Cell Death by Autophagy: Emerging Molecular Mechanisms and Implications for Cancer Therapy

REVIEW Cell death by autophagy: emerging molecular mechanisms and implications for cancer therapy

S Fulda1,2,3 and D Kögel4

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 ﬁndings in this exciting ﬁeld of research.

Oncogene (2015) 34, 5105–5113; doi:10.1038/onc.2014.458; published online 26 January 2015

INTRODUCTION nuclear fragmentation (karyorrhexis), membrane blebbing and the Different modes of programmed cell death formation of apoptotic bodies that are engulfed by neighboring or 5 Programmed cell death is an evolutionary conserved intrinsic specialized cells. At the biochemical level, apoptosis is character- mechanism enabling damaged and unwanted cells to commit ized by phosphatidylserine exposure and (in most cases) activation of effector caspases, the main executors of apoptotic suicide. This cellular suicide may occur either via apoptosis (type I 1 1 2,3 cell death. In extrinsic apoptosis, extracellular death signals act cell death) or via activation of alternative death programs. via specific transmembrane receptors culminating in a caspase- Induction of apoptotic cell death is a major mechanism by which dependent, apoptotic type of cell death.3,6 The mitochondria have most chemotherapeutic drugs and radiation kill tumor cells. In the a pivotal role in intrinsic apoptosis, which is initiated by a wide past couple of years, a tremendous effort has been invested to variety of intracellular stress signals/conditions leading to the develop strategies for triggering cancer cell apoptosis in a target- fi activation of the pro-apoptotic BCL-2 family members Bak and speci c manner. In addition to apoptosis, an ever increasing Bak, mitochondrial outer membrane permeabilization, mitochon- number of studies substantiate the existence of alternative, non- 2,4 drial dysfunction and release of pro-apoptotic factors, such as apoptotic forms of programmed cell death, which may be cytochrome c, apoptosis-inducing factor and Endo G from the exploited for cancer therapy. mitochondria into the cytosol.3,6 Depending on the extent of Until recently, the nomenclature for different cell death types caspase inhibition (for example, by high overexpression of was largely based on morphological criteria and has not Inhibitor of Apoptosis (IAP) family members in tumor cells), been uniformly used and recognized. The emerging knowledge execution of intrinsic apoptosis can occur either in a caspase- on the molecular mechanisms of the different forms of dependent or -independent fashion. programmed cell death now allows the discrimination into In contrast to apoptosis, necrotic cell death has been distinct ‘cell death subroutines’, and this classification is con- traditionally characterized as a passive, that is, non-programmed 3 tinuously refined and further improved. Based on the currently form, of cell death.4,7 Necrosis is the end result of a bioenergetic existing knowledge, the Nomenclature Committee on Cell Death catastrophe resulting from adenosine triphosphate (ATP) deple- (NCCD), consisting of the leading experts in the field of cell tion, which is incompatible with cell survival and is thought to be death research, has proposed the classification into five relatively initiated mainly by cellular injury after toxic insults or physical well-characterized different modes of (programmed) cell damage. Morphologically, necrosis is characterized by vacuoliza- death: (1) extrinsic apoptosis, (2) intrinsic apoptosis, (3) regulated tion of the cytoplasm, breakdown of the plasma membrane and necrosis, (4) mitotic catastrophe (mitosis), and (5) autophagic cell inflammation around dying cells attributable to the release of death (ACD).3 cellular contents and pro-inflammatory molecules.7 However, in Apoptosis (type I cell death) is morphologically defined by addition to passive necrosis, it is now evident that (programmed) cellular and nuclear shrinkage, chromatin condensation (pyknosis), 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 Kögel, Experimental Neurosurgery, Center for Neurology and Neurosurgery, Goethe-University Hospital Frankfurt, Heinrich-Hoffmann Str. 7, 60528 Frankfurt, Germany. E-mail: [email protected] or [email protected] Received 10 November 2014; revised 11 December 2014; accepted 12 December 2014; published online 26 January 2015 Autophagy and cell death S Fulda and D Kögel 5106 alkylating DNA damage, during excitotoxicity and after ligation of Autophagy and ACD 3 death receptors. Regulated necrotic cell death often involves Autophagy is a cellular stress response and a quality control activation of the kinases receptor-interacting protein 1 (RIP1) and mechanism that in general acts in a pro-survival manner. Different RIP3 and can be blocked by the speciﬁc RIP1 inhibitor necrostatin- forms of autophagy can be discriminated, including macroauto- 1. The term ‘necroptosis’ is restricted to these necrostatin-1- phagy (hereafter simply denoted as autophagy), microautophagy 3,4 inhibitable forms of necrotic cell death. Mechanistically, death and chaperone-mediated autophagy. During autophagy, which execution via necroptosis is currently not well understood but serves to regulate the turnover of long-lived proteins and may involve energy failure, oxidative stress and lysosomal damaged organelles, these cellular constituents are engulfed in membrane permeabilization. double-membrane-containing vesicles called autophagosomes The term ‘mitotic catastrophe’ is usually used for cell death (Figure 1).6,8,9 Their vesicular content is subsequently digested induced by aberrant mitosis and executed either during mitosis or by lysosomal proteases after fusion of autophagosomes with in the subsequent interphase (this type of cell death is sometimes lysosomes.8,9 Autophagy is a complex, multistep process that is also called ‘mitoptosis’).3 However, mitotic catastrophe can exhibit genetically regulated by the ~ 30 autophagy-related genes features of apoptosis or necrosis, and the concept that mitotic (ATG) discovered hitherto in mammals. In addition, autophagy catastrophe truly represents a distinct mode of cell death is is subject to posttranscriptional regulation, for example, by currently not generally accepted. microRNAs, as miR-101 has been shown to suppress autophagy

stress conditions cancer drugs (Resveratrol, APO866, TMZ, THC, HDACs)

autophagy mTOR lysosome/ Vps34 induction endosome inhibitors autolysosome Beclin-1 autophagic autophagic depletion fusion initiation flux cell death membrane cargo vesicle degradation elongation autophagosome vesicle nucleation chloroquine BECN1 LC3-II Atg5 BafA1 core complex Betulinic acid B10 Atg5/Atg7 depletion autophagosome

Bcl-2/x Vps34 BECN1 L Mcl-1 membrane Atg5 recruitment

complex FADD formation complex BH3 mimetics disruption RIP1 RIP3

Bcl-2/x Vps34 BECN1 L Mcl-1

necroptosis

Figure 1. Different stages of autophagy and definition of ACD. Autophagy can be induced by multiple stimuli, including metabolic stress, organelle dysfunction, protein aggregation and several cancer drugs, many of which target the central autophagy regulator mTOR. The different stages of this process are tightly regulated by the core autophagy proteins encoded by ATGs. Autophagosome biogenesis starts with the formation of an initiation membrane that can be derived from the ER and several other cellular membrane sources. Vesicle nucleation is 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 regulated by two ubiquitin-like conjugation systems involving several ATG proteins: (1) a large protein complex containing ATG5 (and ATG12/ATG16) and (2) ATG7/ATG3-driven attachment of phosphatidylethanolamine to LC3-I, leading to the generation of LC3-II, which is inserted into the autophagosomal membrane. Following vesicle closure, mature autophagosomes fuse with lysosomes or endosomes to autolysosomes in which the autophagosomal content is digested by lysosomal proteases. Excessive activation of the autophagy pathway can lead to an autophagy-dependent cell death in several paradigms. The term ACD should be exclusively limited to cases of cell death that are mediated and not simply accompanied by autophagy. Therefore, only cases where inhibition of the autophagic pathway suppresses cell death can be considered as true ACD. The effects of autophagy inhibition on cell death can be experimentally addressed at different stages of autophagy, either by Vps34 inhibitors or by knockout/knockdown of core autophagic modulators, such as ATG5, ATG7 or BECN1. It is currently controversially discussed whether the term ACD should be reserved only for cases in which the final cell death process is mediated by an enhanced autophagic flux rather than by alternative forms of cell death, such as necroptosis. In light of this controversy and the cytotoxicity of drugs inhibiting the autophagic flux from autophagosomes to lysosomes (chloroquine, Bafilomycin A1), interference at this stage of the autophagy pathway is currently not a generally accepted approach to analyze ACD. BH3 mimetics have been implicated in several paradigms of ACD. They are capable to induce the release of BECN1 from its inhibitory interaction with BCL-2/BCL-xL and have been shown to recruit necrosome components to the autophagosomal membrane, thereby inducing ACD (for details, please refer to the main text).

Oncogene (2015) 5105 – 5113 © 2015 Macmillan Publishers Limited Autophagy and cell death S Fulda and D Kögel 5107 by downregulation of STMN1, RAB5A and ATG4D.10 The process of mediated by autophagy and can be suppressed by the inhibition autophagy begins with the formation of a nascent initiation of the autophagic pathway (either by Vps34 inhibitors or by membrane, which serves as a prestage of autophagosomes. knockout/knockdown of core autophagic modulators, such as Several ATG proteins (that is, ATG5, ATG7, ATG10 and ATG12) have ATG5, ATG12 or BECN1) (Figure 1).23 Some authors have argued been implicated in autophagosome formation. The mammalian for even more stringent criteria and have proposed to use the ortholog of yeast ATG8 was originally identified as the light chain term ACD only in cases where the final cell death process is 3 (LC3) of microtubule-associated proteins. LC3 exists in two mediated by an enhanced autophagic flux rather than by forms, that is, LC3-I and its lipidated derivative LC3-II, which are apoptosis or necroptosis.6,24 In contrast to the authentic cases of localized in the cytosol (LC3-I) or in autophagosomal membranes ACD as defined by the NCCD, cases of cell death that simply (LC3-II), respectively.11 BECN1 (ATG6) is a core element of cellular exhibit markers of autophagy, such as an increase in autophago- autophagy and a component of the class III phosphatidylinositol somes, the lipidation of LC3 or an increased degradation of the 3′-kinase complex required both for formation and transport of autophagic substrate p62, but cannot be suppressed by autop- autophagosomes. BECN1 is a tumor-suppressor gene monoalleli- hagy inhibition, should not be classified as ACD. Probably, the cally deleted in several types of cancer, including ovarian, breast most convincing examples of bona fide ACD have so far been and prostate cancer.12 In addition, the BECN1-binding protein observed in developmental cell death in lower model organisms, ultraviolet irradiation resistance-associated gene (UVRAG) has including Caenorhabditis elegans, Drosophila melanogaster and been implicated as a tumor-suppressor gene that is monoalleli- Dyctiostelium discoideum.6,25–27 In addition, there are an ever cally mutated at high frequency in human colon cancers.13 Bif-1 increasing number of studies demonstrating true ACD in (also known as Endophilin B1) is another BECN1-interacting mammalian cells. Nevertheless, given the fact that autophagy protein that can act as a tumor suppressor, as knockout of Bif-1 represents a double-edge sword with both tumor-suppressive and has been reported to increase the development of spontaneous -promoting properties, it remains to be determined whether or tumors in mice.14 Furthermore, Ambra1 has been demonstrated to not engagement of autophagy even under conditions of ACD may interact with the BECN1 complex and deficiency of Ambra1 in elicit a more complex cellular response beyond modulation of cell mouse embryos has been shown to result in uncontrolled cell death. Some recent examples supporting the mechanistic concept proliferation,15 suggesting that Ambra1 has some tumor- of ACD are outlined below. suppressive properties. The quality-control function of autophagy, The oncogene H-RAS was shown to trigger upregulation of the especially in the context of genomic integrity, is correlated to its BH3-only proteins Noxa and BECN1 and a caspase-independent role in cancer development: autophagy can act as a tumor- ACD, thereby limiting clonogenic survival.28 In this context, ACD suppressor mechanism, as impaired autophagy, that is, the lack of may represent a safeguard mechanism to limit the oncogenic proper removal of toxic protein aggregates and organelles such as potential of deregulated RAS signals.28 dysfunctional mitochondria, has been shown to promote oxidative Recently, an autophagy-dependent type of cell death termed stress, DNA lesions and genomic instability.16 ‘autosis’ was described.29 In this study, a cell-permeable auto- The net effect of autophagy on cell death is highly contextual, phagy-inducing peptide, that is, Tat-BECN1, was shown to induce and both cytoprotective and cytotoxic functions of autophagy autophagy and, importantly, cell death in a dose-dependent have been reported. Autophagy comprises a primordial pro- manner.29 Autotic cell death was also observed in starved cells survival stress response, for example, under conditions of nutrient in vitro and in hippocampal rat neurons during cerebral ischemia deprivation where it serves to ensure energy balance. In addition, in vivo.29 This type of cell death was blocked by pharmacological there is now substantial evidence showing that enforced over- or genetic inhibition of autophagy, antagonists of the ion pump activation of autophagy can lead to ACD (type II cell death) Na+/K+-ATPase or genetic knockdown of the Na+/K+-ATPase α1 (Figure 1), that is, cellular self-digestion via the autophagosomal– subunit, whereas inhibition of apoptosis or necroptosis provided lysosomal pathway beyond the point of allowing cell no protection.29 The results of this study also suggest that high survival.3,4,8,16,17 It was proposed that the apparent dichotomy amounts of the BH3-only protein BECN1 are sufficient to trigger between pro-survival and pro-death autophagy may be causally ACD in the absence of other cellular stress conditions. In a similar related to the extent and duration of autophagy, indicating that manner, overexpression of the BH3-only protein apolipoprotein L1 this dual function may represent a threshold effect of autophagy, was previously shown to trigger ACD.30 as also observed for other stress responses such as the In another recent study, caspase-10 was identified in an RNAi endoplasmatic reticulum (ER) stress response or activation of screen to be required for suppression of an intrinsic form of ACD p53.17 in multiple myeloma.31 Caspase-10 was demonstrated to be A number of studies suggest that (at least in some instances) it essential for dampening intrinsic autophagy by cleaving the is not simply overactivation of unspecific bulk autophagy but BCL-2-interactor and potent inducer of autophagy BCLAF1, rather the selective removal of autophagy substrates that thereby preventing overactivation of autophagy to avoid ACD.31 promotes cell death. Oxidative stress is a key feature observed Furthermore, the orphan nuclear receptor TR3 was shown to in many paradigms of autophagy-dependent cell death,18,19 and promote ACD in melanoma cells.32 In this study, the TR3-targeting the autophagy-mediated selective degradation of the endogen- compound 1-(3,4,5-trihydroxyphenyl)nonan-1-one was used to ous reactive oxygen species (ROS) scavenger catalase has been trigger TR3 translocation to the mitochondrial inner membrane shown to induce ACD.20 In an analogous manner, inhibition of through Tom40 and Tom70 channel proteins, dissipation of the selective autophagy by targeting the autophagy receptors mitochondrial membrane potential and induction of autophagy p62/SQSTM1 and BNIP3 was proposed to cause ACD.21,22 Further associated with excessive mitochondria clearance and irreversible molecular mechanisms possibly underlying the dual function of cell death. These findings underscore the notion that selective autophagy in promoting either cell death or cell survival are mitophagy, which typically acts in a cytoprotective manner, may currently being addressed. reach a certain threshold level at which it will turn into a death- The exact definition of ACD has been controversially discussed promoting process. in the field. It was suggested that many of the older, descriptive Despite the notion that induction of autophagy in response to cases of the so-called ACD actually reflect death accompanied by anticancer treatments often represents a cytoprotective mechan- the block of the autophagic flux rather than death mediated by ism of cells trying to cope with stress,33 overall there is now an autophagy.6 According to the criteria proposed by the NCCD, the increasing and solid evidence for the existence of true ACD. Given term ‘autophagic cell death’ should be exclusively used from a the fact that disrupted autophagy as well as excess autophagy can functional perspective and limited to cases of cell death that are have detrimental consequences on cell viability, abrogation as

© 2015 Macmillan Publishers Limited Oncogene (2015) 5105 – 5113 Autophagy and cell death S Fulda and D Kögel 5108 well as overactivation of the autophagy pathway may both and in vitro models.38–41 Gossypol acts as a pan-BCL-2 inhibitor represent relevant strategies for cancer therapy. Accordingly, and can inactivate BCL-2, BCL-xL, MCL-1 and BCL-w. There are two induction of autophagy-dependent cell death by pro-autophagic enantiomers of gossypol, (+)-gossypol and ( − )-gossypol (also drugs has emerged as a novel concept to sensitize cancer cells to called AT-101), the latter being more potent as an inhibitor of therapy or to directly kill them with the aim of exploiting caspase- tumor growth. In cancer cells with an intact apoptotic machinery, independent programmed cell death pathways for the develop- ( − )-gossypol has been reported to induce apoptotic cell ment of novel cancer therapies. Recent relevant examples for death.39–42 In contrast, cell death triggered by gossypol largely ACD induced by cancer drugs are outlined in the following seems to depend on induction of ACD in apoptosis-deficient paragraphs (Table 1). prostate cancer and malignant glioma cells.43,44 In glioma cells, ( − )-gossypol was shown to trigger translocation of LC3 to autophagosomes, lysosomal activity and a late ACD INDUCED BY CANCER DRUGS cytochrome c release, but cell death occurred in the absence of Gossypol overt lysosomal damage and effector caspase activation.43 Antiapoptotic BCL-2 family members can form a complex with ( − )-Gossypol and the alkylating agent temozolomide (TMZ) BECN1/ATG634–36 and formation/dissociation of this complex may cooperated to activate ACD in O6-methylguanine-DNA-methyl- have an important role in modulating autophagy in tumor cells. transferase-negative glioma cells, and lentiviral knockdown of BCL-2 and BCL-xL sequester BECN1 via binding to its BH3 domain BECN1 or ATG5 in different glioma cell lines strongly diminished and prevent it from forming a multiprotein complex essential for cell death induced by ( − )-gossypol and combined treatment with vesicle nucleation during the early steps of the autophagic TMZ, indicating that autophagy contributes to this type of cell process, thereby inhibiting autophagy. Consequently, BH3 death.43 In contrast, stable knockdown of the endogenous mimetics are capable to activate both apoptosis and autophagy inhibitor mammalian target of rapamycin (mTOR) autophagy.37 Gossypol is a natural polyphenolic compound and increased ACD.43 BH3 mimetic derived from cotton seeds, which was initially In line with these findings, ( − )-gossypol has been reported to identified as an antifertility agent in China during the 1950s and cause caspase-independent, non-apoptotic cell death in malig- shown to possess cell death-promoting effects in various in vivo 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 mimetics Obatoclax 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 44,76 Gossypol GBM BECN1 and ATG5 siRNA, BafA1 43 Gossypol Peripheral nerve sheath tumor 3-MA 45

Natural products Betulinic acid derivative B10 GBM BECN1 and ATG5, ATG7 siRNA 63 Betulinic acid Cervical carcinoma ATG5 siRNA, ATG5 − / − MEFs 64 Resveratrol Breast carcinoma BECN1 and hVP34, ATG7 siRNA 65 Resveratrol CML ATG5, p62 and LC3 siRNA 66

HDACs SAHA 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, ATG5 − / − MEFs 74 THC, OWH-015 HCC ATG5 siRNA, 3-MA 75

Others Lapatinib HCC BECN1 and ATG5, ATG7 shRNA, 3-MA 54 APO866 Leukemia, lymphoma cells ATG5 and ATG7 siRNA, 3-MA 19,67 Abbreviations: AML = acute myeloid leukemia; BafA1 = Baﬁlomycin A1; CML = chronic myeloid leukemia; CQ = chloroquine; GBM = glioblastoma; HCC = hepatocellular carcinoma; HDAC = histone deacetylase; 3-MA = 3-methyladenine; MEF = mouse embryonic ﬁbroblast; RMS = rhabdomyosarcoma; SAHA = suberoylanilide hydroxamic acid; shRNA = short hairpin RNA; siRNA = small interfering RNA; THC = tetrahydrocannabinol; TMZ = temozolomide.

Oncogene (2015) 5105 – 5113 © 2015 Macmillan Publishers Limited Autophagy and cell death S Fulda and D Kögel 5109 with 3-methyladenine (3-MA) and involved intracellular iron ACD and necroptosis, although the underlying molecular mechan- chelation and hypoxia-inducible factor-1α-induced expression of isms have remained elusive at that point. the Obatoclax subsequently was shown to stimulate the interaction BH3-only protein BNIP3.45 Similarly, BNIP3 was found to be of components of autophagosomal membranes such as involved in autophagy-related cell death induced by combined ATG5 with proteins of the necrosome complex such as RIP1 in treatment of pancreatic adenocarcinoma cells with ( − )-gossypol an ATG5-dependent fashion, as ATG5 silencing inhibited this and BRD4770, a small-molecule inhibitor of the histone methyl- interaction.51 The requirement of autophagy for cell death transferase G9a.46 These observations suggest that the BH3-only induction was demonstrated by genetic silencing, as depletion protein and mitophagy-regulator BNIP3 may have an important of ATG5 or ATG7 inhibited obatoclax-mediated autophagosome function in ACD,47 although it should be noted that the latter formation and cell death.51 Data showing that genetic or study failed to address the question as to whether or not pharmacological ablation of RIP1 inhibited obatoclax-induced cell autophagy does indeed contribute to this particular case of cell death confirmed that RIP1 is a crucial mediator of cell death upon death. In addition to the release of BECN1 from BCL-2/BCL-xL and treatment with obatoclax, while RIP1 turned out to be dispensable activation of BNIP3, oxidative stress is presumably involved in the for obatoclax-stimulated autophagosome formation.51 prominent pro-autophagic and death-promoting effects of Furthermore, Heidari et al.52 reported that obatoclax is able to ( − )-gossypol. Indeed, ( − )-gossypol and its derivative apogossy- bypass glucocorticoid resistance in ALL via the induction of polone have previously been shown to be potent inductors of autophagy in addition to triggering apoptosis. Treatment of ALL oxidative stress in cancer cells.38,48 cells with obatoclax resulted in a rapid LC3 conversion and As indicated above, the effects of ( − )-gossypol-induced degradation of p62 protein, both used as markers of autophagy.52 autophagy on cell death appear to be highly dependent on the Knockdown of ATG5 prevented obatoclax-stimulated autophagy 52 cellular context as also demonstrated in prostate cancer and as well as cell death, underlining that autophagy represents a breast carcinoma cells. In androgen-independent prostate cancer cell death mechanism in this context. By comparison, silencing of cells expressing high levels of BCL-2 and resistant to apoptosis, BECN1 failed to block obatoclax-stimulated autophagy 52 ( − )-gossypol also preferentially induced ACD, which could induction, pointing to a BECN1-independent mode of autop- partially be blocked by knockdown of ATG5 and BECN1.44 In hagy. Similarly, pharmacological inhibition by 3-MA did not affect 52 contrast, apoptosis was preferentially induced in cells with low obatoclax-mediated autophagy. BCL-2 expression.44 In this study, ( − )-gossypol was shown to In colon and breast carcinoma cells, obatoclax has been 53 induce BECN1- and ATG5-dependent autophagy via releasing reported to enhance cell death induced by lapatinib, a small- molecule tyrosine kinase inhibitor targeting epidermal growth BECN1 from BCL-2 and BCL-xL at the ER, thus triggering the autophagic cascade.44 In addition, oral administration of ( − )-gos- factor receptors and Her2/Neu, which is capable to trigger ACD 54 sypol significantly inhibited the growth of androgen-independent either alone or in combination with other drugs. Obatoclax- prostate cancer xenografts, suggesting a potential relevance of mediated sensitization to lapatinib was associated with LC3 conversion and suppression of AKT/mTOR signaling as indicated ACD for the treatment of human hormone-refractory prostate 53 cancer with BCL-2 overexpression.44 In contrast to these observa- by reduced phosphorylation of AKT, mTOR and S6K1. Knock- − down of BECN1 or, alternatively, of ATG5 protected against tions, ( )-gossypol inhibited the interaction between BECN1 and 53 BCL-2 to induce BECN1-dependent autophagy, which was obatoclax-/lapatinib-induced cytotoxicity, underscoring that the followed by the execution of apoptotic cell death in apoptosis- induction of autophagy was necessary for combination treatment- competent MCF-7 human breast adenocarcinoma cells.49 Knock- mediated antitumor activity. Similar to obatoclax, knockdown of − MCL-1 or BCL-xL increased LC3 vesiculation and cell death by down of Vps34 and ATG5 reduced ( )-gossypol-induced autop- 53 55 hagy, and in this particular case, ( − )-gossypol-mediated apoptotic lapatinib. Also, Tang et al. reported that obatoclax acts in cell death was potentiated by treatment with or by small concert with lapatinib to trigger LC3 conversion and cytotoxic interfering RNAs against core autophagy genes (Vps34, BECN1 autophagy in breast carcinoma cells, as genetic inhibition of and ATG5),49 suggesting a cytoprotective function of ( − )-gossy- autophagy by knockdown of BECN1 or pharmacological inhibition fi pol-induced autophagy in this apoptosis-proficient cell model. by using 3-MA signi cantly reduced cell death induced by the combination therapy. Mechanistic studies showed that obatoclax-/ lapatinib-mediated autophagy was associated with Noxa- Obatoclax mediated displacement of BECN1 from MCL-1 as well as inhibition There are a number of studies implicating autophagy as a of mTOR signaling.55 cytotoxic mechanism in obatoclax-induced cell death. In child- Furthermore, obatoclax has been described to enhance the hood acute lymphoblastic leukemia (ALL), subcytotoxic concen- induction of cell death upon treatment with histone deacetylase trations of obatoclax have been reported to overcome (HDAC) inhibitors such as vorinostat or sodium valproate together glucocorticoid resistance by inducing ACD.50 Induction of with the multikinase inhibitor sorafenib via autophagy.56 This autophagy has been associated with disruption of the interaction obatoclax-mediated sensitization to HDAC inhibitor-/sorafenib- of BECN1 with MCL-1 as well as suppression of mTOR activity.50 In triggered cell death was accompanied by LC3 vesiculation and line with the known suppressive function of mTOR on autophagy inhibited by knockdown of BECN1, consistent with an autophagic induction, the mTOR inhibitor rapamycin acted in concert with form of cell death.56 Also, in acute myeloid leukemia obatoclax dexamethasone to induce ACD in this model.50 Concomitant was shown to exert antileukemic activity in concert with HDAC knockdown experiments showing that silencing of BECN1 or ATG7 inhibitors, that is, vorinostat and MGCD0103.57 This synergistic rescued obatoclax-mediated sensitization to glucocorticoids in antileukemic activity by obatoclax and HDAC inhibitors was both cell viability and clonogenic assays confirmed that stimula- associated with the induction of both autophagy and apoptosis.57 tion of autophagy was critical for the ability of obatoclax to The authors’ conclusion that autophagy contributes to the overcome glucocorticoid resistance.50 The authors went on to synergistic antileukemic effects by the combination treatment is, demonstrate that RIP1 as well as cylindromatosis (CYLD) were however, only based on experiments using chloroquine to inhibit required for the execution of cell death upon treatment with autophagy but lacks genetic evidence showing that autophagy obatoclax and glucocorticoids, whereas both RIP1 and CYLD were represents a cell death mechanism under these conditions. dispensable for the initiation of autophagy in this context.50 As Therefore, the question as to whether or not autophagy indeed RIP1 and CYLD are known as critical regulatory proteins in mediates cell death during obatoclax/HDAC inhibitor combination necroptotic signaling, this study provided a genetic link between treatment remains to be clarified.

© 2015 Macmillan Publishers Limited Oncogene (2015) 5105 – 5113 Autophagy and cell death S Fulda and D Kögel 5110 In B-cell lymphoma, obatoclax was reported to stimulate LC3 betulinic acid vary in their ability to modulate the autophagic conversion and ACD in a caspase-independent manner that was pathway. significantly inhibited by BECN1 knockdown.58 Resveratrol represents another naturally occurring compound Importantly, the conclusion that autophagy represents a that has been reported to trigger autophagy-mediated cell death cytotoxic process contributing to obatoclax-induced cell death is in cancer cells. Interestingly, a non-canonical, BECN1-independent based, in most studies, on genetic evidence showing that pathway of autophagy induction was documented upon treat- knockdown of essential autophagy genes such as BECN1, ATG5 ment with resveratrol in human breast carcinoma.65 This or ATG7 rescue cell death upon treatment with obatoclax. conclusion is based on experiments using BECN1 and Vps34 small However, there are also some studies concluding that obatoclax interfering RNAs that failed to inhibit resveratrol-stimulated triggers cell death via autophagy that are solely based on autophagy and cell death, whereas knockdown of ATG7 protected experiments using pharmacological inhibitors of autophagy such breast carcinoma cells against resveratrol-mediated autophagy as chloroquine or 3-MA, which lack absolute specificity and may and cell death.65 Engagement of the autophagic program by also affect additional processes besides autophagy. This calls resveratrol was shown to involve resveratrol-imposed inhibition of for some caution as far as the functional impact of AKT/mTOR signaling as well as conversion of LC3-I to LC3-II.65 In autophagy induction in the context of obatoclax’s cytotoxicity is chronic myeloid leukemia, resveratrol has been demonstrated to concerned. promote ACD via c-Jun N-terminal kinase-mediated p62/SQSTM1 In addition to the pro-death function of autophagy in the expression as well as activation of adenosine monophosphate- course of obatoclax-induced cell death, autophagy has also been activated kinase, which in turn inhibited mTOR signaling.66 implicated as a cytoprotective or bystander mechanism in Knockdown experiments showing that silencing of key autophagy – response to treatment with obatoclax in some studies.59 61 In components such as ATG5, LC3 or p62 protected against lung carcinoma cells, obatoclax-induced autophagy as documen- resveratrol-triggered loss of cell viability confirmed that auto- ted by LC3 processing has been reported to depend on ATG7 but phagy serves as a cytotoxic process in this model.66 not on BECN1.61 However, ATG7 was found to be dispensable for 61 obatoclax-induced cell death in this model, implying that the APO866 induction of autophagy by obatoclax was not required for There is evidence showing that cancer cells have a higher obatoclax-mediated cytotoxicity. In esophageal carcinoma and nicotinamide adenine dinucleotide (NAD+) turnover rate than osteosarcoma cells, treatment with obatoclax caused conversion non-transformed cells, suggesting that this biosynthetic pathway of LC3 and ultrastructural changes consistent with the formation represents an attractive target for cancer treatment.67 Recently, it of autophagosomes.59 Based on experiments showing that the was reported that APO866, an inhibitor of nicotinamide phos- cytotoxicity of obatoclax was significantly reduced by addition of phoribosyltransferase, the rate-limiting enzyme involved in NAD chloroquine or 3-MA, the authors concluded that obatoclax- (+) synthesis, induced an autophagy-dependent type of cell death induced autophagy exerts cytoprotective functions under these in leukemia and lymphoma cells,19,67 although both studies differ conditions.59 In breast carcinoma cells, BECN1 turned out to be with regard to the potential role of apoptosis in APO866-induced required for obatoclax-stimulated autophagosome formation but cell demise. In the study by Cea et al.,67 the authors observed not for obatoclax-induced cell death.60 In addition to engaging APO866-dependent induction of autophagy and cell death, but autophagosome formation in this model, obatoclax was found to the pan-caspase inhibitor zVAD-fmk as well as specific inhibitors of block autophagic degradation of vesicular cargo by attenuating caspase-3 and -9 failed to protect from APO866-induced cell cathepsin activity.60 death. In contrast, the pharmacological autophagy inhibitors wortmannin, LY294002, 3-MA and chloroquine all reduced Natural products APO866-induced cell death. However, genetic ablation of auto- In addition to gossypol, other natural products have been phagy genes was not performed in this study. In the study by reported to engage cell death pathways via autophagy. For Ginet et al.,19 the authors demonstrated that APO866 induces example, the plant-derivative betulinic acid, a pentacyclic autophagosome formation and SQSTM1/p62 degradation in triterpenoid derived from white birch trees,62 has been implicated parallel to caspase-3 activation. Interestingly, APO866 treatment in modulating autophagy-mediated cell death. The semisynthetic evoked depletion of the ROS scavenger catalase, thus leading to glycosylated derivative of betulinic acid B10 has been shown both enhanced ROS levels and cell death. Inhibition of autophagy by to trigger autophagy and to abrogate the autophagic flux in knockdown of ATG5 and ATG7 abrogated catalase degradation, glioblastoma cells, thereby switching autophagy into a cytotoxic ROS production, caspase activation and cell death after APO866 process.63 This B10-induced cell death was found to be associated treatment, which could be rescued by addition of exogenous with destabilization of lysosomes and release of lysosomal catalase. enzymes into the cytoplasm.63 Consistently, the cathepsin inhibitor Ca074Me significantly decreased B10-induced cell death, further supporting that the release of lysosomal enzymes HDAC INHIBITORS (HDACIS) contributes to B10-triggered cell death.63 In line with the notion As aberrant regulation of epigenetic control of gene expression, that destabilization of lysosomes can convert autophagy into a including histone acetylation, represents a hallmark of human detrimental pathway once autophagy has been initiated, inhibi- cancer, small-molecule inhibitors of HDACs are considered as tion of lysosomal enzyme activity was shown to protect against promising cancer therapeutics. Among their various cellular B10-mediated cytotoxicity.63 Genetic studies showing that silen- effects, HDACIs have been reported to engage autophagy that cing of core autophagy genes, including BECN1, ATG5 or ATG7, has also been linked to the induction of cell death. For example, rescues cell death upon exposure to B10 supported the conclusion the HDACI suberoylanilide hydroxamic acid (SAHA) was described that autophagy represents a cell death mechanism in the course to trigger autophagy-associated cell death in chondrosarcoma of B10 treatment.63 By comparison, induction of autophagy by the cells.68 Cell death upon treatment with SAHA was accompanied by parental compound betulinic acid has been implied as a increased lipidation of LC3 as well as ultrastructural changes cytoprotective response, as cervical carcinoma cells with knock- consistent with autophagosome formation.68 Furthermore, the down of ATG5 or mouse embryonic fibroblasts lacking ATG5 or addition of 3-MA significantly restored cell viability upon ATG7 exhibited increased sensitivity to betulinic acid.64 It will treatment with SAHA, leading the authors to the conclusion that therefore be interesting to explore whether different derivatives of autophagy-associated cell death is involved in the antitumor

Oncogene (2015) 5105 – 5113 © 2015 Macmillan Publishers Limited Autophagy and cell death S Fulda and D Kögel 5111 activity of SAHA against chondrosarcoma cells.68 However, the CANNABINOIDS question as to whether or not autophagy represents a cytotoxic Moreover, the main active component of cannabinoids, that is, mechanism in this contest remains to be answered, as genetic tetrahydrocannabinol (THC), has been demonstrated to trigger evidence supporting this conclusion is currently lacking. autophagy-mediated cell death in different cancer entities, Independent studies similarly reported that SAHA stimulates including glioblastoma and hepatocellular carcinoma.74,75 The autophagy-associated cell death in cancer cells. Induction of functional requirement of autophagy to mediate THC-induced autophagy by SAHA in HeLa cervical carcinoma cells was antitumor activity has been established both in vitro and in vivo. associated with the inhibition of mTOR and accompanied by Accordingly, genetic silencing of autophagy-related genes, typical ultrastructural features consistent with autophagy as well including ULK1, ATG5 and Ambra-1, in glioblastoma cells as well as biochemical evidence such as conversion of LC3-I to LC3-II.69 as knockout of ATG5 in fibroblasts rescued THC-imposed 74 Although induction of autophagy was shown to depend on key cytotoxicity. Also in vivo using a tumor xenograft model based genetic components of autophagy signaling, including BECN1 and on transformed mouse embryonic fibroblasts proficient or ATG7,69 the functional relevance of autophagy in SAHA-induced deficient in ATG5, the induction of autophagy has been shown 74 cell death remains to be determined. In hepatocellular carcinoma, to be critical for THC-imposed antitumor activity. In mechanistic HDACIs, including SAHA and OSU-HDAC-42, were described to terms, the authors demonstrated that THC stimulates accumula- trigger ACD based on both pharmacological as well as genetic tion of ceramide and phosphorylation of eukaryotic translation evidence demonstrating that the addition of 3-MA or knockdown initiation factor 2 alpha, which engages an ER stress response that 70 leads to upregulation of ER stress-related proteins, including CHOP of ATG5 reduced SAHA-mediated cytotoxicity. In parallel, 74 treatment with SAHA caused morphological changes typical for and tribbles homologue 3 (TRB3). In turn, TRB3 facilitates autophagy via inhibition of AKT/mTOR signaling.74 Interestingly, autophagy, that is, autophagosome formation as well as lipidation autophagy was shown to be upstream of apoptosis that involved of LC3-I to generate LC3-II, and was accompanied by down- 70 loss of mitochondrial membrane potential and production of regulation of p62, consistent with increased autophagic flux. 74 ROS. Accordingly, THC-induced features of apoptotic cell death, Engagement of autophagy by HDACIs might involve inhibition of including Annexin-V positivity, caspase-3 activation and loss of mTOR signaling, as SAHA and OSU-HDAC-42 resulted in down- 70 mitochondrial membrane potential, were attenuated in cells regulation of AKT/mTOR signaling activity. deficient in key autophagy genes.74 Vice versa, BAX/BAK double knockout mouse embryonic fibroblasts exhibited similar conver- TEMOZOLOMIDE sion of LC3-I to LC3-II compared with wild-type cells, confirming that stimulation of autophagy occurs upstream of apoptosis in Furthermore, the DNA-alkylating agent TMZ, which causes 74 6 THC-induced cancer cell death. It is interesting to note that THC- formation of O -methylguanine in DNA that mispairs with thymine stimulated autophagy-mediated cell death has been documented during the following cycle of DNA replication and is routinely used in different types of cancer cells but not in non-transformed in the treatment of malignant glioma, was shown to stimulate astrocytes,74 pointing to a potential therapeutic window that autophagy associated with the recruitment of LC3 to autophago- could be exploited for cancer therapy. 71 somal membranes. The role of autophagy in TMZ-induced In hepatocellular carcinoma, the cannabinoid receptor glioma cell death is currently controversial. On one side, early 2-selective agonist JWH-015 was reported to trigger autophagy- interference with autophagy by 3-MA inhibited both autophago- mediated cell death in addition to THC.75 In this study, the some formation as well as TMZ-mediated cytotoxicity,71 arguing engagement of the autophagic program by cannabinoids was for a pro-death role of autophagy in TMZ-triggered cell death. In shown to involve activation of adenosine monophosphate- line with these observations, cell death induced by single agent activated kinase via calmodulin-activated kinase beta in addition treatment with TMZ or by TMZ in combination with ( − )-gossypol to the induction of TRB3 and subsequent inhibition of AKT/mTOR was attenuated by early interference with autophagy via lentiviral signaling.75 Genetic or pharmacological inhibition of autophagy depletion of ATG5 and BECN1.43 On the other side, the study by by ATG5 silencing or addition of 3-MA rescued the THC- or Kanzawa et al.71 also demonstrated that interference with JWH-015-imposed suppression of tumor growth of subcutaneous autophagy at a later stage using the vacuolar type H+ (ATPase) hepatocellular carcinoma xenografts in vivo, emphasizing that inhibitor Bafilomycin A1 increased rather than reduced TMZ- activation of autophagy was required for the antitumor activity of 75 induced cell death. These data suggest that concomitant THC and JWH-015. induction of autophagy and disruption of the autophagic flux enhances TMZ-triggered cytotoxicity, possibly through lysosomal OUTLOOK and mitochondrial membrane permeabilization and a shift towards apoptotic cell death.71 To complicate matters even Autophagy has been shown to exert dual functions in human cancers, that is, both as tumor suppressor and as tumor promoter. further, it was also proposed that TMZ stimulates autophagy as As many anticancer drugs can engage autophagy, a better a cytoprotective response in glioblastoma that contributes to 72 understanding of the molecular mechanisms that regulate ACD chemoresistance. This notion is supported by experimental data can pave the avenue for rational exploitation of this cellular demonstrating that (1) TMZ can stimulate an autophagy- program for therapeutic purposes. There are now a number of dependent surge of ATP that limits non-apoptotic glioma cell examples showing that the induction of ACD indeed represents a death and that (2) genetic ablation of BECN1 reduced colony ’ 72 crucial event for the drug s antitumor activity. This opens new formation after TMZ treatment in glioma cells. The opposing perspectives for the development of novel therapeutic strategies fi effects of autophagy described in these studies are dif cult to and drug discovery. In addition, engagement of ACD may offer explain but may be related to the different genetic backgrounds new options to overcome treatment resistance, as autophagy has 72 of the cell models used and/or to dose-dependent changes in been reported to serve as a backup mechanism with important 6 the predominant DNA lesions induced by TMZ (O -methylguanine implications to bypass resistance especially in apoptosis-refractory lesions versus N-alkylations).73 The precise role of autophagy and tumors. However, the molecular determinants that are responsible the relevance of autophagy-dependent mechanisms for the for turning autophagy into a death process are currently still modulation of TMZ-induced glioma cell death await further poorly understood. As autophagy is a double-edged sword at the clarification. interface of cell survival and cell death, an improved

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