C-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis.
ABSTRACT: Resistance to death receptor-mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain-containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1beta-converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-kappaB. Despite expression of other NF-kappaB-dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.
Project description:This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-kappaB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-kappaB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIP(L) cleavage in the death-inducing signaling complex (DISC) and the NF-kappaB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF-kappaB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIP(L) and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95-mediated apoptosis and NF-kappaB signaling.
Project description:To explore the molecular mechanisms by which glioblastomas are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined TRAIL signalling pathways in the tumours. TRAIL has four membrane-anchored receptors, death receptor 4/5 (DR4/5) and decoy receptor 1/2 (DcR1/2). Of these receptors, only DR5 was expressed consistently in glioblastoma cell lines and tumour tissues, ruling out the role of DcR1/2 in TRAIL resistance. Upon TRAIL binding, DR5 was homotrimerized and recruited Fas-associated death domain (FADD) and caspase-8 for the assembly of death-inducing signalling complex (DISC) in the lipid rafts of the plasma membrane. In the DISC, caspase-8 was cleaved and initiated apoptosis by cleaving downstream caspases in TRAIL-sensitive glioblastoma cells. In TRAIL-resistant cells, however, DR5-mediated DISC was modified by receptor-interacting protein (RIP), cellular FADD-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes or in astrocyte-15 (PED/PEA-15). This DISC modification occurred in the non-raft fractions of the plasma membrane and resulted in the inhibition of caspase-8 cleavage and activation of nuclear factor-kappaB (NF-kappaB). Treatment of resistant cells with parthenolide, an inhibitor of inhibitor of kappaB (I-kappaB), eliminated TRAIL-induced NF-kappaB activity but not TRAIL resistance. In contrast, however, targeting of RIP, c-FLIP or PED/PEA-15 with small interfering RNA (siRNA) led to the redistribution of the DISC from non-rafts to lipid rafts and eliminated the inhibition of caspase-8 cleavage and thereby TRAIL resistance. Taken together, this study indicates that the DISC modification by RIP, c-FLIP and PED/PEA-15 is the most upstream event in TRAIL resistance in glioblastomas.
Project description:The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.
Project description:Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.
Project description:CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.
Project description:Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.
Project description:Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression.
Project description:Inability to die by apoptosis is one of the reasons for the deregulated growth of tumour cells and the frequently observed failure of chemotherapy. In this study we thought to identify the common and functionally important characteristics responsible for the apoptosis resistance of pancreatic tumour cells. We analysed cell surface expression level of death receptors CD95 and TRAIL-R1-4 as well as the expression profile of sixteen apoptosis-relevant proteins in five pancreatic carcinoma cell lines Capan1, Colo357, PancTuI, Panc89 and Panc1. These data were evaluated in the context of sensitivity towards anti-CD95 and TRAIL-mediated apoptosis. Here we report that except for resistant Panc1 cells, which only marginally expressed CD95, all other cell lines showed comparable levels of CD95 and TRAIL receptors irrespectively of their apoptotic phenotype. Interestingly, we found that the elevated expression of FLIP, Bcl-x(L) and IAP in parallel with a downregulation of FADD and Bid was common for the resistant cell lines. Consequently, stable overexpression of XIAP, Bcl-x(L) or dominant negative FADD in sensitive cells significantly reduced the death receptor mediated apoptosis while the overexpression of Bid rendered the resistant cells sensitive.
Project description:Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.
Project description:Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarcinoma cells has been defined.Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9. Twenty-four hours after exposure, the activities of extracellular signal-regulated kinase 1/2, AKT, and nuclear factor-kappaB were only modestly modulated by sorafenib and vorinostat treatment. However, 24 h after exposure, sorafenib- and vorinostat-treated cells exhibited markedly diminished expression of c-FLIP-s, full-length BID, BCL-2, BCL-XL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK, and BAD. Expression of eIF2alpha S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP-s levels and overexpression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase-8. Knockdown of CD95 or FADD expression significantly reduced sorafenib/vorinostat-mediated lethality.These data show that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple antiapoptotic proteins and promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the proapoptotic signals from CD95 to promote mitochondrial dysfunction and death.