Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.     

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.     

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.     

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.     

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

The present study aims to understand the mechanism of the lens epithelial cell’s strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.Subject terms: Tumour-necrosis factors, Apoptosis     

Diabetic liver injury from streptozotocin is regulated through the caspase-8 homolog cFLIP involving activation of JNK2 and intrahepatic immunocompetent cells

The endemic occurrence of obesity and the associated risk factors that constitute the metabolic syndrome have been predicted to lead to a dramatic increase in chronic liver disease. Non-alcoholic steatohepatitis (NASH) has become the most frequent liver disease in countries with a high prevalence of obesity. In addition, hepatic steatosis and insulin resistance have been implicated in disease progression of other liver diseases, including chronic viral hepatitis and hepatocellular carcinoma. The molecular mechanisms underlying the link between insulin signaling and hepatocellular injury are only partly understood. We have explored the role of the antiapoptotic caspase-8 homolog cellular FLICE-inhibitory protein (cFLIP) on liver cell survival in a diabetic model with hypoinsulinemic diabetes in order to delineate the role of insulin signaling on hepatocellular survival. cFLIP regulates cellular injury from apoptosis signaling pathways, and loss of cFLIP was previously shown to promote injury from activated TNF and CD95/Apo-1 receptors. In mice lacking cFLIP in hepatocytes (flip^−/−), loss of insulin following streptozotocin treatment resulted in caspase- and c-Jun N-terminal kinase (JNK)-dependent liver injury after 21 days. Substitution of insulin, inhibition of JNK using the SP600125 compound in vivo or genetic deletion of the mitogen-activated protein kinase (MAPK)9 (JNK2) in all tissues abolished the injurious effect. Strikingly, the difference in injury between wild-type and cFLIP-deficient mice occurred only in vivo and was accompanied by liver-infiltrating inflammatory cells with a trend toward increased amounts of NK1.1-positive cells and secretion of proinflammatory cytokines. Transfer of bone marrow from rag-1-deficient mice that are depleted from B and T lymphocytes prevented liver injury in flip^−/− mice. These findings support a direct role of insulin on cellular survival by alternating the activation of injurious MAPK, caspases and the recruitment of inflammatory cells to the liver. Thus, increasing resistance to insulin signaling pathways in hepatocytes appears to be an important factor in the initiation and progression of chronic liver disease.     

Survival function of the FADD-CASPASE-8-cFLIP(L) complex

Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.     

Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD)

In HeLa cells, induction of apoptosis and nuclear factor kappaB (NF-kappaB) activation initiated by TRAIL/Apo2L or the agonistic Apo1/Fas-specific monoclonal antibody anti-APO-1 require the presence of cycloheximide (CHX). Inhibition of caspases prevented TRAIL/anti-APO-1-induced apoptosis, but not NF-kappaB activation, indicating that both pathways bifurcate upstream of the receptor-proximal caspase-8. Under these conditions, TRAIL and anti-APO-1 up-regulated the expression of the known NF-kappaB targets interleukin-6, cellular inhibitor of apoptosis 2 (cIAP2), and TRAF1 (TRAF, tumor necrosis factor receptor-associate factor). In the presence of CHX, the stable overexpression of a deletion mutant of the Fas-associated death domain molecule FADD comprising solely the death domain of the molecule but lacking its death effector domain (FADD-(80-208)) led to the same response pattern as TRAIL or anti-APO-1 treatment. Moreover, the ability of death receptors to induce NF-kappaB activation was drastically reduced in a FADD-deficient Jurkat cell line. TRAIL-, anti-APO-1-, and FADD-(80-208)-initiated gene induction was blocked by a dominant-negative mutant of TRAF2 or the p38 kinase inhibitor SB203580, similar to tumor necrosis factor receptor-1-induced NF-kappaB activation. CHX treatment rapidly down-regulated endogenous cFLIP protein levels, and overexpression of cellular FLICE inhibitory protein (cFLIP) inhibited death receptor-induced NF-kappaB activation. Thus, a novel functional role of cFLIP as a negative regulator of gene induction by death receptors became apparent.     

Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activated protein kinases and NF-kappaB-dependent gene expression

Many Fas-expressing cells do not undergo cell death upon Fas stimulation. In the normal human diploid cell line GM6112, the addition of soluble Fas ligand (sFasL) leads to morphological signs of cell death in less than 1% of cells. Treatment of serum-starved GM6112 fibroblasts with sFasL resulted in a rapid and transient phosphorylation of ERK1/2 without a significant increase in JNK and p38 activities. Unless co-treated with the protein synthesis inhibitor anisomycin, sFasL did not show gene-inducing activity in cells maintained in complete medium. However, when cells were serum-starved for 4 days, treatment with sFasL alone induced interleukin-6 gene expression and, less strongly, interleukin-8 gene expression. Sensitization of the gene-inducing activity by serum starvation correlated with NF-kappaB activation by sFasL. Furthermore, we found that the expression of FADD and caspase-8 was significantly reduced in serum-starved cells, whereas the level of cFLIP remained unchanged. Transfection of GM6112 cells with the antisense caspase-8 expression construct sensitized cells toward sFasL-induced NF-kappaB-dependent reporter activation. Our results support the notion that a change in the ratio of cFLIP and caspase-8 may be responsible for turning on the Fas-activated NF-kappaB pathway, which otherwise is supplanted by the death-inducing pathway.     

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.     

Inhibition of Methyltransferases Accelerates Degradation of cFLIP and Sensitizes B-Cell Lymphoma Cells to TRAIL-Induced Apoptosis

Non-Hodgkin lymphomas (NHLs) are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. It is believed that cancer cells are particularly sensitive to cell death induced by tumor necrosis factor α–related apoptosis-inducing ligand (TRAIL). However, many cancer cells show blocked TRAIL signaling due to up-regulated expression of anti-apoptotic factors, such as cFLIP. This hurdle to TRAIL’s tumor cytotoxicity might be overcome by combining TRAIL-based therapy with drugs that reverse blockages of its apoptotic signaling. In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep) on TRAIL-induced apoptosis in aggressive B-cell NHLs: mantle cell, Burkitt, and diffuse large B-cell lymphomas. We characterized TRAIL apoptosis regulation and caspase activation in several NHL-derived cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. No change in cFLIP mRNA level indicated independence of promoter methylation alterations in methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA stability and protein stability. This appears to be in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346). However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores DZNep’s potential in TRAIL-based therapies for B-cell NHLs.