Ambra1 at the Crossroad Between Autophagy and Cell Death

REVIEW Ambra1 at the crossroad between autophagy and cell death

GM Fimia1, M Corazzari1,2, M Antonioli1 and M Piacentini1,2

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.

Oncogene (2013) 32, 3311–3318; doi:10.1038/onc.2012.455; published online 15 October 2012 Keywords: Ambra1; Bcl-2; Beclin 1; caspases; calpains; apoptosis

AUTOPHAGY: A BRIEF INTRODUCTION vesicles then move along the microtubule network, anchored to The term ‘autophagy’ was coined in the 60s by Christian de Duve, the dynein complex, to fuse with the lysosomes where the the discoverer of lysosomes.1 It is derived from Greek words auto, sequestered material is degraded and the resulting amino acids meaning ’self’, and phagy, ‘to eat’. Autophagy is an evolutionarily and other macromolecular precursors are released into the cytosol 9,10 conserved catabolic process mediating the degradation of to be recycled. Although autophagy was originally described as eukaryotic cells’ own components through lysosomes.2 an unspecific process for bulk degradation of cytosolic materials, Autophagy is responsible for the degradation of long-lived the molecular mechanisms of selectivity are now starting to proteins, protein aggregates, intracellular lipid deposit and emerge with the discovery of a complex class of autophagy 11 entire organelles, as well as intracellular bacteria and viruses.3 receptors that bind to the inner sheath of autophagosomes. Low levels of basal autophagy ensure cellular homeostasis, A similar process of cytosolic sequestration takes place through whereas stressful conditions, including nutrient deprivation, microautophagy, but, in this case, the sequestering membrane is hypoxia and low energy, lead to a rapid increase in autophagy, the lysosomal membrane itself, which invaginates to form 12 which allows the removal of damaged, unwanted or unnecessary microvesicles. This pathway is still characterized to a very constituents and their recycling in order to maintain small extent in mammals. In contrast to macroautophagy and macromolecular synthesis and energy homeostasis.4 Notably, microautophagy, delivery of specific cargo via CMA does not autophagy dysfunction has been associated with cancer, require the formation of intermediate vesicle compartments or muscular diseases and neurodegeneration in higher eukaryotes.5 membrane fusion. CMA is selective for specific cytosolic proteins In recent years, genetic, biochemical and cell biological that expose a pentapeptide motif (KFERQ) that is recognized by approaches have begun to provide a good understanding of the cytosolic and lysosomal chaperones, including heat-shock cognate 13 molecular mechanisms at the basis of autophagy regulation. protein of 70 kDa (HSC70). The substrate–chaperone complex is Autophagic pathways differ in the way that cytosolic components then targeted to the lysosome by binding to lysosomal membrane and organelles are delivered to the lysosome. In fact, three main receptor LAMP-2A. This distinctive mechanism of lysosomal autophagic pathways have been shown to coexist in mammalian transport determines the unique properties of CMA when cells: macroautophagy, microautophagy and chaperone-mediated compared with the other forms of autophagy; only soluble autophagy (CMA).6,7 proteins containing a lysosomal targeting motif, but not Macroautophagy (hereafter autophagy) is the best character- organelles, can be degraded through CMA. ized of these processes. Initially, an isolation membrane, originat- ing from specific regions of the endoplasmic reticulum (ER) called ’omegasomes’, sequesters a portion of the cytoplasm leading to AUTOPHAGY REGULATION autophagosome formation.8 Once formed, this double- The molecular components of autophagy were initially elucidated membraned isolation membrane or phagophore expands to in yeast, and subsequent studies have demonstrated a conserva- form double-membrane vesicles called autophagosomes.6 These tion of the autophagic machinery in eukaryotes.6,14 To date, more

1Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy and 2Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy. Correspondence: Professor M Piacentini, Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, Rome 00173, Italy. E-mail: [email protected] Received 16 July 2012; revised 14 August 2012; accepted 15 August 2012; published online 15 October 2012 The role of Ambra1 in the balance of apoptosis and autophagy GM Fimia et al 3312 than 30 Atg (autophagy-related) genes in yeast have been A key event in the formation of the preautophagic vesicle or identified to be involved in different steps of the assembly line phagophore is the production of phosphatidylinositol-3-phos- that permits the formation of the autophagosome, the recognition phate by the Beclin 1/Class III phosphatidylinositol-3-kinase of cargos and their delivery to the lysosome. (CIII-PI3K) Vps34 complex (Figure 1).18 Phosphatidylinositol-3- Autophagy is directly and tightly controlled by the ‘nutrient phosphate is enriched in specific ER structures, named sensor’ mammalian target of rapamycin (mTOR), which senses and omegasomes because of their O-like shape, formed in the ER integrates signals from numerous sources including growth membranes in starved mammalian cells.19 The omegasome acts as factors, amino acid content, hypoxia and energy levels.15 mTOR a platform for recruiting the specific autophagic proteins that are is a serine/threonine protein kinase present in two different multi- required for the formation of the phagophore. In mammals, the subunit complexes in mammals: mTORC1 and mTORC2.16 Beclin 1/CIII-PI3K complexes consist of a core structure formed by mTORC1 is the protein complex directly involved in autophagy Beclin 1, Vps34 and Vps15.20 Vps15 is a membrane-associated regulation in response to various cellular stresses.17 Under protein that anchors the CIII-PI3K complex to the ER membrane. nutrient-rich conditions, mTORC1 associates with the ULK1 Beclin 1, the mammalian ortholog of yeast Atg6, interacts with complex, which, in addition to ULK1/2 (the mammalian ortholog specific proteins such as Ambra1, Atg14/Barkor, UVRAG or Atg1), contains mammalian Atg13 (mAtg13), FIP200 (the Rubicon, which confer distinct functions to Beclin 1/CIII-PI3K mammalian ortholog of Atg17) and Atg101. Once associated complexes.21–25 Interestingly, different Beclin 1 complexes exist: with the ULK1 complex, mTORC1 phosphorylates ULK1 and the one containing Atg14/Barkor is essential for autophagosome mAtg13 and inhibits ULK1 kinase activity repressing autophagy. formation, whereas UVRAG and Rubicon interactions with Beclin 1 mTORC1 inactivation by upstream signaling (for example, are involved in autophagosome maturation and endocytic traffic.22,24 starvation) causes its dissociation from the ULK1 complex, Beclin 1 was first described as a Bcl-2-interacting protein activating the autophagic cascade. In fact, upon mTORC1 through its BH3 domain.26,27 This interaction has been linked to inhibition, ULK1 relocalizes to the site of autophagosome autophagy inhibition, and the relative amounts of Beclin 1 and formation (Figure 1). Bcl-2 (and also other Bcl-2 family members, for example Bcl-XL)

No Autophagy Autophagy induction

mTOR complex Stress pathways mTOR JNK P Atg13 ULK1/2

Low Microtubules Bcl-2 Atg101 FIP200 Nutrients Endoplasmic Reticulum

Microtubules mTOR PI(3)P Vps15 PI Dynein Beclin1 Vps34/ Complex CIII-PI3K Ambra1 Atg13 ULK1/2 P Vps34/ Ambra1 ATG14 Beclin1 CIII-PI3K Atg101 FIP200

Endoplasmic Reticulum ?

Bcl-2 ? Beclin1 Autophagosome Vps34/ Ambra1 CIII-PI3K

Bcl-2 Beclin1 Lys Ambra1

Mitochondria Parkin

Mitochondria

Ambra1

Bcl-2 Autolysosome

Figure 1. Regulation of autophagy induction. Left panel: In the presence of high levels of nutrients, mTor inhibits ULK1 complex by interacting with and phosphorylating ULK1 and ATG13. A pool of Ambra1/Beclin 1 complex is associated, in an inactive form, with the dynein complex. Bcl-2 contributes to the autophagy repression by interacting with Beclin1 located in the ER, as well as with a pool of Ambra1 localized to the mitochondrial outer membrane. Right panel: when nutrient levels are reduced, mTOR is inactive and lets Ulk1 free to stimulate autophagy. By phosphorylating Ambra1, Ulk1 allows Beclin 1 complex to dissociate from the dynein complex and to translocate to the ER membrane, or alternatively to other intracellular membranes ( not represented in the scheme). The Beclin 1-associated lipid kinase Vps34 phosphorylates the phosphatidylinositol (PI) to produce PI(3)P, which represents the docking site for the autophagosome formation machinery. In parallel, Bcl-2/ Beclin 1 interaction is disrupted by the JNK-mediated phosphorylation of Bcl2 protein. Mitochondrial Ambra1 also dissociates from Bcl-2 following nutrient starvation, but the kinase responsible for this process has not been characterized yet.

Oncogene (2013) 3311 – 3318 & 2013 Macmillan Publishers Limited The role of Ambra1 in the balance of apoptosis and autophagy GM Fimia et al 3313 regulate the transition from cell homeostasis to cell death.28 It has shows a dynamic interaction with the dynein motor complex been proposed that the pool of Bcl-2-like molecules residing during autophagy induction.34 Ambra1 is bound, together with in the ER inhibits Beclin 1 function, thereby preventing Beclin 1 and Vps34, in an autophagy inactive state to the dynein autophagosome formation. Bcl-2/Beclin 1 complexes in the ER complex via a specific interaction with dynein light chains (DLCs) 1 are disrupted by JNK-mediated Bcl-2 phosphorylation permitting and 2 (Figure 1), which is mediated by two DLC-binding consensus autophagosome nucleation (Figure 1).29 To date, a large set of motifs (TQT) at the carboxy-terminal region of Ambra1 (Figure 2). signaling pathways have been described as converging on Beclin Upon autophagy induction, Ambra1 is released from the dynein 1 to modulate its activity and regulate autophagy (see He and complex upon ULK1-dependent phosphorylation and translocates Levine,20 Wirawan et al.,30 Abrahamsen et al.31 and Kang et al.32 to the ER together with Beclin 1-Vps34 to allow autophagosome for comprehensive reviews). 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 ROLE OF Ambra1 IN AUTOPHAGY confirmed that the Beclin 1 complex is sequestered to the dynein Ambra1 is a WD40-containing protein playing an important role in complex and showed that the proapoptotic BH3-only protein Bim the development of the central nervous system. Its functional is also involved in this pathway.35 Autophagic stimuli induce Bim deficiency in mouse embryos leads to neuroepithelial hyperplasia phosphorylation by JNK, which abrogates its binding with DLCs associated with autophagy impairment and excessive apoptotic and leads to the dissociation from Beclin 1. It would be interesting cell death.21 Ambra1 binds to Beclin 1 and stabilizes Beclin 1/ to clarify whether the proposed regulation of autophagy by Bim Vps34 complex, thus potentiating its lipid kinase activity and acts in a synergistic manner with that dependent on Ambra1, or promoting autophagosome formation21 (Figures 1 and 2). The whether there are two distinct pools of Beclin 1 complex at the interaction with Beclin 1 does not require the WD40 domain of level of cytoskeleton, regulated by different upstream pathways.35 Ambra1, but resides in a central region of the protein (aa 533– In addition to the dynein, Ambra1 was found to bind 780), which is also sufficient to prompt autophagy when dynamically to the mitochondrial pool of Bcl-2, further underlining overexpressed.21 On the other hand, Ambra1 interacts with a the relevance of subcellular localization in regulating its func- region of Beclin 1 adjacent to its BH3 domain, and, consistently, is tion.33 Under normal conditions, Bcl-2 docked a pool of Ambra1 at able to compete with the binding of Bcl-2 to Beclin 1.33 the mitochondria, by interacting with both the amino- and the Recent evidence highlighted that Ambra1 is not only a cofactor carboxy-terminal domains of the protein (Figures 1 and 2). After of Beclin 1 in favoring its kinase-associated activity, but also acts as autophagy induction, Ambra1 dissociates from Bcl-2 present on a crucial upstream regulator of autophagy initiation.33,34 In the mitochondrial membrane and increases its binding with a particular, changes in the subcellular localization of Ambra1 fraction of Beclin 1 to enhance Beclin 1-dependent autophagy. during autophagy induction appears to be important in regulating Consistent with this hypothesis, a reduction of Ambra1 colocaliza- autophagosome formation in mammalian cells. Indeed, Ambra1 tion with mitochondria in response to autophagy was observed,

Ambra1 partners and post-translational modifications

Parkin

Bcl-2 Beclin 1 Bcl-2

aa 1-532 aa 533-780 aa 796-1298 aa 51-90 aa 93-133 aa 135-175 TQT 1116 TQT 1104 WD40 WD40 WD40 Dynein aa 268-277 aa 592-639 domain binding Ser-rich Caspase Prolin-rich

K175 Cleavage

Ub PPP D482 PP P DDB1 DLC1 DLC2 Ub S369 S387 S444 S628 Y704 S1192 S372 S391 S458 S629 Y715 T1203 DDA1 Ub T373 S465 S639 Y726 S1204 Cullin S696 Y814 S1205 Ub S1211 4A/B T1238

Other Ambra1-interacting proteins

ATG4B ATG3 Nek9 Map1B USP50

Autophagy Cell cycle Cytoskeleton Ubiquitin/Proteasome

Figure 2. Ambra1 structural domains, post-translational modifications and interacting proteins. Schematic representation of Ambra1 protein describing structural domains, post-translational modifications and identified interacting proteins. Structural information was obtained from the Uniprot database.88 PMTs were obtained from Phosphosite database.89 Interacting proteins were described in the NCBI Gene database.90 P, phosphorylation; Ub, ubiquitination.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3311 – 3318 The role of Ambra1 in the balance of apoptosis and autophagy GM Fimia et al 3314 accompanied by an increased interaction between Ambra1 and autophagic proteins, such as Beclin 1, Atg4D and ATG5, are Beclin 1 in the ER fraction and a reciprocal decrease in binding cleaved by apoptotic executioner proteases and that the between Bcl-2 and Beclin 1 at this site (Figure 1). It remains to be inhibition of their cleavage favors the autophagy prosurvival elucidated which is the mechanism responsible for Ambra1/Bcl-2 functions and counteracts cell death (Figure 3).47–53 Notably, the dissociation and whether a pool of Ambra1 that remains cleaved products of these autophagic proteins were described to associated to the mitochondria is involved in the induction of acquire a new function by translocating to the mitochondria and autophagy directly from the mitochondrial outer membrane, a amplifying mitochondria-mediated apoptosis (Figure 3). Recently, proposed alternative site of autophagosome nucleation.36 another proautophagic factor, ATG12, has been reported to Notably, the binding of Bcl-2 to Ambra1 is not prevented by the contribute to the induction of the apoptotic process, despite not phosphorylation of Bcl-2 in its BH3 domain, at variance with that being a target of apoptotic proteases. Indeed, it was described observed with Beclin 1, thus suggesting that distinct mechanisms that Atg12 is required for an efficient induction of cell death by a may account for the release of Bcl-2 inhibition on Ambra1 and variety of apoptotic stimuli.54,55 This proapoptotic function is Beclin 1 during autophagy induction. Interestingly, the interaction independent of its role in autophagosome elongation, although it between Ambra1 and the mitochondrial Bcl-2 is disrupted by both relies on its ability to bind and inhibit Bcl-2 activity, through a BH3- autophagic and apoptotic stimuli, highlighting the intimate like domain55 (Figure 3). crosstalk between these processes. On the other hand, Hou and colleagues recently demonstrated An interesting recent report has shown that Ambra1 may also the autophagic degradation of the active caspase-8 enzyme have a role in a selective form of autophagy, that is, mitophagy. during TRAIL-induced autophagy, keeping the apoptotic response Indeed, Ambra1 interacts with Parkin, a protein that has a key role at bay (Figure 3).56 These results support the existence of cross- in the mitochondrial quality control by translocating to depolar- regulatory mechanisms between both the cell fate-determining ized mitochondria and inducing mitophagy.37 Interestingly, processes, so that only one process can prevail. prolonged mitochondrial depolarization strongly increases the As previously mentioned, the functional deficiency of Ambra1 in interaction of Parkin with Ambra1, which is recruited in a Parkin- mice causes defects in the development and embryonic death dependent manner to the perinuclear clusters of depolarized associated with autophagy impairment and a large number of mitochondria, where it activates the PtdIns3K complex supernumerary apoptotic cells.21,57 The latter aspect of this around these damaged mitochondria and contributes to their multifaceted phenotype prompted us to investigate more selective autophagic clearance. The ablation of Ambra1 does not deeply the role of Ambra1 during apoptosis.58,59 We found that affect Parkin translocation to depolarized mitochondria, but reduced levels of Ambra1 in a variety of cell lines lead to increased inhibits subsequent mitochondrial clearance. Conversely, susceptibility to different apoptotic stimuli. Moreover, apoptosis Ambra1 overexpression enhances the elimination of depolarized induction causes Ambra1 degradation that occurs in a caspase- mitochondria, but only in the presence of Parkin. 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 Ambra1 IN APOPTOSIS assay confirmed that Ambra1 is a substrate of different caspases The crosstalk between autophagy and apoptosis is highly and allowed us to identify D482 as a major caspase cleavage site in complex.38,39 Factors identified as activators of apoptosis can Ambra1. Significantly, a D482-A Ambra1 mutation prevents the often induce autophagy, whereas factors that negatively regulate formation of cleavage products in Ambra1-overexpressing cells apoptosis also inhibit autophagy induction. The well-known anti- and protects from apoptosis more efficiently than the wild-type apoptotic factor Bcl-2 is a key factor in this context. The pool of protein does. Differently from caspases, calpains completely Bcl-2 resident at the ER is able, indeed with the contribution of degrade Ambra1 in an in vitro assay, thus possibly explaining NAF-1 (nutrient deprivation autophagy factor-1), to negatively why stable endogenous Ambra1 cleavage fragments were not regulate the Beclin 1-dependent autophagic program.28,40 In observed accumulating in vivo upon apoptosis induction. The fact contrast, the mitochondrial pool of Bcl-2 has been proposed to that Ambra1 decline could be prevented by concomitant exert only an anti-apoptotic function41 despite mitochondria inhibition of calpains and caspases, and that a caspase- having been recently demonstrated to be an additional site uncleavable Ambra1 mutant is only partially resistant to for autophagosome formation36 and the further finding that staurosporine-induced degradation, seems to confirm that these Bcl-2/Ambra1 interaction occurs at mitochondria.33 On the other proteases may act in concert to ensure Ambra1 removal during hand, the overexpression of Bcl-2 promotes a protective apoptosis. effect against a wide range of apoptotic inducers. This The prosurvival role for Ambra1 is further demonstrated by the antiapoptotic action derives from the fact that Bcl-2 neutralizes enhanced resistance to apoptosis of Ambra1 overexpressing proapoptotic Bcl-2 family members such as Bax and Bak, as well as cerebellar granule neurons induced to die by trophic factor BH3-only proteins, thus preventing mitochondrial membrane withdrawal.33 permeabilization and consequent cell death.42 Similar to Bcl-2, Taken together, these data strengthen the emerging view that Flip (Flice inhibitory protein), an inhibitor of death receptor- under stressful cellular conditions prosurvival and prodeath mediated apoptosis, can also suppress autophagy by competing processes are concomitantly activated, and that autophagy with LC3 for Atg3 binding, thereby preventing Atg3-mediated impairment by apoptotic machinery could represent a ‘point of autophagosome elongation.43 no return’ toward death. 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- CROSSTALK BETWEEN AUTOPHAGY AND APOPTOSIS IN 2, Bcl-XL, Bad, Bim and Bmf.44–46 Once both processes are induced, CANCER the extent of the damage and the capability of restoring vital Autophagy is a rapidly expanding area of research, and multiple functions will determine the cell fate decision between death or links between deficient or excessive autophagy, oncogenesis, survival, by modulating the complex crosstalk between autophagy cancer therapy and resistance have been reported.60–62 During and apoptosis. Indeed, many functional interactions have been early oncogenesis, carcinogenic aberrations in oncogenes and recently described, which unveiled how these pathways are tumor suppressor genes frequently inhibit the autophagic mutually regulated through the modification of each other’s program, this feature being thought to contribute to the activity. On one hand, it has been demonstrated that crucial genomic instability relevant to oncogenesis and tumor

Oncogene (2013) 3311 – 3318 & 2013 Macmillan Publishers Limited The role of Ambra1 in the balance of apoptosis and autophagy GM Fimia et al 3315

Autophagy induction ΔΨ Apoptosis inhibition Atg4D Casp-8 Beclin1 Atg5 Atg 12 Ambra1

Casp-8 Survival

STRESS Autophagosome Calpains

Atg4D Caspases Beclin1 Proapoptotic Atg5 factors Apoptosis induction Ambra1

Atg Autophagy 12 Bax inhibition Bak

Beclin1 Bcl-2 Death Atg4D

Atg5 Bcl-2 BH3-only

Figure 3. Crosstalk between autophagy and apoptosis. Stress stimuli trigger the parallel induction of autophagy and apoptosis. The ﬁnal outcome (death or survival) depends on the complex network of molecular interactions occurring between these processes. Autophagy attempts to inhibit apoptosis by engulﬁng proapoptotic caspases, such as caspase 8, and damaged or depolarized (DC) mitochondria to prevent the release of cytochrome C. On the other hand, apoptosis dismantles the autophagy machinery by degrading Ambra1, Beclin 1, ATG 3 and ATG5 through caspases and calpains. Notably, proteolytic products of Beclin1, ATG 3 and ATG5 may then translocate to the mitochondrial outer membrane where they exhibit a proapoptotic activity.

DNA damage, Benign Tumors ROS, ER stress…

Low Autophagy

No Apoptosis Impaired Autophagy (p62 increase)

Necrosis Inflammation

Tumor Autophagy Apoptosis Initiation Sustained induction: induction: Autophagy Removal of damage Death of damaged cells

Malignant Tumors

Tumor Suppression

Figure 4. Dysregulation of autophagic and apoptotic pathways in cancer. Different kinds of intracellular damages, such as DNA mutations, accumulation of reactive oxygen species) or impairment of ER function (ER stress), promote the generation of mutant cells prone to transformation. Induction of autophagy and apoptosis blocks the progression of these cells to the tumor by removing damaged intracellular organelles or by killing the ‘abnormal’ cell, respectively. Concomitant inhibition of these processes contribute to tumor formation by increasing the inﬂammatory levels of the microenvironment surrounding the mutant cell, which further increases the probability to accumulate genetic or epigenetic changes. At the late stages of tumorigenesis, the residual basal autophagy of transformed cells is essential for the progression to metastasis, by ensuring survival in nutrient- and oxygen-limiting conditions. progression (Figure 4). Although the molecular mechanisms p62/SQSTM1 has been shown to be a direct link between mediating these events have not been fully determined, autophagy impairment and tumorigenesis.62–64 Moreover, the accumulation of the autophagic cargo-binding protein autophagy provides a protective function to limit necrotic cell

& 2013 Macmillan Publishers Limited Oncogene (2013) 3311 – 3318 The role of Ambra1 in the balance of apoptosis and autophagy GM Fimia et al 3316 death of transformed cells and consequent inflammation favoring vaccines, via the induction of immunogenic cell death.76 One of the tumor progression.65 hallmarks of immunogenic cell death is the release of ATP by cells Particularly important in this respect is the interplay taking place committed to undergoing apoptosis. Notably, knockdown of between Bcl-2 and the Beclin 1 complex. Alteration of Bcl-2 and essential autophagy-related genes (atg3, atg5, atg7 and becn1) Beclin 1 expression has been reported in a large number of human abolishes the preapoptotic secretion of ATP by various cancer cell tumors, underlying their important role in carcinogenesis.60,66 Indeed, lines undergoing immunogenic cell death. Accordingly, autophagy- Beclin 1 gene is monoallelically deleted in a subset of tumors of the proficient, but not autophagy-deficient, tumor cells treated breast, ovary and prostate.67 Moreover, mice heterozygous for the with immunogenic cell death inducers in vitro could induce a beclin 1 mutation are predisposed to multiple tumor types, including tumor-specific immune response in vivo. Altogether, these results lymphoma and liver cancer.68 Similarly, other members of Beclin 1 suggest that autophagy-incompetent tumor cells escape from complex, such as UVRAG and Bif-1, develop multiple spontaneous chemotherapy-induced immunosurveillance because they are cancers.69,70 Although direct evidence of a role of Ambra1 in unable to release ATP. Thus, autophagy is essential for the tumorigenesis has not been demonstrated yet, the described immunogenic release of ATP by dying cells; increased extracellular functional interactions of Ambra1 with the Beclin 1 complex ATP concentrations improve the efficacy of antineoplastic suggest that a dysregulation of Ambra1 function may contribute to chemotherapies when autophagy is disabled. 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 CONCLUDING REMARKS wouldbecrucialinthenearfuturetoelucidatethemolecular Taken as a whole, these data highlight the importance of mechanisms that link the autophagic function of Ambra1 to its ability understanding the mechanisms linking cellular stress/cell death to regulate cell growth, which may be impaired in the early steps of and autophagy in order to design novel therapeutic strategies oncogenesis when cells acquire higher proliferation rates. Moreover, based on the modulation of autophagy in cancer cells. A central Ambra1 may be indirectly involved in tumor formation by regulating question is whether the modulation of autophagy is a relevant the cell death/survival balance through its binding with the cell therapeutic strategy for the selective elimination of tumor cells. death repressor Bcl-2 and altering its ability to modulate the activity Many clinical trials are underway to test the effects of inhibiting or of proapoptotic and proautophagic factors. Indeed, Ambra1 can boosting autophagy as part of treatment regimens for various compete with both mitochondrial and ER-resident Bcl-2 to bind cancers. However, the real challenge now is to gain more 33 Beclin 1. Hence, either the overexpression or the absence of knowledge about the molecular mechanisms involved in autop- Ambra1 molecules can interfere with the Bcl2-binding partners and hagy and the particular deficiencies in these mechanisms decisive possibly with cell death regulation. for oncogenesis. In keeping with these assumptions, of particular Cellular stress occurring in established tumors, be it endogen- interest are recent data concerning the comprehensive character- ous (such as hypoxia or ER stress) or exogenous (such as ization of the interaction networks, as well as of post-translational conventional or targeted chemotherapy and radiotherapy), can modifications that regulate the autophagic pathway.77,78 In induce autophagy, which in turn constitutes a mechanism particular, as regards Ambra1, more than 20 different Ambra1 through which cancer cells protect themselves against stress phosphorylations have been reported, some of which are 4 and thus prolong their survival. Interestingly, only benign tumors modulated in a cell-cycle-regulated manner79,80 (Figure 2). More- developed in the liver of mosaically deleted ATG5 mice, over, Ambra1 has been shown to undergo ubiquitination and to suggesting that sustained levels of autophagy may be required interact with elements of the proteasome-mediated degradation 62 for progression beyond the benign state (Figure 4). system, especially with the E3 ligase Cullin 4 complex.81–84 The E3 Combined treatments of chemotherapeutic drugs and autop- ubiquitin ligases dictate substrate specificity, thus regulating the hagy inhibitors have been shown to increase the rate of apoptosis degradation or the activity of factors involved in a wide range of 71–73 in established tumors. However, there is a subset of cellular processes, including proliferation, differentiation and antitumoral agents that require a functional autophagic apoptosis.85 Dysregulation of E3 ubiquitin ligase activity has machinery to exert their cell death function. For example, been shown to contribute to oncogenesis through the 9 D -tetrahydrocannabinol (THC), the main active component of accumulation of oncoproteins and cell cycle regulators, or the marijuana, induces human glioma cell death through stimula- excessive degradation of tumor suppressors.86,87 In the future, it 74,75 tion of autophagy. THC induced ceramide accumulation and will be important to elucidate whether Ambra1’s interaction with eukaryotic translation initiation factor 2a phosphorylation and E3 ligase family members has a key role in the regulation of thereby activated an ER stress response that promoted autophagy autophagy and whether this function is altered during cellular via tribbles homolog 3-dependent (TRB3-dependent) inhibition transformation. 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 CONFLICT OF INTEREST antitumor action of cannabinoids in vivo. Autophagy preceded The authors declare no conflict of interest. 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 ACKNOWLEDGEMENTS their wild-type counterparts, autophagy-deficient cells underwent This work was supported by grants from the Italian Ministry of University FIRB, neither phosphatidylserine translocation to the outer leaflet of Compagnia di San Paolo to MP, the Ministry of Health of Italy ‘Ricerca Corrente’ and the plasma membrane nor increased production of reactive ‘Ricerca Finalizzata’ to MP and GMF, AIRC to MP and MC, and Telethon to GMF. The oxygen species in response to cannabinoid treatment. 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