Fas/CD95 death receptor and lipid rafts: New targets for ......death ligands. Apoptosis-targeted...

Drug Resistance Updates 9 (2006) 51–73

Fas/CD95 death receptor and lipid rafts: New targets forapoptosis-directed cancer therapy

Faustino Mollinedo a,∗, Consuelo Gajate a,b

a Centro de Investigacion del Cancer, Instituto de Biologıa Molecular y Celular del Cancer, Consejo Superior de Investigaciones Cientıficas(C.S.I.C.)-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain

b Unidad de Investigacion, Hospital Universitario de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain

Received 13 March 2006; received in revised form 3 April 2006; accepted 12 April 2006

Abstract

The development of new drugs able to directly activate the apoptotic machinery in tumors is a promising approach in the treatment of canceras it is independent of sensors and checkpoints, which are frequently mutated in cancer hampering current anti-proliferative chemotherapy.The Fas death receptor (CD95 or APO-1) conveys apoptotic signals through binding to its cognate ligand, FasL (CD95L). Unfortunately, theputative clinical antitumor action of FasL cannot be realized because of severe liver toxicity due to the high presence of Fas in hepatocytes.HasdmgaTo©

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owever, recent evidence for FasL-independent activation of Fas suggests that the death receptor can also be activated intracellularly, in thebsence of its ligand. Unraveling the mechanisms that underlie the intracellular activation of Fas can provide the basis for novel therapeutictrategies and for the development of new compounds able to exploit cytoplasmic triggers of apoptosis. This is of importance in apoptosis-eficient disorders such as cancer and autoimmune diseases. Fas-mediated apoptosis involves translocation of Fas – and downstream signalingolecules – into lipid rafts, a process that can be pharmacologically modulated. FasL-independent clustering of Fas in membrane rafts

enerates high local concentrations of death receptor providing scaffolds for coupling adaptor and effector proteins involved in Fas-mediatedpoptosis. Thus, lipid rafts act as the linchpin from which a potent death signal is launched, becoming a new promising anticancer target.hese findings set a novel framework for the development of more targeted therapies leading to intracellular Fas activation and recruitmentf downstream signaling molecules into Fas-enriched lipid rafts.

2006 Elsevier Ltd. All rights reserved.

eywords: Fas/CD95; FasL-independent Fas activation; Lipid rafts; Apoptosis; Cancer chemotherapy; Antitumor drugs; Edelfosine

. Introduction

There is a continuous need to overcome the rather pooreturns of current cancer chemotherapy with new chemother-peutic agents different from classical cytotoxic drugs usedn clinical practice. The era of chemotherapy was ushered inith the introduction of polyfunctional alkylating agents in

he early 1940s. Since then, a wide array of antitumor drugsas become available, most of them affecting proliferatingells. However, the modest progress in cancer chemotherapyver the past 65 years suggests that some of the premises used

∗ Corresponding author. Tel.: +34 923 294806; fax: +34 923 294795.E-mail address: [email protected] (F. Mollinedo).

for targeting the cancer cell may need reassessment. Percep-tion of the malignant cell as having uncontrolled proliferationis one of such concept. The rather modest impact of anti-proliferative drugs in the clinic is not surprising since manytumors have a low growth capacity. In addition, exposureof normal tissues that have a high rate of cellular prolifera-tion, such as the bone marrow, the gastrointestinal epithelialcells and the cells of the hair follicles, to anti-proliferativedrugs leads to major toxicities. The lack of selectivity ofanti-proliferative antitumor agents translates into a low ther-apeutic index, defined as the ratio of maximum tolerateddose/minimum effective dose.

In contrast, increasing evidence defines the tumor cell asbeing mainly defective in triggering its own death by apop-tosis. The apoptosis deficiency could even be more central to

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52 F. Mollinedo, C. Gajate / Drug Resistance Updates 9 (2006) 51–73

the process of carcinogenesis than unregulated cell prolifera-tion. Further, apoptotic cell death is usually the final commonmechanism by which cytotoxic antitumor agents with diverseprimary targets kill the cancer cell. Hence, the failure of sometumor cells to die following drug treatment may be due totheir resistance to engage apoptosis. The effectiveness of anti-cancer drugs reflects the ability of tumor cells to detect andrespond to the perturbation induced by the drug (Gajate andMollinedo, 2002; Mashima and Tsuruo, 2005). This in turndepends on the presence of sensors and checkpoints whichkeep the genome and cellular fate under surveillance, andwhich are frequently mutated in cancer, leading to drug resis-tance. Understanding the signals modulating survival, cellproliferation, or apoptosis is vital to controlling diseases suchas cancer (Borst and Rottenberg, 2004).

Activating the apoptotic machinery in tumor cells con-stitutes an attractive and promising approach in cancer treat-ment. The approach takes advantage of the apoptotic machin-ery functionally available in tumor cells to direct their owndemise, independently of sensors or checkpoints. Inactivationof sensors and checkpoints is crucial for the development ofcancer since they act as genome guardians. However, oncethe tumor is established, the characteristic genome instabilityand plasticity of cancer cells leads to an increasing number ofadditional mutations and gene expression changes, resultingin a plethora of new mutated genes functioning in an aberrantwcaroeAwttaat

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matin condensation, nuclear fragmentation, cell shrinkage,and breakdown of the cells into small membrane-surroundedfragments (apoptotic bodies), which were cleared by phago-cytosis without prompting an inflammatory response. Apop-tosis is essential for mammalian physiology and apoptosismalfunctioning is critical in the pathogenesis of many humandiseases: cancer and autoimmune diseases where there is toolittle apoptosis; stroke damage and neurodegenerative dis-eases where there is too much.

Carcinogenesis is a multistage process that involves dam-age to the genome accumulating mutations in specific genes.This leads to alterations in either the activity or the amountof the encoded proteins, thereby perturbing normal cell func-tioning. These changes are perceived and appraised by cel-lular “sensors”, involved in restoring normal function. Whenthis is not possible, sensors trigger signaling pathways thatlead eventually to apoptosis. This implies the existence ofa molecular threshold for the engagement of cell death,named “apoptosis threshold” (Gajate and Mollinedo, 2002),in response to damage that is set differently in different celltypes (Fig. 1). Apoptosis thresholds depend on:

a. level of expression or activity of cellular sensors that mustsense genomic or cellular damage and deliver appropriatealert signals;

b. downstream events set in motion by the sensors connecting

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Fcubtpithreshold and thus providing cancer cells with a high survival capacity inadverse conditions. Drugs targeting directly the apoptotic machinery couldhypothetically circumvent these obstacles and lead to rapid demise of cancercells.

ay. This enables tumor cells to endure conditions a normalell could not tolerate, such as hypoxia and chromosomalberrations. Under these circumstances little can be done byepairing the initial mutated sensors that allowed the onsetf these tumorigenic genome changes. At this stage, how-ver, the apoptosis machinery of the tumor cell becomes itschilles’ heel. Thus, apoptosis-targeted therapy can be a neway to kill tumor cells, provided the drug is able to set off

he cancer cell apoptotic program. In the anti-proliferativeherapy, cells sense damage induced by the drug and respondccordingly (Fig. 1). In apoptosis-targeted therapy, the cell’spoptotic machinery is triggered before the cancer cell hashe chance to respond (Fig. 1).

The aim of this review is to discuss the antitumor thera-eutic potential of targeting apoptosis in cancer cells throughhe aggregation of the Fas death receptor (CD95 or APO-1) inipid rafts, independently of its natural ligand FasL (CD95L).his new avenue in cancer therapy may lead to the identifi-ation of novel targets for therapeutic intervention.

.1. Apoptosis threshold and carcinogenesis

The term apoptosis (from the Greek words ��o -apo-“away from, off, detached”, and �� -ptosis-, “fall”;eaning “falling off”, as leaves from a tree), originally coined

y the Australian pathologist John F.R. Kerr together with hiscottish colleagues Andrew H. Wyllie and Alastair R. Currie

n 1972 (Kerr et al., 1972), refer to a type of a physiolog-cal cell death that was initially described by its morpho-ogical characteristics, including membrane blebbing, chro-

with the apoptotic signaling;. expression and functional state of apoptotic molecules.

ig. 1. Induction of apoptosis in cancer chemotherapy. Anti-proliferativeancer chemotherapy engages apoptosis through a multistep pathway. Cellsndergo drug-induced damages on DNA or cell cycle, and sense and cali-rate these lesions through the presence of “sensors”, which set off signalshat eventually trigger apoptosis. Mutation or deletion of sensors in tumorsrecludes or hampers the triggering of downstream signaling events, which,n turn, can also be inhibited in cancer cells, leading to a high apoptosis

F. Mollinedo, C. Gajate / Drug Resistance Updates 9 (2006) 51–73 53

Tumor cells may either inhibit the molecular processesthat lead to their own death by apoptosis and/or increasemarkedly the apoptosis threshold required to sensitize themto apoptotic signals (Gajate and Mollinedo, 2002). In thisregard, the tumor suppressor gene p53, involved in cell-cyclecontrol, apoptosis, and maintenance of genetic stability, ismutated or inactivated in over 50% of all cancers. There aresignificant intrinsic differences among different cell types intheir capacity to invoke an apoptotic response after damage.Most (approximately 86%) of the human cancers derive fromepithelial cells. One explanation might be that epithelial tis-sue has a high apoptosis threshold and can tolerate a highamount of damage before mounting an apoptotic response;hence, mutations might accumulate leading ultimately to can-cer. The high apoptotic threshold of epithelium could bedue to the fact that it is exposed to environmental insultsand some damage risk might be an acceptable trade off foravoiding a high cell death rate. In contrast, the bone marrow,which is protected from external insults, would show a lowerthreshold for engaging apoptosis and this might explain whyhematopoietic tumors are less frequent (about 8%). This lowapoptotic threshold also explains the deleterious effects andthe ease of chemotherapy- and radiation-induced cell deathin the bone marrow.

Impaired apoptosis signaling is common in cancer cellsand plays an important role in tumor initiation, progressionaccreacaetlc

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1.2. Death receptors and cancer chemotherapy

Mammals have evolved a receptor/ligand mechanism thatenables the organism actively to direct individual cells toself-destruct through the presence of cell surface death recep-tors, which transmit apoptosis signals initiated by specificdeath ligands. Apoptosis-targeted therapy through activationof death receptors can engage an apoptotic response thatbypasses the action of sensors, such as p53, and thereforetheir frequent mutant state in cancer should be irrelevantto this therapeutic approach (Fig. 1). Death receptors aremembers of the tumor necrosis factor (TNF) receptor genesuperfamily, which consists of more than 20 proteins with abroad range of biological function, including the regulationof cell death and survival, differentiation or immune regu-lation (Debatin and Krammer, 2004). Death receptors shareregions of high homology including cysteine-rich extracel-lular domains and a cytoplasmic domain of about 80 aminoacids called “death domain”, which plays a crucial role intransmitting the death signal from the cell’s surface to intra-cellular signaling pathways. The best-characterized deathreceptors in their potential to induce apoptosis are Fas, TNFreceptor 1 (TNFR1), TNF-related apoptosis-inducing ligand(TRAIL) receptor 1 (TRAIL-R1) (death receptor 4, DR4) andTRAIL receptor 2 (TRAIL-R2) (death receptor 5, DR5). Dif-ferential expression of TRAIL-R1/DR4 and TRAIL-R2/DR5hTgRetaaHmrllo2atmteucciapht(c

nd metastasis, as cells with genomic damage or deregulatedell cycle are normally eliminated by apoptosis. Resistance ofancer cells to apoptosis is especially deleterious, because itesults in a higher survival capacity under adverse conditions,nhancing the malignant potential of the tumor, favoringccumulation of mutations, metastasis and rendering tumorells resistant to therapy as well as to host defense mech-nisms. The ability to metastasize makes cancers hard toradicate and leads to the high death toll of cancer patients,hough less than 0.1% of the cancer cells released into circu-ation by tumors survive and succeed in founding metastaticolonies.

The gene for caspase-8 (previously known as FADD-ike interleukin-1� converting enzyme -ICE- (FLICE), or

ORT1-associated CED-3 homolog (MACH)), a proapop-otic cysteine protease, is frequently silenced through DNAethylation and gene deletion in neuroblastoma (Teitz et

l., 2000), the most common childhood solid tumor ris-ng from the peripheral nervous system. Caspase-8-nulleuroblastoma cells are resistant to death receptor- andoxorubicin-mediated apoptosis, deficits that are correctedy programmed expression of the enzyme (Teitz et al., 2000).urthermore, loss of caspase-8 has been recently shown tootentiate neuroblastoma metastasis (Stupack et al., 2006).n addition, caspase-8 expression is lost in tumors otherhan neuroblastoma, including small-cell lung carcinoma,habdomyosarcoma, medulloblastoma, and retinoblastomaPingoud-Meier et al., 2003; Shivapurkar et al., 2002). Thesendings highlight the importance of apoptosis in tumor devel-pment, metastasis and treatment.

as been described for various tumor types, usually withRAIL-R2/DR5 being the most prevalent. Recent data sug-est that TRAIL-R2/DR5 may contribute more than TRAIL-1/DR4 to TRAIL-induced apoptosis in cancer cells thatxpress both death receptors (Kelley et al., 2005). Hence,he corresponding death ligands TNF, Fas ligand (FasL)nd TRAIL are interesting candidates for antitumor ther-py (Shankar and Srivastava, 2004; van Geelen et al., 2004).owever, systemic TNF administration has shown low antitu-or activity and higher doses induce a severe inflammatory

esponse syndrome that resembles septic shock. Neverthe-ess, TNF� has been used in human cancer patients throughocal cytokine administration to avoid or reduce the inductionf undesired systemic symptoms (Hohenberger and Tunn,003). Systemic FasL administration in humans has not beenttempted because of severe liver toxicity in mice. Adminis-ration of FasL or agonistic antibody to Fas in tumor-bearing

ice is lethal because of apoptosis induction in hepatocyteshat abundantly express Fas (Ogasawara et al., 1993; Tanakat al., 1997). Unlike TNF and FasL, TRAIL seems to benique, since it has been reported to be nontoxic to normalells at concentrations where it kills a broad range of tumorells (Yagita et al., 2004); hence, this molecule is a promis-ng anticancer therapeutic agent. Agonistic TRAIL-R1/DR4nd TRAIL-R2/DR5 antibodies are also being investigated asotential therapeutic options. Native TRAIL is expressed as aomotrimeric type II transmembrane protein that can be pro-eolytically cleaved into soluble homotrimeric TRAIL cellsAshkenazi et al., 1999). Both membrane and soluble TRAILan interact with TRAIL-R1/DR4 and TRAIL-R2/DR5,


which initiate apoptosis via their intracellular death domains,and with two antagonist receptors TRAIL-R3 (decoy recep-tor 1, DcR1) and TRAIL-R4 (decoy receptor 2, DcR2), whichare unable to engage apoptosis due to the absence or trunca-tion of the cytoplasmic death domain (Ashkenazi and Dixit,1998). The different TRAIL receptors are widely expressed ina variety of normal tissues and malignant cell types. Initially,TRAIL-R3/DcR1 and TRAIL-R4/DcR2 were thought to bepredominantly expressed in normal cells, thus sparing nor-mal cells from apoptosis. However, no correlation betweenTRAIL sensitivity and expression of TRAIL-R3/DcR1 orTRAIL-R4/DcR2 has been found (Lincz et al., 2001). Con-sequently, the mechanism for the tumor-selective activity ofTRAIL remains elusive. Nevertheless, several reports havedescribed apoptotic activity of TRAIL toward various nor-mal human cells, including primary human hepatocytes (Joet al., 2000), keratinocytes (Leverkus et al., 2000), prostateepithelial cells (Nesterov et al., 2002), and brain tissue (Nitschet al., 2000).

Similar to trastuzumab (Herceptin®), the humanized anti-body to the erbB-2/HER2 receptor tyrosine kinase over-expressed in 20–25% of invasive breast cancers, previousassessment of the status of molecular target in the tumorto predict response (Nahta and Esteva, 2006), may also beappropriate for death-receptor-directed therapy. Moreover,because ligands of the TNF family and their cognate recep-t1aiteti

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apoptosis at the cell membrane in mammalian cells througha receptor/ligand interaction.

Mature Fas (Fig. 2) is a 48-kDa type I transmembranereceptor of 319 amino acids with a single transmembranedomain of 17 amino acids (from Leu-158 to Val-174), anN-terminal cysteine-rich extracellular domain (18 cysteineresidues in 157 amino acids) and a C-terminal cytoplasmicdomain of 145 amino acids that is relatively abundant incharged amino acids (28 basic and 20 acidic amino acids).The cytoplasmic portion of Fas contains a domain of about85 amino acids termed “death domain”, which plays a crucialrole in transmitting the death signal from the cell’s surfaceto intracellular pathways (Nagata, 1997). Unlike the intracel-lular regions of other transmembrane receptors involved insignal transduction, the death domain does not possess enzy-matic activity, but mediates signaling through protein–proteininteractions. The death domain has the propensity to self-associate and form large aggregates in solution (Huang etal., 1996) (Gajate and Mollinedo, unpublished results). Thetertiary structure of the Fas death domain, revealed by NMRspectroscopy (Huang et al., 1996), consists of six antipar-allel, amphipathic � helices (Huang et al., 1996). Helices�1 and �2 are centrally located and flanked on each sideby �3/�4 and �5/�6. This leads to an unusual topology inwhich the loops connecting �1/�2 and �4/�5 cross overeach other (Huang et al., 1996). The presence of a highndtraadtc“dcpslvo(p

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ors play a key role in liver pathogenesis (Faubion and Gores,999), hepatotoxicity is a major challenge for the clinicalpplication of death receptor-targeted therapy. If liver toxic-ty could be circumvented, Fas would be a worthy anticancerarget due to its potent proapoptotic activity and widespreadxpression in tumor cells. It is expected that small moleculesargeting these death receptors will be designed to lower tox-city and increase antitumor activity.

.3. Fas death receptor induces apoptosis through itsligomerization and a cascade of protein–proteinnteractions

Yonehara et al. (1989) reported an IgM monoclonal anti-ody that could kill several human cell lines, and termedas (FS7-associated cell surface antigen) the cell surfacerotein recognized by the antibody. In July of the sameear, Peter H. Krammer and his associates reported a mouseonoclonal antibody, named anti-APO-1 antibody, which

romoted apoptosis in human leukemic cells and activatedymphocytes (Trauth et al., 1989). Two years later, Nagatat al. succeeded in cloning the membrane protein recognizedy the killing antibody, the Fas antigen (Itoh et al., 1991)hat turned out to be identical to the APO-1 protein identifiedater in Krammer’s group (Oehm et al., 1992). Then, Nagata’sroup cloned the corresponding physiological ligand of theas death receptor, FasL (Suda et al., 1993) that belongs to theNF family and can be found as a 40-kDa membrane-boundr a 26-kDa soluble protein (Nagata, 1997). Subsequent find-ngs identified the Fas/FasL system as the major regulator of

umber of charged amino acids in the surface of the deathomain is probably responsible for mediating the interac-ions between death domains. Stimulation of Fas by FasLesults in receptor aggregation (Chan et al., 2000), previouslyssembled in trimers (Papoff et al., 1999; Siegel et al., 2000),nd recruitment of the adaptor molecule Fas-associated deathomain-containing protein (FADD) (Chinnaiyan et al., 1995)hrough interaction between its own death domain and thelustered receptor death domains. FADD also contains adeath effector domain” (DED) that binds to an analogousomain repeated in tandem within the zymogen form ofaspase-8 (Boldin et al., 1996). Upon recruitment by FADD,rocaspase-8 oligomerization drives its activation throughelf-cleavage, activating downstream effector caspases andeading to apoptosis (Ashkenazi and Dixit, 1998). Thus, acti-ation of Fas results in receptor aggregation and formationf the so-called “death-inducing signaling complex” (DISC)Kischkel et al., 1995), containing trimerized Fas, FADD androcaspase-8 (Fig. 3).

Mice carrying the lymphoproliferation (lpr) point muta-ion which converts Ile-225 to Asn-225 in the cytoplas-

ic region of the mouse Fas antigen, are characterizedy a deficient Fas antigen that leads to a lymphoprolifer-tion syndrome showing lymphadenopathy and a systemicupus erythematosus-like autoimmune disease (Watanabe-ukunaga et al., 1992). The corresponding mutation in humanas (V238N) leads to inhibition of apoptosis, together withdramatic inhibition in Fas death domain self-association

nd binding to FADD (Huang et al., 1996), suggesting thathis point mutation alters the protein structure of the death


Fig. 2. Schematic diagram of the human Fas death receptor. Mature human Fas protein consists of 319 amino acids (aas) with an N-terminal extracellulardomain of 157 aas, a short transmembrane region (17 aas) and a C-terminal cytoplasmic domain of 145 aas. Relevant domains for Fas oligomerization andapoptotic activity are shown. An N-terminal extracellular oligomerization domain (NOD) of 49 aas (Arg-1 to Pro-49) responsible for the FasL-independentoligomerization of the receptor. Three cysteine-rich domains (CRD1-Gln31 to Val-67, CRD2-Pro-68 to Cys-111-, and CRD3-Arg-112 to Lys150-) containingfour, six and eight Cys residues in each domain, respectively. A cytoplasmic death domain (DD) of 85 aas (Ser-214 to Ile-298) is crucial for apoptotic signaling.The last 15 amino acids (Asp-305 to Val-319) of the Fas amino acid sequence represent a C-terminal inhibitory domain (CID). Domains and membrane are notto scale.

Fig. 3. Schematic representation of Fas activation through its aggregation in membrane rafts. Fas molecules are brought together and concentrated in membranerafts facilitating the formation of DISCs, following protein-protein interactions between Fas-FADD through their respective death domains (DD), and FADD-procaspase-8 through their respective death effector domains (DED). DISC formation leads to activation of unprocessed procaspase-8 by driving its dimerizationand autoproteolysis, resulting in the release of mature, active caspase-8 (composed of a p20/p10 heteromer) into the cytoplasm. The asteriks represent theactive-site cysteine residues of caspase-8, the dash lines indicate proteolytic processing in trans, and the arrowheads point to the sites of proteolytic cleavage.Actin cytoskeleton through ezrin is involved in the clustering of Fas in lipid rafts.


domain. These data suggest that the intracellular portion ofthe Fas molecule is critical for death receptor oligomerizationrequired for apoptotic activity. Current evidence indicates thatthe molecular ordering of the initial events in physiologicalFas-mediated signaling after binding of Fas to its cognateligand include four successive steps (Algeciras-Schimnich etal., 2002):

a. FasL-induced formation of Fas microaggregates at the cellsurface;

b. recruitment of FADD to form a DISC in an actin filament-dependent manner;

c. formation of large Fas surface clusters positively regulatedby DISC-generated caspase-8;

d. actin filament-dependent internalization of activated Fasthrough an endosomal pathway.

2. Fas-mediated apoptosis involves Fas translocationin lipid rafts

While investigating the mechanism of action of the antitu-mor ether lipid edelfosine (ET-18-OCH3), we found that thisdrug induced apoptosis in leukemic cells in a Fas-dependentmanner through translocation and co-clustering of Fas intomembrane rafts, leading to the first demonstration of therTcbalFberaa8rSFtnitt

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brane, and thus they have been implicated in signal transduc-tion from cell surface receptors (Simons and Toomre, 2000;Dimanche-Boitrel et al., 2005).

The sphingolipid ceramide has been implicated in theclustering of Fas into ceramide-rich rafts (Grassme et al.,2003). However, ceramide acts as a mediator of the cluster-ing process not as an initiator of the process, amplifying theprimary Fas signaling events. Thus, C16-ceramide is unableto trigger Fas clustering in the absence of stimulatory anti-Fas antibody or FasL (Grassme et al., 2003). It is suggestedthat Fas–FasL complexes enter initially into small membranerafts and induce a weak formation of the DISC leading tocaspase-8 activation (Grassme et al., 2003). This rather weakcaspase-8 activation then would generate ceramide throughsphingomyelinase translocation and activation to the smalllipid rafts. Due to the high amount of sphingomyelin presentin rafts (about 70% of all cellular sphingomyelin) (Prinettiet al., 2001), the generated ceramide could induce coales-cence of elementary rafts (Grassme et al., 2003) leadingto the formation of big patches containing Fas–FasL com-plexes that would further lead to enhanced DISC formation,thereby potentiating Fas signaling. Thus, sphingomyelinaseand ceramide serve to amplify the signaling of Fas at themembrane level after the initial Fas–FasL interaction.

2.1. Fas oligomerization without interaction with itsl

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ecruitment of Fas in lipid rafts (Gajate and Mollinedo, 2001).his was assessed by both confocal microscopy, showingo-capping of Fas and lipid rafts, and isolation of mem-rane rafts through sucrose gradient centrifugation (Gajatend Mollinedo, 2001). Analysis of the protein content of iso-ated lipid rafts before and after drug treatment indicated thatas was not constitutively present in the raft microdomains,ut it was translocated to these membrane domains afterdelfosine treatment (Gajate and Mollinedo, 2001). Raft dis-uption inhibited both edelfosine-induced Fas clustering andpoptosis (Gajate and Mollinedo, 2001). Subsequent studieslso found that Fas, together with FADD and procaspase-forming the so-called DISC, were translocated into lipid

afts following activation with FasL (Hueber et al., 2002;cheel-Toellner et al., 2002). The importance of lipid rafts inas-mediated apoptosis was further supported by the finding

hat expression of membrane sphingomyelin, a major compo-ent of lipid rafts, enhances Fas-mediated apoptosis throughncreasing DISC formation, activation of caspases, efficientranslocation of Fas into lipid rafts, and subsequent Fas clus-ering (Miyaji et al., 2005).

Membrane rafts are membrane microdomains consist-ng of dynamic assemblies of cholesterol and sphingolipidsMunro, 2003; Simons and Toomre, 2000). The presencef saturated hydrocarbon chains in sphingolipids allows forholesterol to be tightly intercalated, leading to the presencef distinct liquid-ordered phases, membrane rafts, dispersedn the liquid-disordered matrix, and thereby more fluid, lipidilayer. Membrane rafts may serve as foci for recruitmentnd concentration of signaling molecules at the plasma mem-

igand

Several of the early paradigms in Fas activation have beenecently challenged by recent evidence. An early view ofhe molecular events leading to Fas activation consideredhat, upon binding to homotrimers of FasL, the Fas recep-or homotrimerized through the intracellular death domains.owever, Fas is now assumed to constitutively trimerize prior

o FasL binding (Papoff et al., 1999; Siegel et al., 2000). Pre-ssociated Fas complexes were found in living cells by meansf fluorescence resonance energy transfer between variantsf green fluorescent protein (Siegel et al., 2000). A FasL-nd death domain-independent oligomerization domain inhe extracellular region of the Fas receptor, mapping to the-terminal 49 amino acids, mediates homo- and hetero-ligomerization of the death receptor (Papoff et al., 1999)Fig. 2). In addition, the notion that Fas requires interac-ion with its ligand to trigger an apoptotic response has alsoeen challenged. Apoptosis can be triggered in the absencef FasL by overexpression of the Fas cytoplasmic domain orFas receptor lacking the N-terminal 42 amino acids (Papofft al., 1999), suggesting that the extracellular oligomeriza-ion domain of Fas is not required to initiate signaling, andhat self-association of the death domain is necessary andufficient to induce cell death and occurs in the absence ofn intact extracellular oligomerization domain. Thus, twoajor oligomerization domains are present in the Fas receptor

Fig. 2), one mapping to the extracellular region of the recep-or, likely related to the regulation of the non-signaling state,nd another one, involved in apoptotic signaling, mapping


to the intracytoplasmic region, the death domain (Papoff etal., 1999). The intracellular death domains of death receptorsshow a high tendency to self-associate, and when overex-pressed by gene transfer in eukaryotic cells trigger apoptoticsignaling (Boldin et al., 1995). These findings indicate thatthe Fas receptor plays an active role in its own clusteringand suggest the existence of cellular mechanisms that restrictits self-association, thus preventing constitutive signaling.Taken together, these data show that Fas oligomerization canbe achieved in the absence of FasL.

2.2. FasL is dispensable in Fas-mediated apoptosis

Doxorubicin-induced apoptosis in human T-leukemiccells has been proposed to be mediated by FasL expressionwith subsequent autocrine and/or paracrine induction of celldeath through binding of FasL to the membrane Fas recep-tor (Friesen et al., 1996). This led Friesen et al. to postulatethat Fas/FasL interactions could account for chemotherapy-associated apoptosis (Friesen et al., 1996). Additional anti-cancer drugs, such as methotrexate or bleomycin, werealso reported to promote induction of FasL expression andup-regulation of membrane FasL, leading to autocrine orparacrine Fas/FasL-dependent apoptosis (Friesen et al., 1996;Fulda et al., 1997; Muller et al., 1997). Cell lines resistant toFas were reported to be insensitive to anticancer drug-inducedaFtildiaaaebomiarsFew(stwdoi2e

at the level of FADD and caspase-8, respectively (Bertin etal., 1997), protected cells from cisplatin-induced cytotoxicity(Micheau et al., 1999). Nevertheless, incubation with block-ing anti-Fas antibodies (such as ZB4 and SM1/23 antibodies),or with the soluble Fas–IgG fusion protein (to prevent theinteraction of Fas with FasL) failed to inhibit drug-inducedapoptosis and drug-mediated induction of FasL expressionwas not always detected in distinct tumor cells (Gajate etal., 2000a; Micheau et al., 1999). These data suggest that,at least some, anticancer drugs induce cell death through aFas/FADD pathway in a FasL-independent manner.

2.3. Intracellular triggering of Fas activation in aFasL-independent way, a new selective approach to killtumor cells

Combining transfection and microinjection experiments,we successfully demonstrated the intracellular activation ofFas independently of FasL through the elucidation of theunique mechanism of action of the antitumor ether lipidedelfosine (Gajate et al., 2004; Gajate and Mollinedo, 2002;Mollinedo et al., 1997, 2004) (Fig. 4). This antitumor etherlipid mediates selective apoptosis in cancer cells through Fasactivation, independently of FasL, once the drug is insidethe cell (Gajate et al., 2004). Tumor cells take up edelfosineabtemee

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poptosis, and drug-induced cell death was prevented byas-neutralizing antibodies (Friesen et al., 1996). However,

he involvement of Fas/FasL interactions in chemotherapy-nduced apoptosis rapidly became a controversial issue in theate 1990s as several research groups were unable to repro-uce the original findings, showing that blockade of Fas/FasLnteractions did not prevent apoptosis induced by doxorubicinnd other cytotoxic drugs, and that anticancer drug-inducedpoptosis did not require de novo synthesis of FasL (Gajate etl., 2000a; Gamen et al., 1997; Tolomeo et al., 1998; Villungert al., 1997). However, although many cytotoxic drugs haveeen shown to act independently of the Fas system, we andthers detected FasL-independent activation of Fas in theechanism of action of a number of antitumor drugs, includ-

ng edelfosine, cisplatin, etoposide, and vinblastine (Gajate etl., 2000a; Micheau et al., 1999). Cells deficient in Fas wereesistant to the proapoptotic action of edelfosine, but becameensitive to the antitumor ether lipid when transfected withas (Gajate et al., 2000a). The presence or absence of Fasxpression on the cell surface of cancer target cells correlatedith their sensitivity to the proapoptotic activity of edelfosine

Gajate et al., 2000a). Down-regulation of FADD by tran-ient transfection with an antisense FADD construct inhibitedumor cell sensitivity to cisplatin, etoposide or vinblastine,hereas overexpression of FADD sensitized tumor cells torug-induced cell death (Micheau et al., 1999). Transfectionf cells with FADD dominant negative decreased apoptosisnduced by cisplatin or antitumor ether lipids (Matzke et al.,001; Micheau et al., 1999), and transient transfection withither MC159 or E8, two viral proteins that inhibit apoptosis

nd are sensitive to the drug, whereas normal cells are sparedecause they are unable to incorporate significant amounts ofhe drug (Gajate et al., 2000a; Mollinedo et al., 1997). Fas-xpressing cells that do not take up edelfosine from the cultureedium, like normal cells (Gajate et al., 2000a; Mollinedo

t al., 1997), are unaffected by the exogenous addition of thether lipid, but they undergo apoptosis following microinjec-

ig. 4. Selective antitumor action of edelfosine. Normal cells do not takep the drug, and therefore they are spared, whereas cancer cells incorporatedelfosine and undergo apoptosis mediated by intracellular triggering of Fas.


tion of edelfosine (Gajate et al., 2000a). Fas-deficient cellswere spared from the drug action even following microin-jection, but became sensitive after bestowing Fas expressionby gene transfer (Gajate et al., 2000a, 2004). Deletion ofthe Fas 57 C-terminal amino acids that included part of theFas cytoplasmic death domain prevented apoptosis (Gajateet al., 2004). In addition, edelfosine-sensitive Fas-expressingJurkat cells turned drug-resistant when these cells becameFas-deficient (Gajate et al., 2004). Taking together, thesefindings indicate that Fas is required for edelfosine-mediatedapoptosis and that edelfosine must be inside the cell to triggercell death through intracellular activation of Fas (Gajate et al.,2004; Mollinedo et al., 2004). Because edelfosine is selec-tively taken up by the cancer cell, and once inside the cell,edelfosine triggers Fas activation leading to apoptosis, thismechanism represents the first selective activation of Fas intumor cells (Gajate et al., 2004) (Fig. 4). Whether edelfosinetriggers Fas activation by direct interaction with the cytoplas-mic part of the death receptor or through an indirect processremains to be elucidated. However, computational dockingstudies have allowed us to propose a molecular model forthe putative interaction of edelfosine with the intracellularFas death domain (Mollinedo et al., 2004). Thus, edelfos-ine takes advantage of an apparently selective drug uptakein tumor cells (Gajate et al., 2000a, 2004; Mollinedo et al.,1997) and of a general Fas-mediated apoptotic signaling. ThisicoeIm

2Fc

eFMrRawb(eettFweta

antisense FADD, caspase-8 inhibitors, or by MC159 and E8,involving FADD and caspase-8 in this process (Beltinger etal., 1999; Bush et al., 2001; Chen and Lai, 2001; Delmaset al., 2003; Luo et al., 2003; Micheau et al., 1999).These findings suggest that Fas clustering promotes FADDrecruitment and DISC formation, independently of FasL(Fig. 3).

2.5. Aggregation of apoptotic molecules leads toapoptosis

Formation of lipid raft platforms, where a large amountof signaling molecules are brought together, increases DISCformation and therefore potentiates Fas signaling. Becauseactivation of caspase-8 is induced by proximity (Muzio etal., 1998), its concentration in lipid rafts will favor caspase-8activation, triggering downstream apoptotic signaling. It canbe envisaged that the intrinsic enzymatic activity of caspase-8, upon interaction with additional procaspase-8 moleculesmediated by the adapter FADD molecules, attains a suffi-cient concentration to activate the apoptosis pathway. Usingchimeras of caspase-8 with either CD8 or Tac, Martin et al.(1998) found that oligomerization at the cell membrane pow-erfully induces caspase-8 autoactivation and apoptosis. Basedon these findings, it can be envisaged that these oligomeriza-tion processes would be facilitated enormously in the largeFvs

atacfisemeafaaaiTmnap

lm–ar

ntracellular activation of Fas is an attractive way to targetancer cells from within the cell, thus avoiding the deleteri-us systemic activation of Fas death receptor in normal cells,specially in liver (Gajate et al., 2004; Mollinedo et al., 2004).t also sets a conceptual framework for designing novel andore selective proapoptotic antitumor drugs.

.4. FasL-independent translocation and clustering ofas into membrane rafts, a novel approach in cancerhemotherapy

As discussed above, we found that the antitumor drugdelfosine induced the translocation and concentration ofas into lipid rafts (Gajate et al., 2004, 2000a; Gajate andollinedo, 2001), implicating for the first time membrane

afts in Fas-mediated apoptosis and cancer chemotherapy.esveratrol, a polyphenol found mainly in grape skin withntitumor chemopreventive properties (Pervaiz, 2004), asell as the antitumor drugs cisplatin and aplidin have alsoeen found to redistribute Fas in rafts independently of FasLDelmas et al., 2003; Gajate and Mollinedo, 2005; Lacourt al., 2004). Hence Fas/FasL interaction, although it cannhance cell death, is not essential for drug-induced apop-osis; a growing number of agents and experimental condi-ions can induce Fas activation without the participation ofasL (Table 1). These data suggest a common mechanismhereby divergent stimuli can activate membrane-associated

vents that target the Fas apoptotic pathway in a mannerhat precludes its natural ligand FasL. This FasL-independentctivation of Fas is blocked by dominant negative-FADD,

as aggregates formed during stimulation, leading to acti-ation of caspase-8 and generation of downstream apoptoticignals.

Thus, Fas clustering could be an efficient way to elicitpoptosis through recruitment of the DED-containing pro-eins FADD and procaspase-8 into Fas clusters (Fig. 3). Inddition, it has been demonstrated that FADD and caspase-8oalesce into what appear to be perinuclear “death effectorlaments” (DEFs), inducing receptor-independent apoptoticignals and apoptosis (Siegel et al., 1998). Overexpression ofither FADD or caspase-8 induces apoptosis through the for-ation of unique filament structures that contain the death

ffector domains of these proteins (Siegel et al., 1998),ccordingly being named death effector filaments. Thus,ormation of death effector filaments leads to intracellularssemblies of apoptosis-signaling complexes that can initi-te or amplify apoptotic stimuli independently of receptorst the plasma membrane. Cycloheximide has been shown tonduce cell death in human leukemic Jurkat and CEM C7-cell lines in a FADD-dependent and receptor-independentanner through DEF formation (Tang et al., 1999). Also, a

umber of antitumor drugs, including microtubule-disruptinggents, may induce apoptosis via caspase-8 activation inde-endently of the Fas/FasL system (Goncalves et al., 2000).

As stated above, the initial events in Fas signaling areargely dependent on the local concentration of the three

ajor components of the DISC – Fas, FADD and caspase-8oligomerization of each one being sufficient to mount an

poptotic response. Thus, formation of Fas caps leads to theecruitment of these molecules in a limited space, increas-


Table 1FasL-independent activation of Fas by different agents

Inducer Experimental evidence (Refs.)

FasL-independent activation of Fas Fas clustering/capping Co-capping of Fas and rafts

Aplidin Gajate et al. (2003); Gajate andMollinedo (2005)

Gajate and Mollinedo (2005) Gajate and Mollinedo (2005)

Camptothecin Shao et al. (2001)Cisplatin Huang et al. (2003); Micheau et al.

(1999)Huang et al. (2003); Lacour et al. (2004);Micheau et al. (1999)

Lacour et al. (2004)

Curcumin Bush et al. (2001) Bush et al. (2001)Deoxycholic acid Gupta et al. (2004); Qiao et al. (2001)Edelfosine (ET-18-OCH3) Gajate et al. (2004, 2000a); Gajate and

Mollinedo (2001)Gajate et al. (2004, 2000a); Gajate andMollinedo (2001)

Gajate et al. (2004); Gajateand Mollinedo (2001)

-irradiation Huang et al. (2003) Huang et al. (2003)Glutamine

deprivation-mediatedcell shrinkage

Fumarola et al. (2001) Fumarola et al. (2001)

HCV core protein Moorman et al. (2003) Moorman et al. (2003)JNK activation (via

MKK7)Chen and Lai (2001) Chen and Lai (2001)

Mithramycin A Leroy et al. (2006) Leroy et al. (2006)Resveratrol Delmas et al. (2003) Delmas et al. (2003) Delmas et al. (2003)Reactive oxygen species

(ROS)Huang et al. (2003) Huang et al. (2003)

TGF-�1 Kim et al. (2003)TK/GCV Beltinger et al. (1999) Beltinger et al. (1999)Ultraviolet light Aragane et al. (1998); Rehemtulla et al.

(1997); Zhuang and Kochevar (2003)Aragane et al. (1998); Rehemtulla et al.(1997); Zhuang and Kochevar (2003)

Vanadate Luo et al. (2003) Luo et al. (2003)Vinblastine Micheau et al. (1999) Micheau et al. (1999)Etoposide Micheau et al. (1999) Micheau et al. (1999)

Fas clustering was visualized by immunofluorescence confocal microscopy or assessed by immunoprecipitating Fas using limiting antibody concentrations(Rehemtulla et al., 1997). Co-capping of Fas and rafts was visualized by immunofluorescence confocal microscopy and further assessed by identifying Fas inisolated lipid rafts (Gajate and Mollinedo, 2001). HCV, hepatitis C virus. TK/GCV, herpes simplex thymidine kinase/ganciclovir. TGF-�1, transforming growthfactor-�1.

ing the probability of interactions among them, and therebypromoting a strong apoptotic response.

2.6. Recruitment of death receptors and downstreamsignaling apoptotic molecules in lipid rafts

As suggested from the translocation of Fas into membranerafts following antitumor chemotherapy, the concentrationof death receptors in a rather small area of the cell wouldpotentiate death receptor ligands to achieve cell death. In thisregard, resveratrol (Delmas et al., 2004) and aplidin (Gajateand Mollinedo, 2005) have been reported to redistribute Fas,TNFR1, and TRAIL receptor into lipid rafts, and this redis-tribution sensitizes the cells to death receptor stimulationby their cognate ligands or agonistic cytotoxic antibodies(Delmas et al., 2004) (Gajate and Mollinedo, unpublishedresults).

Our recent studies have shown that not only Fas togetherwith FADD and procaspase-8 are recruited into lipid rafts,forming the DISC, but additional downstream apoptotic sig-naling molecules, including procaspase-10, c-Jun N-terminalkinase (JNK), and BH3-interacting domain death agonist(Bid) are also translocated into membrane rafts follow-ing cancer chemotherapy (Gajate et al., 2004; Gajate and

Mollinedo, 2005) (Fig. 5). Persistent JNK activation is asso-ciated with apoptosis (Chen et al., 1996), and Bid has beenshown to act as a bridge between Fas signaling and themitochondrial-dependent pathway of apoptosis (Li et al.,1998). The recruitment in membrane rafts of JNK and Bidfollowing treatment of human leukemic cells with edelfos-ine (Gajate et al., 2004) and aplidin (Gajate and Mollinedo,2005) may explain the dependence of edelfosine- and aplidin-mediated apoptosis on both JNK and mitochondrial signaling(Gajate et al., 1998, 2003). This redistribution of death recep-tors and downstream signaling molecules into lipid rafts doesnot require protein synthesis, and therefore it is achieved fromthe pre-existing protein pool (Gajate et al., 2004). BecauseFas clustering can occur without the participation of FasL,and ceramide enhances Fas clustering, it could be suggestedthat either the different treatments exerting Fas clusteringpromote sphingomyelinase-dependent ceramide generationor cause physical changes in the plasma membrane similar tothose elicited by ceramide, inducing coalescence of rafts lead-ing to large raft platforms and subsequent capping. However,the clustering of Fas and downstream signaling molecules inlipid rafts, leading to Fas-mediated apoptosis, upon treatmentof human leukemic cells with edelfosine was independent ofsphingomyelinase activation (Gajate et al., 2004). Edelfosine


Fig. 5. Clustering of Fas death receptor and downstream signaling molecules in lipid rafts in cancer chemotherapy. Edelfosine and Aplidin induce apoptosisin cancer cells through aggregation of Fas, downstream signaling molecules, including FADD, procaspases 8 and 10, JNK and Bid, and actin-linking proteins(ezrin, moesin) in clusters of lipid rafts. Aplidin also induces recruitment of FasL into lipid rafts, facilitating Fas/FasL killing between neighboring cells.

did not activate either neutral or acidic sphingomyelinase,and did not induce any significant increase in endogenousceramide levels, suggesting that sphingomyelinase activa-tion was not required in edelfosine-induced apoptosis (Gajateet al., 2004). A number of antagonists of ceramide-inducedapoptosis, including cAMP, the free radical scavenger C60,the metal chelator pyrrolidinedithiocarbamate (PDTC), andthe SAPK (stress-activated protein kinase)/ERK (extracellu-lar signal-regulated kinase) kinase (SEK) dominant negativemutant, could not suppress Fas-mediated cell death, sug-gesting that the apoptotic signal of Fas is not mediated byceramide (Hsu et al., 1998).

We hypothesize that accumulation of Fas into aggregatesof stabilized membrane lipid domains from a highly disperseddistribution may represent a general mode of regulating Fasactivation. Thus, membrane rafts could serve, in addition togenerating high local concentration of Fas, as platforms forcoupling adaptor and effector proteins required for Fas signal-ing (Fig. 5). This is of particular importance in Fas-mediatedsignal transduction as the initial signaling events depend onprotein–protein interactions. Furthermore, this could facili-tate and amplify signaling processes by local assembly ofvarious cross-interacting signaling molecules.

3. FasL translocation into lipid rafts and elucidationoc

icFo

potentiating Fas/FasL killing (Gajate and Mollinedo, 2005).The novel antitumor drug aplidin was shown to act throughFasL-independent activation of Fas and Fas/FasL interac-tion, as blocking Fas/FasL interaction partially inhibitedaplidin-induced apoptosis (Gajate et al., 2003; Gajate andMollinedo, 2005). This translocation of Fas and membrane-bound FasL into clusters of lipid rafts gives an explana-tion for the long-standing dilemma on the involvement ofthe Fas/FasL system in cancer chemotherapy first postu-lated by Friesen et al. (1996) in the mid 1990s (Fig. 6A).In contrast to this hypothesis, de novo FasL synthesis isnot essential for the induction of apoptosis upon treatmentwith chemotherapeutic agents. Instead, a redistribution ofpre-existing Fas and membrane-bound FasL into clusters ofmembrane rafts furnishes small areas of the cell surface withpotent cell death promoters (Fig. 6B and C). Fas can inducecell death independently of FasL, once clustered in mem-brane rafts together with downstream signaling molecules(Gajate et al., 2004) (Fig. 6B). Fas/FasL interactions mayenhance this deadly response through binding of the concen-trated Fas and membrane-bound FasL molecules in lipid raftsbetween Fas- and FasL-bearing neighboring cells (Gajate andMollinedo, 2005) (Fig. 6C). Interaction of the correspondingFas/FasL pairs in adjoining cells would lead to their respec-tive apoptotic cell death. Thus, this concentration of Fas andmembrane-bound FasL into specific and small areas of theccrateii

f the dilemma on Fas/FasL involvement in cancerhemotherapy

In early 2005 we found that not only death receptors,ncluding Fas, TRAIL receptor or TNFR1, could be translo-ated into lipid rafts, but the membrane-bound form ofasL was also recruited into lipid rafts following treatmentf leukemic cells with the marine antitumor drug aplidin,

ell membrane may lead to an increase in the cell killingompetence (Gajate and Mollinedo, 2005), and suggests aegulatory mechanism by which cells concentrate receptorsnd ligands at specific regions of the cell surface leadingo a more effective cell response. We have found that thefficiency in promoting the concentration of death receptorss largely dependent on the target cell type and the trigger-ng stimulus (Mollinedo and Gajate, unpublished results). In


Fig. 6. Fas/FasL involvement in cancer chemotherapy. This scheme depicts the three mechanisms by which the Fas/FasL system is involved in cancerchemotherapy: (A) chemotherapeutic drugs induce de novo FasL synthesis and this newly synthesized FasL binds to Fas, killing cancer cells in an autocrineand paracrine manner; (B) chemotherapeutic drugs induce Fas clustering in lipid rafts together with downstream signaling molecules, leading to apoptosis; and(C) chemotherapeutic drugs induce Fas and FasL clustering in lipid rafts together with downstream signaling molecules, and interaction of the correspondingFas/FasL pairs in neighboring cancer cells lead to their respective apoptotic cell death.

this regard, aplidin is an extremely potent proapoptotic agent(Gajate et al., 2003; Gajate and Mollinedo, 2005), and accord-ingly we have recently found that aplidin is able to promotethe translocation of the three major death receptors, namelyFas, DR5 and TNF-R1, together with FasL and downstreamapoptotic signaling molecules in clusters of lipid rafts thatcould explain the remarkable ability of this antitumor drugin promoting apoptosis (Gajate and Mollinedo, 2005).

This mechanism of death receptor concentration in lipidrafts would not be only relevant to the mechanism of action ofanticancer drugs, but we hypothesize this is a physiologicalprocess involved in apoptosis regulation, and some antitumordrugs exacerbate this process. In this regard, clustering of Fasin lipid rafts has been reported during neutrophil spontaneousapoptosis (Scheel-Toellner et al., 2004).

4. Lipid rafts as a novel target in cancerchemotherapy

The antitumor agents edelfosine and aplidin, which pro-mote a potent redistribution of proteins in lipid rafts leadingto apoptosis, are incorporated into lipid rafts (Gajate et al.,2004; Gajate and Mollinedo, 2005; van der Luit et al., 2002).These antitumor drugs reorganize membrane rafts, promot-itbEc

(Zaremberg et al., 2005), but whereas edelfosine induces theconcentration of Fas death receptor and downstream signal-ing molecules into lipid rafts in leukemic cells (Gajate etal., 2004), the drug selectively partitions the essential yeastplasma membrane protein Pma1p out of lipid rafts in Saccha-romyces cerevisiae, as a major mediator of edelfosine toxicityin yeasts (Zaremberg et al., 2005). By using a Pma1p-red fluo-rescent protein chimera and fluorescence microscopy, Pma1pwas found to move from the plasma membrane to intracellularpunctuate regions and finally localized to the yeast vacuole(Zaremberg et al., 2005). This Pma1p redistribution was pre-ceded by the movement of sterols out of the plasma membrane(Zaremberg et al., 2005). Because the activities of proteinsand signaling processes are meaningfully altered by changesin lipid raft biophysical properties, these findings point to anovel mode of action for an anticancer drug through modi-fication of plasma membrane lipid composition resulting inthe displacement of an essential protein from lipid rafts inyeasts (Zaremberg et al., 2005). Current evidence shows thatselective reorganization of lipid rafts, leading to recruitmentor displacement of critical proteins, regulates the cell fate,suggesting that lipid rafts act as controllers of cell death bysubcellular redistribution (Gajate et al., 2004; Garcia et al.,2003; Zaremberg et al., 2005).

4i

i

ng their clustering and redistributing their protein content,o trigger apoptosis in a Fas-dependent manner. This redistri-ution of lipid raft protein composition is cell-type specific.delfosine accumulates in lipid rafts of both human leukemicells (Gajate et al., 2004; van der Luit et al., 2002) and yeasts

.1. How are Fas clusters in lipid rafts formed byntracellular signals?

As shown in Table 1, FasL-independent activation of Fass mediated by Fas clustering, and recent evidence shows co-


Fig. 7. Putative processes involved in the clustering of Fas in lipid rafts. Persistent activation of JNK, increases in ROS or ceramide levels as well as reorganizationof the actin cytoskeleton have been suggested to lead to clustering of Fas. Ezrin links Fas with the actin cytoskeleton.

capping of Fas in membrane rafts (Delmas et al., 2003; Gajateet al., 2004; Gajate and Mollinedo, 2001, 2005; Lacour et al.,2004). How are these Fas clusters generated? Because FasL isnot strictly required, signals from inside the cell must be ableto regulate this process. The formation of Fas clusters and therecruitment of Fas into membrane rafts in a FasL-independentmanner could involve intracellular processes, changes inthe physicochemical properties of cell membranes orboth.

Vanadate-elicited Fas aggregation and Fas–FADD asso-ciation as well as caspase-8 activation, were dependent onJNK activation (Luo et al., 2003). These results highlighta major role for JNK in the signaling mechanisms leadingto FasL-independent Fas activation. In fact, selective JNKactivation by overexpressing the mitogen-activated proteinkinase kinase 7 (MKK7) induced cell death mediated byFADD and Fas activation, independently of FasL (Chen andLai, 2001). Persistent JNK activation is required for apoptosis(Chen et al., 1996; Gajate et al., 2002) and leads to clus-tering of Fas (Chen and Lai, 2001). A JNK-associated pro-tein named JAMP (JNK1-associated membrane protein) hasbeen recently identified as a membrane-anchored regulatorof the duration of JNK1 activity in response to diverse stressstimuli (Kadoya et al., 2005). The extent of JNK activationcan also be determined by several mitogen-activated proteinkinase (MAPK) phosphatases, including MKP5 (Theodosioue(Isos1tnctHF

Another putative mechanism implicated in Fas clusteringinvolves the cytoskeleton (Fig. 7), a dynamic intracellularstructure that due to its continuous assembly/disassemblycould be perfectly equip to translocate proteins and trans-mit signals (Mollinedo and Gajate, 2003). The interactionsbetween plasma membrane and cytoskeleton play an essentialrole in various cellular functions, and a link between raft-mediated signaling and the interaction of actin cytoskeletonwith raft membrane domains has been suggested (Harder andSimons, 1999). Ezrin, a major protein of the ERM (ezrin,radixin, moesin) proteins linking the actin cytoskeleton tothe plasma membrane (Mangeat et al., 1999), interacts withFas and mediates Fas cell membrane polarization duringFas-induced apoptosis in human T lymphocytes (Fais et al.,2005; Parlato et al., 2000) (Fig. 3). Furthermore, interferencewith actin cytoskeleton prevented Fas clustering and apop-tosis triggered by the antitumor agent aplidin (Gajate andMollinedo, 2005).

Treatment with tert-butyl hydrogen peroxide induced arapid Fas aggregation on the surface of Jurkat cells (Huanget al., 2003), involving reactive oxygen species (ROS) in thisprocess (Fig. 7). In addition, free radical scavengers abro-gated apoptosis and Fas aggregation induced by -irradiationor cisplatin (Huang et al., 2003). These data indicate thatROS participate in -irradiation- and cisplatin-induced Fasclustering. However, free radical scavengers did not affectaimFpeaMeanaa

t al., 1999), as well as by scaffold proteins, including JIP-1Whitmarsh et al., 1998) and POSH (Xu et al., 2003, 2006).rrespective of the molecular mechanism involved in JNKustained activity, it is interesting to note that several inducersf FasL-independent Fas capping lead to a rapid and per-istent activation of JNK, such as edelfosine (Gajate et al.,998) and vanadate (Luo et al., 2003). These data suggesthat persistent JNK activation could be at least one of the sig-aling events leading to Fas clustering (Fig. 7). In this regard,eramide, which also favors Fas aggregation, induces apop-osis through sustained JNK activation (Verheij et al., 1996).owever, the molecular events between JNK activation andas clustering remain to be elucidated.

poptosis triggered by the agonistic Fas antibody CH11,ndicating that the ROS-sensitive stage is upstream of a Fas-

ediated apoptotic pathway. ROS has been implicated inasL expression (Bauer et al., 1998), but FasL was dis-ensable for cell death induced by either cisplatin (Huangt al., 2003) or additional stimuli that trigger both ROSnd Fas clustering (Gajate et al., 2000a, 2000b; Gajate andollinedo, 2001). The mechanism by which ROS influ-

nces Fas clustering is unknown at present. Presumably, ROSctivate signals that eventually result in cytoskeleton reorga-ization and membrane protein clustering (van Wetering etl., 2002; Wang et al., 2001). In this context, ROS gener-tion has been shown to regulate actin polymerization by


reversible glutathionylation of 42-kDa actin (Wang et al.,2001).

5. Modulation of Fas-mediated apoptosis throughFas-interacting proteins

So far, we have discussed the concentration of Fas anddownstream signaling molecules in lipid rafts as a majorregulatory process in Fas activation with important con-sequences in cancer chemotherapy. However, a number ofFas-interacting proteins have been identified to modulate Fasapoptotic activity (Table 2), and therefore they could play animportant role in Fas-targeted therapy.

5.1. Daxx

Fas death domain-associated protein (Daxx) was origi-nally identified as a protein that specifically binds to the deathdomain of Fas and potentiates Fas-induced apoptosis (Yanget al., 1997). Daxx contains a region (amino acids 434–493)of 60 amino acids with a high content (71.7%) of glutamicacid and aspartic acid and comprises two small proline-rich regions. Overexpression of Daxx enhances Fas-mediatedapoptosis and activates the JNK pathway. A C-terminal por-tion of Daxx interacts with the Fas death domain, while adbitbeFFfDeeaoJcpt

aaatNptiwto

its subsequent nuclear export (Song and Lee, 2004). Phos-phorylation of Daxx is mediated through activation of theapoptosis signal-regulating kinase 1 (ASK1)-SEK1-JNK1-homeodomain-interacting protein kinase 1 (HIPK1) signaltransduction pathway, and the activated HIPK1 is probablyinvolved in the relocalization of Daxx from the nucleus tothe cytoplasm (Song and Lee, 2003). Phosphorylated Daxxis translocated to the cytoplasm, bind to ASK1, and sub-sequently lead to ASK1 oligomerization (Song and Lee,2003). Daxx has been found to interact with and activatethe upstream JNK kinase kinase ASK1 upon Fas stimulation(Chang et al., 1998). Overexpression of a kinase-deficientASK1 mutant inhibited Fas- and Daxx-induced apoptosisas well as JNK activation. Cellular localization of Daxx isdetermined by the relative concentration of ASK1, whichcontrols the dual function of Daxx as a transcriptional repres-sor in the nucleus and as a proapoptotic signal mediator inthe cytoplasm (Ko et al., 2001). ASK1 sequesters Daxx inthe cytoplasm, and Daxx binds to the activated Fas onlyin the presence of ASK1, accelerating Fas-mediated apop-tosis (Ko et al., 2001). Thus, Daxx requires ASK1 for itscytoplasmic localization and Fas-mediated signaling. Ectopicexpression of Daxx in malignant Jurkat T-cells substantiallyincreases the rate of apoptosis upon incubation with deathreceptor agonists as well as after incubation with the cyto-toxic drug doxorubicin (Boehrer et al., 2005a). Overexpres-scaaebDeo1iaitDs(rto(i2t2

5

m

ifferent region activates both JNK and apoptosis. The Fas-inding domain of Daxx behaves as a dominant-negativenhibitor of both Fas-induced apoptosis and JNK activa-ion, while the FADD death domain partially inhibits death,ut not JNK activation, and hence Daxx and FADD appar-ntly define two distinct apoptotic pathways downstream ofas (Yang et al., 1997). However, the importance of theas–Daxx–JNK pathway has been questioned when trans-ection of a Fas death domain mutant that selectively bindsaxx, activated JNK but failed to induce apoptosis (Chang

t al., 1999). In addition, Jnk1−/−Jnk2−/− primary murinembryonic fibroblasts showed no inhibition of Fas-mediatedpoptosis (Tournier et al., 2000), challenging the putative rolef Daxx in enhancing the Fas pathway of apoptosis throughNK activation. These findings suggest that although Daxxan activate JNK upon Fas ligation, JNK activation is noter se sufficient to trigger cell death, at least in certain cellypes.

Intriguingly, although Daxx was first reported to medi-te the apoptotic signal from Fas to JNK in the cytoplasm,large proportion of Daxx is mainly located in the nucleus

cting as a transcriptional regulator. Daxx associates withhe promyelocytic leukemia (PML) nuclear body (PML-B), which has been proposed to participate in a nuclearathway for apoptosis (Zhong et al., 2000). The cellulararget responsible for the nuclear export of Daxx has beendentified as chromosomal region maintenance 1 (CRM1),hich is a carrier protein for nuclear export and a recep-

or for the nuclear export signal of Daxx. Phosphorylationf Ser-667 is required for Daxx binding to CRM1 and for

ion of Daxx in Jurkat cells slightly sensitized neoplasticells to the apoptosis-inducing effects of specific chemother-peutic agents, including bendamustine, cladribine, cytosine-rabinoside and mitoxantrone (Boehrer et al., 2005b). How-ver, the major role of Daxx in promoting apoptosis haseen challenged by demonstrating that targeted deletion ofaxx in mice results in early embryonic lethality (E9.5), with

xtensive apoptosis, thus supporting an anti-apoptotic rolef Daxx during embryonic development (Michaelson et al.,999). In this regard, specific RNA interference for Daxxn cell lines was reported to increase apoptosis (Michaelsonnd Leder, 2003) and to sensitize cells to the apoptosisnduced by Fas, UV or TNF� (Chen and Chen, 2003), fur-her suggesting an anti-apoptotic role for Daxx. Expression ofaxx also inhibits cell death induced by CD43 (leukosialin,

ialophorin) in the hematopoietic progenitor cell line TF-1Cermak et al., 2002), further supporting an antiapoptoticole for Daxx. Despite a large number of studies attemptingo determine Daxx function in cell death, its precise role isnly partially understood and remains largely controversialSalomoni and Khelifi, 2006). Daxx acts as a promiscuouslynteracting protein, having been found to bind to more than0 proteins, many of them involved in cell death regula-ion and transcriptional regulation (Salomoni and Khelifi,006).

.2. FADD

As shown above FADD is a key adaptor molecule trans-itting the death signal mediated by death receptors, and

64F.M

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Table 2Fas-interacting proteins

Name Protein name Synonyms Aminoacids

Predicted molecularmass (Da)

SDS-PAGE(kDa)

GenBank accession number Reference

Daxx Fas death domain-associated protein Death domain-associated protein 6;ETS1 associated protein 1 (EAP1)

740 81,373 120 AF039136 Yang et al. (1997)

FADD Fas-associated deathdomain-containing protein

Mediator of receptor induced toxicity1 (MORT1)

208 23,279 29 U24231 Chinnaiyan et al. (1995)

FAF1 Fas-associated factor-1 650 73,954 74 AF106798 Ryu et al. (1999)FAIM Fas apoptosis inhibitory molecule FAIM1 179 20,215 20 NM 018147; AK001444 Schneider et al. (1999)FAP-1 Fas-associated phosphatase-1 Fas-associated protein-tyrosine

phosphatase 1; protein-tyrosinephosphatase PTPL1; protein-tyrosinephosphatase 1E (PTPE1); PTP-BAS;tyrosine-protein phosphatasenon-receptor type 13 (PTPN13)

2485 276,906 270 NM 080683; U12128 Banville et al. (1994);Sato et al. (1995)

FIST (HIPK3) Fas-interactingserine/threonine-protein kinase(Homeodomain-interacting proteinkinase 3)

Androgen receptor-interacting nuclearprotein kinase (ANPK); homolog ofprotein kinase YAK1

1215 133,743 130 AF305239 Rochat-Steiner et al.(2000)

LFG Lifeguard Fas apoptotic inhibitory molecule 2(FAIM2); neuronal membrane protein35 (NMP35)

316 35,110 35 AF190461 Somia et al. (1999)

SUMO-1 (Sentrin) Small ubiquitin-related modifier-1 GAP-modifying protein 1; GMP1;SMT3 homolog 3;ubiquitin-homology domain proteinPIC1; ubiquitin-like protein SMT3C;ubiquitin-like protein UBL1

101 11,557 18 U83117 Okura et al. (1996)

Ubc9 Ubiquitin carrier protein 9 p18; SUMO-1-conjugating enzyme;SUMO-1-protein ligase; ubiquitincarrier protein I; ubiquitin carrierprotein 9; ubiquitin-conjugatingenzyme E2 I; ubiquitin-protein ligase I

158 18,007 18 X96427 Becker et al. (1997);Yasugi and Howley(1996)


together with procaspase-8 form the DISC. FADD- orcaspase-8-deficient mouse embryo fibroblasts and lympho-cytes are resistant to Fas-induced apoptosis (Juo et al., 1998;Zhang et al., 1998). These data highlight the importanceof the FADD-caspase-8 route for Fas-induced apoptosis.Human FADD gene has a quite simple organization consist-ing of two exons (286 and 341 bp) separated by a uniqueintron of approximately 2 kb. The 208-amino acid sequenceof human FADD contains two domains that are particularlywell-conserved between species and play a crucial role intransducing the apoptotic signal mediated by death receptors:the death domain at the C-terminus of the protein and the DEDat the N-terminus of the protein (Tourneur et al., 2005). Lackof FADD protein expression in cancer cells has been found tobe a relevant phenomenon in human malignancies, predict-ing resistance to chemotherapy and poor outcome (Tourneuret al., 2004). Post-translational modification of FADD byPKC�, which is up-regulated in a number of tumors, regu-lates Fas receptor-mediated apoptosis in cells by inhibitingDISC formation following Fas receptor activation, this inhi-bition being reversed by overexpressing the PKC inhibitingprotein prostate apoptosis responsive 4 (PAR-4) (de Thonelet al., 2001).

5.3. FAF1

t(ettapronsiFFFntptOdrciraspn

et al., 2004). The NF-B suppressor activity of FAF1 wasalso mapped to the DED-interacting domain (amino acids181–381) (Park et al., 2004). Thus, FAF1 is involved indual signaling mechanisms and could potentiate apoptosisnot only by strengthening apoptotic signaling via DISC, butalso by suppressing the cell’s survival potential by down-regulation of NF-B. FAF1 has been found to be signifi-cantly reduced in gastric carcinomas (Bjorling-Poulsen etal., 2003), suggesting a role for this protein in cancer pro-gression.

5.4. FAIM

Fas apoptosis inhibitory molecule (FAIM) was identi-fied as a Fas antagonist from a differential display strategyto detect cDNAs present in B cells rendered Fas resistant,but absent in those rendered Fas sensitive (Schneider et al.,1999). FAIM behaves as an inducible effector molecule thatmediates Fas resistance produced by surface Ig in B cells(Schneider et al., 1999). However, FAIM is broadly expressedin various tissues and its sequence is highly conserved in evo-lution from Caenorhabditis elegans to humans (Rothstein etal., 2000; Schneider et al., 1999). Recently, an alternativelyspliced isoform of FAIM, FAIM-L, has been identified, whichis predominantly expressed in the brain (Zhong et al., 2001).An additional function of FAIM in the nervous system lies inpvp(F

5

ptoFskdps1ndbccbtttc2

Fas-associated factor-1 (FAF1) was identified by yeastwo-hybrid assay using the cytoplasmic domain of Fas as baitChu et al., 1995). FAF1 is a Fas-associating molecule, whichnhances Fas-mediated apoptosis (Chu et al., 1995). The N-erminus of FAF1 binds to the death domain of Fas evenhough it does not contain the typical death domain (Ryu etl., 1999). Ryu et al. have recently shown that FAF1 is a com-onent of the DISC, formed by interaction of the DED-likeegion (181–381 amino acid region) of FAF1 and the DEDsf procaspase-8 and FADD, providing a molecular expla-ation for the proapoptotic role of FAF1 in Fas-mediatedignaling (Ryu et al., 2003). Overexpression of human FAF1n Jurkat cells caused significant apoptotic death, but theAF1 deletion mutant lacking the N-terminus where Fas,ADD, and procaspase-8 interact protected Jurkat cells fromas-induced apoptosis leading to a dominant-negative phe-otype (Ryu et al., 2003). Park et al. (2005) has foundhat FAF1 mediates chemotherapeutic-induced apoptosis viaarticipation in the formation of the cytoskeleton-like struc-ure DEFs, found in death receptor-independent apoptosis.verexpression of FAF1 enhanced DEF assembly and celleath induced by chemotherapeutic agents such as stau-osporine, cisplatin and etoposide, whereas antisense FAF1onstruct inhibited DEF assembly and chemotherapeutic-nduced apoptosis (Park et al., 2005). Confocal microscopyevealed that FAF1 was present in DEFs together with FADDnd caspase-8 (Park et al., 2005). In addition, FAF1 has beenhown to interact physically with nuclear factor-B (NF-B)65 preventing translocation of RelA (NF-B p65) into theucleus and hence inhibiting its DNA-binding activity (Park

romoting neurite outgrowth by a mechanism involving acti-ation of the Ras-extracellular signal-regulated kinase (ERK)athway and NF-B, but has no effect on neuronal survivalSole et al., 2004), which contrasts with its role in modulatingas signaling and cell survival in the immune system.

.5. FAP-1

Fas-associated phosphatase-1 (FAP-1) is a tyrosine phos-hatase that was identified as a protein that associates withhe negative regulatory domain (C-terminal 15 amino acids)f Fas using the yeast two-hybrid system (Sato et al., 1995).AP-1 is one of the largest known non-receptor protein tyro-ine phosphatases (about 270 kDa) and is the only proteinnown to associate with the C-terminal negative regulatoryomain of Fas. FAP-1 contains an N-terminal leucine zip-er motif, an ezrin-like cytoskeleton binding domain, andix PSD95/Dlg/Zo-1 homology (PDZ) domains (Sato et al.,995). The C-terminal amino acids (SLV) of human Fas areecessary and sufficient for its interaction with the third PDZomain (PDZ3) of FAP-1 (Yanagisawa et al., 1997). Sta-le introduction of an FAP-1 cDNA in Fas-sensitive Jurkatells (Li et al., 2000; Sato et al., 1995) as well as in otherancer cells (Li et al., 2000; Ungefroren et al., 2001) haseen shown to protect these cells from Fas-mediated cyto-oxicity. In addition, FAP-1 is strongly expressed in humanumor cells which are largely refractory to Fas-induced apop-osis, such as pancreatic adenocarcinoma, colon carcinomaells, and astrocytomas (Foehr et al., 2005; Ungefroren et al.,001; Yao et al., 2004), but pretreatment of cells with syn-


thetic SLV tripeptide abolishes Fas resistance (Ungefrorenet al., 2001; Yao et al., 2004). Association of FAP-1 withFas inhibits Fas trafficking to the cell surface, reducing cellsurface Fas levels (Ivanov et al., 2003) and enhancing colo-calization of Fas/FAP-1 in the Golgi complex (Ungefroren etal., 2001). Down-regulation of FAP-1 expression by specificRNA interference restores Fas export (Ivanov et al., 2006),and leads to increased sensitivity to Fas-induced cell death(Foehr et al., 2005). It has been recently reported that FasLinduces tyrosine phosphorylation of Fas in astrocytoma cells,and FAP-1 dephosphorylates tyrosine-275 in the C-terminusof Fas (Foehr et al., 2005). This finding indicates that Fascan be regulated by reversible phosphorylation. This notionis supported by previous observations that showed a physi-cal interaction between Fas and the tyrosine kinase p59fyn(Atkinson et al., 1996), and that Fas ligation induced earlytyrosine phosphorylation of multiple proteins and inhibitorsof protein tyrosine kinases block Fas-induced DNA fragmen-tation and prolong cell survival (Eischen et al., 1994). Theseresults suggest that protein tyrosine kinase activation is anearly and obligatory signal in Fas-induced apoptosis (Eischenet al., 1994).

5.6. FIST/HIPK3

Using the yeast two-hybrid system to screen for proteinstiscFFitoS

5

elowchiaatomdt3(

5.8. Sentrin/SUMO-1

Sentrin was originally isolated by the yeast two-hybridsystem with the death domain of Fas as a bait (Okura etal., 1996). Sentrin (a.k.a. small ubiquitin-related modifier-1, SUMO-1) is a protein of 101 amino acids, that whenoverexpressed provides protection against Fas-mediated celldeath (Okura et al., 1996). Sentrin/SUMO-1, is a ubiquitin-like protein that can covalently modify a large number ofcellular proteins, including signaling and nuclear proteinswith important roles in regulating transcription, chromatinstructure, and DNA repair (Gill, 2004), by a process namedsentrinization (or sumoylation) in a manner analogous toubiquitination. Despite sentrin has a ubiquitin-like domain(amino acids 22–97), it contains four additional amino acids(HSTV) at the C-terminus, which are cleaved so as to allowthe conjugation of sentrin to other proteins via the glycineresidue at the C-terminus (Kamitani et al., 1997). Mod-ification by sentrin requires activation of the Gly-97 bythe ubiquitin-conjugating enzyme Ubc9 and the activatingenzyme complex UBA2/AOS1 (Gong et al., 1999). Unlikeubiquitin modification, sentrinization/sumoylation is not asignal for degradation, but it regulates activity and/or local-ization of proteins, being suggested to be involved in nucleartranslocation of target proteins and obstruction of cell deathsignaling by prevention of FADD binding to Fas (Gill, 2004;Oi

5

ttma1imwUptMUttmt

6

ti

hat bind to the cytososlic domain of murine Fas, another Fas-nteracting protein was identified and named Fas-interactingerine/threonine-protein kinase (FIST), which was identi-al to homeodomain-interacting protein kinase 3 (HIPK3).IST/HIPK3 is a 130-kDa serine/threonine kinase that causesADD phosphorylation. FIST/HIPK3 overexpression inhib-ted FasL-induced JNK activation, but did not affect apop-osis, suggesting that Fas-associated FIST/HIPK3 modulatesne of the two major signaling pathways of Fas (Rochat-teiner et al., 2000).

.7. Lifeguard

Lifeguard (LFG) was isolated from a cDNA library gen-rated in a retroviral vector from a human lung fibroblast celline, MRC5, which was not sensitive to FasL in the absencef the protein synthesis inhibitor cycloheximide. HeLa cellsere transduced with the retroviral vectors containing the

DNA library, maintained in the presence of the mouse anti-uman Fas agonistic antibody CH11, and then LFG wassolated from the genomic DNA of the surviving pool of cellsnd was shown to inhibit death mediated by Fas (Somia etl., 1999). LFG binds directly to the Fas receptor, but not tohe Fas adaptor protein FADD, and does not inhibit bindingf FADD to Fas (Somia et al., 1999). LFG is expressed inost tissues, except spleen and placenta, being highly abun-

ant in the brain (Somia et al., 1999). LFG expression seemso be mediated, at least in part, by the phosphatidylinositol-kinase (PI 3-kinase)-Akt/protein kinase B (PKB) pathwayBeier et al., 2005).

kura et al., 1996). Interestingly, sentrin has been shown tonteract with Daxx (Ryu et al., 2000).

.9. Ubc9

The ubiquitin-conjugating enzyme Ubc9, which is essen-ial for sentrinization/sumoylation, has been found to bindo Fas at the interface between the death domain and the

embrane-proximal region of Fas (Becker et al., 1997). Ubc9lso binds Daxx at the same region as Fas and sentrin/SUMO-(Ryu et al., 2000). MCF7 human breast tumor cells express-

ng a Ubc9 dominant-negative mutant were found to accu-ulate more cytoplasmic Daxx than the control cells andere more sensitive to anticancer agents (Mo et al., 2004).bc9 expression levels are elevated in ovarian tumors com-ared to the matched normal ovarian specimens, suggestinghat Ubc9 may play a role in tumorigenesis. Inoculation of

CF-7 cells overexpressing a dominant-negative mutant ofbc9 and wild-type Ubc9 as xenografts in mice revealed that

umors expressing the wild-type version of Ubc9 grew betterhan the vector control, while tumors expressing the Ubc9

utant reduced growth, further supporting a role for Ubc9 inumorigenesis (Mo et al., 2005).

. DISC-interacting proteins

Because DISC formation plays a critical role in the even-ual cell death response triggered by Fas activation, proteinsnteracting with DISC constituents modulate Fas-mediated


Table 3DISC-interacting proteins

Name Protein name Synonyms Aminoacids

Predicted molecularmass (Da)

SDS-PAGE(kDa)

GenBank accessionnumber

Reference

FLASH FLICE-associatedhuge protein

Caspase-8-associated protein2 (CASP8AP2); RIP25

1982 222,658 220 AF154415 Imai et al. (1999)

c-FLIP CellularFLICE-inhibitoryprotein

Caspase-8 and FADD-likeapoptosis regulator (CFLAR),caspase-8-related protein(Casper), caspase-likeapoptosis regulatory protein(CLARP), MACH-relatedinducer of toxicity (MRIT),caspase homolog (CASH),inhibitor of FLICE(I-FLICE), FADD-likeantiapoptotic molecule-1(FLAME-1), Usurpin

480 55,344 55 U97074 Irmler et al. (1997)

TOSO Toso 390 43,146 59 AF057557 Hitoshi et al. (1998)

apoptotic signaling. Three major proteins regulating theFas signal at the level of the DISC are discussed below(Table 3).

6.1. FLASH

FLICE-associated huge protein (FLASH) is a protein withbinding activity to the DEDs of procaspase-8 and FADDthrough its DED-like domain, being a component of theDISC (Imai et al., 1999). FLASH was identified by usingthe yeast two-hybrid technique and two tandem-repeatedDED domains of procaspase-8 as a probe (Imai et al., 1999).FLASH contains a motif structurally related to C. elegansprotein CED-4/apoptotic protease activating factor-1 (Apaf-1) and two tandem-repeated DED homologous domains (Imaiet al., 1999). FLASH enhances the activation of caspase-8 inFas-mediated apoptosis, indicating that DED-containing pro-teins seem to modulate the apoptotic process.

6.2. FLIP

The cellular FLICE-inhibitory protein (c-FLIP) is anotherDED-containing protein that acts as a major regulator ofapoptosis and can switch life/death signals in tumor cell(Tucker et al., 2004). Although more than 10 isoforms ofFLIP mRNA have been described, only two of them haveb(FNptdtii2h

results in their escape from T-cell immunity (Dutton et al.,2006).

6.3. TOSO

A novel human gene dubbed Toso was identified as amolecule that blocks Fas-mediated apoptosis by Hitoshi etal. using retroviral cDNA library-based functional cloning(Hitoshi et al., 1998). The investigators named the moleculeToso after the Japanese liquor “that is drunk on New Year’sDay to celebrate long life and eternal youth” (Hitoshi et al.,1998). Toso was found to be expressed mainly by lympho-cytes and was suggested to regulate apoptosis by inhibit-ing caspase-8 processing, potentially through up-regulationof cFLIP (Hitoshi et al., 1998). Toso-overexpressing pri-mary T lymphocytes from mouse Toso transgenic miceare resistant to Fas/FasL-induced apoptosis, but sensitive toglucocorticoid-induced apoptosis (Song and Jacob, 2005).Toso knock-out mice are embryonically lethal. Toso is a typeI membrane protein where its C-terminal is involved in FADDbinding, and hence it could interfere with the recruitment ofcaspase-8 to FADD molecule and its activation (Song andJacob, 2005).

7. Conclusions and future perspectives

ilFbpntawe

een significantly studied at the protein level, c-FLIP-longc-FLIPL) (480 amino acids, 55 kDa) and c-FLIP-short (c-LIPS) (221 amino acids, 28 kDa). Both proteins have two-terminal DED motifs that are very similar to the DED’s onrocaspase-8. c-FLIPL in addition has a C-terminal domainhat is homologous to the catalytic domain of caspase-8, butevoid of enzymatic activity. Both proteins can be recruitedo the DISC, binding to the Fas–FADD complex and inhibit-ng the recruitment of caspase-8 to DISC, and preventingnduction of apoptosis mediated by death receptors (Kataoka,005). Interestingly, c-FLIP expression is increased in someuman tumors, and overexpression of cFLIP in tumor cells

Recent evidences indicate that Fas-mediated apoptosiss mediated by the formation of large Fas aggregates inipid rafts. Under physiological conditions FasL triggersas aggregation in caps. Nevertheless, this capping cane also generated by non-physiological agents without thearticipation of FasL (Table 1), raising the possibility forew therapeutic interventions. This is of interest due to theoxic side effects derived from the use of FasL or agonisticnti-Fas antibodies in vivo, leading to a fatal hepatic damageith symptoms similar to fulminant hepatitis (Ogasawara

t al., 1993; Tanaka et al., 1997). Thus, FasL-independent


activation of Fas offers some opportunities to find agents thatcan circumvent the above hepatic effects, but preserve Fasactivating properties. Such a notion has found experimentalsupport in our recent studies on the antitumor ether lipidedelfosine (Gajate et al., 2004). This antitumor compoundis incorporated in significant amounts in tumor cells, andonce inside the cell promotes apoptosis through intracellularactivation and co-capping of Fas, independently of FasL, inlipid rafts (Gajate et al., 2000a, 2004; Gajate and Mollinedo,2001; Mollinedo et al., 1997) (Fig. 4). Because edelfosine isnot taken up by normal cells, including hepatocytes, normalcells are spared and this drug circumvents the hepatic toxicitythat has hampered so far the clinical use of Fas-targetingtherapies in the clinic (Gajate et al., 2000a, 2004; Gajate andMollinedo, 2002; Mollinedo et al., 1997) (Fig. 4). Edelfosineconstitutes the first drug that directly activates the apoptoticsignaling of tumor cells through the selective activation ofFas in cancer cells, by recruitment of Fas and downstreamsignaling molecules in lipid rafts (Gajate et al., 2004) (Fig. 5),and thereby it can constitute the leading compound of a newclass of synthetic drugs targeting apoptotic machinery.

The increasing number of agents that promote FasL-independent activation of Fas through Fas clustering (Table 1)suggests that this process is more general than initiallybelieved. The fact that very different experimental conditionsand diverse agents, targeting distinct molecules and cellularpapcctcfctitmn

mouiafcsmdatolt

is triggered, and as targets for cancer treatment. Future stud-ies must unravel the molecular mechanisms responsible forthe concentration of apoptotic molecules in lipid rafts and itspharmacological modulation, which can lead to new potentanticancer therapies. We are entering an exciting era in whichnovel avenues in apoptosis-targeted therapy may improveefficiency and safety in cancer treatment.

Acknowledgments

Work from the authors’ laboratory, described in thisstudy, was supported by grants from Fondo de Investi-gacion Sanitaria and European Commission (FIS-FEDER04/0843, 02/1199), Ministerio de Educacion y Ciencia(SAF2005-04293), Fundacion de Investigacion MedicaMutua Madrilena (FMM), Fundacion “la Caixa” (BM05-30-0), and Junta de Castilla y Leon (CSI04A05). C.G. wassupported by the Ramon y Cajal Program from the Ministeriode Educacion y Ciencia of Spain.

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