autophagy a potential therapeutic target in lung diseases

Autophagy: a potential therapeutic target in lung diseases

Kiichi Nakahira and Augustine M. K. ChoiDivision of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital, HarvardMedical School, Boston, Massachusetts

Submitted 18 March 2013; accepted in final form 17 May 2013

Nakahira K, Choi AM. Autophagy: a potential therapeutic target in lung diseases.Am J Physiol Lung Cell Mol Physiol 305: L93–L107, 2013. First published May 24,2013; doi:10.1152/ajplung.00072.2013.—Macroautophagy (hereafter referred to asautophagy) is an evolutionally conserved intracellular process to maintain cellularhomeostasis by facilitating the turnover of protein aggregates, cellular debris, anddamaged organelles. During autophagy, cytosolic constituents are engulfed intodouble-membrane-bound vesicles called “autophagosomes,” which are subse-quently delivered to the lysosome for degradation. Accumulated evidence suggeststhat autophagy is critically involved not only in the basal physiological states butalso in the pathogenesis of various human diseases. Interestingly, a diverse varietyof clinically approved drugs modulate autophagy to varying extents, although theyare not currently utilized for the therapeutic purpose of manipulating autophagy. Inthis review, we highlight the functional roles of autophagy in lung diseases withfocus on the recent progress of the potential therapeutic use of autophagy-modifying drugs in clinical medicine. The purpose of this review is to discuss themerits, and the pitfalls, of modulating autophagy as a therapeutic strategy in lungdiseases.

autophagy

IN TIMES OF NUTRIENT OR OXYGEN scarcity, cells survive by eatingand recycling part of themselves (73, 94). These cellularprocesses are collectively named “autophagy” (in Greek, “eat-ing oneself”). Autophagy is a cellular self-degradation processin which cytosolic materials and organelles are sequestered anddelivered to lysosome for degradation and recycling (73, 94).In addition to nutrient starvation, autophagy is induced byvarious physiological and pathological conditions. Impor-tantly, autophagy is regulated in various human diseases suchas cancer (120), metabolic diseases (e.g., obesity and Type 2diabetes) (91), neurodegenerative diseases (e.g., Alzheimer’sdisease and Parkinson disease) (122), and infectious diseases[e.g., human immunodeficiency virus (HIV) and tuberculosis](54, 60). These reports suggest that autophagy can serve as apotential therapeutic target for human diseases. Here, we sum-marize the pathophysiological roles of autophagy in lungdisease, and we explore the possibility that modulating au-tophagy activity using a pharmacological approach can be auseful therapeutic intervention.

MOLECULAR MECHANISM OF AUTOPHAGY

Autophagy encloses cytosolic materials by isolation mem-branes (phagophores) to form double membrane-bound vesicles

called “autophagosomes” (Fig. 1). The isolation membranes areacquired from multiple sources including endoplasmic reticulum,Golgi apparatus, mitochondrial outer membrane, and plasmamembrane (35, 76, 93, 116). Subsequently, the autophagosomecontaining the cytosolic components and organelles fuses with thelysosome to become autolysosome, with subsequent degradationof cytosolic components (Fig. 1). More than 30 autophagy-relatedgene (Atg) proteins have been identified to be involved in au-tophagosome formation (73, 94). The activation of autophagy ismainly regulated by two signaling pathways including mamma-lian target of rapamycin (mTOR)-dependent and mTOR-indepen-dent pathways (94). In normal physiological condition, autophagyactivity is regulated at basal level by activation of mTOR, aserine/threonine kinase that has diverse cellular functions. Whencells encounter nutritional starvation, mTOR is inactivated andpromotes the autophagosome formation (Fig. 2, left). In mTOR-independent pathways, Ca2�–calpain–Gs� and cyclic AMP(cAMP)–phospholipase Cε (PLCε)–inositol-(1,4,5)-trisphosphate(IP3) pathways are involved in autophagy activation (Fig. 2,right).

FUNCTION OF AUTOPHAGY

Whereas autophagy nonselectively engulfs and degradesintracellular proteins for recycling under nutritional starvation,autophagy can selectively target and remove specific subcel-lular components (selective autophagy) (18). This selectivedegradation pathway includes eliminating invading pathogens(xenophagy) (60), dysfunctional cellular organelles such asmitochondria (mitophagy) (126), and polyubiquitinated proteinaggregates (aggrephagy) (56). Autophagy is also involved inlipid metabolism (lipophagy) (108). Selective autophagy

The aim of this series is to provide practicing MDs and researchers anup-to-date physiological understanding of disease. Articles published in thisseries are intended to provide a thoughtful and lucid linkage of science to thepatient. Go here for more information on this series and to browse the content(http://www.the-aps.org/mm/Publications/Journals/PIM).

Address for reprint requests and other correspondence: A. M. K. Choi,Division of Pulmonary and Critical Care Medicine, Brigham and Women’sHospital, Harvard Medical School, 75 Francis St., Boston, MA 02115 (e-mail:[email protected]).

Am J Physiol Lung Cell Mol Physiol 305: L93–L107, 2013.First published May 24, 2013; doi:10.1152/ajplung.00072.2013. Physiology in Medicine

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plays important roles in maintaining cellular homeostasis inbasal physiological states and in response to various cellularstresses.

Cytoprotective roles of autophagy under stress conditionssuch as starvation have been well documented (73). However,when cells receive lethal signals, the cellular stresses causeautophagy and also cell death including apoptosis. Althoughinteractions of autophagy- and apoptosis-related moleculessuch as Beclin 1 and B cell lymphoma 2 (BCL-2), or LC3B andFas have been reported in various models (16, 87), it is stillunclear whether autophagy induced by lethal signals promotescell death or is an independent process from cell death (94).The functional role of autophagy on cell death is likely to bedependent on stress models.

Against invading microbes, autophagy actively participatesin innate immune responses (54, 60). For example, autophagyexerts its host defense role by degrading various pathogens bylysosomal system (xenophagy) (60). The target pathogensinclude bacteria, such as group A Streptococcus pyrogen (60),Mycobacterium tuberculosis (Mtb) (23), Salmonella enterica(54), and Pseudomonas aeruginosa (127); viruses such asherpes simplex virus 1 (54, 60); and parasites such as Toxo-plasma gondii (54). In contrast, recent studies also suggest that

autophagy machinery contributes to the replication and sur-vival of microbes in the host cells. The list of pathogensexploiting autophagy machinery includes clinically importantmicrobes, e.g., bacterial agents such as Brucella abortus (99)and Coxiella burnetii (99) and viruses such as HIV (99),hepatitis B virus (HBV) (54, 99), and avian influenza A H5N1(H5N1) (109). Of note, autophagy exerts both killing andprosurvival properties for HIV (60, 99). Autophagy has diverseeffects on both innate and adaptive immune systems (54, 60).Atg5 is involved with the production of type I interferons inresponse to single-stranded RNA viruses (54, 60). Autophagyproteins are also involved in the regulation of inflammasome,cytosolic multiprotein complexes responsible for activation ofcaspase-1 and its downstream signaling including secretion ofIL-1� and IL-18 (20, 79). Beclin 1 and LC3B negativelyregulate inflammasome activation by preserving mitochondrialhomeostasis (79). The roles of autophagy in adaptive immunityinclude antigen presentation and antibody production. Au-tophagy is required for presenting antigen on both majorhistocompatibility complex class I and II molecules and acti-vating CD8� and CD4� T lymphocytes, respectively (60).Autophagy is also required for B cell differentiation (19) andfor sustainable antibody production in plasma cells (88). Thus

Autophagosome Autolysosome

Initiation, isolation membrane

Elongation Completion Autophagosome and Lysosome fusion

Degradation

Lysosome

Beclin 1

Chloroquine

Bafilomycin A

Azithromycin

Pepstatin A

3-MA

Spautin 1

BCL-2

BH3 mimetics

LC3II Atg5-Atg12-Atg16L

Acid hydrolase Cargos

ULK1 complex

Class III PI3K complex

Class III PI3K

Phagophore

Autophagy inhibitor

Autophagy activator

Cystatin B

Fig. 1. Autophagy process and potential targets for modulating autophagy. Activation of UNC51-like kinase (ULK) complex in response to certain signalsinitiates isolation membrane and the formation of phagophore. (Upstream pathway of ULK1 complex is depicted in Fig. 2.) Class III phosphoinositide 3-kinase(PI3K) complex composed of Beclin 1, class III PI3K, PIK3R4, and activating molecule in BECN1-regulated autophagy protein 1 (AMBRA1) is also requiredfor the phagophore formation. The Atg-Atg12-Atg16L complex and LC3-II phosphatidylethanolamine (PE) conjugate promote the elongation and enwrap thecytosolic cargos including mitochondria, leading to the formation of autophagosome. Subsequently, lysosome fuses with the autophagosome (the formation ofautolysosome) and releases acid hydrases into the interiors to degrade the cytosolic cargos. Autophagy can be activated by drugs such as BCL-2 homology 3(BH3) mimetics that prevent the formation of autophagy inhibitory complex of Beclin 1 and BCL-2. In contrast, ubiquitin-mediated decrease of Beclin 1 proteinby Spautin-1 and inhibition of class III PI3K by 3-methyladenine (3-MA) can inhibit autophagy. In addition, there are drugs to inhibit the late phase of autophagyprocess. Lysosomotropic agents that enhance lysosomal pH such as chloroquine inhibit lysosomal enzymes and also prevent the fusion of autophagosome andlysosome, resulting in inhibition of autophagy. Bafilomycin A1 inhibits the fusion of autophagosome and lysosome by inhibiting vacuolar ATPase (V-ATPase)located in the lysosomal membrane. Compounds such as pepstatin A and cystatin B are inhibitors for lysosomal protease. (Please also see Table 4.)

Physiology in Medicine

L94 AUTOPHAGY MODULATION IN LUNG DISEASES

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00072.2013 • www.ajplung.org

autophagy plays crucial diverse roles in immune systemsincluding pathogen degradation and immune signaling, sug-gesting that the involvement of autophagy in infectious andinflammatory diseases.

Autophagy is critically involved in various metabolic path-ways for maintaining cellular homeostasis (91). For example,autophagy is an important energy generator in the liver. Micewith specific deletion of atg7 gene in liver display decreasedlevel of blood glucose and amino acids after starvation (25).Autophagy is also involved in lipid metabolism by breaking

down lipid droplets to generate energy (lipophagy) (108).Metabolome analysis also suggests autophagy is required formaintenance of tricarboxylic acid cycle-related metabolites(33), suggesting that autophagy is involved in the qualitycontrol of mitochondria. Damaged mitochondria are selec-tively targeted and removed by autophagy to maintain normalmitochondrial function (mitophagy) (126). Although the con-cise molecular mechanism of mitophagy is still unclear, theimportance of preserving mitochondrial homeostasis by au-tophagy is now further linked to the pathogenesis of human

Autophagy

mTOR

Rheb

AMPK TSC1/2

Akt

Class I PI3K

ULK1 complex

Ca2+ channel

cAMP

Epac

Rap2B

PLC-ε

PIP2IP3

IP2

IP1

IMPase

Ins

I1R

Gsα

via cleavage of Atg5

IP3 receptor

Rapamycin and analogues, Torin 1

Metformin

PI103

Calpastatin, calpeptin

L type Ca2+ channel blockers (verapamil, amidarone)

Xestospongin B

Lithium, L-960,330

Valproic acid, Carbamazepine

ClonidineEGFR antagonists (ertinib)

ATP

2’5’ddA Ca2+

ER

Ca2+

AC

Calpain

Cell membrane

CytoplasmReceptor tyrosine kinase

Insulin and IGF receptor

Autophagy inhibitor

Autophagy activator

Autophagy activator

Fig. 2. Overview of the regulation of autophagy pathways and targets for modulating autophagy by drugs/compounds. Two major signaling pathways to regulateautophagy are depicted in this figure: mammalian target of rapamycin (mTOR) signaling pathways and mTOR-independent signaling pathways. Autophagy ismodulated by mTOR in response to certain nutritional stimulations. Insulin or growth factors activate class I PI3K pathway, leading to activation of mTOR andinhibition of autophagy. Glucose starvation activates AMPK and inhibits mTOR, followed by activating of ULK1 complex and activating autophagy. mTOR canbe pharmacologically inhibited by activating AMPK (e.g., metformin) or inhibiting class I PI3K (e.g., EGFR antagonist). Recent new drugs such as PI-103 canactivate autophagy by inhibiting both class I PI3K and mTOR. In the mTOR-independent pathway, increase of intracellular cAMP level and Ca2� inhibitsautophagy. Intracellular level of cAMP is upregulated by adenylyl cyclase (AC) and the calpain-Gs� pathway, which is activated by intracellular Ca2� level.The inhibitory effect of cAMP on autophagy is mediated by increase of synthesis of inositol-(1,4,5)-trisphosphate (IP3) and inositol. Increased level of IP3activates IP3 receptor in endoplasmic reticulum (ER) and release Ca2� into cytosol, leading to inhibition of autophagy. Activation of L-type Ca2� channel triggersCa2� influx and elevates intracellular Ca2� level, which activates cysteine protease called calpain. In addition to elevating cAMP by the calpain-Gs� pathway,calpain inhibits autophagy by cleavage of Atg5. However, the concise mechanism by which cytosolic cAMP, Ca2�, inositol, and IP3 regulate autophagy is stillunclear. In this cyclical mTOR-independent pathway, autophagy can be modulated by mainly targeting intracellular levels of cAMP or Ca2�. L-type Ca2�

receptor blockers such as verapamil inhibit Ca2� influx, which leads inhibition of calpain activity and cAMP synthesis. Activation of autophagy by drugs suchas lithium or carbamazepine is mediated by at least reduction of Ca2� release by IP3 receptor (IP3R). EGFR, epidermal growth factor receptor; IGF, insulin-likegrowth; TSC, tuberous sclerosis 2; AMPK, AMP-activated protein kinase; V-ATPase, vacuolar ATPase; I1R, imidazoline-1 receptor; PLCε, phospholipase Cepsilon; 2=5=ddA, 2=,5=-dideoxyadenosine; PIP2, phosphatidylinositol-4,5-bisphosphate; cAMP, cyclic AMP; Ins, inositol; IP2, inositol-(1,4)-bisphosphate; IP3,Inositol-(1,4,5)-trisphosphate; IMPase, inositol monophosphatase.


L95AUTOPHAGY MODULATION IN LUNG DISEASES


diseases such as Parkinson disease (68, 80, 81, 118). Thus theautophagy process is critically involved in not only physiolog-ical but also pathological metabolic responses.

MEASUREMENT OF AUTOPHAGY

As the research of autophagy continues to evolve, methodsfor monitoring autophagy have been evaluated and discussed indetail. Klionsky et al. (51) have recently reported the guidelinefor monitoring autophagy in higher eukaryotes and describeduseful methods and how to interpret the data. A key tenet toemphasize is that investigators need to recognize whether theyare evaluating the steady-state levels of autophagosomes ordynamic state of autophagy generated by their models (51,101). If they are seeking to assess the change of autophagy fluxor activity (e.g., the rate of delivery of autophagosomes sub-strate to lysosomes) in certain points, it is likely that monitor-ing steady state of autophagosome (e.g., measuring LC3-IIexpression without examining turnover by Western blot, count-ing autophagosomes by using electron microscopy, or countingpuncta formation of LC3 by immunofluorescence microscopy)is not sufficient. Table 1 demonstrates the various methods tomonitor autophagy both in vitro and in vivo. Generally, it isrecommended to use multiple different assays (ideally for bothsteady and dynamic states), rather than relying on the resultsfrom a single method (51, 101).

AUTOPHAGY PATHWAY: POTENTIAL THERAPEUTIC TARGETFOR LUNG DISEASES

Autophagy in Models of Human Lung Diseases

The functional roles of autophagy on various lung diseaseshave been studied both in vitro and in vivo. Tables 2 and 3represent the functions of autophagy or autophagic proteins inlung diseases on the basis of studies using genetic or biochem-ical perturbation of autophagy. Although autophagy has beeninitially thought as a cytoprotective response in pathophysio-logical states, accumulating data reveal diverse functions ofautophagy in lung diseases (Tables 2 and 3). For example,chronic obstructive pulmonary disease (COPD) is one lungdisease that has been shown to be associated with autophagy(15). Increased number of autophagosomes is observed byelectron microscopy analysis, and expression of autophagicprotein LC3B-II is increased in lung tissues from patients withCOPD (15). Several molecules involved in autophagy-medi-ated COPD include FAS (16), Toll-like receptor 4 (5), andcystic fibrosis transmembrane conductance regulator (CFTR)(11). In addition, alveolar macrophages isolated from smokersshow increased of LC3 expression with defect of autophagyflux (77). In vivo, genetic deletion of LC3B displays resistanceto cigarette smoke-induced airway space enlargement com-pared with the control mice (16). Similar adverse effects of

Table 1. Methods for monitoring autophagy in vitro and in vivo

Methods Key Points References

Monitoring static state autophagyQuantifying autophagosomes Electron microscopy • Positive correlation of elevated number of

autophagosomes and increased autophagy flux has notbeen proved reliably in all models.

110

LC3-II level (ratio of LC3-II/LC3-I) Western blot • Increased level of LC3-II (ratio of LC3-II/LC3-I) does notalways correlate with autophagy flux.

75

Puncta formation of LC3 Immunofluorescencemicroscopy

• This method can be applied to in vivo using GFP-LC3transgenic mice.

72, 74

• Increased puncta formation of LC3 does not reliablycorrelate with autophagic flux.

Monitoring dynamic state autophagyTurnover of LC3-II Western blot • LC3-II on the autophagosome membrane is normally

continuously degraded during autophagy process. LC3-IIcan be accumulated by adding lysosomal proteolysisinhibitors such as leupeptin, chloroquine or bafilomycin.

38, 41

• Cells or animals can be treated with lysosomal proteaseinhibitors.

• Autophagic flux can be expressed as the difference inLC3B-II signal on Western blot obtained in the presenceor absence of lysosome protein inhibitors.

Turnover of p62/SQSTMI Western blot • P62/SQSTMI is a cytosolic protein that has an LC3binding domain (REF). It binds to ubiquitinated proteinand carry them to autophagosome. Subsequently, bothp62/SQSTMI and the cargo proteins are degraded byautophagy.

10, 38

• Similar with LC3-II, p62/SQSTMI turnover can bemeasured by adding lysosome protease inhibitors in vitroor in vivo.

Cytosolic protein sequestration assays Western blot • Cells are incubated (or animals are intraperitoneallyadministered) with leupeptin to inhibit lysosomalproteolysis. Autophagosomes are purified via mechanicaldisruption (34) followed by density centrifugation.Autophagy flux is expressed by the amount of cytosolicprotein, such as LDH recovered in the autophagosomefraction (Western blot).

38, 52, 106

LDH, lactate dehydrogenase.




Table 2. The functional role of autophagic proteins in experimental models of lung diseases

Lung Diseases Function of Autophagic Proteins References

COPD • LC3B and Beclin 1 deficiency inhibits CSE-induced cell death in pulmonary epithelial cells. 15, 16, 47LC3B-deficient mice display inhibition of airspace enlargement and apoptosis in lungs after cigarette smoke

exposure.• LC3 deficiency promotes CSE-induced IL-8 secretion in HBEC. 28Autophagy inhibition by bafilomycin A enhances CSE-induced IL-8 secretion in HBEC.

IPF • Autophagy activation by rapamycin inhibits IL-17A-induced collagen production in lung epithelial cells. 71Autophagy inhibition by 3-MA suppresses degradation of collagen in epithelial cells.3-MA reverses the therapeutic effect of IL-17 antagonism on bleomycin-induced lung fibrosis and mortality

of mice.• LC3B and Beclin 1 deficiency promotes TGF-�-induced activation of lung fibroblast in vitro. 85Autophagy activation by rapamycin inhibits TGF-�-induced fibronectin and �-SMA expression in lung

fibroblast.Rapamycin inhibits bleomycin-induced lung fibrosis in mice.• Rapamycin inhibits anti-TLR4 antibody-induced lung fibrosis in mice. 124• Atg5 and LC3 deficiency promotes TGF-�-induced fibroblast activation in vitro. 7Inhibition of mTOR by Torin1 inhibits tunicamycin (ER stress inducer)-induced senescence in HBEC.Atg5 and LC3 deficiency promotes tunicamycin-induced senescence in HBEC.

PH (PAH) • LC3B deficiency promotes hypoxia-induced pulmonary hypertension in mice. 59LC3B deficiency promotes cell proliferation in pulmonary artery endothelial cells and vascular smooth

muscle cells.• Autophagy inhibition by 3-MA and chloroquine increase angiogenesis in PAEC from fetal lambs with

persistent pulmonary hypertension (PPHN-PAEC).114

• Beclin-1 knockdown promotes angiogenesis in PPHN-PAEC.

CF • Beclin 1 deficiency promotes aggresome accumulation in CF epithelia. 65Beclin 1 deficiency promotes macrophage infiltration and MPO activity in nasal mucosa of CF.Autophagy inhibition by 3-MA abrogates CF phenotype in nasal mucosa.• LC3 deficiency promotes growth of B. cepacia and IL-1� secretion in macrophages. 1Autophagy activation by rapamycin inhibits growth of B. cepacia and IL-1� secretion in macrophages.Autophagy activation reduces bacterial burden of lung and inflammation in lung of CF mice after B.

cepacia infection.• Overexpression of SQSTM1/p62 inhibits intracellular survival of B. cepacia in macrophages. 2Deficiency of SQSTM1/p62 promotes intracellular survival of B. cepacia in macrophages.

Lung cancer • Blocking chaperone-mediated autophagy by LAMP-2A depletion suppresses cell proliferation andincreases cell death in lung cancer cells.

53

Injection of lung cancer cells with LAMP-2A deficiency reduces tumor formation in xenograft mousemodel.

• Autophagy inhibition by chloroquine enhances cytotoxicity of gefinitib and erlotinib in lung cancer cells. 36Atg5 and Atg7 deficiency enhances cytotoxicity of gefinitib and erlotinib in lung cancer cells.• Atg7 deficiency reduces cancer cell proliferation in NSCLC cell lines. 46Atg7 deficiency sensitizes the cancer cells to cisplatin-induced apoptosis in NSCLC cell lines.• Atg3 deficiency reverses erlotinib resistance in erlotinib-resistant lung cancer cells. 57

LAM • TSC2 knockdown increases autophagy-dependent cell survival in mouse embryonic fibroblasts. 84Atg5 deficiency inhibits TSC2-null xenograft tumor cell survival.Autophagy inhibition by chloroquine inhibits xenograft tumor size in mice of TSC2-null xenograft tumor.

HALI • LC3B knockdown promotes hyperoxia-induced cell death in lung epithelial cells. 112

Sepsis • Depletion of LC3B and Beclin 1 leads mitochondrial dysfunction and enhances NLRP3 inflammasome-mediated IL-1� and IL-18 secretion in macrophages.

79

LC3B knockdown increases serum level of IL-1� and IL-18 production in CLP-induced polymicrobialsepsis and sensitizes mice to endotoxic shock.

• Autophagy activation by rapamycin restores CLP-induced myocardial dysfunction in mice. 40• LC3 overexpression ameliorates acute lung injury and survival in CLP-induced polymicrobial model of

sepsis.64

• Knockdown of VPS34 increases cell death and liver injury in CLP-induced polymicrobial models ofsepsis.

14

• Autophagy inhibition suppresses releases of neutrophil extracellular trap (NET) induced by E. coli inhuman neutrophils.

45

Nanoparticle-induced ALI • Knockdown of Atg6 improves nanoparticle-induced cell death in lung epithelial cells. 61, 62Autophagy inhibition by 3-MA ameliorates nanoparticle-induced ALI in mice.

COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; HBEC, human bronchial epithelial cells; IPF, idiopathic pulmonary fibrosis;TGF-�, transforming growth factor-�; �-SMA, �-smooth muscle actin; 3-MA, 3-methyladenine; TLR4, Toll-like receptor 4; ER, endoplasmic reticulum; P(A)H,pulmonary (artery) hypertension; PAEC, pulmonary artery endothelial cells; CF, cystic fibrosis; MPO, myeloperoxidase; B. cepacia, Burkholderia cenocepacia;NSCLC, nonsmall cell lung cancer; LAMP2, lysosome-associated membrane protein 2; LAM, Lymphangioleiomyomatosis; TSC, tuberous sclerosis; HALI,hyperoxia-induced acute lung injury; NLRP3, NOD-like receptor family, pyrin domain containing 3; CLP, cecal ligation and puncture; E. coli, Escherichia coli;ALI, acute lung injury.




autophagy are observed in other lung diseases including lungcancer (36, 46, 53) and acute lung injury induced by H5N1infection (109). In contrast, the beneficial roles of autophagyare also observed in the diseases such as cystic fibrosis (CF) (1,2, 65), tuberculosis (23, 34, 42), and sepsis (14, 40, 45, 64, 79).Thus autophagy is an important cellular process to regulate orcontribute to the pathogenesis of lung diseases. Importantly,Fig. 3 demonstrates specificity of the pathophysiological func-tions of autophagy in each lung disease, resulting in eitherfavorable or deleterious phenotype depending in the diseaseprocess.

Autophagy in Clinical Trials

Although drugs modulating autophagy such as rapamycin orchloroquine have been clinically used for years without theintent to modulate autophagy activity, these drugs are nowstudied as autophagy modulators in clinical trials for lungdiseases (17, 99, 120, 125). For example, chloroquine, whichinhibits autophagy-mediated cell survival in tumor cells, isused as an intervention for patients with small cell lung cancerin a clinical trial (phase 1). In addition, the interventions usinghydrochloroquine and other anticancer drugs such as erlotinibare used for non-small cell lung cancer in two clinical trials(phase 1/2 and 2) (125). Although autophagy has been initiallyshown to be associated with anti-tumorigenesis in rodent ani-

mal studies (90, 111), the current strategy for cancer therapy ismore likely to be based on inhibition of autophagy (17, 120,125).

This paradox may be due to the differential roles of au-tophagy in different stages of tumorigenesis (99, 120). Al-though initiation of tumorigenesis in normal cells is associatedwith defect of autophagy, autophagy subsequently may exertprosurvival effect in tumor cells (99, 120). Lymphangioleio-myomatosis (LAM), a progressive lung disease caused bymutation in the tuberous sclerosis genes (tsc), is associatedwith inappropriate activation of mTOR signaling, which reg-ulates cellular growth and lymphangiogenesis (39). The patho-genesis of LAM is in part similar with those of tumorigenesisincluding inappropriate cell growth and survival (39). Chloro-quine is also used with rapamycin for patients with LAM(phase 1). This clinical trial is based on the rationale thatrapamycin blocks mTOR of downstream kinases and restoreshomeostasis in cells with defective tsc function (39, 69, 84).The use of chloroquine aims to inhibit autophagy-mediatedsurvival of LAM cells induced by rapamycin (39, 84). A recentclinical study concludes chloroquine does not prevent infectionwith influenza including H1N1 strain (86). Interestingly, au-tophagy is involved in infection with H5N1, rather than H1N1(109). Therefore, it is possible that chloroquine has a differen-tial effect on infection with H5N1 vs. H1N1, with genetic and

Table 3. The functional role of autophagic proteins on pathogen(s) causing respiratory tract infection

Pathogens Role of Autophagy or Autophagic Proteins References

Tuberculosis (M. tuberculosis) • Autophagy induction by rapamycin or starvation inhibits M. tuberculosis survival ininfected macrophages.

34

• IL-4 and IL-13 inhibit autophagy activation and autophagy-mediated killing ofintracellular M. tuberculosis in macrophages.

37

• Autophagy activation by rapamycin enhances presentation of mycobacterial antigenin macrophages.

42

Subcutaneous injection of mycobacteria-infected dendritic cells pretreated withrapamycin enhances Th1 response and increases vaccine efficacy in mice.

• Autophagy induction by Vitamin D3 promotes killing of M. tuberculosis. 128• Isoniazid-induced autophagy is associated with antimicrobial activity in M.

tuberculosis-infected macrophages.49

Avian influenza A H5N1 • Knockdown of Atg5 and TSC2 inhibits H5N1-induced cell death in lung epithelialcells.

109

Autophagy inhibition by 3-MA ameliorates acute lung injury and mice survival rate inmice infected with H5N1

Knockdown of Atg5 ameliorates acute lung injury and mice survival rate in miceinfected with H5N1.

P. aeruginosa • Autophagy induction by rapamycin promotes P. aeruginosa clearance andautophagy inhibition by 3-MA inhibits the bacterial clearances in macrophages.

127

Knockdown of Beclin 1 inhibits P. aeruginosa clearance in macrophages.

M. abscessus • Autophagy inhibition by Azithromycin inhibits intracellular killing of M. abscessusin macrophages.

96

Autophagy inhibition by Azithromycin promotes M. abscessus infection in mice.

Human rhinovirus 2 • Autophagy activation by rapamycin increases replication of human rhinovirus 2(HRV-2).

50

• Autophagy inhibition by 3-MA suppresses replication of HRV-2.

Human adenovirus type 5 • Knockdown of Atg5 inhibits human adenovirus-mediated cell lysis. 44

S. aureus • Knockdown of Atg5 inhibits replication of S. aureus and S. aureus-mediated celldeath.

105

Respiratory syncytial virus • Knockdown of Beclin 1 or LC3 attenuates respiratory syncytial virus-inducedproinflammatory cytokines production.

78

M. tuberculosis, Mycobacterium tuberculosis; P. aeruginosa, Pseudomonas aeruginosa; M. abscessus, Mycobacterium abscessus; S. aureus, Staphylococcusaureus.




biochemical inhibition of autophagy rescue in mice infectedwith H5N1 (109). Since chloroquine also has autophagy-independent pharmacological effects such as anti-inflamma-tory property, the effect of chloroquine on diseases includingtumor may not be entirely explained by modulating autophagy(120). Although further studies are needed to evaluate theoff-target effects of chloroquine on diseases, these reportssuggest that autophagy modulation by clinically used drugsmay be more accessible to study in clinical trials because oftheir favorable safety profiles. The details of ongoing clinicaltrial targeting modulating autophagy are listed (http://www.clinicaltrials.gov/ct2/results?term�autophagy&Search�Search).

Autophagy Modulators

Given the important association between autophagy andhuman lung diseases, it is worthwhile to review whethermodulating autophagy can serve as potent therapeutic targetsfor various lung diseases. Table 4 demonstrates a list ofautophagy modulating drugs that are clinically used or beingstudied in clinical trials. Table 5 shows conventional and newlydeveloped compounds or molecules modulating autophagyactivity. There are several target signaling pathways whereinautophagy activity is modulated by drugs.

mTOR signaling pathways. The mTOR is one of the maintargets for modulating autophagy by drugs and forms twodistinct complexes to regulate autophagy: mTOR complex 1(mTORC1) and mTOR complex 2 (mTORC2) (94) (Fig. 2,left). Rapamycin and its analogs inhibit mTORC1, a maincomplex responsible for autophagy regulation, and promoteautophagy induction (92, 95, 121). A recently identified com-

pound Torin1 directly inhibits both mTORC1 and mTORC2and induces greater autophagy than does rapamycin (115). Ofnote, another new class of drugs has dual targets in autophagypathways. PI-103 hydrochloride inhibits both mTOR and classI phosphoinositide 3-kinase (class I PI3K), suggesting aneffective autophagy inducer (22).

mTOR-independent pathways. Figure 2, right, shows mTOR-independent pathways.

PHOSPHOINOSITOL SIGNALING PATHWAY. Intracellular inositoland IP3 levels negatively regulate autophagy (94). Drugs suchas lithium (103) or carbamazepine (103, 121) increase au-tophagy activity by decreasing intracellular level of inositol.

CA2�-CAMP-PLC�-IP3 PATHWAY. Intracellular Ca2� and cAMPnegatively regulate autophagy by increasing intracellular ino-sitol level (94). In addition, calpain activated by elevated levelof intracellular Ca2� inhibits autophagy by cleavage of Atg5(94, 99). Antihypertensive drugs including Ca2� receptorblockers (9, 121, 129) or imidazolin receptor agonists (98, 121)induce autophagy by decreasing cAMP.

Degradation steps of autophagy. Inhibiting formation ofautolysosome and lysosomal protease are also important tar-gets to inhibit autophagy (Fig. 1). Chloroquine is a lysosomo-tropic and inhibits the fusion of autophagosome and lysosome(4, 12). Cystatin B is a potent inhibitor of cystatin protease(cathepsin B) in lysosome (99).

Other pathways. BCL-2 homology 3 (BH3) mimics inhibitthe interaction of BCL-2 and BCL-x with Beclin 1, an inhib-itory complex for autophagy (66) (Fig. 1). Resveratrol inducesautophagy by activating sirtuin 1 and inhibits P70 S6 kinase (8,43, 82). There are several compounds or drugs such as L-

Xenophagy

Mitophagy

Aggrephagy

Lipophagy

COPD

Lung cancer

IPF

PAH

Lung infection

Sepsis

Promoting apoptosis

Regulating fibroblast activation and proliferation

Regulating vascular cell proliferation

Regulating inflammatory response (i.e. inflammasome)

Promoting or inhibiting replication of microbes in host cells

Promoting survival and proliferation of cancer cells

Cystic fibrosis

Clearance of protein aggregate and regulating oxidative stressLAM

Promoting survival and proliferation of LAM cells

Tumor suppression

Fig. 3. Diverse effects of autophagy in lungdiseases. The functional modes of autophagyat cellular levels include eliminating intracel-lular microbes (xenophagy), dysfunctionalmitochondria (mitophagy), and protein ag-gregate (aggrephagy). Autophagy also regu-lates lipid metabolism (lipophagy). Au-tophagy is involved in pathogenesis of vari-ous lung diseases. The roles of autophagy ineach disease are shown. In lung cancer, au-tophagy may prevent tumorigenesis, whereasautophagy can promote survival and prolif-eration of tumor cells. In infectious diseases,the roles of autophagy are dependent on mi-crobes. Autophagy may promote bacterialkilling and inhibit intracellular survival ofmicrobes such as Mycobacterium tuberculo-sis. On the other hand, autophagy may pro-mote replication and survival of microbessuch as H5N1.




http://www.clinicaltrials.gov/ct2/results?term=autophagy

http://www.clinicaltrials.gov/ct2/results?term=autophagy

Table 4. Drugs that modulate autophagy activity

Drugs Clinical Application or Pharmacological Class Mechanism of Action in Autophagy Pathway References

Autophagy inducersRapamycin and analogs Immunosuppressant for preventing rejection

in organ transplant and coronary stentcoating for anti-proliferation

Inhibit mTORC1 92, 95, 121

Amidarone Class III antiarrhytmic Inhibits mTORC1 or upstream inmTOR pathway

9

Nicosamide Antiparasitic Inhibits mTORC1 or upstream inmTOR pathway

9

EGFR antagonists (erltinibhydrochlorine)

Anticancer Inhibit PI3K-Akt-mTOR pathway 30, 36

Resveratrol (Stilbenoids) Supplementary diet. Secondary products ofheartwood formation in trees that act asphytoalexins

Activates sirtuin 1 (histone deacetylase)and inhibits S6 kinase

8, 43, 82

Suberoylanilide hydroxamic acid Anticutaneous T cell lymphoma Inhibits mTOR 13Dexamethasone (glucocorticoide) Anti-inflammatory or immunosuppressant Upregulates PML and Akt

dephosphorylation31, 55

Metformin Antidiabetic Upregulates AMPK (and ULK1phosphorylation)

48, 70

Verapamil, nicardipine, nimodipine(L-type Ca2� channel blockers)

Vasodilator Reduce intracellular Ca2� levels 9, 121, 129

Sodium valproate Anticonvulsant Inhibits histone deacetylase and reducesintracellular Ca2� levels

103, 121

Clonidine Antihypertensive, treatment for ADHD andanxiety/panic disorder

Reduces cAMP levels (Imidazoline-1receptor agonist)

121

Rilmenidine Antihypertensive Reduces cAMP levels (imidazoline-1receptor agonist)

98, 121

Lithium, L-690 330 Mood-stabilizing drug Inhibit IMPase and reduce inositol andIP3 levels

103

Carbamazepine Antiepileptics Reduces inositol and IP3 levels 103, 121Tamoxifen (estrogen receptor

antagonist)Anti-breast cancer and anti-bipolar disorder Accumulation of sterol, Increases

Beclin 121, 99

Statin (HMG-CoA reductaseinhibitor)

Lower cholesterol Depletes geranylgeranyl diphosphate 83

Vitamin D3 Supplementary diet to reduce the risk offractures and falls

Increases Beclin 1 expression byupregulating cathelicidin

128

BH3 mimetics (ABT-737) Undergoing clinical trial for ovarian cancer Disrupts interaction between the BH3domain of Beclin 1 and theantiapoptotic proteins BCL-2

66

Carbon monoxide (at 250 ppm) Vasodilator, anti-inflammation andantiproliferation Undergoing clinicaltrials for idiopathic pulmonary fibrosisand sepsis

Increases mitochondrial reactive oxygenspecies

58

Minoxidil (potassium channelopener)

Vasodilator Unknown 121

Salbutamol (�2 adrenergic receptoragonist)

Treatment for asthma by smooth musclerelaxant

Unknown 6

Isoniazid, pyrazinamide Antibiotics for tuberculosis AMPK and intracellular Ca2�

dependent49

Autophagy inhibitorsAzithromycin Macrolide antibiotic Inhibits fusion of autophagosome and

lysosome96

Chloroquine/hydroxychloroquine Treatment for RA, SLE, and malariaUndergoing clinical trials for variouscancers

Inhibits fusion of autophagosome andlysosome

4, 12

Vinblastine Anticancer Inhibits microtubule formation 89IL-4 Undergoing clinical trial for tuberculosis by

blocking IL-4Activates Akt and STAT6 37

IL-13 Undergoing clinical trial for advancedcancers

Activates Akt and STAT6 37

mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; PI3K, phosphoinositide 3-kinase; NF-�B, nuclear factor-�B, AMPK, AMP-activatedprotein kinase: ULK1, UNC51-like kinase; cAMP, cyclic AMP; IP3, inositol-(1,4,5)-trisphosphate; IMPase, inositol monophosphatase; BH3, BCL-2 homology3; BCL-2, B cell lymphoma 2; EGFR, epidermal growth factor receptor; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; STAT6, signal transduction andtranscription 6; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; PML, promyelocytic leukemia protein; ADHD, attention deficit hyperactivitydisorder; IL-4, interleukin 4; IL-13, interleukin 13. For details of clinical trials, please see the website http://www.clinicaltrials.gov/.




NAME (104), trehalose (102), or �2-adrenergic receptor ago-nist (6) whose mechanisms to modulate autophagy are stillunclear (Table 4). Further studies will be needed to identifyconcise pathways by which those drugs or compounds modu-late autophagy.

Since there are several pathways regulating autophagy, itis possible that some drugs can stimulate both proautophagyand antiautophagy pathways. For example, although lithiumhas been shown to activate autophagy via an mTOR-inde-pendent pathway, lithium also inhibits GSK-3�, which canactivate mTOR and lead to inhibition of autophagy (27)(Fig. 2). In addition, some drugs may inhibit Ca2� channel(activating autophagy by mTOR-independent pathways) andalso inhibit EGFR receptor (inhibiting autophagy by acti-vating mTOR), which offset both the cellular signalingpathways against each other and result in no autophagyactivation being observed. Thus it is also important toanalyze the effect of drugs on the individual pathways toregulate autophagy. These profiles may lead to developingthe combined use of different types of autophagy modula-tors such as lithium and rapamycin, which can gain thegreater activation of autophagy (27) (Fig. 2).

Autophagy Modulation as a Potential TherapeuticIntervention for Lung Diseases

Although various classes of clinically used drug modulateautophagy pathways (Table 4), it is still unclear in many caseswhether the therapeutic effects of those drugs arise fromautophagy or autophagy-independent pathways. Even if au-tophagy is involved, there is still an important question remain-ing whether modulating autophagy by drugs contributes totheir beneficial pharmacological effects. For example, met-formin, one of the most commonly used drugs for treatment ofType 2 diabetes, induces autophagy (48, 70). The role ofautophagy on the pathogenesis of diabetes is still controversial(94). Antidiabetic effects of metformin may be mediated by

metformin-induced autophagy activation or conversely may becompromised by the autophagy activation. Recent clinicalstudies show new possible interventions using drugs for lungdiseases including COPD (3) and tuberculosis (67). Azithro-mycin, a macrolide antibiotic, inhibits the frequency of exac-erbation of COPD (3). High-dose supplement of vitamin D3(VitD3) reduces time of sputum culture conversion of Myco-bacterium tuberculosis (Mbt) in patients with polymorphism ofVitD3 receptor (67). Although the concise mechanisms bywhich these drugs exert the beneficial effects are still unclear,they commonly modulate autophagy activity. The pharmaco-logical effect of azithromycin is similar with bafilomycin A1,a family of macrolide antibiotic and also a well-known au-tophagy inhibitor (96) (Fig. 1). The beneficial roles of azithro-mycin on COPD may be associated with the inhibitory effect ofazithromycin on autophagy, since autophagy gene deficiencydisplays protective effects in a mouse model of COPD. Incontrast, VitD3 inhibits replication of Mbt by increasing au-tophagy activity (128). Interestingly, drugs can also increaseautophagy activity in specific pathological condition. Isonia-zid, an antibiotic for Mbt, induces autophagy in macrophagesinfected with Mbt, rather than in uninfected cells (49). Al-though further studies are needed to clarify how autophagy isinvolved in the beneficial effects of those drugs, these reportssuggest that pharmacological manipulation on autophagy ac-tivity can serve as a potential therapeutic strategy in lungdiseases. In addition, investigating the effect of drugs onautophagy activity can reveal the new mechanism of diseasepathogenesis and previously unknown pharmacological actionsof the drugs.

It is also important to explore the possibility to use au-tophagy modulators as an intervention for treatment of lungdiseases. For example, an intervention to enhance autophagyactivity may have beneficial effects in lung diseases such astuberculosis or CF, since the effective drugs reported for thosediseases activate autophagy (32, 67). However, the caution

Table 5. Molecules or compounds that modulate autophagy activity

Molecules or Compounds Mechanism of Action in Autophagy Pathway References

Autophagy activatorsTorin1 Directly inhibits both mTORC1 and mTORC2 115PP242 Inhibits mTORC1 26PI103 hydrochloride Highly selective class I PI3K inhibitor and ATP-competitive mTOR inhibitor 222=,5=- Dideoxyadenosine Reduces cAMP levels (Adenynyl cyclase inhibitor) 121Xestospongin B IP3R antagonist 117Spermidine Postulated to affect expression of Atg genes 24Tat-beclin1 A cell permeable peptide derived from a region (267–284) of Beclin 1 107Calpastatin, calpeptin Inhibit calpain 121Trehalose (an �-linked disaccharide mTOR independent 102L-NAME (inhibition of NOS and decrease of NO

production)Unknown 104

GGTi-298 (inhibition of geranylgeranyl transferase 1) P53 dependent 29Autophagy inhibitors

Spautin-1 Lowers Beclin 1 levels by promoting its ubiquitination 633-MA Inhibits class III PI3K 100Pepstatin A Inhibits aspartyl protease (cathepsin D) 113Cystatin B Inhibits cystatine protease (cathepsin B) 99Leupeptin Inhibits serine and cysteine proteases 38Bafilomycin A1 Inhibits V-ATPase; Inhibits fusion of autophagosome and lysosome 92, 123Nocodazole Inhibits microtubule formation 97, 119

IP3R, inositol-(1,4,5)-trisphosphate receptor; L-NAME, N-L-arginine methyl ester; 3-MA, 3-methyladenine; NO, nitric oxide; NOS, nitric oxide synthase; Atg,autophagy-related; V-ATPase, vacuolar ATPase.




should be taken for this strategy. Autophagy has diverse effectsdepending on diseases or pathological conditions. Althoughactivation of autophagy is beneficial for Mbt infection, theactivated autophagy may also exert protective roles for mi-crobes such as HIV or HBV (99). If patients with tuberculosishave latent infections with HIV or HBV, it is possible thatautophagy activation promotes replication and survival ofthose microbes in the patients (99). On the other hand, au-tophagy inducers may have the additive beneficial effects inpatients enrolled in clinical trials for treatment of tuberculosis,such as patients with Parkinson disease, where autophagy playsprotective roles (99). Finally, therapeutic interventions bymodulating autophagy can add to the current therapeutic strat-egies, rather than be used solely. For example, a recent clinicaltrial for lung cancer utilizes the combined interventions oferlotinib, a tyrosine kinase inhibitor, and chloroquine (125).The anticancer mechanism of erlotinib is likely to be au-tophagy independent (36, 57). Inhibiting autophagy by chlo-roquine enhances anticancer effect of erlotinib by minimizingprosurvival effect of erlotinib-mediated autophagy in tumorcells (36, 57, 120, 125). Modulating autophagy activity mayenhance the beneficial roles of autophagy or minimize theadverse effect of autophagy on each disease. Furthermore,adding autophagy modulators may improve the current phar-macological effect of drugs used for the treatment.

Although clinical trials for investigating the therapeuticeffects of autophagy modulators are most desirable to ob-serve the usefulness of drugs, cohort studies to examine theeffect of autophagy modulator on patients’ prognosis wouldbe more accessible. For example, there are a number ofpatients with COPD who use autophagy modulators such asCa2� blockers, statin, or metformin for their medication;these drugs may influence patients’ quality of life, lungfunction, and frequency of acute exacerbation. In addition,these studies may help to identify drugs which are suitablefor the clinical trials.

CONCLUSION

Until now more than 20 different classes of drug used fortreatment have been identified as autophagy modulators (9,129) (Table 4). Moreover, the new-class autophagy modulatorswith improved specificity and effectiveness have been devel-oped for human disease indications as potential therapeutics(Table 5). Given the important roles of autophagy in variouslung diseases (Fig. 3 and Tables 2 and 3), utilizing autophagymodulators may improve the therapeutic effects of the currentinterventions in various lung diseases. However, there are alsoseveral important points to keep in mind. Although similarsituations are observed in other medical interventions, effectiveand less-invasive methods to monitor autophagy activity forpatients (e.g., biomarkers) are not currently available. Sec-ondly, since autophagy is such a fundamental cellular processthat affects various pathophysiological conditions, it is plausi-ble that modulating autophagy causes other untoward effectsthat may be clinically deleterious. Finally, the autophagy mod-ulators listed in Tables 4 and 5 may have off-target effects inaddition to modulating autophagy. Nonetheless, recent clinicalstudies using autophagy modulators (3, 32, 67) and ongoingclinical trials (more than 15 active trials targeting autophagy)still suggest that autophagy modulators unravel new avenues in

therapeutic interventions of various human diseases includinglung diseases. Although further development of assays forautophagy activity and drugs with improved selectivity areneeded, the day when we use autophagy-modifying drugs totreat lung disease may come sooner than later.

ACKNOWLEDGMENTS

The authors thank Jeffrey Adam Haspel for critical review of this manu-script.

GRANTS

This study is supported by NIH T32HL007633-27 (K. Nakahira) and NIHPO1 HL105339 (A. M. K. Choi) and R01 HL05530 (A. M. K. Choi).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

K.N. and A.M.K.C. conception and design of research; K.N. draftedmanuscript; K.N. and A.M.K.C. edited and revised manuscript; K.N. andA.M.K.C. approved final version of manuscript.

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autophagy a potential therapeutic target in lung diseases

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