Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases

Cell death plays a pivotal role in the maintenance of tissue homeostasis. Key players in the controlled induction of cell death are the Death Receptors (DR). CD95 is a prototypic DR activated by its cognate ligand CD95L triggering programmed cell death. As a consequence, alterations in the CD95/CD95L pathway have been involved in several disease conditions ranging from autoimmune diseases to inflammation and cancer. CD95L-induced cell death has multiple roles in the immune response since it constitutes one of the mechanisms by which cytotoxic lymphocytes kill their targets, but it is also involved in the process of turning off the immune response. Furthermore, beyond the canonical pro-death signals, CD95L, which can be membrane-bound or soluble, also induces non-apoptotic signaling that contributes to its tumor-promoting and pro-inflammatory roles. The intent of this review is to describe the role of CD95/CD95L in the pathophysiology of cancers, autoimmune diseases and chronic inflammation and to discuss recently patented and emerging therapeutic strategies that exploit/block the CD95/CD95L system in these diseases.Subject terms: Drug development, Apoptosis     

Precise Mapping of the CD95 Pre-Ligand Assembly Domain

Pre-association of CD95 at the plasma membrane is mandatory for efficient death receptor signaling. This homotrimerization occurs through self-association of an extracellular domain called the pre-ligand assembly domain (PLAD). Using novel molecular and cellular tools, we confirmed that CD95-PLAD is necessary to promote CD95 multimerization and plays a pivotal role in the transmission of apoptotic signals. However, while a human CD95 mutant deleted of the previously described PLAD domain (amino acids 1 to 66) fails to interact with its wild-type counterpart and trigger autonomous cell death, deletion of amino acids 1 to 42 does not prevent homo- or hetero (human/mouse)-oligomerization of CD95, and thus does not alter transmission of the apoptotic signal. Overall, these findings indicate that the region between amino acids 43 to 66 corresponds to the minimal motif involved in CD95 homotypic interaction and is necessary to convey an efficient apoptotic signal. Interfering with this PLAD may represent a new therapeutic strategy for altering CD95-induced apoptotic and non-apoptotic signals.     

Caspase inhibition activates HIV in latently infected cells. Role of tumor necrosis factor receptor 1 and CD95

Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.     

How CD95 stimulates invasion

CD95 is best known for its capacity to induce apoptosis, but also activates multiple non-apoptotic signalling pathways. In particular, CD95 promotes migration and tissue invasion of apoptosis-resistant cell types, and this plays a central role in inflammation, neurobiology, and tumor biology. CD95 induces invasion by stimulating the expression of extracellular matrix (ECM)-degrading proteases, and by stimulating the formation of actin-driven cell protrusions through Rac and the cofilin pathway. In this review we discuss how CD95-initiated signalling pathways may cooperate to facilitate cell migration and tissue invasion. -     

Non-apoptotic Fas signaling

Fas (Apo-1, CD95) and Fas-Ligand (FasL, CD95L) are typical members of the TNF receptor and TNF ligand family, respectively, with a pivotal role in the regulation of apoptotic processes, including activation-induced cell death, T-cell-induced cytotoxicity, immune privilege and tumor surveillance. Impairment of the FasL/Fas system has been implicated in liver failure, autoimmune diseases and immune deficiency. Thus, the FasL/Fas system was mainly appreciated with respect to its death-inducing capabilities. However, there is increasing evidence that activation of Fas can also result in non-apoptotic responses like cell proliferation or NF-kappaB activation. While the apoptotic features of the FasL/Fas system and the pathways involved are comparably well investigated, the pathways that are utilized by Fas to transduce proliferative and activating signals are poorly understood. This review is focused on the non-apoptotic functions of the FasL/Fas system. In particular, the similarities and differences of the molecular mechanisms of apoptotic and non-apoptotic Fas signaling are addressed.     

The role of CD95 and CD95 ligand in cancer

CD95 (Fas/APO-1) and its ligand, CD95L, have long been viewed as a death receptor/death ligand system that mediates apoptosis induction to maintain immune homeostasis. In addition, these molecules are important in the immune elimination of virus-infected cells and cancer cells. CD95L was, therefore, considered to be useful for cancer therapy. However, major side effects have precluded its systemic use. During the last 10 years, it has been recognized that CD95 and CD95L have multiple cancer-relevant nonapoptotic and tumor-promoting activities. CD95 and CD95L were discovered to be critical survival factors for cancer cells, and were found to protect and promote cancer stem cells. We now discuss five different ways in which inhibiting or eliminating CD95L, rather than augmenting, may be beneficial for cancer therapy alone or in combination with standard chemotherapy or immune therapy.     

CD95 (APO-1/Fas)-associating signalling proteins

The CD95 (APO-1/Fas) receptor has attracted great interest in recent years because it transduces an apoptotic signal in a variety of different tissues. CD95 belongs to the NGF/TNF-receptor superfamily, members of which need to be trimerized by specific protein ligands in order to generate a signal. This review focuses on recent advances in the understanding of the proximal signal transduction mechanism of CD95. The cloning of numerous proteins that interact with CD95 and other members of this receptor family and the in vivo identification of several proteins that associate with CD95 in a ligand-dependent fashion opens the way to delineate the death pathway and to explain crosstalk among these receptors on a molecular basis.     

Regulation of CD95/Fas signaling at the DISC

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.     

CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells

The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.     

Cutting edge: a novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis.

CD95-induced apoptosis contributes to the maintenance of homeostasis in both B and T lymphocyte-mediated immunity. B cells increase CD95 expression in response to activation signals and become susceptible to CD95-induced apoptosis. Protection from CD95-mediated death signals can be induced in mature B cells by signals delivered through the B cell Ag receptor. In this paper we demonstrate for the first time that rescue from apoptosis can occur independently of de novo protein synthesis. This rescue from apoptosis prevents activation of caspase 8, the apical caspase in the CD95 death pathway, and CD95-FADD (Fas-associated death domain containing protein) association does not occur normally. Thus B cell activation signals can biochemically modify proximal elements of the CD95 death pathway and regulate the sensitivity of cells to apoptosis induction at an early stage in programmed cell death.     

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.     

CD95 signaling in colorectal cancer

CD95 and its ligand (CD95L) are widely expressed in colorectal tumors, but their role in shaping tumor behavior is unclear. CD95 activation on tumor cells can lead to apoptosis, while CD95L attracts neutrophils, suggesting a function in tumor suppression. However, CD95 can also promote tumorigenesis, at least in part by activating non-apoptotic signaling pathways that stimulate tumor cell proliferation, invasion and survival. In addition, CD95 signaling in stromal cells and tumor-infiltrating inflammatory cells has to be taken into account when addressing the function of CD95 and its ligand in colorectal tumor biology. We present a model in which the tumor-suppressing and tumor-promoting activities of CD95/CD95L together determine colorectal tumor behavior. We also discuss how these multiple activities are changing our view of CD95 and CD95L as potential therapeutic targets in the treatment of colorectal cancer. We conclude that locking CD95 in apoptosis-mode may be a more promising anti-cancer strategy than simply inhibiting or stimulating CD95.     

CD95/CD95L interactions and their role in autoimmunity

CD95 (Fas/Apo-1) is a broadly expressed death receptor involved in a variety of physiological and pathological apoptotic processes. Since its discovery, defects in CD95/CD95L system have been proposed as major pathogenic factors responsible for impaired immunological tolerance to self antigens and autoimmunity. Later, analysis of altered sensitivity to CD95-induced apoptosis in cells targeted by the immune response has revealed an unexpected role for CD95 and CD95L in organ-specific autoimmunity. CD95 has been shown to be expressed and functional in virtually all cell types that are target of the organ-specific autoimmune response. Here we review some of the major findings concerning the role of CD95 in autoimmunity, in dysfunctions due to increased or decreased CD95-induced apoptosis.     

CD95 tyrosine phosphorylation is required for CD95 oligomerization

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95^Y232,291F), revealed that at least one tyrosine (Y^232,291)-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.     

DICE

The conventional view of CD95 (Fas/APO-1) is that it is a dedicated apoptosis-inducing receptor with important functions in immune cell homeostasis and in viral and tumor defense. There is an emerging recognition, however, that CD95 also has multiple non-apoptotic activities. In the context of cancer, CD95 was shown to have tumor-promoting activities, and the concept of this new function of CD95 in cancer is gaining traction. Recently, we showed that not only is CD95 a growth promoter for cancer cells, but, paradoxically, when either CD95 or CD95 ligand (CD95L) is removed, that virtually all cancer cells die through a process we have named DICE (death induced by CD95R/L elimination). In this perspective, I outline a hypothesis regarding the physiological function of DICE, and why it may be possible to use induction of DICE to treat many, if not most, cancers.