Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.     

c-Myc Blazing a Trail of Death: Coupling of the Mitochondrial and Death Receptor Apoptosis Pathways by c-Myc

TRAIL ligand induces selectively apoptosis in tumor cells by binding to two death receptors (DR4 and DR5) and holds promise as a potential therapeutic agent against cancer. While it has been known for long time that TRAIL receptors are commonly expressed in wide variety of normal tissues, it is not well understood why TRAIL kills tumor cells but leaves normal cells unharmed. The prototypic oncogene c-Myc promotes the cell cycle and simultaneously primes activation of the Bcl-2 family controlled mitochondria apoptosis pathway. A striking reflection of the c-Myc-dependent apoptotic sensitization is the dramatic c-Myc-induced vulnerability of cells to TRAIL and other death receptor ligands. Here we summarize the recent findings regarding the death mechanisms of TRAIL/TRAIL receptor system and the connection of c-Myc to the mitochondrial apoptosis pathway, focusing on our work that couples c-Myc via Bak to the TRAIL death receptor pathway. Finally, we present a mitochondria-priming model to explain how c-Myc-Bak interaction amplifies the TRAIL-induced caspase 8-Bid pathway to induce fullblown apoptosis. We discuss the implications of these findings for understanding the selective cytotoxicity of TRAIL and for the therapeutic exploitation of the death receptor pathway.     

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.     

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.     

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue. This work was supported by a grant from the Wellcome Trust GR071469 (GIO) and the Chilean national science grants FONDECYT 1060495 (GIO) and 1020675 (MC). An erratum to this article is available at .     

Human normal hepatocytes are susceptible to apoptosis signal mediated by both TRAIL-R1 and TRAIL-R2

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells without toxicity to normal cells, but some recombinant versions of TRAIL caused hepatocyte death. We generated fully human monoclonal antibodies (mAbs) that bind specifically to TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), which mediate apoptosis signal when they ligate with TRAIL, to investigate the contribution of each receptor to induce tumor cell apoptosis and hepatocyte toxicity. All of mAbs to TRAIL-R1 and TRAIL-R2 induced cell death in several cancer cell lines susceptible to TRAIL but not in human umbilical vein endothelial cells in vitro. Both anti-TRAIL-R1 mAbs and anti-TRAIL-R2mAbs also caused cell death in hepatocytes. However, a subset of mAbs to TRAIL-R2, which was characterized by the TRAIL blocking activity, did not show strong hepatocyte toxicity. These results indicate that human normal hepatocytes are susceptible to both TRAIL-R1- and TRAIL-R2-mediated apoptosis signal.Cell Death and Differentiation (2004) 11, 203-207. doi:10.1038/sj.cdd.4401331 Published online 24 October 2003     

Claudin-7 suppresses the cytotoxicity of TRAIL-expressing mesenchymal stem cells in H460 human non-small cell lung cancer cells

Evidence suggests that the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics. Studies have also shown that claudin-7 (CLDN7) expression is variably dysregulated in various malignant neoplasms, with a role in lung cancer that has not been definitively decided. This work investigated the differential sensitivity of CLDN7-overexpressing human NSCLC H460 cells to TRAIL in vitro and in mouse xenografts, and explored the molecular mechanisms responsible for these effects. NCI-H460 cells were transfected or not with green fluorescent protein-tagged CLDN7. Each group was then exposed to mesenchymal stem cells (MSCs) or red fluorescent protein-tagged MSCs transduced with lentivirus expressing membrane-bound TRAIL. The effects and related mechanisms of these treatments were evaluated in vitro, and in vivo in murine xenografts. Our results indicate that TRAIL induced apoptosis in H460 cells in vitro, and in established xenograft tumors TRAIL was associated with a decrease in tumor size, tumor weight, and circulating tumor cells. CLDN7 was found to inhibit the MEK/ERK signaling pathway, leading to inhibition of death receptor 5 (TNFRSF10B). The cytotoxicity of TRAIL was confirmed in H460 cells and in vivo, and CLDN7 suppressed the cytotoxicity of TRAIL in H460 cells. Our results indicate that TRAIL may be a useful therapy to enhance apoptosis in CLDN7-negative lung cancer cells.     

The involvement of TNF-alpha-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-beta-stimulated human dendritic cells to tumor cells

TNF-alpha-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34(+) stem cell-derived DCs (CD34DCs) and human CD14(+) monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-beta stimulation. Moreover, IFN-beta could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-beta-stimulated DCs partially contributed to the increased cytotoxicity of DCS: Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-beta-stimulated DCS: In contrast, NF-kappaB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCS: Our studies demonstrate that IFN-beta-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8(+) CTL cells.     

The potential of TRAIL for cancer chemotherapy

Innate and acquired resistance to chemotherapy and radiation therapy has been a major obstacle for clinical oncology. One potential adjunct to such conventional treatments is direct induction of cell death by activation of death receptor-mediated apoptosis. TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand), a recently identified member of the growing TNF superfamily, binds to its cognate death receptors DR4 and DR5 as well as decoy receptors DcR1 and DcR2. Upon binding, rapid apoptosis is enacted in a variety of human cancer cell lines independent of p53 status, but not in normal cell lines. TRAIL treatment results in significant growth suppression of TRAIL-sensitive human cancer xenografts in mice. Furthermore, combination treatment of TRAIL with genotoxic chemotherapeutic agents synergistically suppresses growth of tumor xenografts which are otherwise resistant to treatment with TRAIL or chemotherapy alone. Unlike the other death ligands TNF- or FasL, systemic administration of soluble human TRAIL does not cause toxicity in mice and non-human primates. While further studies are needed to evaluate the possible cytotoxicity of TRAIL especially for human hepatocytes, indications are increasing that TRAIL may be a novel therapeutic agent for human cancer.     

FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic fibroblasts, Yeh et al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryonic fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or FADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.     

TNF-related apoptosis-inducing ligand: signalling of a 'smart' molecule

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor super-family and signals via two death receptors, TRAIL-R1 and TRAIL-R2, and two decoy receptors, TRAIL-R3 and TRAIL-R4, differently expressed in normal and cancer cells. TRAIL is mainly studied for its capacity to induce apoptosis preferentially in cancer cells. TRAIL is expressed in a variety of human tissues, in particular in the lymphoid system, suggesting a strong physiological role in the innate immunity. This review will focus on TRAIL gene structure and regulation, protein folding, tissue expression and molecular signalling. Finally, the potential use of TRAIL as anticancer treatment alone or in combination therapy as well as the use of drugs which signal via TRAIL and its receptors will be analyzed.     

Progesterone induces apoptosis in TRAIL-resistant ovarian cancer cells by circumventing c-FLIPL overexpression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds great potential as an anticancer drug, since it induces selective cell death in cancer cells but not in normal ones. However, cancer cells often acquire resistance to TRAIL, which hinders its clinical efficacy. We previously demonstrated that progesterone triggers apoptosis in human ovarian cancer (OCa) cells. In the present study, we evaluated the prospect of utilizing progestins in combination with TRAIL to enhance cell death in TRAIL-sensitive (OVCA 420, OVCA 429, and OVCA 433) and -resistant (OVCA 432) OCa cell lines. TRAIL sensitivity (60-80% cell kill) bore no correlation with expression of the TRAIL receptors (DR4, DR5) or their decoys (DcR1 and DcR2), but was associated with activation of caspase-8 and -3, and downregulation of the long isoform of FLICE-like inhibitory protein (c-FLIP(L)), an anti-apoptosis mediator. Small interfering RNA-mediated knockdown of c-FLIP(L) expression restored TRAIL sensitivity in OVCA 432 cells. Induction of c-FLIP(L) overexpression increased TRAIL resistance in TRAIL-sensitive lines. Thus, persistent high level of c-FLIP(L) expression likely mediates TRAIL resistance in OCa cells. Treatment of OCa cells with progesterone enhanced TRAIL-induced cell death (>85%), but only in TRAIL-sensitive cell lines. Combined treatment with two progestins was superior to single progestin treatment, with progesterone plus medroxyprogesterone acetate (MPA) achieving over 85% cell kill in both TRAIL-sensitive and -resistant OCa cell lines. Significantly, unlike TRAIL, progestin-induced cell death did not involve c-FLIP(L) downregulation. Hence, combined progestin regimens, with or without TRAIL, may serve as an effective therapy for OCa by circumventing the anti-apoptotic action of c-FLIP(L).     

Induction of necrotic tumor cell death by TRAIL/Apo-2L

A great deal of enthusiasm is being generated for TRAIL (TNF-related apoptosis-inducing ligand)/Apo-2L as a tumor therapeutic agent because it is cytotoxic to a variety of tumor cell types but not normal cells. Moreover, it is well documented that TRAIL/Apo-2L-induced tumor cell death is a caspase-dependent apoptotic process. Through the use of a transfected cell line expressing murine TRAIL/Apo-2L and a recombinant adenovirus encoding the murine TRAIL/Apo-2L cDNA (Ad5-mTRAIL) against two murine tumor cell lines [TRAMP-C2 (prostate adenocarcinoma) and Renca (renal adenocarcinoma)], we found that mTRAIL/Apo-2L also can kill tumor cells by inducing necrosis. Specifically, we observed the default method of mTRAIL/Apo-2L-induced death in TRAMP-C2 cells was via a necrotic process, characterized by the complete lack of an annexin V⁺/PI^– population, SAPK/JNK phosphorylation, caspase activation, Bid cleavage, or cytochrome c release. Moreover, the inclusion of zVAD-fmk, an inhibitor of caspase activation, markedly enhanced mTRAIL/Apo-2L-mediated killing of TRAMP-C2. In contrast, apoptosis was induced in TRAMP-C2 using TNF, as measured by the criteria listed above, as was Renca by mTRAIL/Apo-2L. These results demonstrate the natural occurrence of both TRAIL/Apo-2L-induced apoptotic and necrotic signaling mechanisms within tumor cells.     

Genetically Modified DR5-Specific TRAIL Variant DR5-B Revealed Dual Antitumor and Protumoral Effect in Colon Cancer Xenografts and an Improved Pharmacokinetic Profile

Despite the weak clinical efficacy of TRAIL death receptor agonists, a search is under way for new agents that more efficiently activate apoptotic signaling. We previously created a TRAIL DR5-selective variant DR5-B without affinity for the DR4, DcR1, DcR2, and OPG receptors and increased proapoptotic activity in tumor cells. Here we showed that DR5-B significantly inhibited tumor growth in HCT116 and Caco-2 but not in HT-29 xenografts. The antitumor activity of DR5-B was 2.5 times higher in HCT116 xenografts compared to TRAIL. DR5-B at a dose of 2 or 10 mg/kg/d for 10 days inhibited tumor growth in HCT116 xenografts by 26% or 50% respectively, and increased animal survival. Unexpectedly, DR5-B at a higher dose (25 mg/kg/d) inhibited tumor growth only during the first 8 days of drug exposure, while at the end of the monitoring, no effect or even slight stimulation of tumor growth was observed. The pharmacokinetic parameters of DR5-B were comparable to those of TRAIL, except that the half-life was 3.5 times higher. Thus, enhancing TRAIL selectivity to DR5 may increase both antitumor and proliferative activities depending on the concentration and administration regimens.     

Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.     

Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.     

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.     

Anti-tumor activity of a novel monoclonal antibody, NPC-1C, optimized for recognition of tumor antigen MUC5AC variant in preclinical models

Purpose

NPC-1C is a chimeric immunoglobulin IgG1 developed from antigen tested in the Hollinshead tumor vaccine trials that recognizes an immunogenic MUC5AC-related tumor-associated antigen. In this article, we describe the pre-clinical characterization of this antibody that is currently being tested in human clinical trials.

Experimental design

The specificity of NPC-1C for pancreatic and colorectal cancer cell lines was tested by flow cytometry assays and immunohistochemical staining. Antibody-dependent cell cytotoxicity was measured using a tumor cell line lysis assay. Anti-tumor efficacy and biodistribution were assessed in nude mice bearing human pancreatic tumor xenografts.

Results

Human tumor cell binding measured by flow cytometry ranged from 52 to 94 % of cells stained positive with NPC-1C in three colorectal and one pancreatic cell lines, while IHC demonstrated staining of 43 % of colon cancers and 48 % of pancreatic cancer tissues, with little or no cross-reactivity of NPC-1C with normal colon or pancreas tissues. In vitro NPC-1C-mediated tumor cell killing occurred in a median of 44.5 % of four colorectal and three pancreatic tumor cell lines. In vivo anti-tumor efficacy in a human pancreatic CFPAC-1 tumor xenograft model was demonstrated with a twofold to threefold reduction in tumor growth in the NPC-1C-treated mice compared to saline and human IgG controls. Pharmacodynamic studies indicate NPC-1C localizes in antigen-positive tumors and has minimal uptake in normal mouse tissues.

Conclusions

NPC-1C, a chimeric monoclonal antibody that reacts with a MUC5AC-related antigen expressed by pancreatic and colorectal tumor tissues, has promising preclinical activity in pancreatic and colorectal adenocarcinoma.     

Sensitivity of Malignant Peripheral Nerve Sheath Tumor Cells to TRAIL Is Augmented by Loss of NF1 through Modulation of MYC/MAD and Is Potentiated by Curcumin through Induction of ROS

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.