Apoptosis in Cancer

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  Targeting ER stress induced apoptosis and inflammation in cancer Tom Verfaillie, Abhishek D. Garg, Patrizia Agostinis * Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Catholic University of Leuven, Belgium a r t i c l e i n f o  Article history: Received 3 May 2010Received in revised form 14 July 2010Accepted 19 July 2010Available online xxxx Keywords: ER stressUnfolded protein responseApoptosisCell deathCancerAnticancer therapyInflammationAntitumor immune response a b s t r a c t Disturbance in the folding capacity of the endoplasmic reticulum (ER), caused by a varietyof endogenous and exogenous insults, prompts a cellular stress condition known as ER stress. ER stress is initially shaped to re-establish ER homeostasis through the activationof an integrated intracellular signal transduction pathway termed as unfolded proteinresponse (UPR). However, when ER stress is too severe or prolonged, the pro-survival func-tion of the UPR turns into a toxic signal, which is predominantly executed by mitochondrialapoptosis. Moreover, accumulating evidence implicates ER stress pathways in the activa-tion of various ‘classical’ inflammatory processes in and around the tumour microenviron-ment. In fact, ER stress pathways evoked by certain conventional or experimentalanticancer modalities have been found to promote anti-tumour immunity by enhancingimmunogenicity of dying cancer cells. Thus, the ER functions as an essential sensing orga-nelle capable of coordinating stress pathways crucially involved in maintaining the cross-talk between the cancer cell’s intracellular and extracellular environment. In this reviewwe discuss the emerging link between ERstress, cell fate decisions and immunomodulationand the potential therapeutic benefit of targeting this multifaceted signaling pathway inanticancer therapy.   2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The endoplasmic reticulum (ER) is a central cellularorganelle that fulfils crucial biosynthetic, sensing andsignaling functions in eukaryotic cells. Being responsiblefor the synthesis, folding and posttranslational modifica-tions of proteins destined for the secretory pathway, theER has to maintain a tightly regulated oxidizing and Ca 2+ -rich folding environment. Both protein folding as well asCa 2+ buffering in the ER are assisted by various ER residentchaperones like the glucose-regulated protein GRP78 (alsoknown as BiP), calreticulin, calnexin and protein disulfideisomerases (PDI). Various physiopathological conditionslike hypoxia, ER-Ca 2+ depletion, oxidative injury, hypogly-cemia and viral infections may affect ER homeostasis andinterfere with proper protein folding, ultimately causingan imbalance between protein folding load and capacity.This cellular condition is known as ‘ER stress’. The ER responds to these perturbations by activatingan integrated 0304-3835/$ - see front matter    2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.canlet.2010.07.016  Abbreviations : AP1, activator protein 1; APR, acute-phase response;ATF6, activating transcription factor 6; BFA, Brefeldin A; BiP, bindingimmunoglobulin protein; CHOP, C/EBP homologous protein; COX, cyclo-oxygenase-2; CREBH, cyclic-AMP-responsive-element-binding protein H;CRT, calreticulin; DAMP(s), Damage-associated Molecular Pattern(s); DC,dendritic cells; eIF2 a , eukaryotic initiation factor 2 Alpha; ER, endoplas-mic reticulum; ERAD, endoplasmic reticulum associated proteindegradation; GRP, glucose-regulated protein; HSP, heat shock protein;IL, interleukin; IL-2R, interleukin 2 receptor; IRE1, inositol requiringenzyme 1;JNK,c-jun N-terminalkinases; KEAP1,kelch-likeEch associatedprotein 1; MAPK, mitogen-activated protein kinases; NF- j B, nuclearfactor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclearfactor-E2-related factor 2; PDI, protein disulfide isomerases; PDT, pho-todynamic therapy; PERK,pancreatic ER kinase (PKR)-like ER kinase; ROS,reactive oxygen species; TLR(s), toll-like receptor(s); TNF, tumournecrosis factor; TRAF2, tumour necrosis factor (TNF)-receptor associatedreceptor 2; UPR, unfolded protein response; VEGF, vascular endothelialgrowth factor; XBP1, X-box binding protein 1. *  Correspondingauthor.Address:DepartmentofMolecularCellBiology,Faculty of Medicine, Catholic University of Leuven, Campus GasthuisbergON1, Herestraat 49, B-3000 Leuven Belgium. Tel.: +32 16 345715. E-mail address: (P. Agostinis).Cancer Letters xxx (2010) xxx–xxx Contents lists available at ScienceDirect Cancer Letters journal homepage: Please cite this article in press as: T. Verfaillie et al., Targeting ER stress induced apoptosis and inflammation in cancer, Cancer Lett. (2010),doi:10.1016/j.canlet.2010.07.016  signal transduction pathway, called the unfolded proteinresponse (UPR) [1]. The UPR (Fig. 1) is primarily tailored to re-establish ER homeostasis by coordinating the tempo-ral shut down in protein translation along with a complexprogram of gene transcription that leads to the upregula-tion of components of the ER folding machinery and ER quality control, like the ER-associated degradation (ERAD)pathway. However, when ER stress is too severe or cannotbe solved, the UPR turns from a pro-survival to a pro-deathresponse, usually, but not uniquely, culminating in theactivation of intrinsic apoptosis [2]. In the next sectionswe will briefly discuss the dual role of the UPR in cellularfate’s decision, since this subject has been exhaustivelydiscussed in recent reviews [3,4]. 1.1. The UPR; a double faceted signal in cell death and survival The UPR consists of a complex interplay between threesignaling ‘arms’, each of which emanates from a differentER stress sensor; PERK (pancreatic ER kinase (PKR)-likeER kinase), IRE1 (inositol requiring enzyme 1) and ATF6(activating transcription factor 6). These are ER transmem-brane proteins that are kept in an inactive state throughthe direct association of their luminal domain with theER chaperone BiP/GRP78. Increasing levels of unfolded ormisfolded proteins in the ER lumen titrate BiP/GRP78 awayfrom these three sensors, allowing for the activation of their signaling functions. PERK is a Ser/Thr kinase withtwo known substrates thus far, eIF2 a (eukaryotic initiationfactor 2) and the transcription factor Nrf2 (Nuclear factor-E2-related factor 2) [5]. Under conditions of ER stress, thePERK-mediated phosphorylation of eIF2 a  on Ser51 leadsto the inhibition of cap-dependent translation, therebyreducing the protein load on the ER, while favouringcap-independent translation. One UPR protein that isselectively translated under these circumstances is thetranscription factor ATF4 [5]. The PERK-eIF2 a -ATF4 axisregulates the expression of several genes involved in ER  Fig. 1.  UPR signaling pathways. ER stress is caused by an accumulation of unfolded proteins in the ER lumen. These unfolded proteins tether the ER chaperone BiP/GRP78 away from its interaction with the three ER stress sensors PERK, IRE1 and ATF6 which become subsequently activated. Uponactivation, IRE1 mediates the unconventional splicing of XBP1 mRNA which encodes a transcription factor XBP1s, responsible for the upregulation of genesinvolved in ERAD, ER quality control and redox homeostasis. Concomitantly, IRE1 can recruit TRAF2 and ASK1 to activate MAPK signaling pathways of p38and JNK. PERK-mediated phosphorylation of the translation initiation factor eIF2 a  provides the cell with a temporary ‘rest point’ by suppressing generaltranslation. Under these circumstances, ATF4 is selectively translated via cap-independent translation and upregulates proteins involved in ER homeostasis.Upon BiP/GRP78 release, ATF6 is free to move to the Golgi where it is subsequently cleaved by local site1 and site2 proteases (S1P and S2P). The cleavedfragment forms an active transcription factor that mainly mediates expression of several components for ERAD and ER homeostasis. Finally, persistent ER stress can induce apoptosis. The pro-apoptotic transcription factor CHOP can be up-regulated by XBP1, ATF6 and PERK, and can mediate transcription of thepro-apoptotic BH3-only protein Bim.2  T. Verfaillie et al./Cancer Letters xxx (2010) xxx–xxx Please cite this article in press as: T. Verfaillie et al., Targeting ER stress induced apoptosis and inflammation in cancer, Cancer Lett. (2010),doi:10.1016/j.canlet.2010.07.016  homeostasis (e.g. BiP/GRP78 and GRP94), amino acid bio-synthesis and transport functions, antioxidant stress re-sponses as well as apoptosis. On the other hand, Nrf2phosphorylation by PERK promotes the dissociation fromits cytosolic repressor KEAP1 (kelch-like Ech associatedprotein 1), thereby freeing Nrf2 for nuclear translocation,which ultimately results in the expression of a number of genes implicated in the antioxidant stress response [6].Thus the PERK branch of UPR bifurcates in two parallelbut integrated signaling pathways, PERK-eIF2 a -ATF4 andPERK-Nrf2, which favours the survival of ER stressed cellsby restoring ER quality control and increasing adaptationto oxidative stress. Contrary to PERK, activated IRE1 dis-plays both protein kinase (although there are no other di-rect substrates known besides IRE1 itself) as well asendoribonuclease activity. IRE1 splices  XBP1u  mRNA (ufor unspliced) to form mature  XBP1s  mRNA (s for spliced)which encodes a potent transcription factor XBP1s thatnot only induces genes involved in ER quality control, ER/Golgi biogenesis and certain ERAD components but alsogenes involved in redox homeostasis and oxidative stressresponse [7,8]. Finally, BiP/GRP78’s release from ATF6 in-duces its translocation to the Golgi apparatus where it iscleaved by specific Golgi resident proteases. Processing of ATF6 produces an active transcription factor that apartfrom targeting genes encoding ER chaperones and ERADcomponents, also plays an important role in lipid biogene-sis and ER expansion [9,10].The basic mechanisms outlined thus far define the ini-tial pro-survival side of the UPR. When these early re-sponses do not succeed in restoring ER homeostasis theUPR tends to turn into a pro-death signal. Although themolecular mechanisms underlying this switch remainpoorly understood, each apical UPR sensor holds a dualisticrole in propagating adaptive as well as toxic signals. A po-tential mechanism to explain this dichotomy may involvethe differential stability of pro-survival and pro-deathmRNAs/proteins under conditions of mild or severe ER stress [11]. For instance, under mild ER stress, ATF4-depen-dent pro-survival gene expression is likely to be prevalentsince PERK is activated transiently and to a limited extent.Here, because of the intrinsic instability of certain pro-apoptotic mRNAs and proteins, the apoptotic programwhich is partially mediated by the ATF4 target CHOP (C/EBP homologous protein, an important pro-apoptotic tran-scription factor, see further) would require a more sus-tained PERK activation associated with conditions of more severe ER stress. This concept is supported by recentstudies showing that sustained PERK signaling is requiredfor the transition from protective to pro-apoptotic UPR function[12]. As far as the IRE1 axis is concerned it is inter-esting to note that the general protective role for IRE1-XBP1 signaling during mild ER stress is counterbalancedby the scaffold signaling properties acquired by IRE1 inde-pendent of its XBP1 splicing activity. Here, under condi-tions of ER stress, IRE1 serves as a molecular platform forthe recruitment of the adaptor protein TNF-receptor asso-ciated factor 2 (TRAF2), an E3 ubiquitin ligase, that in turnlinks IRE1 to the activation of the stress-activated ASK1- JNK/p38 MAPK cascades, which have a dominant role incell fate decision as discussed below.Irrespective of the exact mechanisms modulating thefunctional activity of these apical UPR sensors, biochemicaland genetic evidence have assigned a crucial role to thetranscription factor CHOP in ER stress induced apoptosis[13]. In fact, CHOP is a shared target gene of all three armsof the UPR, as it can be induced by ATF4, cleaved ATF6 aswell as XBP1 (Fig. 1). CHOP mediates cell death throughthe induction of various genes including GADD34 andERO1 a , which may tip the balance towards apoptosis,especially under conditions of unresolved ER stress.GADD34, for example, is a regulatory subunit of proteinphosphatase 1 (PP1) that targets PP1 to dephoshorylateeIF2 a , thereby promoting resumption of protein synthesis.However, if the protein folding capacity of the ER fails to bere-established, a premature disinhibition of translationwill increase client protein load in the ER, thus amplifyingthe damage [14]. Besides, induction of ERO1 a  can create ahyperoxidizing ER environment, detrimental to proteinfolding [15]. Thus the PERK-axis, which is involved inmaintaining the redox state during ER stress, as discussedabove, can turn into a pro-oxidant signal when the tran-scriptional program of CHOP is set in motion.In addition, CHOP can also regulate the expression lev-els of some Bcl-2 protein family members [16,17], therebydirectly regulating the apoptotic machinery. Indeed, theBcl-2 family of proteins, known for their essential role inthe control of mitochondrial apoptosis, have been identi-fied as vital regulators of ER homeostasis and participatein UPR sensor mechanisms and cellular fate following ER stress [18]. The pro-apoptotic multidomain proteins Baxand Bak for example regulate ER Ca 2+ levels, modulateIRE1 activity through a direct and specific interaction withthis UPR sensor and are essential for the induction of mito-chondrial apoptosis following ER stress [18]. While Bcl-2proteins can alter IRE1 activity, activation of IRE1 leadsto the activation of MAPK/JNK which in turn can activatecertain BH3-only proteins like Bim or suppress the anti-apoptotic activity of Bcl-2 [3]. In a similar way, CHOP canpromote the transcription of Bim [17] while suppressingthe induction of Bcl-2 [16]. In addition to Bim, otherBH3-only proteins, such as Noxa and Puma are transcrip-tionally activated, through p53-dependent [19] and inde-pendent mechanisms [20] depending on the type of ER stressor. Interestingly, reconstitution of Bak expression atthe ER membranes in Bax/Bak deficient MEFs could re-establish IRE1-TRAF2 activation and mitochondrial apop-tosis mediated by reticular forms of the BH3-only proteinsBim and Puma, thus bypassing the need to be localized tothe mitochondria [21]. This reticular form of Bak also en-gaged an atypical IRE1-TRAF2 activation pathway, whereinthe mobilization of Ca 2+ promoted persistent JNK activa-tion and mitochondrial apoptosis [21]. Furthermore, a re-cent report delineates JNK as a master regulator of bothapoptotic as well as autophagic pathways following ER stress [22], thus providing further evidence of the complexcross-talk between cell death and survival signals engagedby the UPR.In conclusion, the complex interplaybetween Bcl-2 pro-teins and UPR components seems to fine tune the UPR andpoises it as a central mediator of the decision between lifeand death after ER stress. T. Verfaillie et al./Cancer Letters xxx (2010) xxx–xxx  3 Please cite this article in press as: T. Verfaillie et al., Targeting ER stress induced apoptosis and inflammation in cancer, Cancer Lett. (2010),doi:10.1016/j.canlet.2010.07.016  The execution of ER stress–mediated cell death is cru-cially regulated by the cross-talk between pro-apoptoticBcl-2 family members residing both at the ER membraneas well as the mitochondria and in most cases involvesthe apoptosome-induced apoptosis, wherein caspase-9functions as apical caspase in this cascade [18,23]. How- ever, certain ER-associated caspases, which become pro-cessed and activated during ER stress, have beenimplicated as well. For example, in rodents, activatedIRE1 provides a platform for the recruitment of caspase-12 at the ER membrane and results in its proteolytic pro-cessing [24,25]. However, caspase-12 deficient mouseembryonic fibroblasts were found to display either noresistance [25,26] or a partial protection specificallyagainst ER stress inducing agents [27] an observation fur-ther extended to other cell lines exposed to different ER stressors [28]. This suggests that although caspase-12 isprocessed under conditions of ER stress, it may not be akey mediator of ER stress induced apoptosis. Moreover,the general relevance of this event is dubious, as functionalcaspase-12 is only expressed in rodents, while in humansmostly a truncated prodomain-only form or a full-lengthcaspase-12 get expressed but both are catalytically inac-tive [28] Moreover, caspase-12 belongs to inflammatorymediators so it is likely that it becomes processed as a re-sult of apoptosis and may contribute to the regulation of inflammatory processes in cells undergoing ER stress. Assuggested by recent studies, caspase 4 might perform theproposed functional role of caspase-12 in humans and con-tribute to the initiation of apoptosis following ER stress[29,30]. Cleavage of BAP31, an ER transmembrane proteininvolved in the transit of nascent membrane proteins be-tween ER and  cis -Golgi, by ER-associated caspase-8 resultsin a p20 fragment that stimulates Ca 2+ release from the ER followed by uptake of Ca 2+ into mitochondria, mitochon-drial fission and release of cytochrome  c  , precipitatingapoptosis [31]. This mechanism has been shown to con-tribute to apoptosis following ER stress caused by certainanticancer agents, like the alkyl-lysophospholipid ana-logue edelfosine [32]. Interestingly, ER stress engaged byanthracyclines results in the early cleavage of BAP31 by apartially active form of caspase-8. This pre-apoptotic pro-cess is required for the mobilization of calreticulin fromthe ER lumen to the plasma membrane (in case of selectedagents like anthracyclines), an event that has been shownto confer a strong immunogenic character to the apoptoticcell death process [33] (discussed further in Section 3.3.2). 1.2. UPR and autophagy: another way to survive? Another important cellular mechanism that is activatedto cope with ER stress is macroautophagy (hereafter re-ferred to as autophagy), a lysosomal pathway of proteinand organelle degradation in eukaryotic cells [34]. At itsbasal rate, autophagy acts as a major housekeeping mech-anism, crucially involved in the maintenance of normalcellular homeostasis. When stimulated by cellular stressconditions, autophagy can rapidly be up-regulated to copewith an adverse environment. Characteristic for autophagyis the appearance of the autophagosomes, double-mem-braned vesicles carrying cytoplasmic content that willeventually be degraded after fusion with a lysosome.Autophagy is a highly regulated process that is controlledby a subset of evolutionarily conserved autophagy-relatedgenes (Atg). The molecular machinery of autophagy con-sists in its core of two kinase complexes, involving thePI3K-Beclin 1 and ULK1/2-Atg13-mTOR systems, a shut-tling protein and two ubiquitin-like conjugation systems(reviewed in [34]).The role of autophagy in ER stress is not completelyknown and it is possible that when the proteasome-medi-ated degradation system is overloaded by the accumula-tion of unfolded proteins in the ER, autophagy would betriggered to assist in removing them, thus serving as anER protein quality system (reviewed in [35]). Under theseconditions autophagy may have a cytoprotective role inER stress. In mammalian cells autophagy stimulation pro-tects from ER stress induced by thapsigargin and tunica-mycin. Likewise in yeasts, autophagy counterbalances ER expansion, removes aggregated proteins from the ER andimproves overall survival [34,35]. Furthermore, autophagycould prevent ER stress through the clearance of aggre-gated or toxic/diseased proteins accumulating in the cyto-sol, which cannot be removed by the proteasomaldegradation system. In agreement with this, autophagywas found to be activated via a PERK-eIF2 a  mediatedmechanism involving the ATF4-mediated upregulation of the autophagy gene Atg12, after ER stress induced by theaccumulation of polyglutamine (polyQ) proteins [36].However, since in other systems PERK-independent eIF2 a phosphorylation induced by other eIF2 a  kinases likePKR, HRI or GCN2, was also required to induce autophagy[35], it is possible that the eIF2 a -ATF4 axis rather thanPERK itself provides the molecular link between UPR andautophagy.Conversely, other studies implicated predominantly theIRE1-branch of the UPR as mediator of autophagy after ER stress [37,38]. One proposed mechanism relies on the acti-vation of JNK subsequent to IRE1-TRAF2-ASK1 complexformation. JNK has been shown to phosphorylate Bcl-2 atthe ER membrane, thereby disrupting its association withthe autophagy regulator Beclin 1 [39]. Moreover, JNK canalso directly regulate Beclin1 expression [40]. Surprisingly,XBP1 deficiency caused a profound increase in autophagywhich enhanced the removal of superoxide dismutase 1aggregates in an Amyotrophic lateral sclerosis model[41]. This suggests that IRE1 RNase activity (XBP1 splicing)and its scaffold function (activation of the TRAF2-JNK sig-naling) may play a differential role in the regulation of autophagy.ER stress is often accompanied by a release of Ca 2+ intothecytosol.IncreasedcytosolicCa 2+ levelsmayactivatesev-eral Ca 2+ -regulated pathways leading to the activation of various protein kinases, like AMPK, PKC h , and DAPK, whichhavebeenshowntohavearegulatoryroleinbothapoptosisandautophagy[42–44].Inparticular,DAPKhasbeenshownto phosphorylate Beclin 1 on the BH3-only domain, thuspreventing the interaction of this pro-autophagic proteinwith Bcl-2 [45,46]. In this context it is interesting to notethat the scaffold properties of the inositol 1,4,5-trisphos-phate receptor (IP 3 R), which can interact with both Bcl-2and Beclin 1, rather than the Ca 2+ mobilization function of  4  T. Verfaillie et al./Cancer Letters xxx (2010) xxx–xxx Please cite this article in press as: T. Verfaillie et al., Targeting ER stress induced apoptosis and inflammation in cancer, Cancer Lett. (2010),doi:10.1016/j.canlet.2010.07.016
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