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Autophagosomal Membrane Serves as Platform forIntracellular Death-inducing Signaling Complex(iDISC)-mediated Caspase-8 Activation and Apoptosis*

Received for publication, September 29, 2011, and in revised form, February 17, 2012 Published, JBC Papers in Press, February 23, 2012, DOI 10.1074/jbc.M111.309104

Megan M. Young‡1, Yoshinori Takahashi‡1, Osman Khan‡2, Sungman Park‡2, Tsukasa Hori‡, Jong Yun‡,Arun K. Sharma‡, Shantu Amin‡, Chang-Deng Hu§, Jianke Zhang¶, Mark Kester‡, and Hong-Gang Wang‡3

From the ‡Department of Pharmacology and Penn State Hershey Cancer Institute, The Pennsylvania State University College ofMedicine, Hershey, Pennsylvania 17033, the §Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University,West Lafayette, Indiana 47907, and the ¶Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas JeffersonUniversity, Philadelphia, Pennsylvania 19107

Background: It remains a matter of debate whether autophagy contributes to apoptosis.Results:Atg5 and p62 are required for an intracellular death-inducing signaling complex (iDISC) formation on autophagosomalmembranes for caspase-8 self-processing.Conclusion: Autophagosome serves as a platform for the intracellular activation of caspase-8.Significance: Induction of iDISC formationmay shift cytoprotective autophagy to apoptosis formore effective cancer therapies.

Autophagy and apoptosis are two evolutionarily conservedprocesses that regulate cell fate in response to cytotoxic stress.However, the functional relationship between these two pro-cesses remains far from clear. Here, we demonstrate anautophagy-dependent mechanism of caspase-8 activation andinitiation of the apoptotic cascade in response to SKI-I, a pan-sphingosine kinase inhibitor, and bortezomib, a proteasomeinhibitor. Autophagy is induced concomitantly with caspase-8activation, which is responsible for initiation of the caspase cas-cade and the mitochondrial amplification loop that is requiredfor full execution of apoptosis. Inhibition of autophagosomeformation by depletion of Atg5 or Atg3 results in a marked sup-pression of caspase-8 activation and apoptosis. Althoughcaspase-8 self-association depends on p62/SQSTM1, its self-processing requires the autophagosomal membrane. Caspase-8forms a complex with Atg5 and colocalizes with LC3 and p62.Moreover, FADD, an adaptor protein for caspase-8 activation,associates with Atg5 on Atg16L- and LC3-positive autophago-somal membranes and loss of FADD suppresses cell death.Taken together, these results indicate that the autophagosomalmembrane serves as a platform for an intracellular death-induc-ing signaling complex (iDISC) that recruits self-associatedcaspase-8 to initiate the caspase-8/-3 cascade.

Programmed cell death (PCD)4 plays a key role in develop-ment and homeostasis (1). In addition, dysregulation of PCD is

implicated in the pathogenesis of a number of diseases, includ-ing cancer, neurodegenerative disorders, and diabetes. Apopto-sis is a well characterized mechanism of PCD that is defined bythe activation of a family of cysteine proteases known ascaspases. Caspase activation occurs through extrinsic andintrinsic signaling pathways (2, 3). Activation of the extrinsicpathway of apoptosis is initiated at the plasmamembrane by theligation of a death receptor belonging to the tumor necrosisfactor receptor superfamily. Upon activation, the multimeriza-tion of death receptors stimulates the recruitment of Fas-asso-ciated death domain (FADD) and the initiator caspase-8 toform the death-inducing signaling complex (DISC). The forma-tion of DISC results in the oligomerization of caspase-8 andthus facilitates its autoactivation through self-cleavage. In con-trast, the intrinsic pathway of apoptosis is induced in responseto intracellular stress signals, such as DNAdamage or cytotoxicstress, and is characterized by mitochondrial outer-membranepermeabilization followed by the release of apoptogenic factorsincluding cytochrome c. The initiator caspase-9 is activatedupon association with cytochrome c and the apoptotic pro-tease-activating factor 1 (Apaf-1) in a multiprotein complexknown as the apoptosome. Both apoptotic signaling pathwaysconverge upon the activation of effector caspases (caspase-3, -6,and -7), which cleave a number of cytosolic and nuclear sub-strates to execute the cell death pathway. Activated effectorcaspases also directly cleave caspase-8 to amplify the caspasecascade. Furthermore, the extrinsic pathway can initiate themitochondrial pathway through the caspase-8-mediated cleav-age of the BH3-only pro-apoptotic protein Bid.Macroautophagy, hereafter referred as autophagy, is a cata-

bolic process in which cytoplasmic components are seques-tered within membrane-enclosed autophagosomes and deliv-ered to lysosomes for degradation. The degradation throughautophagy is generally considered to be a cytoprotective mech-

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA82197 and CA129682.

1 These authors equally contributed to this work.2 These authors contributed equally to this work.3 To whom correspondence should be addressed: 500 University Dr., Hershey,

PA 17033. Tel.: 717-531-0003 (ext. 285881); Fax: 717-531-5076; E-mail:[email protected].

4 The abbreviations used are: PCD, programmed cell death; AML, acute mye-loid leukemia; Atg, autophagy-related; BiFC, bimolecular fluorescencecomplementation; FADD, Fas-associated death domain; LC3, microtubule-

associated protein light chain 3; p62/SQSTM1, sequestosome 1; SKI-I,sphingosine kinase inhibitor-I.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 15, pp. 12455–12468, April 6, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

APRIL 6, 2012 • VOLUME 287 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 12455

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anism that maintains homeostasis under exposure to environ-mental stresses, such as nutrient deprivation or hypoxia (4, 5).Paradoxically, many studies have shown that the induction ofautophagy can also contribute to caspase-dependent or -inde-pendent PCD (6–8). The formation of autophagosomes beginswith the sequestration of cytoplasmic constituents into cup-shaped membrane structures, known as isolation membranes,which are expanded and eventually sealed to form double-membrane vesicles. Although the origin of autophagosomalmembranes remains unclear, the elongation, expansion, andclosure of autophagosomal membranes have been shown torequire the Atg12-Atg5 and LC3-phosphatidylethanolamine(PE) ubiquitin-like conjugation systems (9). The ubiquitin-likeconjugations are mediated by a common E1-like activatingenzyme, Atg7, followed by E2-like conjugating enzymes, Atg10for Atg12-Atg5 and Atg3 for LC3-PE. The Atg12-Atg5 conju-gate further forms a complex with Atg16L, which acts as anE3-like enzyme to determine the site of LC3-PE conjugation(10). Moreover, loss of Atg3 has been shown to result in amarked reduction of the Atg12-Atg5 conjugates (11). Thus, thetwo conjugation systems work in concert to expand autopha-gosomal membranes.The cross-talk between apoptosis and autophagy exists to

regulate cell death (12, 13). Recent studies have shown thatseveral molecules required for autophagy also play a key role inthe regulation of apoptosis. For example, calpain-mediatedcleavage of Atg5 generates a pro-apoptotic protein fragmentthat translocates to the mitochondria and interacts with theanti-apoptotic Bcl-2 family protein Bcl-xL to stimulate themitochondrial pathway of apoptosis (14). In addition, Atg5 hasbeen shown to directly interact with FADD to stimulatecaspase-dependent cell death (15). Beclin 1, an essentialautophagy-related protein that regulates the nucleation ofautophagosomal membranes, is cleaved by caspases and trans-locates to the mitochondria to enhance apoptosis (16–18).However, the functional relationship between apoptosis andautophagy remains to be further explored. Here, we use thesphingosine kinase (SK) inhibitor SKI-I and the proteasomeinhibitor bortezomib to demonstrate the cross-talk betweenapoptosis and autophagy. SKI-I is a non-lipid pan-SK inhibitorthat inhibits both SK1 and SK2 to suppress the production ofpro-mitogenic sphingosine 1-phosphate and promote celldeath (19–21). We provide evidence that the autophagosomalmembrane serves as a platform for the intracellular activationof caspase-8 to initiate caspase cascade and apoptotic cell death.

EXPERIMENTAL PROCEDURES

Reagents—SKI-I (N�-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) was synthesizedas described (21). Antibodies were obtained from the followingsources: rabbit polyclonal anti-LC3 (Novus Biologicals, NB100-2220 for immunoblot analyses; MBL International, PM046 forimmunostaining), rabbit polyclonal anti-cleaved caspase-3(Cell Signaling, 9661), rabbit polyclonal anti-poly(ADP-ribose)polymerase (PARP) (Cell Signaling, 9542), rabbit polyclonalanti-caspase-8 (Cell Signaling, 4927S; R &D Systems, AF1650),mousemonoclonal anti-caspase-8 (Cell Signaling, 9746), rabbitpolyclonal anti-Atg16L (MBL International, PM040), mouse

monoclonal anti-Atg5 (MBL International, M153-3), mousemonoclonal anti-Bcl-xL (Sigma, B9429), guinea pig polyclonalanti-p62 (American Research Products, Inc., 03-GP62-C),mouse monoclonal anti-FADD (Enzo Life Sciences, AAM-212), rabbit polyclonal anti-DsRed (Clontech, 632496), andmouse monoclonal anti-�-actin (Sigma, A5441).Cell Culture—SV40 large T antigen immortalized Atg5�/�

and Atg5�/� mouse embryonic fibroblasts (MEF) cell lineswere provided by Dr. Noboru Mizushima (Tokyo Medical andDental University, Tokyo, Japan). SV40 large T antigen immor-talized Atg3�/� and Atg3�/� MEF cell lines were provided byDr. Shengkan (Victor) Jin (University of Medicine and Den-tistry of New Jersey-Robert Wood Johnson Medical School,NJ). FADD�/� and FADD�/� MEF cell lines were establishedusing the 3T3 protocol. KG-1 cells were obtained from ATCC(Manassas, VA).MEFs andKG-1 cells were cultured inDulbec-co’s modified Eagle’s medium and RPMI 1640medium, respec-tively, supplemented with 10% fetal bovine serum (FBS), 100�g/ml of streptomycin, 100 units/ml of penicillin, and 250ng/ml of amphotericin B.Virus Production and Transduction—The pLKO.1-based

lentiviral shRNAs targeting ATG5 (TRCN0000150940) and p62/SQSTM1 (MEF, TRCN0000098616; KG-1, TRCN0000098618)were purchased from Open Biosystems (Huntsville, AL). Thescrambled non-targeting shRNAwas purchased fromAddgene(number 1864). The mStrawberry-Atg4B(C74A) cDNA waspurchased from Addgene (number 21076) and subcloned intopCDH1-MCS1-EF1-puro lentiviral vector (NheI and SwaI).Recombinant lentiviruses were produced using the ViraPowerlentiviral expression system (Invitrogen) and transduced intotargeted cells as described previously (22). The pK1-Bcl-xL-IRES-Puro and control pK1-IRES-Puro retroviral vectors weretransfected to Amphotropic 293T cells and retroviruses encod-ing each gene were generated and transduced into targetingcells as described previously (23).Cell Death Assays—Cell viability was measured using a lucif-

erase-coupled ATP quantification assay according to the man-ufacturer’s protocol (CellTiter-Glo viability assay, PromegaG7570). Luminescence intensity was measured using a VictorX5 plate reader (PerkinElmer Life Sciences). To determine apo-ptosis, cells were stained with annexin V-PE (BD Bioscience,number 559763) and analyzed using a Guava EasyCyte PlusFlow Cytometry System (Millipore).Caspase Activity Assays—To determine caspase activity,

total cellular lysates were prepared in CHAPS lysis buffer (150mM NaCl, 10 mM HEPES, pH 7.4, 1% CHAPS) containing pro-tease inhibitors. Caspase-3 activity was determined by meas-uring the DEVDase activity as described previously (24).Caspase-8 activity was determined by measuring the IETDaseactivity using a caspase-8 assay kit (Sigma, number CASP8F)according to the manufacturer’s protocol.Bimolecular Fluorescence Complementation (BiFC) Assay—

Mouse Atg5 (SalI and KpnI) and mouse FADD (EcoRI and XhoI)cDNAs were subcloned into the N- and C-terminal Venus frag-ment expression vectors, pBiFC-VN155(I152L) and pBiFC-VC155, respectively. Human caspase-8 (C360A) catalytic mutant(Addgene, 11818) (SalI and KpnI) was subcloned into pBiFC-VN155 (I152L) and pBiFC-VC155 vectors. MEFs were grown on

Autophagy-dependent iDISC Formation

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gelatinized coverslips or NUNC Lab-Tek II chamber slides andtransfected with BiFC pairs using FuGENE HD (Promega) orLipofectamine LTX (Invitrogen) according to the manufacturer’sprotocol. For Atg5-VN and FADD-VC co-transfection, 50 �M

Z-VAD-fmk was added 1 h post-transfection to prevent caspase-dependent cell death. The BiFC signals were detected using anOLYMPUS IX81 deconvolution microscope and analyzed usingSlideBook 5.0 software (Intelligent Imaging Innovations).Coimmunoprecipitation Assay—Total cell lysates prepared

in DISC IP lysis buffer (1% Triton X-100, 150 mM NaCl, 10%glycerol, 30 mM Tris-HCl, pH 7.5) containing protease inhibi-tors were precleaned by incubating with anti-mouse IgG-con-jugated agarose beads (Sigma, A0919) for 2 h at 4 °C and sub-jected to immunoprecipitation with anti-GFP monoclonalantibodies (Roche Diagnostics, number 11814460001). Theresulting immunocomplexes were washed three times with thelysis buffer and subjected to immunoblot analyses.

RESULTS

SKI-I Induces Caspase-dependent Cell Death Accompaniedwith Induction of Autophagy—Although the antitumor effectsof SKI-I have been largely attributed to the induction of apo-ptosis, the precise mechanism by which SKI-I induces apopto-sis is unknown. To investigate the detailed mechanism of SKI-I-induced cell death, SV40 large T antigen-immortalizedMEFswere treated with SKI-I.MEFs were selected based on the avail-ability of genetic models that permit the study of apoptosis andautophagy. As shown in Fig. 1A, significant cleavage ofcaspase-3 was detected 24 h after SKI-I treatment, supportingthe previous finding that SKI-I triggers the induction of apopto-sis (19). Interestingly, we found that exposure of MEFs to SKI-Iresulted in the accumulation of the autophagymarker LC3-II ina time-dependent manner. Moreover, SKI-I treatmentincreased the number of GFP-LC3 puncta (Fig. 1B), a well char-acterized marker used to visualize autophagosomes (25). As aportion of LC3-II is degraded by autophagosome fusion withlysosomes (25), we next examinedwhether the accumulation ofLC3-II observed during SKI-I treatment was due to an increasein autophagy induction or a decrease in autophagic degradationby treating cells in the presence or absence of lysosomal inhib-itors including bafilomycin A1, chloroquine (CQ), and ammo-nium chloride (NH4Cl). We found that inhibition of lysosomaldegradation resulted in a further accumulation of LC3-II inresponse to SKI-I (Fig. 1C). This clearly indicates that SKI-Ipromotes the induction of autophagy. Furthermore, the addi-tion of lysosomal inhibitors significantly enhanced SKI-I-in-duced caspase-3 processing (Fig. 1C). Notably, cleavedcaspase-3 does not colocalize with the lysosomal markerLamp1 during SKI-I treatment (Fig. 1, D and E), thereby sug-gesting that lysosomal degradation of autophagosomes ratherthan active caspase-3 serves as a cell survivalmechanismduringSKI-I treatment. In contrast, SKI-I-induced cell death was sig-nificantly suppressed by co-treatment with a pan-caspaseinhibitor, Z-VAD-fmk (Fig. 1F). Taken together, these resultsindicate that SKI-I-induced cell death occurs primarily throughthe caspase-dependent apoptotic pathway.

The LC3 Conjugation System Is Required for SKI-I-inducedApoptosis—Although autophagic degradation functions as apro-survival mechanism to maintain cellular homeostasisthrough nutrient recycling and the removal of aggregated pro-teins and malfunctioning organelles (4, 5), autophagy has alsobeen shown to contribute to caspase-dependent and/or -inde-pendent cell death (6–8). Thus, we next examined whether theformation of autophagosomes is involved in cell death inducedby SKI-I. To this end,Atg5�/� andAtg5�/� MEFs were treatedwith SKI-I or control DMSO. Atg5 is a member of theautophagy-related (Atg) protein family that covalently binds toAtg12, an ubiquitin-like protein, and is required for the forma-tion of autophagosomes (26, 27). Consistently, we found thatSKI-I-induced LC3-II conversion was completely abrogated byloss of Atg5 (Fig. 2A). Notably, SKI-I-induced cleavage ofcaspase-3 and PARP, a substrate for caspases, was also sup-pressed by depletion of Atg5. Furthermore, a significant reduc-tion of apoptotic cell death was observed in SKI-I-treatedAtg5�/� MEFs as compared with their control wild-type cells(Fig. 2, B, E, and F).In addition to its role in autophagosome formation, Atg5 has

been shown to promotemitochondrial apoptosis by interactingwith Bcl-xL (14). To determine whether the SKI-I-resistantphenotypeweobserved inAtg5-deficient cells is specifically dueto the impairment of autophagosome formation, we nexttreated Atg3�/� and Atg3�/� MEFs with SKI-I. Atg3 is anE2-like enzyme for the conjugation of LC3-I and PE to formLC3-II, which is indispensable for elongation and closure ofautophagosomal membranes (11). Similar to the resultsobtained using Atg5-deficient cells, loss of Atg3 resulted in amarked inhibition of the cleavages of caspase-3 and PARP andapoptotic cell death in response to SKI-I (Fig. 2, C–F). Collec-tively, these results indicate that the expansion of autophago-somal membranes is critical for the activation of caspase-3 andthe subsequent cell death pathway upon SKI-I treatment.Importantly, SKI-I-induced apoptosis was also inhibited by lossof the pro-apoptotic Bcl-2 family genes, Bax and Bak, suggest-ing that the mitochondrial pathway is important for mediatingSKI-I-induced apoptosis (Fig. 2, E and F).Activation of Caspase-8 Is Critical for Initiation of Atg5-de-

pendentCaspaseCascade during SKI-I Treatment—Our resultsshown above clearly indicate that inhibition of the early stages(i.e. autophagosome formation) but not the late stages ofautophagy (i.e. lysosomal degradation) attenuates SKI-I-in-duced apoptosis. It has recently been reported that autophagyinduction plays a role in regulating caspase-8 activation andsubsequent cell death, although the precise role of autophagy inthis process is not well defined (6, 15, 28, 29).We therefore nextinvestigated whether activation of caspase-8 is involved in SKI-I-induced cell death.We found that SKI-I treatment resulted incleavages of caspase-8 and -3 in wild-type MEFs (Fig. 3A). SKI-I-induced procaspase-8 (pro-Casp-8) processing was inhibitedby the addition of Z-VAD-fmk, indicating that this process ismediated by caspases.Moreover, SKI-I-induced caspase-3 acti-vation was blocked not only by Z-VAD-fmk but also byZ-IETD-fmk, a caspase-8-specific inhibitor (Fig. 3B). Impor-tantly, loss of Atg5 drastically suppressed SKI-I-inducedcaspase-8 cleavage (Fig. 3A). Taken together, these results sug-




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gest that caspase-8 activation is a critical step in Atg5-depen-dent apoptosis induced by SKI-I.Because SKI-I-induced cell death was also suppressed by

loss of Bax and Bak (Fig. 2E), we next investigated whetherthe mitochondrial pathway is required for the initiation oramplification of the caspase cascade triggered by SKI-I. Tothis end, Atg5�/� and Atg5�/� MEFs were infected withrecombinant retroviruses encoding anti-apoptotic Bcl-xL orempty vector. After selection with puromycin, the expres-sion of Bcl-xL was confirmed by Western blot analyses (Fig.3D). SKI-I-induced cleavage and activation of caspase-3

were greatly suppressed by overexpression of Bcl-xL in bothAtg5�/� and Atg5�/� cells (Fig. 3, C and D). Moreover,Bcl-xL overexpression also significantly diminished SKI-I-induced caspase-8 cleavage and activation in wild-type cells(Fig. 3, C and E), indicating that the mitochondrial amplifi-cation loop is required for the full activation of caspase-8upon SKI-I treatment. Notably, little effect of Bcl-xL overex-pression was observed on the activation of caspase-8 inAtg5-deficient cells, indicating that Atg5 acts upstream of themitochondrial pathway to initiate the caspase cascade inresponse to SKI-I (Fig. 3E).

FIGURE 1. SKI-I simultaneously induces autophagy and caspase-dependent cell death. A, MEFs were treated with 2.5 �M SKI-I for the indicated periods oftime and subjected to immunoblot analyses using the indicated antibodies. B, MEFs stably expressing GFP-LC3 were treated with 2.5 �M SKI-I or control DMSOfor 12 h and the number of GFP-LC3 dots per cell area (1000 �m2) was determined using a fluorescence microscope (mean � S.D.; n � 36). Statisticalsignificance was determined by Student’s t test. C, MEFs were treated with 2.5 �M SKI-I or control DMSO for 8 h followed by a 4-h co-treatment with 100 nM

bafilomycin A1, 25 �M CQ, 20 mM NH4Cl, or control PBS and subjected to immunoblot analyses using the indicated antibodies. D, MEFs were treated with 2.5�M SKI-I for 12 h, stained with anti-Lamp1 monoclonal and anti-active caspase-3 (C-Casp-3) polyclonal antibodies, and analyzed by fluorescence deconvolutionmicroscopy. Magnified images are shown as insets. E, the fluorescence intensities along the dotted line in D were quantified using SlideBook software. Thevalues of the vertical axis represent fluorescence intensity units (ADU). The horizontal axis represents distance (S, start point; E, end point). F, MEFs were treatedwith control DMSO or 2.5 �M SKI-I in the presence of 20 �M Z-VAD-fmk or control DMSO for 24 h and cell viability was assessed by measuring cellular ATP levels(mean � S.D.; n � 3). The scale bars represent 10 �m in A and D, and 1 �m in the insets in D.




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SKI-I Promotes Self-association of Caspase-8 in a MannerIndependent of Atg5—Oligomerization is a key step forcaspase-8 processing and activation (30). To determinewhether Atg5-containing autophagosomal membranes arerequired for the recruitment and/or oligomerization ofcaspase-8, we performed BiFC assays. BiFC is based on the for-mation of a fluorescent complex by two non-fluorescent frag-ments of Venus, VN155(I152L) (VN) andVC155 (VC), broughttogether by association of proteins fused with each Venus frag-ment (31). To this end, pro-Casp-8 (C360A) was fused witheach Venus fragment and transfected to Atg5-deficient andcontrol wild-type cells. We utilized a proteolytically inactiveC360A mutant of pro-Casp-8 to prevent apoptosis inductiondue to overexpression of caspase-8 (32). SKI-I induced numer-ous fluorescent puncta as well as large aggregates throughoutthe cytoplasm of wild-type cells, whereas little signal wasdetected before the treatment (Fig. 4A). This result indicatesthat SKI-I promotes self-association and oligomerization of

pro-Casp-8. Importantly, such fluorescent signals were notinduced upon SKI-I treatment in cells transfected with controlempty VN and VC (data not shown). To our surprise, loss ofAtg5 did not suppress SKI-I-induced caspase-8 self-association.Taken together, these results indicate that, whereas SKI-I-in-duced activation of caspase-8 requires Atg5, the autophago-somal membrane is dispensable for caspase-8 self-association.p62-mediated Self-association of Caspase-8 Plays a Regula-

tory Role in Atg5-dependent Caspase-8 Activation and Apopto-sis Induced by SKI-I—p62 is an adapter protein that recruitspolyubiquitinated and aggregated proteins to autophagosomesthrough its direct interaction with LC3-II (33, 34). Recent stud-ies have shown that p62 interacts with polyubiquitinatedcaspase-8 and promotes oligomerization and activation ofcaspase-8 (29, 35). To determine whether p62 is involved inSKI-I-induced caspase-8 oligomerization and subsequent acti-vation of the caspase cascade, Atg5�/� and Atg5�/� MEFs sta-bly expressing p62 shRNA (shp62) or control scrambledshRNA (shScr) were transfected with pro-Casp-8 (C360A)-VNand pro-Casp-8 (C360A)-VC, treated with SKI-I, and stainedwith anti-p62 antibodies. As shown in Fig. 4B, a portion of SKI-I-induced caspase-8 homocomplexes were co-localized withp62 in both wild-type and Atg5-deficient cells. These SKI-I-induced BiFC signals were greatly diminished by knockdown ofp62, indicating that p62 plays a key role in mediating caspase-8self-association during SKI-I treatment.Moreover, by co-trans-fecting the pro-Casp-8 (C360A) BiFC constructs with mRFP-LC3, we found that SKI-I-induced caspase-8 homocomplexeswere also positive for the autophagosomal membrane markerLC3 (Fig. 5A). Furthermore, a portion of endogenous caspase-8colocalized with p62 and LC3 upon SKI-I treatment (Fig. 5, Band C). Taken together, these results suggest that SKI-I pro-motes p62-dependent self-association of pro-Casp-8 and itsrecruitment to autophagosomal membranes.Wenext examined the effect of p62 knockdownon activation

of the caspase cascade during exposure to SKI-I. As expected,knockdown of p62 partially suppressed apoptotic cell death inwild-type cells treated with SKI-I (Figs. 6, A and E). Consis-tently, SKI-I-induced cleavage and activation of caspase-8 and-3 were significantly reduced by knockdown of p62 in wild-typecells (Fig. 6, B–D). Notably, despite nearly complete suppres-sion of p62 expression (Fig. 6B), the effect of p62 knockdown onSKI-I-induced caspase-8 and caspase-3 activation was not asdramatic as that seen in Atg5 knock-out cells (Fig. 6, C and D).These results suggest that a portion of caspase-8 is activatedthrough an additional Atg5-dependent mechanism. Interest-ingly, knockdown of p62 inAtg5-deficient cells failed to furtherreduce, but rather slightly enhanced, SKI-I-induced cell deathand caspase-3 activation (Fig. 6,A–C). In addition tomediatingcaspase-8 self-association, p62 promotes the oligomerization ofTRAF6 to enhance the activation of NF-�B (36). Furthermore,p62 positively regulates the transcription factor Nrf2, which isresponsible for transcription of cytoprotective genes (37).Therefore, knockdown of p62 may also suppress cell survivalpathways to enhance apoptosis in response to SKI-I.SKI-I Induces Translocation of Caspase-8 and FADD to Atg5-

positive Autophagosomal Membranes—It has recently beensuggested that the Atg12-Atg5 complex interacts with the

FIGURE 2. Inhibition of autophagosome formation by depletion of Atg5or Atg3 suppresses SKI-I-induced apoptosis. MEFs with the indicated gen-otypes were treated with 2.5 �M SKI-I or control DMSO for 24 h. A and C, totalcell lysates were subjected to immunoblot analyses using the indicated anti-bodies. B and D, representative micrographs are shown. E, cell viability wasdetermined by ATP assays (mean � S.D.; n � 3). Data are shown as percentageof control DMSO treatment. F, the induction of apoptosis was determined byannexin V staining. Drug-specific apoptosis was calculated by subtracting thepercentage of annexin V-positive cells in DMSO-treated samples (mean �S.D.; n � 3).




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death adaptor protein FADD to recruit caspase-8 (15). Todetermine whether association of FADD and the Atg12-Atg5complex on the autophagosomal membrane serves as an addi-tional mechanism for caspase-8 recruitment and activation inresponse to SKI-I, we first analyzed the spatial association ofAtg5 and FADD by BiFC. We found that strong punctate sig-nals of Venus were accumulated in the cytoplasm of Atg5�/�

MEFs transfected with Atg5-VN and FADD-VC, whereas dif-fuse cytoplasmic and nuclear signals were detected in cellsexpressing control empty VN and VC (Fig. 7A). The BiFC sig-

nals from Atg5-VN and FADD-VC were positive not only forLC3 but also for an isolationmembranemarker, Atg16L. Inter-estingly, the majority of cells transfected with Atg5-VN andFADD-VC but not control vectors eventually underwent apo-ptotic cell shrinkage and death (data not shown). As the fluo-rescent complex formation in BiFC is essentially irreversible(31), formation of the stabilized Atg5�FADD complex mayresult in sustained recruitment and activation of caspase-8leading to cell death. The interaction of Atg5, FADD, and pro-caspase-8 was confirmed by coimmunoprecipitation analyses

FIGURE 3. Loss of Atg5 suppresses SKI-I-induced activation of mitochondrial amplification loop that is initiated by caspase-8. A, Atg5�/� and Atg5�/�

MEFs were treated with 1.5 �M SKI-I or control DMSO in the presence of 20 �M Z-VAD-fmk or control DMSO for 24 h and subjected to immunoblot analyses usingthe indicated antibodies. The cleaved caspase-8 was quantified as the percentage of total caspase-8. B, Atg5�/� MEFs were treated with 1.5 �M SKI-I or controlDMSO in the presence of 20 �M Z-IETD-fmk, 20 �M Z-VAD-fmk, or control DMSO for 24 h and the caspase-3/7-like DEVDase activity was measured (mean � S.D.;n � 3). C–E, Atg5�/� and Atg5�/� MEFs were infected with retroviruses encoding Bcl-xL or empty control. After selection with 1 �g/ml of puromycin, theresultant stable transfectants were subjected to treatment with 1.5 �M SKI-I or control DMSO for 24 h. C, total cell lysates were subjected to immunoblotanalyses using the indicated antibodies. Asterisks indicate nonspecific bands. D, the caspase-3/7-like DEVDase activity and E, the caspase-8-like IETDase activitywere measured (mean � S.D.; n � 3).




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(Fig. 7B). Notably, whereas the association of Atg5 with pro-caspase-8 was enhanced by SKI-I treatment, it decreased uponlonger incubation with SKI-I. This suggests that activated/cleaved caspase-8 was released into the cytosol in a mannersimilar to observed upon TNF-� treatment (38). Indeed,whereas a large portion of FADD signals were found to be colo-calized with Atg5 during SKI-I treatment, only a small fractionof caspase-8 signals were detected on Atg5 and FADD-positivestructures (Fig. 7,C andD). Moreover, loss of FADDwas foundto suppress SKI-I-induced cell death (Fig. 7E). To further exam-ine the role of Atg5-containing autophagosomal membranes inSKI-I-induced apoptosis, we generated MEFs stably overex-pressing a dominant-negative mutant of Atg4B (mStrawberry-

Atg4B (C74A)). The protease Atg4B is one of four mammalianAtg4 homologues required for processing pro-LC3 paraloguesprior to lipidation, a key step in autophagosome biogenesis. Inaddition, Atg4 catalyzes the delipidation and release of LC3from autophagosomal membranes during, or before, autopha-gosome-lysosome fusion. Previous studies have shown thatoverexpression of the inactive mutant Atg4B (C74A) blocksLC3 modification and autophagosome closure to result in theaccumulation of unsealed, Atg5-positive membrane structures(39). Consistently, SKI-I-induced LC3 modification was com-pletely suppressed in cells overexpressing dominant-negativeAtg4B (Fig. 7F). Interestingly, we found that treatment withSKI-I significantly stabilized dominant-negative Atg4B protein

FIGURE 4. SKI-I promotes caspase-8 self-association in a p62-dependent but Atg5-independent manner. A, Atg5�/� and Atg5�/� MEFs were transfectedwith pro-Casp-8 (C360A)-VN and pro-Casp-8 (C360A)-VC for 24 h, treated with 2.5 �M SKI-I for 0 and 6 h, and analyzed by fluorescence microscopy (top) incombination with differential interference contrast microscopy (bottom). B, Atg5�/� and Atg5�/� MEFs were infected with lentiviruses encoding shScr or shp62and selected with 1 �g/ml of puromycin. Cells were transfected with pro-Casp-8 (C360A)-VN and pro-Casp-8 (C360A)-VC for 24 h, treated with 2.5 �M SKI-I for6 h, stained with guinea pig anti-p62 polyclonal antibodies, and analyzed by fluorescence deconvolution microscopy. Arrows indicate colocalization ofcaspase-8 complexes with p62. The scale bars represent 20 �m in A and 10 �m in B.




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expression through an unknown mechanism. Importantly,despite the absence of LC3-II, overexpression of dominant-negative Atg4B substantially enhanced SKI-I-inducedcaspase-8 and caspase-3 cleavage compared with wild-typecells (Fig. 7F), supporting the notion that the autophagosomalmembrane serves as a platform for caspase-8 activation andinduction of apoptosis. Taken together, these results suggestthat the recruitment and activation of caspase-8 in response toSKI-I occurs not only in a p62�LC3-II-dependent manner butalso through the association of Atg5�FADD at the autophago-somal membrane.SKI-I Induces Atg5-dependent Apoptosis in KG-1 Cells

Enhanced by Stabilization of p62 through ProteasomeInhibition—The apoptotic and antitumor effects of SKI-I havepreviously been demonstrated in several human cancer celllines as well as a murine model of mammary adenocarcinoma

(19, 40). We sought to investigate whether SKI-I induces anautophagy-dependent activation of the apoptotic cascade inhuman acute myeloid leukemia (AML), a model in which sus-tained SK activity has been associated with chemoresistance(41). To this end, we treated several human AML cell linesincluding KG-1, HL-60, and multidrug-resistant HL-60/VCRwith SKI-I. We found that SKI-I concomitantly inducesautophagy and apoptosis in all cell lines tested (data notshown). To determine whether Atg5 and p62 are responsiblefor the induction of apoptosis triggered by SKI-I, KG-1 cellsstably expressing ATG5 shRNA (shATG5), shp62, or controlshScrwere generated using a lentiviral transduction system andtreated with SKI-I or control DMSO. We chose KG-1 cells forfurther studies as, in addition to the capability to induce themost prominent autophagy, the most effective knockdown ofthe targeted genes was achieved in this cell line among all AML

FIGURE 5. Caspase-8 homocomplex is recruited to the autophagosomal membrane during SKI-I treatment. A, Atg5�/� MEFs stably expressing shScr orshp62 were transfected with pro-Casp-8 (C360A)-VN, pro-Casp-8 (C360A)-VC, and mRFP-LC3. Twenty hours after transfection, the cells were treated with 2.5 �M

SKI-I for 6 h and subjected to fluorescence deconvolution microscopic analyses. Nuclei were stained with DAPI. B, Atg5�/� MEFs were infected with retrovirusesencoding GFP-LC3 and selected with 1 �g/ml of puromycin for 5 days. The resultant stable transfectants were treated with 2.5 �M SKI-I or control DMSO for 12 h,immunostained with anti-p62 and anti-caspase-8 antibodies, and analyzed by fluorescence deconvolution microcopy. C, the intensity profiles for eachfluorescence along the dotted line in B are shown. Arrows indicate colocalization of caspase-8 complexes with mRFP-LC3 (A) or caspase-8 with p62 and GFP-LC3(B). The scale bars represent 10 �m in A and B, and 1 �m in the inset in B.




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cell lineswe tested (data not shown). Consistentwith the resultsobtained using MEFs, knockdown of either ATG5 or p62 sup-pressed SKI-I-induced caspase-8, caspase-3, and PARP cleav-ages and the induction of apoptosis in KG-1 cells (Figs. 8,A andD). Moreover, co-treatment with CQ enhanced SKI-I-inducedapoptosis in KG-1 cells (Fig. 8B), supporting the notion thatlysosomal degradation of autophagosomes serves as a cell sur-vivalmechanismupon SKI-I treatment.Notably, extensive deg-radation of p62 was observed in KG-1 cells upon SKI-I treat-ment (Fig. 8A). Degradation of p62 occurs by autophagy and theproteasome system (42). As knockdown of ATG5 did not pre-vent, but rather slightly enhanced, p62 degradation in responseto SKI-I, SKI-I-inducedp62degradationwas suggested to occurin a proteasome-dependent manner (Fig. 8A). The enhanceddegradation of p62 observed in KG-1 cells upon SKI-I treat-ment compared with that of MEFs correlates with a significantreduction of SKI-I-induced apoptosis in this cell line (14 versus29%; Figs. 2F and 8B). Therefore, the rapid turnover of p62mayaffect the efficacy of SKI-I in KG-1 cells.

To determine whether stabilization of p62 could enhanceSKI-I-induced apoptosis in KG-1 cells, we next treated cells incombination with SKI-I and the proteasome inhibitor bort-ezomib. As expected, treatment with bortezomib resulted inthe accumulation of p62, indicating that p62 degradation dur-ing SKI-I treatment occursmainly through the proteasome sys-tem rather than autophagy (Fig. 8C). Accordingly, a combina-tion treatment with SKI-I and bortezomib increased thecleavages of caspase-8, caspase-3, and PARP and thus greatlyenhanced apoptosis in control shScr-expressing KG-1 cells(Fig. 8, C and D). Despite the accumulation of p62 upon com-bination treatment, this effect was blocked by knockdown ofATG5 (Fig. 8, C and D). Taken together, these results indicatethat SKI-I induces Atg5-dependent apoptosis in AML cells andthat this process is further enhanced by proteasome inhibitionthrough the stabilization of p62. Notably, similarly to SKI-I,bortezomib-induced caspase-8 and -3 activation was also sup-pressed by knockdown of either p62 or ATG5 (Fig. 8, C and E),a result consistent with previous reports (6, 29).

DISCUSSION

The induction of autophagy is generally considered an adapt-ive and a cytoprotective mechanism for the recycling of nutri-ents and the removal of cytotoxic materials (4, 5). However,mounting evidence has suggested that autophagy is also impli-cated in the induction of caspase-dependent and -independentcell death through pathways that need to be explored further (5,13). In the present study, we have demonstrated a mechanismof apoptosis that is dependent on the autophagosomal mem-brane.We found that activation of caspase-8 is a critical step inthe autophagy-dependent induction of apoptosis in response toSKI-I, a pan-SK inhibitor, and bortezomib, a proteasome inhib-itor. Furthermore, we have shown that the expansion ofautophagosomal membranes is essential for the intracellularactivation of caspase-8. Our data demonstrates that SKI-I-in-duced self-association of caspase-8 is dependent on p62. Con-sistently, p62 has recently been shown to mediate caspase-8oligomerization and activation in response to TRAIL or protea-some inhibition (29, 35). Importantly, we found that SKI-I-in-duced self-association of caspase-8 occurs independently ofAtg5; however, Atg5 is required for the activation of caspase-8.These observations therefore suggest that self-associatedcaspase-8 is recruited to the autophagosomal membrane toform the proper higher order oligomer structure for activation.To support this concept, forced membrane localization andself-association of caspase-8 has been shown to dramaticallypromote apoptosis (43). Furthermore, it has recently been sug-gested that caspase-8 is recruited to the Atg12-Atg5 complexthrough FADD and that autophagic machinery is required foractivation of caspase-8 (6, 15, 28). In this study, we show thatthe caspase-8�FADD complex associates with Atg5 on Atg16-and LC3-positive structures, indicating that the autophago-somal membrane serves as a platform for this interaction. Col-lectively, these results prompt us to propose a model in whichthe autophagosomal membrane serves as a platform for theformation of a dual-armed iDISC that facilitates the activationof caspase-8 and initiation of apoptosis. Elongation of autopha-gosomal membranes occurs in a manner that is dependent on

FIGURE 6. Knockdown of p62 suppresses SKI-I-induced caspase-3 andcaspase-8 activation. Atg5�/� and Atg5�/� MEFs stably expressing shScr orshp62 were treated with 2.5 �M SKI-I or control DMSO for 24 h. A, representa-tive micrographs of SKI-I-treated cells are shown. B, total cell lysates weresubjected to immunoblot analyses using the indicated antibodies. C, thecaspase-3/7-like DEVDase activity and D, the caspase-8-like IETDase activitywere measured (mean � S.D.; n � 3). E, the cells were stained with annexin V,and drug-specific apoptosis was calculated by subtracting the percentage ofannexin V-positive cells in DMSO-treated samples (mean � S.D.; n � 3).




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FIGURE 7. SKI-I induces translocation of caspase-8 and FADD to Atg5-positive autophagosomal membranes. A, Atg5�/� MEFs were transfected incombination with Atg5-VN and FADD-VC or control empty VN and VC for 18 h. Cells were fixed, immunostained with anti-LC3 and anti-Atg16L antibodies, andanalyzed by fluorescence microcopy. B, Atg5�/� MEFs were infected with retroviruses encoding GFP-Atg5 and selected with 1 �g/ml of puromycin for 5 days.The resultant stable transfectants were treated with 2.5 �M SKI-I for the indicated times and subjected to immunoprecipitation with anti-GFP monoclonalantibodies or control mouse IgG followed by immunoblot analyses using the indicated antibodies. C, Atg5�/� MEFs stably expressing GFP-Atg5 were treatedwith 2.5 �M SKI-I or control DMSO for 12 h, immunostained with anti-FADD and anti-caspase-8 antibodies, and analyzed by fluorescence deconvolutionmicrocopy. Arrowheads and arrows indicate colocalization of Atg5 with FADD, and Atg5 and FADD-positive signals with caspase-8, respectively. D, the intensityprofiles for each fluorescence along the dotted line in C are shown. The scale bars represent 10 �m in A and C, and 5 �m in the inset in C. E, FADD�/� and FADD�/�

MEFs were treated with 5 �M SKI-I or control DMSO for 24 h. Representative micrographs are shown. F, Atg5�/� MEFs were infected with lentiviruses encodingmStr-Atg4B(C74A) or empty control. After selection with 1 �g/ml of puromycin, the resultant stable transfectants were subjected to treatment with 2.5 �M SKI-Ior control DMSO for 16 h. Total cell lysates were subjected to immunoblot analyses using the indicated antibodies. Asterisks indicate nonspecific bands.Cleaved caspase-8 was quantified as the percentage of total caspase-8. Cleaved caspase-3 was quantified as the relative expression after normalization to�-actin.




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the Atg12-Atg5 and LC3-PE ubiquitin-like conjugation sys-tems. In our model, FADD is recruited to expanding autopha-gosomalmembranes through interactions with Atg5. In aman-ner analogous to DISC formation upon death receptor ligation(45), FADD recruits caspase-8 to the autophagosomal mem-brane to promote self-activation of caspase-8. Additionally,caspase-8 self-association and recruitment to the autophago-somalmembrane occurs in a p62-dependentmanner. Here, theassociation of LC3-II and p62 mediates the recruitment of self-associated caspase-8 to the autophagosomalmembranes for theformation of proper oligomer structures to facilitate caspase-8self-activation. Furthermore, the mitochondrial amplificationloop is indispensable for the full activation of the iDISC-medi-ated cell death pathway.

Our data clearly indicates that autophagy induction plays akey role in SKI-I-induced apoptosis. However, despite impairedautophagic activity, a slight induction of apoptosis is observedin Atg5- and Atg3-deficient MEFs. Inhibition of SK blocks theformation of pro-mitogenic sphingosine 1-phosphate (S1P) toaccumulate pro-apoptotic precursors, sphingosine and cer-amide. Ceramide accumulation has been shown to induce theintrinsic pathway of apoptosis through several mechanismsincluding direct permeabilization of the mitochondrial mem-brane, enhanced activation of Bax, and direct activation of thelysosomal protease cathepsin D (46). As a result, the accumu-lation of pro-apoptotic lipids, sphingosine and ceramide, islikely responsible for activation of the intrinsic pathway inautophagy deficient cells. Importantly, we also found that SKI-I

FIGURE 8. Inhibition of proteasome-mediated p62 degradation enhances the efficacy of SKI-I to induce Atg5-dependent apoptosis in AML cells. KG-1cells were infected with lentiviruses encoding shScr, shATG5, or shp62 and selected with 1 �g/ml of puromycin. KG-1 cells were infected with lentivirusesencoding shScr, shATG5, or shp62 and selected with 1 �g/ml of puromycin. A and C, the resultant stable transfectants were treated with or without 2.5 �M SKI-Iin the presence or absence of 3 nM bortezomib (Bort) for 24 h and subjected to immunoblot analyses using the indicated antibodies. B and D, parental KG-1 cells(B) or the stable transfectants (D) were treated with 2.5 �M SKI-I or control DMSO in the presence or absence of 25 �M CQ for 24 h (B) or 3 nM bortezomib for 48 h(D), stained with annexin V and 7-amino-actinomycin D (7-AAD), and analyzed flow cytometry. The percentage of annexin V-positive cells is shown. Data shownare representative of two independent experiments. E, the stable transfectants were treated with 5 nM bortezomib for 24 h and subjected to immunoblotanalyses.




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promotes the degradation of p62 through proteasome in AMLcells. Consistently, activation of the proteasome machinery inresponse to SK inhibition has been reported (47). As the addi-tion of bortezomib stabilized p62 and enhanced the autophagy-dependent activation of caspase-8 and apoptosis upon SKI-Itreatment, inhibition of proteasome may be a key factor toincrease the efficacy of SKI-I-induced cell death. In addition,wedetected a rapid autophagic flux in AML cell lines (data notshown). As the Atg12-Atg5 conjugate dissociates from theautophagosomal membrane upon completion (27), a rapidturnover of autophagosomes in cancer cells may limit the for-mation of iDISC and subsequent caspase-8 activation duringSKI-I treatment. Furthermore, rapid autophagic flux may alsolead to lysosomal degradation of the iDISC as well as damagedmitochondria associated with the autophagosomal membrane.Therefore, inhibiting autophagic flux is one approach to stabi-lize pro-apoptotic components on the autophagosomal mem-brane and shift autophagy to caspase-dependent cell death. Tosupport this concept, inhibition of lysosomal degradation wasfound to enhance SKI-I-induced apoptosis in AML cells andcaspase-3 cleavage in MEFs. Interestingly, chloroquine is cur-rently being evaluated as an enhancing agent for cancer therapydue to its ability to selectively sensitize cancer cells to ionizingradiation and several anti-neoplastic drugs (48–52). Moreover,inhibition of the closure of autophagosomalmembranes shouldstabilize the formation of iDISC and enhance apoptosis. Indeed,we have shown that inhibition of autophagosomal closurethrough the overexpression of a dominant-negative mutant ofAtg4B substantially enhanced SKI-I-induced caspase-8 andcaspase-3 cleavage. As there are currently no pharmacologicalinhibitors that target autophagosome completion, this remainsan area to be addressed in future drug discovery.The mechanism behind the induction of autophagy by SKI-I

remains to be determined. Consistent with our results, inhibi-tion of SK by other inhibitors, such as dimethylsphingosine, thepan-SK inhibitor SKI-2, and the sphingosine kinase-2-specificinhibitor ABC294640, have been shown to induce autophagy(53). The induction of autophagy in response to SK inhibitionmay arise from a decrease in S1P production or the accumula-tion of sphingosine and/or ceramide. S1P has been shown tomediate cell survival by activating the phosphoinositide 3-ki-nase/protein kinase B (PI3K/Akt) signaling pathway andnuclear factor �B (NF-�B), both of which exert inhibitoryeffects on autophagy (54, 55). As a result, suppressed S1P pro-duction may relieve the inhibitory effects of these pathways onautophagy. Additionally, S1P protects against apoptosis byblocking the activation of the stress-activated protein kinaseJun amino-terminal kinase (JNK), whereas ceramide activatesJNK (56–58). JNK phosphorylates Bcl-2 to liberate Beclin 1 forthe induction of autophagy (59). Moreover, JNK activates tran-scription factor c-Jun, which has been shown to mediate theup-regulation of Beclin 1 (44, 60). In addition, the induction ofautophagy by ABC294640 has been reported to be accompa-nied not only by a decreased activation of Akt but also by anincreased expression of Beclin 1 (53). However, no obviousBeclin 1 up-regulation was detected during SKI-I treatment inour system (data not shown). Consequently, the mechanism of

autophagy induction in response to SK inhibition is currentlyunder investigation.In conclusion, the present findings, together with those of

recent studies in T cell clonal expansion (15), proteasome inhi-bition (29), and oncolytic adenovirus (28), indicate that theautophagosomal membrane may serve as a platform for iDISCformation, which activates caspase-8 and the caspase cascade,leading to autophagy-dependent apoptosis. As many antican-cer therapies induce both apoptosis and autophagy, establish-ment of an autophagy-dependent mechanism of caspase acti-vation reveals a novel strategy to enhance therapeutic efficacyin tumor cells.

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and Hong-Gang WangJong Yun, Arun K. Sharma, Shantu Amin, Chang-Deng Hu, Jianke Zhang, Mark Kester Megan M. Young, Yoshinori Takahashi, Osman Khan, Sungman Park, Tsukasa Hori,

Signaling Complex (iDISC)-mediated Caspase-8 Activation and ApoptosisAutophagosomal Membrane Serves as Platform for Intracellular Death-inducing

doi: 10.1074/jbc.M111.309104 originally published online February 23, 20122012, 287:12455-12468.J. Biol. Chem.

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