Introduction

Macroautophagy (herein referred to as autophagy) is a mechanism that mediates the sequestration of intracellular entities within double-membraned vesicles, so-called autophagosomes, and their delivery to lysosomes for bulk degradation (He & Klionsky, 2009). Autophagosomes derive from so-called phagophores, membranous structures also known as ‘isolation membranes’ whose precise origin remains a matter of debate (Lamb et al, 2013). Indeed, the plasma membrane, endoplasmic reticulum (ER), Golgi apparatus, ER-Golgi intermediate compartment (ERGIC), and mitochondria have all been indicated as possible sources for phagophores (Lamb et al, 2013). Upon closure, autophagosomes fuse with lysosomes, forming socalled autolysosomes, and their cargo is exposed to the catalytic activity of lysosomal hydrolases (Mizushima & Komatsu, 2011). The degradation products of the autophagosomal cargo, which includes sugars, nucleosides/nucleotides, amino acids and fatty acids, can be transported back to the cytoplasm and presumably re-enter cellular metabolism (Fig 1) (Rabinowitz & White, 2010; Galluzzi et al, 2013). Of note, the molecular machinery that mediates autophagy is evolutionary conserved, and several components thereof have initially been characterized in yeast (He & Klionsky, 2009). In physiological scenarios, autophagy proceeds at basal levels, ensuring the continuous removal of superfluous, ectopic or damaged (and hence potentially dangerous) entities, including organelles and/or portions thereof (Green et al, 2011). Baseline autophagy mediates a key homeostatic function, constantly operating as an intracellular quality control system (Mizushima et al, 2008; Green et al, 2011).

 Moreover, the autophagic flux can be upregulated in response to a wide panel of stimuli, including (but not limited to) nutritional, metabolic, oxidative, pathogenic, genotoxic and proteotoxic cues (Kroemer et al, 2010). Often, stimulusinduced autophagy underlies and sustains an adaptive response to stress with cytoprotective functions (Kroemer et al, 2010; Mizushima & Komatsu, 2011). Indeed, the pharmacological or genetic inhibition of autophagy generally limits the ability of cells to cope with stress and restore homeostasis (Mizushima et al, 2008; Kroemer et al, 2010). This said, regulated instances of cell death that causally depend on the autophagic machinery have been described (Denton et al, 2009; Denton et al, 2012b; Liu et al, 2013b; Galluzzi et al, 2015). The detailed discussion of such forms of autophagic cell death, however, is beyond the scope of this review. Autophagy is tightly regulated. The best characterized repressor of autophagic responses is mechanistic target of rapamycin (MTOR) complex I (MTORCI) (Laplante & Sabatini, 2012). Thus, several inducers of autophagy operate by triggering signal transduction cascades that result in the inhibition of MTORCI (Inoki et al, 2012). Among other effects, this allows for the activation of several proteins that are crucial for the initiation of autophagic responses, such as unc-51-like autophagy-activating kinase 1 (ULK1, the mammalian ortholog of yeast Atg1) and autophagy-related 13 (ATG13) (Hosokawa et al, 2009; Nazio et al, 2013). 

A major inhibitor of MTORCI is protein kinase, AMP-activated (PRKA, best known as AMPK), which is sensitive to declining ATP/AMP ratios (Mihaylova & Shaw, 2011). Besides inhibiting the catalytic activity of MTORCI, AMPK directly stimulates autophagy by phosphorylating ULK1 as well as phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, best known as VPS34) and Beclin 1 (BECN1, the mammalian ortholog of yeast Atg6), two components of a multiprotein complex that produces a lipid that is essential for the biogenesis of autophagosomes, namely phosphatidylinositol 3-phosphate (Egan et al, 2011; Zhao & Klionsky, 2011; Kim et al, 2013). Autophagy also critically relies on two ubiquitin-like conjugation systems, both of which involve ATG7 (Mizushima, 2007). These systems catalyze the covalent linkage of ATG5 to ATG12 and ATG16-like 1 (ATG16L1), and that of phosphatidylethanolamine to proteins of the microtubule-associated protein 1 light chain 3 (MAP1LC3, best known as LC3) family, including MAP1LC3B (LC3B, the mammalian ortholog of yeast Atg8) (Mizushima, 2007). A detailed discussion of additional factors that are involved in the control and execution of autophagic responses can be found in Boya et al (2013). Importantly, autophagosomes can either take up intracellular material in a relatively non-selective manner or deliver very specific portions of the cytoplasm to degradation, mainly depending on the initiating stimulus (Weidberg et al, 2011; Stolz et al, 2014). 

Thus, while non-selective forms of autophagy normally develop in response to cell-wide alterations, most often of a metabolic nature, highly targeted autophagic responses follow specific perturbations of intracellular homeostasis, such as the accumulation of permeabilized mitochondria (mitophagy), the formation of protein aggregates (aggrephagy), and pathogen invasion (xenophagy) (Okamoto, 2014; Randow & Youle, 2014). Several receptors participate in the selective recognition and recruitment of autophagosomal cargoes in the course of targeted autophagic responses (Rogov et al, 2014; Stolz et al, 2014). The autophagy receptor best characterized to date, that is, sequestosome 1 (SQSTM1, best known as p62), recruits ubiquitinated proteins to autophagosomes by virtue of an ubiquitinassociated (UBA) and a LC3-binding domain (Pankiv et al, 2007). Owing to its key role in the preservation of intracellular homeostasis, autophagy constitutes a barrier against various degenerative processes that may affect healthy cells, including malignant transformation. Thus, autophagy mediates oncosuppressive effects. Accordingly, proteins with bona fide oncogenic potential inhibit autophagy, while many proteins that prevent malignant transformation stimulate autophagic responses (Morselli et al, 2011). Moreover, autophagy is involved in several aspects of anticancer immunosurveillance, that is, the process whereby the immune system constantly eliminates potentially tumorigenic cells before they establish malignant lesions (Ma et al, 2013).

 Moreover, carcinogen-induced fibrosarcomas appear at an accelerated pace in autophagy-deficient Atg4c
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mice (Marino et al, 2007), as do KRASG12D-driven and BRAFV600E-driven lung carcinomas in mice bearing lung-restricted Atg5 or Atg7 deletions, respectively (Strohecker et al, 2013; Rao et al, 2014). The pancreas-specific knockout of Atg5 or Atg7 also precipitates the emergence of KRASG12D-driven pre-malignant pancreatic lesions (Rosenfeldt et al, 2013; Yang et al, 2014). Several mechanisms can explain, at least in part, the oncosuppressive functions of autophagy. Proficient autophagic responses may suppress the accumulation of genetic and genomic defects that accompanies malignant transformation, through a variety of mechanisms. Reactive oxygen species (ROS) are highly genotoxic, and autophagy prevents their overproduction by removing dysfunctional mitochondria (Green et al, 2011; Takahashi et al, 2013) as well as redox-active aggregates of ubiquitinated proteins (Komatsu et al, 2007; Mathew et al, 2009). In addition, autophagic responses have been involved in the disposal of micronuclei arising upon perturbation of the cell cycle (Rello-Varona et al, 2012), in the degradation of retrotransposing RNAs (Guo et al, 2014), as well as in the control of the levels of ras homolog family member A (RHOA), a small GTPase involved in cytokinesis (Belaid et al, 2013). Finally, various components of the autophagic machinery appear to be required for cells to mount adequate responses to genotoxic stress (KarantzaWadsworth et al, 2007; Mathew et al, 2007; Park et al, 2014). 

This said, the precise mechanisms underlying such genome-stabilizing effects remain elusive, implying that the impact of autophagy on DNA-damage responses may be indirect. Further investigation is required to shed light on this possibility. Autophagy is intimately implicated in the maintenance of physiological metabolic homeostasis (Galluzzi et al, 2014; Kenific & Debnath, 2015). Malignant transformation generally occurs along with a shift from a predominantly catabolic consumption of glycolysis-derived pyruvate by oxidative phosphorylation to a metabolic pattern in which: (1) glucose uptake is significantly augmented to sustain anabolic reactions and antioxidant defenses, (2) mitochondrial respiration remains high to satisfy increased energy demands; and (3) several amino acids, including glutamine and serine, become essential as a means to cope with exacerbated metabolic functions (Hanahan & Weinberg, 2011; Galluzzi et al, 2013). Autophagy preserves optimal bioenergetic functions by ensuring the removal of dysfunctional mitochondria (Green et al, 2011), de facto counteracting the metabolic rewiring that accompanies malignant transformation. Moreover, the autophagic degradation of p62 participates in a feedback circuitry that regulates MTORCI activation in response to nutrient availability (Linares et al, 2013; Valencia et al, 2014). Autophagy appears to ensure the maintenance of normal stem cells. This is particularly relevant for hematological malignancies, which are normally characterized by changes in proliferation or differentiation potential that alter the delicate equilibrium between toti-, pluri- and unipotent precursors in the bone marrow (Greim et al, 2014). 

The ablation of Atg7 in murine hematopoietic stem cells (HSCs) has been shown to disrupt tissue architecture, eventually resulting in the expansion of a population of bone marrow progenitor cells with neoplastic features (Mortensen et al, 2011). Along similar lines, the tissue-specific deletion of the gene coding for the ULK1 interactor RB1-inducible coiled-coil 1 (RB1CC1, best known as FIP200) alters the fetal HSC compartment in mice, resulting in severe anemia and perinatal lethality (Liu et al, 2010). Interestingly, murine Rb1cc1
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HSCs do not exhibit increased rates of apoptosis, but an accrued proliferative capacity (Liu et al, 2010). The deletion of Rb1cc1 in murine neuronal stem cells (NSCs) also causes a functional impairment that compromises postnatal neuronal differentiation (Wang et al, 2013). However, this effect appears to stem from the failure of murine Rb1cc1
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HNCs to control redox homeostasis, resulting in the activation of a tumor protein p53 (TP53)-dependent apoptotic response (Wang et al, 2013).Finally, Becn1+/
mice display an expansion of progenitor-like mammary epithelial cells (Cicchini et al, 2014). Of note, autophagy also appears to be required for the preservation of normal stem cell compartments in the human system. Indeed, human hematopoietic, dermal, and epidermal stem cells transfected with a short-hairpin RNA (shRNA) specific for ATG5 lose their ability to self-renew while differentiating into neutrophils, fibroblasts, and keratinocytes, respectively (Salemi et al, 2012). 

It has been proposed that autophagy contributes to oncogeneinduced cell death or oncogene-induced senescence, two fundamental oncosuppressive mechanisms. The activation of various oncogenes imposes indeed a significant stress on healthy cells, a situation that is normally aborted through the execution of a cell death program (Elgendy et al, 2011), or upon the establishment of permanent proliferative arrest (cell senescence) that engages the innate arm of the immune system (Iannello et al, 2013). The partial depletion of ATG5, ATG7 or BECN1 limited the demise of human ovarian cancer cells pharmacologically stimulated to express HRASG12V from an inducible construct (Elgendy et al, 2011). Similarly, shRNAs specific for ATG5 or ATG7 prevented oncogene-induced senescence in primary human melanocytes or human diploid fibroblasts (HDFs) expressing BRAFV600E or HRASG12V (Young et al, 2009; Liu et al, 2013a). Accordingly, the overexpression of the ULK1 homolog ULK3 was sufficient to limit the proliferative potential of HDFs while promoting autophagy (Young et al, 2009). Moreover, both pharmacological inhibitors of autophagy and small-interfering RNAs targeting ATG5, ATG7 or BECN1 prevented spontaneous senescence in HDFs while preventing the degradation of an endogenous, dominant-negative TP53 variant (Horikawa et al, 2014). 

Finally, ectopic ATG5 expression reduced the colony-forming ability of melanoma cell lines normally characterized by low ATG5 levels, an effect that could be reproduced by the administration of autophagy inducers (Liu et al, 2013a). Apparently at odds with these results, HRASG12V fails to induce senescence in mouse embryonic fibroblasts (MEFs) lacking transformation-related protein 53 binding protein 2 (Trp53bp2), correlating with the stabilization of Atg5/Atg12 complexes and consequent upregulation of the autophagic flux. In line with this notion, ectopic expression of Atg5 prevented Trp53bp2-sufficient MEFs from entering senescence upon overexpression of HRASG12V (Wang et al, 2012b). Thus, while in some cells autophagy appears to inhibit malignant transformation by favoring oncogene-induced senescence, this may not be a general mechanism of autophagymediated oncosuppression. It has been suggested that autophagy is involved in the degradation of oncogenic proteins, including mutant (but not wild-type) TP53 (Rodriguez et al, 2012; Choudhury et al, 2013; Garufi et al,2014), p62 (Duran et al, 2008; Mathew et al, 2009; Ling et al, 2012), PML-RARA (Isakson et al, 2010; Wang et al, 2011), and BCR-ABL1 (Goussetis et al, 2012). Mutant TP53 often accumulates in neoplastic cells and operates as a dominant-negative factor, thereby interfering with the oncosuppressive function of the wildtype protein (de Vries et al, 2002). Cancer cells depleted of ULK1, BECN1 or ATG5 tend to accumulate increased amounts of mutant TP53, whereas the transgene-driven overexpression of BECN1 or ATG5 results in mutant TP53 depletion (Choudhury et al, 2013).

 Such an autophagy-dependent degradation of mutant TP53 would therefore restore the ability of wild-type TP53 to inhibit malignant transformation, at least in some settings. It is worth noting that both ATG5 and ATG7 have been involved in the regulation of TP53- dependent adaptive responses to stress (Lee et al, 2012; Salemi et al, 2012). However, this activity appears to be independent of autophagy, at least in the case of ATG7 (Lee et al, 2012). Interestingly, p62 itself has been ascribed with potentially oncogenic functions, including a key role in the transduction of RAS-elicited signals as well as in the activation of a feedforward loop involving the cytoprotective transcription factor NF-jB driven by oncogenic stress (Duran et al, 2008; Mathew et al, 2009; Takamura et al, 2011; Ling et al, 2012). Autophagy may therefore inhibit oncogenesis by limiting p62 availability (Mathew et al, 2009), at least in some settings. The t(9;22)(q34;q11) translocation is found in about 90% of chronic myeloid leukemia patients, resulting in the synthesis of a fusion protein that involves breakpoint cluster region (BCR) and ABL proto-oncogene 1 (ABL1) (Ben-Neriah et al, 1986). BCR-ABL1 is a constitutively active kinase and is etiologically involved in leukemogenesis, as demonstrated by the outstanding clinical success of imatinib mesylate, a BCR-ABL1-targeting kinase inhibitor (Druker et al, 2001). Arsenic trioxide, a chemotherapeutic agent commonly employed against various forms of leukemia, appears to trigger the p62-dependent and cathepsin B-dependent degradation of BCR-ABL1 in leukemic progenitors (Goussetis et al, 2012). 

In line with this notion, the pharmacological or genetic inhibition of autophagy or cathepsin B reportedly limits the antileukemic potential of arsenic trioxide (Goussetis et al, 2012). The t(15;17)(q22;q21) translocation can be documented in 95% of promyelocytic leukemia cases, resulting in the expression of a chimera that involves promyelocytic leukemia (PML) and retinoic acid receptor, alpha (RARA) (Goddard et al, 1991). PML-RARA blocks normal retinoic aciddependent myeloid differentiation, de facto driving leukemogenesis (Rousselot et al, 1994). Patients expressing PML-RARA generally benefit from the administration of all-trans retinoic acid (ATRA), resulting in PML-RARA degradation and restored myeloid differentiation (Wang et al, 2011). Pharmacological and genetic evidence suggests that autophagy is implicated in both ATRA- and arsenic trioxide-driven PML-RARA degradation (Isakson et al, 2010; Wang et al, 2011). Further experimentation is required to understand whether autophagy degrades potentially oncogenic proteins in cells not exposed to chemotherapeutic agents. Autophagy is implicated in immune responses that prevent the establishment and proliferation of malignant cells (Ma et al, 2013). At least in some circumstances, dying malignant cells are capable of recruiting antigen-presenting cells (APCs) and other cellular components of the immune system, resulting in the elicitation of innate and/or adaptive antitumor immune responses (Deretic et al, 2013; Kroemer et al, 2013).

 On the one hand, autophagic responses are required for dying neoplastic cells to release ATP in optimal amounts, which not only recruits APCs through purinergic receptor P2Y, G-protein coupled, 2 (P2RY2), but also activates them to release immunostimulatory chemokines through purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7) (Michaud et al, 2011). On the other hand, autophagy in immune cells is implicated in several steps of both adaptive and innate immune responses (Ma et al, 2013). Thus, both cancer cell-intrinsic and systemic defects in autophagy may prevent the host immune system to properly recognize and eliminate pre-malignant and malignant cells. Autophagy mediates potent anti-inflammatory effects (Deretic et al, 2013). At least in some cases, malignant transformation is stimulated by an inflammatory microenvironment, which contains high amounts of potentially genotoxic ROS as well as various mitogenic cytokines (Coussens et al, 2013).

Proficient autophagic responses limit inflammation as: (1) they efficiently dispose of the so-called inflammasomes (the supramolecular platforms that are responsible for the maturation and secretion of pro-inflammatory interleukin-1b and interleukin-18), as well as damaged mitochondria, which would otherwise release endogenous inflammasome activators (Nakahira et al, 2011; Zitvogel et al, 2012); (2) they are linked to the inhibition of pro-inflammatory signals delivered by some pattern recognition receptors, such as RIG-I-like receptors (Jounai et al, 2007); (3) they limit the abundance of B-cell CLL/ lymphoma 10 (BCL10), a protein involved in pro-inflammatory NF-jB signaling (Paul et al, 2012); (4) they are connected to the inhibition of transmembrane protein 173 (TM173, best known as STING), a pattern recognition receptor involved in the delivery of pro-inflammatory cues in response to cytosolic nucleic acids (Saitoh et al, 2009).  Finally, autophagy may suppress carcinogenesis owing to its key role in the first line of defense against viral and bacterial infection (Deretic et al, 2013). Indeed, several potentially carcinogenic pathogens potently activate autophagy upon infection.

 These pathogens include hepatitis B virus (which promotes hepatocellular carcinoma), human herpesvirus 8 (which causes Kaposi’s sarcoma and contributes to the pathogenesis of primary effusion lymphoma and multicentric Castleman’s disease), human papillomavirus type 16 and 18 (HPV-16 and HPV-18, which cause cervical carcinoma), Epstein–Barr virus and Helicobacter pylori (both of which are associated with gastric carcinoma), Streptococcus bovis (which causes colorectal carcinoma), Salmonella enterica (which is associated with an increased incidence of Crohn’s disease, hence sustaining colorectal carcinogenesis, and gallbladder carcinoma), as well as Chlamydia pneumoniae (an etiological determinant in some forms of lung cancer) (Nakagawa et al, 2004; Travassos et al, 2010; Yasir et al, 2011; Conway et al, 2013; Griffin et al, 2013; Zhang et al, 2014). Such a xenophagic response is required for the rapid clearance of intracellular pathogens as well as for the stimulation of pathogen-specific immune responses (Deretic et al, 2013; Ma et al, 2013). Accordingly, epithelial cells bearing molecular defects in the autophagic machinery, such as those provoked by Crohn’s diseaseassociated point mutations in ATG16L1 and nucleotide-binding oligomerization domain containing 2 (NOD2) (Lassen et al, 2014), are more susceptible to infection by intracellular pathogens than their wild-type counterparts. In line with this notion, reduced levels of autophagic markers including BECN1 have recently been correlated with HPV-16 and HPV-18 infection in a cohort of cervical carcinoma patients (Wang et al, 2014).