Autophagy contributes to modulating the cytotoxicities of Bcl-2 homology domain-3 mimetics

Seminars in Cancer Biology 23P (2013) 553–560

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Review

Autophagy contributes to modulating the cytotoxicities of Bcl-2 homology domain-3 mimetics夽 Le Yu, Shuwen Liu ∗ School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China

a r t i c l e Keywords: Autophagy Apoptosis Bcl-2 family BH3 mimetics Cancer therapy

i n f o

a b s t r a c t The dysregulation of apoptosis is a key step in developing cancers, and mediates resistance to cancer therapy. Commitment to apoptosis is caused by permeabilization of the outer mitochondrial membrane, a process regulated by the interactions between different proteins of Bcl-2 family. Furthermore, Bcl-2 family proteins also bind to the endoplasmic reticulum, where they modulate autophagy, another important pathway regulating cell survival and death. Dysregulation of Bcl-2 family has been demonstrated in a wide spectrum of human cancers, including gastrointestinal cancers. Therefore, targeting the Bcl-2 family of proteins represents a promising therapeutic approach for these malignancies. Recent advances have yielded small molecules that have close structural or functional similarity to BH3-only proteins and are therefore named BH3 mimetics. Of these BH3 mimetics, obatoclax, (−)-gossypol, and ABT-263 are currently in clinical trials for multiple cancers. Growing evidence indicates that these BH3 mimetics not only induce apoptosis, but also regulate autophagy which may serve as a pro-survival or pro-death mechanism to counteract or mediate the cytotoxicity of BH3 mimetics. This review discusses the role of autophagy in cell-fate decision upon BH3 mimetics treatment. Further exploration of our understanding of the association between autophagy and cellular outcomes in response to BH3 mimetics treatment will likely offer improved therapies for patients with cancer. © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction The ability of cancer cells to evade apoptosis is a hallmark of human cancers and a major cause of treatment failure [1,2]. Apoptosis occurs via activation of two different pathways, the extrinsic pathway, triggered by the activation of the cell-surface death receptors, and the intrinsic pathway, followed by the perturbation of mitochondrial membrane integrity. Structural and functional studies have demonstrated that the intrinsic pathway is tightly controlled by the interactions between the pro- and anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins which control the integrity of the outer mitochondrial membrane. The anti-apoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL have been shown to be among the most commonly amplified oncogenes in the cancer genome [3]. Moreover, the anti-apoptotic group of Bcl-2 family proteins is frequently found to be over-expressed in a wide range of cancers including gastrointestinal cancers, causing both evasion of apoptosis and resistance to treatment [4–7]. As a result,

夽 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel.: +86 020 61648538; fax: +86 020 61648655. E-mail addresses: [email protected] (L. Yu), [email protected] (S. Liu).

targeting the Bcl-2 family of proteins is especially attractive clinically either in single agent therapy or combination treatment in many cancers [8]. Accordingly, recently identified small molecules that have close structural or functional similarity to BH3-only proteins and are therefore named BH3 mimetics, constitute a novel class of potentially important targeted therapeutics [9]. In addition to inducing apoptosis by directly or indirectly stimulating the mitochondrion-permeabilizing activity of pro-apoptotic multidomain proteins from the Bcl-2 family as expected, accumulating evidence indicates that BH3 mimetics also regulate another cellular process, macroautophagy (hereafter referred to as autophagy). Autophagy, which literally means ‘to eat oneself’, is an evolutionarily conserved, ubiquitous and multi-step process by which cytosolic material is sequestered in a double-layered membrane, delivered to the lysosome for degradation and recycled to sustain cell viability. Autophagy starts with the formation of an isolation membrane also called phagophore that elongates, encapsulates cytoplasmatic cargo and seals to form the autophagosome [10]. Unlike apoptosis, which is characterized by nuclear condensation and fragmentation without major ultrastructural changes in cytoplasmic organelles, autophagy is a caspase-independent process characterized by partial chromatin condensation, cell membrane blebbing, and the appearance of autophagosomes [11]. For obvious reasons the process is tightly regulated as it operates as a major homeostatic mechanism to eliminate damaged organelles,

1044-579X/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.semcancer.2013.08.008

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long-lived or aberrant proteins and superfluous portions of the cytoplasm. In response to stress signals caused by decreased intracellular metabolite concentrations, autophagy prevents cell death by replenishing metabolites [12]; however, autophagy can also cause cell death, depending on the stimuli and environment [13]. This review will focus on gastrointestinal cancers. We will initially describe the dysregulation of Bcl-2 family in gastrointestinal cancers. In the major part of this review, we will discuss how autophagy is regulated by Bcl-2 family proteins and BH3 mimetics. We will also concentrate upon the function of autophagy as a cellfate decision machinery and explore molecular mechanisms that link autophagy to cellular outcome in response to BH3 mimetics treatment. 2. Dysregulation of Bcl-2 family in gastrointestinal cancers Cancers of the gastrointestinal tract account for more than a third of total cancer incidence and nearly half of the cancer-related deaths in the world [14]. Bcl-2 family dysregulation has been demonstrated in gastrointestinal cancers (Table 1). The altered expression of Bcl-2 family members has been reported in transcriptional, translational and post-translational levels. Mutations of Bcl-2 family members have also been documented. Notably, some of these abnormalities have been shown to correlate with clinicopathological parameters and disease outcomes including overall survival in cancer patients [15–37]. These findings not only underscore a pivotal role of Bcl-2 family in tumorigenesis, but also highlight the possibility of targeting the Bcl-2 family members as a potential therapeutic avenue for the treatment of gastrointestinal cancers. 3. Bcl-2 family proteins are common players in apoptosis The mitochondrion serves not only as the cell’s powerhouse, but also as a center for integration of signals for apoptosis vs. survival. Capable of releasing a plethora of pro-apoptotic proteins into the cytoplasm, mitochondrion is a necessary site of extensive regulation in apoptosis. The Bcl-2 family is an important group of proteins that was first noted to regulate the release of apoptotic proteins from the mitochondria, specifically by causing mitochondrial outer membrane permeabilization (MOMP). A consequence of MOMP is the release of intermembrane space proteins such as cytochrome c into the cytoplasm, where they allosterically activate the adaptor protein Apaf-1 to initiate the cascade of caspases that cleave substrates leading to cellular apoptosis. Even without caspase activation, the reduced respiration following cytochrome c release soon triggers a backup cell death [38]. In this way, the Bcl-2 proteins family consists of upstream activators of apoptotic signaling in relation to MOMP [39]. On the basis of various structural and functional characteristics, the Bcl-2 family proteins can be divided into three functional subgroups (Fig. 1). Bax, Bak, and the much less studied Bok (Bcl2-related ovarian killer) represent the multidomain pro-apoptotic subgroup of the family and possess three (BH1, BH2, and BH3) out of the four evolutionarily conserved ␣-helical Bcl-2 homology (BH) domains. When exerting their cell-killing activity, Bax and Bak damage mitochondria, and either protein suffices for MOMP, indicative of functional redundancy [40]. A second subset of the family, possessing four BH domains (BH1, BH2, BH3, and BH4), includes five apoptosis-inhibitory proteins, i.e., the multidomain anti-apoptotic proteins Bcl-2, Bcl-xL (B-cell lymphoma-extra large), Mcl-1 (myeloid cell leukemia sequence 1), Bcl-w/Bcl2L2 (Bcl-2-like protein 2), Bcl2A1 (Bcl-2-related protein A1), and, in human only, Bcl-B. Although the five anti-apoptotic proteins share extensive similarity with their multidomain pro-apoptotic relatives, including three-dimensional structure, they protect rather

Fig. 1. According to conserved domain (BH) and its anti or pro-apoptotic action, the Bcl-2 family can be divided into three subgroups: anti-apoptotic multidomain, proapoptotic multidomain and pro-apoptotic BH3-only. Most family members contain a C-terminal hydrophobic transmembrane domain (TM) for membrane anchoring.

than damage mitochondria [41]. Both the pro-apoptotic effectors and anti-apoptotic Bcl-2 proteins are regulated by a third subgroup of Bcl-2 proteins, the BH3-only proteins (so named because of the four BH domains, they contain only BH3). At least eleven BH3-only proteins have been described in mammals, including Bcl2-interating mediator of cell death (Bim), BH3-interatingdomain death agonist (Bid), Bcl-2-associated death promoter (Bad), Bcl2-modifying factor (Bmf), Noxa (the Latin word for damage; also known as PMAIP1), p53-upregulated modulator of apoptosis (Puma), Bcl2-interacting killer (Bik), and Harakiri (Hrk) [42]. The BH3-only proteins function as apoptosis initiators, which bind and inactivate their pro-survival relatives [43] and perhaps also transiently bind and activate Bax and Bak [44,45]. The BH3-only proteins are activated by distinct cytotoxic stimuli in various ways, including enhanced transcription and post-translational modifications [46]. The Bcl-2 family can be regarded as a tripartite switch that sets the threshold for commitment to apoptosis, primarily by interactions within the family [47]. With regard to how the Bcl-2 apoptotic switch is flipped, different models, including ‘direct activation model’ [44,48], ‘derepression model’ [49,50] and ‘embedded model’ [51], have been proposed to describe how the interplay between the three Bcl-2 subgroups activates Bax and Bak and hence induces MOMP. The common feature of these models is that the heterodimetic interactions among different subgroups of the Bcl2 family occur through the BH3 ‘ligand’ domain of pro-apoptotic proteins which bind to a ‘receptor’ BH3-binding groove formed by BH1-3 regions on the anti-apoptotic proteins. This rational was successfully employed for the development of new anticancer therapies, in which small molecules acting as BH3-peptide mimetics fit into the ‘receptor’ binding groove of anti-apoptotic Bcl-2 family members. Such compounds hold promise for the development of new anticancer therapies (See below). 4. Autophagy is another cellular process modulated by the Bcl-2 family In addition to their anti-apoptotic property, anti-apoptotic members of Bcl-2 family also function as anti-autophagy proteins via its inhibitory interaction with Beclin 1 [52,53]. Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, is part of a Class III PI3K complex that participates in autophagosome formation, mediating the localization of other autophagy proteins to the preautophagosomal membrane [54]. It was first identified in a yeast

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Table 1 Dysregulation of Bcl-2 family in human gastrointestinal cancers. Cancer types

Nature of dysregulation

Clinical correlations

References

Colorectal cancer

Overexpression of Bcl-2 was observed in 30–94% of cases, whereas Bcl-2 was weakly expressed in the basal proliferative layer of normal colonic epithelium Expression of Bcl-xL mRNA in tumor tissues was higher than that in non-tumor colon tissues Bcl-w was observed in greater than 90% (69/75) of colorectal adenocarcinomas whereas adenomas demonstrated a much lower frequency of Bcl-w expression (1/17) Mcl-1 IHC staining was limited to apical areas in normal mucosa cells whereas in 50–70% of tumors samples diffuse expression was observed Inactivated phosphorylated Bad was detected in 60.1% of cases whereas it was not observed in normal colonic mucosal epithelial cells Inactivating genetic mutations of Bad gene were detected in colon cancer tissues Frameshift mutations of bax gene were detected in approximately half of MSI(+) cases High Bax expression was observed in all normal colon tissue samples whereas it occurred with a lower frequency (66.4%) in colon cancer tissues

Bcl-2 overexpression is a negative prognostic factor with respect to recurrence and survival

[15]

High Bcl-xL expression correlated with poor overall survival Increased Bcl-w expression was related to tumor progression

[16]

Diffuse IHC staining of Mcl-1 was associated with poorly differentiated tumors

[18]

ND

[19]

ND

[20]

The inactivating mutations of bax were correlated with poor prognosis Low Bax expression was correlated with poor overall survival

[21]

Bcl-2 expression correlated with histopathological grading and low apoptotic index Low expression of Bax was correlated with lack of clinical complete response and poor overall survival ND

[23]

No notable relationship was observed between apoptosis and overexpression of Bcl-2 ND

[26]

High Mcl-1 expression was correlated with poor 5-year overall survival Bcl-w expression was associated with the infiltrative morphotypes Low expression of Bax was associated with poor survival Combined p53/Bax mutation was associated with a very aggressive clinical course and an extremely poor prognosis Bcl-2 expression did not correlate with survival

[28]

Overexpression of Bcl-xL was associated with poor overall survival

[33]

ND

[34]

ND

[35]

Overexpression of Bax was associated with longer survival

[36]

Bax expression was correlated with longer survival

[37]

Esophageal cancer

Bcl-2 immunoreactivity was higher in poorly differentiated tumors than well-differentiated tumors A high expression of Bax was observed in 67% of cases Bcl-xL was highly positive in dysplasia and tumor cells but not in intestinal metaplasia

Gallbladder cancer

Overexpression of Bcl-2 was observed in 18.4% of cases. Bax expression in adenocarcinoma was decreased compared to benign diseased gallbladders

Gastric cancer

The expression of Mcl-1 was higher in gastric cancer tissues than corresponding noncancerous tissues Bcl-w was expressed in cancer cells but not in the neighboring normal mucosa in 46% of cases A high expression of Bax was detected in 31% of cases Frameshift Bax mutation was detected in 18.2% of cases

Tumor tissues expressed Bcl-2 more frequently compared with normal tissues Bcl-xL overexpression was observed in 63.6% of cases

Liver cancer

Pancreatic cancer

Mcl-1 expression was enhanced in HCC tissue compared to adjacent non-tumor tissue Tumor tissues expressed Bcl-2 more frequently than tissues of pancreatitis and normal pancreas A strong positive correlation was identified between Bax expression and p53 expression Bax mRNA was overexpressed in 61% of cases compared with normal pancreas

[17]

[22]

[24] [25]

[27]

[29] [30] [31]

[32]

Abbreviations: ND, not determined; MSI, microsatellite instability; ESCC, esophageal squamous cell carcinoma; HCC, hepatocellular carcinoma; IHC, immunohistochemistry.

two-hybrid screen as a Bcl-2-interacting protein [55]. Beclin 1 possesses a BH3 domain that dictates its interaction with the BH3 receptor domain of anti-apoptotic proteins of the Bcl-2 family. Association of Beclin 1 with Bcl-2 family proteins blocks the autophagy-promoting function of Beclin 1 [56]. It has been reported that Beclin 1 directly interacts with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 [57]. Overexpression of Bcl-2 or BclxL has been shown to inhibit autophagy by sequestering Beclin 1 [58]. Accordingly, increased expression or stabilization of BH3-only proteins (such as Bid, Bad or Bik), which bind to anti-apoptotic proteins through their BH3 domain, reduces the availability of anti-apoptotic proteins, thereby disinhibiting Beclin 1-dependent autophagy [56,57,59]. Of note, emerging evidences reveal that the competitive displacement of Beclin 1 by pro-apoptotic proteins

may not be of significance at all in turning on autophagy, for only the endoplasmic reticulum (ER)-targeted but not the mitochondriontargeted Bcl-2 has been shown to inhibit autophagy [39]. For instance, studies have shown that Bcl-2 targeted to the ER but not mitochondrial outer membrane inhibits starvation-induced autophagy [58]. This suggests that binding of Bcl-2 to Beclin 1 prevents it from assembling the pre-autophagosomal vesicles at an ER-associated location. 5. The cytotoxicity of BH3 mimetics is modulated by autophagy BH3 mimetics are small molecules that have close structural or functional similarity to BH3-only proteins. Most BH3 mimetics that

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are currently under preclinical and clinical development bind to and antagonize anti-apoptotic members of the Bcl-2 family of proteins [60]. Several excellent reviews on the apoptosis-promoting effects of these BH3 mimetics have recently been published [51,61–64]. Here, we review the three BH3 mimetics (obatoclax, (−)-gossypol, and ABT-263) that have advanced furthest clinically in relation to autophagy in a variety of cancers including gastrointestinal cancers. 5.1. Obatoclax Obatoclax (GX15-070) is a novel BH3 mimetic pan Bcl-2 inhibitor [65]. In vitro, obatoclax binds to all anti-apoptotic Bcl2 family members and by fluorescence polarization assays has IC50 s in the 1–7 ␮M range for Bcl-2, Bcl-xL, Bcl-w and Mcl-1 [66]. Given the inhibitory effect of anti-apoptotic Bcl-2 family proteins on Beclin 1, obatoclax is proposed to induce autophagy by disrupting anti-apoptotic Bcl-2 family proteins and Beclin 1 interactions. Consistent with this idea, obatoclax has been shown to induce interruption of a complex of Mcl-1 and Beclin 1, which contributes to autophagy induction [67,68]. However, the mechanism by which obatoclax deliberates Beclin 1 from Mcl-1 is unclear. Interestingly, a recent study demonstrates that obatoclax combined with laptatinib increases the protein level of Noxa, which competes away Mcl-1 from Beclin 1 leading to autophagy induction [69]. Till now, there is rare evidence indicating obatoclax liberates Beclin 1 from its restraint by direct interacting with anti-apoptotic Bcl-2 family proteins. Therefore, the mechanism by which obatoclax deliberates Beclin 1 from its Bcl-2 binding partners appears to be more complicated than expected. Notably, several studies have shown that Beclin 1 independent induction of autophagy by obatoclax, as knockdown of Beclin 1 failed to blunt obatoclax-induced autophagy [70,71]. In this regard, inhibition of mTOR signaling pathway by obatoclax may provide an alternative mechanism for autophagy induction [67,69]. Although obatoclax has been shown to engage autophagy in various cancer cells (Table 2), the critical question whether or not obatoclax-stimulated autophagic activity promotes or inhibits cell death has been controversially discussed. On one hand, obatoclaxinduced autophagy has been linked to cell death, as genetic or pharmacological blockage of autophagy was found to inhibit cell death [67,68,70,72–77]. On the other hand, inhibition of autophagy was demonstrated to enhance obatoclax-induced cell death, supporting a pro-survival function of autophagy [78]. Interestingly, obatoclax-induced autophagy also appears to act as a bystander, as blockade of autophagy by silencing Atg7 does not impair obatoclaxtriggered cytotoxicity [71]. Besides the above controversial points on the functional relevance of autophagy for obatoclax-induced cytotoxicity, the molecular mechanisms that link autophagy to cell death upon treatment with obatoclax is in exploration up till now. In this scenario, subcytotoxic dose of obatoclax (100 nM) combined with dexamethasone has been shown to activate autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. Execution of cell death was strictly dependent on expression of receptorinteracting protein (RIP-1) kinase, a key regulator of necroptosis [77]. However, it was unclear how RIP-1 was activated and how the autophagic machinery was connected to the necroptotic pathway. It is noteworthy that a very recent study seems to have addressed these questions. In this study, obatoclax (200 nM) has been shown to stimulate assembly of the necrosome on autophagosomal membranes, which functions as a key event connecting the induction of autophagy to cell death via necroptosis. Obtoclax promotes the interaction of Atg5, a component of autophagosomal membranes, with key constituents of the necrosome, that is, FADD, RIP1 and RIP3. This leads to the formation of a cytosolic cell death signaling

complex that initiates necroptotic cell death [72]. These findings provide new insights into the mechanisms of obatoclax-induced cell death by linking autophagy induction to necroptosis signaling. Nevertheless, on contrast to these observations, obatoclax at much higher concentrations (0.5 and 1 ␮M) has been demonstrated to dramatically reduce cell viability and induce autophagy without inducing loss of plasma membrane integrity, which would not be the case if necrosis was being induced [71]. Given RIP1 is critically required for obatoclax-induced necroptosis, it is tempting to speculate that the availability and/or function of signaling proteins (e.g. Atg5, FADD, RIP1) may contribute to determine whether necroptosis is engaged for the execution phase. In contrary to its autophagy-promoting function, obatoclax has been reported to inhibit the completion of autophagic flux [69,79]. For instance, obatoclax combined with lapatinib has been shown to impair autophagic degradation reflected by accumulation of undigested large autophagosomes and p62 proteins and unliquidated damaged mitochondria in breast cancer cells [69]. Given the importance of mitochondrial clearance in the regulation of cell homeostasis, it was proposed that impaired autophagic degradation disturbed the proper autophagy flux with pro-survival function, leading to the accumulation of sequestered but undigested defective mitochondria and precipitating cell death [80]. Consistent with this observation, a recent study shows that obatoclax’s anticancer efficacy is associated with an increase in autophagy initiation without the complete digestion of autophagosomes in breast cancer cells [79]. Mechanistically, impaired autophagy flux is due to decreased cathepsin protein expression with concomitant reduced proteolytic activity. Cathepsins are lysosomal hydrolases, which are known to degrade autolysosomal content [81]. Therefore, obatoclax-induced attenuation of cathepsin activities limits the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Of note, the concentrations of obatoclax used in the above studies are 50 and 100 nM respectively, which are pharmacologically achievable and are clinically relevant, as the approximate peak plasma concentration of obatoclax is 100 ng/ml (∼250 nM) [71]. Thus, these findings raise the critical question as to whether obatoclax can impair autophagic degradation not limited to the tested breast cancer cell lines, if so, whether it can improve the treatment of cancer when administered in combination with anticancer drugs. Since adaptive autophagy in cancer cells sustains tumor growth and survival in face of the toxicity of caner therapy, it is intriguing to know whether blocking autophagy while giving standard treatment will improve treatment outcome. It is noteworthy that some autophagy inhibitors have already been applied in clinical trails (http://clinicaltrials.gov/ct2/results?term=autophagy). Currently, autophagy inhibitors hydrooxychloroquine (HCQ) and chloroquine (CQ) have attracted extensive attention, since they are clinically used drugs [82]. The toxicological and pharmacokinetic profiles of these agents are well established and, therefore, these agents can be easily incorporated into existing regiments of cancer therapy. In comparison with HCQ and CQ, obatoclax per se possesses multiple killing mechanisms involving apoptosis and necroptosis in addition to its autophagy-suppressing activity, it is tempting to hypothesize that obatoclax may have more potential advantages than HCQ and CQ when administered in combination with anticancer drugs. Taken together, it appears that obatoclax plays a dual function in autophagy, in which this reagent exhibits both autophagypromoting activity and autophagy-suppressing activity. Data from recent studies are beginning to unveil the apparently paradoxical role of obatoclax in autophagy. In this respect, deliberating Beclin 1 from anti-apoptotic Bcl-2 family proteins and inhibition of mTOR signaling pathway by obatoclax in the initial phase of autophagy may contribute to its autophagy-promoting function,

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Table 2 Modulation of autophagy in cancer cells by BH3 mimetics. Cancer cell types

Roles in autophagy

Fates of autophagy

Autophagy inhibitor used

References

Obatoclax Rhabdomyosarcoma cells Colon cancer cells ALL Leukemia cells Leukemic B cells B-cell lymphoma cells Hepatoma cells Esophageal cancer cells Osteosarcoma cancer cells Breast cancer cells Lung cancer cells

Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Inhibits autophagy Induces autophagy

Pro-death Pro-death Pro-death Pro-death Pro-death Pro-death Pro-death Pro-survival Pro-survival Pro-survival Bystander

Baf, Atg5 shRNA, Atg7 siRNA Atg5, Beclin 1 siRNA Atg5, Beclin 1 siRNA CQ Beclin 1 siRNA Beclin 1 siRNA Beclin 1 siRNA 3-MA, CQ 3-MA, CQ 3-MA, HCQ, Baf, Beclin 1, Atg7 siRNA Beclin 1 shRNA, Beclin 1, Atg7 siRNA

[72] [73] [70] [75,76] [68] [77] [74] [78] [78] [79] [71]

gossypol Burkitt lymphoma cells Breast cancer cells Cervical cancer cells Prostate cancer cells Glioma cells

Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy

Pro-survival Pro-survival Pro-survival Pro-death Pro-death

3-MA Wort, Vps34, Beclin 1, Atg5 siRNA Wort, Vps34, Beclin 1, Atg5 siRNA 3-MA, Beclin 1, Atg5 siRNA Baf, Beclin 1, Atg5 siRNA

[88] [85] [85] [86,87] [90]

ABT-737 Lung cancer cells Lymphocytic leukemia cells Cervical cancer cells Colon cancer cells Prostate cancer cells Melanoma cells

Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy Induces autophagy

Uncertain Uncertain Bystander Bystander Pro-survival Pro-survival

3-MA, CQ, Beclin 1 shRNA 3-MA, CQ ND ND HCQ, Beclin 1 siRNA Atg7 siRNA

[102] [101] [98] [98] [99] [100]

Abbreviations: ALL, acute lymphoblastic leukemia; Baf, bafilomycin A1; BH3, Bcl-2 homology domain 3; CQ, chloroquine; HCQ, hydoxychloroquine; ND, not determined; Wort, wortmannin; siRNA, small interfering RNA, shRNA, short hairpin RNA; 3-MA, 3-methyladenine. In these studies, the role of autophagy has been characterized through the use of pharmacological inhibitors or RNA interference targeting genes that are essential to autophagy. As a pro-survival or pro-death mechanism, blockade of autophagy enhances or attenuates the inhibitory effect of the BH3 mimetic, respectively. ‘Bystander’ refers to the situation that blockade of autophagy does not affect the inhibitory effect.

whereas attenuation of cathepsin activities by obatoclax in the final step of autophagy may contribute to its autophagy-suppressing function. However, the molecular mechanisms those determine whether obatoclax promotes autophagy or inhibits autophagy have remained obscure. Further investigations are needed to achieve mechanistic insights on this issue. 5.2. Gossypol Gossypol is a natural polyphenolic compound derived from cottonseeds, which has been identified as a small molecule paninhibitor of anti-apoptotic Bcl-2 family members including Bcl-2, Bcl-xL, and Mcl-1, and Bcl-w [83,84]. Natural gossypol is a racemic mixture of (+)-gossypol and (−)-gossypol, the latter being more potent as an inhibitor of tumor growth. Accordingly, (−)-gossypol is the most clinically acceptable form of gossypol and has been brand named as AT-101 (Ascenta) [64]. Gossypol and (−)-gossypol have been reported to induce autophagy in various cancer cells (Table 2). Functioning as a panBcl-2 inhibitor, gossypol inhibits the interaction between Beclin 1 and Bcl-2, antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy [85–87]. Interestingly, (−)-gossypol is also able to induce Beclin 1-dependent autophagy by additional mechanisms. For instance, (−)-gossypol has been shown to increase ROS generation, subsequently leading to cytosolic translocation of high mobility group box 1 (HMGB1) [88]. HMGB1 directly interacts with Beclin 1, displacing Bcl-2, and thereby induces autophagy [89]. Furthermore, (−)-gossypol has been shown to downregulate expression of Mcl-1 and Bcl-2, which possibly leads to less anti-apoptotic Bcl-2 proteins available for sequestering Beclin 1 and therefore induces autophagy [86,87,90]. Of note, gossypol also induces Beclin 1-independent autophagy, which may result from its inhibitory action on mTOR signaling pathway [85]. Consistent with the dual functions of autophagy in cell-fate decision, both pro-survival and pro-death effects have been implicated

in gossypol-induced autophagy (Table 2). The accumulative evidence indicates that the pro-survival function of autophagy has been linked to its ability to suppress various forms of cell death, including apoptosis [91–94]. In support of this idea, gossypol and (−)-gossypol have been observed to induce cytoprotective autophagy, for suppression of autophagy using either pharmacological inhibitors or RNA interference with essential autophagy genes enhances apoptosis induced by these compounds [85,88]. In contrast, several studies also demonstrate that (−)-gossypol induces autophagic cell death in prostate cancer cells and glioma cells [86,87,90]. It is interesting to note that (−)-gossypol induced pro-survival autophagy in MCF-7 and Hela cells, whereas triggered autophagic cellular death in glioma cells with similar dosage and identical treatment time [85,90]. Moreover, mitochondrial dysfunction featured with mitochondrial fragmentation, swelling, or loss of cristae has been observed in both cells occurring self-defensive autophagy and cells occurring self-destructive autophagy in these studies [85,90]. These findings raise the question about the critical determinants for cells to be driven toward autophagy with different functions in response to (−)-gossypol treatment. Considering the important role of mitochondria in controlling the fate of cell, it appears reasonable to speculate that upon (−)-gossypol treatment, self-defensive autophagy occurs to ensure the turnover of damaged mitochondria. However, if increased mitochondrial damage reaches a “threshold” level above which excessive autophagy occurs and is followed by cell death. Therefore, the degree of mitochondrial damage and the capability of cells to deal with damaged mitochondria appear to contribute to determine which type of autophagy will occur upon gossypol treatment. 5.3. ABT-737 and ABT-263 Of the Bcl-2 inhibitors discovered to date, one of the most promising candidates that selectively kills cancer cells through

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direct interaction with the Bcl-2 family is the BH3 mimetic ABT737. It selectively binds to and inhibits Bcl-2, Bcl-xL and Bcl-w with nanomolar affinities, while it binds with poor affinity to Mcl-1 and Bfl-1 with a dissociation constant in the micro-molar range [95]. ABT-263 (navitoclax) is the orally applicable version of ABT-737, which has a similar binding profile and affinities to anti-apoptotic Bcl-2 family proteins as ABT-737 [64]. Till now, there is rare study investigating the association between ABT-263 and autophagy. Therefore, we focus on ABT-737 in relation to autophagy. ABT-737 has been shown to competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2 or Bcl-xL, thus allowing Beclin 1 to accomplish its autophagic stimulatory functions [96,97]. Notably, a recent study measures the possible effects of ABT-737 on multiple distinct autophagy regulators beyond that organized by Beclin 1. According to this study, ABT-737 causes the activation of AMPK, the inhibition of mTOR, dephosphorylates p53, and deactivates the autophagy-inhibitory Akt kinase. These results point to unexpected and pleiotroic pro-autophagic effects of ABT-737 involving the modulation of multiple signaling pathways [98]. With regard to the function of ABT-737-induced autophagy in relation to cell-fate decision (Table 2), it has been shown that induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cells. Therapeutic inhibition of autophagy with HCQ increased cytotocixity of ABT-737 both in vitro and in vivo [99]. Similarly, ABT-737 promoted autophagy and hence cell survival in melanoma cells, as abrogation autophagy by Atg7 knockdown resulted in a significant increase in cell death [100]. Interestingly, autophagy induced by ABT-737 also appears to act as a bystander, whose induction does not interfere with cell death [98]. However, in some scenarios, the role of autophagy in cell-fate decision is uncertain whose inhibition by different inhibitors yields controversial results. For instance, the cytotoxicity of ABT-737 in combination with vesicular stomatitis virus (VSV) was partially reversed by CQ, however, inhibition of autophagy with 3-MA led to increased apoptosis [101]. Similar to this study, ABT-737 has been shown to induce cytoprotective autophagy, since two inhibitors of autophagy (CQ and 3-MA) augmented cytototoxic action of ABT737. Surprisingly, and in sharp contrast to the results obtained with the pharmacological inhibitors, knockdown of Beclin 1 diminished ABT-737-induced cytotoxicity, indicating that cellular destructive rather than cytoprotective autophagy occurred [102]. Several possible explanations are proposed for these seemingly contradictory results. First, pharmacological inhibitors of autophagy used could have biological effects on the regulation of cell survival independent of the autophagy pathway. Second, Beclin 1 and Bcl-2 are known to directly interact, and knockdown of Beclin 1 may affect Bcl-2 function or localization independent of any effect on induction of autophagy. Discussing the advantages and pitfalls of these autophagy inhibitors is beyond the scope of this article. Nevertheless, it may be advisable to interrogate possible cases of autophagic cell death or cytoprotective autophagy by knocking down at least two distinct essential autophagic proteins in addition to pharmacological inhibitors.

6. Conclusions and future perspectives Much progress has been made in the last few years on the mechanisms by which the Bcl-2 family proteins function through selective interactions to control mitochondrial apoptosis. Recently, small molecules capable of inhibiting the interactions of the antiapoptotic Bcl-2 protein family have been developed and three BH3 mimetics, obatoclax, (−)-gossypol and ABT-263, have progressed into clinical studies. Apart from their pro-apoptotic property, accumulating evidence indicates that BH3 memetics, including obatoclax, (−)-gossypol and ABT-737, also regulate autophagy

both in Beclin 1-dependent and Beclin 1-independent pathways. There has been no publication regarding the association between autophagy and ABT-263 during preparation of this review. Further investigations are needed on this issue. In cancer therapy, autophagy is induced in cancer cells as an adaptive response to promote cell survival. However, in certain circumstances, autophagy is required for the cytotoxic action of some anticancer agents [103]. In line with this concept, BH3 mimetics have been shown to induce both self-defensive autophagy and self-destructive autophagy in various cancer cells. In the case that autophagy contributes to survival, pharmacological blockage of autophagy by clinically available autophagy inhibitors such as HCQ and CQ has been shown to enhance BH3 mimetics-induced cell death [78,99]. Of note, in addition to its autophagy-promoting function, obatoclax has been reported to inhibit the completion of autophagic flux, leading to the accumulation of sequestered but undigested defective mitochondria and precipitating cell death. Whether ABT compounds and (−)-gossypol also exert similar autophagy-inhibitory action is unclear. In the case that autophagy contributes to both death and survival, it is important to determine if the pro-death or pro-survival action of autophagy is induced upon a BH3 mimetic treatment. Equally important, is the molecular mechanism governing cell-fate decision during autophagy upon BH3 mimetics treatment. Such knowledge will likely foster the future development of strategies in relation to autophagy to better direct the use of these BH3 mimetics in the clinic. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This study was supported by the National Science Foundation of China (81273551), the Program for Pearl River New Stars of Science and Technology in Guanghzou, China (2012J2200035), and Guangdong Pearl River Scholar Funded Scheme. References [1] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2000;100:57–70. [2] Fulda S. Tumor resistance to apoptosis. International Journal of Cancer 2009;124:511–5. [3] Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature 2010;463:899–905. [4] Youle RJ, Strasser A. The Bcl-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology 2008;9:47–59. [5] Buggins AG, Pepper CJ. The role of Bcl-2 family proteins in chronic lymphocytic leukaemia. Leukemia Research 2010;34:837–42. [6] Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996;88:386–401. [7] Coultas L, Strasser A. The role of the Bcl-2 protein family in cancer. Seminars in Cancer Biology 2003;13:115–23. [8] Letai AG. Diagnosing and exploiting cancer’s addiction to blocks in apoptosis. Nature Reviews Cancer 2008;8:121–32. [9] Zhang L, Ming L, Yu J. BH3 mimetics to improve cancer therapy; mechanisms and examples. Drug Resistance Updates 2007;10:207–17. [10] Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nature Reviews Molecular Cell Biology 2009;10:458–67. [11] Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, et al. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Natural Cell Biology 2004;6: 1221–8. [12] Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005;120:237–48. [13] Gonzalez-Polo RA, Boya P, Pauleau AL, Jalil A, Larochette N, Souquere S, et al. The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. Journal of Cell Science 2005;118:3091–102.

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