Cytokine & Growth Factor Reviews 24 (2013) 311–318

Crosstalk between ER stress and immunogenic cell death

Oliver Kepp a,b,c,1, Laurie Menger a,b,c,1, Erika Vacchelli a,b,c, Clara Locher c,d,e,Sandy Adjemian a,b,c, Takahiro Yamazaki c,d,e, Isabelle Martins a,b,c,Abdul Qader Sukkurwala a,b,c, Michael Michaud a,b,c, Laura Senovilla a,b,c,Lorenzo Galluzzi c,f,g, Guido Kroemer e,f,g,h,i,*, Laurence Zitvogel a,c,d,e,**a Universite Paris-Sud/Paris XI, Le Kremlin-Bicetre, Franceb INSERM, U848, Villejuif, Francec Institut Gustave Roussy, Villejuif, Franced INSERM, U1015, Villejuif, Francee Center of Clinical Investigations CBT507, Institut Gustave Roussy, Villejuif, Francef Universite Paris Descartes/Paris V, Sorbonne Paris Cite, Paris, Franceg Equipe 11 labellisee Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, Franceh Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, Francei Pole de Biologie, Hopital Europeen Georges Pompidou, AP-HP, Paris, France

A R T I C L E I N F O

Article history:

Available online 17 June 2013

Keywords:

Anthracyclines

Apoptosis

ATP

Calcium

Calreticulin

PERK

A B S T R A C T

Preclinical and clinical findings suggest that tumor-specific immune responses may be responsible – at

least in part – for the clinical success of therapeutic regimens that rely on immunogenic cell death (ICD)

inducers, including anthracyclines and oxaliplatin. The molecular pathways whereby some, but not all,

cytotoxic agents promote bona fide ICD remain to be fully elucidated. Nevertheless, a central role for the

endoplasmic reticulum (ER) stress response has been revealed in all scenarios of ICD described thus far.

Hence, components of the ER stress machinery may constitute clinically relevant druggable targets for

the induction of ICD. In this review, we will summarize recent findings in the field of ICD research with a

special focus on ER stress mechanisms and their implication for cancer therapy.

� 2013 Elsevier Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Cytokine & Growth Factor Reviews

jo ur n al ho mep ag e: www .e lsev ier . c om / loc ate /c yto g f r

1. Introduction

Systemic anthracycline-, oxaliplatin- or cyclophosphamide-based anticancer chemotherapy is particularly efficient whenadministered to tumor-bearing immunocompetent, as opposed toimmunodeficient, hosts, mediating antineoplastic effects in theformer, but not in the latter, scenario [1–3]. In addition, murinecancer cells treated with anthracyclines, oxaliplatin and UVCirradiation in vitro and then injected into syngenic mice are able toconfer long-term protection against a subsequent challenge withlive cells of the same type [1,2,4]. Importantly, such a vaccinationeffect strictly relies on an intact immune system, as it vanishes inmice depleted of T cells by means of neutralizing antibodies as well

* Corresponding author at: INSERM, U848, Institut Gustave Roussy, Pavillon de

Recherche 1, 39 rue Camille Desmoulins, F-94805 Villejuif, France. Tel.: +33 1 4211

6046; fax: +33 1 4211 6047.

** Corresponding author at: INSERM, U1015, Institut Gustave Roussy, University

Paris XI, 114 rue Edouard Vaillant, F-94805 Villejuif, France. Tel.: +33 1 4211 6046;

fax: +33 1 4211 6047.

E-mail addresses: kroemer@orange.fr (G. Kroemer), zitvogel@igr.fr (L. Zitvogel).1 Equally contributed to this work.

1359-6101/$ – see front matter � 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.cytogfr.2013.05.001

in mice genetically deficient for critical components of adaptiveimmunity, including interleukin (IL)-1b, IL-17, interferon g (IFNg)and the IFNg receptor 1, among others [1,2,5–7]. These observa-tions indicate that some therapeutics offer a benefit over others asthey are intrinsically endowed with the ability to kill cancer cellswhile eliciting a tumor-specific immune response, which de facto

ameliorates the long-term efficacy of the treatment.The ability of anthracyclines, oxaliplatin and g irradiation to

stimulate anticancer immune responses relies – at least in part –on the fact that these agents can kill malignant cells in a fashionthat is perceived as immunogenic by the host, that is, they canpromote so-called immunogenic cell death (ICD) [8–11]. Thus, cellssuccumbing in response to ICD inducers emit a spatiotemporallydefined combination of signals that must be decoded by the hostimmune system to activate an antitumor immune response [7,12].This implies that the clinical efficacy of anthracyclines and otherICD inducers is linked not only to the intrinsic physicochemical andpharmacodynamic properties of these agents, but also (i) to theability of cancer cells to properly emit all the signals that arerequired for cell death to be interpreted as immunogenic, (ii) to thecapacity of the host immune system to properly recognize anddecode such signals, and (iii) to the ability of the host immune

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318312

system to translate such signals into a robust cell-mediatedimmune response [7,12].

Thus far, three cell death-associated processes resulting in theemission of three distinct damage-associated molecular patterns(DAMPs) have been shown to be critical for the demise ofneoplastic cells to be perceived as an immunogenic event: (i) thepre-apoptotic exposure of the endoplasmic reticulum (ER)-sessilemolecular chaperone calreticulin (CRT) on the cell surface [2,13];(ii) the active secretion of ATP, the most prominent energy-storingmolecule among all living organisms [6,14,15]; the release of thenon-histone nuclear protein high-mobility group box 1 (HMGB1)into the extracellular space following the permeabilization of boththe nuclear and the plasma membrane [5,16,17]. The spatiotem-porally defined emission of these DAMPs initiates a molecular andcellular cascade that ultimately results in the activation of acognate tumor-specific immune response. First, by binding toCD91 (and perhaps other hitherto uncharacterized receptors) ondendritic cells (DCs) and other antigen-presenting cells (APCs),surface-exposed CRT serves as a bona fide ‘‘eat-me’’ signal, hencefacilitating the engulfment of cell debris, protein aggregates andother potential sources of tumor-associated antigens [2,11,18]. Inthis setting, CRT is functionally antagonized by CD47, whichdelivers ‘‘don’t eat me’’ signals to DCs and APCs [19]. Second, thesecretion of ATP from dying tumor cells, which has recently beenshown to obligatorily rely on the autophagic machinery [14,20],stimulates purinergic P2RX7 receptors on the surface of DCs todeliver intracellular signals that culminate in the activation of theNLRP3 inflammasome [6,21]. In turn, this promotes the proteolyticmaturation of pro-inflammatory cytokines, mainly IL-1b and IL-18, and their release into the extracellular space [6,22,23]. Amongvarious effects, IL-1b promotes the functional polarization of IFNg-secreting cytotoxic T lymphocytes (CTLs), which are fundamentalplayers in the cell-mediated antitumor immune response elicitedby ICD inducers. In addition, IL-1b can stimulate the production ofIL-17 (and IL-22) by TH17 gd T cells, which further contribute toCTL activation [24,25]. Third, once released in extracellular space,HMGB1 can bind Toll-like receptor 4 (TLR4) on DCs [26], henceigniting a myeloid differentiation primary response gene 88(MYD88)-dependent signaling cascade that supports the optimalprocessing of tumor antigens and their presentation to CTLs [5,27].Altogether, the emission of these DAMPs along a specificspatiotemporal pattern facilitates the development of tumor-specific adaptive immune responses by the host. These can controland sometimes even eradicate therapy-resistant tumor (stem)cells [19,28,29].

In this review, we will provide a brief background on themolecular mechanisms underlying ICD and summarize recentresults on the discovery/development of novel ICD inducers, with aparticular focus on the links between ICD and the ER stress.

2. Molecular mechanisms underlying the immunogenicity ofcell death

The mechanisms that are molecularly responsible for theemission of ICD-associated DAMPs have only recently begun toemerge [12,30], and appear to exhibit at least some degree ofcontext dependence. Among all, the signal transduction cascadesthat underpin the exposure of CRT in response to ICD inducers areprobably the best characterized, followed by the signaling path-ways promoting the secretion of ATP and the release of HMGB1.

CRT exposure following anthracycline-based chemotherapy isimperatively paralleled by the co-translocation to the cell surfaceof the ER-sessile disulfide isomerase ERp57, even though the latter– by itself – fails to exert any pro-immunogenic effect [31].Conversely, ERp57 appears to be dispensable for the exposure ofCRT as triggered by hypericin-based photodynamic therapy (PDT)

[11,15]. The anthracycline-stimulated co-translocation of CRT andERp57 to the outer leaflet of the plasma membrane has beendeconvoluted into three sequentially activated but functionallydistinguishable signaling modules: (i) an ER stress module, (ii) anapoptotic module, and (iii) a translocation module [32,33].

Malignant cells responding to anthracyclines, oxaliplatin andUVC irradiation rapidly undergo a phase of intense ER stresscharacterized by the overgeneration of reactive oxygen species(ROS) [32], increased cytoplasmic Ca2+ concentrations [34], and theactivation of the protein kinase, RNA-activated (PKR)-like ERkinase PERK [32]. Similar to PKR, which reacts to the presence ofviral RNA in the cytoplasm [35,36], activated PERK phosphorylatesthe eukaryotic translation initiation factor 2a (eIF2a), henceshutting down protein translation [37–39]. In line with afundamental role for PERK-mediated eIF2a phosphorylation inICD, both the siRNA-mediated downregulation of PERK and theknock-in of a non-phosphorylatable variant of eIF2a (i.e.,eIF2aS51A) reportedly prevent the anthracycline-induced co-exposure of CRT and ERp57, hence compromising the vaccinationeffects of ICD in vivo [32]. Similar inhibitory effects have beendemonstrated for antioxidants and intracellular Ca2+ chelators[34]. Downstream of the ER stress module, anthracyclines andoxaliplatin engage several regulators of cell death [32], includingcaspase-8, the most prominent initiator caspase of the extrinsicpathways of apoptosis, as well as BAX and BAK, two pro-apoptoticmembers of the Bcl-2 protein family that play a central role inmitochondrial outer membrane permeabilization [40–42]. Theonly ICD-relevant substrate for caspase-8 identified thus far isanother ER-resident protein, i.e., BAP31 [32,43]. In line with thisnotion, the pharmacological blockade of caspase-8 with the broadspectrum caspase inhibitor Z-VAD.fmk, the knockout of Casp8, Bax

and Bak, as well as the expression of a non-cleavable mutant ofBAP31 inhibit CRT exposure and the immunogenicity of cell deathas triggered by anthracyclines and oxaliplatin [2,32]. Finally, asdemonstrated in pharmacological and genetic inhibition models,phosphoinositide-3-kinase (PI3K) activity, the anterograde ER-to-Golgi transport, soluble N-ethylmaleimide-sensitive factor (NSF)attachment protein (SNAP) receptor (SNARE)-dependent mem-brane transport and lipid rafts, all appear to be required for theterminal translocation module that underpins the ICD-related co-exposure of CRT and ERp57 on the cell surface [32].

Of note, CRT translocation as triggered by hypericin-based PDTproceeds not only independent of ERp57, but also irrespective ofincreased cytosolic Ca2+ concentrations, eIF2a phosphorylation,caspase-8 activation and lipid rafts [7,11,18]. Conversely, hyper-icin-based PDT promotes ICD via a molecular cascade that relies onROS overgeneration, PERK, BAX and BAK, PI3K activity and the ER-to-Golgi transport apparatus [7,11,18]. These observations suggestthat while some of the molecular players implicated in CRTexposure operate in a context-dependent manner, others may bepart of a core machinery underpinning all instances of ICD.

Recently, autophagy has been shown to play an essential role inthe active secretion of ATP that accompanies ICD [14,20]. Thisseems to involve a two-step mechanism, as only the activation ofthe autophagic machinery along with the execution of apoptosis,but neither of these processes alone, results in the secretion of ATPby dying cells [14]. Thus, on one hand, both the siRNA-mediateddepletion of essential autophagic factors such as ATG5 or ATG7 inhuman cells and the knock-out of the corresponding genes (Atg5

and Atg7) in mouse-embryonic fibroblasts have been shown toprevent the active secretion of ATP in the course of ICD [14]. On theother hand, experimental maneuvers that potently activate theautophagic flux but (at least initially) do not trigger cell death (e.g.,nutrient deprivation, the administration of the mTOR inhibitorrapamycin) fail to promote ATP secretion by cancer cells [14]. Ofnote, the extracellular concentration of ATP decreases along a

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318 313

rather steep gradient due to the presence of both plasmamembrane-associated and plasmatic ectonucleotidases [44,45].Besides promoting the activation of the NLRP3 inflammasome (seeabove), extracellular ATP functions as a potent chemoattractant forseveral cell types involved in immune responses, including (butnot limited to) monocytes, granulocytes and dendritic cells (DCs)[46]. Such a chemotactic effect, however, is mostly mediated by theG protein-coupled purinergic receptor P2Y2 [46].

The molecular mechanisms that allow for the release of HMGB1by cancer cells succumbing to ICD remain largely obscure. Resultsfrom video- and time-lapse fluorescence microscopy-basedexperiments suggest that HMGB1 is liberated in a two-stepprocess: it translocates first from the nucleus to the cytoplasm andthen from the cytoplasm to the extracellular space [47]. Whetherthis involves an active regulation or only reflects the passivediffusion of a concentrated pool of HMGB1 that ensues the celldeath-associated permeabilization of the nuclear compartmentand the plasma membrane remains to be elucidated.

3. Discovery of bona fide ICD inducers

Until recently, the ability of a given agent to trigger bona fide ICDcould be assessed only empirically, in vivo, following theexperimental approach that led to the discovery of the first ICDinducers, i.e., doxorubicin, mitoxantrone, oxaliplatin, cyclophos-phamide and UVC irradiation [1–3]. In this setting, cancer cellssuccumbing to a hypothetic ICD inducer were subcutaneouslyinoculated into syngenic immunocompetent hosts, and theirability to prevent the outgrowth of a subsequent challenge withlive tumor cells of the same type (performed 7–14 days later) wasinterpreted as an indicator of bona fide ICD [1,2]. Since then, greatexperimental efforts have been dedicated to the identification of

Fig. 1. High-throughput strategy for the identification of novel inducers of immunogen

screened for their capacity to elicit the hallmarks of ICD with cell lines engineered to ex

ATP-sensitive chemical dye (e.g., quinacrine), or genetically modified to express a high-

cumulative assessment of ICD-related hallmarks, that is, CRT exposure, ATP secretion an

low-throughput assays in vitro, and in vivo, in suitable murine models. ELISA, enzyme-

the hallmarks of ICD (see above), not only to gain further insightsinto ICD as a clinically relevant entity, but also to identifyparameters that could be monitored in vitro to predict theimmunogenicity of cell death in vivo. Importantly, it soon turnedout that structurally similar compounds such as oxaliplatin andcisplatin (CDDP) do not share the ability to promote ICD,suggesting that no simple structure–activity relationship existsin this respect [47]. The side-by-side comparison of oxaliplatin andCDDP revealed that both agents trigger cell death along with thesecretion of ATP and the release of HGMB1, yet only the former alsostimulates the pre-apoptotic exposure of CRT on the cell surface[47,48]. In an attempt to get further insights into this discrepancy,a panel of 480 different compounds with defined biological activitywas screened for their ability to promote CRT exposure in thepresence of CDDP. The ER stressor thapsigargin (employed atsublethal doses) turned out to be the most efficient of thesechemicals at rescuing the ability of CDDP to stimulate CRTexposure [48]. Moreover, neoplastic cells succumbing to CDDP plusthapsigargin efficiently protected mice against a subsequent tumorchallenge, while cells dying in response to either compound usedas standalone treatment failed to do so [48]. These findingsdemonstrated that CRT exposure, ATP secretion and HGMB1release can be conveniently assessed in vitro to predict the abilityof a given intervention to promote ICD, a notion that has beencomforted by subsequent studies investigating the immunogenicpotential of hypericin-based PDT [11].

Intrigued by the possibility to discover novel inducers of ICD,we have recently screened (by an epifluorescence microscopy-based system implemented with human osteosarcoma U2OS cells)more than 1000 chemicals (including all FDA-approved com-pounds as well as investigational agents that are currentlyauthorized by US authorities for use in clinical trials) for their

ic cell death (ICD). Compounds from large chemical libraries can be conveniently

press a calreticulin-green fluorescence protein (CRT-GFP) chimera, stained with an

mobility group box 1 (HMGB1)-GFP fusion protein. In this experimental setup, the

d HMGB1 release, allows for the identification of hits to be validated in subsequent,

linked immunosorbent assay; FACS, fluorescence-activated cell sorter.

Fig. 2. Molecular mechanisms underlying the unfolded-protein response (UPR). In physiological conditions, inositol-requiring enzyme 1 (IRE1a), activating transcription

factor 6 (ATF6) and protein kinase, RNA-activated (PKR)-like endoplasmic reticulum (ER) kinase (PERK) are bound and held in check by the ER-resident molecular chaperone

binding immunoglobulin protein, BIP (A–C, left panels). When misfolded proteins accumulate in the ER lumen, BIP is competitively displaced by IRE1a, ATF6 and PERK,

allowing for the activation of a multi-pronged, adaptive response (A–C, right panels). In this scenario, (A) IRE1a gets phosphorylated and becomes capable of processing the

unspliced mRNA coding for X-box binding protein 1 (XBP1s), hence generating a splicing variant (XBP1s) that encodes a functional b-ZIP transcription factor; (B) ATF6 is

transported to the Golgi apparatus, by a coat protein II (COPII)-dependent mechanism, where it is processed to release a transcriptionally active fragment (ATF6f); and (C)

PERK becomes activated by trans-autophosphorylation and acquires the capacity to phosphorylate the eukaryotic translation initiation factor 2a (eIF2a), hence shutting

down general protein translation while selectively favoring the synthesis of activating transcription factor 4 (ATF4). XBP1s, ATF6 and ATF4 transactivate several genes that

code for factors involved in protein folding and degradation, including GRP78 (the BIP-coding gene), hence (at least potentially) ameliorating the degree of ER stress imposed

by the accumulation of misfolded proteins.

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318314

ability to promote (i) the intracellular redistribution of a CRT-greenfluorescent protein (GFP) chimera; (ii) a decrease in intracellularATP, monitored with the ATP-sensitive dye quinacrine; and (iii) therelease of a HMGB1-GFP fusion protein (Fig. 1) [49]. Moreover, inthe attempt to minimize the rate of false positive results due totechnical bias, CRT exposure, ATP secretion and HMGB1 release asinduced by the constituents of our chemical library were allmonitored with up to two additional distinct biosensors, again in aU2OS cell-based setup [49]. Doxorubicin, epirubicin and mitox-antrone, three anthracyclines that are well known for theircapacity to promote ICD [2], ranked among the top 10 hits ofsuch a screening effort, comforting us on the reliability of ourexperimental system. Unexpectedly, this shortlist also containedfour distinct cardiac glycosides (CGs), the clinically employeddrugs digoxin and digitoxin as well as the experimental agentsouabain and lanatoside C [49].

In low-throughput validation experiments, CGs elicited all thehallmarks of ICD in a large panel of human cancer cell lines. Inaddition, CGs promoted the exposure on the cell surface of theheat-shock 90 kDa protein (HSP90), which had recently beenshown to be required for the induction of a DC-driven immuneresponse against myeloma cells by the proteasomal inhibitorbortezomib [50]. Similar to all ICD inducers discovered to date, thecapacity of CGs to stimulate ICD was shown to obligatorily rely onthe activation of caspases [49]. Moreover, a detailed mechanisticcharacterization revealed that CGs elicit CRT exposure, ATPsecretion and HMGB1 release by inhibiting their established,therapeutically relevant target, that is, the plasma membrane Na+/K+-ATPase [49,51,52]. Indeed, human cells overexpressing themurine a1 subunit of the Na+/K+-ATPase, whose sensitivity to CGsis significantly lower than that of its human homologue [53],became virtually unable to emit any of the hallmarks of ICD in

response to digoxin [49]. In addition, the CRT exposure-promotingactivity of CGs could be mimicked by the Ca2+ ionophore A23187(but neither by the K+ ionophore nigericin nor by the protonophorecarbonyl cyanide m-chlorophenylhydrazone), and could beblunted with both intra- and extracellular Ca2+ chelators [49].Altogether, these findings demonstrate that CGs promote ICD via

an on-target effect that involves alterations in the intracellularhomeostasis of Ca2+ ions.

In vivo experiments demonstrated that CGs ameliorate theantineoplastic effects of the DNA-damaging agents cisplatin andmitomycin C, which per se are unable to induce ICD, inimmunocompetent, but not in immunodeficient, mice [49].Moreover, murine cancer cells succumbing in vitro to digoxinplus cisplatin or mitomycin C, but not to cisplatin or mitomycin Calone, became able to vaccinate syngenic mice against asubsequent challenge with live cells of the same type [49]. Finally,we undertook a large retrospective clinical study involving morethan 145 cancer patients who – concomitant to antineoplastictherapy – received CGs (due to a subjacent, cancer-unrelatedcardiac condition) and a double number of case-matched controlpatients (who did not receive CGs). Surprisingly, we found that theco-administration of CGs along with anticancer therapy prolongedthe overall survival of subjects affected by a large panel ofneoplasms, including head and neck, breast, hepatocellular andcolorectal carcinoma (but not lung and prostate cancer), Of note,breast and colorectal carcinoma patients appeared to benefit fromthe co-administration of CGs only when they were treated withtherapeutic agents other than anthracyclines and oxaliplatin [49].Taken together, these data indicate that CGs not only elicit thehallmarks of ICD in vitro, but also (i) promote ICD in vivo, in rodentmodels, when combined with non-immunogenic chemotherapy,and (ii) may ameliorate disease outcome in specific subsets of

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318 315

cancer patients. A prospective clinical trial will shortly be launchedto investigate the safety and efficacy of a chemotherapeuticregimen combining digoxin and cisplatin in subjects affected byhead and neck carcinoma.

4. ER stress and Ca2+ fluxes as common denominators of ICD

The term unfolded protein response (UPR) refers to the multi-pronged adaptive reaction of cells to conditions that promote theaccumulation of misfolded proteins in the lumen of the ER, thusinterfering with its functions, first of all Ca2+ homeostasis [54,55].In mammalian cells, the UPR is activated by three distinct sensors:endoplasmic reticulum to nucleus signaling 1 (ERN1, best knownas inositol-requiring enzyme 1, IRE1a), activating transcriptionfactor 6 (ATF6) and eIF2a kinase 3 (EIF2AK3, best known as PERK)[37,56–59]. Under homeostatic conditions, all these proteins arebound and held in check by the ER-resident molecular chaperoneglucose-regulated protein, 78 kDa (GRP78, also known as bindingimmunoglobulin protein, BIP) [37,60]. Conversely, when unfoldedproteins accumulate, GRP78 is competitively displaced by IRE1a,ATF6 and PERK, a setting that allows for their activation (Fig. 2)[37,54,59].

Owing to its enzymatic activity of endoribonuclease, activeIRE1a splices 26 nucleotides out of the mRNA coding for X-boxbinding protein 1 (XBP1), hence causing a frameshift that results inthe synthesis of an active b-ZIP transcription factor, XBP1s [61,62].In turn, XBP1s activates an adaptive transcriptional program aimedat re-establishing ER homeostasis, which involves several factorspromoting protein folding and degradation [61,63]. Moreover,IRE1a appears to elicit a c-JUN-dependent signaling pathway thatcouples the UPR to the activation of autophagy, a prominentcytoprotective mechanism [64,65]. The dissociation of ATF6 fromGRP78 unmasks a Golgi apparatus-translocation signal, henceinitiating a sequence of post-translational modifications thateventually generate an active transcription factor. Among multiplegenes, ATF6 transactivates GRP78, de facto (i) maximizing thecapacity of the ER to handle unfolded proteins and (ii) igniting anegative feedback loop that extinguishes the UPR [66]. Upondissociation from GRP78, PERK dimerizes and becomes activatedby trans-autophosphorylation at Thr980, hence acquiring thecapacity to phosphorylate eIF2a to shut off protein synthesis [57].

While the activation of PERK (and in some instance thephosphorylation of eIF2a) appears to be quintessential for ICD-associated CRT exposure and ATP secretion, in vitro [11,32], as wellas for the immunogenicity of cell death in vivo [32], similarconsiderations may not apply to IRE1a and ATF6. Indeed, while afull-blown UPR (involving PERK, IRE1a and ATF6 activation) hasbeen documented in cancer cells responding to CGs in vitro [49],the genetic inhibition of IRE1a and ATF6 failed to affect CRTexposure as promoted by mitoxantrone, oxaliplatin and UVCirradiation [32]. Of note, both the prototypic ER stressorsthapsigargin and tunicamycin (which inhibit the sarcoplasmic/ER Ca2+ ATPase, SERCA, and generalized protein glycosylation,respectively) induce a global ER stress response involving PERKactivation, yet only the former is able to promote CRT exposure onits own [67]. This said, both thapsigargin and tunicamycin are ableto convert otherwise non-immunogenic lethal stimuli, such asCDDP, into bona fide ICD inducers [48] Thus, the induction of an ERstress is required, but not sufficient, for the ICD-associatedexposure of CRT.

In some, but not all, experimental settings [11,32,34,49], anincrease in the cytosolic concentrations of Ca2+ appears to berequired for the ICD-associated exposure of CRT on the outer leafletof the plasma membrane. Among multiple lines of evidence insupport of this notion: (i) the Ca2+ ionophore A23187, but not K+

and H+ ionophores, mimics the CRT-exposing activity of CGs [49];

(ii) both extracellular and intracellular Ca2+ chelators block CRTexposure as promoted by CGs [49]; (iii) experimental manipula-tions that promote the cytosolic leakage of ER Ca2+ stores promoteICD-associated CRT exposure [34]; (iv) anthracyclines, the firstbona fide ICD inducers to be characterized [1], have a well-documented effect on Ca2+ homeostasis, hence exerting prominentcardiotoxic effects (which is the main factor limiting their clinicaluse) [32]; (v) thapsigargin (which used as a single drug promotesCRT exposure under certain experimental conditions) and tunica-mycin (which does not) both elicits an ER stress, yet only theformer does so while provoking alterations in Ca2+ homeostasis[67]; and (vi) murine fibroblasts responding to Ca2+ ionophoressecrete ‘‘reticuloplasmins’’ (including CRT) into the extracellularspace [68], a phenomenon associated with ultrastructural changesof the ER that can be taken advantage of for the generation of high-throughput screening (HTS)-compatible fluorescent biosensors[49]. However, while hypericin-based PDT promotes the leakage ofER Ca2+ into the cytosol, intracellular Ca2+ chelators reportedly donot influence CRT exposure as triggered by this intervention [11].Hence, deregulations in Ca2+ homeostasis may not constitute auniversal requirement for the ICD-associated translocation of CRTto the cell surface.

Of note, ER stress and autophagy appear to be intimately linked.Thus, most compounds currently used in clinical oncology induceATP secretion (when used at cytotoxic concentrations) [20] whilepromoting a generalized ER stress response. This said, the exactsequence (and perhaps magnitude) by which the differentbranches of the ER stress must be activated to allow for thefunctionally productive emission of the hallmarks of ICD remainselusive.

5. Conclusions and perspectives

As discussed in this review, the ER stress response constitutes acentral hub in the signaling cascades leading to the emission ofICD-promoting signals. The accumulation of misfolded proteins,the inhibition of protein glycosylation and the deregulation ofredox and Ca2+ homeostasis all can disrupt the delicate homeo-stasis of the ER-Golgi network, resulting in the activation ofadaptive cell responses [69]. At present, one simple cause-effectrelationship cannot be drawn between any of these processes andthe immunogenicity of cell death. Indeed, neither agents thatinterrupt the ER-to-Golgi transport (e.g., brefeldin A), hencefavoring the accumulation of misfolded polypeptides [32], neitherchemicals that block N-linked protein glycosylation (e.g., tunica-mycin) [32], nor compounds that prevent the formation ofdisulfide bridges (e.g., dithiothreitol) [70,71] are capable ofpromoting CRT exposure. In addition, while both thapsigarginand hypericin-based PDT stimulate the translocation of CRT to theouter leaflet of the plasma membrane, only the former does so in astrictly Ca2+-dependent fashion [11,67]. Further studies arerequired to elucidate in detail this central aspect of ICD, perhapsresulting in the identification of novel druggable targets. In themeanwhile, GCs stand out as promising candidates to amelioratethe efficacy of chemotherapeutic regimens based on non-immunogenic compounds such as CDDP. We have recentlylaunched the recruitment of head and neck cancer patients toappropriately investigate this possibility in a clinical setting. Inaddition, several clinical studies are currently testing theantineoplastic potential of various ER stressors, including thapsi-gargin and bortezomib (reviewed in [72]).

PERK activation has been involved in all instances of ICDdescribed thus far [11,32,49], indicating that – irrespective ofthe initiating stimulus (see above) – ER stress responsesmay be converted into the emission of ICD-promoting signalsvia PERK. Intriguingly, PERK is highly enriched at so-called

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318316

‘‘mitochondria-associated ER membranes’’ (MAMs) [73], which aresites of physical and functional interconnection between mitochon-dria and the ER [74,75]. According to recent observations, PERKwouldbe required not only for the structural stability of MAMs, but also forthe translation of ROS-dependent ER stress signals, such as thoseelicited by hypericin-based PDT, into mitochondrial apoptosis [73]. Itis therefore tempting to speculate that – at least in some instances –the crosstalk between the ER and mitochondria might determinewhether one specific type of ER stress results in the emission of ICDstimuli or not.Further insights into this question are urgently awaitedfor the development of novel and perhaps clinically useful inducers ofICD.

Acknowledgments

GK and LZ are supported by the Ligue Nationale contre le Cancer(Equipes labellisees), SIRIC Socrates, Agence Nationale pour laRecherche (ANR AUTOPH, ANR Emergence), European Commission(ArtForce), European Research Council Advanced Investigator Grant(to GK), Fondation pour la Recherche Medicale (FRM), InstitutNational du Cancer (INCa), Fondation de France, Canceropole Ile-de-France, Fondation Bettencourt-Schueller, the LabEx Immuno-Oncology, and the Paris Alliance of Cancer Research Institutes.

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Oliver Kepp received his Ph.D. in 2006 from the Hum-boldt University of Berlin and the Max Planck Institutefor Infection Biology in Berlin, Germany. He is currentlya Senior Scientist in the laboratory of Guido Kroemer,where he investigates several aspects of immunogeniccell death, focusing on systems biology approaches.

Laurie Menger received her Ph.D. in 2012 from theUniversity of Paris SUD-XI, France. She is currentlyworking as a postdoctoral at UCL Cancer Institute Lon-don where she investigates the involvement of B7 fami-ly in regulation of anti-tumour immune responses.

Erika Vacchelli is currently a Ph.D. student in thelaboratory of Guido Kroemer, where she is involved inthe identification of new potential immune targets thatmight play a role in immunogenic cell death by per-forming high throughput genotyping studies.

Clara Locher studied pharmacy and is currently work-ing as a PhD student in the Lab of Laurence Zitvogelwhere she is analyzing certain aspects of immunogenictumor cell death.

Sandy Adjemian received her Ph.D. in 2012 from theUniversity of Paris Sud/Paris XI, France. She is currentlyworking as a Post-Doc in the Immunology department,at the University of Sao Paulo, Brazil, where she inves-tigates cell death pathways.

Takahiro Yamazaki received his Ph.D. in 2009 fromToho University, Japan. He is currently a postdoctoralfellow in the laboratory of Laurence Zitvogel, and focus-es on immunogenic cell death and immunochemother-apeutic approaches in vitro and in vivo.

Isabelle Martins received her Ph.D. in 2008 from theUniversity Denis Diderot of Paris VII, France. She hasbeen working as a postdoctoral researcher in the labo-ratory of Guido Kroemer and now works in the labora-tory of Eric Deutsch. She is particularly interested in celldeath and senescence.

O. Kepp et al. / Cytokine & Growth Factor Reviews 24 (2013) 311–318318

Abdul Qader Sukkurwala recieved his M.D. from DowMedical College, University of Karachi, Pakistan in 2006.He is currently working as PhD student and is about todefend his thesis titled ‘‘Autophagy: A new modulator ofimmunogenic cell death for cancer therapy’’ in thelaboratory of Guido Kroemer.

Michael Michaud received his Ph.D. in 2007 from theLyon 1 University in France, awarding his work oncancer chemoresistance. He is currently a senior post-doc in the laboratory of Guido Kroemer, where heinvestigates the links between autophagy and immu-nogenic cell death.

Laura Senovilla received her Ph.D. in 2006 from theUniversity of Valladolid, Spain. She was working as aPostDoc in the laboratory of Guido Kroemer and, cur-rently, she is a researcher at the Institut Gustave Roussyfocused on the links between ploidy and cancer immu-nosurveillance in collaboration with the laboratory ofGuido Kroemer and the laboratory of Laurence Zitvogel.

Lorenzo Galluzzi received his Ph.D. in 2008 from theUniversity of Paris Sud/Paris XI, France, and now worksas a Research Manager in the laboratory of Guido Kroe-mer. He is particularly fascinated by several aspects ofmitochondrial cell death, metabolism and tumor immu-nology.

Guido Kroemer received his M.D. in 1985 from theUniversity of Innsbruck, Austria, and his Ph.D. in 1992from the Autonomous University of Madrid, Spain. Henow works as a Research Director at the French MedicalResearch Council (INSERM) in Villejuif, France, as a FullProfessor at the University of Paris Descartes, France,and as a hospital practitioner at the Hopital EuropeenGeorge-Pompidou, Paris, France. His research interestsencompass mitochondrial cell death, autophagy and theimmune response to dying cancer cells.

Laurence Zitvogel is Research Director at Institut Na-tional de la Sante et Recherche Medicale and Co-Directorof a Center of Clinical Investigations at Institut GrustaveRoussy, Villejuif, France. She graduated in 1992 with adegree in Medical Oncology from the School of Medicineof the University of Paris, France. She began her scientificcareer at the University of Pittsburgh, USA working underMichael Lotze. Her expertise is mainly dendritic cell andinnate effector biology and relevance during tumor de-velopment and immunotherapy of cancer. She currentlyinvestigates the immunogenicity of cell death with drugsof the oncological armamentarium and conducts a PhaseII trail using DC-derived autologous exosome- vaccines.