Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was independently identified by two different groups as the third member of the tumor necrosis factor (TNF) super-family (Wiley et al. 1995; Pitti et al. 1996). It was soon described as capable of inducing apoptosis in transformed cells while sparing normal cells (LeBlanc and Ashkenazi 2003). Apo2L/TRAIL is a type-II membrane protein, composed of 281 amino acids and its gene, designated as TNFSF10, is located on human chromosome 3 at locus 3q26. Its C-terminal extracellular domain shares significant homology with other members of the TNF super-family, whereas the N-terminal does not. The polypeptide moiety of the Apo2L/TRAIL monomer has a predicted molecular mass of 32.5 kDa, but its mature, fully glycosylated form has a molecular mass of around 41 kDa. There is a potential cleavage site in the extracellular domain of Apo2L/TRAIL at amino acid position 114, which would generate a soluble form of 24 kDa, suggesting that it could be cleaved by a putative metalloprotease releasing the extracellular portion. However, to date no evidence of the existence of this metalloprotease has been found in vivo. Like other members of TNF super-family, Apo2L/TRAIL is a homotrimer, with each monomer composed of two antiparallel β-sheets. Interestingly, unlike other members of the TNF super-family, native Apo2L/TRAIL contains a central Zn atom buried at the trimer interface, which is crucial for the stability, solubility, and biological activity of the protein.
Apo2L/TRAIL and Its Receptors
Among them, only TRAIL-R1 and TRAIL-R2 are able to transduce the apoptotic signal, since both TRAIL-R3/DcR1and TRAIL-R4/DcR2 lack functional cytoplasmic death domains (DD) (Merino et al. 2006). In fact, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 have been suggested to act as decoy receptors that inhibit apoptosis induction by Apo2L/TRAIL as a consequence of ligand scavenging. In addition, Apo2L/TRAIL-R4 has been proposed to be capable of inhibiting Apo2L/TRAIL-induced apoptosis by forming ligand-independent inactive complexes with TRAIL-R2 or the induction of pro-survival pathways such as NF-κB. However, there is still controversy concerning the physiological role of TRAIL-R3 and TRAIL-R4, and their function might depend on the cell type.
Finally, as afore mentioned, Apo2L/TRAIL can bind, but with lower affinity, to a soluble receptor called OPG. The main function of OPG is the binding and modulation of the interaction of receptor activator of NF-κB ligand (RANKL), another TNF super-family member, with its cell-surface receptor, RANK. RANKL-RANK interaction induces osteoclast activation, differentiation, and bone resorption. Since Apo2L/TRAIL- or DR5-deficient mice (the only TRAIL receptor expressed in mouse) are viable and exhibit normal bone density, it is unlikely that Apo2L/TRAIL may have a role in bone remodeling.
The initial step of apoptosis induced by Apo2L/TRAIL is the binding of the trimeric ligand to DR4 and/or DR5. This interaction causes the clustering of the death receptor followed by the recruitment of the adaptor protein Fas-associated death domain (FADD) to the intracellular DD of the receptors, promoting the assembly of death-inducing signaling complex DISC. The homotypic interaction of FADD with the DRs through their respective DDs exposes the death-effector domain (DED) of FADD and allows the recruitment of the inactive forms of caspases-8 or -10 through the binding to DED caspase pro-domains (Sprick et al. 2000). Recruitment of procaspase-8 to the DISC induces its activation by conformational change followed by auto-processing and release into the cytosol. Once in the cytosol, active caspases-8 and -10 can either directly cleave and activate the effector caspases (mainly caspase-3) or the BH3-only protein Bid, rendering truncated-Bid (tBid) (Kantari and Walczak 2011).
Caspases-8 and -10 are recruited to and activated at the DISC with similar kinetics and may function independently of each other. Some authors have reported that caspase-10 may substitute for caspase-8, but, in some cell types, caspase-10 does not restore the sensitivity to Apo2L/TRAIL in caspase-8-deficient cells.
Apart from triggering the canonical extrinsic apoptotic pathway, Apo2L/TRAIL is able to trigger nonapoptotic signaling pathways, namely, pro-inflammatory pathways and necroptosis (Fig. 2). In the first case, the mechanism by which Apo2L/TRAIL triggers pro-inflammatory pathways involves the formation of a secondary cytoplasmic complex (Complex II) following DISC formation (Varfolomeev et al. 2005). This secondary complex would retain the DISC components FADD, caspase-8, and cFLIP and, additionally, would recruit RIPK1, TRAF2, and NEMO. This secondary complex would be responsible for the activation pro-inflammatory pathways involving NF-κB, MAPK, and JNK. Initially, Apo2L/TRAIL-induced NF-κB activation was suggested to be a mechanism for negative regulation of Apo2L/TRAIL-induced apoptosis. However, it seems that it might fulfill other functions in TRAIL-resistant cells, where it has been show to mediate RIP1-dependent Apo2L/TRAIL-induced survival and proliferation. The biological significance of NF-κB activation by Apo2L/TRAIL is not fully understood but has been implicated in the restriction of apoptosis induction, proliferation, cell migration, and invasion in certain tumor cell lines resistant to Apo2L/TRAIL-induced apoptosis. It has also been reported that Apo2L/TRAIL induces activation of other signaling pathways such as PI3K/Akt pathway, involved in survival and cell motility, and others involving MAPKs which promotes cell proliferation and differentiation (Azijli et al. 2013).
Recently, a newly discovered mechanism by which endogenous Apo2L/TRAIL/TRAIL-R signaling promotes dissemination and invasion of KRAS-mutated tumors has been described. In this context, autocrine activation of TRAIL-R2 by endogenous tumor Apo2L/TRAIL triggers activation of Rac1 and induces cell migration (von Karstedt et al. 2015). Interestingly, this activation was found to be independent of the DD, but require the membrane proximal domain (MPD) of TRAIL-R2 instead. On the other hand, in conditions where caspases are inhibited and apoptosis blocked, Apo2L/TRAIL has been described to trigger necroptosis in several cell types. For that, Apo2L/TRAIL induced the formation of a complex called necrosome in which RIPK1 and RIPK3 are recruited (Jouan-Lanhouet et al. 2012). The necrosome complex can activate MLKL by phosphorylation which, in turn, promotes its oligomerization. Finally, active MLKL inserts into and permeabilizes plasma membrane leading to necrotic cell death.
In summary, although the physiological relevance of the Apo2L/TRAIL-induced nonapoptotic signaling is not fully established, these observations must be considered in particular in cell types in which Apo2L/TRAIL does not induce a rapid apoptosis.
Regulation of Apo2L/TRAIL-Induced Apoptosis
Several mechanisms of different nature have been described that may regulate Apo2L/TRAIL signaling. Posttranslational modifications of DR4 and DR5 by glycosylation or palmitoylation seem to be important modulators of the initial events of Apo2L/TRAIL signaling. O-Glycosylation promoted ligand-stimulated clustering of DR4 and DR5, the first step in DISC formation, essential for recruitment and activation of the initiator caspase-8. Conversely, depletion of certain O-glycosylation enzymes attenuates caspase-8 activation at the DISC, whereas overexpression of GALNT14, the O-glycosylation-initiating enzyme, enhances the formation of the DISC and sensitizes cells to Apo2L/TRAIL (Wagner et al. 2007). At the DISC level, the main regulator protein is cFLIP. This protein, which shares high sequence homology with caspase-8 and -10 except for lacking protease activity, is also recruited to the DISC similarly to caspases -8 and -10. There are a number of splicing variants of cFLIP, but only a longer (cFLIPL) and a shorter version (cFLIPS) can be usually detected at the protein level. Both isoforms of cFLIP are recruited to the DISC by homotypic DED interactions. The C-terminal part of cFLIPL consists of two catalytically inactive caspase-like domains, whereas the C-terminal portion of cFLIPS is neither homologous to procaspase-8 nor -10. However, modulation of caspase-8 activation depends on the relative amounts of cFLIPS, cFLIPL, and caspase-8. More specifically, cFLIPS can inhibit caspase-8 activation in a dominant-negative manner by competing for binding to FADD. The role of cFLIPL is, however, more complex and seems to be dependent on the relative amounts of both caspase-8 and cFLIPL. Although cFLIPL was first reported to act as an antiapoptotic protein in a similar manner to cFLIPS, later studies demonstrated that the cFLIPL-caspase-8 heterodimer, apart from retaining enzymatic activity, also displays an enhanced and more localized activity towards certain substrates when compared to the caspase-8 homodimer, somehow modulating caspase-8 substrate specificity. Accordingly, it was subsequently shown that the activity of the FLIPL-caspase-8 heterodimer is indeed crucial to negatively regulate necroptosis. Nevertheless, it should be noted that when expressed at high levels, cFLIPL can also completely block apoptosis. Several studies have demonstrated that cancer cells exploit overexpression of cFLIP to evade Apo2L/TRAIL-induced apoptosis and, consequently, downregulation of cFLIP by different means can sensitize many cancer cells to Apo2L/TRAIL-induced apoptosis (Hellwig and Rehm 2012).
On the other hand, ubiquitination has been described to regulate the full activation of caspase-8 upon Apo2L/TRAIL activation (Jin et al. 2009). Immunoprecipitation of the Apo2L/TRAIL-DISC, followed by mass spectrometry, revealed the presence of the E3 ubiquitin ligase subunit Cullin 3 (CUL3) and the deubiquitinase (DUB) A20 within the DISC. Biochemical analysis indicated that caspase-8 is polyubiquitinated on its C-terminal region (within the p10 subunit) and identified K461 as the likely main conjugation site. Both K48- and K63-linked polyubiquitin chains were detected on caspase-8 after Apo2L/TRAIL stimulation, and both linkages were required for caspase-8 activation. More specifically, ubiquitination of caspase-8 promoted its translocation from the receptor-based DISC to intracellular ubiquitin-rich protein aggregates or foci. This localized concentration of caspase-8 seemed to facilitate its full activation, ultimately leading to effector-caspase engagement and apoptosis. On the contrary, deubiquitination by A20 counteracted this effect.
An additional level of regulation is provided by endocytosis of the ligand-receptor complexes. Upon binding of Apo2L/TRAIL to its plasma membrane receptor(s), both DR4 and DR5 are rapidly internalized in lipid vesicles through a dynamin-dependent mechanism. However, contrary to Fas signaling, internalization of the ligand-receptor complex is not required for Apo2L/TRAIL-mediated DISC formation and apoptosis signaling.
Finally, sensitivity to Apo2L/TRAIL-induced apoptosis may also be modulated by X chromosome-linked inhibitor of apoptosis (XIAP), an endogenous inhibitor of caspases-3, -7, and -9 which prevents unintended caspase activation in living cells. Interfering with XIAP expression renders cells highly sensitive to Apo2L/TRAIL bypassing the requirement for mitochondrial amplification in type II cells. Second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), a protein released from mitochondria during apoptosis, binds to and antagonizes XIAP. It has been proposed that the ratios of cFLIP to caspase-8 and XIAP to Smac/DIABLO together may determine whether the cells respond in a type I or type II way to DRs-Apo2L/TRAIL interactions (Fig. 2) (Gonzalvez and Ashkenazi 2010).
Biological Role of Apo2L/TRAIL
Although the ability of Apo2L/TRAIL to kill certain transformed cells by apoptosis is well established, its physiological role is not fully understood. Studies with mice deficient for Apo2L/TRAIL or its apoptosis-inducing receptor (DR5/TRAIL-R), as well as experiments carried out with Apo2L/TRAIL-blocking agents, have led to unraveling diverse functions of Apo2L/TRAIL in vivo. Apo2L/TRAIL-deficient mice do not display any overt developmental defects. Similarly, TRAIL-R knockout mice are viable and normally develop, indicating that Apo2L/TRAIL signaling is not essential for normal embryonic development.
The major roles of Apo2L/TRAIL are exerted in the immune system, shaping and regulating the immune response. This was early suggested by the inducible expression of Apo2L/TRAIL in immune cells. At least in human-activated T cells, Apo2L/TRAIL is stored inserted in the inner membrane vesicles of cytoplasmic multivesicular bodies, also known as secretory lysosomes. When T-lymphocytes receive activation signals, native Apo2L/TRAIL is secreted to extracellular medium in the form of microvesicles (exosomes) after fusion of the outer membrane of secretory lysosomes with the plasma membrane. This membrane-bound form of Apo2L/TRAIL displays full pro-apoptotic activity (Anel et al. 2007) (Figs. 1 and 2).
The immunoregulatory role of Apo2L/TRAIL is dependent on two different mechanisms: (1) Apo2L/TRAIL can inhibit IL2-dependent human CD8+ T cell blast proliferation through a cell-cycle arrest in G 2/M and (2) Apo2L/TRAIL is also able to induce apoptosis of CD8+ T cell blasts, but in this case, an additional re-stimulation is needed. It has also been clearly demonstrated that Apo2L/TRAIL regulates CD8+ T cell memory (Anel et al. 2007). The key role of Apo2L/TRAIL in the regulation of T cell responses has been confirmed in Apo2L/TRAIL knockout mice, which are much more susceptible to develop experimentally induced autoimmune diseases, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, than wild-type mice (Lamhamedi-Cherradi et al. 2003). In fact, Apo2L/TRAIL has been proposed to be used as a treatment in several experimental models of those autoimmune diseases with good results, especially in the case of animal models of rheumatoid arthritis. The efficiency of Apo2L/TRAIL for the treatment of this inflammatory disease is greatly improved through its association with liposomes, mimicking its physiological released form in exosomes.
Apo2L/TRAIL is one of the effector arms of natural killer (NK) cells and plays a key role in NK cell-mediated, interferon (IFN)-γ-dependent, suppression of tumor cell growth and prevention of metastasis formation. Indeed, one of the physiological roles of Apo2L/TRAIL seems to be the tumor immune surveillance (Takeda et al. 2001). TRAIL knockout mice are more susceptible to cell-inoculated and chemically induced tumors as well as to metastasis dissemination than wild-type mice. In addition, TRAIL-deficient mice show a high rate of spontaneous hematological tumors appearing at old age (Zerafa et al. 2005). The low systemic toxicity of Apo2L/TRAIL on normal cells, while exerting a potent pro-apoptotic activity on a variety of human tumors has led to the development of different recombinant versions of Apo2L/TRAIL and agonistic monoclonal antibodies to its signaling receptors (anti-DR4 or anti-DR5). These agents, alone or in combination with chemotherapeutic drugs, provide new and promising approaches to cancer treatment (de Miguel et al. 2016).
The membrane protein Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) is a cytokine which interacts with a complex system of membrane receptors and triggers the extrinsic pathway of apoptosis. The ability of Apo2L/TRAIL to kill tumor cells while sparing normal cells makes this cytokine a promising antitumor agent. In fact, numerous clinical trials using recombinant forms or Apo2L/TRAIL or agonistic antibodies are currently underway. During the last decade, it has become apparent that Apo2L/TRAIL is a key molecule in the immune system function, mainly in tumor immune surveillance and in downregulation of immune response. Apo2L/TRAIL exerts immunosuppressive and immunoregulatory functions, important for immune homeostasis, tumor control, and prevention of autoimmunity. Hence, apart from the intended use of Apo2L/TRAIL as a chemotherapeutic agent, a recent body of evidence suggests that this cytokine may also be a promising therapeutic agent for autoimmune diseases. However, distinct aspects of the physiological role of Apo2L/TRAIL in cancer and autoimmunity need to be unraveled to fully understand the importance of this signaling pathway. Among these aspects for future research, it stands out to fully unravel the role of Apo2L/TRAIL in the immune system, as well as to establish the true role of nonapoptotic signaling by Apo2L/TRAIL in vivo.
Further investigations on the mechanism of action of this cytokine will unravel the complexities of its in vivo function and ultimately apply this knowledge to a better treatment of cancer and autoimmune diseases.
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