Amélie Fouqué

Laure Debure

Patrick Legembre
email: patrick.legembre@inserm.fr

The CD95/CD95L Signaling Pathway: A Role in Carcinogenesis

Keywords: Fas, apoptosis, cytokine, inflammation, carcinogenesis

Apoptosis is a fundamental process that contributes to tissue homeostasis, immune responses, and development. The receptor CD95, also called Fas, is a member of the tumor necrosis factor receptor (TNF-R) superfamily. Its cognate ligand, CD95L, is implicated in immune homeostasis and immune surveillance, and various lineages of malignant cells exhibit loss-offunction mutations in this pathway; therefore, CD95 was initially classified as a tumor suppressor gene. However, more recent data indicate that in different pathophysiological contexts, this receptor can transmit non-apoptotic signals, promote inflammation, and contribute to carcinogenesis. A comparison with the initial molecular events of the TNF-R signaling pathway leading to non-apoptotic, apoptotic, and necrotic pathways reveals that CD95 is probably using different molecular mechanisms to transmit its non-apoptotic signals MAPK, and PI3K). As discussed in this review, the molecular process by which the receptor switches from an apoptotic function to an inflammatory role is unknown. More importantly, the biological functions of these signals remain elusive.

Introduction

The two main apoptotic signaling pathways are distinguished by the origins of the initiating signals. The intrinsic pathway is triggered by accumulation of DNA damage, deregulation of mitochondrial function, or viral infection, and induces the release of pro-apoptotic factors from the mitochondria. By contrast, the extrinsic pathway is activated by the binding of apoptotic ligands to death receptors on the cell surface. The pathways are interconnected, and both converge on activation of a family of cysteine proteases specific for aspartic acid residues, the caspases [START_REF] Alnemri | Human ICE/CED-3 protease nomenclature[END_REF]. The apoptotic role of the mitochondrion is associated with a reduction in transmembrane potential and the loss of extracellular membrane integrity, leading to the release of various apoptogenic factors into the cytosol. One of these factors, cytochrome c, associates with the caspase-9/APAF1 complex to form the apoptosome and trigger apoptosis [START_REF] Hengartner | The biochemistry of apoptosis[END_REF].

The intrinsic and extrinsic pathways share common features, and both require the aggregation of initiator caspases as an early event. During interactions with their respective ligands, members of the death-receptor superfamily recruit adaptor proteins such as Fasassociating protein with a death domain (FADD) [START_REF] Boldin | A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain[END_REF][START_REF] Chinnaiyan | FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis[END_REF] or Tumor Necrosis Factor (TNF) Receptor 1-Associated Death Domain Protein (TRADD) [START_REF] Hsu | The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation[END_REF], resulting in the aggregation and activation of initiator caspases (caspase-8 and -10) to form the death-inducing signaling complex (DISC) [START_REF] Kischkel | Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor[END_REF]. In a similar manner, release of cytochrome c and ATP from mitochondria promotes the formation of the apoptosome (along with cytosolic APAF-1), resulting in aggregation and activation of initiator caspase-9, which in turn cleaves caspase-3 [START_REF] Li | Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade[END_REF].

It should be kept in mind that death receptors CD95 [START_REF] Itoh | The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis[END_REF], TNFR1 [START_REF] Loetscher | Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor[END_REF], DR4 [START_REF] Pan | The receptor for the cytotoxic ligand TRAIL[END_REF], DR5 [START_REF] Walczak | TRAIL-R2: a novel apoptosismediating receptor for TRAIL[END_REF], and DR6 [START_REF] Pan | Identification and functional characterization of DR6, a novel death domain-containing TNF receptor[END_REF] were cloned based on their ability to elicit apoptosis. Although the abilities of Fas/CD95, DR4, and DR5 to trigger non-apoptotic signaling pathways were observed immediately after the corresponding genes were cloned [START_REF] Alderson | Fas transduces activation signals in normal human T lymphocytes[END_REF][START_REF] Schulze-Osthoff | Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death[END_REF], most if not all studies of these proteins have been focused on characterizing the molecular events leading to cell death. Accordingly, several agonistic molecules were developed in order to kill cancer cells, neglecting the impact of non-apoptotic signals in pathophysiological contexts. More recent data has altered this vision by highlighting the biological role of death receptormediated non-apoptotic signaling pathways in chronic inflammatory disorders and carcinogenesis.

TNF Receptor Family

Death receptors TNFR1, CD95, DR3, DR4, DR5, and DR6 belong to the tumor necrosis factor receptor (TNF-R) superfamily. These type I transmembrane proteins share common features: extracellular amino-terminal cysteine-rich domains (CRDs) [START_REF] Smith | The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death[END_REF][START_REF] Locksley | The TNF and TNF receptor superfamilies: integrating mammalian biology[END_REF], which contribute to ligand specificity [START_REF] Bodmer | The molecular architecture of the TNF superfamily[END_REF]; pre-association of the receptor at the plasma membrane [START_REF] Edmond | Precise Mapping of the CD95 Pre-Ligand Assembly Domain[END_REF][START_REF] Papoff | Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF]; and the death domain (DD), a conserved 80 amino-acid sequence located in the cytoplasmic tail, which is necessary for DISC formation and initiation of the apoptotic signal [START_REF] Itoh | A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen[END_REF][START_REF] Tartaglia | A novel domain within the 55 kd TNF receptor signals cell death[END_REF].

TNFR1 signaling pathways

TNF-α exerts its effects by binding to two receptors, TNFR1 and TNFR2 [START_REF] Locksley | The TNF and TNF receptor superfamilies: integrating mammalian biology[END_REF]. Recently, progranulin was identified as a ligand of TNFR with a higher affinity than TNF-α.

Progranulin antagonizes TNF-α signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice [START_REF] Tang | The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice[END_REF]. TNFR1, a 55 kDa protein with a DD in its intracellular region, is expressed in almost all cell types, whereas, TNFR2, a 75 kDa protein, is mainly expressed in oligodendrocytes, astrocytes, T cells, myocytes, thymocytes, endothelial cells, and human mesenchymal stem cells [START_REF] Cabal-Hierro | Signal transduction by tumor necrosis factor receptors[END_REF]. Considerable uncertainty persists regarding the TNFR2 signaling pathway, which has been reviewed previously [START_REF] Cabal-Hierro | Signal transduction by tumor necrosis factor receptors[END_REF]. The CRD1 domains of CD95, TNFR1, and TNFR2 are involved in homotypic interactions, leading to pre-association of the receptor as a homotrimer in the absence of ligand [START_REF] Papoff | Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF][START_REF] Chan | A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling[END_REF]. Thus, this domain has been designated the pre-ligand binding assembly domain (PLAD) [START_REF] Chan | A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling[END_REF]. Receptors of the TNFR superfamily do not possess any enzymatic activity on their own, and therefore rely on the recruitment of adaptor proteins for signaling. Among these adaptor proteins, TRADD or FADD are instrumental in the implementation of cell death processes [START_REF] Boldin | A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain[END_REF][START_REF] Chinnaiyan | FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis[END_REF][START_REF] Hsu | The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation[END_REF][START_REF] Kischkel | Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor[END_REF].

TNF-α is synthesized as a 26 kDa transmembrane type II protein (m-TNF-α) of 233 amino acids [START_REF] Pennica | Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin[END_REF], which can be cleaved by the metalloprotease TACE [START_REF] Black | A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells[END_REF][START_REF] Moss | Cloning of a disintegrin metalloproteinase that processes precursor tumournecrosis factor-alpha[END_REF] to release the 17 kDa soluble form of the cytokine (cl-TNF-α). In contrast to cl-TNF-α, which only activates TNFR1, m-TNF-α can bind and activate both TNFR1 and TNFR2 [START_REF] Grell | The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor[END_REF].

Activation of TNFR1 induces cellular processes ranging from cell death (apoptosis or necroptosis) to cell proliferation, migration, and differentiation; the implementation of these cellular responses reflects the formation of different molecular complexes following receptor activation [START_REF] Cabal-Hierro | Signal transduction by tumor necrosis factor receptors[END_REF]. Binding of TNF to TNFR1 causes formation of two consecutive complexes, resulting in the divergence of their kinetic and spatial distributions. Whereas the plasma membrane complex (complex I) elicits a non-apoptotic signaling pathway, a second, internalized complex (complex II or DISC) triggers cell death [START_REF] Micheau | Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[END_REF]. In the presence of TNF-α, the adaptor protein TRADD interacts with TNFR1 and recruits other proteins involved in the signaling of the receptor, such as TRAF2, cIAP1, cIAP2, and RIP1, to form complex I. At the plasma membrane, this complex activates the NF-κB signaling pathway, which in turn promotes transcription of anti-apoptotic genes such as cIAP-1, cIAP-2, and c-FLIP [START_REF] Wang | NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation[END_REF]. The linear ubiquitin chain assembly complex (LUBAC) is also recruited to complex I via cIAPgenerated ubiquitin chains [START_REF] Haas | Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNFmediated gene induction[END_REF]. The LUBAC complex consists of HOIL-1, HOIP, and sharpin; HOIL-1 and HOIP add a linear ubiquitin chain by catalyzing the head-to-tail ligation of ubiquitin [START_REF] Kirisako | A ubiquitin ligase complex assembles linear polyubiquitin chains[END_REF] to RIP1 and NEMO (IKKγ) in complex I [START_REF] Gerlach | Linear ubiquitination prevents inflammation and regulates immune signalling[END_REF], thereby activating NF-κB. TNF-α-induced caspase activation is mediated by a second intracellular complex, known as complex II, which is formed when complex I dissociates from the receptor along with FADD and caspase-8 recruitment [START_REF] Micheau | Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[END_REF]. NF-κB activation leads to c-FLIP overexpression, preventing formation of complex II. Contrariwise, when NF-κB activation is blocked, the short-lived c-FLIP protein is depleted [START_REF] Poukkula | Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail[END_REF], and cells undergo programmed death [START_REF] Micheau | Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[END_REF]. In this context, RIP1 is deubiquitinated by enzymes such as Cezanne [START_REF] Enesa | NF-kappaB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling[END_REF] and CYLD [START_REF] Green | RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts[END_REF]. In addition, the complex composed of TRADD and RIP1 moves to the cytosol to form complex II. FADD is recruited to TRADD by the DD-DD interaction and binds caspase-8 [START_REF] Micheau | Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[END_REF]. Notably, when caspase-8 activity is inhibited or its expression is extinguished, DISC is unable to trigger the apoptotic signaling pathway, but TNFR1 or CD95 stimulation leads to the activation of another cell death signal, necroptosis [START_REF] Holler | Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule[END_REF][START_REF] Vercammen | Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor[END_REF]. To prevent the induction of the necroptotic signal, caspase-8 cleaves and inactivates RIP1 and RIP3 [START_REF] Cho | Phosphorylationdriven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation[END_REF]. The fine-tuned control of necroptosis by members of the apoptotic signaling pathway has been elegantly confirmed by experiments showing that the embryonic lethality of mice harboring single KO of caspase-8 or FADD can be rescued by an additional KO of the RIP3 gene [START_REF] Kaiser | RIP3 mediates the embryonic lethality of caspase-8-deficient mice[END_REF][START_REF] Oberst | Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis[END_REF][START_REF] Welz | FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation[END_REF].

TNF/TNFR: a gold mine for therapeutic tools

Many studies of TNF-α have demonstrated its pivotal role in fueling inflammation, a multistep process that promotes autoimmunity (e.g., rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, and refractory asthma) and cancer. Many TNF inhibitors, such as neutralizing monoclonal antibodies (mAbs) (e.g., infliximab, adalimumab, and golimumab) have been developed to treat these chronic inflammatory disorders, demonstrating that altering ligand/receptor interactions with neutralizing mAbs is an invaluable strategy for treating certain chronic inflammatory disorders. Other TNF-α antagonists, such as etanercept, a TNFR2-immunoglobulin Fc fusion protein, can improve the clinical course of rheumatoid arthritis [START_REF] Feldmann | TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases[END_REF].

A large and growing body of evidence has contributed to elucidation of the molecular mechanisms underlying induction of apoptotic and non-apoptotic signaling pathways by TNFR1, and also provided clues regarding how the receptor can switch from one signal to the other. However, the mechanistic links involved in implementation of non-apoptotic signaling pathways by CD95 remain elusive. However, several recent findings have revealed its proinflammatory effects [START_REF] Desbarats | Fas engagement induces neurite growth through ERK activation and p35 upregulation[END_REF][START_REF] Desbarats | Fas engagement accelerates liver regeneration after partial hepatectomy[END_REF][START_REF] Letellier | CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site[END_REF][START_REF] O' Reilly | Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[END_REF][START_REF] Ruan | A novel juxtamembrane domain in tumor necrosis factor receptor superfamily molecules activates Rac1 and controls neurite growth[END_REF][START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Tauzin | CD95-mediated cell signaling in cancer: mutations and post-translational modulations[END_REF].

CD95: a death receptor?

In 1989, identification of the mAb APO-1 by Peter Krammer et al. revealed the existence of a 52 kDa protein whose aggregation resulted in transmission of an apoptotic signal in cancer cells [START_REF] Trauth | Monoclonal antibody-mediated tumor regression by induction of apoptosis[END_REF]. This receptor was cloned in 1991 by Nagata and colleagues, who named it Fas (CD95 or APO-1) [START_REF] Itoh | The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis[END_REF]. Its ligand, CD95L, was cloned in 1993 by the same group, and was found to be primarily expressed at the surface of activated T lymphocytes [START_REF] Suda | Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family[END_REF] and natural killer (NK) cells [START_REF] Oshimi | Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells[END_REF]; however, its expression was also detected in tissues in which the presence of acute or chronic inflammation is highly undesirable, including the eyes [START_REF] Griffith | Fas ligand-induced apoptosis as a mechanism of immune privilege[END_REF] and testes [START_REF] Bellgrau | A role for CD95 ligand in preventing graft rejection[END_REF].

Structure /function

The CD95 gene (APT1) consists of nine exons encoding a type I transmembrane protein harboring three CRDs, with exon 6 encoding the transmembrane domain [START_REF] Behrmann | Structure of the human APO-1 gene[END_REF] (Figure 1). Under denaturing conditions, CD95 migrates as a 40-50 kDa protein on SDS-PAGE. Similar to the TNF receptor [START_REF] Chan | A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling[END_REF], CD95 is pre-associated at the plasma membrane as a homotrimer, and this quaternary structure is mandatory for transmission of apoptotic signals in the presence of CD95L [START_REF] Papoff | Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF]. Homotrimerization of CD95 occurs mainly through homotypic interactions involving the CD95-CRD1 domain [START_REF] Edmond | Precise Mapping of the CD95 Pre-Ligand Assembly Domain[END_REF][START_REF] Papoff | Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF]. Binding of CD95L or agonistic anti-CD95 mAbs to CD95 alters the receptor"s conformation and the extent to which the receptor is multimerized at the plasma membrane [START_REF] Lang | Signaling active CD95 receptor molecules trigger co-translocation of inactive CD95 molecules into lipid rafts[END_REF]. The intracellular region of CD95 encompasses an 80 amino-acid stretch designated as the DD (Figure 1), which consists of six anti-parallel α-helices [START_REF] Huang | NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain[END_REF]. Upon addition of CD95L, CD95 undergoes conformational modification of the DD, inducing a shift of helix 6 and fusion with helix 5, promoting both oligomerization of the receptor and recruitment of the adaptor protein FADD [START_REF] Scott | The Fas-FADD death domain complex structure unravels signalling by receptor clustering[END_REF]. One consequence of the opening of the globular structure of CD95 is that the receptor becomes connected through this bridge, which increases the extent of its homo-aggregation. This long helix allows stabilization of the complex by recruitment of FADD. The CD95-DD:FADD-DD crystal structure provides several insights into the formation of the large CD95 clusters observed by imaging or biochemical methods in cells stimulated with CD95L. In addition, the structure also confirms that alteration in the conformation of CD95 plays an instrumental role in signal induction [START_REF] Scott | The Fas-FADD death domain complex structure unravels signalling by receptor clustering[END_REF]. However, the idea of an elongated C-terminal α-helix favoring the cis-dimerization of CD95-DD was challenged by Driscoll et al., who did not observe the fusion of the last two helices at a more neutral pH (pH 6.2), in contrast to the acidic condition (pH 4) used in the initial study in which Scott et al. resolved the CD95-DD:FADD-DD structure [START_REF] Scott | The Fas-FADD death domain complex structure unravels signalling by receptor clustering[END_REF]. At pH 6.2, association of CD95 predominantly interacted with FADD in a 5:5 complex, which arose via a polymerization mechanism involving three types of asymmetric interactions, but without major alteration of the DD globular structure [START_REF] Esposito | Solution NMR investigation of the CD95/FADD homotypic death domain complex suggests lack of engagement of the CD95 C terminus[END_REF][START_REF] Wang | The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations[END_REF]. It is likely that the low-pH condition used by Scott et al. altered the conformation of CD95, resulting in the formation of nonphysiological CD95:FADD oligomers [START_REF] Scott | The Fas-FADD death domain complex structure unravels signalling by receptor clustering[END_REF]. Nonetheless, we cannot rule out the possibility that a local decrease in intracellular pH affects the initial steps of the CD95 signaling pathway in vivo, e.g., by promoting the opening of the CD95-DD and eventually contributing to formation of a complex that elicits a sequence of events distinct from that occurring at physiologic pH.

Once docked on CD95-DD, FADD self-associates [START_REF] Muppidi | Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis[END_REF], and binds procaspases-8 and -10, which are auto-processed and released in the cytosol as active caspases. Once activated, these caspases cleave many substrates, ultimately leading to the execution of the apoptotic program and cell death. The complex CD95/FADD/caspase-8/-10 is called DISC (Figure 2) [START_REF] Kischkel | Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor[END_REF]. Due to the importance of DISC formation to cell fate, it is not surprising that numerous cellular and viral proteins have evolved to hamper the formation of this structure: for example, both FLIP [START_REF] Irmler | Inhibition of death receptor signals by cellular FLIP[END_REF][START_REF] Thome | Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors[END_REF] and PED/PEA-15 [START_REF] Condorelli | PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis[END_REF] interfere with the recruitment of caspase-8/-10 (Figure 2).

Type I /II signaling pathways

Following the discovery of CD95 and the elucidation of the initial steps in its signaling pathway, Peter and colleagues reported that cells can be divided in to two groups with regard to the kinetics with which they respond to CD95-mediated apoptotic signals, the magnitude of DISC formation, and the role played by the mitochondrion in this pathway [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. DISC formation occurs rapidly and efficiently in type I cells, resulting in the release of a large amount of activated caspase-8 in the cytosol, whereas type II cells have difficulty forming this complex, and the amount of active caspase-8 is insufficient to directly activate the effector caspases-3 and -7 [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF]. Nonetheless, type II cells experience cell death upon CD95 engagement and are even more sensitive to CD95-mediated apoptotic signals than type I cells [START_REF] Scaffidi | Two CD95 (APO-1/Fas) signaling pathways[END_REF][START_REF] Algeciras-Schimnich | Two CD95 tumor classes with different sensitivities to antitumor drugs[END_REF][START_REF] Chaigne-Delalande | CD95 engagement mediates actin-independent and -dependent apoptotic signals[END_REF]. This discrepancy can be partly explained by the fact that the low level of activated caspase-8 in type II cells is sufficient to cleave BID, a BH3-only protein, which constitutes the molecular link between caspase-8 activation and the apoptotic activity of mitochondria. Indeed, after cleavage by caspase-8, truncated BID (tBID) translocates to mitochondria, where it triggers the release of pro-apoptotic factors (Figure 2) [START_REF] Yin | Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways[END_REF][START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF]. Although CD95 stimulation activates the mitochondrion-dependent apoptotic signal in type I and type II cells, it seems that only type II cells are addicted to this signal, because they contain higher levels of the caspase-3 inhibitor XIAP than type I cells [START_REF] Jost | XIAP discriminates between type I and type II FAS-induced apoptosis[END_REF]. Several members of the inhibitor of apoptosis (IAP) protein family, XIAP, c-IAP1, and c-IAP2 inhibit caspase-3, -7 [START_REF] Roy | The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases[END_REF][START_REF] Deveraux | X-linked IAP is a direct inhibitor of celldeath proteases[END_REF], and pro-caspase-9 [START_REF] Deveraux | IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases[END_REF] activity by direct binding, thereby preventing access to substrates. Furthermore, XIAP can function as an E3 ligase; this activity is involved in the ubiquitination of active caspase-3 and its subsequent degradation by the proteasome [START_REF] Suzuki | Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death[END_REF]. To detach XIAP from caspase-3 and restore the apoptotic signal, cells require the release of SMAC/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) by the mitochondrion [START_REF] Du | Smac, a mitochondrial protein that promotes cytochrome cdependent caspase activation by eliminating IAP inhibition[END_REF][START_REF] Sun | Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein[END_REF], explaining why type II cells are more "addicted" to this organelle than type I cells (Figure 2).

To summarize, DISC formation and IAP amount are two cellular markers that allow a clear discrimination between type I and type II cells. Even though IAP overexpression can account for the mitochondrial dependency observed in type II cells, it remains unclear why DISC formation is hampered in type II cells and/or augmented in their type I counterparts.

Recently, high activity of the lipid kinase phosphoinositide 3-kinase (PI3K) or downregulation of its neutralizing phosphatase, phosphatase and tensin homologue on chromosome 10 (PTEN), were observed in type II cells, whereas this signal is blocked in type I cell lines [START_REF] Beneteau | Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway[END_REF][START_REF] Peacock | PTEN loss promotes mitochondrially dependent type II Fas-induced apoptosis via PEA-15[END_REF]. The PI3K signaling pathway prevents the aggregation of CD95 [START_REF] Varadhachary | Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95[END_REF], probably by retaining the receptor outside of lipid rafts [START_REF] Beneteau | Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway[END_REF][START_REF] Pizon | Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis[END_REF]. PEA-15, also known as PED, is a protein containing a death effector domain (DED) that inhibits the CD95 and TNFR1 apoptotic signals (Figure 2) [START_REF] Condorelli | PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis[END_REF]. Activation of PI3K and its downstream effector, serine-threonine kinase Akt, leads to phosphorylation of PEA-15 at serine 116 [START_REF] Beneteau | Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway[END_REF][START_REF] Pizon | Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis[END_REF]; this post-translational modification promotes its interaction with FADD, ultimately inhibiting DISC formation [START_REF] Renganathan | Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD[END_REF][START_REF] Trencia | Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action[END_REF].

Notably, the existence of type I and type II cells is not only an in vitro observation, but has been identified physiologically in the human body. CD95-mediated apoptotic signals cannot be altered in thymocytes or activated T cells expressing a Bcl-2 transgene, consistent with the type I nature of these cells [START_REF] Strasser | Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis[END_REF], whereas hepatocytes expressing the same transgene resist CD95-induced apoptosis and thus behave as type II cells [START_REF] Lacronique | Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice[END_REF][START_REF] Rodriguez | A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection[END_REF].

What can we learn from CD95 mutations?

Human.

Germinal mutations in APT1 have been reported in patients with autoimmune lymphoproliferative syndrome type Ia (ALPS, also called Canale-Smith syndrome) [START_REF] Drappa | Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity[END_REF][START_REF] Fisher | Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome[END_REF][START_REF] Rieux-Laucat | Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity[END_REF].

ALPS patients exhibit chronic lymphadenopathy and splenomegaly and expanded populations of double-negative α/β T lymphocytes CD3 + CD4 -CD8 -), and often develop autoimmunity [START_REF] Drappa | Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity[END_REF][START_REF] Fisher | Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome[END_REF][START_REF] Canale | Chronic lymphadenopathy simulating malignant lymphoma[END_REF][START_REF] Rieux-Laucat | Lymphoproliferative syndrome with autoimmunity: A possible genetic basis for dominant expression of the clinical manifestations[END_REF]. In agreement with the notion that CD95 behaves as a tumor suppressor, ALPS patients display an increased risk of Hodgkin and non-Hodgkin lymphoma [START_REF] Straus | The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis[END_REF]. The predominance of post-germinal center (GC) lymphomas in patients with either germ line or somatic CD95 mutations can be explained by the fact that, inside germinal centers of the secondary lymphoid follicles, the CD95 signal plays a pivotal role in the deletion of selfreactive maturing B lymphocytes [START_REF] Hennino | FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis[END_REF]; in addition, APT1 belongs to a set of rare genes (i.e., PIM1, c-myc, PAX5, RhoH/TTF, and Bcl-6) subject to somatic hypermutation [START_REF] Montesinos-Rongen | Primary diffuse large B-cell lymphomas of the central nervous system are targeted by aberrant somatic hypermutation[END_REF][START_REF] Muschen | The origin of CD95-gene mutations in B-cell lymphoma[END_REF], which may affect its biological function. In addition to post-GC lymphomas, tumors of various histological origins have been shown to exhibit significant numbers of mutations in the CD95 gene (reviewed in [START_REF] Tauzin | CD95-mediated cell signaling in cancer: mutations and post-translational modulations[END_REF]). Extensive analysis of CD95 mutations and their distribution in APT1 reveals that, with some exceptions, most are gathered in exons 8 and 9, which encode the CD95 intracellular region (Figure 3) [START_REF] Peter | Does CD95 have tumor promoting activities?[END_REF]. Remarkably, most of these mutations are heterozygous, mainly localized in CD95-DD, and lead to inhibition of the CD95-mediated apoptotic signal. Indeed, in agreement with the notion that CD95 is expressed at the plasma membrane as a pre-associated homotrimer [START_REF] Papoff | Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor[END_REF][START_REF] Siegel | Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations[END_REF], formation of heterocomplexes containing wild-type and mutated CD95 prevents FADD recruitment and dominantly abrogates the initiation of the apoptotic signal.

Extensive analysis of the positions of CD95 mutations described in the literature has revealed mutation "hot spots" in the CD95 sequence (Figure 3). Among these hot spots, arginine 234, aspartic acid 244, and valine 251 account for a considerable proportion of the documented CD95 mutations. Indeed, among the 189 mutations annotated in the 335 amino acids of CD95, 30 (~16%) are localized in one of these three amino acids (Figure 3). The pivotal roles played by these amino acids in stabilization or formation of intra-and inter-bridges between CD95 and FADD may explain the existence of these hot spots. For instance, both R234 and D244 contribute to homotypic aggregation of the receptor and FADD recruitment [START_REF] Huang | NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain[END_REF].

Nevertheless, the observation of death-domain hot spots contradicts the study of Scott and colleagues, who demonstrated that the region of CD95-DD that interacts with the FADD-DD extends over a dispersed surface and is mediated by a large number of low-affinity interactions [START_REF] Scott | The Fas-FADD death domain complex structure unravels signalling by receptor clustering[END_REF].

Most ALPS type Ia patients affected by malignancies do not undergo loss of heterozygosity (LOH), leading some authors to hypothesize that preservation of a wild-type allele may contribute to carcinogenesis [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF][START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF]. In the same vein, expression of a unique mutated CD95 allele blocks the induction of apoptotic signals, but fails to block non-apoptotic signals such as NF-κB and MAPK [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF][START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF], whose induction promotes invasiveness in tumor cells [START_REF] Peter | Does CD95 have tumor promoting activities?[END_REF][START_REF] Barnhart | CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells[END_REF]. In addition, mutations in the intracellular CD95-DD result in more highly penetrant ALPS phenotype features in mutation-bearing relatives than mutations in the extracellular domain. These results suggest that unlike DD mutations, CD95 mutations localized outside the DD somehow block apoptotic signaling but fail to promote non-apoptotic pathways that may contribute to disease aggressiveness.

Mouse models.

Three mouse models exist in which either CD95L affinity for CD95 is reduced (due to the germline mutation F273L in CD95L, called generalized lymphoproliferative disease [gld], which decreases CD95L binding to CD95) [START_REF] Kimura | Autoimmunity in mice bearing lprcg: a novel mutant gene[END_REF][START_REF] Takahashi | Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[END_REF]), the level of CD95 expression is downregulated (due to an insertion of a retrotransposon in intron 2 of the receptor gene, these mice are called lymphoproliferation [Lpr] [START_REF] Watanabe-Fukunaga | Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis[END_REF][START_REF] Adachi | Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice[END_REF][START_REF] Chu | The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn[END_REF]), or DISC formation is hampered (due to a spontaneous mutation inside the CD95 DD at position 238, specifically, replacement of the valine 238 with asparagine; these mice are called lpr cg for lpr gene complementing gld [START_REF] Matsuzawa | A new allele of the lpr locus, lprcg, that complements the gld gene in induction of lymphadenopathy in the mouse[END_REF]).

These mice have provided valuable insights into the pivotal role played by CD95 and CD95L in immune surveillance and immune tolerance [START_REF] Strasser | The many roles of FAS receptor signaling in the immune system[END_REF]. In an attempt to simplify, some authors associated the phenotypes observed in these lpr, lpr cg or gld mice with the complete loss of CD95 or CD95L [START_REF] Heinzelmann | Irradiation-induced pneumonitis mediated by the CD95/CD95-ligand system[END_REF]. However, conclusions must be drawn with caution, due to subtle differences between the phenotypes of spontaneous mouse models and genetically engineered mice. Indeed, in Lpr mice, insertion of an early transposon in intron 2 of CD95 causes premature termination of the CD95 transcript [START_REF] Adachi | Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice[END_REF], which is leaky; consequently, CD95 mRNA and protein can be detected in mice homozygous for the spontaneous mutation [START_REF] Kobayashi | Transcriptional repression and differential splicing of Fas mRNA by early transposon (ETn) insertion in autoimmune lpr mice[END_REF][START_REF] Wu | Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene[END_REF]. Also, the DD mutation in Lpr cg mice reduces FADD recruitment but does not abrogate it [START_REF] Martin | Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia[END_REF]. Furthermore, CD95 can still interact with CD95L harboring the gld mutation, albeit somewhat more weakly than wild-type CD95L [START_REF] Orlinick | Separate domains of the human fas ligand dictate selfassociation and receptor binding[END_REF]. Finally, lpr, lpr cg , and gld mice overexpress CD95L relative to their wild-type counterparts [START_REF] Chu | Massive upregulation of the Fas ligand in lpr and gld mice: implications for Fas regulation and the graftversus-host disease-like wasting syndrome[END_REF]. Using T lymphocytes from ALPS type Ia patients or Lpr mice, we confirmed that far less intact CD95 is required to activate NF-κB than to induce apoptosis; therefore, although a single wild-type allele cannot restore cell death induction in these cells, it is sufficient to transduce NF-κB and MAPK cues [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF][START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF]. Overall, these observations support the idea that the biological roles ascribed to the CD95/CD95L pair, based on the analysis of these patients and mouse models, may correspond to the additive effects of the receptor"s inability to induce cell death and its tendency to implement non-apoptotic signals.

A recent study elegantly showed that elimination of the remaining allele in cancer cells leads to the induction of an unconventional cell death program called "death induced by CD95R/L elimination" (DICE) [START_REF] Hadji | Death Induced by CD95 or CD95 Ligand Elimination[END_REF].

These findings highlight the fact that distinct activation thresholds exist in the process of CD95 engagement. Although complete loss of CD95 expression in cancer cells leads to cell death, one wild-type allele (low activation threshold) is sufficient to elicit non-apoptotic signaling pathways, and the second allele (high activation threshold) is required to implement the canonical apoptotic signal [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF]115]. However, this rule suffers from an exception: metalloprotease-cleaved CD95L implements non-apoptotic signals in cells expressing two wild-type alleles of CD95 [START_REF] O' Reilly | Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[END_REF][START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF][START_REF] Malleter | CD95L cell surface cleavage triggers a prometastatic signaling pathway in triple-negative breast cancer[END_REF] (further discussed in 3.6.2). In summary, because the characterization of CD95/CD95L biological roles has been carried out mainly by considering the default of apoptosis in ALPS type Ia patients and mouse models, we believe it is important to carefully reconsider these conclusions by integrating the notion that exposure of these cells to CD95L will also lead to a chronic activation of non-apoptotic signaling pathways [START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF]. To better appreciate the complexity of the pathophysiological roles of CD95 and its ligand, it is therefore more appropriate to use conditional and tissue-specific CD95 and CD95L KO mice.

Regulation of the Initial Steps of CD95-mediated Signaling

Lipid rafts

In addition to CD95 down-regulation or expression of a mutated allele of the receptor, alteration of the plasma membrane distribution of CD95 represents an additional mechanism by which tumor cells could develop resistance to CD95L-expressing immune cells. The plasma membrane is a heterogeneous lipid bilayer comprising compacted or liquid-ordered domains, called microdomains, lipid rafts, or detergent-resistant microdomains (DRMs).

These domains, which are enriched in ceramides, have been described as floating in a more fluid or liquid-disordered two-dimensional (2-D) lipid bilayer [START_REF] Grassme | CD95 signaling via ceramide-rich membrane rafts[END_REF]. A series of elegant experiments showed that although CD95 is mostly excluded from lipid rafts in activated T lymphocytes, TCR-dependent re-activation of these cells leads to rapid distribution of the death receptor into lipid rafts [START_REF] Muppidi | Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death[END_REF]. This CD95 compartmentalization contributes to a reduction in the apoptotic threshold, leading to clonotypic elimination of activated T lymphocytes through activation of the CD95-mediated apoptotic signal [START_REF] Muppidi | Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death[END_REF]. Similarly, the reorganization of CD95 into DRMs can occur independently of ligand upon addition of certain chemotherapeutic drugs (e.g., rituximab [START_REF] Stel | Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis[END_REF], resveratrol [START_REF] Delmas | Resveratrol-induced apoptosis is associated with Fas redistribution in the rafts and the formation of a death-inducing signaling complex in colon cancer cells[END_REF][START_REF] Delmas | Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells[END_REF], edelfosine [START_REF] Beneteau | Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway[END_REF][START_REF] Gajate | Intracellular triggering of Fas aggregation and recruitment of apoptotic molecules into Fas-enriched rafts in selective tumor cell apoptosis[END_REF][START_REF] Gajate | Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts[END_REF], aplidin [START_REF] Gajate | Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy[END_REF], perifosine [START_REF] Gajate | Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts[END_REF], and cisplatin [START_REF] Lacour | Cisplatin-induced CD95 redistribution into membrane lipid rafts of HT29 human colon cancer cells[END_REF]). The molecular cascades underlying this process remain elusive. Nevertheless, a growing body of evidence leads us to postulate that alteration of intracellular signaling pathway(s), such as the aforementioned PI3K signal [START_REF] Beneteau | Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway[END_REF][START_REF] Pizon | Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis[END_REF], may change biophysical properties of the plasma membrane, such as membrane fluidity, which in turn may facilitate CD95 clustering into large lipid raft-enriched platforms, favoring DISC formation and induction of the apoptotic program [START_REF] Pizon | Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis[END_REF].

Post-translational modifications

Accumulation of CD95 mutations is not the only mechanism by which malignant cells inhibit the extrinsic signaling pathway. Post-translational modifications in the intracellular tail of CD95, such as reversible oxidation or covalent attachment of palmitic acid, alter the plasma membrane distribution of CD95 and thereby its downstream signaling. For instance, Sglutathionylation of mouse CD95 at cysteine 294 promotes clustering of CD95 and its distribution into lipid rafts [START_REF] Anathy | Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas[END_REF]. This amino acid is conserved in the human CD95 sequence and corresponds to cysteine 304 (or C288 when the 16 amino-acid signal peptide is taken into consideration [START_REF] Itoh | The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis[END_REF][START_REF] Oehm | Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen[END_REF]). Interestingly, Janssen-Heininger and colleagues emphasize that death receptor gluthationylation occurs downstream of activation of caspase-8 and -3; the catalytic activities of these caspases damage the thiol transferase glutaredoxin 1 (Grx1) [START_REF] Anathy | Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas[END_REF]. One consequence of Grx1 inactivation is accumulation of glutathionylated CD95, which clusters into lipid rafts, thereby sensitizing cells to CD95-mediated apoptotic signals. Based on these findings, caspase-8 activation occurs prior to aggregation of CD95 and redistribution into lipid rafts, both of which are required to form the DISC and subsequently activate larger amounts of caspase-8. In agreement with these observations, activation of caspase-8 occurs in a two-step process. First, a small amount of activated caspase-8 (<1%) is generated immediately when CD95L interacts with CD95, resulting in acid sphingomyelinase (ASM) activation, ceramide production, and CD95 clustering; these in turn promote DISC formation and the burst of caspase-8 processing that is essential for implementation of the apoptotic program [START_REF] Grassme | Ceramide-mediated clustering is required for CD95-DISC formation[END_REF]. S-glutathionylation consists of a bond between a reactive Cys-thiol and reduced glutathione (GSH), a tripeptide consisting of glycine, cysteine, and glutamate. Attachment of this group to a protein alters its structure and function in a manner similar to the addition of a phosphate [START_REF] Chen | S-glutathionylation uncouples eNOS and regulates its cellular and vascular function[END_REF]. S-glutathionylation is not the only post-translational modification of a cysteine in CD95: S-nitrosylation of cysteine 199 (corresponding to C183 after subtraction of signal peptide sequence) and 304 (C288) in colon and breast tumor cells also promotes the redistribution of CD95 into DRMs, formation of the DISC, and the transmission of the apoptotic signal [START_REF] Leon-Bollotte | S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells[END_REF].

Two reports have demonstrated that covalent coupling of a 16-carbon fatty acid (palmitic acid) to cysteine 199 (C183) elicits the redistribution of CD95 into DRMs, the formation of SDS-stable CD95 microaggregates resistant to denaturing and reducing treatments, and internalization of the receptor [START_REF] Chakrabandhu | Palmitoylation is required for efficient Fas cell death signaling[END_REF][START_REF] Feig | Palmitoylation of CD95 facilitates formation of SDSstable receptor aggregates that initiate apoptosis signaling[END_REF]. Although the order of these events remains to be precisely determined, it is clear that these molecular steps play a critical role in the implementation of apoptotic signals.

As with S-nitrosylation, both the aforementioned S-glutathionylation at C304 (C288) and palmitoylation at C199 (C183) promote the partition of CD95 into lipid rafts and augment the subsequent apoptotic signal. Further investigation is required to determine whether these posttranslational modifications are redundant, and occur simultaneously in dying cells, or instead are elicited in a cell-specific and/or in a microenvironment-specific manner. Understanding the molecular mechanisms controlling these post-translational modifications would be of great value in efforts to identify the mechanisms by which tumor cells block them, leading to resistance to the extrinsic signaling pathway.

Soon after CD95 was cloned, several groups investigated phosphorylation of this protein on serine/threonine and tyrosine and explored its biological role. Although serine/threonine phosphorylation may participate in the implementation of the CD95 signal, these authors mainly focused on the role of tyrosine phosphorylation in the cell death pathway.

Phosphorylation can occur on two tyrosines located in the first (Y232, corresponding to Y216 starting from the first amino acid after the signal peptide) and fifth (Y291/Y275) α-helices of CD95-DD [START_REF] Gradl | The CD95 (Fas/APO-1) receptor is phosphorylated in vitro and in vivo and constitutively associates with several cellular proteins[END_REF]. Y275 is located within a conserved YXXL motif reminiscent of the conserved "I/VxYxxL" motif, termed the immunoreceptor tyrosine-based inhibitory motif (ITIM), which is responsible for the recruitment and activation of inhibitory phosphatases [START_REF] Daigle | Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils[END_REF]. By recruiting the src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), Y275 phosphorylation promotes CD95-mediated cell death in T cells [START_REF] Su | Defective expression of hematopoietic cell protein tyrosine phosphatase (HCP) in lymphoid cells blocks Fas-mediated apoptosis[END_REF] and counteracts the GM-CSF-driven pro-survival signals in neutrophils [START_REF] Daigle | Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils[END_REF]. Notably, this Y 275 DTL cytoplasmic domain is also a putative consensus YXXF sequence for AP-2 binding [START_REF] Ohno | Interaction of tyrosine-based sorting signals with clathrin-associated proteins[END_REF], which is instrumental in CD95 internalization (see below and [START_REF] Lee | The role of receptor internalization in CD95 signaling[END_REF]). Consistent with this, replacement of Y275 by a phenylalanine inhibits CD95 internalization and thereby blocks the induction of apoptosis, but does not affect non-apoptotic responses [START_REF] Lee | The role of receptor internalization in CD95 signaling[END_REF]. In addition, tyrosine phosphorylation of CD95 promotes the recruitment of the src kinases Fyn and Lyn through their SH2 domains, thereby promoting cell death [START_REF] Atkinson | A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT[END_REF][START_REF] Wang | Altered antigen receptor signaling and impaired Fasmediated apoptosis of B cells in Lyn-deficient mice[END_REF]. Accordingly, it is tempting to speculate that Y275 phosphorylation may guide the receptor through the induction of the apoptotic signal at the expense of non-apoptotic pathways. These data raise some questions about the identity of the tyrosine kinase involved in Y275 phosphorylation, the order of the molecular events leading to phosphatase and src kinase recruitment, and their respective roles in the CD95 signaling pathway.

CD95 internalization

A powerful magnetic method for isolating receptor-containing endocytic vesicles was used to show that CD95 promptly associates with endosomal and lysosomal markers upon incubation of cells with agonistic anti-CD95 mAb [START_REF] Lee | The role of receptor internalization in CD95 signaling[END_REF]. In addition, expression of a CD95 mutant in which the DD-located tyrosine 291 (Y275) is changed to phenylalanine does not seem to alter the capacity to bind FADD, but instead compromises CD95L-mediated CD95 internalization occurring through an AP-2/clathrin-driven endocytic pathway [START_REF] Lee | The role of receptor internalization in CD95 signaling[END_REF]. More strikingly, expression of the internalization-defective CD95 mutant Y291F abrogates the transmission of apoptotic signals, but fails to block the non-apoptotic signaling pathways (i.e., NF-κB and ERK); indeed, the mutant even promotes these pathways (Figure 3). These findings highlight the presence of a region in the DD, which interacts with AP2 and promotes a clathrindependent endocytic pathway in a FADD-independent manner. The role of palmitoylation in the AP2/clathrin-driven internalization of CD95 remains to be elucidated.

Ca 2+ response

A recent study demonstrated that CD95 engagement evokes rapid and transient Ca 2+ signaling, which stimulates the recruitment of protein kinase C-β2 (PKC-β2) from the cytosol to the DISC [START_REF] Khadra | CD95 triggers Orai1mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) beta2, and prevents death-inducing signaling complex formation[END_REF]. This kinase transiently halts DISC formation, providing a checkpoint before the irreversible commitment to cell death [START_REF] Penna | The CD95 signaling pathway: To not die and fly[END_REF]. These findings raised two important questions: what are the Ca 2+ -dependent molecular mechanisms transiently inhibiting DISC formation, and do tumor cells use this signal to escape the immune response and/or resist chemotherapy?

Programmed necrosis, also known as necroptosis

In 1998, inhibition of caspase activity was shown to sensitize the fibroblastic L929 cell line to TNF-mediated necrotic cell death [START_REF] Vercammen | Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor[END_REF]. With respect to the CD95 signal, Tschopp et al.

showed that FADD and RIP1 participate in the implementation of a non-apoptotic signaling pathway that leads to a necrotic morphology associated with loss of plasma membrane integrity but not with chromatin condensation [START_REF] Holler | Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule[END_REF]. Of note, BID cleavage was not observed in the context of this necrotic signal. Whereas FADD plays a crucial role in both apoptotic and necrotic pathways, RIP1 recruitment to CD95 occurs independently of this adaptor protein. Indeed, yeast two-hybrid experiments showed that RIP1 can bind directly to the CD95 DD, whereas this interaction is lost when a bait corresponding to mutated CD95-DD (replacement of Val 238 to Asn) is used [START_REF] Stanger | RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death[END_REF]. In addition, RIP3 (RIPK3, a member of the RIP kinase family) is an indispensable factor for the induction of the necrotic signaling pathway [START_REF] Yin | Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways[END_REF][START_REF] Yin | Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis[END_REF][START_REF] Jost | XIAP discriminates between type I and type II FAS-induced apoptosis[END_REF]. Identification of necrostatin, a chemical inhibitor of necroptosis [START_REF] Degterev | Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury[END_REF] that specifically inhibits RIP1 kinase activity [START_REF] Degterev | Identification of RIP1 kinase as a specific cellular target of necrostatins[END_REF], has accelerated the pace of discovery in this field of cell death. The apoptosis and necroptosis pathways interact: for instance, caspase-8 exerts a potent inhibitory effect on CD95 and TNFR1-mediated necroptosis [START_REF] Lee | The roles of FADD in extrinsic apoptosis and necroptosis[END_REF] through its ability to process and inactivate RIP1 and RIP3 [START_REF] Lin | Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis[END_REF][START_REF] Feng | Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain[END_REF]. At least in the case of TNF signaling, the necrotic signal relies on the activity of CYLD, a deubiquitinating enzyme that is also cleaved and inactivated by caspase-8 [START_REF] O'donnell | Caspase 8 inhibits programmed necrosis by processing CYLD[END_REF].

Overall, these findings suggest that the apoptotic machinery controls the necrotic pathway. This concept was recently confirmed by the results of in vivo double-KO experiments [START_REF] Kaiser | RIP3 mediates the embryonic lethality of caspase-8-deficient mice[END_REF][START_REF] Oberst | Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis[END_REF][START_REF] Welz | FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation[END_REF][START_REF] Peter | Programmed cell death: Apoptosis meets necrosis[END_REF]. Indeed, FADD and caspase-8 can be considered to be a prosurvival factor, mainly because both of these two "apoptotic" molecules inhibit the RIP1/RIP3-dependent necrotic signal; consequently, their loss unleashes the necroptotic program and leads to embryonic lethality. However, most studies on necroptosis have focused on the TNF signaling pathway, whereas the mechanism by which CD95 elicits this cell death pathway, as well as how this receptor switches between non-apoptotic, apoptotic, and necroptotic signals, remains to be elucidated. Importantly, the impact of each type of cell death on antigen presentation, and on the efficiency of immune response after elimination of infected or transformed cells, also remains unclear.

CD95L, an

Inflammatory/Oncogenic Cytokine?

A ligand that creates immune privilege

The transmembrane CD95L (CD178/FasL) is present on the surface of activated lymphocytes [START_REF] Takahashi | Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand[END_REF] and NK cells [START_REF] Montel | Fas involvement in cytotoxicity mediated by human NK cells[END_REF], where it orchestrates the elimination of transformed and infected cells. In addition, CD95L is expressed on the surface of neurons [START_REF] Saas | Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?[END_REF], corneal epithelia and endothelia [START_REF] Griffith | Fas ligand-induced apoptosis as a mechanism of immune privilege[END_REF][START_REF] Stuart | CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival[END_REF], and Sertoli cells [START_REF] Bellgrau | A role for CD95 ligand in preventing graft rejection[END_REF], where it acts to prevent the infiltration of immune cells and thereby inhibit the spread of inflammation in these sensitive organs (i.e., brain, eyes, and testis, respectively), commonly referred to as "immune-privileged" sites. The initial description of physiological immune privilege was followed by an understanding of tumormediated immune privilege: two groups independently reported that the ectopic expression of CD95L by malignant cells participated in the elimination of infiltrating T lymphocytes, and could thus play a role in the establishment of a tumor site to which immune cells are denied access [START_REF] Hahne | Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape[END_REF][START_REF] O'connell | The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand[END_REF]. However, these observations are controversial, because ectopic expression of CD95L in allogenic transplant of β-islets [START_REF] Allison | Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts[END_REF][START_REF] Kang | Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction[END_REF] and in tumor cell lines [START_REF] Chen | Regulation of the proinflammatory effects of Fas ligand (CD95L)[END_REF] led to a more rapid elimination of these cells relative to control cells, at least in part due to increased infiltration of neutrophils and macrophages endowed with antitumor activity.

(At least) two different ligands and two different signals

Among the weapons at the disposal of immune cells is transmembrane CD95L, which contributes to the elimination of pre-tumor cells. Therefore, pre-tumor cells that escape immune surveillance will tend to have developed resistance to CD95, a process termed immunoediting [START_REF] Bui | Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes?[END_REF]. In other words, the influence of the immune system on pre-tumor cells will select for malignant cells with elevated resistance to the CD95L-induced signal. As previously mentioned, these alterations to the CD95 signal not only block the CD95-mediated apoptosis, but also promote the transmission of non-apoptotic signals by CD95L, which may also play a critical role in carcinogenesis [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF][START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF][START_REF] Barnhart | CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells[END_REF][START_REF] Beneteau | Dominant-negative Fas mutation is reversed by down-expression of c-FLIP[END_REF]. In support of this hypothesis, complete loss of CD95 expression is rarely observed in malignant cells [START_REF] Chen | CD95 promotes tumour growth[END_REF].

Accumulating evidence indicates that the apoptotic ligand CD95L behaves as a chemoattractant for neutrophils, macrophages [START_REF] Letellier | CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site[END_REF][START_REF] Kang | Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction[END_REF][START_REF] Chen | Regulation of the proinflammatory effects of Fas ligand (CD95L)[END_REF], T lymphocytes [START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF], and malignant cells in which the CD95-mediated apoptotic signal is non-productive [START_REF] Barnhart | CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells[END_REF][START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF].

Nonetheless, the biological role of CD95L must be further clarified, given that in pathophysiological settings, the ligand is present in at least two forms with different stoichiometries. CD95L is a transmembrane cytokine whose ectodomain can be cleaved by metalloproteases such as MMP3 [START_REF] Matsuno | Stromelysin-1 (MMP-3) in synovial fluid of patients with rheumatoid arthritis has potential to cleave membrane bound Fas ligand[END_REF], MMP7 [START_REF] Vargo-Gogola | Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand[END_REF], MMP9 [START_REF] Kiaei | Matrix metalloproteinase-9 regulates TNF-alpha and FasL expression in neuronal, glial cells and its absence extends life in a transgenic mouse model of amyotrophic lateral sclerosis[END_REF], and ADAM-10 (A disintegrin and metalloproteinase 10) [START_REF] Kirkin | The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells[END_REF][START_REF] Schulte | ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death[END_REF], and then released into the bloodstream as a soluble ligand. Based on data showing that hexameric CD95L represents the minimal level of self-association required to signal apoptosis [START_REF] Holler | Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex[END_REF], and that cleavage by metalloproteases releases an homotrimeric ligand [START_REF] Holler | Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex[END_REF][START_REF] Schneider | Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF], this soluble ligand has long been considered to be an inert molecule that competes with its membrane-bound counterpart for CD95 binding, thereby antagonizing the death signal [START_REF] Schneider | Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF][START_REF] Suda | Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing[END_REF]. However, recent work has demonstrated that this metalloprotease-cleaved CD95L (cl-CD95L) actively aggravates inflammation and autoimmunity in patients affected by systemic lupus erythematosus (SLE) by inducing the non-apoptotic NF-κB and PI3K [START_REF] O' Reilly | Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[END_REF][START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF] signaling pathways (Figure 4). In contrast to the case of transmembrane CD95L, induction of the PI3K signaling pathway by its metalloproteasecleaved counterpart occurs through the formation of a molecular complex devoid of FADD and caspase-8 that instead recruits the src kinase c-yes [START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF]; this unconventional receptosome was designated the motility-inducing signaling complex (MISC) [START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Malleter | CD95L cell surface cleavage triggers a prometastatic signaling pathway in triple-negative breast cancer[END_REF](Figure 4). Even though we did not detect any trace of caspase-8 in MISC, this enzyme has been shown to participate in cell migration. The protease activity of caspase-8 can be abolished by phosphorylation at tyrosine 380 by src kinase [START_REF] Cursi | Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression[END_REF]. This post-translational modification was observed in cells stimulated with EGF and in colon cancer cells with constitutive activation of src; from a molecular standpoint, the modification does not alter caspase homodimerization or recruitment in DISC [START_REF] Cursi | Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression[END_REF]. Moreover, the epidermal growth factor receptor (EGFR)-driven phosphorylation of caspase-8 at Y380 turns out to potently induce the PI3K signaling pathway by recruiting the PI3K adaptor p85 alpha subunit [START_REF] Senft | Caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility[END_REF].

Ultimately, caspase-8 phosphorylation triggers cell migration. Nonetheless, it is noteworthy that CD95-induced migration and invasion does not appear to require an intact DD (reviewed in [START_REF] Steller | How CD95 stimulates invasion[END_REF]), suggesting either that the caspase-8-dependent mode of cell migration reflects the action of an alternative signal mediated by death receptors or that it only participates in nondeath receptor-induced cell motility. It would be interesting to address this question in the future. At present, we can only surmise that phosphorylation of caspase-8 at Y380 following EGFR stimulation primes certain cancer cells to become unresponsive to the apoptotic signal triggered by cytotoxic CD95L, and meanwhile promotes cell migration, an essential event in cancer cell metastasis (Figure 4). We recently showed that CD95 implements the PI3K signaling pathway by recruiting EGFR. This CD95-dependent EGFR activation relies on the recruitment of the NADPH oxidase 3 (Nox3), the production of reactive oxygen species, which in turn activate the src kinase c-yes [START_REF] Malleter | CD95L cell surface cleavage triggers a prometastatic signaling pathway in triple-negative breast cancer[END_REF]. In triple-negative breast cancer cells exposed to metalloprotease-cleaved CD95L, c-yes activation is instrumental in forming an EGFR-containing MISC, and this receptor tyrosine kinase (RTK) orchestrates the activation of PI3K in an EGF-independent manner (Figure 4). These data are in accordance with a recent study showing that another RTK, platelet-derived growth factor receptor-β, is recruited in colon cancer cells exposed to CD95L and thereby triggers cell migration [START_REF] Steller | The death receptor CD95 activates the cofilin pathway to stimulate tumour cell invasion[END_REF]. Accordingly, we postulate that RTK recruitment is a common process in CD95 stimulation that can simultaneously inhibit the apoptotic signal by phosphorylating DISC-recruited caspase-8, and promote cell motility by activating the PI3K signaling pathway. This hypothesis will be investigated further in future work.

In a similar manner, a reduction in the plasma membrane level of CD95 or expression of a mutated CD95 allele, as observed in ALPS patients and malignant cells, inhibits the implementation of the apoptotic signal but does not affect the transmission of non-apoptotic signals, e.g., via NF-κB, MAPK, and PI3K [START_REF] Legembre | The relevance of NF-kappaB for CD95 signaling in tumor cells[END_REF][START_REF] Legembre | Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds[END_REF][START_REF] Chen | CD95 promotes tumour growth[END_REF], suggesting that these signals may stem from a domain other than CD95-DD, or are elicited at different thresholds. In summary, although the CD95/CD95L interaction can eliminate malignant cells by promoting formation of the DISC, or contribute to carcinogenesis by sustaining inflammation and/or inducing metastatic dissemination [START_REF] Letellier | CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site[END_REF][START_REF] O' Reilly | Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[END_REF][START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Barnhart | CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells[END_REF][START_REF] Kleber | Yes and PI3K bind CD95 to signal invasion of glioblastoma[END_REF][START_REF] Chen | CD95 promotes tumour growth[END_REF][START_REF] Bivona | FAS and NF-kappaB signalling modulate dependence of lung cancers on mutant EGFR[END_REF], the molecular mechanisms underlying the switch between these different signaling pathways remain enigmatic. An important question to be addressed is how the magnitude of CD95 aggregation regulates the formation of "Death"vs. "Motility"-ISCs. Answering these questions will lead to the development of new therapeutic agents with the ability to contain the spread of inflammation or impede carcinogenesis, at least in pathologies associated with increased soluble CD95L, such as cancers (e.g., pancreatic cancer [START_REF] Bellone | Production and pro-apoptotic activity of soluble CD95 ligand in pancreatic carcinoma[END_REF], large granular lymphocytic leukemia, breast cancer [START_REF] Malleter | CD95L cell surface cleavage triggers a pro-metastatic signaling pathway in triple negative breast cancer[END_REF], and NK-cell lymphoma, [START_REF] Tanaka | Fas ligand in human serum[END_REF]) or autoimmune disorders (e.g., rheumatoid arthritis and osteoarthritis [START_REF] Hashimoto | Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis[END_REF], and graft-versus-host-disease (GVDH) [START_REF] Das | Levels of soluble FasL and FasL gene expression during the development of graft-versushost disease in DLT-treated patients[END_REF][START_REF] Kanda | Increased soluble Fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease[END_REF] or SLE [START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Tomokuni | Serum levels of soluble Fas ligand in patients with silicosis[END_REF]). Together, these studies support the notion that the death function of CD95 corresponds to its "day job" while the receptor may act as "a night killer" by fueling inflammation in certain pathophysiological contexts.

Strikingly, although the soluble form of CD95L generated by MMP7 (cleavage site inside the 113 ELR 115 sequence, Figure 5) induces apoptosis [START_REF] Vargo-Gogola | Identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human Fas ligand[END_REF], its counterpart processed between serine 126 and leucine 127 does not [START_REF] O' Reilly | Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[END_REF][START_REF] Tauzin | The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway[END_REF][START_REF] Schneider | Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity[END_REF]. To explain this discrepancy, we speculate that the different quaternary structures of naturally processed CD95L molecules underlie the implementation of death-inducing vs. non-death-inducing signaling complexes. Consistent with this notion, soluble CD95L incubated in the bronchoalveolar lavage (BAL) fluid of patients suffering from acute respiratory distress syndrome (ARDS) undergoes oxidation at methionines 224 and 225 (Figure 5), promoting aggregation of the soluble ligand and boosting its cytotoxic activity [START_REF] Herrero | The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region[END_REF]. The same authors observed that the stalk region of CD95L, corresponding to amino acids 103-136 and encompassing the metalloprotease cleavage sites (Figure 5), participates in the multimerization of CD95L, accounting for the damage to the lung epithelium that occurs in ARDS [START_REF] Herrero | The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region[END_REF]. Of note, in ARDS BAL fluid, additional oxidation occurs at methionine 121 (Figure 5), which prevents the processing of CD95L by MMP7, potentially explaining why this cytotoxic ligand retains its stalk region [START_REF] Herrero | The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region[END_REF]. Nonetheless, preservation of this region in soluble CD95L raises the possibility that an unidentified MMP7-independent cleavage site exists in the juxtamembrane region of CD95L, near the plasma membrane, or alternatively that the ligand detected in ARDS patients corresponds to full-length CD95L embedded in exosomes [START_REF] Alonso | Diacylglycerol kinase alpha regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes[END_REF][START_REF] Bianco | Modulation of the immune response using dendritic cell-derived exosomes[END_REF]. Indeed, this peculiar exosome-bound CD95L is sometimes expressed by human prostate cancer cells (i.e., LNCaP), and it evokes apoptosis in activated T lymphocytes [START_REF] Abusamra | Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis[END_REF].

Overall, these findings emphasize the importance of finely characterizing the quaternary structure of naturally processed CD95L from the sera of patients affected by cancers or chronic/acute inflammatory disorders. Such investigations will improve our understanding of the molecular mechanisms set in motion by this ligand, and thus our appreciation of its downstream biological functions. increases the aggregation level of the soluble ligand, whereas oxidation at methionine 121 prevents the processing of CD95L by metalloproteases [START_REF] Herrero | The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region[END_REF]. TM: transmembrane domain.

Figure 1 .Figure 2 .

 12 Figure 1. CD95: mRNA to protein.

Figure 5 .

 5 Figure 5. CD95L: metalloprotease cleavage sites and domains.

Acknowledgements This work was supported by grants from INCa, Ligue Contre le Cancer (Comités d"Ille-et-Vilaine/du Morbihan/des Côtes d"Armor/du Finistère/du Maine et Loire), ARC, Cancéropole Grand Ouest, Région Bretagne, Rennes Métropole.

Binding of transmembrane CD95L to CD95 leads to formation of the DISC, which consists of FADD and procaspase-8. C-FLIP and PEA-15 bind to FADD, thereby preventing caspase-8 recruitment. At the level of the DISC, aggregation of procaspase-8 promotes its auto-cleavage and activation. Cleaved caspase-8 is then released to the cytosol, where it promotes the