Combining proteasome inhibition with TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) for cancer therapy

Apoptosis has an essential role in embryogenesis, adult tissue homeostasis and cellular responses to stressful stimuli. Therefore, increased apoptosis is involved in the pathogenesis of various ischaemic, degenerative and immune disorders. Conversely, genetic aberration that results in a reduction or abolition of apoptosis can promote tumorigenesis and underlie the resistance of cancer cells to various genotoxic anticancer agents. Therefore, a detailed knowledge of the control of apoptotic pathways could aid in the rational design of effective therapeutics for a variety of human diseases including cancer. One major way to promote apoptosis involves signaling through members of the tumor necrosis factor (TNF) superfamily. On binding to their appropriate receptors, some TNF family members can promote caspase activation and apoptosis. Early studies on TNF indicated that a limited number of tumor cell lines could be induced to undergo apoptosis on exposure to TNF. Another member of the TNF family Fas ligand (FasL) is also known to induce apoptosis in a variety of tumor cells. Although TNF and FasL can efficiently induce apoptosis in a limited number of tumor cells, administration of either of these agents is associated with extreme toxicity. This toxicity has precluded further development of either TNF or FasL for cancer therapy. However, within the last 8 years another member of the TNF family, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has been characterized, which induces apoptosis of a wider range of cancer cells than either TNF or FasL. Surprisingly, most normal non-transformed cells are quite resistant to the apoptotic effects of Apo2L/TRAIL. This selective toxicity for cancer cells is the basis for the current enthusiasm for Apo2L/TRAIL as a potential novel anticancer therapy. In this symposium report, we provide a brief overview of Apo2L/TRAIL, its receptors and their signaling pathways. We discuss findings on the antitumor effects of Apo2L/TRAIL alone or in combination with radiotherapy or chemotherapy. In addition, we present recent information from our groups concerning the possible therapeutic benefits of combining Apo2L/TRAIL with the proteasome inhibitor bortezomib. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Apo2L/TRAIL and its death and decoy receptors

Apo2 ligand or tumour necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is one of the several members of the tumour necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs). Apo2L/TRAIL interacts with an unusually complex receptor system of two DRs and three decoys. This protein has garnered intense interest as a potential candidate for cancer therapy because as a trimer it selectively induces apoptosis in many transformed cells but not in normal cells. While much of the early characterisation of Apo2L/TRAIL and its receptors relied on overexpression studies, recent work using untransfected cells has clarified how endogenous proteins transmit apoptotic signals from this ligand. In this review, we focus on the apoptotic signalling pathways stimulated by Apo2L/TRAIL and summarise what is known about its physiological role.     

The apoptotic ligands TRAIL,TWEAK, and Fas ligand mediate monocyte death induced by autologous lupus T cells

Individuals with systemic lupus erythematosus show evidence of a significant increase in monocyte apoptosis. This process is mediated, at least in part, by an autoreactive T cell subset that kills autologous monocytes in the absence of nominal Ag. We have investigated the apoptotic pathways involved in this T cell-mediated process. Expression of the apoptotic ligands TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and Fas ligand on lupus T cells was determined, and the role of these molecules in the monocyte apoptotic response was examined. We report that these apoptotic ligands mediate the autologous monocyte death induced by lupus T cells and that this cytotoxicity is associated with increased expression of these molecules on activated T cells, rather than with an increased susceptibility of lupus monocytes to apoptosis induced by these ligands. These results define novel mechanisms that contribute to increased monocyte apoptosis characterizing patients with lupus. We propose that this mechanism could provide a source of potentially antigenic material for the autoimmune response and interfere with normal clearing mechanisms.     

Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients' stroma-conditioned medium, we determined that soluble factor(s) produced by stroma-myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.     

Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.     

Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors.     

Mechanisms of resistance of human glioma cells to Apo2 ligand/TNF-related apoptosis-inducing ligand.

BACKGROUND: Many tumor cells are resistant to Apo2L/TRAIL-induced apoptosis in the absence of inhibitors of protein synthesis. Apo2L/TRAIL, in addition to induction of apoptosis, may therefore also activate survival pathways. METHODS: Here we investigated whether such survival pathways mediate resistance to Apo2L.0-induced apoptosis in human glioma cells. RESULTS: Apo2L.0 induced the phosphorylation of ERK1/2, but not of Akt. This effect was unaffected by caspase inhibition. Inhibitors of protein synthesis, PI3 kinase, ERK kinase, NF-kappaB or casein kinase 2 sensitized for Apo2L.0-induced apoptosis to a different extent in a panel of human malignant glioma cell lines. However, none of the sensitizers overcame resistance mediated by ectopic expression of the viral caspase 8 inhibitor, crm-A. Primary glioma cultures were almost completely resistant to Apo2L.0-induced cell death even in the presence of the inhibitors. Caspase-8 was expressed in these cells whereas only weak expression of DR5 was detected. Transient expression of DR5 conferred sensitivity to Apo2L.0. CONCLUSION: These data challenge the view that specific cell lines harbour specific mechanisms of resistance to Apo2L/TRAIL. Weak expression of DR5 in primary glioma might limit the therapeutic application of Apo2L/TRAIL in human glioblastoma patients.     

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.     

Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.     

PTHrP Overexpression Increases Sensitivity of Breast Cancer Cells to Apo2L/TRAIL

Parathyroid hormone-related protein (PTHrP) is a key component in breast development and breast tumour biology. PTHrP has been discovered as a causative agent of hypercalcaemia of malignancy and is also one of the main factors implicated in breast cancer mediated osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been demonstrated to cause tumour cell death both in vitro and in vivo. Apo2L/TRAIL is a promising new anti-cancer agent, due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. The effects of PTHrP on cell death signalling pathways initiated by Apo2L/TRAIL were investigated in breast cancer cells. Expression of PTHrP in Apo2L/TRAIL resistant cell line MCF-7 sensitised these cells to Apo2L/TRAIL-induced apoptosis. The actions of PTHrP resulted from intracellular effects, since exogenous treatment of PTHrP had no effect on Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP expressing cells occurred through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. PTHrP increased cell surface expression of Apo2L/TRAIL death receptors, TRAIL-R1 and TRAIL-R2. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL mediated its apoptotic signals through activation of the TRAIL-R2 in PTHrP expressing breast cancer cells. These studies reveal a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer.     

Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation

Upon activation, na?ve T helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of costimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate the differentiation program, the selective action of death effectors determines the end point balance of differentiating T helper subsets. We examined the expression of TNF-related apoptosis-inducing ligand (TRAIL) and CD95L in cloned and in vitro differentiated Th1 and Th2 cells. We found that activation-induced expression of TRAIL is exclusively observed in Th2 clones and primary T helper cells differentiated under the Th2 condition, while the expression of CD95L is mainly in Th1 cells. Furthermore, these two subsets exhibit distinct susceptibilities to TRAIL- and CD95L-mediated apoptosis. Th2 cells are more resistant to either TRAIL- or CD95L-induced apoptosis than Th1 cells. More importantly, both Th1 and Th2 cells could induce apoptosis in labeled Th1 but not Th2 cells. Blocking TRAIL and CD95L significantly enhance IFN-gamma production in vitro. Likewise, young MRL/MpJ-lpr/lpr mice also showed more Th1 response to ovalbumin immunization as compared to MRL/MpJ+/+. Therefore, apoptosis mediated by CD95L and TRAIL is critical in determining the fate of differentiating T helper cells.     

Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and splenomegaly and suffer from autoimmune disease. The lpr mice have a defect in a cell-surface receptor, Fas, that mediates apoptosis, while gld mice have a mutation in the Fas ligand (FasL). Northern hybridization with the FasL cDNA as probe indicated that the cells accumulating in lpr and gld mice abundantly express the FasL mRNA without stimulation. By means of in situ hybridization and immunohistochemistry, we identified the cells expressing the FasL mRNA as CD4-CD8- double negative T cells. The T cells from lpr mice were specifically cytotoxic against Fas-expressing cells. Since FasL is normally expressed in activated mature T cells these results indicate that the double negative T cells accumulating in lpr and gld mice are activated once, and support the notion that the Fas/FasL system is involved in activation-induced suicide of T cells. Furthermore, the graft-versus host disease caused by transfer of lpr bone marrow to wild-type mice can be explained by the constitutive expression of the FasL in lpr-derived T cells.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

Identification of a potential HIV-induced source of bystander-mediated apoptosis in T cells: Upregulation of TRAIL in primary human macrophages by HIV-1 tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.     

Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.     

Direct binding of Fas-associated death domain (FADD) to the tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 is regulated by the death effector domain of FADD

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.