onc2014458aartigo

892019 onc2014458aArtigo

httpslidepdfcomreaderfullonc2014458aartigo 19

REVIEW

Cell death by autophagy emerging molecular mechanisms

and implications for cancer therapyS Fulda123 and D Koumlgel4

Autophagy is a tightly-regulated catabolic process of cellular self-digestion by which cellular components are targeted to lysosomes

for their degradation Key functions of autophagy are to provide energy and metabolic precursors under conditions of starvation

and to alleviate stress by removal of damaged proteins and organelles which are deleterious for cell survival Therefore autophagy

appears to serve as a pro-survival stress response in most settings However the role of autophagy in modulating cell death is

highly dependent on the cellular context and its extent There is an increasing evidence for cell death by autophagy in particular in

developmental cell death in lower organisms and in autophagic cancer cell death induced by novel cancer drugs The death-

promoting and -executing mechanisms involved in the different paradigms of autophagic cell death (ACD) are very diverse and

complex but a draft scenario of the key molecular targets involved in ACD is beginning to emerge This review provides an up-to-

date and comprehensive report on the molecular mechanisms of drug-induced autophagy-dependent cell death and highlights

recent key 1047297ndings in this exciting 1047297eld of research

Oncogene advance online publication 26 January 2015 doi101038onc2014458

INTRODUCTION

Different modes of programmed cell death

Programmed cell death is an evolutionary conserved intrinsicmechanism enabling damaged and unwanted cells to commitsuicide This cellular suicide may occur either via apoptosis (type Icell death)1 or via activation of alternative death programs23

Induction of apoptotic cell death is a major mechanism by whichmost chemotherapeutic drugs and radiation kill tumor cells In thepast couple of years a tremendous effort has been invested to

develop strategies for triggering cancer cell apoptosis in a target-speci1047297c manner In addition to apoptosis an ever increasingnumber of studies substantiate the existence of alternative non-apoptotic forms of programmed cell death24 which may beexploited for cancer therapy

Until recently the nomenclature for different cell death typeswas largely based on morphological criteria and has notbeen uniformly used and recognized The emerging knowledgeon the molecular mechanisms of the different forms of programmed cell death now allows the discrimination intodistinct lsquocell death subroutinesrsquo a n d this classi1047297cation is con-tinuously re1047297ned and further improved3 Based on the currentlyexisting knowledge the Nomenclature Committee on Cell Death(NCCD) consisting of the leading experts in the 1047297eld of celldeath research has proposed the classi1047297cation into 1047297ve relativelywell-characterized different modes of (programmed) celldeath (1) extrinsic apoptosis (2) intrinsic apoptosis (3) regulatednecrosis (4) mitotic catastrophe (mitosis) and (5) autophagic celldeath (ACD)3

Apoptosis (type I cell death) is morphologically de1047297ned bycellular and nuclear shrinkage chromatin condensation (pyknosis)

nuclear fragmentation (karyorrhexis) membrane blebbing and theformation of apoptotic bodies that are engulfed by neighboring orspecialized cells5 At the biochemical level apoptosis is character-ized by phosphatidylserine exposure and (in most cases)activation of effector caspases the main executors of apoptoticcell death1 In extrinsic apoptosis extracellular death signals actvia speci1047297c transmembrane receptors culminating in a caspase-dependent apoptotic type of cell death36 The mitochondria havea pivotal role in intrinsic apoptosis which is initiated by a widevariety of intracellular stress signalsconditions leading to the

activation of the pro-apoptotic BCL-2 family members Bak andBak mitochondrial outer membrane permeabilization mitochon-drial dysfunction and release of pro-apoptotic factors such ascytochrome c apoptosis-inducing factor and Endo G from themitochondria into the cytosol36 Depending on the extent of caspase inhibition (for example by high overexpression of Inhibitor of Apoptosis (IAP) family members in tumor cells)execution of intrinsic apoptosis can occur either in a caspase-dependent or -independent fashion

In contrast to apoptosis necrotic cell death has beentraditionally characterized as a passive that is non-programmedform of cell death47 Necrosis is the end result of a bioenergeticcatastrophe resulting from adenosine triphosphate (ATP) deple-tion which is incompatible with cell survival and is thought to beinitiated mainly by cellular injury after toxic insults or physicaldamage Morphologically necrosis is characterized by vacuoliza-tion of the cytoplasm breakdown of the plasma membrane andin1047298ammation around dying cells attributable to the release of cellular contents and pro-in1047298ammatory molecules7 However inaddition to passive necrosis it is now evident that (programmed)necrosis can also occur in a regulated fashion for example after

1Institute for Experimental Cancer Research in Pediatrics Goethe-University Frankfurt Germany 2German Cancer Consortium (DKTK) Heidelberg Germany 3German Cancer

Research Center (DKFZ) Heidelberg Germany and 4Experimental Neurosurgery Center for Neurology and Neurosurgery Goethe-University Hospital Frankfurt Germany

Correspondence Professor S Fulda Institute for Experimental Cancer Research in Pediatrics Goethe-University Komturstr 3a 60528 Frankfurt Germany or Professor D Koumlgel

Experimental Neurosurgery Center for Neurology and Neurosurgery Goethe-University Hospital Frankfurt Heinrich-Hoffmann Str 7 60528 Frankfurt Germany

E-mail simonefuldakgude or koegelemuni-frankfurtde

Received 10 November 2014 revised 11 December 2014 accepted 12 December 2014

Oncogene (2015) 1ndash9

copy 2015 Macmillan Publishers Limited All rights reserved 0950-923215

wwwnaturecomonc



alkylating DNA damage during excitotoxicity and after ligation of death receptors3 Regulated necrotic cell death often involvesactivation of the kinases receptor-interacting protein 1 (RIP1) andRIP3 and can be blocked by the speci1047297c RIP1 inhibitor necrostatin-1 The term lsquonecroptosisrsquo is restricted to these necrostatin-1-inhibitable forms of necrotic cell death34 Mechanistically deathexecution via necroptosis is currently not well understood butmay involve energy failure oxidative stress and lysosomalmembrane permeabilization

The term lsquomitotic catastrophersquo is usually used for cell deathinduced by aberrant mitosis and executed either during mitosis orin the subsequent interphase (this type of cell death is sometimes

also called lsquomitoptosisrsquo)3 However mitotic catastrophe can exhibitfeatures of apoptosis or necrosis and the concept that mitoticcatastrophe truly represents a distinct mode of cell death iscurrently not generally accepted

Autophagy and ACD

Autophagy is a cellular stress response and a quality controlmechanism that in general acts in a pro-survival manner Differentforms of autophagy can be discriminated including macroauto-phagy (hereafter simply denoted as autophagy) microautophagyand chaperone-mediated autophagy During autophagy whichserves to regulate the turnover of long-lived proteins anddamaged organelles these cellular constituents are engulfed in

double-membrane-containing vesicles called autophagosomes(Figure 1)689 Their vesicular content is subsequently digestedby lysosomal proteases after fusion of autophagosomes withlysosomes89 Autophagy is a complex multistep process that isgenetically regulated by the ~ 30 autophagy-related genes(ATG) discovered hitherto in mammals In addition autophagyis subject to posttranscriptional regulation for example bymicroRNAs as miR-101 has been shown to suppress autophagy

autophagic

cell death

autophagosome

lysosomeendosome

fusion

autophagic

initiation

membrane

LC3-II

autophagyinduction

stress conditions

cancer drugs

(Resveratrol APO866 TMZ THC HDACs)

vesicleelongation

flux

chloroquine

BafA1

Betulinic acid B10

Vps34

inhibitors

Atg5Atg7

depletion

Beclin-1

depletion

BECN1

core complex

vesicle

nucleation

Vps34 Bcl-2xLMcl-1

BECN1

Bcl-2xL

Mcl-1BECN1Vps34

complexdisruption

complex

formation

BH3 mimetics

autophagosome

Atg5membrane

recruitment

Atg5

FADD

RIP3RIP1

necroptosis

mTOR

autolysosome

cargo

degradation

Figure 1 Different stages of autophagy and de1047297nition of ACD Autophagy can be induced by multiple stimuli including metabolic stressorganelle dysfunction protein aggregation and several cancer drugs many of which target the central autophagy regulator mTOR Thedifferent stages of this process are tightly regulated by the core autophagy proteins encoded by ATGs Autophagosome biogenesis startswith the formation of an initiation membrane that can be derived from the ER and several other cellular membrane sources Vesicle nucleationis promoted by a large macromolecular complex containing the lipid kinase Vps34 (BECN1 core complex) BECN1 (ATG6) serves to activate

Vps34 leading to formation of PtdIns3P which is required for this stage of the autophagic pathway Vesicle elongation is regulatedby two ubiquitin-like conjugation systems involving several ATG proteins (1) a large protein complex containing ATG5 (and ATG12ATG16)and (2) ATG7ATG3-driven attachment of phosphatidylethanolamine to LC3-I leading to the generation of LC3-II which is inserted intothe autophagosomal membrane Following vesicle closure mature autophagosomes fuse with lysosomes or endosomes to autolysosomes inwhich the autophagosomal content is digested by lysosomal proteases Excessive activation of the autophagy pathway can leadto an autophagy-dependent cell death in several paradigms The term ACD should be exclusively limited to cases of cell death that aremediated and not simply accompanied by autophagy Therefore only cases where inhibition of the autophagic pathway suppresses celldeath can be considered as true ACD The effects of autophagy inhibition on cell death can be experimentally addressed at differentstages of autophagy either by Vps34 inhibitors or by knockoutknockdown of core autophagic modulators such as ATG5 ATG7 or BECN1It is currently controversially discussed whether the term ACD should be reserved only for cases in which the 1047297nal cell death process ismediated by an enhanced autophagic 1047298ux rather than by alternative forms of cell death such as necroptosis In light of this controversyand the cytotoxicity of drugs inhibiting the autophagic 1047298ux from autophagosomes to lysosomes (chloroquine Ba1047297lomycin A1)interference at this stage of the autophagy pathway is currently not a generally accepted approach to analyze ACD BH3 mimeticshave been implicated in several paradigms of ACD They are capable to induce the release of BECN1 from its inhibitory interaction withBCL-2BCL-xL and have been shown to recruit necrosome components to the autophagosomal membrane thereby inducing ACD (for detailsplease refer to the main text)

Autophagy and cell death

S Fulda and D Koumlgel

2

Oncogene (2015) 1 ndash 9 copy 2015 Macmillan Publishers Limited



by downregulation of STMN1 RAB5A and ATG4D10 The process of autophagy begins with the formation of a nascent initiationmembrane which serves as a prestage of autophagosomesSeveral ATG proteins (that is ATG5 ATG7 ATG10 and ATG12) havebeen implicated in autophagosome formation The mammalianortholog of yeast ATG8 was originally identi1047297ed as the light chain3 (LC3) of microtubule-associated proteins LC3 exists in twoforms that is LC3-I and its lipidated derivative LC3-II which arelocalized in the cytosol (LC3-I) or in autophagosomal membranes(LC3-II) respectively11 BECN1 (ATG6) is a core element of cellularautophagy and a component of the class III phosphatidylinositol3prime-kinase complex required both for formation and transport of autophagosomes BECN1 is a tumor-suppressor gene monoalleli-cally deleted in several types of cancer including ovarian breastand prostate cancer12 In addition the BECN1-binding proteinultraviolet irradiation resistance-associated gene (UVRAG) hasbeen implicated as a tumor-suppressor gene that is monoalleli-cally mutated at high frequency in human colon cancers13 Bif-1(also known as Endophilin B1) is another BECN1-interactingprotein that can act as a tumor suppressor as knockout of Bif-1has been reported to increase the development of spontaneoustumors in mice14 Furthermore Ambra1 has been demonstrated tointeract with the BECN1 complex and de1047297ciency of Ambra1 inmouse embryos has been shown to result in uncontrolled cellproliferation15 suggesting that Ambra1 has some tumor-suppressive properties The quality-control function of autophagyespecially in the context of genomic integrity is correlated to itsrole in cancer development autophagy can act as a tumor-suppressor mechanism as impaired autophagy that is the lack of proper removal of toxic protein aggregates and organelles such asdysfunctional mitochondria has been shown to promote oxidativestress DNA lesions and genomic instability16

The net effect of autophagy on cell death is highly contextualand both cytoprotective and cytotoxic functions of autophagyhave been reported Autophagy comprises a primordial pro-survival stress response for example under conditions of nutrientdeprivation where it serves to ensure energy balance In additionthere is now substantial evidence showing that enforced over-activation of autophagy can lead to ACD (type II cell death)(Figure 1) that is cellular self-digestion via the autophagosomalndashlysosomal pathway beyond the point of allowing cellsurvival3481617 It was proposed that the apparent dichotomybetween pro-survival and pro-death autophagy may be causallyrelated to the extent and duration of autophagy indicating thatthis dual function may represent a threshold effect of autophagyas also observed for other stress responses such as theendoplasmatic reticulum (ER) stress response or activation of p5317

A number of studies suggest that (at least in some instances) itis not simply overactivation of unspeci1047297c bulk autophagy butrather the selective removal of autophagy substrates thatpromotes cell death Oxidative stress is a key feature observedin many paradigms of autophagy-dependent cell death1819 and

the autophagy-mediated selective degradation of the endogen-ous reactive oxygen species (ROS) scavenger catalase has beenshown to induce ACD20 In an analogous manner inhibition of selective autophagy by targeting the autophagy receptorsp62SQSTM1 and BNIP3 was proposed to cause ACD2122 Furthermolecular mechanisms possibly underlying the dual function of autophagy in promoting either cell death or cell survival arecurrently being addressed

The exact de1047297nition of ACD has been controversially discussedin the 1047297eld It was suggested that many of the older descriptivecases of the so-called ACD actually re1047298ect death accompanied bythe block of the autophagic 1047298ux rather than death mediated byautophagy6 According to the criteria proposed by the NCCD theterm lsquoautophagic cell deathrsquo should be exclusively used from afunctional perspective and limited to cases of cell death that are

mediated by autophagy and can be suppressed by the inhibitionof the autophagic pathway (either by Vps34 inhibitors or byknockoutknockdown of core autophagic modulators such asATG5 ATG12 or BECN1) (Figure 1)23 Some authors have arguedfor even more stringent criteria and have proposed to use theterm ACD only in cases where the 1047297nal cell death process ismediated by an enhanced autophagic 1047298ux rather than byapoptosis or necroptosis624 In contrast to the authentic cases of ACD as de1047297ned by the NCCD cases of cell death that simplyexhibit markers of autophagy such as an increase in autophago-somes the lipidation of LC3 or an increased degradation of theautophagic substrate p62 but cannot be suppressed by autop-hagy inhibition should not be classi1047297ed as ACD Probably themost convincing examples of bona 1047297de ACD have so far beenobserved in developmental cell death in lower model organismsincluding Caenorhabditis elegans Drosophila melanogaster andDyctiostelium discoideum625ndash27 In addition there are an everincreasing number of studies demonstrating true ACD inmammalian cells Nevertheless given the fact that autophagyrepresents a double-edge sword with both tumor-suppressive and-promoting properties it remains to be determined whether ornot engagement of autophagy even under conditions of ACD mayelicit a more complex cellular response beyond modulation of celldeath Some recent examples supporting the mechanistic conceptof ACD are outlined below

The oncogene H-RAS was shown to trigger upregulation of theBH3-only proteins Noxa and BECN1 and a caspase-independentACD thereby limiting clonogenic survival28 In this context ACDmay represent a safeguard mechanism to limit the oncogenicpotential of deregulated RAS signals28

Recently an autophagy-dependent type of cell death termedlsquoautosisrsquo was described29 In this study a cell-permeable auto-phagy-inducing peptide that is Tat-BECN1 was shown to induceautophagy and importantly cell death in a dose-dependentmanner29 Autotic cell death was also observed in starved cellsin vitro and in hippocampal rat neurons during cerebral ischemiain vivo29 This type of cell death was blocked by pharmacologicalor genetic inhibition of autophagy antagonists of the ion pumpNa+K+-ATPase or genetic knockdown of the Na+K+-ATPase α1subunit whereas inhibition of apoptosis or necroptosis providedno protection29 The results of this study also suggest that highamounts of the BH3-only protein BECN1 are suf 1047297cient to triggerACD in the absence of other cellular stress conditions In a similarmanner overexpression of the BH3-only protein apolipoprotein L1was previously shown to trigger ACD30

In another recent study caspase-10 was identi1047297ed in an RNAiscreen to be required for suppression of an intrinsic form of ACDin multiple myeloma31 Caspase-10 was demonstrated to beessential for dampening intrinsic autophagy by cleaving theBCL-2-interactor and potent inducer of autophagy BCLAF1thereby preventing overactivation of autophagy to avoid ACD31

Furthermore the orphan nuclear receptor TR3 was shown topromote ACD in melanoma cells32 In this study the TR3-targeting

compound 1-(345-trihydroxyphenyl)nonan-1-one was used totrigger TR3 translocation to the mitochondrial inner membranethrough Tom40 and Tom70 channel proteins dissipation of themitochondrial membrane potential and induction of autophagyassociated with excessive mitochondria clearance and irreversiblecell death These 1047297ndings underscore the notion that selectivemitophagy which typically acts in a cytoprotective manner mayreach a certain threshold level at which it will turn into a death-promoting process

Despite the notion that induction of autophagy in response toanticancer treatments often represents a cytoprotective mechan-ism of cells trying to cope with stress33 overall there is now anincreasing and solid evidence for the existence of true ACD Giventhe fact that disrupted autophagy as well as excess autophagy canhave detrimental consequences on cell viability abrogation as



3

copy 2015 Macmillan Publishers Limited Oncogene (2015) 1 ndash 9



well as overactivation of the autophagy pathway may bothrepresent relevant strategies for cancer therapy Accordinglyinduction of autophagy-dependent cell death by pro-autophagicdrugs has emerged as a novel concept to sensitize cancer cells totherapy or to directly kill them with the aim of exploiting caspase-independent programmed cell death pathways for the develop-ment of novel cancer therapies Recent relevant examples forACD induced by cancer drugs are outlined in the following

paragraphs ( Table 1)

ACD INDUCED BY CANCER DRUGS

Gossypol

Antiapoptotic BCL-2 family members can form a complex withBECN1ATG634ndash36 and formationdissociation of this complex mayhave an important role in modulating autophagy in tumor cellsBCL-2 and BCL-xL sequester BECN1 via binding to its BH3 domainand prevent it from forming a multiprotein complex essential forvesicle nucleation during the early steps of the autophagicprocess thereby inhibiting autophagy Consequently BH3mimetics are capable to activate both apoptosis andautophagy37 Gossypol is a natural polyphenolic compound andBH3 mimetic derived from cotton seeds which was initially

identi1047297ed as an antifertility agent in China during the 1950s andshown to possess cell death-promoting effects in various in vivo

and in vitro models38ndash41 Gossypol acts as a pan-BCL-2 inhibitorand can inactivate BCL-2 BCL-xL MCL-1 and BCL-w There are twoenantiomers of gossypol (+)-gossypol and (minus )-gossypol (alsocalled AT-101) the latter being more potent as an inhibitor of tumor growth In cancer cells with an intact apoptotic machinery(minus )-gossypol has been reported to induce apoptotic celldeath39ndash42 In contrast cell death triggered by gossypol largelyseems to depend on induction of ACD in apoptosis-de1047297cient

prostate cancer and malignant glioma cells

4344

In glioma cells ( minus )-gossypol was shown to trigger translocationof LC3 to autophagosomes lysosomal activity and a latecytochrome c release but cell death occurred in the absence of overt lysosomal damage and effector caspase activation43

(minus )-Gossypol and the alkylating agent temozolomide (TMZ)cooperated to activate ACD in O6-methylguanine-DNA-methyl-transferase-negative glioma cells and lentiviral knockdown of BECN1 or ATG5 in different glioma cell lines strongly diminishedcell death induced by ( minus )-gossypol and combined treatment with

TMZ indicating that autophagy contributes to this type of celldeath43 In contrast stable knockdown of the endogenousautophagy inhibitor mammalian target of rapamycin (mTOR)increased ACD43

In line with these 1047297ndings (minus )-gossypol has been reported to

cause caspase-independent non-apoptotic cell death in malig-nant peripheral nerve sheath tumor cells which could be blocked

Table 1 Examples of autophagic cell death-inducing anticancer drugs

Substances Cancer types Autophagy inhibition used References

BH3 mimeticsObatoclax ALL BECN1 and ATG7 siRNA 3-MA 50

Obatoclax RMS ATG5 shRNA ATG7 siRNA BafA1 51

Obatoclax ALL BECN1 and ATG5 siRNA 3-MA 52

Obatoclax Colon carcinoma BECN1 and ATG5 siRNA 53

Obatoclax Breast carcinoma BECN1 siRNA 3-MA 55

Obatoclax Pancreatic carcinoma BECN1 siRNA 56

Obatoclax AML CQ 57

Obatoclax B-cell lymphoma BECN1 siRNA 58

Gossypol Prostate carcinoma BECN1 and ATG5 siRNA 3-MA 4476

Gossypol GBM BECN1 and ATG5 siRNA BafA1 43

Gossypol Peripheral nerve sheath tumor 3-MA 45

Natural productsBetulinic acid derivative B10 GBM BECN1 and ATG5 ATG7 siRNA 63

Betulinic acid Cervical carcinoma ATG5 siRNA ATG5minus minus MEFs 64

Resveratrol Breast carcinoma BECN1 and hVP34 ATG7 siRNA 65

Resveratrol CML ATG5 p62 and LC3 siRNA 66

HDACsSAHA Chondrosarcoma 3-MA 68

SAHA Cervical carcinoma BECN1 and ATG7 siRNA 69

SAHA OSU-HDAC 42 HCC ATG5 siRNA 3-MA 70

Chemotherapeutics TMZ GBM 3-MA 71

TMZ GBM BECN1 and ATG5 siRNA 43

Cannabinoids THC GBM ULK1 ATG5 and Ambra-1 siRNA ATG5minus minus MEFs 74

THC OWH-015 HCC ATG5 siRNA 3-MA 75

OthersLapatinib HCC BECN1 and ATG5 ATG7 shRNA 3-MA 54

APO866 Leukemia lymphoma cells ATG5 and ATG7 siRNA 3-MA 1967

Abbreviations AML=acute myeloid leukemia BafA1=Ba1047297lomycin A1 CML=chronic myeloid leukemia CQ=chloroquine GBM=glioblastoma

HCC=hepatocellular carcinoma HDAC=histone deacetylase 3-MA=3-methyladenine MEF=mouse embryonic 1047297broblast RMS= rhabdomyosarcoma

SAHA= suberoylanilide hydroxamic acid shRNA= short hairpin RNA siRNA= small interfering RNA THC= tetrahydrocannabinol TMZ= temozolomide



4




with 3-methyladenine (3-MA) and involved intracellular ironchelation and hypoxia-inducible factor-1α-induced expression of theBH3-only protein BNIP345 Similarly BNIP3 was found to beinvolved in autophagy-related cell death induced by combinedtreatment of pancreatic adenocarcinoma cells with (minus )-gossypoland BRD4770 a small-molecule inhibitor of the histone methyl-transferase G9a46 These observations suggest that the BH3-onlyprotein and mitophagy-regulator BNIP3 may have an importantfunction in ACD47 although it should be noted that the latterstudy failed to address the question as to whether or notautophagy does indeed contribute to this particular case of celldeath In addition to the release of BECN1 from BCL-2BCL-xL andactivation of BNIP3 oxidative stress is presumably involved in theprominent pro-autophagic and death-promoting effects of (minus )-gossypol Indeed (minus )-gossypol and its derivative apogossy-polone have previously been shown to be potent inductors of oxidative stress in cancer cells3848

As indicated above the effects of (minus )-gossypol-inducedautophagy on cell death appear to be highly dependent on thecellular context as also demonstrated in prostate cancer andbreast carcinoma cells In androgen-independent prostate cancercells expressing high levels of BCL-2 and resistant to apoptosis

(minus

)-gossypol also preferentially induced ACD which couldpartially be blocked by knockdown of ATG5 and BECN144 Incontrast apoptosis was preferentially induced in cells with lowBCL-2 expression44 In this study (minus )-gossypol was shown toinduce BECN1- and ATG5-dependent autophagy via releasingBECN1 from BCL-2 and BCL-xL at the ER thus triggering theautophagic cascade44 In addition oral administration of ( minus )-gos-sypol signi1047297cantly inhibited the growth of androgen-independentprostate cancer xenografts suggesting a potential relevance of ACD for the treatment of human hormone-refractory prostatecancer with BCL-2 overexpression44 In contrast to these observa-tions (minus )-gossypol inhibited the interaction between BECN1 andBCL-2 to induce BECN1-dependent autophagy which wasfollowed by the execution of apoptotic cell death in a poptosis-competent MCF-7 human breast adenocarcinoma cells49 Knock-

down of Vps34 and ATG5 reduced (minus

)-gossypol-induced autop-hagy and in this particular case ( minus )-gossypol-mediated apoptoticcell death was potentiated by treatment with or by smallinterfering RNAs against core autophagy genes (Vps34 BECN1and ATG5)49 suggesting a cytoprotective function of ( minus )-gossy-pol-induced autophagy in this apoptosis-pro1047297cient cell model

Obatoclax

There are a number of studies implicating autophagy as acytotoxic mechanism in obatoclax-induced cell death In child-hood acute lymphoblastic leukemia (ALL) subcytotoxic concen-trations of obatoclax have been reported to overcomeglucocorticoid resistance by inducing ACD50 Induction of autophagy has been associated with disruption of the interactionof BECN1 with MCL-1 as well as suppression of mTOR activity50 Inline with the known suppressive function of mTOR on autophagyinduction the mTOR inhibitor rapamycin acted in concert withdexamethasone to induce ACD in this model50 Concomitantknockdown experiments showing that silencing of BECN1 or ATG7rescued obatoclax-mediated sensitization to glucocorticoids inboth cell viability and clonogenic assays con1047297rmed that stimula-tion of autophagy was critical for the ability of obatoclax toovercome glucocorticoid resistance50 The authors went on todemonstrate that RIP1 as well as cylindromatosis (CYLD) wererequired for the execution of cell death upon treatment withobatoclax and glucocorticoids whereas both RIP1 and CYLD weredispensable for the initiation of autophagy in this context50 AsRIP1 and CYLD are known as critical regulatory proteins innecroptotic signaling this study provided a genetic link between

ACD and necroptosis although the underlying molecular mechan-isms have remained elusive at that point

Obatoclax subsequently was shown to stimulate the interactionof components of autophagosomal membranes such asATG5 with proteins of the necrosome complex such as RIP1 inan ATG5-dependent fashion as ATG5 silencing inhibited thisinteraction51 The requirement of autophagy for cell deathinduction was demonstrated by genetic silencing as depletionof ATG5 or ATG7 inhibited obatoclax-mediated autophagosomeformation and cell death51 Data showing that genetic orpharmacological ablation of RIP1 inhibited obatoclax-induced celldeath con1047297rmed that RIP1 is a crucial mediator of cell death upontreatment with obatoclax while RIP1 turned out to be dispensablefor obatoclax-stimulated autophagosome formation51

Furthermore Heidari et al52 reported that obatoclax is able tobypass glucocorticoid resistance in ALL via the induction of autophagy in addition to triggering apoptosis Treatment of ALLcells with obatoclax resulted in a rapid LC3 conversion anddegradation of p62 protein both used as markers of autophagy52

Knockdown of ATG5 prevented obatoclax-stimulated autophagyas well as cell death52 underlining that autophagy represents acell death mechanism in this context By comparison silencing of BECN1 failed to block obatoclax-stimulated autophagyinduction52 pointing to a BECN1-independent mode of autop-hagy Similarly pharmacological inhibition by 3-MA did not affectobatoclax-mediated autophagy52

In colon and breast carcinoma cells obatoclax has beenreported to enhance cell death induced by lapatinib53 a small-molecule tyrosine kinase inhibitor targeting epidermal growthfactor receptors and Her2Neu which is capable to trigger ACDeither alone54 or in combination with other drugs Obatoclax-mediated sensitization to lapatinib was associated with LC3conversion and suppression of AKTmTOR signaling as indicatedby reduced phosphorylation of AKT mTOR and S6K153 Knock-down of BECN1 or alternatively of AT G5 protected againstobatoclax-lapatinib-induced cytotoxicity53 underscoring that theinduction of autophagy was necessary for combination treatment-mediated antitumor activity Similar to obatoclax knockdown of MCL-1 or BCL-x

L

increased LC3 vesiculation and cell death bylapatinib53 Also Tang et al55 reported that obatoclax acts inconcert with lapatinib to trigger LC3 conversion and cytotoxicautophagy in breast carcinoma cells as genetic inhibition of autophagy by knockdown of BECN1 or pharmacological inhibitionby using 3-MA signi1047297cantly reduced cell death induced by thecombination therapy Mechanistic studies showed that obatoclax- lapatinib-mediated autophagy was associated with Noxa-mediated displacement of BECN1 from MCL-1 as well as inhibitionof mTOR signaling55

Furthermore obatoclax has been described to enhance theinduction of cell death upon treatment with histone deacetylase(HDAC) inhibitors such as vorinostat or sodium valproate togetherwith the multikinase inhibitor sorafenib via autophagy56 Thisobatoclax-mediated sensitization to HDAC inhibitor-sorafenib-

triggered cell death was accompanied by LC3 vesiculation andinhibited by knock down of BECN1 consistent with an autophagicform of cell death56 Also in acute myeloid leukemia obatoclaxwas shown to exert antileukemic activity in concert with HDACinhibitors that is vorinostat and MGCD010357 This synergisticantileukemic activity by obatoclax and HDAC inhibitors wasassociated with the induction of both autophagy and apoptosis57

The authorsrsquo conclusion that autophagy contributes to thesynergistic antileukemic effects by the combination treatment ishowever only based on experiments using chloroquine to inhibitautophagy but lacks genetic evidence showing that autophagyrepresents a cell death mechanism under these conditions

Therefore the question as to whether or not autophagy indeedmediates cell death during obatoclaxHDAC inhibitor combinationtreatment remains to be clari1047297ed



5




In B-cell lymphoma obatoclax was reported to stimulate LC3conversion and ACD in a caspase-independent manner that wassigni1047297cantly inhibited by BECN1 knockdown58

Importantly the conclusion that autophagy represents acytotoxic process contributing to obatoclax-induced cell death isbased in most studies on genetic evidence showing thatknockdown of essential autophagy genes such as BECN1 ATG5or ATG7 rescue cell death upon treatment with obatoclaxHowever there are also some studies concluding that obatoclaxtriggers cell death via autophagy that are solely based onexperiments using pharmacological inhibitors of autophagy suchas chloroquine or 3-MA which lack absolute speci1047297city and mayalso affect additional processes besides autophagy This callsfor some caution as far as the functional impact of autophagy induction in the context of obatoclaxrsquos cytotoxicity isconcerned

In addition to the pro-death function of autophagy in thecourse of obatoclax-induced cell death autophagy has also beenimplicated as a cytoprotective or bystander mechanism inresponse to treatment with obatoclax in some studies59ndash61 Inlung carcinoma cells obatoclax-induced autophagy as documen-ted by LC3 processing has been reported to depend on ATG7 butnot on BECN161 However ATG7 was found to be dispensable for

obatoclax-induced cell death in this model

61

implying that theinduction of autophagy by obatoclax was not required forobatoclax-mediated cytotoxicity In esophageal carcinoma andosteosarcoma cells treatment with obatoclax caused conversionof LC3 and ultrastructural changes consistent with the formationof autophagosomes59 Based on experiments showing that thecytotoxicity of obatoclax was signi1047297cantly reduced by addition of chloroquine or 3-MA the authors concluded that obatoclax-induced autophagy exerts cytoprotective functions under theseconditions59 In breast carcinoma cells BECN1 turned out to berequired for obatoclax-stimulated autophagosome formation butnot for obatoclax-induced cell death60 In addition to engagingautophagosome formation in this model obatoclax was found toblock autophagic degradation of vesicular cargo by attenuatingcathepsin activity60

Natural products

In addition to gossypol other natural products have beenreported to engage cell death pathways via autophagy Forexample the plant-derivative betulinic acid a pentacyclictriterpenoid derived from white birch trees62 has been implicatedin modulating autophagy-mediated cell death The semisyntheticglycosylated derivative of betulinic acid B10 has been shown bothto trigger autophagy and to abrogate the autophagic 1047298ux inglioblastoma cells thereby switching autophagy into a cytotoxicprocess63 This B10-induced cell death was found to be associatedwith destabilization of lysosomes and release of lysosomalenzymes into the cytoplasm63 Consistently the cathepsininhibitor Ca074Me signi1047297cantly decreased B10-induced cell deathfurther supporting that the release of lysosomal enzymescontributes to B10-triggered cell death63 In line with the notionthat destabilization of lysosomes can convert autophagy into adetrimental pathway once autophagy has been initiated inhibi-tion of lysosomal enzyme activity was shown to protect againstB10-mediated cytotoxicity63 Genetic studies showing that silen-cing of core autophagy genes including BECN1 ATG5 or ATG7rescues cell death upon exposure to B10 supported the conclusionthat autophagy represents a cell death mechanism in the courseof B10 treatment63 By comparison induction of autophagy by theparental compound betulinic acid has been implied as acytoprotective response as cervical carcinoma cells with knock-down of ATG5 or mouse embryonic 1047297broblasts lacking A TG5 orATG7 exhibited increased sensitivity to betulinic acid64 It willtherefore be interesting to explore whether different derivatives of

betulinic acid vary in their ability to modulate the autophagicpathway

Resveratrol represents another naturally occurring compoundthat has been reported to trigger autophagy-mediated cell deathin cancer cells Interestingly a non-canonical BECN1-independentpathway of autophagy induction was documented upon treat-ment with resveratrol in human breast carcinoma65 Thisconclusion is based on experiments using BECN1 and Vps34 smallinterfering RNAs that failed to inhibit resveratrol-stimulatedautophagy and cell death whereas knockdown of ATG7 protectedbreast carcinoma cells against resveratrol-mediated autophagyand cell death65 Engagement of the autophagic program byresveratrol was shown to involve resveratrol-imposed inhibition of AKTmTOR signaling as well as conversion of LC3-I to LC3-II65 Inchronic myeloid leukemia resveratrol has been demonstrated topromote ACD via c-Jun N-terminal kinase-mediated p62SQSTM1expression as well as activation of adenosine monophosphate-activated kinase which in turn inhibited mTOR signaling66

Knockdown experiments showing that silencing of key autophagycomponents such as ATG5 LC3 or p62 protected againstresveratrol-triggered loss of cell viability con1047297rmed that auto-phagy serves as a cytotoxic process in this model66

APO866 There is evidence showing that cancer cells have a highernicotinamide adenine dinucleotide (NAD+) turnover rate thannon-transformed cells suggesting that this biosynthetic pathwayrepresents an attractive target for cancer treatment67 Recently itwas reported that APO866 an inhibitor of nicotinamide phos-phoribosyltransferase the rate-limiting enzyme involved in NAD(+) synthesis induced an autophagy-dependent type of cell deathin leukemia and lymphoma cells1967 although both studies differwith regard to the potential role of apoptosis in APO866-inducedcell demise In the study by Cea et al67 the authors observedAPO866-dependent induction of autophagy and cell death butthe pan-caspase inhibitor zVAD-fmk as well as speci1047297c inhibitors of caspase-3 and -9 failed to protect from APO866-induced celldeath In contrast the pharmacological autophagy inhibitors

wortmannin LY294002 3-MA and chloroquine all reducedAPO866-induced cell death However genetic ablation of auto-phagy genes was not performed in this study In the study byGinet et al19 the authors demonstrated that APO866 inducesautophagosome formation and SQSTM1p62 degradation inparallel to caspase-3 activation Interestingly APO866 treatmentevoked depletion of the ROS scavenger catalase thus leading toenhanced ROS levels and cell death Inhibition of autophagy byknockdown of ATG5 and ATG7 abrogated catalase degradationROS production caspase activation and cell death after APO866treatment which could be rescued by addition of exogenouscatalase

HDAC INHIBITORS (HDACIS)

As aberrant regulation of epigenetic control of gene expressionincluding histone acetylation represents a hallmark of humancancer small-molecule inhibitors of HDACs are considered aspromising cancer therapeutics Among their various cellulareffects HDACIs have been reported to engage autophagy thathas also been linked to the induction of cell death For examplethe HDACI suberoylanilide hydroxamic acid (SAHA) was describedto trigger autophagy-associated cell death in chondrosarcomacells68 Cell death upon treatment with SAHA was accompanied byincreased lipidation of LC3 as well as ultrastructural changesconsistent with autophagosome formation68 Furthermore theaddition of 3-MA signi1047297cantly restored cell viability upontreatment with SAHA leading the authors to the conclusion thatautophagy-associated cell death is involved in the antitumor



6




activity of SAHA against chondrosarcoma cells68 However thequestion as to whether or not autophagy represents a cytotoxicmechanism in this contest remains to be answered as genetic

evidence supporting this conclusion is currently lackingIndependent studies similarly reported that SAHA stimulates

autophagy-associated cell death in cancer cells Induction of

autophagy by SAHA in HeLa cervical carcinoma cells wasassociated with the inhibition of mTOR and accompanied by

typical ultrastructural features consistent with autophagy as wellas biochemical evidence such as conversion of LC3-I to LC3-II69

Although induction of autophagy was shown to depend on keygenetic components of autophagy signaling including BECN1 and

ATG769 the functional relevance of autophagy in SAHA-inducedcell death remains to be determined In hepatocellular carcinomaHDACIs including SAHA and OSU-HDAC-42 were described to

trigger ACD based on both pharmacological as well as geneticevidence demonstrating that the addition of 3-MA or knockdownof ATG5 reduced SAHA-mediated cytotoxicity70 In parallel

treatment with SAHA caused morphological changes typical forautophagy that is autophagosome formation as well as lipidationof LC3-I to generate LC3-II and was accompanied by down-

regulation of p62 consistent with increased autophagic 1047298ux70

Engagement of autophagy by HDACIs might involve inhibition of mTOR signaling as SAHA and OSU-HDAC-42 resulted in down-

regulation of AKTmTOR signaling activity70

TEMOZOLOMIDE

Furthermore the DNA-alkylating agent TMZ which causesformation of O6-methylguanine in DNA that mispairs with thymineduring the following cycle of DNA replication and is routinely used

in the treatment of malignant glioma was shown to stimulateautophagy associated with the recruitment of LC3 to autophago-somal membranes71 The role of autophagy in TMZ-induced

glioma cell death is currently controversial On one side earlyinterference with autophagy by 3-MA inhibited both autophago-some formation as well as TMZ-mediated cytotoxicity71 arguing

for a pro-death role of autophagy in TMZ-triggered cell death Inline with these observations cell death induced by single agenttreatment with TMZ or by TMZ in combination with (minus )-gossypolwas attenuated by early interference with autophagy via lentiviral

depletion of AT G5 and BECN143 On the other side the study byKanzawa et al71 also demonstrated that interference withautophagy at a later stage using the vacuolar type H+ (ATPase)

inhibitor Ba1047297lomycin A1 increased rather than reduced TMZ-induced cell death These data suggest that concomitantinduction of autophagy and disruption of the autophagic 1047298ux

enhances TMZ-triggered cytotoxicity possibly through lysosomaland mitochondrial membrane permeabilization and a shifttowards apoptotic cell death71 To complicate matters even

further it was also proposed that TMZ stimulates autophagy as

a cytoprotective response in glioblastoma that contributes tochemoresistance72 This notion is supported by experimental data

demonstrating that (1) TMZ can stimulate an autophagy-dependent surge of ATP that limits non-apoptotic glioma celldeath and that (2) genetic ablation of BECN1 reduced colony

formation after TMZ treatment in glioma cells72 The opposingeffects of autophagy described in these studies are dif 1047297cult toexplain but may be related to the different genetic backgrounds

of the cell models used72 andor to dose-dependent changes inthe predominant DNA lesions induced by TMZ (O6-methylguaninelesions versus N -alkylations)73 The precise role of autophagy and

the relevance of autophagy-dependent mechanisms for themodulation of TMZ-induced glioma cell death await furtherclari1047297cation

CANNABINOIDS

Moreover the main active component of cannabinoids that istetrahydrocannabinol (THC) has been demonstrated to triggerautophagy-mediated cell death in different cancer entitiesincluding glioblastoma and hepatocellular carcinoma7475 Thefunctional requirement of autophagy to mediate THC-inducedantitumor activity has been established both in vitro and in vivoAccordingly genetic silencing of autophagy-related genes

including ULK1 ATG5 and Ambra-1 in glioblastoma cells as wellas knockout of ATG5 in 1047297broblasts rescued THC-imposedcytotoxicity74 Also in vivo using a tumor xenograft model basedon transformed mouse embryonic 1047297broblasts pro1047297cient orde1047297cient in ATG5 the induction of autophagy has been shownto be critical for THC-imposed antitumor activity74 In mechanisticterms the authors demonstrated that THC stimulates accumula-tion of ceramide and phosphorylation of eukaryotic translationinitiation factor 2 alpha which engages an ER stress response thatleads to upregulation of ER stress-related proteins including CHOPand tribbles homologue 3 (TRB3)74 In turn TRB3 facilitatesautophagy via inhibition of AKTmTOR signaling74 Interestinglyautophagy was shown to be upstream of apoptosis that involvedloss of mitochondrial membrane potential and production of ROS74 Accordingly THC-induced features of apoptotic cell death

including Annexin-V positivity caspase-3 activation and loss of mitochondrial membrane potential were attenuated in cellsde1047297cient in key autophagy genes74 Vice versa BAXBAK doubleknockout mouse embryonic 1047297broblasts exhibited similar conver-sion of LC3-I to LC3-II compared with wild-type cells con1047297rmingthat stimulation of autophagy occurs upstream of apoptosis in

THC-induced cancer cell death74 It is interesting to note that THC-stimulated autophagy-mediated cell death has been documentedin different types of cancer cells but not in non-transformedastrocytes74 pointing to a potential therapeutic window thatcould be exploited for cancer therapy

In hepatocellular carcinoma the cannabinoid receptor2-selective agonist JWH-015 was reported to trigger autophagy-mediated cell death in addition to THC75 In this study theengagement of the autophagic program by cannabinoids was

shown to involve activation of adenosine monophosphate-activated kinase via calmodulin-activated kinase beta in additionto the induction of TRB3 and subsequent inhibition of AKTmTORsignaling75 Genetic or pharmacological inhibition of autophagyby ATG5 silencing or addition of 3-MA rescued the THC- orJWH-015-imposed suppression of tumor growth of subcutaneoushepatocellular carcinoma xenografts in vivo emphasizing thatactivation of autophagy was required for the antitumor activity of

THC and JWH-01575

OUTLOOK

Autophagy has been shown to exert dual functions in humancancers that is both as tumor suppressor and as tumor promoterAs many anticancer drugs can engage autophagy a betterunderstanding of the molecular mechanisms that regulate ACDcan pave the avenue for rational exploitation of this cellularprogram for therapeutic purposes There are now a number of examples showing that the induction of ACD indeed represents acrucial event for the drugrsquos antitumor activity This opens newperspectives for the development of novel therapeutic strategiesand drug discovery In addition engagement of ACD may offernew options to overcome treatment resistance as autophagy hasbeen reported to serve as a backup mechanism with importantimplications to bypass resistance especially in apoptosis-refractorytumors However the molecular determinants that are responsiblefor turning autophagy into a death process are currently stillpoorly understood As autophagy is a double-edged sword at theinterface of cell survival and cell death an improved



7




understanding of the underlying signaling pathways that regulatethe impact of autophagy on cell death versus survival decisionswill be critical for the further exploitation of autophagy as astrategy for cancer therapy

ABBREVIATIONS

THC tetrahydrocannabinol TMZ temozolomide

CONFLICT OF INTEREST

The authors declare no con1047298ict of interest

ACKNOWLEDGEMENTS

The expert secretarial assistance of C Hugenberg is greatly appreciated This work ha s

been partly supported by a grant from the BMBF (to SF)

REFERENCES

1 Hengartner MO The biochemistry of apoptosis Nature 2000 407 770ndash776

2 Kroemer G Martin SJ Caspase-independent cell death Nat Med 2005 11

725ndash730

3 Galluzzi L Vitale I Abrams JM Alnemri ES Baehrecke EH Blagosklonny MV et al

Molecular de1047297nitions of cell death subroutines recommendations of theNomenclature Committee on Cell Death 2012 Cell Death Differ 2012 19 107ndash120

4 Nikoletopoulou V Markaki M Palikaras K Tavernarakis N Crosstalk between

apoptosis necrosis and autophagy Biochim Biophys Acta 2013 1833 3448ndash3459

5 Kerr JF Wyllie AH Currie AR Apoptosis a basic biological phenomenon with

wide-ranging implications in tissue kinetics Br J Cancer 1972 26 239ndash257

6 Marino G Niso-Santano M Baehrecke EH Kroemer G Self-consumption the

interplay of autophagy and apoptosis Nat Rev Mol Cell Biol 2014 15 81ndash94

7 Rami A Kogel D Apoptosis meets autophagy-like cell death in the ischemic

penumbra two sides of the same coin Autophagy 2008 4 422ndash426

8 Codogno P Meijer AJ Autophagy and signaling their role in cell survival and

cell death Cell Death Differ 2005 12 1509ndash1518

9 He C Klionsky DJ Regulation mechanisms and signaling pathways of autophagy

Annu Rev Genet 2009 43 67ndash93

10 Frankel LB Wen J Lees M Hoyer-Hansen M Farkas T Krogh A et al microRNA-101

is a potent inhibitor of autophagy EMBO J 2011 30 4628ndash4641

11 Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T et al LC3 a

mammalian homologue of yeast Apg8p is localized in autophagosome mem-

branes after processing EMBO J 2000 19 5720ndash5728

12 Sinha S Levine B The autophagy effector Beclin 1 a novel BH3-only protein

Oncogene 2008 27 S137ndashS148

13 Liang C Feng P Ku B Dotan I Canaani D Oh BH et al Autophagic and tumour

suppressor activity of a novel Beclin1-binding protein UVRAG Nat Cell Biol 2006

8 688ndash699

14 Takahashi Y Coppola D Matsushita N Cualing HD Sun M Sato Y et al Bif-1

interacts with Beclin 1 through UVRAG and regulates autophagy and tumor-

igenesis Nat Cell Biol 2007 9 1142ndash1151

15 Fimia GM Stoykova A Romagnoli A Giunta L Di Bartolomeo S Nardacci R et al

Ambra1 regulates autophagy and development of the nervous system Nature

2007 447 1121ndash1125

16 Gozuacik D Kimchi A Autophagy as a cell death and tumor suppressor

mechanism Oncogene 2004 23 2891ndash2906

17 Munoz-Pinedo C Martin SJ Autosis a new addition to the cell death tower

of babel Cell Death Dis 2014 5 e131918 Chen Y McMillan-Ward E Kong J Israels SJ Gibson SB Oxidative stress induces

autophagic cell death independent of apoptosis in transformed and cancer cells

Cell Death Differ 2008 15 171ndash182

19 Ginet V Puyal J Rummel C Aubry D Breton C Cloux AJ et al A critical role of

autophagy in antileukemialymphoma effects of APO866 an inhibitor of NAD

biosynthesis Autophagy 2014 10 603ndash617

20 Yu L Wan F Dutta S Welsh S Liu Z Freundt E et al Autophagic programmed cell

death by selective catalase degradation Proc Natl Acad Sci USA 2006 103

4952ndash4957

21 Nihira K Miki Y Ono K Suzuki T Sasano H An inhibition of p62SQSTM1 caused

autophagic cell death of several human carcinoma cells Cancer Sci 2014 105

568ndash575

22 Azad MB Chen Y Henson ES Cizeau J McMillan-Ward E Israels SJ et al Hypoxia

induces autophagic cell death in apoptosis-competent cells through a mechan-

ism involving BNIP3 Autophagy 2008 4 195ndash204

23 Galluzzi L Bravo-San Pedro JM Vitale I Aaronson SA Abrams JM Adam D et al

Essential versus accessory aspects of cell death recommendations of the

NCCD 2015 Cell Death Differ 2014 22 58ndash73

24 Shen HM Codogno P Autophagic cell death Loch Ness monster or endangered

species Autophagy 2011 7 457ndash465

25 Berry DL Baehrecke EH Growth arrest and autophagy are required for salivary

gland cell degradation in Drosophila Cell 2007 131 1137ndash1148

26 Calvo-Garrido J Carilla-Latorre S Kubohara Y Santos-Rodrigo N Mesquita A

Soldati T et al Autophagy in Dictyostelium genes and pathways cell death and

infection Autophagy 2010 6 686ndash701

27 Samara C Syntichaki P Tavernarakis N Autophagy is required for necrotic cell

death in Caenorhabditis elegans Cell Death Differ 2008 15 105ndash112

28 Elgendy M Sheridan C Brumatti G Martin SJ Oncogenic Ras-induced expression

of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic

survival Mol Cell 2011 42 23ndash35

29 Liu Y Shoji-Kawata S Sumpter RM Jr Wei Y Ginet V Zhang L et al Autosis is

a Na+K+-ATPase-regulated form of cell death triggered by autophagy-inducing

peptides starvation and hypoxia-ischemia Proc Natl Acad Sci USA 2013 110

20364ndash20371

30 Zhaorigetu S Wan G Kaini R Jiang Z Hu CA ApoL1 a BH3-only lipid-binding

protein induces autophagic cell death Autophagy 2008 4 1079ndash1082

31 Lamy L Ngo VN Emre NC Shaffer AL 3rd Yang Y Tian E et al Control of

autophagic cell death by caspase-10 in multiple myeloma Cancer Cell 2013 23

435ndash449

32 Wang WJ Wang Y Chen HZ Xing YZ Li FW Zhang Q et al Orphan nuclear

receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway

Nat Chem Biol 2014 10 133ndash14033 Shen S Kepp O Michaud M Martins I Minoux H Metivier D et al Association and

dissociation of autophagy apoptosis and necrosis by systematic chemical study

Oncogene 2011 30 4544ndash4556

34 Maiuri MC Le Toumelin G Criollo A Rain JC Gautier F Juin P et al Functional and

physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1 EMBO J

2007 26 2527ndash2539

35 Pattingre S Levine B Bcl-2 inhibition of autophagy a new route to cancer Cancer

Res 2006 66 2885ndash2888

36 Pattingre S Tassa A Qu X Garuti R Liang XH Mizushima N et al Bcl-2 anti-

apoptotic proteins inhibit Beclin 1-dependent autophagy Cell 2005 122

927ndash939

37 Hetschko H Voss V Senft C Seifert V Prehn JH Koumlgel D BH3 mimetics reactivate

autophagic cell death in anoxia-resistant malignant glioma cells Neoplasia 2008

10 873ndash885

38 Friesen C Glatting G Koop B Schwarz K Morgenstern A Apostolidis C et al

Breaking chemoresistance and radioresistance with [213Bi]anti-CD45 antibodies

in leukemia cells Cancer Res 2007 67 1950ndash1958

39 Meng Y Tang W Dai Y Wu X Liu M Ji Q et al Natural BH3 mimetic (-)-gossypol

chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by

increase of Puma and Noxa Mol Cancer Ther 2008 7 2192ndash2202

40 Paoluzzi L Gonen M Gardner JR Mastrella J Yang D Holmlund J et al Targeting

Bcl-2 family members with the BH3 mimetic AT-101 markedly enhances the

therapeutic effects of chemotherapeutic agents in in vitro and in vivo models of

B-cell lymphoma Blood 2008 111 5350ndash5358

41 Wolter KG Wang SJ Henson BS Wang S Grif 1047297th KA Kumar B et al (-)-gossypol

inhibits growth and promotes apoptosis of human head and neck squamous cell

carcinoma in vivo Neoplasia 2006 8 163ndash172

42 Balakrishnan K Wierda WG Keating MJ Gandhi V Gossypol a BH3 mimetic

induces apoptosis in chronic lymphocytic leukemia cells Blood 2008 112

1971ndash1980

43 Voss V Senft C Lang V Ronellen1047297tsch MW Steinbach JP Seifert V et al The pan-

bcl-2 inhibitor (-)-gossypol triggers autophagic cell death in malignant glioma

Mol Cancer Res 2010 8 1002ndash101644 Lian J Wu X He F Karnak D Tang W Meng Y et al A natural BH3 mimetic induces

autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1

interaction at endoplasmic reticulum Cell Death Differ 2011 18 60ndash71

45 Kaza N Kohli L Graham CD Klocke BJ Carroll SL Roth KA BNIP3 regulates AT101

[(-)-gossypol] induced death in malignant peripheral nerve sheath tumor cells

PLoS ONE 2014 9 e96733

46 Yuan Y Tang AJ Castoreno AB Kuo SY Wang Q Kuballa P et al Gossypol and an

HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells

Cell Death Dis 2013 4 e690

47 Kanzawa T Zhang L Xiao L Germano IM Kondo Y Kondo S Arsenic trioxide

induces autophagic cell death in malignant glioma cells by upregulation of

mitochondrial cell death protein BNIP3 Oncogene 2005 24 980ndash991

48 Cheng P Ni Z Dai X Wang B Ding W Rae Smith A et al The novel BH-3 mimetic

apogossypolone induces Beclin-1- and ROS-mediated autophagy in human

hepatocellular carcinoma [corrected] cells Cell Death Dis 2013 4 e489



8




49 Gao P Bauvy C Souquere S Tonelli G Liu L Zhu Y et al The Bcl-2 homology

domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-

independent cytoprotective autophagy in cancer cells J Biol Chem 2010 285

25570ndash25581

50 Bonapace L Bornhauser BC Schmitz M Cario G Ziegler U Niggli FK et al

Induction of autophagy-dependent necroptosis is required for childhood acute

lymphoblastic leukemia cells to overcome glucocorticoid resistance J Clin Invest

2010 120 1310ndash1323

51 Basit F Cristofanon S Fulda S Obatoclax (GX15-070) triggers necroptosis by

promoting the assembly of the necrosome on autophagosomal membranes Cell

Death Differ 2013 20 1161ndash1173

52 Heidari N Hicks MA Harada H GX15-070 (obatoclax) overcomes glucocorticoid

resistance in acute lymphoblastic leukemia through induction of apoptosis and

autophagy Cell Death Dis 2010 1 e76

53 Martin AP Mitchell C Rahmani M Nephew KP Grant S Dent P Inhibition of

MCL-1 enhances lapatinib toxicity and overcomes lapatinib resistance via BAK-

dependent autophagy Cancer Biol Ther 2009 8 2084ndash2096

54 Chen YJ Chi CW Su WC Huang HL Lapatinib induces autophagic cell death and

inhibits growth of human hepatocellular carcinoma Oncotarget 2014 5

4845ndash4854

55 Tang Y Hamed HA Poklepovic A Dai Y Grant S Dent P Poly(ADP-ribose) poly-

merase 1 modulates the lethality of CHK1 inhibitors in mammary tumors Mol

Pharmacol 2012 82 322ndash332

56 Martin AP Park MA Mitchell C Walker T Rahmani M Thorburn A et al BCL-2

family inhibitors enhance histone deacetylase inhibitor and sorafenib lethality via

autophagy and overcome blockade of the extrinsic pathway to facilitate killing

Mol Pharmacol 2009 76 327ndash

34157 Wei Y Kadia T Tong W Zhang M Jia Y Yang H et al The combination of a histone

deacetylase inhibitor with the Bcl-2 homology domain-3 mimetic GX15-070 has

synergistic antileukemia activity by activating both apoptosis and autophagy Clin

Cancer Res 2010 16 3923ndash3932

58 Brem EA Thudium K Khubchandani S Tsai PC Olejniczak SH Bhat S et al Distinct

cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant

lymphomas Br J Haematol 2011 153 599ndash611

59 Pan J Cheng C Verstovsek S Chen Q Jin Y Cao Q The BH3-mimetic GX15-070

induces autophagy potentiates the cytotoxicity of carboplatin and 5-1047298uorouracil

in esophageal carcinoma cells Cancer Lett 2010 293 167ndash174

60 Schwartz-Roberts JL Shajahan AN Cook KL Warri A Abu-Asab M Clarke R GX15-

070 (obatoclax) induces apoptosis and inhibits cathepsin D- and L-mediated

autophagosomal lysis in antiestrogen-resistant breast cancer cells Mol Cancer

Ther 2013 12 448ndash459

61 McCoy F Hurwitz J McTavish N Paul I Barnes C OHagan B et al Obatoclax

induces Atg7-dependent autophagy independent of beclin-1 and BAXBAK Cell

Death Dis 2010 1 e108

62 Fulda S Kroemer G Targeting mitochondrial apoptosis by betulinic acid in human

cancers Drug Discov Today 2009 14 885ndash890

63 Gonzalez P Mader I Tchoghandjian A Enzenmuller S Cristofanon S Basit F et al

Impairment of lysosomal integrity by B10 a glycosylated derivative of betulinic

acid leads to lysosomal cell death and converts autophagy into a detrimental

process Cell Death Differ 2012 19 1337ndash1346

64 Potze L Mullauer FB Colak S Kessler JH Medema JP Betulinic acid-induced

mitochondria-dependent cell death is counterbalanced by an autophagic salvage

response Cell Death Dis 2014 5 e1169

65 Scarlatti F Maffei R Beau I Codogno P Ghidoni R Role of non-canonical Beclin 1-

independent autophagy in cell death induced by resveratrol in human breast

cancer cells Cell Death Differ 2008 15 1318ndash1329

66 Puissant A Robert G Fenouille N Luciano F Cassuto JP Raynaud S et al

Resveratrol promotes autophagic cell death in chronic myelogenous leukemia

cells via JNK-mediated p62SQSTM1 expression and AMPK activation Cancer Res

2010 70 1042ndash1052

67 Cea M Cagnetta A Fulciniti M Tai YT Hideshima T Chauhan D et al Targeting

NAD+ salvage pathway induces autophagy in multiple myeloma cells via

mTORC1 and extracellular signal-regulated kinase (ERK12) inhibition Blood 2012

120 3519ndash3529

68 Yamamoto S Tanaka K Sakimura R Okada T Nakamura T Li Y et al Sub-

eroylanilide hydroxamic acid (SAHA) induces apoptosis or autophagy-associated

cell death in chondrosarcoma cell lines Anticancer Res 2008 28 1585ndash1591

69 Cao Q Yu C Xue R Hsueh W Pan P Chen Z et al Autophagy induced by

suberoylanilide hydroxamic acid in Hela S3 cells involves inhibition of protein

kinase B and up-regulation of Beclin 1 Int J Biochem Cell Biol 2008 40 272ndash283

70 Liu YL Yang PM Shun CT Wu MS Weng JR Chen CC Autophagy potentiates the

anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carci-

noma Autophagy 2010 6 1057ndash

106571 Kanzawa T Germano IM Komata T Ito H Kondo Y Kondo S Role of autophagy in

temozolomide-induced cytotoxicity for malignant glioma cells Cell Death Differ

2004 11 448ndash457

72 Katayama M Kawaguchi T Berger MS Pieper RO DNA damaging agent-induced

autophagy produces a cytoprotective adenosine triphosphate surge in malignant

glioma cells Cell Death Differ 2007 14 548ndash558

73 Knizhnik AV Roos WP Nikolova T Quiros S Tomaszowski KH Christmann M et al

Survival and death strategies in glioma cells autophagy senescence and apop-

tosis triggered by a single type of temozolomide-induced DNA damage PLoS One

2013 8 e55665

74 Salazar M Carracedo A Salanueva IJ Hernandez-Tiedra S Lorente M Egia A et al

Cannabinoid action induces autophagy-mediated cell death through stimulation

of ER stress in human glioma cells J Clin Invest 2009 119 1359ndash1372

75 Vara D Salazar M Olea-Herrero N Guzman M Velasco G Diaz-Laviada I Anti-

tumoral action of cannabinoids on hepatocellular carcinoma role of AMPK-

dependent activation of autophagy Cell Death Differ 2011 18 1099ndash1111

76 Lian J Karnak D Xu L The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced

autophagy versus apoptosis in prostate cancer cells Autophagy 2010 6

1201ndash1203



9




alkylating DNA damage during excitotoxicity and after ligation of death receptors3 Regulated necrotic cell death often involvesactivation of the kinases receptor-interacting protein 1 (RIP1) andRIP3 and can be blocked by the speci1047297c RIP1 inhibitor necrostatin-1 The term lsquonecroptosisrsquo is restricted to these necrostatin-1-inhibitable forms of necrotic cell death34 Mechanistically deathexecution via necroptosis is currently not well understood butmay involve energy failure oxidative stress and lysosomalmembrane permeabilization

The term lsquomitotic catastrophersquo is usually used for cell deathinduced by aberrant mitosis and executed either during mitosis orin the subsequent interphase (this type of cell death is sometimes

also called lsquomitoptosisrsquo)3 However mitotic catastrophe can exhibitfeatures of apoptosis or necrosis and the concept that mitoticcatastrophe truly represents a distinct mode of cell death iscurrently not generally accepted

Autophagy and ACD

Autophagy is a cellular stress response and a quality controlmechanism that in general acts in a pro-survival manner Differentforms of autophagy can be discriminated including macroauto-phagy (hereafter simply denoted as autophagy) microautophagyand chaperone-mediated autophagy During autophagy whichserves to regulate the turnover of long-lived proteins anddamaged organelles these cellular constituents are engulfed in

double-membrane-containing vesicles called autophagosomes(Figure 1)689 Their vesicular content is subsequently digestedby lysosomal proteases after fusion of autophagosomes withlysosomes89 Autophagy is a complex multistep process that isgenetically regulated by the ~ 30 autophagy-related genes(ATG) discovered hitherto in mammals In addition autophagyis subject to posttranscriptional regulation for example bymicroRNAs as miR-101 has been shown to suppress autophagy

autophagic

cell death

autophagosome

lysosomeendosome

fusion

autophagic

initiation

membrane

LC3-II

autophagyinduction

stress conditions

cancer drugs

(Resveratrol APO866 TMZ THC HDACs)

vesicleelongation

flux

chloroquine

BafA1

Betulinic acid B10

Vps34

inhibitors

Atg5Atg7

depletion

Beclin-1

depletion

BECN1

core complex

vesicle

nucleation

Vps34 Bcl-2xLMcl-1

BECN1

Bcl-2xL

Mcl-1BECN1Vps34

complexdisruption

complex

formation

BH3 mimetics

autophagosome

Atg5membrane

recruitment

Atg5

FADD

RIP3RIP1

necroptosis

mTOR

autolysosome

cargo

degradation

Figure 1 Different stages of autophagy and de1047297nition of ACD Autophagy can be induced by multiple stimuli including metabolic stressorganelle dysfunction protein aggregation and several cancer drugs many of which target the central autophagy regulator mTOR Thedifferent stages of this process are tightly regulated by the core autophagy proteins encoded by ATGs Autophagosome biogenesis startswith the formation of an initiation membrane that can be derived from the ER and several other cellular membrane sources Vesicle nucleationis promoted by a large macromolecular complex containing the lipid kinase Vps34 (BECN1 core complex) BECN1 (ATG6) serves to activate

Vps34 leading to formation of PtdIns3P which is required for this stage of the autophagic pathway Vesicle elongation is regulatedby two ubiquitin-like conjugation systems involving several ATG proteins (1) a large protein complex containing ATG5 (and ATG12ATG16)and (2) ATG7ATG3-driven attachment of phosphatidylethanolamine to LC3-I leading to the generation of LC3-II which is inserted intothe autophagosomal membrane Following vesicle closure mature autophagosomes fuse with lysosomes or endosomes to autolysosomes inwhich the autophagosomal content is digested by lysosomal proteases Excessive activation of the autophagy pathway can leadto an autophagy-dependent cell death in several paradigms The term ACD should be exclusively limited to cases of cell death that aremediated and not simply accompanied by autophagy Therefore only cases where inhibition of the autophagic pathway suppresses celldeath can be considered as true ACD The effects of autophagy inhibition on cell death can be experimentally addressed at differentstages of autophagy either by Vps34 inhibitors or by knockoutknockdown of core autophagic modulators such as ATG5 ATG7 or BECN1It is currently controversially discussed whether the term ACD should be reserved only for cases in which the 1047297nal cell death process ismediated by an enhanced autophagic 1047298ux rather than by alternative forms of cell death such as necroptosis In light of this controversyand the cytotoxicity of drugs inhibiting the autophagic 1047298ux from autophagosomes to lysosomes (chloroquine Ba1047297lomycin A1)interference at this stage of the autophagy pathway is currently not a generally accepted approach to analyze ACD BH3 mimeticshave been implicated in several paradigms of ACD They are capable to induce the release of BECN1 from its inhibitory interaction withBCL-2BCL-xL and have been shown to recruit necrosome components to the autophagosomal membrane thereby inducing ACD (for detailsplease refer to the main text)



2


















3







Gossypol





4344














Obatoclax AML CQ 57























4






(minus




Obatoclax







L








5








61


Natural products











6
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9


















3







Gossypol





4344














Obatoclax AML CQ 57























4






(minus




Obatoclax







L








5








61


Natural products











6
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9







Gossypol





4344














Obatoclax AML CQ 57























4






(minus




Obatoclax







L








5








61


Natural products











6
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9






(minus




Obatoclax







L








5








61


Natural products











6
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9








61


Natural products











6
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9
















TEMOZOLOMIDE














CANNABINOIDS







THC and JWH-01575

OUTLOOK




7





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9





ABBREVIATIONS




ACKNOWLEDGEMENTS



REFERENCES



725ndash730
























8 688ndash699






2007 447 1121ndash1125












4952ndash4957



568ndash575






















20364ndash20371





435ndash449








2007 26 2527ndash2539


Res 2006 66 2885ndash2888



927ndash939



10 873ndash885
















1971ndash1980








PLoS ONE 2014 9 e96733












8







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9







25570ndash25581




2010 120 1310ndash1323












4845ndash4854





















Ther 2013 12 448ndash459



















2010 70 1042ndash1052




120 3519ndash3529












2004 11 448ndash457







2013 8 e55665









1201ndash1203



9


onc2014458aartigo

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