Tumor Necrosis Factor-related Apoptosis-inducing Ligand Stimulation Research Articles

TRAIL Triggers CRAC-Dependent Calcium Influx and Apoptosis through the Recruitment of Autophagy Proteins to Death-Inducing Signaling Complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Diffuse Intrinsic Pontine Glioma (DIPG): Breakthrough and Clinical Perspective.

Diffuse intrinsic pontine glioma (DIPG) mainly affects children with a median age of 6-7 years old. It accounts for 10% of all pediatric tumors. Unfortunately, DIPG has a poor prognosis, and the median survival is generally less than 16-24 months independently from the treatment received. Up to now, children with DIPG are treated with focal radiotherapy alone or in combination with antitumor agents. In the last decade, ONC201 known as dopamine receptor antagonist was uncovered, by a high throughput screening of public libraries of compounds, to be endowed with cytotoxic activity against several cancer cell lines. Efforts were made to identify the real ONC201 target, responsible for its antiproliferative effect. The hypothesized targets were the Tumor necrosis factor-Related Apoptosis-Inducing Ligand stimulation (TRAIL), two oncogenic kinases (ERK/AKT system) that target the same tumor-suppressor gene (FOXO3a), dopamine receptors (DRD2 and DRD3 subtypes) and finally the mitochondrial Caseynolitic Protease P (ClpP). ONC201 structure-activity relationship is extensively discussed in this review, together with other two classes of compounds, namely ADEPs and D9, already known for their antibiotic activity but noteworthy to be discussed and studied as potential "leads" for the development of new drugs to be used in the treatment of DIPG. In this review, a detailed and critical description of ONC201, ADEPs, and D9 pro-apoptotic activity is made, with particular attention to the specific interactions established with its targets that also are intimately described. Pubmed published patents and clinical trial reports of the last ten years were used as the bibliographic source.

Defining rules for cancer cell proliferation in TRAIL stimulation

Owing to their self-organizing evolutionary plasticity, cancers remain evasive to modern treatment strategies. Previously, for sensitizing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant human fibrosarcoma (HT1080), we developed and validated a dynamic computational model that showed the inhibition of protein kinase (PK)C, using bisindolylmaleimide (BIS) I, enhances apoptosis with 95% cell death. Although promising, the long-term effect of remaining ~ 5% cells is a mystery. Will they remain unchanged or are they able to proliferate? To address this question, here we adopted a discrete spatiotemporal cellular automata model utilizing simple rules modified from the famous “Conway’s game of life”. Based on three experimental initializations: cell numbers obtained from untreated (high), treatment with TRAIL only (moderate), and treatment with TRAIL and BIS I (low), the simulations show cell proliferation in time and space. Notably, when all cells are fixed in their initial space, the proliferation is rapid for high and moderate cell numbers, however, slow and steady for low number of cells. However, when mesenchymal-like random movement was introduced, the proliferation becomes significant even for low cell numbers. Experimental verification showed high proportion of mesenchymal cells in TRAIL and BIS I treatment compared with untreated or TRAIL only treatment. In agreement with the model with cell movement, we observed rapid proliferation of the remnant cells in TRAIL and BIS I treatment over time. Hence, our work highlights the importance of mesenchymal-like cellular movement for cancer proliferation. Nevertheless, re-treatment of TRAIL and BIS I on proliferating cancers is still largely effective.

Targeting KPNB1 overcomes TRAIL resistance by regulating DR5, Mcl-1 and FLIP in glioblastoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin β1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4–induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.

Abstract LB-193: TRAIL promotes entosis through Caspase-8

Abstract Despite its apoptosis-inducing activity, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also activates survival signaling pathways. Survival signaling not only interferes with apoptosis and causes resistance, but can also lead to gain of several tumor-promoting functions such as proliferation, invasion and metastasis. Here we demonstrate that colorectal cancer cells surviving from TRAIL treatment can initiate a cell-in-cell invasion process known as entosis. Entosis refers to a unique cell-in-cell phenomenon, in which one or more viable cells are actively internalized into another cell of the same type. After formation of cell-in-cell structures (CICs), internalized cells can divide inside the host cell or they can escape, however, the vast majority of cells undergo cell death inside their hosts, through a lysosome-dependent mechanism. Although the clinical relevance of entosis is highly context-dependent, the frequency of entosis has been shown associated with tumor grade and poor outcome in several malignancies. While monitoring single-cell response upon TRAIL stimulation, we noted that some cells internalized into their neighboring cells and died inside a vacuole. We confirmed CICs by fluorescence staining of cytoplasm, nucleus, lysosome and cell membrane, in addition to TEM. Further quantification revealed that TRAIL indeed increased the number of CICs in colorectal cancer cell lines (HCT116 and LS180) and spheroids. Moreover, both pre-treatment with ROCK inhibitor (Y-27632) and knockout of Caspase-8 resulted in a significant reduction in the number of TRAIL-induced CICs. After forming cell-in-cell structures, the majority of internalized cells appeared positive for lysotracker staining and underwent cell death inside their hosts. These cells showed an increase in IETD FRET probe cleavage, additionally, we observed increased levels of cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 in these cells in presence of TRAIL. Pre-treatment with z-VAD-fmk reduced the number of internalized cells undergoing cell death and Bax/Bak double knockout cells escaped from their hosts without undergoing cell death in response to TRAIL. Our findings demonstrate that entosis can be induced by TRAIL in a caspase-8-dependent manner, and may give new insights in understanding the death, survival and escape mechanisms in internalized cells. Citation Format: Emir Bozkurt, Heiko Dussmann, Jochen H. Prehn. TRAIL promotes entosis through Caspase-8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-193.

Cisplatin or LA-12 enhance killing effects of TRAIL in prostate cancer cells through Bid-dependent stimulation of mitochondrial apoptotic pathway but not caspase-10

Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action.

Marine actinomycete crude extracts with potent TRAIL-resistance overcoming activity against breast cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, as it can kill tumor cells selectively. In our search of bioactive natural products to overcome TRAIL-resistance, we isolated 47 actinomycete strains from different sediments and seawater samples collected from the Red Sea coast in Egypt and found four crude extracts (EGY1, EGY3, EGY24 and EGY34) displaying TRAIL sensitizing activity in the resistant breast cancer cell line MDA-MB-231. None of these crude extracts exhibited cytotoxic effect on normal mouse embryonic fibroblasts (MEF), with the exception of EGY34. Analysis of the signaling pathways underlying the sensitization of MDA-MB-231 cells to TRAIL-induced apoptosis, by western blotting, revealed that all crude extracts facilitated initiator caspase‑8/-10 activation upon TRAIL stimulation, but that in addition, EGY3 and EGY34, alone, induced strong ER-stress activation, with the appearance of BiP in the cytosolic extracts. Our results pave the way to the discovery and the development of marine-derived drugs for cancer therapy.

TRAIL signaling is proinflammatory and proviral in a murine model of rhinovirus 1B infection.

the aim of this study is to elucidate the role of TRAIL during rhinovirus (RV) infection in vivo. Naïve wild-type and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-deficient (Tnfsf10-/-) BALB/c mice were infected intranasally with RV1B. In separate experiments, Tnfsf10-/- mice were sensitized and challenged via the airway route with house dust mite (HDM) to induce allergic airways disease and then challenged with RVIB or UV-RVIB. Airway hyperreactivity (AHR) was invasively assessed as total airways resistance in response to increasing methacholine challenge and inflammation was assessed in bronchoalveolar lavage fluid at multiple time points postinfection. Chemokines were quantified by ELISA of whole lung lysates and viral load was determined by quantitative RT-PCR and tissue culture infective dose (TCID50). Human airway epithelial cells (BEAS2B) were infected with RV1B and stimulated with recombinant TRAIL or neutralizing anti-TRAIL antibodies and viral titer assessed by TCID50 HDM-challenged Tnfsf10-/- mice were protected against RV-induced AHR and had suppressed cellular infiltration in the airways upon RV infection. Chemokine C-X-C-motif ligand 2 (CXCL2) production was suppressed in naïve Tnfsf10-/- mice infected with RV1B, with less RV1B detected 24 h postinfection. This was associated with reduced apoptotic cell death and a reduction of interferon (IFN)-λ2/3 but not IFN-α or IFN-β. TRAIL stimulation increased, whereas anti-TRAIL antibodies reduced viral replication in RV1B-infected BEAS2B cells in vitro. In conclusion, TRAIL promotes RV-induced AHR, inflammation and RV1B replication, implicating this molecule and its downstream signaling pathways as a possible target for the amelioration of RV1B-induced allergic and nonallergic lung inflammation and AHR.

STAT3 activity regulates sensitivity to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cervical cancer cells.

In cervical cancer, p53-induced apoptosis is abrogated by human papilloma virus (HPV)-derived oncoproteinE6. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) provides tumor-specific apoptosis in various cancers, including cervical cancer, the sensitivity differs depending on the cell lines. Signal transducer and activator of transcription 3 (STAT3) is a hub molecule that shifts the cellular fate to apoptosis or survival in response to cellular stresses. However, the contribution of STAT3 activity to TRAIL-induced apoptosis in cervical cancer remains unknown. We examined the TRAIL sensitivity in cervical cancer cells, using TRAIL-resistant (SiHa) and -sensitive (CaSki) cervical cancer cell lines and focused on STAT3 function involving the apoptotic pathway. STAT3 was inactivated by TRAIL stimulation in the CaSki cell line, but not in the SiHa cell line. We then inhibited STAT3 expression in the SiHa cell line using siRNA against STAT3 and suppressed STAT3 activity using a STAT3 inhibitor; both these treatments sensitized TRAIL-induced apoptosis in the SiHa cell line. Furthermore, the SiHa cells were exposed to tunicamycin (TM), an endoplasmic reticulum (ER) stress inducer that inactivates STAT3, with or without TRAIL. Accompanied by STAT3 inactivation, TM pretreatment significantly enhanced TRAIL-induced apoptosis. We therefore concluded that TRAIL-induced apoptosis was regulated by STAT3 in response to TRAIL stimulation. Our results also suggest that STAT3 inhibition increases the sensitivity of malignancies, particularly HPV-related cancer, to TRAIL-based therapy.

β-arrestin2 regulates TRAIL-induced HepG2 cell apoptosis via the Src-extracellular signal-regulated signaling pathway.

β-arrestins, including β-arrestin1 and β‑arrestin2, two ubiquitously expressed members of the arrestin family in various types of tissue, are adaptor proteins that modulate the desensitization and trafficking of seven membrane‑spanning receptors. Recently, β‑arrestins have been shown to bind to numerous signaling molecules, including c‑Src and mitogen‑activated protein kinase family members. In addition, accumulating evidence has suggested that β‑arrestins are involved in the anti‑apoptosis signaling pathway by associating with kinases, such as Akt and ERK, and altering their activities. However, the role of β‑arrestins in tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑induced apoptosis remains unclear. In the present study, β‑arrestin2, but not β‑arrestin1, was observed to modulate TRAIL‑triggered HepG2 cell apoptosis by regulating activation of the Src‑extracellular signal‑regulated kinase (ERK) signaling pathway. Using overexpression and RNA interference experiments, β‑arrestin2 was demonstrated to prevent TRAIL‑induced HepG2 cell apoptosis. Additionally, β‑arrestin2 exerted an additive effect on TRAIL‑induced activation of Src and ERK. Furthermore, downregulating β‑arrestin2 expression attenuated the TRAIL‑induced activation of Src and ERK survival signaling and enhanced TRAIL‑induced apoptosis. PP2, a pharmacological inhibitor of Src, reduced activation of the Src‑ERK signaling pathway and enhanced TRAIL‑induced HepG2 cell apoptosis. Co-immunoprecipitation experiments demonstrated a physical association between β‑arrestin2 and Src, and TRAIL stimulation resulted in enhanced quantities of the β‑arrestin2/Src complex. A notable interaction was identified between β‑arrestin2 and death receptors (DR)4and5, but only in the presence of TRAIL stimulation. To the best of our knowledge, these findings are the first to demonstrate that β‑arrestin2 mediates TRAIL‑induced apoptosis by combing with DRs and Src, and regulates the activation of Src‑ERK signaling in HepG2 cells. It is hypothesized that the formation of a signaling complex comprising DR, β‑arrestin2 and Src is required for the action of TRAIL on HepG2 cell apoptosis, which provides a novel insight into analyzing the effects of β‑arrestin2 on protecting cells from TRAIL‑induced apoptosis.

Characterization of MUDENG, a novel anti-apoptotic protein.

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60–180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells.

Systems Biology Strategy Reveals PKCδ is Key for Sensitizing TRAIL-Resistant Human Fibrosarcoma.

Cancer cells are highly variable and largely resistant to therapeutic intervention. Recently, the use of the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced treatment is gaining momentum due to TRAIL’s ability to specifically target cancers with limited effect on normal cells. Nevertheless, several malignant cancer types still remain non-sensitive to TRAIL. Previously, we developed a dynamic computational model, based on perturbation-response differential equations approach, and predicted protein kinase C (PKC) as the most effective target, with over 95% capacity to kill human fibrosarcoma (HT1080) in TRAIL stimulation (1). Here, to validate the model prediction, which has significant implications for cancer treatment, we conducted experiments on two TRAIL-resistant cancer cell lines (HT1080 and HT29). Using PKC inhibitor bisindolylmaleimide I, we demonstrated that cell viability is significantly impaired with over 95% death of both cancer types, in consistency with our previous model. Next, we measured caspase-3, Poly (ADP-ribose) polymerase (PARP), p38, and JNK activations in HT1080, and confirmed cell death occurs through apoptosis with significant increment in caspase-3 and PARP activations. Finally, to identify a crucial PKC isoform, from 10 known members, we analyzed each isoform mRNA expressions in HT1080 cells and shortlisted the highest 4 for further siRNA knock-down (KD) experiments. From these KDs, PKCδ produced the most cancer cell death in conjunction with TRAIL. Overall, our approach combining model predictions with experimental validation holds promise for systems biology based cancer therapy.

STAT-6 mediates TRAIL induced RANK ligand expression in stromal/preosteoblast cells

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis, and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein, we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly, TRAIL treatment induced RANKL mRNA expression in these cells. In addition, TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition, siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus, our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells.

TRAIL-induced expression of uPA and IL-8 strongly enhanced by overexpression of TRAF2 and Bcl-xL in pancreatic ductal adenocarcinoma cells

The death ligand, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), induces apoptosis and non-apoptotic signaling in some tumor cells. The purpose of this study was to investigate the roles of the pro-apoptotic TRAIL receptors, TRAIL-R1 and TRAIL-R2, as well as Bcl-xL and TRAF2 in TRAIL-induced expression of the pro-inflammatory cytokine IL-8 and the invasion-promoting protein urokinase (uPA) in pancreatic ductal adenocarcinoma (PDAC) cells. Colo357wt, Colo357/TRAF2, Colo357/Bcl-xL, Panc89 and PancTuI cells were stimulated with TRAIL and uPA and IL-8 expression was detected using real-time PCR. Antagonistic, receptor-specific antibodies were used to investigate the effects of TRAIL-R1 or TRAIL-R2 inhibition. Dose-dependent increases in uPA and IL-8 expression were detected following TRAIL stimulation in PDAC cells. These effects were inhibited when TRAIL-R1 but not TRAIL-R2 was blocked. Overexpression of TRAF2 or Bcl-xL strongly increased TRAIL-mediated upregulation of uPA and IL-8. In PDAC cells, TRAIL strongly induced uPA and IL-8 via TRAIL-R1. This response was further enhanced in cells overexpressing TRAF2 and Bcl-xL. Therefore, inhibition of the non-apoptotic "side-effects" of TRAIL treatments by inactivation of TRAF2 and Bcl-xL might represent additional relevant strategies for the treatment of pancreatic cancer.

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

KSR1 Is Overexpressed in Endometrial Carcinoma and Regulates Proliferation and TRAIL-Induced Apoptosis by Modulating FLIP Levels

The Raf/MEK/extracellular signal-regulated kinase (ERK) pathway participates in many processes altered in development and progression of cancer in human beings such as proliferation, transformation, differentiation, and apoptosis. Kinase suppressor of Ras 1 (KSR1) can interact with various kinases of the Raf/MEK/extracellular signal-regulated kinase pathway to enhance its activation. The role of KSR1 in endometrial carcinogenesis was investigated. cDNA and tissue microarrays demonstrated that expression of KSR1 was up-regulated in endometrial carcinoma. Furthermore, inhibition of KSR1 expression by specific small hairpin RNA resulted in reduction of both proliferation and anchorage-independent cell growth properties of endometrial cancer cells. Because inhibition of apoptosis has a pivotal role in endometrial carcinogenesis, the effects of KSR1 in regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis were investigated. KSR1 knock-down sensitized resistant endometrial cell lines to both TRAIL- and Fas-induced apoptosis. Sensitization to TRAIL and agonistic anti-Fas antibody was caused by down-regulation of FLIP (FLICE-inhibitory protein). Also investigated was the molecular mechanism by which KSR1 regulates FLIP protein levels. It was demonstrated that KSR1 small hairpin RNA did not affect FLIP transcription or degradation. Rather, FLIP down-regulation was caused by Fas-associated death domain protein-dependent inhibition of FLIP translation triggered after TRAIL stimulation in KSR1-silenced cells. Re-expression of heterologous KSR1 in cells with down-regulated endogenous KSR1 restored FLIP protein levels and TRAIL resistance. In conclusion, KSR1 regulates endometrial sensitivity to TRAIL by regulating FLIP levels.

TRAIL activates acid sphingomyelinase via a redox mechanism and releases ceramide to trigger apoptosis

We have previously shown that activation of the acid sphingomyelinase (ASM), the release of ceramide and the formation of ceramide-enriched membrane domains are central for the induction of apoptosis by CD95. Here, we demonstrate that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 activate the ASM via a redox mechanism resulting in release of ceramide and formation of ceramide-enriched membrane platforms. Ceramide-enriched membrane platforms serve to cluster DR5 upon stimulation. Antioxidants prevent TRAIL-mediated stimulation of ASM, the release of ceramide, the formation of ceramide-enriched membrane platforms and the induction of apoptosis by TRAIL. Further, ASM-deficient splenocytes fail to cluster DR5 in ceramide-enriched membrane domains upon TRAIL stimulation and resist TRAIL-induced apoptosis, events that were restored by addition of natural C(16)-ceramide. A dose-response analysis indicates that ceramide-enriched membrane platforms greatly sensitized tumor cells to TRAIL-induced apoptosis. Our data indicate that ceramide-enriched membrane platforms are required for the signaling of TRAIL-DR5 complexes under physiological conditions.

Inhibition of DNA Methylation Sensitizes Glioblastoma for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Destruction

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments.

Induction of costimulation of human CD4 T cells by tumor necrosis factor-related apoptosis-inducing ligand: possible role in T cell activation in systemic lupus erythematosus.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently been shown to induce costimulation of mouse T cells in conjunction with signals from the T cell receptor. This study was undertaken to investigate TRAIL-induced costimulation of human T cells in order to determine the role of TRAIL-induced T cell activation in human systemic lupus erythematosus (SLE). An in vitro T cell stimulation system with immobilized anti-CD3 and recombinant TRAIL receptor DR4-Fc proteins was used to activate human T cells purified from healthy individuals and from patients with SLE. The T cells were stimulated in vitro to assay their proliferation response by (3)H-thymidine incorporation, and their cytokine production by enzyme-linked immunosorbent assay. Activation of p38 MAPK after TRAIL stimulation was detected with specific anti-phospho-p38 MAPK monoclonal antibodies in Western blots. Enhanced T cell proliferation and increased interleukin-2 and interferon-gamma (IFNgamma) production were demonstrated in human T cells after stimulation with immobilized DR4-Fc and anti-CD3 in vitro. TRAIL engagement selectively activated human CD4, rather than CD8, T cells and augmented IFNgamma production. Activation of p38 MAPK was detected after TRAIL-induced T cell activation. T cells isolated from patients with SLE demonstrated a stronger response to TRAIL-induced costimulation, in terms of proliferation and increased up-regulation of CD25 after activation, when compared with T cells from healthy subjects. TRAIL engagement induces costimulation of human CD4 T cells via a p38 MAPK-dependent pathway. The results suggest that enhanced reactivity of T cells to autoantigens as a result of TRAIL-induced costimulation may play a role in the development of human autoimmune diseases.

TRAIL-mediated enhancement of collagen production by human lung fibroblasts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes in vitro. The expression of TRAIL was found in CD8 T cells that had undergone oligoclonal expansion in the lungs of patients with systemic sclerosis (scleroderma) and were able to stimulate collagen production in lung fibroblasts in vitro. Among the family members, TRAIL displays highest homology to CD95 ligand, a receptor of which may not only mediate apoptosis of T cells, but also mediate the proliferation of normal human fibroblasts. Considering structural and functional similarities between TRAIL and CD95 ligand, we examined the effects of soluble TRAIL on normal human lung fibroblasts. Collagen α2(I) mRNA expression was assessed by real-time RT-PCR, with ribosomal protein S9 as an internal standard. Total soluble collagen was measured in culture supernatants using the Sircol Biocolor Assay. Both normalized α2(I) collagen mRNA expression and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (>5-fold increase) at 10 ng/ml TRAIL. DNA microarray hybridization revealed 78 genes involved in signal transduction, DNA transcription, and tissue remodeling, whose expression level increased, and 8 genes whose expression level decreased >2.5-fold in comparison with quiescent fibroblasts. Augmented expression of a number of genes involved in the TGFβ pathway suggested that TRAIL might induce TGFβ-mediated autocrine and/or paracrine stimulation of fibroblasts. This was confirmed by the ability of anti-TGFβ antibody to inhibit the effects of TRAIL on collagen production by fibroblasts. DNA mobility shift assay also revealed TRAIL-induced increase in protein binding to the collagen promoter that was substantially inhibited by consensus oligonucleotide for Smad3/4, mediators of TGFβ signaling. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGFβ production, which acts in autocrine manner.