Endogenous TNF-related Apoptosis-inducing Ligand Research Articles

A Dual Role for Death Receptor 5 in Regulating Cardiac Fibroblast Function.

The fibrotic response is involved in nearly all forms of heart failure and dysregulated responses can lead to enhanced cardiac dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor (DR) 5, are associated with multiple forms of heart failure, but their role in the heart is poorly defined. Our previous study identified DR5 expression on cardiac fibroblasts however, the impact of DR5 on fibroblast function remains unexplored. To investigate the role of DR5 in cardiac fibroblasts, a variety of fibroblast functions were examined following treatment with the endogenous ligand, TRAIL, or small molecule agonist, bioymifi. DR5 activation did not induce apoptosis in naïve fibroblasts but activated ERK1/2 signaling to increase proliferation. However, upon activation and differentiation to myofibroblasts, DR5 expression was elevated, and DR5 agonists induced caspase 3 activation resulting in myofibroblast apoptosis. To investigate the impact of DR5 regulation of fibroblasts in vivo, a chronic isoproterenol administration model of heart failure was used. Wild-type (WT) mice receiving isoproterenol had increased hypertrophy, cardiomyocyte death, and fibrosis and decreased contractility compared to vehicle treated animals. DR5 knockout (KO) mice had no overt baseline phenotype however, following isoproterenol infusion, increased cardiomyocyte death and hypertrophy in comparison to isoproterenol treated WT animals was observed. DR5KO mice had an augmented fibrotic response with isoproterenol treatment compared with WT, which corresponded with additional decreases in contractility. These findings identify a dual role for DR5 in cardiac fibroblast function through enhanced naïve fibroblast proliferation, which switches to a pro-apoptotic function upon differentiation to myofibroblasts. This is important in heart failure where DR5 activation suppresses maladaptive remodeling and may represent a novel therapeutic target for the treatment of heart failure.

Importance of TRAIL Molecular Anatomy in Receptor Oligomerization and Signaling. Implications for Cancer Therapy.

(TNF)-related apoptosis-inducing ligand (TRAIL) is able to activate the extrinsic apoptotic pathway upon binding to DR4/TRAIL-R1 and/or DR5/TRAIL-R2 receptors. Structural data indicate that TRAIL functions as a trimer that can engage three receptor molecules simultaneously, resulting in receptor trimerization and leading to conformational changes in TRAIL receptors. However, receptor conformational changes induced by the binding of TRAIL depend on the molecular form of this death ligand, and not always properly trigger the apoptotic cascade. In fact, TRAIL exhibits a much stronger pro-apoptotic activity when is found as a transmembrane protein than when it occurs as a soluble form and this enhanced biological activity is directly linked to its ability to cluster TRAIL receptors in supra-molecular structures. In this regard, cells involved in tumor immunosurveillance, such as activated human T cells, secrete endogenous TRAIL as a transmembrane protein associated with lipid microvesicles called exosomes upon T-cell reactivation. Consequently, it seems clear that a proper oligomerization of TRAIL receptors, which leads to a strong apoptotic signaling, is crucial for inducing apoptosis in cancer cells upon TRAIL treatment. In this review, the current knowledge of oligomerization status of TRAIL receptors is discussed as well as the implications for cancer treatment when using TRAIL-based therapies.

Abstract B18: Characterization and therapeutic harnessing of TRAIL’s pro-tumorigenic and pro-apoptotic functions in cancer

Abstract Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is known for specifically killing many cancer cells. This has led to the clinical development of several agonists for TRAIL-Rs. However, while some cancer cells die in response to TRAIL, most primary cancer cells are resistant to TRAIL-based monotherapy. In resistant cells, TRAIL signaling can induce non-apoptotic, tumor-supporting effects. Hence, efficient therapy design requires understanding of the bivalent nature of TRAIL signaling. Here we demonstrate that, on the one hand, TRAIL–TRAIL-R signaling in cancer cells elicits the production of cytokines, most importantly CCL2, which promote the accumulation of alternatively activated, M2-like myeloid cells in the tumor microenvironment, consequently promoting tumor growth via a CCL2/CCR2 axis. On the other hand, we identified CDK9 inhibition as a powerful means to render many TRAIL-resistant cancer cells highly sensitive to TRAIL-induced apoptosis. Importantly, this combination was also highly effective in vivo and led to significant tumor regression in mouse models of lung cancer. Therefore, in certain cancer patients antagonizing endogenous TRAIL–TRAIL-R signaling may modulate the tumor microenvironment in favour of tumor regression, while in other cancer patients the combination of TRAIL-R agonists with CDK9 inhibitors should be considered as a highly active alternative therapy. Citation Format: Antonella Montinaro, Torsten Hartwig, Silvia von Karstedt, Itziar Areso Zubiaur, Johannes Lemke, Lucia Taraborelli, Silvia Surinova, Mona A. El-Bahrawy, Henning Walczak. Characterization and therapeutic harnessing of TRAIL’s pro-tumorigenic and pro-apoptotic functions in cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B18.

Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiopathogenesis characterized by fibrous cholangiopathy of large and small bile ducts. Systemic administration of a murine TNF-related apoptosis-inducing ligand (TRAIL) receptor agonist induces a sclerosing cholangitis injury in C57BL/6 mice, suggesting endogenous TRAIL may contribute to sclerosing cholangitis syndromes. Cellular inhibitor of apoptosis proteins (cIAP-1 and cIAP-2) are negative regulators of inflammation and TRAIL receptor signaling. We hypothesized that if endogenous TRAIL promotes sclerosing cholangitis, then cIAP depletion should also induce this biliary tract injury. Herein, we show that cIAP protein levels are reduced in the interlobular bile ducts of human PSC livers. Downregulation of cIAPs in normal human cholangiocytes in vitro by use of a SMAC mimetic (SM) induces moderate, ripoptosome-mediated apoptosis and RIP1-independent upregulation of proinflammatory cytokines and chemokines. Cytokine and chemokine expression was mediated by the non-canonical activation of NF-κB. To investigate whether downregulation of cIAPs is linked to generation of a PSC-like phenotype, an SM was directly instilled into the mouse biliary tree. Twelve hours after biliary instillation, TUNEL-positive cholangiocytes were identified; 5 days later, PSC-like changes were observed in the SM-treated mice, including a fibrous cholangiopathy of the interlobular bile ducts, portal inflammation, significant elevation of serum markers of cholestasis and cholangiographic evidence of intrahepatic biliary tract injury. In contrast, TRAIL and TRAIL-receptor deficient mice showed no sign of cholangiopathy following SM intrabiliary injection. We conclude that in vivo antagonism of cIAPs in mouse biliary epithelial cells is sufficient to trigger cholangiocytes apoptosis and a proinflammatory response resulting in a fibrous cholangiopathy resembling human sclerosing cholangitis. Therefore, downregulation of cIAPs in PSC cholangiocytes may contribute to the development of the disease. Our results also indicate that inhibition of TRAIL signaling pathways may be beneficial in the treatment of PSC.

Enhanced Metastasis Suppression by Targeting TRAIL Receptor 2 in a Murine Model of Triple-Negative Breast Cancer

Metastatic breast cancer is a deadly disease which requires new therapeutic strategies. Endogenous TNF-related apoptosis-inducing ligand (TRAIL) functions as a metastasis suppressor by activating proapoptotic TRAIL receptors (TRAIL-R1/DR4 and/or TRAIL-R2/DR5) in transformed cells, making it an attractive pathway for antimetastatic therapies. However, it is unclear whether TRAIL-R1 or TRAIL-R2 is a better therapeutic target in metastatic breast cancer. Several metastatic, triple (estrogen receptor, progesterone receptor, and HER2)-negative cancer cell lines were treated with human agonistic monoclonal antibodies targeting TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumumab). The effects on cell viability, apoptosis, and caspase-8 activation were determined. An orthotopic model of triple-negative breast cancer in which fluorescently labeled breast cancer cells metastasize from the mammary gland to lymph nodes and lung was utilized to evaluate the effects of mapatumumab, lexatumumab, or doxorubicin on primary and metastatic tumor burden in vivo. Lexatumumab was more effective than mapatumumab in activating caspase-8, inducing apoptosis and inhibiting long-term survival of metastatic cancer cells, which expressed both TRAIL-R1 and TRAIL-R2. Human mammary epithelial cells transformed by oncogenic Ras were more sensitive to lexatumumab than nontransformed cells. Lexatumumab inhibited lymph node and lung metastases more robustly than mapatumumab in an orthotopic model of triple-negative breast cancer; both agents inhibited mammary tumor growth. In addition, lexatumumab was more effective than doxorubicin at suppressing metastases at doses of doxorubicin that were associated with toxicity, even though doxorubicin reduced primary tumor burden more robustly than lexatumumab. Targeting TRAIL-R2 receptor may be an effective therapeutic strategy for metastatic breast cancer.

A critical role for TRAIL in resolution of granulomatous experimental autoimmune thyroiditis

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTG)-sensitized splenocytes activated in vitro with MTG and IL-12. Thyroid lesions reach maximal severity 20 days after cell transfer, and usually resolve or progress to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Our previous studies indicated that neutralization of TNF-alpha or FasL had no effect on G-EAT induction, but neutralization of TNF-alpha promoted, while neutralization of FasL inhibited, G-EAT resolution. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. This study was undertaken to define the role of endogenous TRAIL in G-EAT development and/or resolution. Neutralization of endogenous TRAIL had little effect on G-EAT induction, but significantly inhibited G-EAT resolution and increased thyroid fibrosis. This correlated with higher expression of pro-inflammatory cytokines and preferential expression of the pro-apoptotic molecule TRAIL, and anti-apoptotic molecules FLIP and Bcl-xL on inflammatory cells in thyroids of anti-TRAIL-treated recipients. The results suggest that endogenous TRAIL is not required for G-EAT development in recipients, but is critical for G-EAT resolution. Endogenous TRAIL might promote resolution, at least in part, through modulation of the balance between pro- and anti-inflammatory cytokines, and the expression pattern of pro- and anti-apoptotic molecules of thyroid epithelial cells (TECs) and inflammatory cells.

TRAIL limits excessive host immune responses in bacterial meningitis

Apart from potential roles in anti-tumor surveillance, the TNF-related apoptosis-inducing ligand (TRAIL) has important regulatory functions in the host immune response. We studied antiinflammatory effects of endogenous and recombinant TRAIL (rTRAIL) in experimental meningitis. Following intrathecal application of pneumococcal cell wall, a TLR2 ligand, we found prolonged inflammation, augmented clinical impairment, and increased apoptosis in the hippocampus of TRAIL(-/-) mice. Administration of rTRAIL into the subarachnoid space of TRAIL(-/-) mice or reconstitution of hematopoiesis with wild-type bone marrow cells reversed these effects, suggesting an autoregulatory role of TRAIL within the infiltrating leukocyte population. Importantly, intrathecal application of rTRAIL in wild-type mice with meningitis also decreased inflammation and apoptosis. Moreover, patients suffering from bacterial meningitis showed increased intrathecal synthesis of TRAIL. Our findings provide what we believe is the first evidence that TRAIL may act as a negative regulator of acute CNS inflammation. The ability of TRAIL to modify inflammatory responses and to reduce neuronal cell death in meningitis suggests that it may be used as a novel antiinflammatory agent in invasive infections.

On the TRAIL Toward Death Receptor–Based Cancer Therapeutics

Identifying targeted agents capable of selectively inducing death of cancer cells, while sparing normal host cells, has been the ultimate goal of cancer therapeutic drug development. In the late 1980s, monoclonal antibodies (mAbs) that were found to have antitumor activity via induction of tumor cell apoptosis were developed by several laboratories. These mAbs were found to ligate a tumor necrosis factor (TNF) receptor family member named Fas (APO-1/CD95), and investigation into the biochemical mechanism by which anti-Fas Abs induce cell death has revealed incredible detail regarding the apoptotic machinery. Unfortunately, agonistic antimurine Fas mAbs, when tested in preclinical models in vivo, were extremely toxic, because of rapid induction of hepatocyte apoptosis. In addition, a critical role for Fas/FasL interactions in the regulation of T-cell homeostasis was uncovered through genetic analyses of the autoimmune-prone mouse strains Lpr and Gld. It was, therefore, not possible to move nonspecific Fas-based therapeutics into the clinical arena, although engagement of Fas on tumor cells by Fas ligand expressed by specific CD8 T cells can promote tumor cell death in an antigen-specific fashion, and alterations in Fas signaling pathways in cancer have been reported to contribute to tumor cell resistance to immunemediated destruction. These observations support the notion that Fas engagement still may be an important component of antitumor immune effector mechanisms in patients. An additional TNF family member that also could promote apoptosis of tumor cells, called TNF-related apoptosis-inducing ligand (TRAIL), was subsequently identified. TRAIL can interact with five distinct receptors, two of which are death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Ligation of these receptors initiates a caspase-driven apoptotic pathway that is similar to what is induced through Fas. In contrast to FasL-deficient mice, TRAIL-deficient mice generated by gene targeting do not show global perturbation in T-cell development and homeostasis. Preclinical in vivo experiments of tumor growth in TRAIL-deficient mice (or in mice with endogenous TRAIL blocked) indicated a critical role in immune surveillance, because increased susceptibility to tumor formation— both spontaneously and in response to carcinogens—was observed. Constitutive expression of TRAIL receptors has been observed in a wide variety of human tumor types. These observations thus provided a sound rationale for the development of therapeutic agents that engage or mimic the TRAIL pathway on cancer cells. Several approaches have been explored to target TRAIL receptors therapeutically. One avenue of research has been the use of trimeric TRAIL itself as a recombinant natural ligand. Preclinical mouse models have shown effective antitumor activity in vivo, without significant toxicity. However, clinical development was stalled when hepatotoxicity was observed with some TRAIL preparations. It was subsequently determined that hepatocyte apoptosis occurred only with polyhistidine-tagged versions of recombinant TRAIL, a manufacturing artifact that could be avoided, which then reopened the door toward clinical application. In addition to TRAIL itself, an alternative therapeutic strategy is based on agonistic mAbs that specifically target the TRAIL receptors DR4 or DR5. The existence of decoy receptors that can bind TRAIL, yet not deliver a death signal, suggests a potential disadvantage of using the recombinant ligand and indicates a theoretical advantage to the use of specific anti-DR4 or -DR5 mAbs. All of these strategies are currently in clinical testing. In this issue of the Journal of Clinical Oncology, Tolcher et al report a phase I clinical trial of a fully human mAb against human TRAIL-R1/DR4. A careful dose escalation was performed, in which patients first received a single dose of mAb, which was an important step for safety concerns, given the potential for hepatotoxicity. The dose and frequency of administration were increased to 10 mg/kg every 14 days. A terminal half-life (t1/2 ) of 18 days was observed, suggesting that dosing every 2 to 3 weeks is a reasonable schedule. The concentration of anti-TRAIL-R1 mAb that is effective for tumor apoptosis in vitro is approximately 1 g/mL, and all dose levels above 1 mg/kg achieved this concentration as a trough level in this phase I study. Importantly, no grade 3 elevations in transaminases and no bilirubin elevation clearly attributable to the drug were observed, although several patients did show grade 1 to 2 transaminase elevation, and two patients with pre-existing liver metastases with elevated transaminase levels at baseline did increase to grade 3. Therefore, although the overwhelming hepatotoxicity previously observed with anti-Fas mAbs in murine studies did not occur, these detectable effects on the liver from this trial warrant caution in moving forward in combination with other anticancer agents having hepatic effects, or in the context of patients receiving hepatotoxic drugs for other indications. There were no global effects on lymphocyte subsets seen in this study. However, recent work has indicated a clear role for the activity of TRAIL in determining whether CD4 T-cell help for JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 11 APRIL 1

TRAIL receptor–mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Conditionally replicative adenovirus vector carrying TRAIL gene for enhanced oncolysis of human hepatocellular carcinoma

Conditionally replicative adenovirus (CRAd) mediated tumor specific gene therapy based on transcriptional control is considered a new direction for the treatment of cancer. Our previous studies showed that an HS4 insulator increased the alpha-fetoprotein (AFP) promoter-driven expression in the context of an adenovirus (Ad) vector, while retaining the highly specific gene expression in hepatoma cells in vitro and in vivo. In this study, we constructed two HS4-AFP promoter based CRAd vectors (Ad.HS4.AFP.E1a/TRAIL and Ad.HS4.AFP.E1a) with and without the expression cassette of TNF-related apoptosis-inducing ligand (TRAIL). The TRAIL-expressing virus vector, Ad.HS4.AFP.E1a/TRAIL, exhibited more obvious oncolytic effect than Ad.HS4.AFP.E1a in both high-AFP-producing HCC cell lines (Hep3B and HUH7) and a low-AFP-producing HCC cell line (PLC/PRF/5) examined, indicating endogenous TRAIL over-expression increased CRAd potency. The enhanced hepatoma cell death was mainly mediated through apoptotic mechanism, as evidenced by the activation of caspase-3, binding of annexin V and inhibition by caspase inhibitor z-vad-fmk. In s.c. xenograft of low-AFP-producing PLC/PRF/5 hepatoma model, the administration of Ad.HS4.AFP.E1a/TRAIL resulted in a more potent oncolytic effect compared with the same dose of Ad.HS4.AFP.E1a 28 days after virus exposure. This study demonstrated that the TRAIL in the context of a CRAd vector was able to increase the oncolytic activity in low-AFP-producing HCC cells in vitro and in vivo. Considering that oncolytic viruses destroy tumor cells expressing low levels of the tumor marker is a clinical concern, TRAIL might be a useful tool to improve the efficacy of these vectors.

Aberrant Expression of Trail in B Chronic Lymphocytic Leukemia (B-CLL) Cells.

Chronic lymphocytic leukemia (CLL) is a quintessential example of human malignancies that are caused primarily by defect in apoptosis. Defects in apoptotic pathways contribute also to chemoresistance and can promote resistance to cellular immune responses. TNF-related apoptosis inducing ligand (TRAIL), interacts with four high affinity membrane receptors (TRAIL-R1 – R4): R1 and R2 are thought to transducer apoptotic signals, while R3 and R4 may act as “decoy” receptors, protecting cells from apoptosis. Despite its potential anti–cancer activity, TRAIL alone has a low cytotoxic activity on B-CLL, and no data are available on the expression of TRAIL and its biological potential function in B-CLL.We examined the expression of TRAIL in peripheral blood CD19+/CD5+ B lymphocytes from 44 patients affected by CLL at diagnosis, the susceptibility of B-CLL cells to recombinant TRAIL and the role of endogenous membrane-bound TRAIL on autologous cell survival.Each B-CLL patient had a follow up time of 12 to 24 months from diagnosis and was clinically stable. None of the patients received chemotherapy.Lymphocytes from normal PBMC revealed an absent or dim expression of TRAIL. Surface TRAIL was detected in all 44 B-CLL examined, at variable intensity from patient to patient, but with an overall significantly greater MFI with respect to normal lymphocytes. Higher levels of TRAIL mRNA and protein were documented in purified CLL CD19+ B lymphocytes, confirming that TRAIL was overexpressed at the transcriptional level.The addition in culture of a TRAIL-R1-Fc chimera, which binds at high affinity to surface TRAIL, significantly decreased the percentage of viable cells with respect to untreated cultures. No effects were noted when TRAIL-R1-Fc chimera was added to normal CD19+ B cells. Preincubation of TRAIL-R1-Fc chimera with recombinant TRAIL significantly counteracted the decrease of viability induced by TRAIL-R1-Fc chimera. These data indicate that surface TRAIL might be involved in a promoting a prosurvival activity, at least in some B-CLL samples.We then investigated the response in terms of cell viability to exogenously added recombinant TRAIL (1 μg/ml) of all 44 B-CLL samples examined in this study. On the basis of their response to recombinant TRAIL, B-CLL samples could be subdivided in 3 groups. In 11 patients (group 1), B-CLL cells showed a faster decline in the number of viable cells upon treatment with recombinant TRAIL. In 19 samples (group 2),no significant variation in terms of viable cell number in TRAIL-treated cultures was observed. In 14 patients (group 3), TRAIL-treatment results in a significantly higher numbers of viable cells compared to untreated cultures.At flow cytometry, B-CLL lymphocytes expressed at variable levels TRAIL-R1, TRAIL-R2 and TRAIL-R4, while TRAIL-R3 was never detected. TRAIL-R1 was expressed at low levels in a subset of B-CLL samples, while TRAIL-R2 was expressed in almost the totality of the B-CLL samples. TRAIL-R4 was detected in the majority of B-CLL lymphocytes, but at lower levels than in normal lymphocytes. Of note, the different apoptotic/survival response to recombinant TRAIL among the three groups of B-CLL samples described above, apparently did not depend on differences in death and/or decoy receptor patterns of expression. A progressive increase of TRAIL MFI was noticed from group 1 to group 3.

TNF‐related apoptosis‐inducing ligand (TRAIL)/Apo2L suppresses experimental autoimmune encephalomyelitis in mice

Studies have suggested that endogenous TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L may suppress the induction of some autoimmune diseases in mice. Here, we show that TRAIL/Apo2L suppresses autoimmune damage in relapsing-remitting, and non-remitting models of experimental autoimmune encephalomyelitis (EAE). TRAIL/Apo2L-deficient mice and wild-type mice treated with neutralizing anti-TRAIL/Apo2L antibody displayed enhanced clinical score, increased T-cell proliferative responses to myelin oligodendrocyte glycoprotein (MOG), and increased numbers of inflammatory lesions in the spinal cord and central nervous system. TRAIL neutralization immediately before disease onset was most effective at exacerbating disease score. More importantly, therapeutic intervention with recombinant soluble TRAIL/Apo2L delayed the onset and reduced the severity of MOG-induced EAE. These data are the first to illustrate the potential therapeutic value of recombinant TRAIL/Apo2L in suppressing T-cell-mediated autoimmune diseases.