Enhanced adhesion of monocytes via reverse signaling triggered by decoy receptor 3

Decoy receptor 3 (DcR3), a newly identified soluble protein belonging to the tumor necrosis factor receptor (TNFR) superfamily, is a receptor for Fas ligand (FasL), LIGHT and TL1A. It has been demonstrated that DcR3 is frequently overexpressed by malignant tumors arising from lung, gastrointestinal tract, neuronal glia and virus-associated leukemia. Recently, we demonstrated that DcR3 is able to modulate the differentiation and activation of dendritic cells (DCs), and that DcR3-treated DCs skew naive T cell differentiation towards a Th2 phenotype. In this study, we further demonstrate that DcR3 is able to induce actin reorganization and enhance the adhesion of monocytes and THP-1 cells by activating multiple signaling molecules, such as protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK) and Src kinases. This provides the first evidence that the soluble DcR3, like other immobilized members of TNFR superfamily, is able to trigger 'reverse signaling' to modulate cell function.     

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.     

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.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

The role of apoptosis signal-regulating kinase 1 in lymphotoxin-beta receptor-mediated cell death

LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-beta receptor (LTbetaR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTbetaR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTbetaR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTbetaR monoclonal antibody, we found that signaling triggered by LTbetaR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTbetaR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTbetaR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTbetaR-induced cell death.     

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.     

Potentiation of Fas-mediated apoptosis by an engineered glycosylphosphatidylinositol-linked Fas

FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI and transmembrane Fas were localized in distinct membrane compartments, since Fas-GPI, but not transmembrane Fas, was found in the glycolipid-rich membrane microdomains. These results suggest that apoptosis induced by members of this ligand/receptors family may be differentially modulated through other and parallel signalling pathways.     

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.     

The combination of TRAIL and luteolin enhances apoptosis in human cervical cancer HeLa cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for cancer therapeutics. However, some tumor cells are resistant to TRAIL-induced apoptosis. Our previous studies have shown that luteolin, a naturally occurring flavonoid, induces the up-regulation of death receptor 5 (DR5), which is a receptor for TRAIL. Here, we show for the first time that luteolin synergistically acts with exogenous soluble recombinant human TRAIL to induce apoptosis in HeLa cells, but not in normal human peripheral blood mononuclear cells. The combined use of luteolin and TRAIL induced Bid cleavage and the activation of caspase-8. Also, human recombinant DR5/Fc chimera protein, caspase inhibitors, and DR5 siRNA efficiently reduced apoptosis induced by co-treatment with luteolin and TRAIL. These results raise the possibility that this combined treatment with luteolin and TRAIL might be promising as a new therapy against cancer.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na(+)/H(+) exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sensitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephosphorylation and caspase-8 activation via the intracellular acidification and that Na(+)/H(+) exchanger inhibitors may play an important role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt.     

GDP-mannose-4,6-dehydratase (GMDS) deficiency renders colon cancer cells resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor- and CD95-mediated apoptosis by inhibiting complex II formation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors.     

Overexpression of bcl-2 enhances LIGHT- and interferon-gamma -mediated apoptosis in Hep3BT2 cells

LIGHT is a member of the tumor necrosis factor superfamily and is the ligand for LT-betaR, HVEM, and decoy receptor 3. LIGHT has a cytotoxic effect, which is further enhanced by the presence of interferon-gamma (IFN-gamma). Although LIGHT/IFN-gamma can activate caspase activity, neither benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can completely inhibit LIGHT/IFN-gamma-mediated apoptosis. Moreover, overexpression of Bcl-2 further enhances LIGHT/IFN-gamma-mediated apoptosis. It appears that LIGHT and IFN-gamma act synergistically to activate caspase-3, with the resultant cleavage of Bcl-2, removal of the BH4 domain, leading to conversion of Bcl-2 from an antiapoptotic to a proapoptotic form in p53-deficient hepatocellular carcinoma Hep3BT2 cells. Thus, LIGHT seems to be able to override the protective effect of Bcl-2 and induce cell death. Although benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can prevent the cleavage of Bcl-2 by LIGHT/IFN-gamma, they only partially inhibit apoptosis in Hep3BT2 cells that are overexpressing Bcl-2. In contrast, both LIGHT/IFN-gamma-mediated apoptosis and Bcl-2 cleavage are inhibited by free radical scavengers, indicating that free radicals may play an essential role in LIGHT/IFN-gamma-mediated apoptosis at a step upstream of caspase-3 activation. These results suggest that LIGHT signaling may diverge into multiple, separate processes.