Autophagy is a self-digesting mechanism responsible for the degradation and recycling of most intracellular macromolecules and the removal of damaged organelles by the lysosome. An impressive number of recent studies have provided key information about the regulation of autophagy and its role in cell survival during nutrient depletion and many other stressful situations. In particular, many evidences have highlighted a crucial role of dysregulated autophagy in oncogenesis. Perturbations of the autophagic pathway have been shown to contribute to tumor development. Moreover, cancer cells have developed several mechanisms that allow them to evade chemotherapy-induced cell death, as well as to use autophagy-associated pathways, to potentiate their survival. In this regard, a complex crosstalk between autophagy and apoptosis has recently emerged; the understanding of the molecular mechanisms regulating this interplay may provide new hints on how to properly modulate these processes to halt cancer. Indeed, key proteins originally thought to be apoptosis-specific inhibitors also block autophagy, while apoptosis proteolytic enzymes hamper autophagy by cleaving autophagy-specific proteins and, in some cases, converting them into proapoptotic factors. This review is focused on the role that Ambra1, a central component of the autophagosome formation machinery, has in the switch between autophagy and apoptosis and its implication in cancer development and chemotherapy resistance.
Autophagy: a brief introduction
The term ‘autophagy’ was coined in the 60s by Christian de Duve, the discoverer of lysosomes.1 It is derived from Greek words auto, meaning 'self', and phagy, ‘to eat’. Autophagy is an evolutionarily conserved catabolic process mediating the degradation of eukaryotic cells' own components through lysosomes.2 Autophagy is responsible for the degradation of long-lived proteins, protein aggregates, intracellular lipid deposit and entire organelles, as well as intracellular bacteria and viruses.3 Low levels of basal autophagy ensure cellular homeostasis, whereas stressful conditions, including nutrient deprivation, hypoxia and low energy, lead to a rapid increase in autophagy, which allows the removal of damaged, unwanted or unnecessary constituents and their recycling in order to maintain macromolecular synthesis and energy homeostasis.4 Notably, autophagy dysfunction has been associated with cancer, muscular diseases and neurodegeneration in higher eukaryotes.5
In recent years, genetic, biochemical and cell biological approaches have begun to provide a good understanding of the molecular mechanisms at the basis of autophagy regulation. Autophagic pathways differ in the way that cytosolic components and organelles are delivered to the lysosome. In fact, three main autophagic pathways have been shown to coexist in mammalian cells: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA).6, 7
Macroautophagy (hereafter autophagy) is the best characterized of these processes. Initially, an isolation membrane, originating from specific regions of the endoplasmic reticulum (ER) called 'omegasomes', sequesters a portion of the cytoplasm leading to autophagosome formation.8 Once formed, this double-membraned isolation membrane or phagophore expands to form double-membrane vesicles called autophagosomes.6 These vesicles then move along the microtubule network, anchored to the dynein complex, to fuse with the lysosomes where the sequestered material is degraded and the resulting amino acids and other macromolecular precursors are released into the cytosol to be recycled.9, 10 Although autophagy was originally described as an unspecific process for bulk degradation of cytosolic materials, the molecular mechanisms of selectivity are now starting to emerge with the discovery of a complex class of autophagy receptors that bind to the inner sheath of autophagosomes.11
A similar process of cytosolic sequestration takes place through microautophagy, but, in this case, the sequestering membrane is the lysosomal membrane itself, which invaginates to form microvesicles.12 This pathway is still characterized to a very small extent in mammals. In contrast to macroautophagy and microautophagy, delivery of specific cargo via CMA does not require the formation of intermediate vesicle compartments or membrane fusion. CMA is selective for specific cytosolic proteins that expose a pentapeptide motif (KFERQ) that is recognized by cytosolic and lysosomal chaperones, including heat-shock cognate protein of 70 kDa (HSC70).13 The substrate–chaperone complex is then targeted to the lysosome by binding to lysosomal membrane receptor LAMP-2A. This distinctive mechanism of lysosomal transport determines the unique properties of CMA when compared with the other forms of autophagy; only soluble proteins containing a lysosomal targeting motif, but not organelles, can be degraded through CMA.
The molecular components of autophagy were initially elucidated in yeast, and subsequent studies have demonstrated a conservation of the autophagic machinery in eukaryotes.6, 14 To date, more than 30 Atg (autophagy-related) genes in yeast have been identified to be involved in different steps of the assembly line that permits the formation of the autophagosome, the recognition of cargos and their delivery to the lysosome.
Autophagy is directly and tightly controlled by the ‘nutrient sensor’ mammalian target of rapamycin (mTOR), which senses and integrates signals from numerous sources including growth factors, amino acid content, hypoxia and energy levels.15 mTOR is a serine/threonine protein kinase present in two different multi-subunit complexes in mammals: mTORC1 and mTORC2.16 mTORC1 is the protein complex directly involved in autophagy regulation in response to various cellular stresses.17 Under nutrient-rich conditions, mTORC1 associates with the ULK1 complex, which, in addition to ULK1/2 (the mammalian ortholog Atg1), contains mammalian Atg13 (mAtg13), FIP200 (the mammalian ortholog of Atg17) and Atg101. Once associated with the ULK1 complex, mTORC1 phosphorylates ULK1 and mAtg13 and inhibits ULK1 kinase activity repressing autophagy. mTORC1 inactivation by upstream signaling (for example, starvation) causes its dissociation from the ULK1 complex, activating the autophagic cascade. In fact, upon mTORC1 inhibition, ULK1 relocalizes to the site of autophagosome formation (Figure 1).
A key event in the formation of the preautophagic vesicle or phagophore is the production of phosphatidylinositol-3-phosphate by the Beclin 1/Class III phosphatidylinositol-3-kinase (CIII-PI3K) Vps34 complex (Figure 1).18 Phosphatidylinositol-3-phosphate is enriched in specific ER structures, named omegasomes because of their Ω-like shape, formed in the ER membranes in starved mammalian cells.19 The omegasome acts as a platform for recruiting the specific autophagic proteins that are required for the formation of the phagophore. In mammals, the Beclin 1/CIII-PI3K complexes consist of a core structure formed by Beclin 1, Vps34 and Vps15.20 Vps15 is a membrane-associated protein that anchors the CIII-PI3K complex to the ER membrane. Beclin 1, the mammalian ortholog of yeast Atg6, interacts with specific proteins such as Ambra1, Atg14/Barkor, UVRAG or Rubicon, which confer distinct functions to Beclin 1/CIII-PI3K complexes.21, 22, 23, 24, 25 Interestingly, different Beclin 1 complexes exist: the one containing Atg14/Barkor is essential for autophagosome formation, whereas UVRAG and Rubicon interactions with Beclin 1 are involved in autophagosome maturation and endocytic traffic.22, 24
Beclin 1 was first described as a Bcl-2-interacting protein through its BH3 domain.26, 27 This interaction has been linked to autophagy inhibition, and the relative amounts of Beclin 1 and Bcl-2 (and also other Bcl-2 family members, for example Bcl-XL) regulate the transition from cell homeostasis to cell death.28 It has been proposed that the pool of Bcl-2-like molecules residing in the ER inhibits Beclin 1 function, thereby preventing autophagosome formation. Bcl-2/Beclin 1 complexes in the ER are disrupted by JNK-mediated Bcl-2 phosphorylation permitting autophagosome nucleation (Figure 1).29 To date, a large set of signaling pathways have been described as converging on Beclin 1 to modulate its activity and regulate autophagy (see He and Levine,20 Wirawan et al.,30 Abrahamsen et al.31 and Kang et al.32 for comprehensive reviews).
Role of ambra1 in autophagy
Ambra1 is a WD40-containing protein playing an important role in the development of the central nervous system. Its functional deficiency in mouse embryos leads to neuroepithelial hyperplasia associated with autophagy impairment and excessive apoptotic cell death.21 Ambra1 binds to Beclin 1 and stabilizes Beclin 1/Vps34 complex, thus potentiating its lipid kinase activity and promoting autophagosome formation21 (Figures 1 and 2). The interaction with Beclin 1 does not require the WD40 domain of Ambra1, but resides in a central region of the protein (aa 533–780), which is also sufficient to prompt autophagy when overexpressed.21 On the other hand, Ambra1 interacts with a region of Beclin 1 adjacent to its BH3 domain, and, consistently, is able to compete with the binding of Bcl-2 to Beclin 1.33
Recent evidence highlighted that Ambra1 is not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also acts as a crucial upstream regulator of autophagy initiation.33, 34 In particular, changes in the subcellular localization of Ambra1 during autophagy induction appears to be important in regulating autophagosome formation in mammalian cells. Indeed, Ambra1 shows a dynamic interaction with the dynein motor complex during autophagy induction.34 Ambra1 is bound, together with Beclin 1 and Vps34, in an autophagy inactive state to the dynein complex via a specific interaction with dynein light chains (DLCs) 1 and 2 (Figure 1), which is mediated by two DLC-binding consensus motifs (TQT) at the carboxy-terminal region of Ambra1 (Figure 2). Upon autophagy induction, Ambra1 is released from the dynein complex upon ULK1-dependent phosphorylation and translocates to the ER together with Beclin 1-Vps34 to allow autophagosome formation (Figure 1). In keeping with this hypothesis, both DLCs’ downregulation and Ambra1 mutations in its DLC-binding sites strongly enhance autophagosome formation. A very recent report confirmed that the Beclin 1 complex is sequestered to the dynein complex and showed that the proapoptotic BH3-only protein Bim is also involved in this pathway.35 Autophagic stimuli induce Bim phosphorylation by JNK, which abrogates its binding with DLCs and leads to the dissociation from Beclin 1. It would be interesting to clarify whether the proposed regulation of autophagy by Bim acts in a synergistic manner with that dependent on Ambra1, or whether there are two distinct pools of Beclin 1 complex at the level of cytoskeleton, regulated by different upstream pathways.35
In addition to the dynein, Ambra1 was found to bind dynamically to the mitochondrial pool of Bcl-2, further underlining the relevance of subcellular localization in regulating its function.33 Under normal conditions, Bcl-2 docked a pool of Ambra1 at the mitochondria, by interacting with both the amino- and the carboxy-terminal domains of the protein (Figures 1 and 2). After autophagy induction, Ambra1 dissociates from Bcl-2 present on the mitochondrial membrane and increases its binding with a fraction of Beclin 1 to enhance Beclin 1-dependent autophagy. Consistent with this hypothesis, a reduction of Ambra1 colocalization with mitochondria in response to autophagy was observed, accompanied by an increased interaction between Ambra1 and Beclin 1 in the ER fraction and a reciprocal decrease in binding between Bcl-2 and Beclin 1 at this site (Figure 1). It remains to be elucidated which is the mechanism responsible for Ambra1/Bcl-2 dissociation and whether a pool of Ambra1 that remains associated to the mitochondria is involved in the induction of autophagy directly from the mitochondrial outer membrane, a proposed alternative site of autophagosome nucleation.36 Notably, the binding of Bcl-2 to Ambra1 is not prevented by the phosphorylation of Bcl-2 in its BH3 domain, at variance with that observed with Beclin 1, thus suggesting that distinct mechanisms may account for the release of Bcl-2 inhibition on Ambra1 and Beclin 1 during autophagy induction. Interestingly, the interaction between Ambra1 and the mitochondrial Bcl-2 is disrupted by both autophagic and apoptotic stimuli, highlighting the intimate crosstalk between these processes.
An interesting recent report has shown that Ambra1 may also have a role in a selective form of autophagy, that is, mitophagy. Indeed, Ambra1 interacts with Parkin, a protein that has a key role in the mitochondrial quality control by translocating to depolarized mitochondria and inducing mitophagy.37 Interestingly, prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1, which is recruited in a Parkin-dependent manner to the perinuclear clusters of depolarized mitochondria, where it activates the PtdIns3K complex around these damaged mitochondria and contributes to their selective autophagic clearance. The ablation of Ambra1 does not affect Parkin translocation to depolarized mitochondria, but inhibits subsequent mitochondrial clearance. Conversely, Ambra1 overexpression enhances the elimination of depolarized mitochondria, but only in the presence of Parkin.
Ambra1 in apoptosis
The crosstalk between autophagy and apoptosis is highly complex.38, 39 Factors identified as activators of apoptosis can often induce autophagy, whereas factors that negatively regulate apoptosis also inhibit autophagy induction. The well-known anti-apoptotic factor Bcl-2 is a key factor in this context. The pool of Bcl-2 resident at the ER is able, indeed with the contribution of NAF-1 (nutrient deprivation autophagy factor-1), to negatively regulate the Beclin 1-dependent autophagic program.28, 40 In contrast, the mitochondrial pool of Bcl-2 has been proposed to exert only an anti-apoptotic function41 despite mitochondria having been recently demonstrated to be an additional site for autophagosome formation36 and the further finding that Bcl-2/Ambra1 interaction occurs at mitochondria.33 On the other hand, the overexpression of Bcl-2 promotes a protective effect against a wide range of apoptotic inducers. This antiapoptotic action derives from the fact that Bcl-2 neutralizes proapoptotic Bcl-2 family members such as Bax and Bak, as well as BH3-only proteins, thus preventing mitochondrial membrane permeabilization and consequent cell death.42 Similar to Bcl-2, Flip (Flice inhibitory protein), an inhibitor of death receptor-mediated apoptosis, can also suppress autophagy by competing with LC3 for Atg3 binding, thereby preventing Atg3-mediated autophagosome elongation.43
Importantly, stress-activated pathways, such as those activating JNK, can concomitantly induce the autophagic and apoptotic pathways by targeting different Bcl-2 family members, such as Bcl-2, Bcl-XL, Bad, Bim and Bmf.44, 45, 46 Once both processes are induced, the extent of the damage and the capability of restoring vital functions will determine the cell fate decision between death or survival, by modulating the complex crosstalk between autophagy and apoptosis. Indeed, many functional interactions have been recently described, which unveiled how these pathways are mutually regulated through the modification of each other’s activity. On one hand, it has been demonstrated that crucial autophagic proteins, such as Beclin 1, Atg4D and ATG5, are cleaved by apoptotic executioner proteases and that the inhibition of their cleavage favors the autophagy prosurvival functions and counteracts cell death (Figure 3).47, 48, 49, 50, 51, 52, 53 Notably, the cleaved products of these autophagic proteins were described to acquire a new function by translocating to the mitochondria and amplifying mitochondria-mediated apoptosis (Figure 3). Recently, another proautophagic factor, ATG12, has been reported to contribute to the induction of the apoptotic process, despite not being a target of apoptotic proteases. Indeed, it was described that Atg12 is required for an efficient induction of cell death by a variety of apoptotic stimuli.54, 55 This proapoptotic function is independent of its role in autophagosome elongation, although it relies on its ability to bind and inhibit Bcl-2 activity, through a BH3-like domain55 (Figure 3).
On the other hand, Hou and colleagues recently demonstrated the autophagic degradation of the active caspase-8 enzyme during TRAIL-induced autophagy, keeping the apoptotic response at bay (Figure 3).56 These results support the existence of cross-regulatory mechanisms between both the cell fate-determining processes, so that only one process can prevail.
As previously mentioned, the functional deficiency of Ambra1 in mice causes defects in the development and embryonic death associated with autophagy impairment and a large number of supernumerary apoptotic cells.21, 57 The latter aspect of this multifaceted phenotype prompted us to investigate more deeply the role of Ambra1 during apoptosis.58, 59 We found that reduced levels of Ambra1 in a variety of cell lines lead to increased susceptibility to different apoptotic stimuli. Moreover, apoptosis induction causes Ambra1 degradation that occurs in a caspase- and calpain-dependent manner. Ambra1 cleavage occurs early during the apoptotic process and was observed to precede the decline of other autophagic proteins. In vitro caspase cleavage assay confirmed that Ambra1 is a substrate of different caspases and allowed us to identify D482 as a major caspase cleavage site in Ambra1. Significantly, a D482→A Ambra1 mutation prevents the formation of cleavage products in Ambra1-overexpressing cells and protects from apoptosis more efficiently than the wild-type protein does. Differently from caspases, calpains completely degrade Ambra1 in an in vitro assay, thus possibly explaining why stable endogenous Ambra1 cleavage fragments were not observed accumulating in vivo upon apoptosis induction. The fact that Ambra1 decline could be prevented by concomitant inhibition of calpains and caspases, and that a caspase-uncleavable Ambra1 mutant is only partially resistant to staurosporine-induced degradation, seems to confirm that these proteases may act in concert to ensure Ambra1 removal during apoptosis.
The prosurvival role for Ambra1 is further demonstrated by the enhanced resistance to apoptosis of Ambra1 overexpressing cerebellar granule neurons induced to die by trophic factor withdrawal.33
Taken together, these data strengthen the emerging view that under stressful cellular conditions prosurvival and prodeath processes are concomitantly activated, and that autophagy impairment by apoptotic machinery could represent a ‘point of no return’ toward death.
Crosstalk between autophagy and apoptosis in cancer
Autophagy is a rapidly expanding area of research, and multiple links between deficient or excessive autophagy, oncogenesis, cancer therapy and resistance have been reported.60, 61, 62 During early oncogenesis, carcinogenic aberrations in oncogenes and tumor suppressor genes frequently inhibit the autophagic program, this feature being thought to contribute to the genomic instability relevant to oncogenesis and tumor progression (Figure 4). Although the molecular mechanisms mediating these events have not been fully determined, the accumulation of the autophagic cargo-binding protein p62/SQSTM1 has been shown to be a direct link between autophagy impairment and tumorigenesis.62, 63, 64 Moreover, autophagy provides a protective function to limit necrotic cell death of transformed cells and consequent inflammation favoring tumor progression.65
Particularly important in this respect is the interplay taking place between Bcl-2 and the Beclin 1 complex. Alteration of Bcl-2 and Beclin 1 expression has been reported in a large number of human tumors, underlying their important role in carcinogenesis.60, 66 Indeed, Beclin 1 gene is monoallelically deleted in a subset of tumors of the breast, ovary and prostate.67 Moreover, mice heterozygous for the beclin 1 mutation are predisposed to multiple tumor types, including lymphoma and liver cancer.68 Similarly, other members of Beclin 1 complex, such as UVRAG and Bif-1, develop multiple spontaneous cancers.69, 70 Although direct evidence of a role of Ambra1 in tumorigenesis has not been demonstrated yet, the described functional interactions of Ambra1 with the Beclin 1 complex suggest that a dysregulation of Ambra1 function may contribute to oncogenesis. In support of this hypothesis, the functional deficiency of Ambra1 in mouse embryos leads to uncontrolled cell proliferation, in addition to severe autophagy impairment.21 In keeping with this, it would be crucial in the near future to elucidate the molecular mechanisms that link the autophagic function of Ambra1 to its ability to regulate cell growth, which may be impaired in the early steps of oncogenesis when cells acquire higher proliferation rates. Moreover, Ambra1 may be indirectly involved in tumor formation by regulating the cell death/survival balance through its binding with the cell death repressor Bcl-2 and altering its ability to modulate the activity of proapoptotic and proautophagic factors. Indeed, Ambra1 can compete with both mitochondrial and ER-resident Bcl-2 to bind Beclin 1.33 Hence, either the overexpression or the absence of Ambra1 molecules can interfere with the Bcl2-binding partners and possibly with cell death regulation.
Cellular stress occurring in established tumors, be it endogenous (such as hypoxia or ER stress) or exogenous (such as conventional or targeted chemotherapy and radiotherapy), can induce autophagy, which in turn constitutes a mechanism through which cancer cells protect themselves against stress and thus prolong their survival.4 Interestingly, only benign tumors developed in the liver of mosaically deleted ATG5 mice, suggesting that sustained levels of autophagy may be required for progression beyond the benign state62 (Figure 4).
Combined treatments of chemotherapeutic drugs and autophagy inhibitors have been shown to increase the rate of apoptosis in established tumors.71, 72, 73 However, there is a subset of antitumoral agents that require a functional autophagic machinery to exert their cell death function. For example, Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy.74, 75 THC induced ceramide accumulation and eukaryotic translation initiation factor 2α phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mTORC1 axis. Interestingly, autophagy induction is upstream of apoptosis in cannabinoid-induced cancer cell death, and the activation of this pathway is necessary for the antitumor action of cannabinoids in vivo. Autophagy preceded the appearance of apoptotic features in THC-treated cells; the selective knockdown of Ambra1 or other ATG genes prevented THC-induced caspase-3 activation. In fact, unlike their wild-type counterparts, autophagy-deficient cells underwent neither phosphatidylserine translocation to the outer leaflet of the plasma membrane nor increased production of reactive oxygen species in response to cannabinoid treatment. These findings indicate that activation of the autophagy-mediated cell death pathway occurs upstream of apoptosis in cannabinoid antitumoral action.
An intriguing role of autophagy in the immune response to cancer cells has recently emerged: autophagy activation has been shown to be essential to render anticancer therapies more efficient by converting the patient’s own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death.76 One of the hallmarks of immunogenic cell death is the release of ATP by cells committed to undergoing apoptosis. Notably, knockdown of essential autophagy-related genes (atg3, atg5, atg7 and becn1) abolishes the preapoptotic secretion of ATP by various cancer cell lines undergoing immunogenic cell death. Accordingly, autophagy-proficient, but not autophagy-deficient, tumor cells treated with immunogenic cell death inducers in vitro could induce a tumor-specific immune response in vivo. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced immunosurveillance because they are unable to release ATP. Thus, autophagy is essential for the immunogenic release of ATP by dying cells; increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.
Taken as a whole, these data highlight the importance of understanding the mechanisms linking cellular stress/cell death and autophagy in order to design novel therapeutic strategies based on the modulation of autophagy in cancer cells. A central question is whether the modulation of autophagy is a relevant therapeutic strategy for the selective elimination of tumor cells. Many clinical trials are underway to test the effects of inhibiting or boosting autophagy as part of treatment regimens for various cancers. However, the real challenge now is to gain more knowledge about the molecular mechanisms involved in autophagy and the particular deficiencies in these mechanisms decisive for oncogenesis. In keeping with these assumptions, of particular interest are recent data concerning the comprehensive characterization of the interaction networks, as well as of post-translational modifications that regulate the autophagic pathway.77, 78 In particular, as regards Ambra1, more than 20 different Ambra1 phosphorylations have been reported, some of which are modulated in a cell-cycle-regulated manner79, 80 (Figure 2). Moreover, Ambra1 has been shown to undergo ubiquitination and to interact with elements of the proteasome-mediated degradation system, especially with the E3 ligase Cullin 4 complex.81, 82, 83, 84 The E3 ubiquitin ligases dictate substrate specificity, thus regulating the degradation or the activity of factors involved in a wide range of cellular processes, including proliferation, differentiation and apoptosis.85 Dysregulation of E3 ubiquitin ligase activity has been shown to contribute to oncogenesis through the accumulation of oncoproteins and cell cycle regulators, or the excessive degradation of tumor suppressors.86, 87 In the future, it will be important to elucidate whether Ambra1’s interaction with E3 ligase family members has a key role in the regulation of autophagy and whether this function is altered during cellular transformation.
Klionsky DJ . Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8: 931–937.
Mizushima N . Physiological functions of autophagy. Curr Top Microbiol Immunol 2009; 335: 71–84.
Deretic V, Levine B . Autophagy, immunity, and microbial adaptations. Cell Host Microbe 2009; 5: 527–549.
Kroemer G, Marino G, Levine B . Autophagy and the integrated stress response. Mol Cell 2010; 40: 280–293.
Mizushima N, Levine B, Cuervo AM, Klionsky DJ . Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069–1075.
Mizushima N, Yoshimori T, Ohsumi Y . The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011; 27: 107–132.
Orenstein SJ, Cuervo AM . Chaperone-mediated autophagy: molecular mechanisms and physiological relevance. Semin Cell Dev Biol 2010; 21: 719–726.
Burman C, Ktistakis NT . Regulation of autophagy by phosphatidylinositol 3-phosphate. FEBS Lett 2010; 584: 1302–1312.
Mehrpour M, Esclatine A, Beau I, Codogno P . Overview of macroautophagy regulation in mammalian cells. Cell Res 2010; 20: 748–762.
Jahreiss L, Menzies FM, Rubinsztein DC . The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 2008; 9: 574–587.
Dikic I, Johansen T, Kirkin V . Selective autophagy in cancer development and therapy. Cancer Res 2010; 70: 3431–3434.
Li WW, Li J, Bao JK . Microautophagy: lesser-known self-eating. Cell Mol Life Sci 2012; 69: 1125–1136.
Arias E, Cuervo AM . Chaperone-mediated autophagy in protein quality control. Curr Opin Cell Biol 2011; 23: 184–189.
Yang Z, Klionsky DJ . Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 2010; 22: 124–131.
Jung CH, Ro SH, Cao J, Otto NM, Kim DH . mTOR regulation of autophagy. FEBS Lett 2010; 584: 1287–1295.
Laplante M, Sabatini DM . mTOR signaling in growth control and disease. Cell 2012; 149: 274–293.
Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 2009; 20: 1981–1991.
Simonsen A, Tooze SA . Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. J Cell Biol 2009; 186: 773–782.
Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008; 182: 685–701.
He C, Levine B . The Beclin 1 interactome. Curr Opin Cell Biol 2010; 22: 140–149.
Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R et al. Ambra1 regulates autophagy and development of the nervous system. Nature 2007; 447: 1121–1125.
Itakura E, Kishi C, Inoue K, Mizushima N . Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol Biol Cell 2008; 19: 5360–5372.
Sun Q, Fan W, Chen K, Ding X, Chen S, Zhong Q . Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 2008; 105: 19211–19216.
Matsunaga K, Saitoh T, Tabata K, Omori H, Satoh T, Kurotori N et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol 2009; 11: 385–396.
Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT . Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol 2009; 11: 468–476.
Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G et al. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 1998; 72: 8586–8596.
Sinha S, Levine B . The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene 2008; 27 (Suppl 1): S137–S148.
Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122: 927–939.
Wei Y, Pattingre S, Sinha S, Bassik M, Levine B . JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell 2008; 30: 678–688.
Wirawan E, Lippens S, Vanden Berghe T, Romagnoli A, Fimia GM, Piacentini M et al. Beclin1: a role in membrane dynamics and beyond. Autophagy 2012; 8: 6–17.
Abrahamsen H, Stenmark H, Platta HW . Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression. FEBS Lett 2012; 586: 1584–1591.
Kang R, Zeh HJ, Lotze MT, Tang D . The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 2011; 18: 571–580.
Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM et al. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. EMBO J 2011; 30: 1195–1208.
Di Bartolomeo S, Corazzari M, Nazio F, Oliverio S, Lisi G, Antonioli M et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J Cell Biol 2010; 191: 155–168.
Luo S, Garcia-Arencibia M, Zhao R, Puri C, Toh PP, Sadiq O et al. Bim inhibits autophagy by recruiting Beclin 1 to microtubules. Mol Cell 2012; 47: 359–370.
Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 2010; 141: 656–667.
Van Humbeeck C, Cornelissen T, Hofkens H, Mandemakers W, Gevaert K, De Strooper B et al. Parkin interacts with Ambra1 to induce mitophagy. J Neurosci 2011; 31: 10249–10261.
Maiuri MC, Zalckvar E, Kimchi A, Kroemer G . Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007; 8: 741–752.
Fimia GM, Piacentini M . Regulation of autophagy in mammals and its interplay with apoptosis. Cell Mol Life Sci 2010; 67: 1581–1588.
Chang NC, Nguyen M, Germain M, Shore GC . Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1. EMBO J 2010; 29: 606–618.
Levine B, Sinha S, Kroemer G . Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 2008; 4: 600–606.
Adams JM, Cory S . Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 2007; 19: 488–496.
Lee JS, Li Q, Lee JY, Lee SH, Jeong JH, Lee HR et al. FLIP-mediated autophagy regulation in cell death control. Nat Cell Biol 2009; 11: 1355–1362.
Wei Y, Sinha S, Levine B . Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation. Autophagy 2008; 4: 949–951.
Lei K, Davis RJ . JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc Natl Acad Sci USA 2003; 100: 2432–2437.
Donovan N, Becker EB, Konishi Y, Bonni A . JNK phosphorylation and activation of BAD couples the stress-activated signaling pathway to the cell death machinery. J Biol Chem 2002; 277: 40944–40949.
Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC . Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett 2009; 274: 95–100.
Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I et al. Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis 2010; 1: e18.
Li H, Wang P, Sun Q, Ding WX, Yin XM, Sobol RW et al. Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1. Cancer Res 2011; 71: 3625–3634.
Luo S, Rubinsztein DC . Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL. Cell Death Differ 2010; 17: 268–277.
Russo R, Berliocchi L, Adornetto A, Varano GP, Cavaliere F, Nucci C et al. Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis 2011; 2: e144.
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 2006; 8: 1124–1132.
Betin VM, Lane JD . Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J Cell Sci 2009; 122: 2554–2566.
Radoshevich L, Murrow L, Chen N, Fernandez E, Roy S, Fung C et al. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell 2010; 142: 590–600.
Rubinstein AD, Eisenstein M, Ber Y, Bialik S, Kimchi A . The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis. Mol Cell 2011; 44: 698–709.
Hou W, Han J, Lu C, Goldstein LA, Rabinowich H . Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis. Autophagy 2010; 6: 891–900.
Cecconi F, Piacentini M, Fimia GM . The involvement of cell death and survival in neural tube defects: a distinct role for apoptosis and autophagy? Cell Death Differ 2008; 15: 1170–1177.
Corazzari M, Fimia GM, Piacentini M . Dismantling the autophagic arsenal when it is time to die: concerted AMBRA1 degradation by caspases and calpains. Autophagy 2012; 8: 1255–1257.
Pagliarini V, Wirawan E, Romagnoli A, Ciccosanti F, Lisi G, Lippens S et al. Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response. Cell Death Differ 2012; 19: 1495–1504.
Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999; 402: 672–676.
Mathew R, White E . Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr Opin Genet Dev 2011; 21: 113–119.
Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al. Autophagy-deficient mice develop multiple liver tumors. Genes Dev 2011; 25: 795–800.
Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009; 137: 1062–1075.
Inami Y, Waguri S, Sakamoto A, Kouno T, Nakada K, Hino O et al. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J Cell Biol 2011; 193: 275–284.
Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006; 10: 51–64.
Placzek WJ, Wei J, Kitada S, Zhai D, Reed JC, Pellecchia M . A survey of the anti-apoptotic Bcl-2 subfamily expression in cancer types provides a platform to predict the efficacy of Bcl-2 antagonists in cancer therapy. Cell Death Dis 2010; 1: e40.
Aita VM, Liang XH, Murty VV, Pincus DL, Yu W, Cayanis E et al. Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics 1999; 59: 59–65.
Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003; 112: 1809–1820.
Liang C, Feng P, Ku B, Dotan I, Canaani D, Oh BH et al. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat Cell Biol 2006; 8: 688–699.
Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat Cell Biol 2007; 9: 1142–1151.
Amaravadi RK, Thompson CB . The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 2007; 13: 7271–7279.
Livesey KM, Tang D, Zeh HJ, Lotze MT . Autophagy inhibition in combination cancer treatment. Curr Opin Investig Drugs 2009; 10: 1269–1279.
Armstrong JL, Corazzari M, Martin S, Pagliarini V, Falasca L, Hill DS et al. Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition. Clin Cancer Res 2011; 17: 2216–2226.
Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Lorente M, Egia A et al. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J Clin Invest 2009; 119: 1359–1372.
Velasco G, Sanchez C, Guzman M . Towards the use of cannabinoids as antitumour agents. Nat Rev Cancer 2012; 12: 436–444.
Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science 2011; 334: 1573–1577.
Behrends C, Sowa ME, Gygi SP, Harper JW . Network organization of the human autophagy system. Nature 2010; 466: 68–76.
Yi C, Ma M, Ran L, Zheng J, Tong J, Zhu J et al. Function and molecular mechanism of acetylation in autophagy regulation. Science 2012; 336: 474–477.
Dephoure N, Zhou C, Villen J, Beausoleil SA, Bakalarski CE, Elledge SJ et al. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci USA 2008; 105: 10762–10767.
Daub H, Olsen JV, Bairlein M, Gnad F, Oppermann FS, Korner R et al. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. Mol Cell 2008; 31: 438–448.
Kim W, Bennett EJ, Huttlin EL, Guo A, Li J, Possemato A et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 2011; 44: 325–340.
Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 2011; 10: 013284.
Bennett EJ, Rush J, Gygi SP, Harper JW . Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 2010; 143: 951–965.
Jin J, Arias EE, Chen J, Harper JW, Walter JC . A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 2006; 23: 709–721.
Schwartz AL, Ciechanover A . Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 2009; 49: 73–96.
Hoeller D, Dikic I . Targeting the ubiquitin system in cancer therapy. Nature 2009; 458: 438–444.
Lipkowitz S, Weissman AM . RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis. Nat Rev Cancer 2011; 11: 629–643.
UniProt Consortium. Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 2012; 40 (Database issue): D71–D75.
Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res 2012; 40 (Database issue): D261–D270.
Maglott D, Ostell J, Pruitt KD, Tatusova T . Entrez Gene: gene-centered information at NCBI. Nucleic Acids Res 2011; 39 (Database issue): D52–D57.
This work was supported by grants from the Italian Ministry of University FIRB, Compagnia di San Paolo to MP, the Ministry of Health of Italy ‘Ricerca Corrente’ and ‘Ricerca Finalizzata’ to MP and GMF, AIRC to MP and MC, and Telethon to GMF. The support of the EU grant ‘Transpath ‘ Marie Curie project to MP is also acknowledged.
The authors declare no conflict of interest.
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Fimia, G., Corazzari, M., Antonioli, M. et al. Ambra1 at the crossroad between autophagy and cell death. Oncogene 32, 3311–3318 (2013) doi:10.1038/onc.2012.455
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