05). Fn14 mRNA levels in muscle tissue positively correlated with muscle disease activity (r = 0.512, P < 0.01). Patients with oropharyngeal dysphagia had significantly higher Fn14 mRNA levels than patients without oropharyngeal dysphagia (P < 0.05). The results of immunofluorescence staining showed that 19 out of 23 PM/DM patients were TWEAK-positive, and 20 out of 23 PM/DM patients were Fn14-positive. No detectable expressions of TWEAK or Fn14 were observed in the healthy controls.
TWEAK-Fn14 axis may be involved in the pathogenesis of PM/DM. Further understanding of TWEAK-Fn14 function in PM/DM may help to define therapeutic targets for PM/DM.
PMCID: PMC3978894  PMID: 24467773
11.  Reduced sTWEAK and Increased sCD163 Levels in HIV-Infected Patients: Modulation by Antiretroviral Treatment, HIV Replication and HCV Co-Infection 
PLoS ONE  2014;9(3):e90541.
Patients infected with the human immunodeficiency virus (HIV) have an increased risk of cardiovascular disease due to increased inflammation and persistent immune activation. CD163 is a macrophage scavenger receptor that is involved in monocyte-macrophage activation in HIV-infected patients. CD163 interacts with TWEAK, a member of the TNF superfamily. Circulating levels of sTWEAK and sCD163 have been previously associated with cardiovascular disease, but no previous studies have fully analyzed their association with HIV.
The aim of this study was to analyze circulating levels of sTWEAK and sCD163 as well as other known markers of inflammation (hsCRP, IL-6 and sTNFRII) and endothelial dysfunction (sVCAM-1 and ADMA) in 26 patients with HIV before and after 48 weeks of antiretroviral treatment (ART) and 23 healthy subjects.
Patients with HIV had reduced sTWEAK levels and increased sCD163, sVCAM-1, ADMA, hsCRP, IL-6 and sTNFRII plasma concentrations, as well as increased sCD163/sTWEAK ratio, compared with healthy subjects. Antiretroviral treatment significantly reduced the concentrations of sCD163, sVCAM-1, hsCRP and sTNFRII, although they remained elevated when compared with healthy subjects. Antiretroviral treatment had no effect on the concentrations of ADMA and sTWEAK, biomarkers associated with endothelial function. The use of protease inhibitors as part of antiretroviral therapy and the presence of HCV-HIV co-infection and/or active HIV replication attenuated the ART-mediated decrease in sCD163 plasma concentrations.
HIV-infected patients showed a proatherogenic profile characterized by increased inflammatory, immune-activation and endothelial-dysfunction biomarkers that partially improved after ART. HCV-HIV co-infection and/or active HIV replication enhanced immune activation despite ART.
PMCID: PMC3942443  PMID: 24594990
12.  Is TWEAK a Biomarker for Autoimmune/Chronic Inflammatory Diseases? 
The TWEAK/Fn14 pathway is now well-known for its involvement in the modulation of inflammation in various human autoimmune/chronic inflammatory diseases (AICID) including lupus, rheumatoid arthritis, and multiple sclerosis. A panel of data is now available concerning TWEAK expression in tissues or biological fluids of patients suffering from AICID, suggesting that it could be a promising biological marker in these diseases. Evidences from several teams support the hypothesis that blocking TWEAK/Fn14 pathway is an attractive new therapeutic lead in such diseases and clinical trials with anti-TWEAK-blocking antibodies are in progress. In this mini-review we discuss the potential use of TWEAK quantification in AICD management in routine practice and highlight the challenge of standardizing data collection to better estimate the clinical utility of such a biological parameter.
PMCID: PMC3873518  PMID: 24409182
TWEAK; biomarker; auto-immunity; disease monitoring; serum levels; urinary levels
13.  TWEAK/Fn14 and Non-Canonical NF-kappaB Signaling in Kidney Disease 
The incidence of acute kidney injury (AKI) and chronic kidney disease (CKD) is increasing. However, there is no effective therapy for AKI and current approaches only slow down, but do not prevent progression of CKD. TWEAK is a TNF superfamily cytokine. A solid base of preclinical data suggests a role of therapies targeting the TWEAK or its receptor Fn14 in AKI and CKD. In particular TWEAK/Fn14 targeting may preserve renal function and decrease cell death, inflammation, proteinuria, and fibrosis in mouse animal models. Furthermore there is clinical evidence for a role of TWEAK in human kidney injury including increased tissue and/or urinary levels of TWEAK and parenchymal renal cell expression of the receptor Fn14. In this regard, clinical trials of TWEAK targeting are ongoing in lupus nephritis. Nuclear factor-kappa B (NF-κB) activation plays a key role in TWEAK-elicited inflammatory responses. Activation of the non-canonical NF-κB pathway is a critical difference between TWEAK and TNF. TWEAK activation of the non-canonical NF-κB pathways promotes inflammatory responses in tubular cells. However, there is an incomplete understanding of the role of non-canonical NF-κB activation in kidney disease and on its contribution to TWEAK actions in vivo.
PMCID: PMC3857575  PMID: 24339827
acute kidney injury; fibrosis; inflammation; kidney; lupus nephritis; podocyte; proteinuria
14.  Involvement of Tweak in Interferon γ–Stimulated Monocyte Cytotoxicity 
The Journal of Experimental Medicine  2000;192(9):1373-1380.
TWEAK, a new member of the tumor necrosis factor (TNF) family, induces cell death in some tumor cell lines, but its physiological functions are largely unknown. In this study, we investigated the expression and function of TWEAK in human peripheral blood mononuclear cells (PBMCs) by using newly generated anti–human TWEAK mAbs. Although freshly isolated PBMCs expressed no detectable level of TWEAK on their surfaces, a remarkable TWEAK expression was rapidly observed on monocytes upon stimulation with interferon (IFN)-γ but not with IFN-α or lipopolysaccharide. Cytotoxic activity of IFN-γ–stimulated monocytes against human squamous carcinoma cell line HSC3 was inhibited partially by anti-TWEAK mAb alone and almost completely by combination with anti-TRAIL (TNF-related apoptosis-inducing ligand) mAb. These results revealed a novel pathway of monocyte cytotoxicity against tumor cells that is mediated by TWEAK and potentiated by IFN-γ.
PMCID: PMC2193363  PMID: 11067885
TWEAK; TRAIL; IFN-γ; monocyte; cytotoxicity
15.  Therapeutic potential of the TWEAK/Fn14 pathway in intractable gastrointestinal cancer 
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily. It has been suggested that it plays a pivotal role in various physiological and pathological conditions due to its proinflammatory properties. Fibroblast growth-inducible 14 (Fn14) has been identified as a TWEAK receptor. A number of studies have suggested that TWEAK-Fn14 interaction results in the promotion of apoptosis, cell growth as well as angiogenesis. Although recent studies have indicated that TWEAK and Fn14 are expressed in a number of tumor lines and tissues, the therapeutic potential of this pathway has yet to be elucidated. This study investigated the potential of TWEAK and Fn14 in esophageal and pancreatic cancer as novel molecular targets for anti-cancer therapy. TWEAK and Fn14 protein expression was evaluated in 43 patients with esophageal cancer and 51 patients with pancreatic cancer by immunohistochemistry. As a result, either TWEAK or Fn14 expression was observed in 58.1% of the cases with esophageal cancer and 74.5% of the cases with pancreatic cancer. Furthermore, TWEAK/Fn14 gene expression was identified in the majority of the human esophageal and pancreatic cancer cell lines. Therapeutic efficacies of blocking TWEAK and Fn14 were evaluated by tumor growth inhibition assay in TWEAK- and Fn14-expressing human esophageal and pancreatic cancer cell lines. Coculture with anti-TWEAK or -Fn14 mAb was found to induce a 22–65% cell growth inhibition of these cells. Finally, the significant therapeutic effect of targeting this pathway under in vivo physiological conditions was confirmed using a murine gastrointestinal cancer model. In conclusion, the TWEAK/Fn14 pathway may be functional and critical in intractable gastrointestinal cancers. Therefore, TWEAK and/or Fn14 may be novel molecular targets for anti-cancer therapy.
PMCID: PMC3440634  PMID: 22977477
esophageal cancer; pancreatic cancer; tumor necrosis factor-like weak inducer of apoptosis; fibroblast growth-inducible 14; molecular target therapy
16.  TWEAK/Fn14 Axis-Targeted Therapeutics: Moving Basic Science Discoveries to the Clinic 
The TNF superfamily member TWEAK (TNFSF12) is a multifunctional cytokine implicated in physiological tissue regeneration and wound repair. TWEAK is initially synthesized as a membrane-anchored protein, but furin cleavage within the stalk region can generate a secreted TWEAK isoform. Both TWEAK isoforms bind to a small cell surface receptor named Fn14 (TNFRSF12A) and this interaction stimulates various cellular responses, including proliferation and migration. Fn14, like other members of the TNF receptor superfamily, is not a ligand-activated protein kinase. Instead, TWEAK:Fn14 engagement promotes Fn14 association with members of the TNFR associated factor family of adapter proteins, which triggers activation of various signaling pathways, including the classical and alternative NF-κB pathways. Numerous studies have revealed that Fn14 gene expression is significantly elevated in injured tissues and in most solid tumor types. Also, sustained Fn14 signaling has been implicated in the pathogenesis of cerebral ischemia, chronic inflammatory diseases, and cancer. Accordingly, several groups are developing TWEAK- or Fn14-targeted agents for possible therapeutic use in patients. These agents include monoclonal antibodies, fusion proteins, and immunotoxins. In this article, we provide an overview of some of the TWEAK/Fn14 axis-targeted agents currently in pre-clinical animal studies or in human clinical trials and discuss two other potential approaches to target this intriguing signaling node.
PMCID: PMC3870272  PMID: 24391646
TWEAK; Fn14; inflammatory disease; cancer; clinical trial
17.  A Bioinformatics Resource for TWEAK-Fn14 Signaling Pathway 
Journal of Signal Transduction  2012;2012:376470.
TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders.
PMCID: PMC3357548  PMID: 22649723
18.  The cytokine tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 have a neuroprotective effect in the central nervous system 
Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. However, the brain has the ability to detect and accommodate to hypoxic conditions. This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. Accordingly, in this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions.
Here we used in vitro and in vivo models of hypoxic and ischemic preconditioning, an animal model of transient middle cerebral artery occlusion and mice and neurons genetically deficient in TWEAK, Fn14, or tumor necrosis factor alpha (TNF-α) to investigate whether treatment with recombinant TWEAK or an increase in the expression of endogenous TWEAK renders neurons tolerant to lethal hypoxia. We used enzyme-linked immunosorbent assay to study the effect of TWEAK on the expression of neuronal TNF-α, Western blot analysis to investigate whether the effect of TWEAK was mediated by activation of mitogen-activated protein kinases and immunohistochemical techniques and quantitative real-time polymerase chain reaction analysis to study the effect of TWEAK on apoptotic cell death.
We found that either treatment with recombinant TWEAK or an increase in the expression of TWEAK and Fn14 induce hypoxic and ischemic tolerance in vivo and in vitro. This protective effect is mediated by neuronal TNF-α and activation of the extracellular signal-regulated kinases 1 and 2 pathway via phosphorylation and inactivation of the B-cell lymphoma 2-associated death promoter protein.
Our work indicate that the interaction between TWEAK and Fn14 triggers the activation of a cell signaling pathway that results in the induction of tolerance to lethal hypoxia and ischemia. These data indicate that TWEAK may be a potential therapeutic strategy to protect the brain from the devastating effects of an ischemic injury.
PMCID: PMC3311607  PMID: 22394384
TWEAK; Cerebral ischemia; Inflammation; Ischemic tolerance; Preconditioning
19.  TWEAK-Independent Fn14 Self-Association and NF-κB Activation Is Mediated by the C-Terminal Region of the Fn14 Cytoplasmic Domain 
PLoS ONE  2013;8(6):e65248.
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.
PMCID: PMC3672086  PMID: 23750247
20.  TWEAK/Fn14 pathway modulates properties of a human microvascular endothelial cell model of blood brain barrier 
The TNF ligand family member TWEAK exists as membrane and soluble forms and is involved in the regulation of various human inflammatory pathologies, through binding to its main receptor, Fn14. We have shown that the soluble form of TWEAK has a pro-neuroinflammatory effect in an animal model of multiple sclerosis and we further demonstrated that blocking TWEAK activity during the recruitment phase of immune cells across the blood brain barrier (BBB) was protective in this model. It is now well established that endothelial cells in the periphery and astrocytes in the central nervous system (CNS) are targets of TWEAK. Moreover, it has been shown by others that, when injected into mice brains, TWEAK disrupts the architecture of the BBB and induces expression of matrix metalloproteinase-9 (MMP-9) in the brain. Nevertheless, the mechanisms involved in such conditions are complex and remain to be explored, especially because there is a lack of data concerning the TWEAK/Fn14 pathway in microvascular cerebral endothelial cells.
In this study, we used human cerebral microvascular endothelial cell (HCMEC) cultures as an in vitro model of the BBB to study the effects of soluble TWEAK on the properties and the integrity of the BBB model.
We showed that soluble TWEAK induces an inflammatory profile on HCMECs, especially by promoting secretion of cytokines, by modulating production and activation of MMP-9, and by expression of cell adhesion molecules. We also demonstrated that these effects of TWEAK are associated with increased permeability of the HCMEC monolayer in the in vitro BBB model.
Taken together, the data suggest a role for soluble TWEAK in BBB inflammation and in the promotion of BBB interactions with immune cells. These results support the contention that the TWEAK/Fn14 pathway could contribute at least to the endothelial steps of neuroinflammation.
PMCID: PMC3570290  PMID: 23320797
CCL-2; hCMEC/D3; HMEC; IL-8; MMP-9; Neuroinflammation; TNFSF12; ZO-1
21.  PGC1α Plays a Critical Role in TWEAK–Induced Cardiac Dysfunction 
PLoS ONE  2013;8(1):e54054.
Inflammatory cytokines play an important role in the pathogenesis of heart failure. We have recently found the cytokine TWEAK (tumor necrosis factor (TNF)-like weak inducer of apoptosis), a member of the TNF superfamily, to be increased in patients with cardiomyopathy and result in the development of heart failure when overexpressed in mice. The molecular mechanisms underlying TWEAK-induced cardiac pathology, however, remain unknown.
Methodology and Critical Finding
Using mouse models of elevated circulating TWEAK levels, established through intravenous injection of adenovirus expressing TWEAK or recombinant TWEAK protein, we find that TWEAK induces a progressive dilated cardiomyopathy with impaired contractile function in mice. Moreover, TWEAK treatment is associated with decreased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and genes required for mitochondrial oxidative phosphorylation, which precede the onset of cardiac dysfunction. TWEAK-induced downregulation of PGC1α requires expression of its cell surface receptor, fibroblast growth factor-inducible 14 (Fn14). We further find that TWEAK downregulates PGC1α gene expression via the TNF receptor-associated factor 2 (TRAF2) and NFκB signaling pathways. Maintaining PGC1α levels through adenoviral-mediated gene expression is sufficient to protect against TWEAK-induced cardiomyocyte dysfunction.
Collectively, our data suggest that TWEAK induces cardiac dysfunction via downregulation of PGC1α, through FN14-TRAF2-NFκB-dependent signaling. Selective targeting of the FN14-TRAF2-NFκB-dependent signaling pathway or augmenting PGC1α levels may serve as novel therapeutic strategies for cardiomyopathy and heart failure.
PMCID: PMC3546975  PMID: 23342071
22.  Soluble TWEAK independently predicts atherosclerosis in renal transplant patients 
BMC Nephrology  2013;14:144.
Cardiovascular risk is increased in the early stages of chronic kidney disease (CKD) and also found to be ongoing in renal transplant (Rtx) patients. As a sign of atherosclerosis, increased carotid intima-media thickness (CIMT) has been widely accepted as a strong predictor of cardiovascular disease (CVD) and mortality in CKD patients. A novel markers, soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and neutrophil-to-lymphocyte ratio (NLR) were introduced as potential markers in inflammatory disorders including CKD. The role of Rtx in terms of atherogenesis is still unclear. We aimed to investigate the relationship between sTWEAK, NLR and CIMT in Rtx patients without overt CVD and to compare these results with those obtained from healthy subjects.
Cross-sectional analysis in which CIMT measurements, NLR and serum TWEAK levels were assessed in 70 Rtx patients (29 females; mean age, 40.6 ± 12.4 years) and 25 healthy subjects (13 females, mean age; 37.4±8.8 years).
sTWEAK levels were significantly decreased (p=0.01) and hs-CRP, NLR and CIMT levels of Rtx patients were significantly increased compared to healthy subjects (p<0.0001, p=0.001, p<0.0001, respectively). sTWEAK was also found to be decreased when eGFR was decreased (p=0.04 between all groups). CIMT was positively correlated with sTWEAK and NLR in Rtx patients (r=0.81, p<0.0001 and r=0.33, p=0.006, respectively). sTWEAK was also positively correlated with NLR (r=0.37, p=0.002). In the multivariate analysis only sTWEAK was found to be an independent variable of increased CIMT.
sTWEAK might have a role in the pathogenesis of ongoing atherosclerosis in Rtx patients.
PMCID: PMC3711729  PMID: 23849432
sTWEAK; Neutrophil-to-lymphocyte ratio; Carotid intima-media thickness; Renal transplantation
23.  Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice 
Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12−/−ApoE−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12−/−ApoE−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12+/+ApoE−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12−/−ApoE−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12+/+ApoE−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12−/−ApoE−/− or anti-TWEAK treatment in TNFSF12+/+ApoE−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.
PMCID: PMC4000122  PMID: 24479820
TWEAK; atherosclerosis; inflammation; stability
24.  Serum Levels of TWEAK and Scavenger Receptor CD163 in Type 1 Diabetes Mellitus: Relationship with Cardiovascular Risk Factors. A Case-Control Study 
PLoS ONE  2012;7(8):e43919.
To test the usefulness of serum concentrations of tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and soluble scavenger receptor CD163 (sCD163) as markers of subtle inflammation in patients with type 1 diabetes mellitus (T1DM) without clinical cardiovascular (CV) disease and to evaluate their relationship with arterial stiffness (AS).
Sixty-eight patients with T1DM and 68 age and sex-matched, healthy subjects were evaluated. Anthropometrical variables and CV risk factors were recorded. Serum concentrations of sTWEAK and sCD163 were measured. AS was assessed by aortic pulse wave velocity (aPWV). All statistical analyses were stratified by gender.
T1DM patients showed lower serum concentrations of sTWEAK (Men: 1636.5 (1146.3–3754.8) pg/mL vs. 765.9 (650.4–1097.1) pg/mL; p<0.001. Women: 1401.0 (788.0–2422.2) pg/mL vs. 830.1 (562.6–1175.9) pg/mL; p = 0.011) compared with their respective controls. Additionally, T1DM men had higher serum concentrations of sCD163 (285.0 (247.7–357.1) ng/mL vs. 224.8 (193.3–296.5) ng/mL; p = 0.012) compared with their respective controls. sTWEAK correlated negatively with aPWV in men (r = −0.443; p<0.001). However, this association disappeared after adjusting for potential confounders. In men, the best multiple linear regression model showed that the independent predictors of sTWEAK were T1DM and WHR (R2 = 0.640; p<0.001). In women, T1DM and SBP were the independent predictors for sTWEAK (R2 = 0.231; p = 0.001).
sTWEAK is decreased in T1DM patients compared with age and sex-matched healthy subjects after adjusting for classic CV risk factors, although sTWEAK levels may be partially influenced by some of them. Additionally, T1DM men have higher serum concentrations of sCD163. These results point out an association between the inflammatory system and CV risk in T1DM.
PMCID: PMC3427173  PMID: 22937125
25.  TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK 
Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NFκB signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.
PMCID: PMC3927163  PMID: 24600451

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