TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kappaB.

TRAIL induces apoptosis through two closely related receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Here we show that TRAIL-R1 can associate with TRAIL-R2, suggesting that TRAIL may signal through heteroreceptor signaling complexes. Both TRAIL receptors bind the adaptor molecules FADD and TRADD, and both death signals are interrupted by a dominant negative form of FADD and by the FLICE-inhibitory protein FLIP. The recruitment of TRADD may explain the potent activation of NF-kappaB observed by TRAIL receptors. Thus, TRAIL receptors can signal both death and gene transcription, functions reminiscent of those of TNFR1 and TRAMP, two other members of the death receptor family.

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4), pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Loss of TRAIL-receptors is a recurrent feature in pancreatic cancer and determines the prognosis of patients with no nodal metastasis after surgery

INTRODUCTION Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity. AIMS AND METHODS Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated. RESULTS The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22-0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance. CONCLUSION This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.

Characterization of two receptors for TRAIL.

Two receptors for TRAIL, designated TRAIL-R2 and TRAIL-R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL-R2 is structurally similar to the death-domain-containing receptor TRAIL-R1 (DR-4), and is capable of inducing apoptosis. In contrast, TRAIL-R3 does not promote cell death. TRAIL-R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL-R3 is due to the presence of multiple threonine-, alanine-, proline- and glutamine-rich repeats (TAPE repeats). TRAIL-R2 shows a broad tissue distribution, whereas the expression of TRAIL-R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL-mediated signals.

The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation.

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Apoptosis induced by death receptors.

Death receptors belong to the TNF receptor family and are characterised by an intracellular death domain that serves to recruit adapter proteins such as TRADD and FADD and cysteine proteases such as Caspase-8. Activation of Caspase-8 on the aggregated receptor leads to apoptosis. Triggering of death receptors is mediated through the binding of specific ligands of the TNF family, which are homotrimeric type-2 membrane proteins displaying three receptor binding sites. There are various means of modulating the activation of death receptors. The status of the ligand (membrane-bound vs. soluble) is critical in the activation of Fas and of TRAIL receptors. Cleavage of membrane-bound FasL to a soluble form (sFasL) does not affect its ability to bind to Fas but drastically decreases its cytotoxic activity. Conversely, cross-linking epitope-tagged sFasL with anti-tag antibodies to mimic membrane-bound ligand results in a 1000-fold increase in cytotoxicity. This suggests that more than three Fas molecules need to be aggregated to efficiently signal apoptosis. Death receptors can also be regulated by decoy receptors. The cytotoxic ligand TRAIL interacts with five receptors, only two of which (TRAIL-R1 and -R2) have a death domain. TRAIL-R3 is anchored to the membrane by a glycolipid and acts as a dominant negative inhibitor of TRAIL-mediated apoptosis when overexpressed on TRAIL-sensitive cells. Intracellular proteins interacting with the apoptotic pathway are potential modulators of death receptors. FLIP resembles Caspase-8 in structure but lacks protease activity. It interacts with both FADD and Caspase-8 to inhibits the apoptotic signal of death receptors and, at the same time, can activate other signalling pathways such as that leading to NF-kappa B activation.

Reovirus-induced apoptosis is mediated by TRAIL.

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8% (TRAIL) or 17% (ADM) to 38.7%, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by &gt;1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Induction of synthetic lethality in mutant KRAS cells for non-small cell lung cancers chemoprevention and therapy

Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Despite improvement in treatment strategies, the 5-year survival rate of lung cancer patients remains low. Thus, effective chemoprevention and treatment approaches are sorely needed. Mutations and activation of KRAS occur frequently in tobacco users and the early stage of development of non-small cell lung cancers (NSCLC). So they are thought to be the primary driver for lung carcinogenesis. My work showed that KRAS mutations and activations modulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors by up-regulating death receptors and down-regulating decoy receptors. In addition, we showed that KRAS suppresses cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of ERK/MAPK-mediated activation of c-MYC which means the mutant KRAS cells could be specifically targeted via TRAIL induced apoptosis. The expression level of Inhibitors of Apoptosis Proteins (IAPs) in mutant KRAS cells is usually high which could be overcome by the second mitochondria-derived activator of caspases (Smac) mimetic. So the combination of TRAIL and Smac mimetic induced the synthetic lethal reaction specifically in the mutant-KRAS cells but not in normal lung cells and wild-type KRAS lung cancer cells. Therefore, a synthetic lethal interaction among TRAIL, Smac mimetic and KRAS mutations could be used as an approach for chemoprevention and treatment of NSCLC with KRAS mutations. Further data in animal experiments showed that short-term, intermittent treatment with TRAIL and Smac mimetic induced apoptosis in mutant KRAS cells and reduced tumor burden in a KRAS-induced pre-malignancy model and mutant KRAS NSCLC xenograft models. These results show the great potential benefit of a selective therapeutic approach for the chemoprevention and treatment of NSCLC with KRAS mutations.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

T cell-specific FADD-deficient mice: FADD is required for early T cell development

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD−/− T cells from RAG-1−/− reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8− stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis, In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2, The location of TRAIL-R1, -R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells, Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in decoy receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cell.