DISC formation is the first step toward apoptosis after engagement of DR4 and DR5 by their ligands. Not surprisingly, resistance to TRAIL-mediated apoptosis is often initiated at the level of the DISC . We observed that upon stimulation with TRAIL, all canonical DISC proteins were recruited to DR4 and DR5 in SW948 and SW948-TR cells. In relation to the other DISC proteins, smaller amounts of cleaved caspase 8 were found in the TRAIL-DISC of SW948-TR cells as compared with SW948 cells. These results were in agreement with the lower basal caspase 8 protein levels found in SW948-TR. Altogether, these findings suggest less active recruitment and processing of caspase 8 in our resistant cell line. We also describe a difference in apoptosis-inducing ability between DR4 and DR5. Despite equivalent initial DISC formation at the level of each receptor, DR4 stimulation achieves superior caspase 8 processing than DR5.
Antagonistic DR4 or DR5 antibodies were used to specifically block the function of their respective receptors during rhTRAIL-induced apoptotic signaling. Experiments with these blocking antibodies proved that DR4 was critical to rhTRAIL-induced apoptotic signaling in both cell lines; DR5 was not. It was previously reported that apoptosis induction in keratinocytes with leucine zipper TRAIL was also mainly mediated by DR4 . In contrast, Kelley et al. generated receptor-selective mutants of TRAIL, with three to six ligand amino acid substitutions , and found DR5 to be more important for apoptotic signaling than DR4 in cancer cells expressing both receptors (including colon cancer cells). Van der Sloot et al. demonstrated that DR5-selective TRAIL variants did not induce apoptosis in cell lines mostly responsive to DR4 stimuli, while they greatly stimulated apoptosis in DR5-responsive cancer cell lines . Little is known about the mechanisms underlying these differences. Blocking of both DR4 and DR5 prevented apoptosis more efficiently than single blocking of DR4, which suggests that DR5 also contributes to rhTRAIL-induced apoptosis. While DR4 was more potent in transducing apoptosis, DR5 might, at least in our model, increase the overall apoptotic stimulus compared with DR4 stimulation alone.
We found that in SW948 and SW948-TR cells, sensitivity to agonistic DR4 and DR5 antibody mostly reflected the sensitivity of each receptor to rhTRAIL. Although the agonistic DR4 antibody was more potent than its anti-DR5 counterpart, agonistic DR5 antibody could induce apoptosis to some extent in SW948 cells, and also in SW948-TR following pre-sensitization with CHX. This strongly suggests that DR5 is functional in both cell lines, as also indicated by rhTRAIL binding and comparable DISC formation between DR4 and DR5. Georgakis et al. found that in primary non-Hodgkin's lymphoma samples, an agonistic DR4 antibody was also more effective than an agonistic DR5 antibody .
We investigated the ability of each receptor to initiate DISC formation and caspase 8 cleavage in SW948 by co-immunoprecipitating DR4- and DR5-DISC. Similar DISC formation and caspase 8 cleavage were triggered at the level of both receptors, although the rate of caspase 8 processing was much higher in whole cell lysates when cells were stimulated with agonistic DR4 antibody instead of agonistic DR5 antibody. This is consistent with our recent findings that caspase 8 cleavage at the level of DR5-DISC in SW948 is limited due to a lower turn-over of DISC components rather than to decreased DISC formation . Data on such DISC protein turn-over following TRAIL receptor stimulation are limited. However, studies by McDonald et al. and Jin et al. have suggested that caspase 8 ubiquitination influences processing of the available caspase 8 cellular pool [46, 47]. Brief pre-incubation with CHX could also restore DR5-mediated caspase 8 cleavage and sensitivity; a short-lived protein might, therefore, be involved in this process.
IFN-γ is known to increase caspase 8 expression and, consequently, to sensitize cancer cells (including colon cancer cells) to rhTRAIL [24, 41, 48–50]. Our results are in slight contrast to these findings, as upregulation of caspase 8 by IFN-γ did not further enhance apoptosis induction in the highly sensitive SW948 cells. Caspase 8 levels might not be a limiting factor in these cells, since procaspase 8 recruitment to the DISC is first determined by the amount of FADD molecules available there . IFN-γ did, however, increase apoptosis induction by rhTRAIL and agonistic DR4 antibody in SW948-TR cells, which express lower caspase 8 levels than their parental cells. In SW948-TR, IFN-γ induced a marked increase in both pro- and intermediate forms of caspase 8, which were cleaved to the active form upon DR4 stimulation. Downregulation of caspase 8 levels in SW948 - that is, to a level comparable to that which is normally observed in SW948-TR cells - was sufficient to induce TRAIL resistance, indicating the importance of the caspase-8/c-FLIP ratio in these cells . It is noteworthy that IFN-γ, while inducing an increase in caspase 8 levels, did not change c-FLIP expression in SW948-TR. In contrast, IFN-γ failed to enhance the appearance of active caspase 8 following DR5 stimulation. This was shown using agonistic DR5 antibody, but also rhTRAIL in combination with DR4 blocking antibodies. The slight reduction in DR5 surface expression may explain the lack of increase in DR5-mediated apoptosis.
Unlike IFN-γ, MG-132 markedly increased both DR4- and DR5-mediated sensitivity in SW948-TR cells. Another proteasome inhibitor, bortezomib, was previously reported to enhance the effect of agonistic DR4 and agonistic DR5 antibody in Hodgkin's disease cell lines [18, 52]. MG-132 could increase TRAIL-induced apoptosis in both Bax-deficient and proficient colon cancer cells, which is suggestive of a sensitization mechanism independent of the mitochondrial pathway of apoptosis . DR4 and DR5 upregulation by proteasome inhibitors is thought to be a major factor contributing to TRAIL-induced apoptosis sensitization. However, this hypothesis has been subject to an extensive debate . We found that upon MG-132 treatment, DR4-mediated sensitivity increased in the absence of DR4 upregulation. The intermediate form of caspase 8 was upregulated by MG-132, an event which we found was sufficient to restore sensitivity to agonistic DR4 antibody or rhTRAIL following IFN-γ treatment. Importantly, MG-132 also increased caspase 8 cleavage and enhanced DR5-mediated apoptosis. MG-132 has previously been shown to modulate DR5 expression [24, 54]. Our flow cytometry analysis only showed negligible DR5 upregulation by MG-132, which again points to a mechanism of sensitization independent of this effect. MG-132 upregulated caspase 8 in SW948-TR, but we demonstrated using IFN-γ that restoring caspase 8 levels was not sufficient to increase DR5-mediated caspase 8 cleavage and apoptosis. Taken together, these results support the existence of additional sensitizing mechanisms of MG-132 at the level of DR5-DISC, beyond DR5 and caspase 8 upregulation. Many of the proteins involved in the DR-mediated apoptotic pathways are regulated by ubiquitination , and some of these proteins could play a crucial role in specific regulation of DR5 signaling.
In particular, understanding how MG-132 can overcome DR5-resistance may be of crucial importance, since Johnsen et al. previously reported that IFN-γ failed to increase TRAIL-induced apoptosis in 3 out of 8 neuroblastoma cancer cell lines; this result occurred despite caspase 8 upregulation and in the presence of all the proteins known to be necessary for DISC formation . It would be interesting to verify whether DR4 signaling remains functional in the non-responding cells. Furthermore, in view of the large number of agents in clinical development targeting DR5, as opposed to DR4, a deeper knowledge on DR5-mediated signaling regulation is critical for a more rational design of targeted therapies.