The autophagic paradox in cancer therapy

Abstract

Autophagy, hallmarked by the formation of double-membrane bound organelles known as autophagosomes, is a lysosome-dependent pathway for protein degradation. The role of autophagy in carcinogenesis is context dependent. As a tumor-suppressing mechanism in early-stage carcinogenesis, autophagy inhibits inflammation and promotes genomic stability. Moreover, disruption of autophagy-related genes accelerates tumorigenesis in animals. However, autophagy may also act as a pro-survival mechanism to protect cancer cells from various forms of cellular stress. In cancer therapy, adaptive autophagy in cancer cells sustains tumor growth and survival in face of the toxicity of cancer therapy. To this end, inhibition of autophagy may sensitize cancer cells to chemotherapeutic agents and ionizing radiation. Nevertheless, in certain circumstances, autophagy mediates the therapeutic effects of some anticancer agents. Data from recent studies are beginning to unveil the apparently paradoxical nature of autophagy as a cell-fate decision machinery. Taken together, modulation of autophagy is a novel approach for enhancing the efficacy of existing cancer therapy, but its Janus-faced nature may complicate the clinical development of autophagy modulators as anticancer therapeutics.

Introduction

Protein degradation and normal turnover of organelles are essential for cellular homeostasis. There are two major evolutionarily conserved intracellular pathways for protein degradation, namely, the ubiquitin–proteasome system and macroautophagy (hereafter referred to as ‘autophagy’). The former degrades short-lived proteins through an elaborate set of ubiquitinating enzymes and a multimeric protein complex known as the proteasome, whereas the latter degrades long-lived proteins in a lysosome-dependent manner (Figure 1) (Mizushima et al., 2008; Wu et al., 2010a). Autophagy is also involved in the removal of damaged organelles and protein aggregates. Autophagy, which literally means ‘to eat oneself’, is initiated by the nonselective seizing of cytosolic proteins and organelles such as mitochondria, endoplasmic reticulum, peroxisomes and ribosomes by an expanding membranous structure known as the phagophore or isolation membrane. The closure of phagophores sequestering these cytosolic cargos results in the formation of double-membrane bound autophagosomes (Mizushima et al., 2008). Autophagy may also occur in a selective fashion in which aggregated proteins and damaged mitochondria are recognized by specific receptors for incorporation into autophagosomes (Gu et al., 2004; Bjorkoy et al., 2005; Wild and Dikic, 2010). In the late stage of autophagy, autophagosomes fuse with lysosomes to form autolysosomes, in which the engulfed content is digested by acidic hydrolases and the engendered amino acids are shuttled back to the cytoplasm via lysosomal membrane permeases for reuse (Mizushima et al., 2008). In this way, autophagy acts as an important internal source of cellular energy through self-cannibalism, especially in time of nutrient deprivation.

Figure 1
figure1

Cellular mechanism and regulators of autophagy. Cytoplasmic proteins and damaged organelles are sequestered by the phagophore to form the double-membrane bound autophagosome. The autophagosome is then fused with lysosome to produce autolysosome where the cargos are digested by lysosomal hydrolases. Free amino acids are released during the process and then returned to the cytoplasm for reuse. Autophagy is regulated by multiple upstream signaling components, some of which are well-established oncoproteins and tumor-suppressor proteins.

Dysregulation of autophagy, which alters the rate of protein degradation and the metabolic state of the cells, has severe consequences and is associated with several pathophysiological conditions, such as cancer, infection, autoimmunity, inflammatory diseases, neurodegeneration and aging (Mizushima et al., 2008; Levine et al., 2011). In relation to carcinogenesis, malignant tissues very often exhibit altered level of autophagic activity. Moreover, autophagy is regulated by various well-established cancer-related signaling pathways (Figure 1). Nevertheless, a coherent understanding of the role of autophagy in the pathogenesis of cancer is still lacking. Depending on context, autophagy can act as a bona fide oncogenic or tumor-suppressing mechanism. In cancer therapy, the role of autophagy is also paradoxical, in which this cellular process may serve as a pro-survival or pro-death mechanism to counteract or mediate the cytotoxic effect of anticancer agents. In the major part of this review, we will discuss how autophagy is deranged in human cancers and its regulation by different oncogenic and tumor-suppressing pathways. We will also focus on the function of autophagy as a cell-fate decision machinery in the context of cancer therapy and explore possible factors that may affect the cellular outcome of autophagy.

Autophagy and cancer

Dysregulation of autophagy in human cancers

Autophagy is dysregulated in a wide spectrum of human cancers (Table 1). For instance, the altered expression of several autophagy markers such as microtubule-associated protein light chain 3 (LC3) and Beclin 1 has been reported in brain, esophageal, colon, gastric, liver and pancreatic cancers as well as osteosarcoma and melanoma. Mutations of various autophagy-related genes have also been documented in a subset of gastrointestinal cancers. Importantly, some of these abnormalities have been shown to correlate with clinicopathological parameters and disease outcomes including overall survival in cancer patients. These findings not only underscore a pivotal role of autophagy in tumorigenesis, but also highlight the possibility of using autophagy-associated molecules as novel prognostic markers in clinical settings (Russell et al., 1990; Eccles et al., 1992; Futreal et al., 1992; Cliby et al., 1993; Saito et al., 1993; Gao et al., 1995; Ahn et al., 2007; Miracco et al., 2007, 2010; Ding et al., 2008; Fujii et al., 2008; Yoshioka et al., 2008; Kim et al., 2008a; Kang et al., 2009; Lazova and Pawelek, 2009; Nomura et al., 2009; Othman et al., 2009; Pirtoli et al., 2009; Li et al., 2009, 2010; Zhang et al., 2009a; Huang et al., 2010; Koukourakis et al., 2010; Lazova et al., 2010; Miao et al., 2010; Negri et al., 2010; Sivridis et al., 2010; Wan et al., 2010; Karpathiou et al., 2010; Chang et al., 2011; Ma et al., 2011).

Table 1 Dysregulation of autophagy in human cancers

Regulation of autophagy by oncogenic and tumor-suppressing pathways

The importance of autophagy in carcinogenesis can also be underlined by the fact that this cellular process is meticulously regulated by many cancer-related signaling pathways.

PI3K/Akt/mTOR

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a major oncogenic cascade. Abnormal activation of this pathway as a result of gain-of-function mutations or amplifications of the oncogenes PI3K and Akt as well as the loss of the tumor-suppressor phosphatase and tensin homolog (PTEN; an antagonist of PI3K) are frequently detected in tumor tissues. Somatic mutations of TSC1 (tuberous sclerosis protein 1), which is a negative regulator of mTOR, may also contribute to bladder and liver cancers (Platt et al., 2009; Totoki et al., 2011). In addition, heightened mTOR activity has been reported in many cancer types (Zhang et al., 2009b; Xu et al., 2010; Liu et al., 2011). The PI3K/Akt/mTOR cascade can also be transactivated by receptor tyrosine kinases that mediate the mitogenic signal of growth factor stimulation. The PI3K/Akt/mTOR pathway is a central repressor of autophagy. PTEN overexpression has been shown to promote autophagy (Arico et al., 2001), whereas the targeted deletion of PTEN in mouse liver causes a strong inhibition of autophagy (Ueno et al., 2008). Akt inhibition also strongly promotes autophagy whereas constitutively active Akt has the opposite action (Laane et al., 2009). Inhibitors of mTOR have also been shown to induce autophagy in various cell types (Paglin et al., 2005; Cao et al., 2006; Iwamaru et al., 2007). In addition, stabilization of TSC2, which inhibits the mTOR signaling, promotes autophagy and suppresses tumorigenesis (Kuo et al., 2010). The inhibitory effect of PI3K/Akt/mTOR axis on autophagy is mainly mediated through the ULK1/2 (unc-51-like kinase 1/2)/mAtg13/FIP200 (focal adhesion kinase family interacting protein of 200 kDa) complex (Ganley et al., 2009; Jung et al., 2009). Under growth-permissive conditions, mTOR inhibits the ULK1/2/mAtg13/FIP200 complex to repress autophagy. However, when the cells are deprived of growth factors or nutrients, mTOR dissociates from the ULK1/2/mAtg13/FIP200 complex, thereby unmasking the kinase activities of ULK1/2, leading to FIP200 phosphorylation and recruitment of Atg proteins to the autophagosome formation site. Although the link between mTOR inhibition and autophagy is well established, it is worthwhile to notice that in some situations, mTOR may stimulate autophagy. In this regard, Zeng and Kinsella (2008) demonstrated that mTOR and its downstream mediator S6 kinase 1 may positively regulate autophagy in 6-thioguanine-treated cells, possibly through the negative feedback inhibition of Akt.

Bcl-2 family

Evasion of apoptosis is linked to the development of cancer. The antiapoptotic members (for example, Bcl-2, Mcl-1, Bcl-xL, Bcl-w, A1) of B-cell lymphoma 2 (Bcl-2) family are essential BH domain-containing proteins that exert their action through antagonism of pro-apoptotic members (for example, Bak and Bax). Growing evidence supports that, in addition to their antiapoptotic property, Bcl-2, Mcl-1 and Bcl-xL (B-cell lymphoma-extra large) are potent inhibitors of autophagy (Zhou et al., 2011). Beclin 1, the mammalian ortholog of Atg6, is a Bcl-2-interacting protein that exists in complexes of at least three different configurations (Beclin 1/hVps34/p150/Atg14, Beclin 1/hVps34/p150/UVRAG/Bif1 and Beclin 1/hVps34/p150/Rubicon/UVRAG). It is now known that the Atg14-containing complex functions at the early stage of autophagosome formation, whereas the UVRAG/Bif1-containing complex facilitates membrane curvature of autophagosomes. In contrast, the Rubicon/UVRAG-containing complex promotes the maturation phase (Yang and Klionsky, 2010). Regardless of the specific functions of these complexes, the interaction between hVps34 and Beclin 1 is required for autophagosome formation. Overexpression of Bcl-2 or Bcl-xL has been shown to disrupt hVps34/Beclin 1 interactions by sequestering Beclin 1 (Pattingre et al., 2005). Bcl-2 or Bcl-xL also inhibits the heterodimerization between UVRAG and Beclin 1 (Noble et al., 2008). Accordingly, overexpression of BH3 (Bcl-2 homology domain 3)-only proteins or addition of BH3 mimetics, which bind to antiapoptotic members of the Bcl-2 family through their BH domains, reduce the availability of Bcl-2 and Bcl-xL, thereby disinhibiting Beclin 1-dependent autophagy (Maiuri et al., 2007a).

Ras/Raf/MEK/ERK

Abnormal activation of the oncogenic Ras is frequently documented in cancer. Recent findings have revealed that active Ras may promote autophagy that, depending on the cellular context, limits or enables the oncogenic effect of Ras. Previous studies have reported that expressing active Ras in the absence of other cooperating mutations may induce proliferative arrest or senescence in some cell lines, indicating that increased Ras activity per se is insufficient to transform cells (Serrano et al., 1997; Yaswen and Campisi, 2007). In this regard, a recent study reported that human ovarian surface epithelial cells expressing HRASV12 undergo caspase-independent cell death with features of autophagy (Elgendy et al., 2011). In this connection, HRASV12-induced cell death is prevented by knockdown of Beclin 1 or Atg5, both of which are essential for the formation of autophagosomes in mammalian cells. Mechanistically, HRASV12 upregulates the expression of Beclin 1 through the downstream mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade. Meanwhile, HRASV12 induces the expression of Noxa (a BH3-only protein) that displaces Beclin 1 from Mcl-1 (a Bcl2 family member). The unopposed Beclin 1 then promotes autophagy that eventually leads to cell death. These findings suggest that autophagy as a tumor-suppressing mechanism may restrict Ras-induced oncogenesis, especially when other oncogenic signals are unavailable. Paradoxically, autophagy is also required for the survival of Ras-transformed cells. In immortalized baby mouse kidney (iBMK) cells, expression of HRASV12 or KRASV12 increases rates of basal autophagy, whereas genetic ablation of Atg5 or Atg7 impairs the tumorigenicity of these cells in nude mice (Guo et al., 2011). In Ras-expressing iBMK cells, defects in autophagy promote the accumulation of dysmorphic mitochondria, reduce oxygen consumption and cause depletion of tricarboxylic acid cycle metabolites. Consistent with these findings, Yang et al. (2011) demonstrated that treatment with the autophagy inhibitor chloroquine leads to prolonged survival in KRAS-driven genetic mouse model of pancreatic ductal adenocarcinoma. Genetic or pharmacologic inhibition of autophagy also reduces mitochondrial oxidative phosphorylation in pancreatic ductal adenocarcinoma cells. These findings indicate that autophagy enables Ras-induced oncogenesis by maintaining functional mitochondria and oxidative metabolism. The importance of the Ras/Raf/MEK/ERK cascade in the regulation of autophagy is further corroborated by the finding that hyperactivation of oncogenic BRAF induces autophagy whereas specific inhibition on MEK or depletion of ERK inhibits autophagy (Wang et al., 2009; Maddodi et al., 2010).

p53

The TP53 tumor-suppressor gene, also known as the guardian of human genome, is mutated in 50% of human cancers. It encodes the 53 kDa phosphoprotein p53 that accumulates in cells in response to DNA damage, oncogene activation and other stresses. Activation of p53 induces DNA repair genes and arrests the cell cycle to allow enough time for fixation of DNA damage. Nevertheless, if DNA damage is beyond repair, p53 induces apoptosis to prevent accumulation of potentially oncogenic mutations. Accumulating evidence suggests that p53 can regulate autophagy in a dual fashion, the outcome of which depends on its subcellular localization (Maiuri et al., 2010). Nuclear p53 as a transcription factor stimulates autophagy through transcriptional upregulation of its pro-autophagic target genes, including damage-regulated autophagy modulator (DRAM; a lysosomal protein), Sestrin2, Skp2, PUMA, Bax, ULK1/2 and ISG20L1 (Crighton et al., 2006; Maiuri et al., 2009; Yee et al., 2009; Barre and Perkins, 2010; Eby et al., 2010; Gao et al., 2011). In addition, p53 promotes the dissociation of the Beclin 1/Bcl-2 complex and inhibits mTOR signaling (Feng and Levine, 2010; Lorin et al., 2010). In contrast to nuclear p53, cytoplasmic p53 exerts a negative regulatory effect on autophagy. In cells of different species, depletion or inhibition of p53 stimulates pro-survival autophagy that improves cellular fitness under conditions of hypoxia and nutrient depletion. Importantly, the pro-autophagic effect of p53 depletion can be reproduced in enucleated cells. Moreover, cytoplasmic, but not nuclear, p53 represses the enhanced autophagy in p53-deficient cells, indicating a transcription-independent inhibitory effect of p53 on autophagy (Tasdemir et al., 2008). In line with these findings, Morselli et al. (2008) have demonstrated that one-third mutants among a panel of 22 cancer-associated p53 mutants inhibit autophagy when transfected into p53−/− cells, in which most of these mutants preferentially localize to the cytoplasm. The regulation of autophagy by p53 has also been complicated by the finding that p53 post-transcriptionally downregulates LC3 during prolonged nutrient deprivation to enable a reduced, yet sustainable autophagic flux (Scherz-Shouval et al., 2010). To this end, p53 may increase cell fitness by maintaining better autophagic homeostasis that partially explains why some cancer cells retain wild-type p53.

Nuclear factor-κB

Nuclear factor-κB (NF-κB) is a family of dimeric transcription factors. The most common form in human cells is the p50/RelA but other forms, such as p50/p50, p52/p52, p52/RelA, p50/c-Rel, c-Rel/c-Rel, p52/RelB and p50/RelB, have also been identified. This transcription factor is normally sequestered in the cytoplasm by its inhibitor IκB. Upon activation, IκB is phosphorylated by the IκB kinase (IKK) complex and subject to proteasomal degradation, causing the liberation and translocation of NF-κB into the nucleus where it alters gene expression. Activation of NF-κB is linked to inflammation and may be implicated in the pathogenesis of inflammation-associated cancers. In the past two decades, owing to the frequent detection of its activation in human cancers, much attention has been given to the oncogenic property of NF-κB. Nevertheless, recent evidence suggests that this transcription factor may also function as a tumor suppressor in some scenarios, for example, during liver carcinogenesis (Chen and Castranova, 2007). Evolving evidence demonstrates that NF-κB can positively regulate autophagy. For instance, RelA upregulates Beclin 1 through direct binding to the NF-κB-binding site on the Beclin 1 gene promoter to induce autophagy (Copetti et al., 2009). NF-κB-dependent upregulation of Beclin 1 is also responsible for the induction of pro-survival autophagy after heat shock (Nivon et al., 2009). Gangliosides and oridonin also activate NF-κB and autophagy in astrocytes and HT1080 cells, respectively, in which their pro-autophagic effects are blocked by specific NF-κB inhibitors (Hwang et al., 2010). In addition, NF-κB, in concert with p53, regulates autophagy through modulating the expression of its target gene Skp2 (Barre and Perkins, 2010). The multilevel control of autophagy by the IKK/IκB/NF-κB axis is also highlighted by the finding that IKK promotes the autophagic pathway in an NF-κB-independent manner. All these findings support the idea that NF-κB activation promotes autophagy (Criollo et al., 2010). Nevertheless, contrary evidence also exists in the literature. It has been shown that NF-κB represses tumor necrosis factor-α-induced autophagy in Ewing sarcoma cells through activation of mTOR (Djavaheri-Mergny et al., 2006). The reason for these discrepancies remains unclear, but it is possible that the dimeric composition of NF-κB, as in other physiological contexts, may determine the functional outcome of NF-κB activation (Bren et al., 2001; Lovas et al., 2008).

Cyclin-dependent kinase inhibitors and E2F1

E2F1 is a pivotal transcription factor that controls cell proliferation through inducing a number of genes required for G1–S transition. Paradoxically, E2F1 may in some scenarios facilitate cell cycle arrest and apoptosis. Thus, E2F1 displays dual functions of an oncogene and a tumor-suppressor protein. In growth-arrested cells, the transcriptional activity of E2F1 is inhibited by its binding partner retinoblastoma protein (pRB), whose degradation depends on its phosphorylation by cyclin-dependent kinases (CDK) 4 and 6. The activity of CDK 4 and 6 are in turn controlled by the relative abundance of G1 cyclins (cyclin D, cyclin E) and CDK inhibitors (p15, p16, p18, p19, p21, p27). E2F1 is recently shown to be linked to autophagy in which, depending on cellular context, this transcription factor may positively or negatively regulate autophagy. In U2OS osteosarcoma cells, Polager et al. (2008) demonstrated that E2F1 binds to the promoter regions of Atg1, LC3 and DRAM and enhances their expression. E2F1 also indirectly increases Atg5 expression. Overexpression of E2F1 increases the number of LC3-positive autophagosomes, whereas RNA interference targeting E2F1 abolishes DNA damage-induced autophagy. In contrast to this finding, Jiang et al. (2010a) reported that overexpression of pRB, which antagonizes E2F1, induces autophagy in various cell lines accompanied with increased mRNA expression of Atg4a and Atg7. pRB mutants with impaired binding to E2F are deficient for autophagy induction whereas enforced expression of E2F1 antagonizes pRB-mediated autophagy. It has also been shown that two CDK inhibitors (that is, p16 and p27) are able to enhance autophagic activity. The authors postulated that the pro-autophagic effect of pRB is mediated through repression of E2F1-dependent Bcl-2 expression (Jiang et al., 2010b). In line with these findings, several groups of investigators have demonstrated that ectopic expression of p27 or stabilization of p27 by targeting the E3 ligase Spk2 is sufficient to induce autophagy (Komata et al., 2003; Liang et al., 2007; Chen et al., 2008). As opposed to p27, the role of another CDK inhibitor p21 is more equivocal. Although p21 has been shown to impair C(2)-ceramide- and CD40-induced autophagic activity, enforced expression of cytoplasmic p21 induces autophagy (Fujiwara et al., 2008; Portillo et al., 2010; Yang et al., 2010). These findings indicate that, similar to p53, the regulatory effect of p21 on autophagy may depend on its subcellular localization.

LKB1/AMPK

LKB1 is a tumor suppressor that is mutated in the Peutz–Jeghers cancer syndrome. Somatic mutations of LKB1 are also observed in non-small cell lung carcinoma (Sanchez-Cespedes, 2007). LKB1 activates the downstream adenine monophosphate-activated protein kinase (AMPK) to control cell proliferation and apoptosis under metabolic stress conditions. It has been demonstrated that activation of the LKB1/AMPK energy-sensing cascade stimulates autophagic activity through multiple pathways, including stabilization of p27 (Liang et al., 2007), repression of mTOR signaling (Alexander et al., 2010) and direct phosphorylation of ULK1 (Egan et al., 2011; Kim et al., 2011). In addition to metabolic stress, AMPK signaling is regulated by multiple upstream signaling components. For instance, the LKB1/AMPK cascade mediates the pro-autophagic effect of reactive oxygen species-dependent activation of ATM (ataxia telangiectasia mutated; Alexander et al., 2010). Independent of LKB1, transforming growth factor-β activated kinase 1 (TAK1) activates AMPK to induce cytoprotective autophagy in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated epithelial cells (Herrero-Martin et al., 2009). LKB1/AMPK is also downstream of calmodulin-dependent kinase kinase-β that mediates autophagy induced by elevation of intracellular Ca2+ levels (Hoyer-Hansen and Jaattela, 2007).

Transforming growth factor-β

Transforming growth factor (TGF)-β is a cytokine with pleiotropic actions. In early-stage carcinogenesis, TGF-β signaling suppresses tumor formation by inhibiting cell proliferation or by inducing cell differentiation and apoptosis. However, in later stages of cancer, TGF-β signaling promotes cancer progression by fostering cell motility and invasiveness, angiogenesis and immune evasion. A recent study has demonstrated that TGF-β signaling induces autophagy in cancer cells. In hepatocellular carcinoma cells, TGF-β induces accumulation of autophagosomes and the lipidation of LC3 and enhances the degradation of long-lived proteins. TGF-β also enhances the expression of Beclin 1, Atg5, Atg7 and death-associated protein kinase (DAPK), the latter of which is a calmodulin-regulated serine/threonine kinase that possesses tumor-suppressing property. The induction of autophagy by TGF-β in hepatocellular carcinoma cells can be abolished by knockdown of Smad2/3, Smad4 or DAPK as well as pharmacological inhibition of c-Jun N-terminal kinase, indicating the involvement of both Smad and non-Smad signals (Kiyono et al., 2009). In addition to cancer cells, TGF-β has been shown to induce autophagy in mammary and renal epithelial cells as well as mesangial cells (Gajewska et al., 2005; Ding et al., 2010; Koesters et al., 2010).

Other tumor suppressors

The ARF (alternate reading frame) tumor-suppressor protein activates p53 through antagonism of Mdm2, the ubiquitin ligase that targets p53 for proteasome-dependent degradation. Evolving evidence supports that ARF is linked to autophagy through p53-dependent and p53-independent mechanisms. On the one hand, the full-length ARF induces p53 activation to promote autophagy. In this respect, ARF-mediated autophagy can be attenuated by knockdown of p53 (Abida and Gu, 2008). On the other hand, initiation of translation at alternative start codon on ARF mRNA gives rise to a short isoform known as short mitochondrial ARF. The short mitochondrial ARF causes dissipation of mitochondrial membrane potential and induces autophagic cell death that can be prevented by knockdown of Atg5 and Beclin 1 (Reef et al., 2006). The pro-autophagic effect of short mitochondrial ARF is known to be enhanced by the mitochondrial p32 protein that stabilizes short mitochondrial ARF but not the full-length ARF (Reef et al., 2007). It has been shown that the full-length ARF that normally resides in the nucleolus is incapable of inducing p53-independent autophagy (Reef and Kimchi, 2008).

Rab7 is a late endosome/lysosome-associated small GTPase that is involved in endosomal sorting, biogenesis of lysosome and phagocytosis. Rab7 is also a potential tumor suppressor. The RAB7 gene is located in a region that is frequently deleted in solid tumors (Kashuba et al., 1997). Dominant-negative Rab7 also cooperates with E1A to promote the transformation of p53-deleted mouse embryonic fibroblasts. In this connection, Rab7 inhibition prolongs cell survival after growth factor withdrawal by preventing the clearance of glucose and amino-acid transporter proteins from the cell surface (Edinger et al., 2003). Stable knockdown of Rab7 also promotes cell invasion whereas overexpression of Rab7 has an opposite effect (Steffan and Cardelli, 2010; Steffan et al., 2010). It has been demonstrated that Rab7 plays a role in the final maturation of late autophagic vacuoles and is essential for the normal progression of autophagy in mammalian cells (Gutierrez et al., 2004; Jager et al., 2004). In this regard, Rab7 associates with the membrane of autophagic vesicles and through its effector FYCO-1 (FYVE and coiled-coil domain containing 1) promotes the transport of autophagosomes along microtubule tracks to fuse with late endosomes or lysosomes (Pankiv and Johansen, 2010; Pankiv et al., 2010).

Tumor-suppressing and oncogenic action of autophagy

As a housekeeping mechanism, autophagy is involved in the elimination of damaged organelles, the failure of which may lead to cellular dysfunction and, eventually, tumorigenesis. For example, removal of damaged mitochondria by autophagy reduces the production of reactive oxygen species that lead to genomic instability (Gu et al., 2004; Mathew et al., 2007; Stenmark, 2010). Autophagy also promotes oncogene-induced cellular senescence and restricts necrosis that is associated with inflammation and accelerated tumor growth (Degenhardt et al., 2006; Narita and Young, 2009; Young et al., 2009). In two different animal models, monoallelic loss of Beclin 1 promotes the development of spontaneous malignancies and hepatitis B virus-induced premalignant lesions whereas mice deficient in Atg4C/autophagin-3 show an increased susceptibility to develop carcinogen-induced fibrosarcomas (Yue et al., 2003; Marino et al., 2007). Autophagy has also been shown to promote cancer cell death or cell cycle arrest induced by various tumor suppressors, such as, FoxO1 (forkhead box protein O1), EphB2 (ephrin receptor B2) and TGF-β (Kandouz et al., 2010; Suzuki et al., 2010; Zhao et al., 2010). A recent study also demonstrated that activation of autophagy inhibits invasion and migration of MDA-MB-231 cells, in which the effect could be blocked by LC3 silencing, suggesting that autophagy may negatively regulate cancer invasiveness (Indelicato et al., 2010).

As an oncogenic process, autophagy has been exploited by cancer cells to sustain tumor growth and survival, especially in time of nutrient deprivation or other stressful conditions. In this regard, autophagy protects cancer cells against various forms of cellular insults, including endoplasmic reticulum stress, loss of contact with the extracellular matrix and toxicities of cancer therapy (Fung et al., 2008; Levine and Kroemer, 2008; Wu et al., 2010b). Using Cre-mediated excision, Altman et al. (2011) showed that Atg3 deletion prevents BCR-Abl-mediated leukemogenesis. To this end, autophagy is essential to suppress a stress response that leads to p53 phosphorylation and upregulation of p21 and PUMA. Autophagy may also contribute to tumor dormancy. For instance, ARHI enables human ovarian cancer cells grown in mice to become dormant by inducing pro-survival autophagy (Lu et al., 2008). In addition, autophagy facilitates glycolysis and helps to maintain functional mitochondria and thus oxidative phosphorylation to satisfy the metabolic requirement of oncogene-induced transformation (Guo et al., 2011; Lock et al., 2011).

Autophagy and its outcome determinants in cancer therapy

Autophagy is induced by different forms of cancer therapy, including conventional chemotherapeutic drugs, novel targeted cancer therapeutics and ionizing radiation, in various types of solid and hematological malignancies (Table 2) (Han et al., 2007, 2008; Kim et al., 2007, 2008b, 2009a; Maiuri et al., 2007b; Kamitsuji et al., 2008; Meschini et al., 2008; Qadir et al., 2008; Turcotte et al., 2008; Fu et al., 2009; Lorin et al., 2009; Tormo et al., 2009; Fan et al., 2010; Gupta et al., 2010; Huang and Sinicrope, 2010; Li and Fan, 2010; Voss et al., 2010; Yacoub et al., 2010; Wu et al., 2010c; Lian et al., 2011; O’Donovan et al., 2011). In some circumstances, autophagy mediates the cytotoxic or cytostatic effect of anticancer agents, in which blockade of autophagy abolishes the therapeutic actions. On the other hand, autophagy can be triggered as a pro-survival response to withstand the toxicity of the therapy. In this case, inhibition of autophagy enhances drug- or radiation-induced cytotoxicity or cytostasis. However, in some scenarios, autophagy is merely a bystander whose induction or inhibition does not interfere with cell death or survival. The molecular mechanisms governing the switch between these different modes of action remain largely elusive but recent findings have shed new light on several factors that may potentially affect the cell-fate decision process of autophagy.

Table 2 Activation of autophagy in cancer cells by conventional chemotherapeutic drugs, novel molecular therapeutics or ionizing radiation

Genetic composition

The genetic composition of cancer cells may play a deterministic role in controlling the functional outcome of autophagy. For instance, autophagy has been shown to protect HCT116 colon cancer cells and DU145 prostate cancer cells against cell death caused by inducers of endoplasmic reticulum stress, such as A23187, tunicamycin, thapsigargin and brefeldin A, whereas autophagy contributes to cell death in normal colonic epithelial cells and in the nontransformed murine embryonic fibroblasts treated by the same chemicals (Ding et al., 2007). Impairment of autophagy by dominant-negative Vps34, the class III PI3K, also induces apoptotic cell death in neuroblastoma cells expressing a C98X vasopressin mutant but not in the wild-type vasopressin-expressing counterpart (Castino et al., 2008). Moreover, the pan-Bcl-2 inhibitor (–)-gossypol paradoxically induces cytoprotective autophagy in MCF-7 breast cancer cells but autophagic cell death in glioma cells, in which the effects on cell viability can be blocked by RNA interference against Beclin 1 and Atg5 in both cases (Gao et al., 2010; Voss et al, 2010). The molecular mechanism underlying these disparities is presently unknown, but it is speculated that these cells may differentially express some proteins with cell death- or cell survival-specific function in autophagy. In agreement with this notion, a protein known as Draper has been shown to be required for autophagic cell death during the development of salivary gland in Drosophila but not autophagic survival in larval fat body cells following starvation (McPhee et al., 2010). It is believed that similar cell fate-specific proteins are conserved in human and may contribute to the decision between life and death during autophagy in cancer therapy.

Growth factor stimulation

Apart from the genetic composition of cancer cells, growth factor stimulation also significantly affects the cellular consequence of autophagy. For example, various cytokines and growth factors, including interleukin-1, macrophage colony-stimulating factor, insulin growth factor-1, heregulin, interleukin-8 and vascular endothelial growth factor, significantly rescue ARHI-induced reduction of colony formation in autophagic ovarian cancer cells, although by themselves they have no effect on the colony-forming ability of control cells. Exposure of cells to different extracellular matrix components, including poly-D-lysine, poly-L-lysine and fibronectin, also produces a similar effect (Lu et al., 2008). In another study, the chemokine MCP-1 (monocyte chemoattractant protein-1) protects prostate cancer cells from rapamycin-induced autophagic cell death by upregulating survivin expression (Roca et al., 2008). Moreover, in many circumstances, deprivation of growth factor signals by itself triggers autophagy (Lum et al., 2005; Altman et al., 2009). These findings highlight the importance of growth factor signals in the modulation of the functional consequence of autophagy.

Signaling pathway

As a crucial metabolic response at the cellular level, autophagy is regulated by multiple signaling pathways in a widely interacting manner. Emerging evidence supports the concept that the pro-survival or pro-death nature of autophagy may be pathway specific. For instance, curcumin, a natural phytochemical that is used as a chemoprophylactic agent, induces autophagy in cancer cells through concurrent activation of ERK1/2 and inactivation of the Akt/mTOR pathway. Although Akt activation and ERK1/2 inhibition both abolish curcumin-induced autophagy, only the former attenuates the cytotoxicity of curcumin. In contrast, ERK1/2 inhibition induces apoptotic cell death, suggesting that the cellular outcome of autophagy may be determined by the pathway that initiates autophagy (Aoki et al., 2007). This finding also reiterates the importance of pro-survival signaling in cell-fate decision during autophagy as ERK1/2 is a common mediator of growth factor stimulation.

Extent of autophagy

The level of autophagy has also been proposed to determine the die-or-survive decision of autophagy. Kang et al. (2007) found that in Caenorhabditis elegans, physiological levels of autophagy act to promote survival during starvation, whereas insufficient or excessive levels of autophagy render C. elegans starvation hypersensitive and lead to death. It is believed that unrestrained autophagy or unwarranted activation of autophagy in the midst of nutritional abundance may result in premature exhaustion of internal feeding materials, thereby making the cell vulnerable in face of real nutritional challenges. Excessive autophagy may also compromise the integrity of lysosomal membrane, resulting in the release of cathepsins into the cytosol (Park et al., 2008; Hsu et al., 2009; Bhoopathi et al., 2010). In fact, lysosomal membrane permeabilization (LMP) may underlie the therapeutic actions of some pro-autophagic drugs, such as temozolomide and brevinin-2R (Mathieu et al., 2007; Ghavami et al., 2008). The causational relationship between autophagy and LMP is exemplified by the finding that inhibition of resveratrol-induced autophagy by wortmannin or asparagine results in reduced LMP and resveratrol-associated cytotoxicity (Hsu et al., 2009). The induction of Atg5 and Beclin 1 together with formation of LC3-positive autophagosomes have also been shown to precede lysosomal dysfunction, cathepsin activation and cell death in cells treated with OSU-03012, a celecoxib derivative (Park et al., 2008). Cathepsin B also facilitates autophagy-mediated apoptosis in SPARC (secreted protein, acidic and rich in cysteine)-overexpressed primitive neuroectodermal tumor cells (Bhoopathi et al., 2010). These findings suggest that LMP may be an effector pathway of autophagic cell death, the blockade of which may prevent cellular demise. Nevertheless, it is noteworthy that the linkage from autophagy to LMP is not always unidirectional. In this regard, some lysosomotropic detergents, such as siramesine, which trigger cell death via a direct destabilization of lysosomes, have been shown to induce cytoprotective accumulation of autophagosomes (Ostenfeld et al., 2008).

Bcl-2 expression

There are some situations in which induction of autophagy only acts as a bystander, whose inhibition does not enhance or attenuate cell survival. In this respect, Bcl-2, an inhibitor of both apoptosis and autophagy, may limit both the pro-death and pro-survival action of autophagy. In growth factor-deprived conditions, autophagy sensitizes cells with moderate Bcl-2 expression to apoptosis. However, inhibition of autophagy by Atg5- or Beclin 1 short hairpin RNA has no effect on the survival of apoptosis-resistant, high Bcl-2-expressing cells, although these cells undergo a similar extent of autophagy upon growth factor withdrawal (Altman et al., 2009). In line with this notion, silencing of Bcl-2 expression by small interfering RNA (siRNA) induces autophagic cell death in breast cancer cells, in which knockdown of Atg5 significantly inhibits Bcl-2 siRNA-induced loss of cell viability (Akar et al., 2008). Concurrent inhibition of Bcl-2 by ABT-737 and induction of autophagy by rapamycin, a mTOR inhibitor, also significantly enhance the therapeutic effect of ionizing radiation in non-small cell lung tumor xenograft model (Kim et al., 2009b). During pro-survival autophagy, inhibition of Bcl-2 by the natural BH3 mimetic gossypol has also been shown to sensitize cancer cells to apoptosis following suppression of autophagy by wortmannin or Vps34, Atg5 or Beclin 1 short hairpin RNAs (Gao et al., 2010). It is therefore suggested that Bcl-2, in some circumstances, may mask the functional consequences of autophagy, either cell death or survival, with or without the effect on the formation of autophagosomes.

Choice of autophagy inhibitors

The complexity of the duality problem of autophagy is further increased by the fact that using different methods to inhibit autophagy could lead to entirely different outcomes. There are two major approaches, namely pharmacological inhibitors and RNA interference, to inhibit autophagy in the context of cancer therapy, both of which have been widely adopted by investigators in the field. The former consists of the use of chloroquine and bafilomycin A1, which interfere with lysosomal functions and prevent autophagy at the late stage. The RNA interference approach encompasses the use of siRNA or short hairpin RNA that knocks down proteins core to the autophagic machinery, including Vps34, LC3, Atg5, Atg7 and Beclin 1. In addition, some investigators use embryonic fibroblasts derived from Atg5- or Atg7-knockout mice to corroborate results derived from the former approaches.

In the literature, there are many instances in which the use of different inhibitors or the selection of different knockdown targets results in conflicting results. For example, inhibition of imatinib-induced autophagy by Atg5 siRNA attenuates imatinib-induced cytotoxicity whereas inhibition of autophagy by bafilomycin A1 enhances imatinib-induced cytotoxicity (Shingu et al., 2009). In hepatoma cells, LC3 siRNA, but not Atg5 or Beclin 1 siRNAs, abolishes concanavalin A-induced cell death (Chang et al., 2007). These disparities can be partly attributed to the limitation that lysosomotropic agents, such as bafilomycin A1 and chloroquine, may interfere with other lysosome-dependent cellular processes, including endosomal transport and nonautophagic lysosome-dependent cell death. As for the RNA interference approach, LC3 can have autophagy-independent function whereas autophagy can occur independent of Beclin 1 and Atg5/Atg7 (Scarlatti et al., 2008; Nishida et al., 2009; de Haan et al., 2010). Atg5 is also involved in calpain-induced apoptotic cell death (Yousefi et al., 2006). It is therefore recommended that multiple approaches should be used to confirm the role of autophagy.

Concluding remarks and future perspectives

Dysregulation of autophagy, a process regulated by many cancer-related signaling pathways and involved in both tumor suppression and oncogenesis, is a common phenomenon in human cancers. 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 effect of some anticancer agents. As modulation of autophagy represents a novel approach for enhancing the therapeutic efficacies of cancer therapy, research efforts have been put forth to identify agents that induce or inhibit autophagy. Several FDA (Food and Drug Administration)-approved drugs have been identified as inducers (for example, rapamycin, fluspirilene, trifluoperazine, pimozide, niguldipine, nicardipine and amiodarone, loperamide) or inhibitors (for example, chloroquine, hydroxychloroquine, verteporfin) of autophagy (Zhang et al., 2007; Donohue et al., 2011). 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. Nevertheless, the molecular mechanism governing cell-fate decision during autophagy is still poorly understood and the Janus-faced nature of autophagy may complicate the clinical development of its modulators. It is important to determine if the pro-death or pro-survival action of autophagy is associated with a particular class of cancer therapeutics. Moreover, blockade of growth factor signals may enhance the therapeutic outcome of autophagy modulators. It is anticipated that, with more information on the cell fate-specific proteins or pathways essential to autophagy in human, the mystery of the autophagic paradox will be unlocked. Until then, the successful application of autophagy modulators as cancer therapeutics in clinical settings is more hype than hope.

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Acknowledgements

This work was supported by research grant from the CUHK Group Research Scheme (3110043) and CUHK Focused Investments Scheme-Scheme C.

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Correspondence to W K K Wu or J Yu or J J Y Sung.

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Wu, W., Coffelt, S., Cho, C. et al. The autophagic paradox in cancer therapy. Oncogene 31, 939–953 (2012). https://doi.org/10.1038/onc.2011.295

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Keywords

  • autophagy
  • cell survival
  • cell death

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