TWEAK signals through JAK–STAT to induce tumor cell apoptosis

The TWEAK receptor Fn14 (TNFRSF12), a member of the TNF Receptor superfamily, can mediate many processes, including apoptosis. Fn14 agonists have therefore been the subject of interest as potential cancer therapeutics. In cell culture experiments, interferon gamma (IFNγ) is typically required for induction of apoptotic activity by either TWEAK or Fn14 agonistic antibodies in most cell lines. We have investigated the mechanism of IFNγ signaling and the role of JAK–STAT signaling in TWEAK/Fn14-mediated tumor cell killing. We found that IFNγ-mediated enhancement of tumor cell killing is JAK–STAT dependent, as JAK inhibitors block IFNγ?dependent TWEAK induced apoptosis. Exposure of tumor cells to IFNγ results in an increase in Fn14 expression on the cell surface, which may be a mechanism by which IFNγ induces sensitivity to TWEAK. In a reciprocal fashion, we observed that IFNγ receptor levels increase in response to TWEAK treatment in WiDr cells. Significantly, we found that TWEAK alone can induce STAT1 phosphorylation in WiDr tumor cells. Moreover, TWEAK induction of tumor cell apoptosis in WiDr cells in the absence of IFNγ is mediated by the JAK–STAT pathway. Correspondingly, we show that treatment of tumor bearing mice with mBIIB036, an Fn14 agonistic antibody, results in STAT1 phosphorylation in the tumors. Notably, the level of STAT1 phosphorylation appears to correlate with the degree of tumor growth inhibition by BIIB036 in vivo. Additionally, in WiDr cells, TWEAK induces a soluble factor, which we have identified as IFNβ, capable of independently inducing STAT1 phosphorylation when transferred to naïve cells. Finally, either IFNα or IFNβ can partially substitute for IFNγ in sensitizing tumor cells to Fn14 agonists. In summary, we show that TWEAK/Fn14 can signal through the JAK–STAT pathway to induce IFNβ, and that the ability of TWEAK to induce tumor cell apoptosis is mediated by JAK-STAT signaling. We also demonstrate that IFNγ enhancement of TWEAK/FN14-mediated tumor cell death is JAK-dependent and may occur by IFNγ-dependent upregulation of Fn14 on tumor cells. These findings may have implications for the appropriately targeted clinical development of Fn14 agonists as anti-cancer therapy.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

Inhibition of suppressor of cytokine signaling 1 mediates the profibrotic effect of TWEAK/Fn14 signaling on kidney cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) engagement with the receptor Fn14 contributes to the fibrotic process of kidney cells in systemic lupus erythematosus. Downregulation of the protein suppressor of cytokine signaling 1 (SOCS1) correlates with amplified production of proinflammatory factors and cell apoptosis, which participate in the pathogenesis of lupus nephritis. To elucidate the potential role of SOCS1 in TWEAK/Fn14 signaling, we determined the SOCS1 levels in primary kidney cells from MRL/MpJ (control strain) or MRL/lpr (lupus-prone) mice. These cells (mesangial cells, glomerular endothelial cells, and tubular epithelial cells) were also evaluated after stimulation with TWEAK (0 to 250 ng/mL). The results showed that the lupus-prone cells exhibited reduced SOCS1 expression. TWEAK induced the production of profibrotic factors (laminin, fibronectin, (CC motif) ligand 20, etc.) in kidney cells from both mouse strains. TWEAK stimulation also decreased both the mRNA and protein levels of SOCS1 in all cells. Moreover, the effect of TWEAK on mesangial cells was amplified by pre-transfection of SOCS1 siRNA but was partly reduced with SOCS1 overexpression by adenoviral delivery. Therefore, TWEAK/Fn14 activation contributes to renal fibrosis in lupus nephritis involving the depression of SOCS1 function.     

TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the chronic graft-versus-host model of systemic lupus erythematosus

TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a prominent inducer of proinflammatory cytokines in vitro and in vivo. We previously found that kidney cells display the TWEAK receptor Fn14, and that TWEAK stimulation of mesangial cells and podocytes induces a potent proinflammatory response. Several of the cytokines up-regulated in the kidney in response to TWEAK are instrumental in Lupus nephritis; we therefore hypothesized that TWEAK/Fn14 interactions may be important in the cascade(s) leading to renal damage in systemic Lupus erythematosus. In this study, we analyzed the effects of Fn14 deficiency in the chronic graft-vs-host model of SLE, and the benefits of treatment with an anti-TWEAK mAb in this mouse model. We found that anti-nuclear Ab titers were no different between C57BL/6 Fn14 wild-type and deficient mice injected with alloreactive bm12 splenocytes. However, kidney disease was significantly less severe in Fn14 knockout mice. Furthermore, kidney IgG deposition, IL-6, MCP-1, RANTES, and IP-10, as well as macrophage infiltration, were significantly decreased in Fn14-deficient mice with induced lupus. Similarly, mice with induced Lupus treated with an anti-TWEAK neutralizing mAb had significantly diminished kidney expression of IL-6, MCP-1, IL-10, as well as proteinuria, but similar autoantibody titers, as compared with control-treated mice. We conclude that TWEAK is an important mediator of kidney damage that acts by promoting local inflammatory events, but without impacting adaptive immunity in this experimental LN model. Thus, TWEAK blockade may be a novel therapeutic approach to reduce renal damage in SLE.     

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor–inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor κB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK–Fn14 system is an important target for preventing skeletal muscle wasting.     

Fibroblast growth factor inducible 14 (Fn14) is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes. Evidence for TWEAK-independent functions of Fn14 during myogenesis

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal alpha-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.     

Fn14?Trail Effectively Inhibits Hepatocellular Carcinoma Growth

Background

New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL''s capacity to inhibit HCC growth was tested.

Results

Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL''s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice.

Conclusions

In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein’s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.     

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims

Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH.

Methods

To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies.

Results

In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12–8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes.

Conclusions

TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.     

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-kappaB (NF-kappaB), 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-kappaB 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.     

Functional Expression of TWEAK and the Receptor Fn14 in Human Malignant Ovarian Tumors: Possible Implication for Ovarian Tumor Intervention

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.     

Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr¹⁷⁶, but not Trp²³¹, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr¹⁷⁶ as the anchoring residue. Importantly, mutation of TWEAK at Tyr¹⁷⁶ did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.     

TWEAK-Independent Fn14 Self-Association and NF-κB Activation Is Mediated by the C-Terminal Region of the Fn14 Cytoplasmic Domain

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.     

A Further TWEAK to Multiple Sclerosis Pathophysiology

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF super family that controls many cellular activities including proliferation, migration, differentiation, apoptosis, and inflammation by binding to fibroblast growth factor-inducible 14 (Fn14), a highly inducible cell surface receptor. Recent studies have indicated that TWEAK–Fn14 axis signaling may contribute to chronic autoimmune diseases. TWEAK expression via microglia in cortical lesions, presence of TWEAK⁺ macrophages in inflamed leptomeninges, and absence of TWEAK/Fn14 expression in healthy brain implicates importance of this pathway in pathogenesis of multiple sclerosis lesions. TWEAK–Fn14 axis blockade has also shown promise in various multiple sclerosis animal models. Stimulation of the TWEAK/Fn14 pathway can result in activation of both canonical and noncanonical NF-κB signaling and could also stimulate mitogen-activated protein kinase (MAPK) signaling pathways. Here, we have reviewed evidence of the possible role of TWEAK–Fn14 axis in pathophysiology of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) via neuroinflammation, tissue remodeling, blood–brain barrier (BBB) disruption, neurodegeneration, and astrogliosis.     

Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-gamma-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-alpha + IL-1beta. CXCL11/interferon-inducible T cell alpha chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Ikappa-B, while pretreatment with an Ikappa-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-kappaB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.