Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhanced proliferation and increased IFN-gamma production in T cells by signal transduced through TNF-related apoptosis-inducing ligand

TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.     

The role of the TRAIL/TRAIL receptors system in hematopoiesis and endothelial cell biology

TRAIL is a member of the tumor necrosis factor superfamily that interacts with an unusually complex receptor system, comprising transmembrane (TRAIL-R1, -R2, -R3 and -R4) and soluble (osteoprotegerin) receptors. TRAIL has received considerable attention because of the finding that many cancer cell types are sensitive to TRAIL-induced apoptosis. However, increasing experimental evidence shows that TRAIL exhibits regulatory roles in various normal tissues, as well. Although the best-characterized biological activity of TRAIL is in the homeostatic regulation of the immune system, in this review we have summarized and discussed the physiological function of TRAIL and its receptors, in normal hematopoiesis and vascular physiopathology.     

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG‐DSPE/TRAIL nanoparticles

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single‐walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1‐pyrenebutyric acid N‐hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1‐pyrenebutyric acid N‐hydroxysuccinimide ester nanotubes were mixed with methoxy‐poly(ethylene glycol)‐1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH ) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand‐receptor interface. It was shown that their rank‐ordered affinities were strongly different in the sequence TRAIL_DR4 < NPT_DR4 < TRAIL_DR5 < NPT_DR5, and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.     

Compartmentalization of TNF-related apoptosis-inducing ligand (TRAIL) death receptor functions: emerging role of nuclear TRAIL-R2

Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.     

Death to the bad guys: Targeting cancer via Apo2L/TRAIL

All higher organisms consist of an ordered society of individual cells that must communicate to maintain and regulate their functions. This is achieved through a complex but highly regulated network of hormones, chemical mediators, chemokines and other cytokines, acting as ligands for intra or extra-cellular receptors. Ligands and receptors of the tumor necrosis factor (TNF) superfamilies are examples of signal transducers, whose integrated actions influence the development, homeostasis and adaptive responses of many cells and tissue types. Apo2L/TRAIL is one of several members of the tumour necrosis factor superfamily that induce apoptosis through the engagement of death receptors. Apo2L/TRAIL interacts with an unusually complex receptor system, which in humans comprises two death receptors and three decoy receptors. This molecule has received considerable attention recently because of the finding that many cancer cell types are sensitive to Apo2L/TRAIL-induced apoptosis, while most normal cells appear to be resistant to this action of Apo2L/TRAIL. In this review, we specifically emphasise on the actions of Apo2L/TRAIL with respect to its apoptotic signaling pathways and summarise what is known about its physiological role. The potential therapeutic usefulness of Apo2L/TRAIL, especially in combination with chemotherapeutic agents, is also discussed in some detail.     

Modulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors in a human osteoclast model in vitro

TRAIL (TNF-related apoptosis-inducing ligand) has been shown to induce apoptosis by binding to TRAIL-R1 and -R2 death receptors, but not to TRAIL-R3 or -R4, its decoy receptors that lack the internal death domain. Osteoclasts (Ocs) are sensitive to TRAIL-induced apoptosis, and modulation of these receptors may change Oc sensitivity to TRAIL. Using human Oc cultures, we first investigated the gene expression profile of these receptors (TNFRSF10 -A, -B, -C, -D encoding TRAIL-Rs 1–4) by real time PCR after adding osteotropic factors during the last week of Oc cultures. We observed a significant decrease in the expression of TNFRSF10-A after the addition of TGFβ, and an increase in that of TNFRSF10-A and -B post-PTH stimulation. Protein expression of TRAIL-R1 and -R3 was upregulated in the presence of MIP-1α, but down-regulated in the presence of TGFβ (R1), TRAIL (R2) or OPG (R3). The percentage of Ocs expressing the TRAIL-R1 and/or -R2 at their surface was increased by MIP-1α and TRAIL, increased (R2) or decreased (R1) by TGFβ, and the percentage expressing TRAIL-R3 was increased by MIP-1α, TRAIL and RANKL. Although significant, the magnitude of all these changes was of about 10–15%. While a direct correlation between these changes and TRAIL-induced Oc apoptosis was less clear, a protective effect was observed in Ocs that had been treated with OPG, and an additive effect in Ocs pre-treated with TRAIL or TGFβ increased TRAIL sensitivity.     

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-kappaB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-kappaB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a transducer for proinflammatory and angiogenic signals in human brain tumors.     

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus‐infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV‐1‐infected cells. By combining an unbiased large‐scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV‐1‐infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor‐mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL‐mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL‐mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti‐HIV‐1 activity of NK cells but also possesses a multifunctional role beyond receptor‐mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.     

It's not over until the FAT lady sings

The death receptors FAS, TRAIL‐Rs and TNFR1 play critical roles in programmed cell death, particularly in the immune system. Upon ligation of death receptors, caspase‐8 is activated within the so‐called ‘Death Induced Signalling Complex’ (DISC) but the mechanisms that mediate and modulate the activation of caspase‐8 are still not fully understood. This is an important issue because caspase‐8 is essential for apoptosis induced by death receptors. In this issue of The EMBO Journal, Kranz and Boutros ( 2014 ) describe their findings from a whole genome siRNA screen for the identification of novel regulators of death receptor induced apoptosis signalling. They identified the atypical cadherin FAT1 as a negative regulator of TRAIL‐R‐mediated caspase‐8 activation and consequent induction of apoptosis, although it had no impact on NF‐κB activation. The authors also show that FAT1 depletion substantially increased TRAIL‐induced killing of glioblastoma‐derived cell lines, suggesting a potential novel approach for treatment of this highly aggressive cancer.     

Involvement of TRAIL/TRAIL-receptors in human intestinal cell differentiation

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.     

The three Rs along the TRAIL: Resistance,re-sensitization and reactive oxygen species (ROS)

Abstract

Ligation of the Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors has been associated with cancer specific apoptotic execution in a number of model systems. This has generated tremendous interest in the use of TRAIL as a potential therapeutic modality. However, recent evidence indicates that resistance to TRAIL might present with a therapeutic challenge. In this short report, we review the basic biology of TRAIL signalling in cancer cells, highlight the mechanisms underlying resistance to TRAIL and the ability of small molecule compounds to re-sensitize cells to TRAIL-mediated apoptosis. In particular, we provide evidence that intracellular reactive oxygen species could be critical in regulating the response of cancer cells to TRAIL.     

Conjugation of doxorubicin to cell penetrating peptides sensitizes human breast MDA-MB 231 cancer cells to endogenous TRAIL-induced apoptosis

Previous work from our laboratory has shown that coupling doxorubicin (Dox) to cell penetrating peptides (Dox–CPPs) is a good strategy to overcome Dox resistance in MDA-MB 231 breast cancer cells. We also reported that, in contrast to unconjugated Dox-induced cell death, the increase in apoptotic response does not involve the mitochondrial apoptotic pathway. In this study, we demonstrate that both Dox and Dox–CPPs can increase the density of the TRAIL receptors DR4 and DR5 at the plasma membrane and moderately sensitize MDA-MB 231 cells to exogeneously added recombinant TRAIL, as has already been shown for other chemotherapeutic drugs. Moreover, we show that Dox–CPPs, used alone, induce the clustering of TRAIL receptors into ceramide-enriched membrane lipid rafts, a property not shared by unconjugated Dox and that this process is due to the generation of ceramide during Dox–CPPs treatment. In addition, MDA-MB 231 cells were found to express TRAIL and we show that the increased apoptotic rate induced by Dox–CPPs is due to the sensitization of MDA-MB 231 cells to endogenous TRAIL. The capacity of Dox–CPPs to sensitize cancer cells to physiologic amounts of TRAIL suggests that, in addition to their efficiency in combination chemotherapy, these compounds might increase the response of tumor cells to cytotoxic lymphocyte-mediated killing via TRAIL.     

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a modelin vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL-induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.     

Selective TRAIL‐induced cytotoxicity to lung cancer cells mediated by miRNA response elements

Lung cancer is among the most common cancers, and the current therapeutic strategies are still inefficient in most cases. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising biological agent for cancer treatment because of its potent pro‐apoptotic effect on cancer cells. However, TRAIL also induces apoptosis in normal cells and therefore may cause toxicity to normal tissues if clinically applied. To address this issue, we inserted microRNA response elements (MREs) of miR‐133a, miR‐137 and miR‐449a, which are all underexpressed in lung cancer cells, into an adenoviral vector to regulate TRAIL expression. This MRE‐regulated vector (Ad‐TRAIL‐MRE) was able to express TRAIL in a lung‐cancer‐specific fashion. No TRAIL expression was detected in normal cells. Consistently, Ad‐TRAIL‐MRE exerted cytotoxicity to lung cancer cells, rather than normal cells, perhaps via inducing selective apoptosis. The selective TRAIL‐mediated growth‐inhibiting effect was further confirmed in a tumour xenograft model. Also, Ad‐TRAIL‐MRE only resulted in very low hepatotoxicity when applied. Collectively, we generated a novel TRAIL‐expressing adenoviral vector that was regulated by MREs. This strategy permits TRAIL expression in a lung‐cancer‐specific manner and is worth further studying for clinical trials. Copyright © 2014 John Wiley & Sons, Ltd.     

TRAIL stimulates proliferation of vascular smooth muscle cells via activation of NF-kappaB and induction of insulin-like growth factor-1 receptor

TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) is a multifunctional protein regulating homeostasis of the immune system, infection, autoimmune diseases, and apoptosis. However, its function in normal, nontransformed tissues is not clear. Here we show that TRAIL increases vascular smooth muscle cell (VSMC) proliferation in vitro, effects that can be blocked with neutralizing antibodies to TRAIL receptors DR4 and DcR1. In aortocoronary saphenous vein bypass grafts in vivo, TRAIL co-localizes with VSMC, proliferating cell nuclear antigen, and insulin-like growth factor type 1 receptor (IGF1R) expression but not active caspase-3. TRAIL is required for serum-inducible IGF1R expression, and antisense IGF1R inhibits TRAIL-induced VSMC proliferation. At 1 ng/ml, TRAIL stimulates IGF1R mRNA expression greater than insulin-like growth factor-1 and also activates the IGF1R promoter 7-fold. TRAIL-inducible IGF1R expression requires NF-kappaB activation. Consistent with this, ammonium pyrrolidine dithiocarbamate, a pharmacological inhibitor of NF-kappaB, blocks TRAIL-induced IGF1R expression, and p65 overexpression increases IGF1R protein levels. In addition, NF-kappaB binds a novel TRAIL-responsive element on the IGF1R promoter. Our findings suggest that the biological functions of TRAIL in VSMC extend beyond its role in promoting apoptosis. Thus, TRAIL may play an important role in atherosclerosis by regulating IGF1R expression in VSMC in an NF-kappaB-dependent manner.