Ambra1 at the crossroad between autophagy and cell death

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Abstract

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.

Autophagy regulation

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).

Figure 1
figure1

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.

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

Figure 2
figure2

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.

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).

Figure 3
figure3

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 (ΔΨ) 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.

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

Figure 4
figure4

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.

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.

Concluding remarks

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.

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Acknowledgements

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.

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Correspondence to M Piacentini.

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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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Keywords

  • Ambra1
  • Bcl-2
  • Beclin 1
  • caspases
  • calpains
  • apoptosis

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