Autophagy function and its relationship to pathology

Review article Received: 25 August 2016,

Revised: 30 August 2016,

Accepted: 30 August 2016

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3393

Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity† Dayton M. Petibonea*, Waqar Majeedb and Daniel A. Cascianob ABSTRACT: Autophagy is a cellular process that facilitates nutrient turnover and removal of expended macromolecules and organelles to maintain homeostasis. The recycling of cytosolic macromolecules and damaged organelles by autophagosomes occurs through the lysosomal degradation pathway. Autophagy can also be upregulated as a prosurvival pathway in response to stress stimuli such as starvation, hypoxia or cell damage. Over the last two decades, there has been a surge in research revealing the basic molecular mechanisms of autophagy in mammalian cells. A corollary of an advanced understanding of autophagy has been a concurrent expansion of research into understanding autophagic function and dysfunction in pathology. Recent studies have revealed a pivotal role for autophagy in drug toxicity, and for utilizing autophagic components as diagnostic markers and therapeutic targets in treating disease and cancer. In this review, advances in understanding the molecular basis of mammalian autophagy, methods used to induce and evaluate autophagy, and the diverse interactions between autophagy and drug toxicity, disease progression and carcinogenesis are discussed. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: autophagy; drug toxicity; amphiphilic cationic drugs; phospholipidosis; disease progression; carcinogenesis; clinical applications

Introduction Recycling of cellular macromolecules and organelles is accomplished through three autophagic pathways: (1) macroautophagy involving autophagosomes; (2) microautophagy involving invagination of the lysosomal membrane to engulf cytoplasmic components; and (3) chaperone-mediated autophagy, which delivers cytosolic proteins to lysosomes for degradation. Macroautophagy (autophagy, henceforth) in mammals is the focus of this review. Autophagy is a fundamental cell biology pathway involving sequestration of cytoplasmic components into double-membrane vesicles, called autophagosomes, which are delivered to lysosomes for degradation and recycling nutrients (Fig. 1). Autophagy is a dynamic intracellular recycling system for cytoplasmic components, which despite its relative simplicity and only involving a limited set of genes (approximately 32 core genes in mammals), has a significant effect in maintaining healthy cell physiology (Choi et al., 2013). Autophagy functions at a basal state in cells under ordinary conditions, but can be upregulated in response to environmental stressors or signaling stimuli. Upregulation of autophagy can occur through numerous physiological stimuli such as starvation, amino acid depletion, hypoxia, cell overcrowding, oxidative stress, pathogens or through several pharmacological agents, of which rapamycin (sirolimus) is the most commonly used (Mizushima et al., 2010). Through autophagic degradation and recycling activities, basal autophagy provides a pathway for normal cell growth by balancing the protein synthesis and organelle biogenesis activities of a cell with protein degradation and organelle turnover (Qu et al., 2003). Cytoplasmic materials recycled by autophagosomes can occur in either a selective or a non-selective manner. Under starvation conditions, bulk degradation primarily occurs in a non-selective fashion (Stolz et al., 2014). In other stress

J. Appl. Toxicol. 2016

situations such as cell damage, however, selective autophagy of protein aggregates (aggresomes) or damaged organelles largely occurs through targeting of these polyubiquitinated cytoplasmic components for degradation (Kirkin et al., 2009b). The size of autophagosomes is also dynamic and flexible, thus providing the ability to vary with the type of cargo engulfed. Nanometer scale autophagosomes can contain cytoplasmic materials such as macromolecules, while micrometer scale autophagosomes in selective autophagy can contain organelles, organelle fragments or bacteria ( Jin & Klionsky, 2014; Stolz et al., 2014). As a prosurvival mechanism, upregulated autophagy removes damaged cytoplasmic components and provides metabolic precursors to cells in response to stress stimuli. Although autophagy is often observed during apoptosis, it is generally accepted that autophagy is a “programmed survival” mechanism that rarely (if ever) is a mechanism of cell death (Choi et al., 2013; Kroemer & Levine, 2008). Yet, autophagy and apoptosis are intrinsically linked pathways and a role for autophagy in cell death is debatable (Kroemer & Levine, 2008).

*Correspondence to: Dayton Petibone, National Center for Toxicological Research, US FDA, Division of Genetic and Molecular Toxicology, Jefferson, AR 72079, USA. E-mail: [email protected] † Disclaimer: The views presented in this article are those of the authors and do not necessarily reflect those of the U.S. Food and Drug Administration. a National Center for Toxicological Research, US FDA, Division of Genetic and Molecular Toxicology, Jefferson, AR, 72079, USA b Center of Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, 72204, USA

Copyright © 2016 John Wiley & Sons, Ltd.

D. M. Petibone et al.

Figure 1. Autophagy is initiated following inhibition of mTOR through starvation or rapamycin exposure. This releases mTOR-mediated repression of the ULK complex, which is then free to phosphorylate Beclin 1, dissociating it from BCL2. Beclin 1 is necessary for initiating the PtdIns3k complex along with p150, VPS34 and ATG14L, and subsequently joined by other proteins. The PtdIns3k complex initiates nucleation of the phagophore, consisting of a double membrane and autophagosomal LC3 and gamma-aminobutyric acid receptor-associated proteins. The phagophore elongates around cytoplasmic components; some such as polyubiquitinated aggresomes are targeted for engulfment by the p62 protein. The phagophore then closes to form a mature autophagosome. Next, the autophagosomes fuse with lysosomes to form an autolysosome. However, if chloroquine is present, it can block autolysosome acidification preventing autophagic flux. Finally, the autophagosome inner membrane, along with its cytoplasmic components, is degraded by acid hydrolases. Autophagic flux is completed by efflux of nutrients back into the cytoplasm through permeases in the autolysosomes. mTOR, mechanistic (or mammalian) target of rapamycin; PtdIns3k, class III phosphatidylinositol 3–kinase; ULK, unc-51-like kinase.

Molecular mechanisms of mammalian autophagy While much of the early ultrastructural and molecular work characterizing autophagy was performed in yeast, this review is focused primarily on mammalian autophagy. There are five stages of autophagy categorized as initiation, elongation, membrane closure, maturation and degradation of cytoplasmic cargo. The four core molecular groups essential for autophagosome progression through the five stages of autophagy (Fig. 2). These core groups are the unc-51-like kinase (ULK) complex, the class II phosphatidylinositol 3–kinase (PtdIns3k) complex, the two ATG12ATG5-ATG16L and LC3 ubiquitin-like protein conjugation complexes, and the ATG9 and VMP1 transmembrane proteins involved in vesicle trafficking and Beclin 1 interaction, respectively (Meijer & Codogno, 2009; Papackova & Cahova, 2014; Xie & Klionsky, 2007). Initiation The most studied mechanism for autophagy induction in mammalian cells occurs through inhibition of the mechanistic (or mammalian) target of rapamycin (mTOR) (Yang et al., 2013). A potent suppressor of autophagy, the mTOR protein along with RAPTOR and LST8 proteins are part of the mTOR complex 1 (mTORC1) that conducts sensing for nutrient, particularly amino acids, availability

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to regulate protein translation, cell growth and proliferation (Guertin & Sabatini, 2007; Nobukuni et al., 2005). Currently, it is not fully understood how cells can sense amino acid levels and communicate with mTOR. However, potential candidates include involvement of the tRNA-binding protein kinase, GCN2, the VPS34 PtdIns3k, and the key autophagy regulator, Beclin 1 (Byfield et al., 2005; Nobukuni et al., 2005; Talloczy et al., 2002). By targeting the ULK complex, the mTORC1 complex suppresses autophagy through its kinase activity (Hosokawa et al., 2009a). The ULK complex, comprised of ULK1, ULK2, ATG13, ATG101 and FIP200 proteins, is a key initiator of autophagy (Fig. 2a) (Ganley et al., 2009). FIP200 is a scaffold protein that binds to ULK1 and ULK2 and is required for formation of the pre-autophagosome membrane, called the phagophore (Hara et al., 2008). ATG13 interacts with ULK1/2 and FIP200. Knockdown of the ATG13 protein has been shown to inhibit autophagosome formation ( Jung et al., 2009), while the ATG101 protein functions to protect ATG13 from proteasomal degradation (Hosokawa et al., 2009b). Several phosphorylation events are involved in the regulation of the ULK complex. Inactivation of mTORC1 by nutrient deprivation dissociates it from the ULK complex, thus allowing ULK1/2-mediated phosphorylation of FIP200 ( Jung et al., 2009). This allows the ULK complex to phosphorylate Beclin 1, releasing it from BCL2 suppression to form the PtdIns3k complex and initiate phagophore nucleation (Russell et al., 2013).



Mammalian Autophagy

Figure 2. (a) mTORC suppression of ULK1 and ULK2 in the ULK complex is inactivated during starvation, hypoxia, energy depletion or following treatment with rapamycin. Release from mTORC frees ULK1/2 in the ULK complex to phosphorylate ATG13 and FIP200. The FIP200 kinase then phosphorylates Beclin 1 dissociating it from BCL2. (b) Following dissociation from BCL2, Beclin 1 recruits autophagic proteins p150, ATG14L, VPS34 and Ambra1 (complex I) to the omegasome (see text) to form the PtdIns3k complex to initiate phagophore nucleation and incorporate phosphatidylinositol 3-phosphate signaling phospholipids into expanding phagophore. PtdIns3k complex II containing UVRAG and BIF1 is thought to mediate endosome trafficking and possibly maturation of autophagosomes. Rubicon can also bind UVRAG in complex II and inhibit autophagy. (c) There are two transmembrane proteins required for autophagosome formation in mammals, ATG9 and VMP1 that might function in delivering membrane to the nascent autophagosome and recruiting Beclin 1 and associated PtdIns3k proteins to the phagophore assembly site, respectively. (d) Two lipid conjugation systems are involved in mammalian autophagy, the LC3 system and the ATG16L complex that conjugate phosphatidylethanolamine to the ubiquitin-like LC3 proteins. ATG4 cleaves the C-terminus of LC3 proteins to reveal a glycine residue of cytosolic LC3-I, and the ATG3 and ATG7 proteins conjugate the phosphatidylethanolamine to LC3-I to form the autophagosome membrane-bound LC3-II variant. mTORC, mechanistic (or mammalian) target of rapamycin complex; PE, phosphatidylethanolamine; PtdIns3k, class III phosphatidylinositol 3–kinase; ULK, unc-51-like kinase.

Phagophore nucleation Most of the proteins involved in assembling the autophagosome are recruited to the phagophore assembly site, and it is generally accepted that de novo phagophore assembly originates from nucleation on pre-existing membranes. An omegasome is a membranous structure enriched with phosphatidylinositol 3-phosphate (PI3P) produced on an existing organelle located near the phagophore assembly site. Omegasomes were originally identified as associated with the endoplasmic reticulum (ER) (Axe et al., 2008). It does not appear, however, that phagophore assembly site formation necessitates a specific location for an omegasome within mammalian cells. At the phagophore assembly site and near the omegasome, nucleation of a phagophore is initiated from the Beclin 1-VPS34-ATG14L-p150 platform, the stable core of a PtdIns3k complex (Axe et al., 2008; He et al., 2015; Itakura et al., 2008; Kihara et al., 2001; Russell et al., 2013; Tassa et al., 2003). The core PtdIns3k complex can then form either complex I, with association of Ambra 1 involved in phagophore nucleation, or complex II, with association of UVRAG involved in late autophagosome maturation (Fig. 2b) (He & Levine, 2010; Itakura et al., 2008). Rubicon can bind to UVRAG in PtdIns3k complex II and serves to inhibit autophagy (Matsunaga et al., 2009).


Nucleation of the phagophore is thought to occur following PtdIns3k generation of PI3P signaling molecules on an omegasome (Axe et al., 2008). The PI3P molecules recruit WIPI2 and DFCP1 proteins to the phagophore assembly site, which signals autophagy related (ATG) proteins that function to elongate further the omegasome into a phagophore (Polson et al., 2010). The PtdIns3k complex kinase activity continues to produce PI3P signaling molecules that recruit molecular modeling proteins to add lipidated LC3-II proteins and additional phospholipids to the expanding phagophore (Devereaux et al., 2013). Evidence from a 14 C-methionine- and 3H-glycerol-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine study supports upregulation of de novo phospholipids biogenesis in the ER during autophagy (Girardi et al., 2011). However, the precise lipid source, their composition and the membrane modeling proteins involved in autophagosome formation have not yet been elucidated (Carlsson & Simonsen, 2015).

Phagophore elongation and closure As previously mentioned, expansion and curvature of the phagophore membrane involves incorporation of de novo



D. M. Petibone et al. synthesized lipids and lipidated proteins. However, evidence from eukaryote models suggest the phagophore membrane expanding from the phagophore assembly site also incorporates existing membranes from ER complexes and other organelles such as the Golgi body, the plasma membrane and endosomes, into its own expanding membrane (Ge et al., 2013; Graef et al., 2013; Longatti et al., 2012; Ravikumar et al., 2010; van der Vaart et al., 2010). There are two transmembrane proteins essential to autophagy, ATG9 and VMP1, which contribute to the elongating phagophore membrane (Fig. 2c). Data suggest that ATG9 might function by delivery of membrane segments to the nascent phagophore (Feng et al., 2016). ATG9 is cycled between the phagophore assembly site and peripheral sites to transport these membrane segments to the elongating phagophore (Feng et al., 2016). The VMP1 protein co-localizes with LC3 and Beclin 1 to mediate interaction with TP53INP2 during autophagy induction (Nowak et al., 2009; Ropolo et al., 2007), and might function by recruiting Beclin 1 to the phagophore assembly site (Molejon et al., 2013). The VMP1 protein has also been shown to recruit ATG5, part of the ATG16L complex involved in LC3 lipidation, as part of phagophore elongation (Koyama-Honda et al., 2013). Elongation of the phagophore membrane is regulated by two systems involving ubiquitin-like conjugation proteins: the ATG12– ATG5–ATG16L1 complex and the LC3 proteins (Fig. 2d). There are five reported members of the LC3 protein family, which consists of LC3A (variant 1 and variant 2), LC3B, LC3B2 and LC3C, all having a similar function. Of these proteins, LC3B is most widely used as a marker to study autophagy (Bai et al., 2012; Koukourakis et al., 2015). The LC3 family proteins have three isoforms: LC3; LC3-I; and LC3-II. The LC3 isoform has its C-terminus processed to include a glycine residue to produce LC3-I. Next the LC3 I is lipidated through conjugation with a phosphatidylethanolamine to produce LC3-II (Hamasaki et al., 2013; Ichimura et al., 2000; Mizushima et al., 2011). The LC3-II proteins are incorporated into both inner and outer phagophore membranes, but are removed from the outer membrane before fusion with lysosomes (Yang & Klionsky, 2010). ATG12 is an E3-like ligase covalently bound to ATG5 by ATG7 and ATG10, and the ATG12ATG5 heterodimer non-covalently interacts with ATG16L to form the ATG16L complex (Fig. 2d) (Hanada et al., 2007). The ATG16L complex can facilitate lipidation of LC3 in a site-specific fashion in the expanding phagophore (Fujita et al., 2008). The phagophore double membrane expansion continues to surround cytoplasmic cargo including ER segments, mitochondria, aggresomes and peroxisomes in both non-selective and selective autophagy (Mizushima, 2007). Selective autophagy occurs primarily through p62 proteins that target polyubiquitinated cytoplasmic components and bind autophagosome membranespecific LC3-II proteins to facilitate sequestration (Lippai & Low, 2014; Stolz et al., 2014). Other proteins that enable selective autophagy include NIX binding mitochondria (mitophagy), NBR1 targeting ubiquitinated peroxisomes (pexophagy) and aggresomes (aggrepathy), and OPTN and NDP52 proteins targeting bacteria (xenophagy) for degradation (Kirkin et al., 2009a,b; Mostowy et al., 2011; Stolz et al., 2014; Wild et al., 2011; Zhang & Ney, 2009). Elongation of the autophagosome membrane with either selective or non-selective sequestration of cytosolic components is followed by closure of the membranes to form a mature autophagosome (He et al., 2015). Another family of proteins associated with the autophagosome membrane is ubiquitin-like gamma-aminobutyric acid receptor-associated proteins


(GABARAP) that consists of two homologs, GABARAP and GABARAPL1 (Slobodkin & Elazar, 2013; Weidberg et al., 2010). While the LC3 proteins are involved in expansion of the phagophore, GABARAP functions primarily in late autophagosome maturation. Similar to LC3 proteins, GABARAP exist in two additional isoforms following post-translational modifications. GABARAP and GABARAP I are cytosolic proteins. Following conjugation to phosphatidylethanolamine to produce GABARAP-II, the protein incorporates into the autophagosome membrane (Kabeya et al., 2004). As with LC3 proteins, GABARAP-II is upregulated during starvation conditions; however, GABARAP-II is likely involved in sealing the autophagosomes during the final stages of maturation (Slobodkin & Elazar, 2013; Weidberg et al., 2010). Albeit, little is currently known about the precise functions of GABARAP-II or the molecular mechanisms involved in phagophore closure to form a discreet autophagosome compartment. Closure of the doublemembrane phagophore is likely different from maturation of other endosomes originating from budding organelle membranes. Phagophore closure might be similar to that of multivesicular endosomes that result from invagination of organelle membranes (Carlsson & Simonsen, 2015). As the autophagosome is a double membrane body, closure likely involves sealing of a narrow opening. Phagophore closure might involve the endosomal sorting complex proteins necessary for transport and formation of other multivesicle bodies, as loss of these proteins results in accumulation of autophagosomes (Rusten et al., 2012). While others have hypothesized that formation of the double-membrane phagophores might involve a membrane scission (one membrane splitting into two) event followed with a fusion process that results in two similarly sized vesicles, one within the other (Knorr et al., 2015). Autolysosome maturation and degradation To complete autophagic flux, autophagosomes fuse with lysosomes to digest their cargo and release nutrients back into the cytoplasm (Fig. 1). Lysosomes contain an assortment of acid hydrolases and protons (H+) that degrade the cytosolic cargos within their membrane. The autophagosomes fuse with lysosomes to form an autolysosome where the autophagosome inner membrane and its cargo are degraded for the efflux of amino acids and other nutrients back into the cytoplasm (Kang et al., 2011). Protein complexes consisting of STX17, ATG14 and STX17 facilitate autophagosome and autolysosome tethering and fusion (Diao et al., 2015). Some data suggest that autophagosomes fuse with endosomes, a process that supplies the autophagosomes with factors that enable fusion with lysosomes (Mizushima, 2007). Additionally, the Rab7, LAMP1 and LAMP2 proteins are essential to autolysosome maturation and acidification (Chua et al., 2011; Huynh et al., 2007; Wang et al., 2013). However, the half-life of autolysosomes is approximately 10 min, making detailed analysis of their structure and function rather difficult (Schworer et al., 1981). Nevertheless, the mechanisms responsible for autophagosome and lysosome fusion and autolysosomal cargo degradation are key steps in autophagic flux that make understanding this process crucial.

Methods used to measure autophagy Multiple approaches have been developed to manipulate and control the dynamic process of autophagy in research settings, for both inducing autophagy and inhibiting autophagic flux



Mammalian Autophagy (Vakifahmetoglu-Norberg et al., 2015; Yang et al., 2013). While many pharmacological methods to manipulate autophagy have clinical applications (discussed later), induction of autophagy for research purposes has primarily relied on starvation to deplete amino acid pools or through treatment with rapamycin, a potent inhibitor of mTORC kinase activity, which is a suppressor of autophagy. Autophagic flux, which is defined as completion of autolysosomal cargo degradation and release of nutrients back into the cytoplasm, is typically blocked through chloroquine treatment. Chloroquine is lysomotropic compound that upon entering the lysosome blocks acidification, thereby preventing autophagosome–lysosome fusion and degradation of their cargo (Shintani & Klionsky, 2004). Numerous additional compounds are available for finely tuned studies of the autophagic process that either induce autophagy or inhibit autophagic flux at multiple steps including: phagophore assembly site formation, initiation, phagophore elongation, autophagosome closure, autolysosome formation or autolysosome acidification. Conventional methods used to measure autophagy include, but are not limited to, Southern blot, western blot, electron microscopy and immunohistochemical staining of autophagy related proteins and structures (Klionsky et al., 2016). While invaluable to basic science research, many of these techniques do not lend themselves readily to toxicological screening or clinical assessments of autophagy, given the highly specialized expertise needed to conduct these methods and interpret the results. Flow cytometry has the capacity to measure multiple phenotypic markers in a single cell to assess autophagy. The use of flow cytometry is currently the most sensitive, reproducible and highest throughput means used to detect and evaluate toxicity, and is the premier technique advancing the modernization of toxicology approaches and clinical diagnostics. Over the past two decades, research into understanding the molecular basis of autophagy and investigation into its roles in physiology and pathophysiology has intensified. Thus, an understanding of autophagic molecular processes has led to the development of new and innovative tools for rapid and precise detection and quantification of autophagy marker profiles (Degtyarev et al., 2014; Klionsky et al., 2016; Mizushima et al., 2010; Phadwal et al., 2012; Shen et al., 2011; Warnes 2015). Several methods for assessing autophagy have been designed specifically for flow cytometric analysis utilizing autophagic markers (Chan et al., 2012; Klionsky et al., 2016; Phadwal et al., 2012; Warnes, 2015). These flow cytometry based methods have immense potential in screening for relationships between agent toxicity and alterations in autophagic processes. Flow cytometry based methods have additional utility in measuring the effect compromised autophagy has on pathology phenotypes, or to elucidate the molecular mechanisms of autophagy under normal physiological conditions. In addition, flow cytometry has the capability to measure multiple phenotypic markers in a single cell, making it particularly useful for measuring dynamic processes such as autophagic flux. Commonly used and well-characterized autophagosome markers are available, which includes identifying and measuring the levels of autophagosomes, aggresomes, LC3-I turnover, LC3B-II puncta and p62 protein levels (Klionsky et al., 2016; Mizushima et al., 2010; Warnes, 2015). The LC3 autophagosome proteins, localized to the autophagosome inner and outer membranes, serve as consistent markers in many autophagy-profiling endpoints (Klionsky et al., 2016; Mizushima et al., 2010). During autophagy, LC3-I (cytosolic) is converted into LC3-II (lipidated, membrane bound) and


incorporated into autophagosomes. This biochemical processing results in decreased levels of LC3-I and increased levels of autophagic LC3-II during autophagy progression. The formation of autophagosomes and large LC3B-II puncta were found to be significantly different from the wild-type controls in two studies using both BECN1!/! HeLa cell cultures and using BECN1+/! mice (He et al., 2015; Yue et al., 2003). Studies using gene transfection techniques indicated significantly lower autophagosome levels as measured through LC3-GFP positive foci markers in murine muscle and bronchial epithelia histological samples in BECN1+/! mice compared to wild-type controls (Qu et al., 2003). Measurements of autophagy levels with both autophagosome specific dyes and LC3B antibodies as candidate markers are possible for toxicological assessments and clinical applications (Chan et al., 2012; Lee & Lee, 2012; Warnes, 2015). Basal autophagy is responsible for clearing long-lived proteins and aggresomes by aggrephagy, and Beclin 1 along with p62 are key regulators of aggrephagy (Lamark & Johansen, 2012). Research suggests autophagy is capable of selectively degrading aggresomes and long-lived proteins (Bjorkoy et al., 2005; Knaevelsrud & Simonsen, 2010; Lamark & Johansen, 2012), although the precise mechanism through which this occurs is not well understood. Inhibiting autophagosome formation results in aggresomes that are observed in some diseases (Choi et al., 2013; Lamark & Johansen, 2012). Increases in cellular granularity or internal cell complexity due to aggresome accumulation can be measured through shifts in a cell’s side scatter profile by flow cytometry, and may provide a cursory means to differentiate autophagy-deficient cells from autophagy-competent cells. Furthermore, newly emerging fluorescent rotor dye technology that fluoresces only when bound to aggresome structural features allows for precise labeling and quantification of protein aggregates in cells (Shen et al., 2011). The p62 protein is associated with the autophagosome inner membrane through its interaction and binding to LC3 proteins and is degraded selectively and efficiently by autophagy (Bjorkoy et al., 2005). The p62 protein also has an ubiquitin binding domain and it is hypothesized that p62 might target ubiquitinated organelles and aggresomes to facilitate their conveyance into autophagosomes (Bjorkoy et al., 2005; Pankiv et al., 2007; Stolz et al., 2014). Thus, p62 accumulation, which inversely correlates with autophagic levels, has been measured in multiple studies (He et al., 2015; Klionsky et al., 2016; Komatsu et al., 2007; Mizushima et al., 2010). Following starvation, accumulation in p62 levels were observed in cells of autophagy-deficient mice compared with cells of wild-type mice (He et al., 2015; Komatsu et al., 2007), and thus can serve as a potential marker to discriminate between autophagy-competent and autophagy-deficient cells. Detailed research quantifying cellular autophagic responses suggests that both p62 and aggresome levels could potentially serve as reliable markers to measure autophagy as markers for pathology or drug toxicity. The techniques to quantify autophagic markers described in this section are currently being developed to evaluate the role of autophagy in toxicology and monitoring autophagy in clinical samples. The combination of rapid throughput with precise quantification of autophagy within a cell population is also well suited for the development of drug therapies that alter autophagic flux (Warnes, 2015). However, modifications to improve the specificity and sensitivity of these methods are likely to occur with their continued development and implementation. Finally, care should be taken that quantifying autophagic data



D. M. Petibone et al. adhere to international guidelines for interpreting autophagy (Klionsky et al., 2016).

Drug toxicity induced autophagic dysfunction As previously mentioned the autophagic pathway plays a critical role in maintaining cellular homeostasis via normal turnover of macromolecules and membranous organelles. In mammalian cells, this process involves degradation of cytoplasmic components by the lysosomal pathway, but this process can be perturbed by a variety of environmental stressors. Initially, cytoplasmic components are sequestered in autophagosomes that then fuse with lysosomes resulting in chemical and enzymatic degradation of these cellular macromolecules to their respective monomers (Fig. 1). During nutrient deprivation, cells may recycle these monomers for reincorporation into newly synthesized components that function to restore normal homeostasis. Thus, physiologically functioning autophagy is dependent on a number of genes performing properly, and on lysosomes whose integrity to complete autophagic flux has not been compromised by environmental stressors. The major source for lysosome dysfunction is lysosomal storage disorders, a prevalent side effect of many drugs (Anderson & Borlak, 2006). Early investigations considered autophagy a cell death mechanism characterized by the presence of cytoplasmic vacuoles resulting from cellular stress (Kroemer & Levine, 2008). A number of drug and environmental toxins that induced cellular death were also identified as capable of inducing cellular stress resulting in the activation of autophagy. These autophagic stressors include growth factor withdrawal, glucocorticoids, cytokines, viral infection, bacterial toxins, cytostatics, chemical toxicants, ER stressors, DNA damaging agents, ionophores, mutagens, oxidants, ionizing radiation, hypoxia and hyperthermia (Bolt & Klimecki, 2012; Orrenius et al., 2011, 2013). In addition, toxicants such as arsenic, and other xenobiotics including dexamethasone, histone deacetylase inhibitors, tamoxifen, etoposide and staurosporine, 3-methyladenine, wortmannin, vinblastine, rapamycin, verapamil and lithium were among a number of drugs identified that activated or inhibited the autophagic process (Orrenius et al., 2013). These drugs and chemicals were demonstrated to direct their activity to specific genes or gene products responsible for the constitutive performance of the autophagic pathway. While other drugs or chemicals were directly or indirectly responsible for inducing lysosomal dysfunction. Many of the drugs that altered lysosomal function are amphiphilic cationic compounds whose therapeutic action includes anorectic, antidepressant, neuroleptic, inhibitors of cholesterol synthesis, antimalarial, antianginal, antihistaminic and antiestrogen (Lüllmann-Rauch, 1979; Shayman & Abe, 2013). Included in the amphiphilic cationic compound chemicals are chlorphentermine, imipramine, iprindole, clozapine, chloroquine, amiodarone, chlorcyclizine and tamoxifen. Cells concentrate cationic drugs or chemicals (weak bases with a pKa ~8–10) into acidic organelles and vesicles that increases intracompartmental pH, preventing normal fusion and resulting in accumulation of autophagosomes (Marceau et al., 2012, 2014). Autophagy also serves to degrade intracellular lipid stores thereby regulating lipid metabolism (Singh, 2010; Zamani et al., 2016). Concentration of cationic drugs can also result in the accumulation of lamellar filled vesicles, a condition commonly referred to as phospholipidosis (Anderson & Borlak, 2006; Halliwell, 1997; Reasor & Kacew, 2001). Reasor and Kacew (2001), among other investigators, indicate that


the primary mechanism responsible for the induction of phospholipidosis is an inhibition of lysosomal phospholipase activity. Drug-induced phospholipidosis is regarded as an intracellular accumulation of phospholipids with vesicle enclosed lamellar bodies that ultrastructurally, very much resemble a membrane-filled autophagosome. The cationic amine drug chloroquine is a potent inducer of phospholipidosis and inhibitor of autophagic flux. It appears that many drugs and other environmental chemicals inducing autophagy and phospholipidosis do so through a common mechanism, lysosomal dysfunction, and may not only share a similar pathway but may result in the same pathology, suggesting that these induced phenotypes are one and the same. Quinicrine, a tertiary amine antihelmintic or antiprotozoal used in the treatment of malaria, giardiasis and tapeworm, was used in a study by Parks et al. (2015) to evaluate autophagic flux inhibition and lysomogenesis by a cationic drug in several murine models. These investigators found quinicrine to be present in perinuclear granules that are positive for Rab7 and LAMP1 proteins (Parks et al., 2015). The Rab7 and LAMP1 proteins are both essential for autolysosome maturation (Chua et al., 2011; Wang et al., 2013). Additionally, it was discovered that both quinicrine uptake and retention was inhibited by an inhibitor of the proton pump vacuolar (V)ATPase (Parks et al., 2015). Parks and colleagues revealed that the elevated autophagy induced by quinicrine was in fact due to blocking autophagic flux and accumulation of autolysosomes. Thus, they suggested that V-ATPase-mediated cationic sequestration is associated with autophagic-flux inhibition and feedback lysomogenesis. Methapyrilene (MP), a tertiary amine histamine H1-receptor antagonist, was the active ingredient in several allergy medications, cold medications and sleep aids. Lijinsky et al. (1980) identified MP as a potent liver carcinogen in Fischer 344 rats, and this study was the basis for its removal from the market by the Food and Drug Administration. Follow-up studies included ultrastructural examination of MP-exposed liver tissue that indicated MP induced a significant increase in mitochondria in periportal hepatocytes, as well as a distinct conformational change of these organelles. Additional findings revealed that lipid, glycogen and smooth ER levels were greatly reduced following MP exposures (Reznik-Schuller & Lijinsky, 1981; Reznik-Schuller & Reuber, 1986). Also observed, only in the exposed rats, were several vesicles that appeared to contain lamellar bodies, perhaps suggesting that MP induced inhibition of autophagic flux in vivo resulting in an accumulation of autophagosomes or phospholipidosis. Lijinsky’s work and a large number of additional studies (Budroe et al., 1984; Casciano, 2007; Casciano & Schol, 1984; Casciano et al., 1988, 1991) suggested that MP interacts with cell constituents other than DNA and it was postulated that MP induced cancer in rats by an indirect, epigenetic mechanism. During the studies to detect MP-induced DNA damage in rat hepatocytes in primary culture, phase contrast microscopy of the living cells revealed a peculiar unique vacuolization within the exposed cells that was not seen in the control population (Casciano, 2007). Although the cells possessed this unusual morphology for at least 24 h in culture, they remained viable. When these cultures were examined ultrastructurally using transmission electron microscopy, it was apparent that the tertiary amine, MP, and several of its congeners, altered autophagic flux causing an accumulation of autophagosomes within the cell (Casciano, 2007). At high magnification, it was observed that the vesicles also contained mitochondria, ER, other membranous organelles and, likely, soluble macromolecules indigenous to the cytoplasm that were eventually destined for normal macromolecular turnover. Most often, autophagy serves as a protective



Mammalian Autophagy mechanism; however, persistent activation and perturbation of autophagy can result in cell death (Orrenius et al., 2013). Chronic perturbation of the autophagy pathway by drugs or chemicals may lead to organ failures or in the case of the liver, through inappropriate regenerative mitosis, can lead to cancer.

Autophagy in disease and cancer progression Growing evidence suggests that autophagy plays an important role in maintaining human health. Autophagic deficiencies have been identified in the pathophysiology of neurodegenerative disorders, pulmonary and cardiovascular disease, aging, obesity and cancer (Beau et al., 2011; Chen & Debnath, 2010; Choi et al., 2013; Mizushima et al., 2008). Thus, there has been a growing awareness of autophagy deficiencies and the resulting accumulation of defective proteins or organelles that contribute to, or result in, a variety of pathologies. Neurodegenerative diseases Neurons reliance on autophagy is evident by the fact that any defect in autophagy or endosomal pathways lead to neurodegenerative disorders (Nixon, 2013). Under normal circumstances, neurons effectively degrade any defective misfolded proteins through both the ubiquitin–proteasome system and the autophagic pathway (Boland et al., 2008). However, any impediment to proteolytic clearance of autolysosomal substrates leads to pathophysiological conditions. Several studies in neuron-specific Atg5 and Atg7 knockout mice demonstrated that the loss of either Atg5 or Atg7 caused severe neurodegeneration, loss of motor function and accumulation of polyubiquitinated protein aggresomes in cytoplasmic inclusion bodies (Hara et al., 2006; Komatsu et al., 2005, 2006). In the case of Huntington’s disease, huntingtin proteins with expanded polyglutamine tracts aggregate and appear to sequester mTOR, thus inducing autophagy by constraining mTOR kinase activity (Ravikumar et al., 2004). Aggregated huntingtin proteins activating autophagy through sequestration of mTOR is contradictory, given that autophagy clears mutant huntingtin proteins. It is therefore unclear what role the expanded polyglutamine proteins play in Huntington’s disease. However, Ravikumar and colleagues deduced that the length of the polyglutamine tract might be a factor. These investigators found that longer polyglutamine tracts of 74 glutamines coprecipitated with mTOR, whereas tracts of 23 glutamines did not co-precipitate with mTOR (Ravikumar et al., 2004). Albeit, the binding of mutant huntingtin aggregated proteins with Beclin 1 also hinders the autophagic process (Shibata et al., 2006). Rapamycin treatment has demonstrated time-dependent protection against neurodegeneration through inhibiting the synthesis of huntingtin protein and is thought to contribute to the clearance of cytosolic aggregates (Ravikumar et al., 2004; Sarkar et al., 2009). Elevated autophagy is also found to be associated with Parkinson’s disease (Anglade et al., 1997). Parkinson’s disease is characterized at the molecular level by increased intracellular concentrations of α-synuclein that form Lewy bodies (proteinaceous cytoplasmic aggregates). Elevated α-synuclein levels are a consequence of alterations in the ubiquitin–proteasome system or the autophagy–lysosomal pathway that obstruct α-synuclein degradation (Cuervo et al., 2004; Webb et al., 2003). Ebrahimi-Fakhari et al. (2011) demonstrated that degradation by the ubiquitin– proteasome system is independent of the pre-existing α-synuclein burden and is likely active both under normal conditions and in


the disease state. The investigators also demonstrated that autophagy functions to degrade α-synuclein only when intracellular α-synuclein levels are elevated (Ebrahimi-Fakhari et al., 2011). This was the first report that proposed the linkage between the proteasome, autophagy and α-synuclein pathology, and the relationship between protein burden and the pathways recruited to maintain homeostasis. As observed with other autophagy-related neurodegenerative diseases, treatment with rapamycin has been observed to inhibit mTOR, facilitating clearance of α-synuclein both in vitro and in vivo (Dehay et al., 2010). Alzheimer’s disease is also linked to altered autophagy due to disrupted autolysosomal proteolysis (Nixon, 2013; Nixon & Yang, 2011). This disruption in autophagic flux produces an accumulation of autophagosomes, suggesting obstructed autophagy either at a stage of autophagosome and lysosome fusion or at the autolysosomal degradation stage. The aberrant accumulation of microtubule associated protein tau, promotes the formation of neurofibrillary tangles and the accumulation of beta amyloid peptide in neuronal plaques observed in Alzheimer’s disease (Choi et al., 2013; Jellinger, 2010). The inhibition of lysosomal proteolysis has been shown to produce neuropathological conditions in wildtype mice and exacerbate the accumulation of beta-amyloid peptides in mouse models of Alzheimer’s disease (Nixon & Yang, 2011). However, induction of autophagy through therapeutic intervention has shown to clear the mutant tau with reduced neurotoxicity in both mice and Drosophila models (Rubinsztein et al., 2007). Cardiovascular diseases Autophagy also has a key function in maintaining a healthy heart by regulating nutrient availability and cellular quality control (Hamacher-Brady et al., 2006; Kuma et al., 2004; Matsui et al., 2008; Nakai et al., 2007; Nishida et al., 2009). Autophagy possibly regulates physiological responses during cardiac stress such as ischemia, cardiac hypertrophy and heart failure (Hamacher-Brady et al., 2006; Levine & Kroemer, 2008; Matsui et al., 2008; Meijer & Codogno, 2009; Nakai et al., 2007; Nishida et al., 2009). Atg5 knockout mice have been shown to develop cardiac hypertrophy and contractile dysfunction (Nakai et al., 2007). Loss of Atg5 function also resulted in increased levels of ubiquitinated proteins and abnormal mitochondria in cardiomyocytes and abnormal sarcomere structures (Nakai et al., 2007). Growing evidence continues to support autophagy’s protective functions on cardiac health (De Meyer & Martinet, 2009; Hamacher-Brady et al., 2006; Matsui et al., 2008; Nakai et al., 2007; Nishida et al., 2009). Although, there are also contradictory reports based on experimental evidence that autophagy exerts detrimental effects on cardiac function through degradation of crucial proteins and organelles (Gustafsson & Gottlieb, 2008; Matsui et al., 2008). Therefore, additional studies are still needed to strengthen the mechanistic understanding of the protective and pathophysiological consequences of autophagy in cardiac disease (Gatica et al., 2015). Autophagy in carcinogenesis Numerous studies have indicated that autophagy is an important factor in tumorigenesis. Autophagy influences the initiation, progression and therapeutic responsiveness of cancer, thus distinguishing autophagy as a critical pathway in tumor development (Chen & Debnath, 2010; Choi et al., 2013; Qian & Yang, 2016). Yet, how autophagy regulates tumorigenesis is not at all clear-cut, and at times can present a contradiction, as autophagy



D. M. Petibone et al. can have either a negative or a positive effect depending on the cell type or stage of tumor progression. In healthy cells, autophagy inhibits tumorigenesis by removing damaged organelles and aggresomes that might induce reactive oxygen species, or through degradation of mutagenic compounds, thereby preventing DNA damage and genetic instability (Marino et al., 2007; Mathew et al., 2007; Takamura et al., 2011). During advanced stages of tumor progression, autophagy can provide nutrients to cancer cells that are rapidly dividing or located within poorly vascularized tumors that might result in nutrient depravation (Chen & Debnath, 2010). Autophagy can also alter the prognosis of certain chemotherapy regimens through promoting tumor cell survival and metastasis, preventing apoptosis and introducing drug resistance (Carew et al., 2007; Fung et al., 2008; Lu et al., 2008). Mutations in several autophagy genes have been implicated in cancer progression, including BECN1, UVRAG and ATG genes in experimental rodent models and human tumors (Maiuri et al., 2009; Zhi & Zhong, 2015). BECN1 is an evolutionarily conserved gene in mammals that codes for the Beclin 1 protein, a key autophagic component necessary for the induction and regulation of the pre-autophagosomal complex (Sahni et al., 2014; Yue et al., 2003). It is hypothesized that Beclin 1, via promoting autophagosome formation, exerts its tumor suppressor function through delivery of xenobiotics, damaged proteins and damaged organelles to lysosomes, which otherwise could result in cytotoxic or genotoxic stress (Chen & Debnath, 2010; Choi et al., 2013; Karantza-Wadsworth et al., 2007). BECN1 is also distinctive in that it is a haploinsufficient tumor suppressor gene. Haploinsufficiency refers to an incidence where the gene dosage of a BECN1+/! locus, with only one functional gene copy, is not sufficient to maintain the wild-type autophagy phenotype observed with two functional gene copies. Mono-allelic deletions of the BECN1 gene, mapped to a tumor susceptibility region at chromosome 17q21 (Aita et al., 1999; Tangir et al., 1996), occur in 40–75% of breast, ovarian and prostate cancers (Gao et al., 1995; Liang et al., 1999; Saito et al., 1993). Autophagy is also a crucial cellular process involved in maintaining lymphocyte homeostasis (McLeod et al., 2012). In agreement with Beclin 1 maintaining lymphocyte homeostasis, lowered expression of BECN1 has been observed at high incidence in lymphomas (He et al., 2014; Huang et al., 2010). Beclin 1 expression also predicts a favorable outcome for B-cell lymphoma following chemotherapy (Huang et al., 2011, Nicotra et al., 2010). Homozygous deletions of the BECN1 gene (BECN!/!) resulted in the death of mouse embryos (Yue et al., 2003), and BECN1 haploinsufficient (BECN1+/!) mice had higher incidence of spontaneous tumorigenesis and impaired autophagy compared with BECN1 wild-type mice (Qu et al., 2003; Yue et al., 2003). Evidence from HeLa cells suggest that UVRAG is not necessary for early autophagosome formation from the PtdIns3k complex (Itakura et al., 2008), but competes with ATG14 for binding to Beclin 1, and is associated with the Rab9-positive endosomes and late autophagosome maturation (Itakura et al., 2008). The Bif-1 protein interacts with Beclin 1 through its association with UVRAG to form PtdIns3k complex II (Takahashi et al., 2007). UVRAG is thought to promote late autophagosome development, endosome trafficking and acidification of autolysosomes (Funderburk et al., 2010; Liang et al., 2008; Vanhaesebroeck et al., 2010). UVRAG protein interacts with Beclin 1 in PtdIns3k complex II and exhibits tumor suppressor activity in human colon cancer cells by promoting autophagy (Liang et al., 2006). In addition, UVRAG has been found to be mono-allelically deleted in


colon cancer (Liang et al., 2007; Maiuri et al., 2009). However, the scope of how UVRAG functions in autophagy is not entirely clear. While UVRAG appears to be a tumor suppressor and UVRAG deficiencies are associated with cancers, the precise role for UVRAG in autophagy is still a topic of debate and further studies are required to elucidate its functions fully. Mutated ATG genes have also been found in both human cancers and involved in tumorigenesis in gene knockout rodent models. The ATG5 protein is part of the ATG16L1 complex that functions as an E3-like ligase transferring LC3 from ATG3 to a phosphatidylethanolamine in the expanding phagophore (Fujita et al., 2008). Mutations in the ATG5 gene, and decreased levels of ATG5 gene expression, have been frequently identified in patient samples with gastric cancer, colorectal cancer and hepatocellular gastrointestinal cancer (An et al., 2011; Cho et al., 2012). Additionally, frameshift mutations in ATG5 alone or with concurrent mutations in ATG2B and ATG9B have been observed in patients with gastric and colorectal cancers (Kang et al., 2009). Evidence from Atg5 conditional gene knockout mouse models also suggests that ATG5 has an important role in carcinogenesis. Murine Atg5flox/flox mutants, displaying an Atg5 mosaic phenotype, developed small tumors at 6–9 months exclusively in livers (Takamura et al., 2011). Hepatic tumors were detected in all Atg5flox/flox mutant mice at 9 months, and by 19 months, numerous larger tumors were observed in the livers of Atg5flox/flox mutant mice (Takamura et al., 2011). The role of Atg5 in tumorigenesis was investigated in a mouse model genetically modified to be representative of human pancreatic ductal adenocarcinoma (PDAC), capable of inducible oncogenic Kras expression and inducible ablation of p53 expression. When oncogenic Kras was activated, mice also lacking Atg5 had pancreatic precancerous lesions, but progression to PDAC was blocked (Rosenfeldt et al., 2013). Rosenfeldt et al. (2013) next observed that with both oncogenic Kras activation and p53 deficiency, Atg5 loss attenuated autophagy but no longer blocked pancreatic tumor progression and accelerated p53-dependent tumor onset. Similarly, in mice with Kras-driven lung cancer, loss of Atg5 function impaired cancer progression, while in mice with oncogenic Kras and p53 deficiency, loss of Atg5 and autophagy reinstated cancer progression in lung tumors (Rao et al., 2014). These results suggest that loss of ATG5 mutation contributes to tumor progression through loss of autophagy in mice with oncogenic Kras activation and is driven by p53 deficiency. The ATG7 protein, responsible for activating ATG12 to facilitate its dimerization with ATG5 as part of the ubiquitin-like Atg16L complex, was also demonstrated to have a role in cancer suppression. In the same Takamura et al. (2011) study investigating Atg5flox/flox mutant mice, Atg7flox/flox mutant mice were also analyzed for liver tumor formation. Several Atg7flox/flox mutant mice died of liver dysfunction at 3 months, while those surviving 1 year developed liver tumors (Takamura et al., 2011). However, in an Atg7flox/flox and p62!/! double knockout model, depletion of p62, which mediates autophagic degradation of polyubiquitinated structures, developed tumors albeit with reduced size (Takamura et al., 2011). These results suggest p62 accumulation or accumulation of its polyubiquitinated targets might contribute to hepatic tumor progression. In the Rosenfeldt et al. (2013) study discussed previously, loss of Atg7 in mice with oncogenic Kras activation developed neoplastic lesions but progression to PDAC was blocked. Similarly, mice expressing oncogenic Kras but also lacking p53, as observed with loss of Atg5, Atg7 loss also inhibited autophagy and accelerated tumor onset and progression (Rosenfeldt et al., 2013). These results suggest



Mammalian Autophagy that loss of Atg7, as with Atg5 deficiency, contributes to tumor progression, through defective autophagy in mice with oncogenic Kras activation and is driven by p53 deficiency. Autophagy in cisplatin toxicity resistant cancer Autophagy plays a fundamental role in cellular responses to drug efficacy and toxicity, including metabolizing pharmaceuticals and recycling drug-damaged cytoplasmic components (lipids, proteins, organelles, etc.). While autophagy can confer resistance to numerous chemotherapeutic regimens in cancer cells, resistance to cisplatin toxicity is discussed here as one important example. Cisplatin is a toxic metal widely employed as an effective chemotherapeutic to treat numerous human tumors. Unfortunately, in many cases the clinical responses to cisplatin are often temporary as cancer cells readily acquire cisplatin resistance. Autophagy has been implicated as a key mechanism associated with acquired cisplatin toxicity resistance in numerous cancers. However, the mechanism for how autophagy counteracts the anticancer effect of cisplatin remains elusive. Human ovarian cancer cells demonstrated resistance to cisplatin conferred by autophagy (Bao et al., 2015; Wang & Wu, 2014). In human ovarian cancer cell lines with acquired cisplatin resistance, cisplatin exposure induced ERK expression that subsequently upregulated autophagy (Wang & Wu, 2014). Wang and Wu (2014) found that ovarian cancer cells were sensitized to cisplatin-induced apoptosis by MEK inhibitors or ERK knockdown with siRNA, which decreased cisplatin-induced autophagy and reinstated cisplatin sensitivity in human ovarian cancer cells. Human esophageal cancer cells have also demonstrated that cisplatin resistance was associated with induction of autophagy (O’Donovan et al., 2011). In human esophageal cancer cells with acquired cisplatin resistance, cisplatin increased ERK phosphorylation and suppression of mTOR kinase activity leading to induction of autophagy (Yu et al., 2014). The acquired resistance to cisplatin in esophageal cancer cells was suppressed by cotreatment with chloroquine and cisplatin, as compared to cisplatin treatment alone (Yu et al., 2014). Autophagy has been demonstrated to confer cisplatin resistance in human non-small cell lung cancers. By providing the metabolic precursors necessary to produce energy, autophagy might permit tumor cell survival under hypoxic conditions often found within tumors. Evidence also suggests that autophagy induced by hypoxic conditions can also confer cisplatin resistance in non-small cell lung cancers (Lee et al., 2015; Wu et al., 2015). Cisplatin treatment induced apoptosis under both hypoxia and normoxia conditions in non-small cell lung cancer cell lines, but apoptosis under hypoxic conditions was significantly reduced (Wu et al., 2015). A similar study demonstrated that hypoxia-induced autophagy conferred cisplatin resistance in non-small cell lung cancer, but that the cells could be re-sensitized by LC3B siRNA knockdown (Lee et al., 2015). The mechanism through which autophagy protects non-small cell lung cancer cells through cisplatin resistance is not fully understood. Some evidence suggests that hypoxia-induced autophagy protects cells from apoptosis in a manner dependent on Hif-1α and Hif-2α hypoxia-inducible transcription factors, and by suppressing BNIP3 death pathways (Wu et al., 2015). The role of autophagy in chemotherapy is still controversial and as to whether it exerts a chemoresistant or chemo-sensitizing effect appears to depend on the cancer cell type and the stage of tumor malignancy. However, autophagy still offers a promising therapeutic target to enhance the efficacy of chemotherapy (Sui et al., 2013).


Clinical applications for autophagy as a diagnostic or therapeutic target Investigations into autophagic proteins as clinical diagnostic markers for cancer progression have focused primarily on Beclin 1, LC3 and p62 proteins, and were conducted in numerous cancer types. In a study evaluating the prognostic value of Beclin 1 in diffuse large B-cell lymphoma, 118 tumor samples from patients treated with doxorubicin, vincristine and rituximab plus cyclophosphamide were analyzed for Beclin 1 protein expression (Huang et al., 2011). It was determined that expression of Beclin 1 was a predictor for positive clinical outcome for patients with diffuse large B-cell lymphoma treated with this regimen (Huang et al., 2011). In another study conducted by Huang et al. (2010), analysis of 65 tumor specimens from patients with natural killer T-cell lymphoma, nasal type, a lowered Beclin 1 protein expression was found to be a prognostic risk indicator for newly diagnosed patients and could be used to predict differential low- and high-risk survival outcomes. Autophagic function is perturbed in gastric cancer cells and can affect metastasis (Qian & Yang, 2016). Several autophagic proteins have promise as prognostic markers in gastric cancer, including Beclin 1, LC3 and p62. LC3 expression was evaluated in early stage gastrointestinal cancers, to include esophageal, gastric and colorectal cancers. The results indicated that LC3 expression was upregulated in gastrointestinal cancers as compared to noncancerous cells, thus suggesting promising prognostic value (Yoshioka et al., 2008). Additionally, LC3A expression patterns in 188 patients with gastric cancer indicated high LC3A positive structures were associated with a poor prognosis and lower survival in late-stage gastric cancer (Liao et al., 2014). Evaluation of p62 expression in patients with gastric cancer revealed that high p62 expression levels, indicative of suppressed autophagy, correlated with poor differentiation but less lymph node metastasis (Mohamed et al., 2015). Likewise, studies of gastric tumors found that with high levels of Beclin 1 protein there was a favorable prognosis for patient survival. Zhou et al. (2012) evaluated 153 patients with gastric cancer for Beclin 1 and the anti-apoptotic Bcl-xL protein expression determined an inverse relationship between the two proteins. Low Beclin 1 and high Bcl-xL expression indicated a poor survival compared to patients with high Beclin 1 with low Bcl-xL expression (Zhou et al., 2012). These findings were in agreement with corroborating studies that also found decreased Beclin 1 expression to be associated with metastasis and poor clinical outcomes (Chen et al., 2012; Geng et al., 2012). Worldwide, gastric cancer is one of the most pervasive, with a poor prognosis in patients with advanced disease due to recurrence and metastasis (Digklia & Wagner, 2016). The autophagy related proteins Beclin 1, LC3 and p62 may prove useful as markers for prognosis of gastric cancer and enable design of more effective treatment regimens (Masuda et al., 2016). Beclin 1 and LC3 proteins have also been identified as prognostic markers in breast (Chen et al., 2013; Zhao et al., 2013), ovarian (Valente et al., 2014), hepatocellular (Ding et al., 2008; Shi et al., 2009) and brain (Miracco et al., 2007) cancers. He et al. (2014) conducted a meta-analysis on 23 such cancer case studies, including some of the studies listed above, for the prognostic value of Beclin 1 and LC3B on overall survival and disease-free survival in cancers. The results indicated that high expression of Beclin 1 was a favorable predictor of overall survival associated with gastric cancer, lymphoma and breast cancer (He et al., 2014). In contrast, high expression of LC3B was predictive of an adverse prognosis in breast



D. M. Petibone et al. cancer (He et al., 2014). Data from these studies suggest there is an immense potential for Beclin 1, LC3 and p62 as prognostic indicators for low risk and high-risk cancer survival differentiation. Pharmaceuticals used to alter autophagy Numerous pharmaceuticals are available that specifically target autophagy or have been reported to modulate the autophagic activity of cells by targeting ATG proteins at multiple steps during autophagic flux (Vakifahmetoglu-Norberg et al., 2015; Yang et al., 2013). Modulating autophagy activation or inhibition may have clinical benefits in treating a variety of diseases. Thus, evaluation of several compounds that alter autophagy show promise in treating cancer and neurodegenerative diseases for use as part of therapeutic regimens. Clinical applications for autophagy activators Sirolimus (rapamycin) is a common tool for activating autophagy by suppressing mTOR function in both in vitro and in vivo models, but sirolimus also induces alterations in other cellular processes such as repressing protein translation and mitochondrial biogenesis (Sarbassov et al., 2005). In addition, sirolimus acts as an immunosuppressant following long-term exposure, and thus is not appropriate for many therapies. However, several derivatives of sirolimus are available that offer promising clinical applications. Temsirolimus, a soluble rapamycin ester approved for treatment of renal cell carcinoma, shows effectiveness for treatment of mantle cell lymphoma, and Huntington’s disease (Galimberti & Petrini, 2010; Piha-Paul et al., 2011; Ravikumar et al., 2004; Rini, 2008; Wang et al., 2011). Other sirolimus derivatives with promise in clinical applications include Everolimus that has been FDA approved for the treatment of cancer, as an immunosuppressant for solid organ transplants recipients and in the treatment of coronary artery disease (Gabardi & Baroletti, 2010). Deforolimus, an intravenous formulation also inhibits mTOR and has a mild toxicity profile compared to sirolimus with promise in clinical settings (Mita et al., 2008; Vignot et al., 2005). Other activators of autophagy function through an mTORindependent mechanism. Lithium chloride, long used to treat bipolar disorders, can induce autophagy by inhibiting inositol monophosphatase leading to depletion of inositol levels (Sarkar et al., 2005). Another compound with clinical significance that activate autophagy by inhibiting inositol monophosphatases include rilmenidine, FDA approved as an antihypertensive (Widimsky & Sirotiakova, 2006), shows promise for the treatment of Huntington’s disease (Hochfeld et al., 2013). Additional pharmaceuticals that deplete inositol levels and activate autophagy are L-690330, carbamazepine and valproic acid used as mood stabilizers (Atack et al., 1993; Fu et al., 2010; Sarkar et al., 2005). The disaccharide (non-mammalian) trehalose, another mTORindependent autophagy inducer, has promise as a safe treatment for neurodegenerative diseases. In neurons, tau proteins are microtubule stabilizers that become defective, often being hyperphosphorylated and forming aggregates associated with Alzheimer’s and Parkinson’s diseases (Alonso et al., 2001). Sprague–Dawley rat primary neurons and a murine neuron cell line both overexpressing tau protein were treated with trehalose, which inhibited tau aggregation and enhanced autophagic removal of tau protein (Kruger et al., 2012). Prion diseases are infectious and fatal neurodegenerative disorders for which there is currently no therapeutic intervention. Treatment of neuronal cells


with trehalose enhanced autophagy resulting in dose- and timedependent clearance of prion proteins, suggesting a potential treatment for prion infections (Aguib et al., 2009). Evidence from numerous mammalian cell lines suggests that trehalose can induce mTOR-independent autophagy and facilitate clearance of mutant huntingtin proteins associated with Huntington’s disease and mutant α-synuclein protein associated with Parkinson’s disease (Aguib et al., 2009; Kruger et al., 2012; Sarkar et al., 2007). However, currently few data available address the hazards associated with trehalose-induced and sustained upregulation of autophagy and whether the beneficial effects involve health risks.

Clinical applications for autophagy inhibitors There are numerous pharmaceuticals used for inhibiting autophagic flux being investigated for clinical applications. Chloroquine and its analog hydroxychloroquine are lysosomal lumen alkalizers conventionally used as antimalarial agents (Homewood et al., 1972; Slater, 1993), and are currently the only widely employed clinical autophagy inhibitors. Chloroquine is uncharged and free to diffuse through the cell membrane at neutral pH. Once within the lysosome lumen chloroquine is protonated, which serves to neutralize the acidic pH of lysosomes, thus preventing the acid hydrolases from degrading the lysosomal cargo (Pasquier, 2016). In turn, chloroquine, by alkalizing the lysosomes, results in accumulation of autophagosomes by preventing autophagic flux. Retinal toxicity is a side effect stemming from chloroquine treatment (Finbloom et al., 1985). Hydroxychloroquine functions in much the same way as chloroquine, but has an additional hydroxyl moiety added to the side chain of the quinolone group that prevents it from crossing the blood–retinal barrier, thereby limiting retinal toxicity (Wolfe & Marmor, 2010). Additional new therapeutic applications for chloroquine and hydroxychloroquine are emerging that include treating rheumatoid arthritis, systemic lupus erythematosus and HIV-1 activity (Rainsford et al., 2015; Romanelli et al., 2004). Chloroquine also has a potential application as an autophagy inhibitor in the treatment of numerous cancers including lymphoma, ovarian cancer, gliomas and pancreatic cancer (Amaravadi et al., 2007; Fan et al., 2010; Lu et al., 2008; Yang et al., 2011). A number of clinical studies have already used chloroquine and hydroxychloroquine as part of the chemotherapeutic regimens (Pasquier, 2016). With a long history of use as recognized antimalarial agents and new clinical applications continually being investigated, the potential benefits of the well-tolerated lysosomal alkalizers chloroquine and hydroxychloroquine are likely to expand. Additionally, a drug has been developed that targets Beclin 1 for degradation, thereby inhibiting autophagosome formation. The specific and potent inhibitor of autophagy 1 (spautin 1) degrades Beclin 1 by promoting its ubiquitination and proteosomal degradation, and studies with spautin 1 and imatinib demonstrated synergistic enhancement of imatinib-induced apoptosis in chronic myeloid leukemia (Shao et al., 2014). Several other compounds are being investigated as autophagy inhibitors that target PI3K, the ULK complex, proteases and lysosomes with promise in clinical settings for treatment of pathologies. For thorough reviews of the clinically relevant drugs and other experimental approaches currently investigated as a means for activating or inhibiting autophagy see the references (i.e., Pasquier, 2016; Yang et al., 2013; Vakifahmetoglu-Norberg et al., 2015). Pharmaceuticals targeting both autophagy induction and autophagic flux have promising



Mammalian Autophagy applications in numerous regimens designed to treat a variety of diseases and cancers.

Conclusions Investigation into the molecular mechanisms of autophagy performed over nearly two decades has greatly benefited research designed to improve human health. The role autophagy has in drug toxicity, pathophysiology and therapeutic outcomes, can be leveraged to design more effective treatment regimens. Methods for detecting and quantifying autophagy have immense potential in toxicological evaluations, and for the development of pharmaceuticals that specifically target autophagy for clinical applications. Autophagic function in cancer is complex, as autophagy is considered to have a tumor-suppressive function in healthy cells, but autophagy also promotes malignancy in tumor cells. As our knowledge of the autophagic process continues to grow, undoubtedly novel and innovative application of this information will serve to improve human health through the development of autophagy proteins as prognostic markers and drugs that target autophagy in pathologies. Acknowledgments The authors would like to thank Dr. Meagan Myers for editorial assistance during preparation of this manuscript.

Conflict of interest The authors did not report any conflict of interest.

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