Evidence that BJcuL, a C-type lectin from Bothrops jararacussu venom, influences deubiquitinase activity, resulting in the accumulation of anti-apoptotic proteins in two colorectal cancer cell lines.

BJcuL is a snake venom C-type lectin (SVCTL) purified from the snake's venom Bothrops jararacussu. It has been previously demonstrated that BJcuL induces the accumulation of pro-apoptotic proteins of the extrinsic pathway, such as FADD and caspase-8, in the colorectal cancer cell line HT29, suggesting that the lectin may be able to enhance TRAIL-induced apoptosis. To test this hypothesis, we exposed two colorectal cancer cell lines, HT29 and HCT116, to increasing concentrations of BJcuL (1-20 µg/mL) in the presence or absence of TRAIL. Contrary to our expectations, however, BJcuL was unable to induce apoptosis in these cells, as shown by annexin-V/7AAD, clonogenic assays, and immunoblotting. Nevertheless, BJcuL was able to induce the accumulation of FADD and caspase-8, as well as anti-apoptotic proteins such as c-FLIP and survivin and poly-ubiquitinated proteins. Incubation with the deubiquitinase inhibitor WP1130 (10 µM) resulted in decreased BJcuL-induced survivin levels. Altogether, our results evince the effects of SVCTL on the ubiquitin-proteasome system in vitro for the first time. Compounds that can influence such system are important tools in the search for new therapeutic or diagnostic targets in cancer since they can elucidate the molecular mechanisms involved in determining cell fate as well as contributing to drug-development strategies in partnership with the pharmaceutical industry.

FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation.

Fas is a cell surface death receptor that signals apoptosis. Several proteins have been identified that bind to the cytoplasmic death domain of Fas. Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain. We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

E2F1 induces apoptosis and sensitizes human lung adenocarcinoma cells to death-receptor-mediated apoptosis through specific downregulation of c-FLIP(short).

E2F1 is a transcription factor that plays a well-documented role during S phase progression and apoptosis. We had previously postulated that the low level of E2F1 in primary lung adenocarcinoma contributes to their carcinogenesis. Here, we show that E2F1 triggers apoptosis in various lung adenocarcinoma cell lines by a mechanism involving the specific downregulation of the cellular FLICE-inhibitory protein short, leading to caspase-8 activation at the death-inducing signaling complex. Importantly, we also provide evidence that E2F1 sensitizes tumor as well as primary cells to apoptosis mediated by FAS ligand or tumor necrosis factor-related apoptosis-inducing ligand, and enhances the cytotoxic effect of T lymphocytes against tumor cells. Finally, we describe the specific overexpression of c-FLIP(S) in human lung adenocarcinomas with low level of E2F1. Overall, our data identify E2F1 as a critical determinant of the cellular response to death-receptor-mediated apoptosis, and suggest that its downregulation contributes to the immune escape of lung adenocarcinoma tumor cells.

NF-kappaB signals induce the expression of c-FLIP.

Activation of the transcription factor NF-kappaB is a major effector of the inducible resistance to death receptor-mediated apoptosis. Previous evidence indicates that the combined transcriptional activation of TRAF-1, TRAF-2, IAP-1, and IAP-2 is required to suppress cell death by tumor necrosis factor (TNF). Here we show that NF-kappaB activation upregulates the caspase 8 inhibitor FLIP, resulting in increased resistance to Fas ligand (FasL) or TNF. Restoration of either the full-length 55-kDa long form of FLIP or an alternatively spliced short form of FLIP in NF-kappaB null cells inhibits TNF- and FasL-induced cell death efficiently, whereas the expression of IAP or TRAF family members only partially rescues cells from death. Resistance to either FasL- or TNF-induced apoptosis is overcome when cells are incubated in the presence of the protein synthesis inhibitor cycloheximide. This treatment leads to the rapid downregulation of FLIP but not to that of TRAF2. Our findings suggest that FLIP is an important mediator of NF-kappaB-controlled antiapoptotic signals.

TRAIL-NP hybrids for cancer therapy: a review.

Cancer is a worldwide health problem. It is now considered as a leading cause of morbidity and mortality in developed countries. In the last few decades, considerable progress has been made in anti-cancer therapies, allowing the cure of patients suffering from this disease, or at least helping to prolong their lives. Several cancers, such as those of the lung and pancreas, are still devastating in the absence of therapeutic options. In the early 90s, TRAIL (Tumor Necrosis Factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF superfamily, attracted major interest in oncology owing to its selective anti-tumor properties. Clinical trials using soluble TRAIL or antibodies targeting the two main agonist receptors (TRAIL-R1 and TRAIL-R2) have, however, failed to demonstrate their efficacy in the clinic. TRAIL is expressed on the surface of natural killer or CD8+ T activated cells and contributes to tumor surveillance. Nanoparticles functionalized with TRAIL mimic membrane-TRAIL and exhibit stronger antitumoral properties than soluble TRAIL or TRAIL receptor agonist antibodies. This review provides an update on the association and the use of nanoparticles associated with TRAIL for cancer therapy.

Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity.

BACKGROUND: The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. PURPOSE: This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. METHODS: Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. RESULTS: Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. CONCLUSION AND IMPLICATIONS: In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.

Cancer cell sensitization to fas-mediated apoptosis by sodium butyrate.

Cancer cells often resist Fas-mediated apoptosis even when the Fas receptor is expressed at the cell surface. We show here that human and rat colon cancer cells undergo massive apoptosis when they are exposed to soluble Fas ligand in the presence of sodium butyrate, an agent that induces by itself only a low rate of apoptosis. Sodium butyrate potentiates Fas-dependent apoptosis in seven out of eight colon cancer cell lines. Sodium butyrate does not increase Fas receptor cell surface expression and does not modify cell levels of Bcl-2, Bcl-xL, Bcl-xS and Bax. Sodium butyrate also induces tumor cell sensitization to the apoptotic effect of the combination of TNF-alpha and IFN-gamma, but it does not modify the level of the FADD/Mort1 adaptator molecule, at the connection between Fas- and TNF-dependent apoptosis pathways. Because the clinical toxicity of butyrate is low, its ability to enhance Fas-signal delivery in cancer cells could be of therapeutic interest.

Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion.

TRAIL is involved in immune tumor surveillance and is considered a promising anti-cancer agent owing to its limited side effects on healthy cells. However, some cancer cells display resistance, or become resistant to TRAIL-induced cell death. Hyperthermia can enhance sensitivity to TRAIL-induced cell death in various resistant cancer cell lines, including lung, breast, colon or prostate carcinomas. Mild heat shock treatment has been proposed to restore Fas ligand or TRAIL-induced apoptosis through c-FLIP degradation or the mitochondrial pathway. We demonstrate here that neither the mitochondria nor c-FLIP degradation are required for TRAIL-induced cell death restoration during hyperthermia. Our data provide evidence that insolubilization of c-FLIP, alone, is sufficient to enhance apoptosis induced by death receptors. Hyperthermia induced c-FLIP depletion from the cytosolic fraction, without apparent degradation, thereby preventing c-FLIP recruitment to the TRAIL DISC and allowing efficient caspase-8 cleavage and apoptosis. Hyperthermia-induced c-FLIP depletion was independent of c-FLIP DED2 FL chain assembly motif or ubiquitination-mediated c-FLIP degradation, as assessed using c-FLIP point mutants on lysine 167 and 195 or threonine 166, a phosphorylation site known to regulate ubiquitination of c-FLIP. Rather, c-FLIP depletion was associated with aggregation, because addition of glycerol not only prevented the loss of c-FLIP from the cytosol but also enabled c-FLIP recruitment within the TRAIL DISC, thus inhibiting TRAIL-induced apoptosis during hyperthermia. Altogether our results demonstrate that c-FLIP is a thermosensitive protein whose targeting by hyperthermia allows restoration of apoptosis induced by TNF ligands, including TRAIL. Our findings suggest that combining TRAIL agonists with whole-body or localized hyperthermia may be an interesting approach in cancer therapy.

Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-kappaB activation.

Bcl10, a caspase recruitment domain (CARD)-containing protein identified from a breakpoint in mucosa-associated lymphoid tissue (MALT) B lymphomas, is essential for antigen-receptor-mediated nuclear factor kappaB (NF-kappaB) activation in lymphocytes. We have identified a novel CARD-containing protein and interaction partner of Bcl10, named Carma1. Carma1 is predominantly expressed in lymphocytes and represents a new member of the membrane-associated guanylate kinase family. Carma1 binds Bcl10 via its CARD motif and induces translocation of Bcl10 from the cytoplasm into perinuclear structures. Moreover, expression of Carma1 induces phosphorylation of Bcl10 and activation of the transcription factor NF-kappaB. We propose that Carma1 is a crucial component of a novel Bcl10-dependent signaling pathway in T-cells that leads to the activation of NF-kappaB.

P27KiP1 overexpression inhibits the growth and doxorubicin sensitivity of HT29 human colon cancer cells in vivo.

We have previously shown that p27KiP1 plays a role in the tumor cell resistance of HT29 confluent monolayers to cytotoxic drugs in vitro. To determine whether p27KiP1 was a resistance factor to cytotoxic drugs in vivo we tested the effect of doxorubicin on p27KiP1-overexpressing HT29 tumors in nude mice. In this study we show that ectopic overexpression of p27KiP1 in HT29 human colon cancer cells decreases their tumorigenicity in vivo in nude mice. This decreased tumor growth was associated with increased p27KiP1 protein expression, studied by Western blotting in tumor extracts. Interestingly, the overexpressing-p27KiP1 tumors were significantly more resistant to intraveneous doxorubicin treatment than the control tumors. These results indicate that p27KiP1, which delays tumor growth could also increase tumor resistance to cytotoxic drugs in vivo.

[The Fas/Fas-ligand system: implications in the antitumor immune response and in the activity of cytotoxic agents]. / Le système Fas/Fas-ligand: implications dans la réponse immunitaire antitumorale et dans l'activité des agents cytotoxiques.

Interaction of Fas-ligand (Fas-L) with the extracytoplasmic domain of the Fas receptor can induce Fas trimerization and activation of the apoptotic cell death process. Several molecular pathways that lead to apoptosis and some of their regulatory mechanisms have been identified. Fas-related membrane receptors that contain a death domain in their intracytoplasmic domain have been identified. They constitute a death receptor family (DR1 to DR5) whose first member is the TNFR1 receptor for TNF alpha. The Fas/Fas-L system plays a role in the cytotoxic activity of immune cells and the regulation of immune response amplitude. This system could be involved in the immune response to tumor cells and the cytotoxic activity of drugs and radiations. The expression of Fas-L on the plasma membrane of numerous tumor cells allow them, in vitro, to kill Fas-expressing immune cells. This observations has suggested that tumor cells used Fas-L to induce a specific immune tolerance. However, in vivo, Fas-L expression rather induces tumor cell rejection. The quantity of Fas-L expressed on tumor cells could determine whether tumor cells are tolerated or rejected. Cytotoxic drugs and radiations modulate Fas and Fas-L expression on tumor cells. The role of Fas/Fas-L interactions in the cytotoxicity of these agents remains poorly defined. It has been clearly shown, however, that low doses of cytotoxic drugs increase Fas expression on tumor cells, thereby improving their elimination by immune cells. Drug-induced modulation of Fas expression could provide new therapeutic strategies combining chemotherapy with immunotherapy.

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells.

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.

Death receptors as targets in cancer.

UNLABELLED: Anti-tumour therapies based on the use pro-apoptotic receptor agonists, including TNF-related apoptosis-inducing ligand (TRAIL) or monoclonal antibodies targeting TRAIL-R1 or TRAIL-R2, have been disappointing so far, despite clear evidence of clinical activity and lack of adverse events for the vast majority of these compounds, whether combined or not with conventional or targeted anti-cancer therapies. This brief review aims at discussing the possible reasons for the lack of apparent success of these therapeutic approaches and at providing hints in order to rationally design optimal protocols based on our current understanding of TRAIL signalling regulation or resistance for future clinical trials. LINKED ARTICLES: This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity?

The cytoskeleton is a highly complex network of three major intracellular filaments, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). This network plays a key role in the control of cell shape, division, functions and interactions in animal organs and tissues. Dysregulation of the network can contribute to numerous human diseases. Although small HSPs (sHSPs) and in particular HSP27 (HSPB1) or αB-crystallin (HSPB5) display a wide range of cellular properties, they are mostly known for their ability to protect cells under stress conditions. Mutations in some sHSPs have been found to affect their ability to interact with cytoskeleton proteins, leading to IF aggregation phenotypes that mimick diseases related to disorders in IF proteins (i.e. desmin, vimentin and neuro-filaments). The aim of this review is to discuss new findings that point towards the possible involvement of IFs in the cytoprotective functions of sHSPs, both in physiological and pathological settings, including the likelihood that sHSPs such as HSPB1 may play a role during epithelial-to-mesenchymal transition (EMT) during fibrosis or cancer progression. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Identification of a novel pro-apoptotic role of NF-κB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells.

We recently reported that nuclear factor-kappa B (NF-κB) promotes DNA damage-triggered apoptosis in glioblastoma, the most common brain tumor. In the present study, we investigated the role of NF-κB in death receptor-mediated apoptosis. Here, we identify a novel pro-apopotic function of NF-κB in TRAIL- and CD95-induced apoptosis. Inhibition of NF-κB by overexpression of the dominant-negative IκBα-superrepressor (IκBα-SR) significantly decreases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- or CD95-induced apoptosis. Vice versa, activation of NF-κB via overexpression of constitutively active IκB kinase complex (IKK)ß (IKK-EE) significantly increases TRAIL-mediated apoptosis. Intriguingly, NF-κB inhibition reduces the recruitment of Fas-associated death domain and caspase-8 and formation of the death-inducing signaling complex (DISC) upon stimulation of TRAIL receptors or CD95. This results in reduced TRAIL-mediated activation of caspases, loss of mitochondrial potential and cytochrome c release in IκBα-SR-expressing cells. In comparison, NF-κB inhibition strongly enhances TNF-α-mediated apoptosis. Comparative studies revealed that TNF-α rapidly stimulates transcriptional activation and upregulation of anti-apoptotic proteins, whereas TRAIL causes apoptosis before transcriptional activation. Thus, this study demonstrates for the first time that NF-κB exerts a pro-apoptotic role in TRAIL- and CD95-induced apoptosis in glioblastoma cells by facilitating DISC formation.

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8.

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.

Chemotherapy overcomes TRAIL-R4-mediated TRAIL resistance at the DISC level.

TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.