Autophagy Modulators in Cancer: Focus on Cancer Treatment

life

Review Autophagy Modulators in Cancer: Focus on Cancer Treatment

Hye Jin Nam

Drug Discovery Platform Research Center, Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea; [email protected]; Tel.: +82-42-860-7456

Abstract: Uncontrolled autophagy has been associated with the development and progression of various cancers that are resistant to cancer therapy. Therefore, many efforts to modulate uncontrolled autophagy as a cancer treatment have been attempted, from basic science to clinical trials. However, it remains difﬁcult to equally apply autophagy modulators to cancer therapy because autophagy is a double-edged sword in cancer: it can be tumor-suppressive or tumor-protective. Therefore, the precise mechanisms of autophagy modulators and their varied responsiveness to each cancer type should be addressed in detail. This study will describe the precise mechanisms of developing various autophagy modulators, their current therapeutic applications and future perspectives.

Keywords: autophagy; autophagy enhancer; autophagy inhibitor; cancer treatment

1. Introduction Autophagy (self-eating) is defined as a homeostatic process that enables the lysosomal degradation of unnecessary or dysfunctional organelles or proteins [1]. There are three types of autophagy: microautophagy, chaperone-mediated autophagy (CMA) and Citation: Nam, H.J. Autophagy macroautophagy [2]. Microautophagy is involved with the direct uptake of cargo by the Modulators in Cancer: Focus on lysosome, and little is known about its mechanism [3]. CMA is a selective process and Cancer Treatment. Life 2021, 11, 839. involves substrates with specific motifs (KFERQ or similar KFERQ generated by modi- https://doi.org/10.3390/ fication). Heat-shock cognate chaperone of 70 kDa (Hsc70) recognizes the KFERQ motif life11080839 of the substrate and binds and moves together to the lysosomal membrane. Chaperone selectively binds to LAMP2A on the lysosomal membrane and internalizes the substrate Academic Editors: Evangelos Koustas into the lysosome through LAMP2A. Thus, CMA has not only selective substrates but also and Panagiotis Sarantis specific machinery components [4]. Macroautophagy, usually considered as “autophagy” and thereafter referred to as Received: 28 July 2021 autophagy, is primarily induced by nutrient starvation (e.g., glucose starvation or amino Accepted: 13 August 2021 acid starvation) [5]. Cells deal with starvation by recycling cellular organelles and proteins Published: 17 August 2021 and then generating energy. Autophagy is initiated with the formation of a phagophore, which is also known as an isolation membrane. The membrane source of phagophore has Publisher’s Note: MDPI stays neutral been proposed to originate from the smooth endoplasmic reticulum (ER), mitochondria or with regard to jurisdictional claims in plasma membrane [6]. The conjugation of LC3 to phosphatidylethanolamine (PE) yields published maps and institutional affil- iations. recruitment to the nascent phagophore structure [7]. The precursor proLC3 is cleaved by Atg4 protease into LC3-I with a C-terminal glycine residue, and the C-terminus is then con- jugated to the polar head of PE by Atg complex to form LC3-II. The lipidated LC3 (LC3-II) is localized on the membrane’s inner and outer sides and contributes to the expansion and closure of the phagophore. As LC3-II migrates faster than LC3-I in gel electrophoresis ex- Copyright: © 2021 by the author. periments, it is widely used as a marker for assessing autophagy induction [5]. Phagophore Licensee MDPI, Basel, Switzerland. grows, engulfs cytosolic components and closes to become an autophagosome. Then, This article is an open access article the autophagosome fuses with a lysosome and degrades sequestered contents. Cellular distributed under the terms and conditions of the Creative Commons response to nutrient deprivation is thought to be non-selective autophagy and usually Attribution (CC BY) license (https:// involves random uptake of cytoplasm. Conversely, selective autophagy selectively elimi- creativecommons.org/licenses/by/ nates specific cellular components, such as damaged organelles, or protein aggregates [8]. 4.0/). Moreover, pathogens are targeted for degradation by selective autophagy. Cargo-specific

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Life 2021, 11, 839 2 of 19 or protein aggregates [8]. Moreover, pathogens are targeted for degradation by selective autophagy. Cargo-specific names have been used to classify the various types of selective autophagynames have (e.g., been usedpexophagy, to classify mitophagy, the various typesaggrephagy, of selective glycophagy, autophagy (e.g., lipophagy pexophagy, ribophagy andmitophagy, xenophagy) aggrephagy, [9,10]. In glycophagy, particular, lipophagy mitophagy ribophagy is strongly and associated xenophagy) with [9, 10tumorigenesis]. In [11,12]particular, (Figure mitophagy 1). is strongly associated with tumorigenesis [11,12] (Figure1).

FigureFigure 1. SchematicSchematic diagramdiagram ofof autophagicautophagic progression. progression. There There are are three three types types of of autophagy autophagy in in mam- malianmammalian cells: cells:macroautophagy, macroautophagy, micr microautophagyoautophagy and and chaperone-mediated chaperone-mediated autophagy. autophagy. When When macro- autophagymacroautophagy is induced, is induced, cytoplasmic cytoplasmic materials materials ar aree engulfed engulfed by by double double membranesmembranes (phagophore), (phagophore), closedclosed (autophagosome), fused fused with with the the lysosome lysosome (autolysosome) (autolysosome) and degraded.and degraded. The KFERQ The KFERQ motif motif isis recognized recognized byby Hsc70Hsc70 and and binds binds with with LAMP2A LAMP2A in CMA.in CMA. LAMP2A LAMP2A mediates mediates the translocation the translocation of of thethe substrate substrate intointo lysosomes. lysosomes. Microautophagy Microautophagy directly directly uptakes uptakes the cytoplasmic the cytoplasmic cargo. cargo.

Autophagy-modulating drugs have been increasingly used in clinical trials; simul- Autophagy-modulating drugs have been increasingly used in clinical trials; simulta- taneously, many phenotypic screens are being conducted for new drug discovery, which neously,can modulate many autophagy phenotypic for therapeuticscreens are purposes. being conducted However, itfor is notnew easy drug to screendiscovery, and which canidentify modulate new autophagy-modulating autophagy for therapeutic chemicals. purposes. The brief However, analysis of theit is role not of easy autophagy to screen and identifymodulators new through autophagy-modulating LC3-II or autophagosome chemicals. accumulation The briefduring analysis drug of the screening role of may autophagy modulatorsbe misleading. through An increase LC3-II in LC3-IIor autophagosome or autophagosomes accumulation can be interpreted during drug as autophagy screening may beinduction misleading. and blockade: An increase an increase in LC3-II in the or rateauto ofphagosomes autophagosome can formation be interpreted or a decrease as autophagy inductionin the rate and of autophagosome blockade: an increase clearance in following the rate of fusion autophagosome with lysosomes. formation To solve or this a decrease inproblem, the rate autophagic of autophagosome flux probes, suchclearance as GFP-LC3-RFP-LC3 following fusion∆G or with mRFP-GFP-tagged lysosomes. To LC3, solve this were developed and used for high-throughput screening to identify autophagy modula- problem, autophagic flux probes, such as GFP-LC3-RFP-LC3ΔG or mRFP-GFP-tagged tors [13,14]. Autophagy flux was quantitatively monitored by calculating the GFP/RFP LC3,ratio were or LC3 developed puncta: the and decreased used for GFP/RFP high-throughput ratio is detected screening by the to autophagyidentify autophagy inducer, mod- ulatorswhereas [13,14]. the autophagy Autophagy inhibitor flux detects was quantitati the increasedvely GFP/RFP monitored ratio. by However,calculating an the assay GFP/RFP ratiousing or an LC3 autophagic puncta: flux the probedecreased has a GFP/RFP limitation, ra astio it doesis detected not distinguish by the autophagy whether the inducer, whereasautophagy the modulators autophagy inhibit inhibitor the initiation detects the step in orcreased lysosomal GFP/RFP fusion stepratio. [15 However,]. To apply an assay usingautophagy an autophagic modulators flux as therapeutics, probe has a more limitation, research as beyond it does screening not distinguish is required whether to the autophagydetermine which modulators steps of inhibit autophagy the areinitiation regulated step by or these lysosomal chemicals. fusion Through step various[15]. To apply autophagyautophagy screeningmodulators methods, as therapeutics, researchers more are discovering research beyond novel autophagy screening modulators is required to de- and/or adding new functions as autophagy modulators to existing drugs [15]. termine which steps of autophagy are regulated by these chemicals. Through various au- tophagy2. Autophagy: screening Tumor methods, Suppressor researchers or Promoter? are discovering novel autophagy modulators and/orIn normaladding cells, new autophagy functions contributesas autopha togy the modulators maintenance to of existing homeostasis. drugs Autophagy [15]. is a mechanism to deal with starvation, but it also plays an essential role in removing substances that can be toxic to cells. For instance, autophagy is induced in response to

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reactive oxygen species (ROS) to remove them and protect the cells from apoptosis, whereas autophagy impairment causes the accumulation of oxidative stress [16,17]. Moreover, since ROS is involved in DNA damage and genetic instability, the removal of ROS by autophagy may be critical for blocking the transformation of normal cells [18,19]. However, in cancer cells, the role of autophagy has remained controversial. Autophagy has been reported to have both antitumor and tumor-promoting effects in cancers. Since the role of autophagy varies according to the cancer stage and tumor type, it is necessary first to check how autophagy is dysregulated in target cancers. Due to the opposite effects of autophagy, either inhibitors or inducers of autophagy could be exploited for cancer therapy depending on cancer context, respectively. In the early stages of cancer development, autophagy is believed to play a protective role against cancer initiation. Inhibition of autophagy or defects of autophagy can lead to impaired removal of toxic materials such as damaged organelles, unfolded proteins or ROS. Here, inducing autophagy might serve as a cancer prevention or treatment. Additionally, Beclin1, which is the core subunit of the PI3K complex and is involved in initiation of autophagosome [20], heterozygote mice exhibited spontaneous development of malignant tumors, indicating that autophagy inhibition can induce tumorigenesis. Furthermore, decreased expression of autophagic genes (Atg5, Beclin1, Atg7) and autophagic activity was observed in hepatocellular carcinoma (HCC) [21]. In glioblastoma (GBM), low levels of ULK2 transcripts by DNA methylation were reported [22]. ULK2 overexpression induced autophagy and inhibited astrocyte transformation and tumor growth in glioblastoma. Altogether, these studies indicate that autophagy genes function as tumor suppressors in several tumors. However, tumor-promoting effects of autophagy are more prominent in various cancers. Once the carcinogenic phenotype is established, cancer cells exploit autophagy mechanisms to satisfy their energy requirements. Particularly, autophagy contributes to the adaptation and survival of cancer cells in unfavorable conditions such as hypoxia or nutrient-deficient conditions [23]. Therefore, inducing autophagy in this situation may rather promote tumor progression. Notably, enhanced autophagy flux was observed in various tumors. Additionally, autophagy has also been determined to promote the invasion of hepatocellular carcinoma cells through the activation of epithelial mesenchymal transition (EMT) [24]. Autophagy is also associated with the metastatic ability of pancreatic cancer [25]. These studies suggest that autophagy is critical for cancer metastasis [24]. However, it was reported that autophagy induction in glioblastoma cells rather impairs migration and invasion [26]. Interestingly, SNAIL, which is responsible for glioblastoma cell move- ment, was degraded upon autophagy induction. This is another example showing that autophagy does not have the same effect on all cancer types. Autophagy is also related to cancer stem cells (CSCs) in various cancers such as breast, ovarian, liver and brain cancer. Basal levels of autophagy are required to maintain the balance between pluripotency and differentiation in CSCs. Intriguingly, changes in the basal levels of autophagy by either autophagy inducer or inhibitor can result in a decrease in pluripotency or an increase in differentiation and senescence in CSCs [27]. Several studies have already reported that autophagy flux is enhanced in various CSCs, and, in this case, suppressing autophagy has a positive effect in terms of cancer treatment. In mammosphere conditions, a culture system in which mammary CSCs/progenitor cells can be propagated, a greater autophagy flux was displayed than in normal adher- ent culture conditions [28]. The knockdown of Beclin1 impaired CSC maintenance and proliferations [28]. Additionally, ovarian CSCs have enhanced autophagic flux, and the inhibition of autophagy decreased the self-renewal ability and chemoresistance of ovarian CSCs [29]. It has been reported that the enhanced autophagy flux in liver CSCs contributes to the adaptation of liver CSCs to the tumor microenvironment, such as hypoxia and nutrient-deficient conditions [30]. Therefore, autophagy inhibitors may make liver CSCs Life 2021, 11, x FOR PEER REVIEW 4 of 20

Life 2021, 11, 839 4 of 19 contributes to the adaptation of liver CSCs to the tumor microenvironment, such as hypoxia and nutrient-deficient conditions [30]. Therefore, autophagy inhibitors may make liver CSCs difficult to survive in an unfavorable tumor microenvironment, which may difﬁculthelp improve to survive anticancer in an unfavorable therapeutic tumoreffects. microenvironment, which may help improve anticancerCSC resistance therapeutic to effects.chemotherapy is also associated with autophagy [31]. Many studies haveCSC shown resistance that combining to chemotherapy cytotoxic is alsodrugs associated and autophagy with autophagy inducers [31 or]. Manyinhibitors studies in- havecreases shown the thatsensitivity combining of CSCs. cytotoxic For drugsinstance, and autophagyautophagy inducersinduction or inhibitorsby rapamycin increases pro- the sensitivity of CSCs. For instance, autophagy induction by rapamycin promoted cell moted cell differentiation and made GSCs sensitive to radiation therapy [32]. Azathio- differentiation and made GSCs sensitive to radiation therapy [32]. Azathioprine, an im- prine, an immunosuppressant for rheumatoid arthritis or Crohn’s disease, induces au- munosuppressant for rheumatoid arthritis or Crohn’s disease, induces autophagy through tophagy through mTORC1 [33] and also sensitizes GBM to chemotherapy or radiotherapy mTORC1 [33] and also sensitizes GBM to chemotherapy or radiotherapy [34]. In terms of [34]. In terms of autophagy inhibition, chloroquine was able to increase the chemosensi- autophagy inhibition, chloroquine was able to increase the chemosensitivity of glioma cells tivity of glioma cells to temozolomide [35]. Altogether, autophagy modulators can be used to temozolomide [35]. Altogether, autophagy modulators can be used for cancer treatment for cancer treatment because the imbalance in autophagy leads to cancer cell death. because the imbalance in autophagy leads to cancer cell death.

3.3. ModulatorsModulators ofof InitialInitial AutophagyAutophagy andand CancerCancer TreatmentTreatment TheThe initiationinitiation stagestage ofof autophagyautophagy isis controlledcontrolled byby aa seriesseries ofof autophagy-relatedautophagy-related genesgenes (ATGs)(ATGs) [[36].36]. Specifically, Speciﬁcally, AMPK, AMPK, mTOR, mTOR, and and unc-51-like unc-51-like kinase kinase 1 1(ULK1, (ULK1, also also known known as asAtg1) Atg1) play play a central a central role role in inautophagy autophagy initiation initiation [37] [37 (Figure] (Figure 2).2 Under). Under conditions conditions of nu- of nutrienttrient sufficiency, sufﬁciency, active active mTOR mTOR inhibits inhibits AM AMPKPK activation activation and phosphorylates ULK1ULK1 atat thethe S758S758 sitesite inin humanshumans (Ser757(Ser757 inin mouse)mouse) [38[38].]. PhosphorylationPhosphorylation ofof ULKULK S758S758 waswas alsoalso foundfound toto inhibitinhibit the the interaction interaction between between ULK1 ULK1 and and AMPK. AMPK. Upon Upon nutrient nutrient starvation starvation condi- con- tions,ditions, activated activated AMPK AMPK phosphorylates phosphorylates ULK1 ULK1 at various at various sites sites and initiatesand initiates autophagy autophagy [39]. Additionally,[39]. Additionally, autophagy autophagy is accelerated is accelerated by AMPK-induced by AMPK-induced phosphorylation phosphorylation of Raptor of Rap- and TSC1,tor and which TSC1, is which associated is associated with mTOR with inhibition mTOR inhibition [40]. Firstly, [40]. an Firstly, autophagy an autophagy activator acti- can regulatevator can autophagy regulate autophagy initiation andinitiation serve and as a serve treatment as a treatment for cancer. for This cancer. is described This is de- in Figurescribed2 andin Figure Table 12. and Table 1.

FigureFigure 2.2. SignalingSignaling cascadescascades andand modulatorsmodulators thatthat regulateregulate autophagyautophagy initiation.Theinitiation.The energyenergy sensors,sensors, mTORmTOR andand AMP-AMP- activatedactivated kinase kinase (AMPK), (AMPK), are are the the primary primary regulators regulators of of autophagy. autophagy. AMPK AMPK activation activation or mTORor mTOR inhibition inhibition induces induces autophagy. autoph- Activatedagy. Activated AMPK AMPK phosphorylates phosphorylates ULK1/2, ULK1/2, and autophagy and autophagy is then is initiated. then initiated. Various Various autophagy autophagy modulators modulators and their and targets their aretargets shown are in shown the ﬁgure. in the figure.

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Life 2021, 11, x FOR PEER REVIEW 5 of 20 Table 1. Autophagy modulators associated with autophagy initiation. Life 2021, 11, x FOR PEER REVIEW 5 of 20 Life 2021, 11, x FOR PEER REVIEW Table 1. Autophagy modulators associated with autophagy initiation. 5 of 20 Life 2021, 11 , 839 Table 1. Autophagy modulators associated with autophagy initiation.Clinical Trials in5 ofProgress 19 or Compound Mechanism/UseTable 1. Autophagy modulators associated Structure with autophagy initiation.ClinicalCompleted Trials for in Cancer Progress Treatment or Compound Mechanism/UseTable 1. Autophagy modulators Structure associated with autophagyCompleted initiation.Clinical Trials for Cancer in Progress Treatment or Table 1. Autophagy modulators associated with autophagy initiation. (NCT Number) Compound Mechanism/Use Structure CompletedClinical(NCT Trials for Number)Cancer in Progress Treatment or Table 1. Autophagy modulators associated with autophagy initiation. ClinicalPhase 3 in Trials breast in cancer Progress (NCT01101438) or Compound Mechanism/Use Structure PhaseCompletedClinical 3 in breast(NCT Trials for Cancercancer Number) in Progress (NCT01101438) Treatment or Compound Mechanism/UseAMPK Structure CompletedPhase for 3 Cancerin colorectal Treatment cancer Compound Mechanism/UseAMPK Structure ClinicalPhaseCompleted Phase3 Trials in (NCTbreast 3 infor in Progress colorectalNumber)Cancercancer or(NCT01101438) Treatment cancer CompoundMetformin activation/autophagy Mechanism/Use Structure Completed for Cancer(NCT02614339) Treatment AMPK Phase 3Phase in breast(NCT 3 in cancer colorectalNumber) (NCT01101438) cancer Metformin activation/autophagyinducer (NCTPhase(NCT Number)(NCT02614339) 3 in Number) endometrial cancer Metformin activation/autophagyAMPKinducer Phase Phase3 in breast 3(NCT02614339) in endometrial colorectalcancer (NCT01101438) cancer cancer PhasePhase 3 in 3 inbreast breast(NCT02065687) cancer cancer (NCT01101438) Metformin activation/autophagyAMPKinducer PhasePhase (NCT02614339)33 inin endometrialcolorectal cancer cancer AMPK Phase(NCT01101438) 3(NCT02065687) in colorectal cancer Metformin activation/autophagyinducer Phase 3(NCT02614339) in endometrial cancer Metformin AMPKactivation/autophagyAMPK activation/autophagy activation, AMP Phase 3 in colorectal(NCT02614339)(NCT02065687) cancer Metformin inducer Phase 3(NCT02065687) in endometrial cancer AICAR AMPKanalog/autophagy induceractivation,inducer AMP Phase(NCT02614339) 3 in endometrialPreclinical cancer AICAR AMPKanalog/autophagy activation, AMP Phase 3 in endometrial(NCT02065687)Preclinical cancer inducer (NCT02065687)(NCT02065687) AICAR AMPKanalog/autophagy activation,inducer AMP Preclinical AICAR AMPKanalog/autophagy activation, AMP Preclinical AMPK activation,inducer AMP AICAR AMPKanalog/autophagyinducer activation, AMP Preclinical AICARAICAR analog/autophagy PreclinicalPreclinical analog/autophagyinducer inducer inducerAMPK A-769662 activation/autophagyAMPK Preclinical AMPK A-769662 activation/autophagyinducer Preclinical A-769662 activation/autophagyAMPKinducer Preclinical A-769662 AMPKactivation/autophagy activation/autophagyAMPK Preclinical A-769662 AMPKinducer Preclinical A-769662 activation/autophagyinducerinducer Preclinical A-769662 activation/autophagy Preclinical inducer inducer AMPK

GSK621 activation/autophagyAMPK Preclinical AMPK GSK621 activation/autophagyinducer Preclinical AMPK activation/autophagy GSK621GSK621 activation/autophagyAMPKinducer PreclinicalPreclinical GSK621 activation/autophagyAMPKinducerinducer Preclinical AMPK GSK621 activation/autophagyinducer Preclinical GSK621 activation/autophagy Preclinical inducer inducer

mTOR Rapamycin/siroli mTOR Approved to treat Rapamycin/siroli inhibition/autophagy Approved to treat mus mTOR inhibition/autophagymTOR Approvedlymphangioleiomyomatosis to treat (LAM) Rapamycin/sirolimusRapamycin/siroli inhibition/autophagyinducer Approved to treat mus inhibition/autophagymTORinducer lymphangioleiomyomatosislymphangioleiomyomatosis (LAM) (LAM) Rapamycin/sirolimus inducer lymphangioleiomyomatosisApproved to treat (LAM) inhibition/autophagymTORinducer Rapamycin/sirolimus mTOR lymphangioleiomyomatosisApproved to treat (LAM) Rapamycin/siroli inhibition/autophagyinducer Approved to treat mus inhibition/autophagy lymphangioleiomyomatosis (LAM) mus inducer lymphangioleiomyomatosis (LAM) inducer

mTOR inhibition, Temsirolimus/C Approved for the treatment of renal cell mTORmTOR inhibition, Approved for the treatment of renal Temsirolimus/CCI-779Temsirolimus/C rapalog/autophagy Approved for the treatment of renal cell CI-779 rapalog/autophagymTOR inhibition, inducer cell carcinomacarcinoma Temsirolimus/C rapalog/autophagyinducer Approved for the treatment of renal cell CI-779 mTORrapalog/autophagy inhibition, carcinoma Temsirolimus/CCI-779 inducer Approved for thecarcinoma treatment of renal cell rapalog/autophagymTORinducer inhibition, Temsirolimus/CCI-779 mTOR inhibition, Approved for thecarcinoma treatment of renal cell Temsirolimus/C rapalog/autophagyinducer Approved for the treatment of renal cell CI-779 rapalog/autophagy carcinoma CI-779 inducer carcinoma inducer

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Life 2021, 11, x FOR PEER REVIEW 6 of 20 Table 1. Cont. Life 2021, 11, x FOR PEER REVIEW 6 of 20 Approved for the treatment of renal cell Clinical Trials in Progress or Approved for the treatment of renal cell Compound Mechanism/Use Structure Completedcarcinoma, for Cancer advanced Treatment breast cancer, mTOR inhibition, Approvedcarcinoma,(NCTpancreatic for Number) the advanced treatment cancer, renalbreast of renal cancer, cell Everolimus/RA mTOR inhibition, Approvedcarcinoma, forpancreatic advanced the treatment cancer, breast of renal cancer,renal cell Everolimus/RA rapalog/autophagy angiomyolipoma, tuberous sclerosis D001 mTORrapalog/autophagy inhibition, Approvedcarcinoma,angiomyolipoma,pancreatic for advanced the treatmentcancer, tuberous breast renal of cancer, renal sclerosis cell Everolimus/RAD001 inducer complex, neuroendocrine tumors rapalog/autophagymTOR inhibition,inducer angiomyolipoma,Approvedcarcinoma,complex,pancreatic for advanced neuroendocrine the cancer, tuberoustreatment breast renal sclerosis ofcancer, tumors renal cell Everolimus/RAD001 Approved(NET), for theadvanced treatment gastrointestinal of renal rapalog/autophagymTORinducer inhibition, angiomyolipoma,complex,carcinoma,(NET),pancreatic neuroendocrine advanced advanced cancer,tuberous gastrointestinal breast renal tumorssclerosis cancer, Everolimus/RAD001 cell carcinoma, advancedtumors breast rapalog/autophagymTOR inhibition, angiomyolipoma,pancreatic tuberous cancer, renal sclerosis Everolimus/RA inducer cancer,(NET),complex, pancreatic advanced neuroendocrine cancer,tumors gastrointestinal renal tumors D001 mTOR inhibition, Everolimus/RAD001 rapalog/autophagyinducer angiomyolipoma,(NET),complex,angiomyolipoma, advanced neuroendocrine tuberous gastrointestinal sclerosistuberous tumors sclerosis D001 rapalog/autophagy inducer tumors inducer complex, (NET),complex, neuroendocrine advanced neuroendocrinetumors gastrointestinal tumors tumors (NET), advanced(NET), advanced gastrointestinaltumors gastrointestinal tumors tumors

mTOR inhibition, Ridaforolimus/ mTOR inhibition, Withdrawn from the European Union Ridaforolimus/ rapalog/autophagy Withdrawn from the European Union MK-8669 mTORrapalog/autophagy inhibition, market in 2012 Ridaforolimus/MK-8669 inducer Withdrawn frommarket the European in 2012 Union rapalog/autophagymTOR inhibition,inducer Ridaforolimus/MK-8669 Withdrawnmarket from the in European2012 Union rapalog/autophagymTOR inhibition, Ridaforolimus/ inducer Withdrawn from the European Union Ridaforolimus/MK-MK-8669 mTORmTOR inhibition, inhibition, Withdrawn frommarket the European in 2012 Ridaforolimus/ rapalog/autophagyinducer Withdrawn from the European Union 8669MK-8669 rapalog/autophagyrapalog/autophagy inducer Union marketmarket in 2012 in 2012 MK-8669 inducer market in 2012 inducer

Phase 1 in recurrent gliomas mTORC1/2 Phase(NCT01316809) 1 in recurrent gliomas

AZD8055 inhibition/autophagymTORC1/2 PhasePhase 1 in liver 1 in(NCT01316809) recurrentcancer (NCT00999882) gliomas AZD8055 inhibition/autophagymTORC1/2inducer PhasePhasePhasePhase 1 in 1 1 recurrentin in(NCT01316809)1 advanced inliver recurrent gliomascancer solid gliomas(NCT00999882) tumors Phase 1 in recurrent gliomas AZD8055 inhibition/autophagymTORC1/2inducer Phase(NCT00731263,Phase (NCT01316809)1 in liver (NCT01316809)1 in canceradvanced NCT00973076) (NCT00999882) solid tumors mTORC1/2 PhasePhase 1 in liver 1 in cancer recurrent gliomas AZD8055AZD8055 inhibition/autophagymTORC1/2inducer PhasePhase 1(NCT00731263, in1 inliver(NCT01316809) advanced cancer NCT00973076)(NCT00999882)solid tumors inhibition/autophagy inducer (NCT00999882) AZD8055 inhibition/autophagyinducermTORC1/2 PhasePhase(NCT00731263, 1 1in in liver advanced(NCT01316809) cancer NCT00973076) solid(NCT00999882) tumors Phase 1 in advanced solid tumors AZD8055 inhibition/autophagyinducer PhasePhase 11 inin liveradvanced cancer solid (NCT00999882) tumors (NCT00731263,(NCT00731263, NCT00973076) NCT00973076) inducer (NCT00731263,Phase 1 in advanced NCT00973076) solid tumors (NCT00731263, NCT00973076)

mTORC1/2 Torin 1 inhibition/autophagymTORC1/2 Preclinical Torin 1 inhibition/autophagymTORC1/2inducer Preclinical mTORC1/2 TorinTorin 1 1 inhibition/autophagymTORC1/2inducer PreclinicalPreclinical Torin 1 inhibition/autophagyinhibition/autophagymTORC1/2inducer inducer Preclinical mTORC1/2 Torin 1 inhibition/autophagyinducer Preclinical Torin 1 inhibition/autophagyinducer Preclinical ULK1/2inducer ULK1/2 MRT-68921 inhibition/autophagyULK1/2 Preclinical MRT-68921 inhibition/autophagyULK1/2 Preclinical MRT-68921 inhibition/autophagyinhibitor Preclinical ULK1/2 MRT-68921 inhibition/autophagyinhibitorinhibitor Preclinical MRT-68921 inhibition/autophagyULK1/2 Preclinical inhibitor ULK1/2 MRT-68921 inhibition/autophagyinhibitor Preclinical MRT-68921 inhibition/autophagyULK1 Preclinical ULK1 inhibition/autophagyinhibitorULK1 SBI-0206965SBI-0206965 inhibition/autophagyinhibitor PreclinicalPreclinical SBI-0206965 inhibition/autophagyinhibitorULK1inhibitor Preclinical SBI-0206965 inhibition/autophagyULK1inhibitor Preclinical

SBI-0206965 inhibition/autophagyinhibitorULK1 Preclinical

SBI-0206965 inhibition/autophagyinhibitor Preclinical

inhibitor

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Life 2021, 11, x FOR PEER REVIEW 7 of 20 Table 1. Cont. Life 2021, 11, x FOR PEER REVIEW 7 of 20

Class I and III PI3K Clinical Trials in Progress or Compoundinhibition/autophagyClass Mechanism/Use I and III PI3K Structure Completed for Cancer Treatment 3-Methyladenine inhibitor,inhibition/autophagyClass I andbut autophagyIII PI3K (NCT Number) 3-Methyladenine inhibitor,inhibition/autophagyClass I andbut autophagyIII PI3K Preclinical (3-MA) effects depend on Preclinical 3-Methyladenine inhibitor,inhibition/autophagyClass but I and autophagy III PI3K (3-MA) concentrationeffects depend or oncell Preclinical 3-Methyladenine(3-MA) inhibitor,concentrationeffectsinhibition/autophagy butdepend autophagy or on cell 3-Methyladenine (3-MA) inhibitor,type but autophagy PreclinicalPreclinical (3-MA) concentrationeffects depend or oncell effectstype depend on concentration or cell concentrationtype or cell type Non-specific,type covalent Non-specific,inhibition covalent of Wortmannin Preclinical Non-specific,PI3K/autophagyinhibition covalent of Wortmannin Non-specific, covalent Preclinical Non-specific,PI3K/autophagyinhibitioninhibitor covalentof WortmanninWortmannin inhibition of PI3K/autophagy PreclinicalPreclinical PI3K/autophagyinhibitioninhibitor of Wortmannin inhibitor Preclinical PI3K/autophagyinhibitor Modulatinginhibitor balance betweenModulating mTOR balance and ModulatingbetweenAMPK/autophagy mTOR balance and ModulatingbetweenModulatingAMPK/autophagy mTOR balance balance and between EGCG mTORinducer and AMPK/autophagyor inhibitor Nutraceutical betweenAMPK/autophagy mTOR and EGCGEGCG inducerinducer(different or inhibitor or inhibitor roles (different NutraceuticalNutraceutical AMPK/autophagy EGCG inducerroles(differentdepending depending or inhibitor roles on on Nutraceutical EGCG inducer(differentdependingconcentration) or inhibitor roles on Nutraceutical concentration) (differentdepending roles on concentration) depending on concentration) concentration)mTOR mTOR inhibition/autophagymTOR FisetinFisetin inhibition/autophagy NutraceuticalNutraceutical Fisetin inhibition/autophagyinducermTORinducer Nutraceutical Fisetin inhibition/autophagymTOR Nutraceutical inducer Fisetin inhibition/autophagy Nutraceutical inducer inducer 3.1. AMPK Activator 3.1. AMPK Activator 3.1. AMPK Activator ActivatingActivating AMPK is AMPK believed is believed to be a promisingto be a prom cancerising therapeuticcancer therapeutic because because AMPK AMPK is 3.1. AMPK Activator involvedActivating in regulating AMPK iscell believed growth-related to be a prom pathwaysising cancer such therapeuticas cell proliferation, because AMPK protein is is involved3.1. in AMPK regulating Activator cell growth-related pathways such as cell proliferation, protein synthesissynthesisinvolved andActivating lipid andin biosynthesis regulating lipid AMPK biosynthesis is [cell41 believed]. growth-related AMPK [41]. to actsbe AMPK a asprom apathways sensoractsising as ofcancera AMP/ATPsensorsuch therapeutic as of cell AMP/ATP or proliferation, ADP/ATP because or ADP/ATP AMPK protein is ratio andinvolvedratiosynthesis contributes Activatingand incontributesand regulating to lipid maintainingAMPK biosynthesis to is cellmaintaining believed homeostasisgrowth-related [41]. to homeostasisbe AMPK a when prom pathways acts cellularising when as cancer a such energysensor cellular therapeuticas is ofcell energy lowAMP/ATP proliferation, [42 isbecause]. low or [42]. ADP/ATP AMPK protein is Metformin,synthesisinvolvedratioMetformin, and which inandcontributes regulating lipid has which biosynthesis already to cellhasmaintaining growth-relatedalready been [41]. used beenhomeostasis AMPK for used apathways acts long for whenas timea along suchsensor cellular as time aas drugof cell energyasAMP/ATP a againstproliferation, drug is low against typeor [42].ADP/ATP protein type 2 2 diabeteratiosynthesisdiabetes,s, induces Metformin,and contributesinducesand autophagy lipid which autophagy biosynthesis to by maintaininghas activating already by activating[41]. AMPK homeostasisbeen AMPK used indirectly.AM actsPK for when asindirectly. a aInhibitionlong sensor cellular time Inhibitionof ofenergy asAMP/ATP mitochondrial a drug is of low mitochondrialagainst or [42]. ADP/ATP type 2 respiratoryratiorespiratorydiabetes, chain Metformin,and complexcontributesinduces chain which autophagy Icomplex by to metformin hasmaintaining alreadyI byby activatingmetformin increases beenhomeostasis used AM theincreases PKfor AMP/ATP when indirectly.a long thecellular timeAMP/ATP ratio, Inhibition energyas which a drug ratio,is of then low againstmitochondrial which in-[42]. type then 2 duces AMPKdiabetes,inducesrespiratory activationMetformin, AMPK induces chain [ 43activation which ].autophagy complex Several has [43]. studiesalreadyI byby Several activating metformin have been studie suggested used AM sincreases havePK for thatindirectly. asuggested long metforminthe time AMP/ATP Inhibition that as suppresses metformina drug ratio, of againstmitochondrial cell whichsuppresses type then 2 proliferationrespiratorydiabetes,cellinduces proliferation and inducesAMPK induces chain activation apoptosisand autophagycomplex induces [43]. in I severalbyapoptosisby Several activatingmetformin cancer studie in severalAM cells, increasess havePK including cancerindirectly. suggested the cells, renal,AMP/ATP Inhibition includingthat colorectal, metformin ratio, of renal, mitochondrial liver which suppresses colorectal, then and pancreaticinducesrespiratorylivercell proliferation and cancer AMPK pancreaticchain cells activation and [complex21, 44 inducescancer–46 [43]. ].I An byapoptosis Severalcells inmetformin vivo[21,44–46]. studie instudy several sincreases have demonstrated An cancer suggested in thevivo cells, AMP/ATP that thatstudyincluding administration metformin demonstrated ratio, renal, which suppresses colorectal, thenthat of metformincellinducesadministrationliver proliferation toand hamstersAMPK pancreatic ofactivation and withmetformin induces high-fatcancer [43]. to apoptosis Severalcells diethamsters has[21,44–46]. studie diminishedin with severals high-fathave An cancer suggested the in diet occurrence cells,vivo has thatincludingdiminishedstudy metformin of pancreaticdemonstrated renal, the suppressesoccurrence colorectal, that cancer followinglivercellofadministration pancreatic proliferation and exposure pancreatic cancer of and metformin to induces following pancreaticcancer to apoptosiscells hamsters exposure carcinogens [21,44–46]. in with severalto pancreatichigh-fat [47 An]. cancer Additionally,in diet vivocells,carcinogens has includingstudydiminished patients demonstrated[47]. renal, the withAdditionally, occurrencecolorectal, that type 2 diabetesadministrationliverpatientsof pancreatic andwho with pancreatic were typecancer of metformin given 2 diabetes followingcancer metformin to who cellshamsters exposure were had[21,44–46]. give a with significantly ton high-fatmetforminpancreatic An in diet lower vivo hadcarcinogens has riska diminishedstudy significantly of developing demonstrated[47]. the Additionally,lower occurrence risk that of pancreaticofadministrationdevelopingpatients andpancreatic hepatocellular with pancreatic typecancer of metformin 2 diabetes following cancer and to thanhepatocellularwho hamsters exposure patientswere give with to who n cancer pancreatichigh-fatmetformin received than diet othercarcinogens had haspatients a diminished medicationssignificantly who [47]. received theAdditionally, [lower48 occurrence]. riskother of These studiespatientsofmedicationsdeveloping pancreatic suggest with [48].pancreatic type thatcancer These AMPK2 diabetes following studiesand activation whohepatocellular suggestexposure were and thatgive subsequent to n AMPK cancerpancreaticmetformin activation than autophagy hadcarcinogens patients aand significantly induction subsequent who [47]. received Additionally, maylower autophagy risk other of have a preventivedevelopingpatientsinductionmedications with role may pancreatic[48]. type in cancerhave These 2 diabetes a incidence. studiesandpreventive whohepatocellular suggest Basedwere role givethaton in these nAMPKcancerca metforminncer studies, activationincidence.than had metforminpatients a and significantlyBased subsequent who is currentlyon receivedthese lower autophagy studies, riskother of undergoingmedicationsdevelopingmetformininduction various may is clinical[48].pancreatic currently have These trials a studies andundergoingpreventive for cancerhepatocellular suggest patients rolevarious that in AMPK withoutcancer caclinicalncer activation thanincidence. diabetes trials patients for and [49 Basedcancer]. subsequent Interestingly,who onpatients received these autophagy withoutstudies, other the additioninductionmedicationsdiabetesmetformin of metformin [49]. may is [48]. Interestingly,currently have toThese standard a studies preventiveundergoing the EGFR-TKIs suggestaddition role various that of therapyin metformin AMPKca cncerlinical in activation patientsincidence. totrials standard withfor and Basedcancer advanced subsequentEGFR-TKIs on patients these lung autophagytherapy studies,without in adenocarcinomametformininductionpatientsdiabetes significantly with[49]. mayis Interestingly,currentlyadvanced have improveda undergoingpreventivelung the adenocarcino progression-freeaddition rolevarious ofin metformin ma caclinicalncer significantly survival incidence. trialsto instandard phasefor improved Basedcancer 2EGFR-TKIs randomized on patientsprogression-free these therapy withoutstudies, in clinical trialsdiabetesmetforminsurvivalpatients [50]. [49].inwith Additionally,phaseis Interestingly, currentlyadvanced 2 randomized undergoing variouslung the adenocarcinoaddition clinical phase various 1oftrials clinical metforminma c[50].linical significantly trials Additionally, trialsto werestandard for conductedimproved cancer various EGFR-TKIs patientsprogression-free forphase pa- therapy 1without clinical in tients withpatientsdiabetestrialssurvival glioblastoma were with[49].in conductedphase Interestingly,advanced or advanced/refractory2 randomized for lung patients the adenocarcino addition clinical with cancer, glio oftrials metforminblastomama and[50]. significantly the Additionally, or stabilityto advanced/refractorystandard improved of metforminvarious EGFR-TKIs progression-free phase was cancer, therapy 1 clinical and in confirmedsurvivalpatientsthetrials [51 stability, 52were]. inwith phaseconducted of advancedmetformin 2 randomized for lung waspatients confirmedadenocarcino clinical with gliotrials [51,52].blastomama [50]. significantly Additionally, or advanced/refractory improved various progression-free phase cancer, 1 clinical and trialssurvivalthe stability5-Aminoimidazole-4-carboxamide-1- were in conductedphase of metformin 2 randomized for patientswas confirmed clinical with gliotrials [51,52].β-blastomaD-ribofuranoside [50]. Additionally, or advanced/refractory (AICAR), various another phase cancer, 1activator clinical and thetrialsof AMPK, stability 5-Aminoimidazole-4-carboxamide-1-were inducesconducted of metformin apoptosis for waspatients ofconfirmed renal with cancer glio [51,52].β-blastomaD-ribofuranoside cells; moreover, or advanced/refractory (AICAR), AICAR isanother also cancer, known activator and to theof AMPK,stability5-Aminoimidazole-4-carboxamide-1- induces of metformin apoptosis was ofconfirmed renal cancer [51,52].β-D-ribofuranoside cells; moreover, (AICAR), AICAR anotheris also known activator to of AMPK,5-Aminoimidazole-4-carboxamide-1- induces apoptosis of renal cancerβ-D-ribofuranoside cells; moreover, (AICAR), AICAR isanother also known activator to of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to

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5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), another activator of AMPK, induces apoptosis of renal cancer cells; moreover, AICAR is also known to inhibit autophagy by a pathway independent of AMPK [53]. Similar to other AMPK activators such as metformin, phenformin, salicylate and 2-deoxy-D-glucose (2-DG), AICAR inhibits cell growth; however, it also inhibits cell growth in AMPK-deficient cells, suggesting that AMPK-independent regulation exists [54,55]. In other words, AICAR may have nonspecific effects on AMPK or control independent of autophagy. Recently developed AMPK activators are more specific. A-769662, an AMPK activator, showed specific AMPK-dependent retardation of cell growth and metabolism [54]. GSK621, a direct and specific activator of AMPK, induces cytotoxicity and autophagy in acute myeloid leukemia (AML) [56]. Continuous efforts are made to develop specific and potent AMPK activators and use them for cancer treatment [57].

3.2. mTOR Inhibitor Rapamycin/sirolimus, a representative mTOR inhibitor and autophagy inducer, was initially approved as an immunosuppressant to prevent allograft rejection. Due to its poor solubility and pharmacokinetics of rapamycin, several rapamycin analogs (rapalogs) were developed. Autophagy is induced in cancer cells not only by rapamycin but also by rapalogs [58]. Temsirolimus, a rapalog, was approved by the Food and Drug Administration (FDA) in 2007 for treating advanced renal cancer carcinoma (RCC). Thereafter, everolimus was approved for RCC treatment by FDA in 2009. Additionally, everolimus was later approved as a treatment for various cancers, including astrocytoma, breast cancer, angiomyolipoma and neuroendocrine cancer. However, rapalogs have achieved only modest effects in clinical practice. The reasons for clinical limit have not been fully established, but it may involve multiple mTORC1 regulatory negative feedback loops in cancer cells. To overcome this limitation, multiple clinical trials are currently evaluating the efficacy of rapalogs plus chemotherapy combination therapy [59]. AZD-8055 is a potent bioavailable ATP-competitive mTORC1 and mTORC2 inhibitor and is under phase I clinical trial in patients with various tumors. In preclinical data, AZD8055 exhibited a more potent anticancer effect than rapamycin in the brain tumor xenograft model [60]. Efforts focused on developing novel mTOR inhibitors or optimal combinations with rapalogs will have great potential to yield an improved efficacy. Nutraceutical products obtained from normal food can control autophagy. Epigallocat- echin gallate (EGCG), a polyphenol compound obtained from green tea, has been reported to modulate autophagy by affecting the balance of the mTOR-AMPK pathway [61–63]. Fisetin, a member of the flavonoid group of polyphenols, inhibits mTOR activity and induces autophagic-programmed cell death in several cancer cells, including prostate carcinoma and oral squamous cell carcinoma [64,65]. Various inhibitors of autophagy initiation (autophagy blockers) were developed and actively used in biological experiments to determine the role of autophagy in cancer cells. However, autophagy blockers have not entered clinical trials yet. Since various preclinical experiments are in progress, it is expected that autophagy blockers that enter clinical trials will emerge when numerous data are accumulated. Autophagy blockers are discussed below in terms of cancer treatment (Figure2 and Table1).

3.3. ULK1/2 Inhibitors Several ULK1 or 2 activators, such as BL-918, typically target Parkinson’s disease but not cancers. Rather, ULK1/2 inhibitors have been developed as oncolytic drugs in certain cancers. In preclinical data, SBI-0206965, ULK1 inhibitor, reduced cell growth and promoted apoptosis in neuroblastoma [66]. SBI-0206965 and MRT-68921, another ULK1 inhibitor, was able to induce apoptosis in AML with FLT3-ITD mutation [67]. However, ULK1 inhibitors alone are not sufﬁcient for tumor suppression in certain cancers. Recently, a paper was published explaining the reason why the anticancer effect of ULK inhibition monotherapy was ineffective in pancreatic ductal adenocarcinoma (PDAC) [68]. Intriguingly, autophagy Life 2021, 11, 839 9 of 19

Life 2021, 11, x FOR PEER REVIEW inhibition induced micropinocytosis, which is the uptake of extracellular ﬂuid droplets10 of 20 containing proteins and other macromolecules. As micropinocytosis provides energy to autophagy-compromised cells, ULK inhibition alone could not induce apoptosis in PDAC. Indeed, combination treatment of MRT-68921 and macropinosome formation inhibitor 3.4.synergistically PI3K Inhibitors induced tumor regression in an in vivo model. Although the PI3K-AKT-mTOR axis is also critical for autophagy regulation, modulators3.4. PI3K Inhibitors of upstream kinases are not specific in autophagy regulation and show mixed autophagyAlthough readouts the PI3K-AKT-mTORdepending on cell axis type issalso or treatment critical for concentration autophagy regulation, and time. mod-For example,ulators of3-methyladenine upstream kinases (3-MA) are not is used speciﬁc as an in autophagy autophagy inhibitor, regulation but and it also show inhibits mixed PI3Kautophagy class I and readouts class III. depending PI3K class on III cell stimul typesates or treatmentautophagic concentration sequestering, and whereas time. ForPI3K ex- classample, I inhibits 3-methyladenine it [5]. Although (3-MA) the is used overall as an effect autophagy of 3-MA inhibitor, is considered but it also an inhibitsautophagy PI3K inhibitor,class I and PI3K class inhibitors III. PI3K are class not III suitable stimulates for autophagicclinical setting sequestering, due to the whereas complexity PI3K of class the I results.inhibits it [5]. Although the overall effect of 3-MA is considered an autophagy inhibitor, PI3K inhibitors are not suitable for clinical setting due to the complexity of the results. 4. Modulators of Lysosomal Activity 4. Modulators of Lysosomal Activity Autophagosome fuses with a lysosome and eventually degrades the contents, includingAutophagosome cargo proteins fuses or organelles. with a lysosome Chloroquine and eventually(CQ) and adegrades derivative the of contents,CQ, that is, in- hydroxychloroquinecluding cargo proteins (HCQ), or organelles. are the most Chloroquine well-known (CQ) and aclinically derivative used of CQ,chemical that is, blockershydroxychloroquine (Figure 3). CQ (HCQ), and HCQ are the have most been well-known used as antimalarial and clinically drugs. used chemicalCQ and HCQ block- inhibiters (Figure autophagy3). CQ by and increasing HCQ have intralysosomal been used as pH, antimalarial but the exact drugs. mechanism CQ and HCQ is not inhibit well autophagy by increasing intralysosomal pH, but the exact mechanism is not well under- understood. Autophagy inhibition limits the energy supply for cancer growth. stood. Autophagy inhibition limits the energy supply for cancer growth. Additionally, Additionally, several damaged proteins or mitochondria can be accumulated in CQ- or several damaged proteins or mitochondria can be accumulated in CQ- or HCQ-treated HCQ-treated cells, resulting in the ER stress. Triggering excessive ER stress is an excellent cells, resulting in the ER stress. Triggering excessive ER stress is an excellent strategy to strategy to kill cancer cells [69]. Apoptosis is eventually induced because cancer cells kill cancer cells [69]. Apoptosis is eventually induced because cancer cells cannot tolerate cannot tolerate excessive ER stress. In this context, CQ and HCQ show synergistic excessive ER stress. In this context, CQ and HCQ show synergistic enhancement when enhancement when combined with various therapeutic agents in cancer treatment. combined with various therapeutic agents in cancer treatment. Treatment with CQ or HCQ Treatment with CQ or HCQ along with radiation therapy or standard therapy is already along with radiation therapy or standard therapy is already in clinical trials. There are in clinical trials. There are multiple reports that CQ sensitizes glioblastoma to radiation or multiple reports that CQ sensitizes glioblastoma to radiation or chemotherapy. Favorable chemotherapy. Favorable toxicity of CQ in patients was determined [70], and phase III toxicity of CQ in patients was determined [70], and phase III clinical trials are ongoing clinical trials are ongoing (NCT00224978) [71]. According to the results of a recently (NCT00224978) [71]. According to the results of a recently published phase II clinical trial, publishedHCQ along phase with II gemcitabineclinical trial, andHCQ nab-paclitaxel along with gemcitabine resulted in aand greater nab-paclitaxel pathological resulted tumor inresponse a greater in pathological pancreatic cancer tumor [ 72response] (Table in2). pancreatic cancer [72] (Table 2).

FigureFigure 3. 3. LysosomalLysosomal inhibitors. inhibitors. Autophagy Autophagy is is inhibited inhibited by by lysosomal lysosomal inhibitors. inhibitors. Lysosomal Lysosomal activity activityis inhibited is inhibited by increasing by increasing intralysosomal intralysosomal pH or inhibitingpH or inhibiting v-ATPase. v-ATPase.

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Life 2021, 11, x FOR PEER REVIEW 11 of 20 Table 2. Autophagy modulators that control lysosomal activity. Life 2021, 11, 839 10 of 19 Table 2. Autophagy modulators that control lysosomal activity. Table 2. Autophagy modulators that control lysosomal activity. Clinical Trials in Progress or Compound Mechanism/UseTable 2. Autophagy modulators Structure that control lysosomal activity.Clinical Completed Trials in for Progress Cancer or Clinical Trials in Progress or Compound Mechanism/UseTable 2. Autophagy modulators Structure that control lysosomal activity. Completed for Cancer ClinicalTreatment Trials in(NCT Progress Number) or Compound Mechanism/Use Structure TreatmentCompleted (NCT for Cancer Number) Compound Mechanism/Use Structure ClinicalTreatmentCompletedPhase Trials (NCT 3 in in Progressfor glioblastoma Number)Cancer or Compound Mechanism/Use Structure Completed for Cancer Treatment TreatmentPhase (NCT00224978)/3 in(NCT glioblastoma Number) (NCT Number) PhasePhase(NCT00224978)/ 3 1/2 in glioblastomain IDH1/2-mutated Inhibition of lysosomal Phase 3 in glioblastoma Phasesolid(NCT00224978)/ 1/2 tumors in IDH1/2-mutated (NCT02496741) CQ Inhibitionacidification/autophag of lysosomal PhasePhase 1/2(NCT00224978)/ 3 in in glioblastoma IDH1/2-mutated Inhibition of lysosomal solidPhase tumors 1/2 in ductal(NCT02496741) carcinoma in CQ acidification/autophagy inhibitor solidPhase(NCT00224978)/ tumors 1/2 in IDH1/2-mutated(NCT02496741) CQ acidification/autophagInhibitionInhibition of lysosomal of lysosomal PhasePhase 1/2situ in1/2 IDH1/2-mutated(DCIS) in ductal (NCT01023477) carcinoma solid in y inhibitor Phasesolid 1/2 tumors in ductal (NCT02496741) carcinoma in CQ acidification/autophagy inhibitoracidiﬁca- tumorssitu Phase(DCIS) (NCT02496741) 2 (NCT01023477)in breast cancer CQ Phase 1/2 in ductal carcinoma in tion/autophagy Phasesitu 1/2Phase (DCIS) in ductal (NCT01446016)2 in (NCT01023477) breast carcinoma cancer in y inhibitor inhibitor situsituPhase (DCIS) (DCIS)(NCT01446016) 2 (NCT01023477)in (NCT01023477)breast cancer PhasePhasePhase(NCT01446016) 2 in2 2 in breast in breast Pancreatic cancer cancer cancer (NCT01446016) Phase(NCT01273805,(NCT01446016) 2 in Pancreatic NCT01494155, cancer

Phase(NCT01273805,NCT01978184, 2 in Pancreatic NCT01494155, NCT01128296) cancer Phase 2 in Pancreatic cancer (NCT01273805,PhaseNCT01978184,Phase 2 in Pancreatic 2 in colorectalNCT01494155, NCT01128296) cancer cancer (NCT01273805, NCT01494155, Inhibition of lysosomal (NCT01273805,NCT01978184,Phase(NCT01006369, 2 in colorectal NCT01494155, NCT01128296) NCT01206530 cancer NCT01978184, NCT01128296) HCQ Inhibitionacidification/autophag of lysosomal NCT01978184,Phase(NCT01006369,NCT02316340, 2 in colorectal NCT01128296) NCT01206530 NCT03215264) cancer Phase 2 in colorectal cancer InhibitionInhibition of lysosomal of lysosomal (NCT01006369,Phase 2 in colorectal NCT01206530 cancer HCQ acidification/autophagy inhibitor (NCT01006369,NCT02316340,Phase NCT012065302 in NCT03215264) lung cancer HCQ acidification/autophagInhibition acidiﬁca-of lysosomal NCT02316340,(NCT01006369, NCT03215264) NCT01206530 HCQ y inhibitor NCT02316340,(NCT00977470,Phase 2 NCT03215264)in lung NCT01649947, cancer HCQ acidification/autophagtion/autophagy NCT02316340, NCT03215264) y inhibitor (NCT00977470,PhasePhase 2 in2NCT02470468) in lung lung NCT01649947, cancer cancer y inhibitorinhibitor Phase 2 in lung cancer (NCT00977470,(NCT00977470,PhaseNCT02470468) 2 in NCT01649947,renal NCT01649947, cell carcinoma NCT02470468) (NCT00977470,Phase 2NCT02470468) in renal NCT01649947, cell carcinoma Phase(NCT01510119, 2 in renal cell carcinoma NCT01550367) Phase 2 inNCT02470468) renal cell carcinoma (NCT01510119,(NCT01510119, NCT01550367) NCT01550367) (NCT01510119,Phase 2 in renal NCT01550367) cell carcinoma (NCT01510119, NCT01550367) Inhibition of v- Bafilomycin InhibitionATPase/autophagy of v- Preclinical BafilomycinA1 InhibitionInhibition of v- of BaﬁlomycinBafilomycin A1 ATPase/autophagyv-ATPase/autophagyinhibitor PreclinicalPreclinical A1 ATPase/autophagyInhibition of v- Preclinical BafilomycinA1 inhibitorinhibitor ATPase/autophagyinhibitor Preclinical A1 inhibitor

Inhibition of v- Concanamyci Inhibition of Inhibition of v- Concanamycin A v-ATPase/autophagyATPase/autophagy PreclinicalPreclinical Concanamycin A Inhibition of v- Concanamyci ATPase/autophagyinhibitorinhibitor Preclinical Life 2021n, 11A , x FOR ATPase/autophagyPEERInhibition REVIEW of v- Preclinical 12 of 20 Concanamyci n A inhibitor ATPase/autophagyinhibitor Preclinical n A inhibitor

Inhibition of v- Salicylihalami Inhibition of ATPase/no autophagy No autophagy research Salicylihalamidede A A v-ATPase/no No autophagy research autophagyresearch research

Vacuolar-typeVacuolar-type proton proton adenosine adenosine triphosphatase triphosphatase (v-ATPase) (v-ATPase) is anis an ATP ATP-driven driven proton proton pump responsible for controlling the acidification of lysosomes. Bafilomycin A1 and pump responsible for controlling the acidiﬁcation of lysosomes. Baﬁlomycin A1 and concanamycin A are v-ATPase inhibitors known to prevent the acidification of lysosomes

(Figure 3). Several in vivo xenograft mouse studies revealed that bafilomycin A1 can inhibit tumor growth, including bladder, breast and liver cancers [73–75]. Although bafilomycin A1 is more famous and commonly used in many preclinical experiments to identify an inhibitory role in cancer growth, several more selective and potent v-ATPase inhibitors exist (e.g., salicylihalamide A, lobatamides and oximidines). Unlike bafilomycin A1 and concanamycin A, these chemicals inhibit mammalian V-ATPases at low concentrations but do not affect fungi and yeast v-ATPases [76,77]. However, there are few reports that these chemicals inhibit tumor growth and autophagy, but further studies are still needed to confirm these findings (Table 2).

5. Modulation of CMA in Cancer The recognition of substrates initiates CMA with KFERQ-like motifs by cytosolic chaperone Hsc70, and the substrates are then delivered to the lysosomal surface. The targeted substrates bind to lysosomal-associated membrane protein 2A (LAMP2A) and are translocated inwardly across the lysosomal membrane and are eventually degraded [4] (Figure 1). The role of CMA in oncology has not been fully understood as encompassing both pro-survival and pro-death parts in different contexts [78]. Similar to macroautophagy, CMA plays a critical supportive role in cancer cell proliferation because it satisfies the excessive energy needs of cancer cells by degradation of substrates. However, given CMA substrates, CMA degrades not only oncogenic proteins (e.g., PKM2, mutant p53, HK2, HIF-1α) but also tumor suppressors (unphosphorylated PED, Rnd3) [79]. These observations suggest that the role of CMA in oncology is more controversial than macroautophagy, especially regarding cancer treatment. The distinct and notable characteristics of CMA are the selectivity of the target substrate and specificity of core components. CMA degrades only substrates with KFERQ-like motifs. Oncoproteins or oncoprotein activators with the KFERQ motif might be a promising therapeutic target through CMA modulation [45]. Hsc70 and LAMP2A are identified as the core components required for CMA. Specifically, LAMP2A is the rate- limiting component of the CMA pathway [80]. LAMP2A is under the negative regulation by nuclear retinoic acid receptor-α (RAR-α) [81]. Indeed, RAR-α inhibition by synthetic derivatives of all-trans-retinoic acid activated only CMA without regulating other RAR- α-dependent transcriptions [81]. However, RAR-α derivatives have the disadvantage that they can be used only when cancer cells are under the regulation of RAR-α. The application of RAR-α derivatives to cancer cells has not been published. Development of more diverse CMA modulators is required for application to anticancer treatment.

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concanamycin A are v-ATPase inhibitors known to prevent the acidification of lysosomes (Figure3). Several in vivo xenograft mouse studies revealed that bafilomycin A1 can inhibit tumor growth, including bladder, breast and liver cancers [73–75]. Although bafilomycin A1 is more famous and commonly used in many preclinical experiments to identify an inhibitory role in cancer growth, several more selective and potent v ATPase inhibitors exist (e.g., salicylihalamide A, lobatamides and oximidines). Unlike bafilomycin A1 and concanamycin A, these chemicals inhibit mammalian V-ATPases at low concentrations but do not affect fungi and yeast v-ATPases [76,77]. However, there are few reports that these chemicals inhibit tumor growth and autophagy, but further studies are still needed to confirm these findings (Table2).

5. Modulation of CMA in Cancer The recognition of substrates initiates CMA with KFERQ-like motifs by cytosolic chaperone Hsc70, and the substrates are then delivered to the lysosomal surface. The targeted substrates bind to lysosomal-associated membrane protein 2A (LAMP2A) and are translocated inwardly across the lysosomal membrane and are eventually degraded [4] (Figure1). The role of CMA in oncology has not been fully understood as encompassing both pro-survival and pro-death parts in different contexts [78]. Similar to macroautophagy, CMA plays a critical supportive role in cancer cell proliferation because it satisfies the excessive energy needs of cancer cells by degradation of substrates. However, given CMA substrates, CMA degrades not only oncogenic proteins (e.g., PKM2, mutant p53, HK2, HIF-1α) but also tumor suppressors (unphosphorylated PED, Rnd3) [79]. These observations suggest that the role of CMA in oncology is more controversial than macroautophagy, especially regarding cancer treatment. The distinct and notable characteristics of CMA are the selectivity of the target substrate and specificity of core components. CMA degrades only substrates with KFERQ-like motifs. Oncoproteins or oncoprotein activators with the KFERQ motif might be a promising therapeutic target through CMA modulation [45]. Hsc70 and LAMP2A are identified as the core components required for CMA. Specifically, LAMP2A is the rate-limiting component of the CMA pathway [80]. LAMP2A is under the negative regulation by nuclear retinoic acid receptor-α (RAR-α)[81]. Indeed, RAR-α inhibition by synthetic derivatives of all-trans-retinoic acid activated only CMA without regulating other RAR-α-dependent transcriptions [81]. However, RAR-α derivatives have the disadvantage that they can be used only when cancer cells are under the regulation of RAR-α. The application of RAR-α derivatives to cancer cells has not been published. Development of more diverse CMA modulators is required for application to anticancer treatment.

6. Autophagy Modulators Associated with Epigenetics Although autophagy is rapidly regulated and occurs in the cytoplasm by starvation signal, transcriptional and epigenetic regulation in the nucleus are also deemed essential for the overall autophagy process [82]. Nutrient starvation regulates not only upstream kinase of autophagy, such as AMPK or mTOR, but also the translocation of transcription factors to the nucleus or histone modiﬁcations of target genes. Transcriptional regulation of autophagy is required to maintain prolonged autophagy. Furthermore, because autophagy receptors and LC-3 proteins are degraded with cargo, cells replenish autophagy proteins with transcriptional upregulation of autophagy-related genes.

6.1. MiT/TFE Family The major transcription factor of autophagy regulation is the transcription factor EB (TFEB) [83]. TFEB belongs to the microphthalmia/transcription factor E (MiT/TFE) family of transcription factors that bind coordinated lysosomal expression and regulation element [84]. Under starvation conditions, TFEB translocates to the nucleus to promote the transcription of lysosome biogenesis and autophagy-related genes. Its phosphorylation status at various sites tightly controls the nuclear import or export of TFEB and induced by Life 2021, 11, 839 12 of 19

mTORC1 or GSK3β [85,86]. According to an interesting publication, increased mRNA and protein expression of MITF, TFE3 and TFEB was detected in pancreatic ducal adenocarci- Life 2021, 11, x FOR PEER REVIEW noma (PDA) cell lines and patient tumors [87]. PDA is one of the most lethal of cancers with14 of 20 a 5-year survival rate of only 6% after diagnosis [88]; it is a cancer determined to be highly dependent on autophagy for the supply of essential nutrients. Overexpressed MiT/TFE family transcription factor activates autophagy and maintains intracellular amino acid enhancingpools. However, α-tubulin small acetylation molecules and that alleviated can directly Cockayne modulate syndrome the MiT/TFE (CS),family a condition have of prematurenot yet been aging developed. mainly Sincecaused the by MiT/TFE mutations family in the is an csb attractive gene [98]. target Currently, for PDA SAHA treat- is approvedment, it is expectedto treat toT-cell be an lymphoma excellent therapeutic (CTCL), agentmalignant if potent mesothelioma and selective modulatorsand multiple myeloma.are developed. In the case of cancer in which reduced autophagy promotes tumorigenesis, treatment with SAHA can be effective. Another HDAC inhibitor, that is, 6.2. Histone Modifications MGCD0103/mocetinostat, has been reported to inhibit autophagy [99]. MGCD0103 decreasedAutophagy the autophagic regulates, directlyflux in orprimary indirectly, lymphocytic histone modifications leukemia cells to control by activating transcrip- the mTORtion. Negative pathway regulation and downregulating occurs through autophagy histone H3 gene K9 or transcription K27 methylation [99]. (Figure This 4suggests and Table3). Under nutrient-rich conditions, H3K9 methylation is maintained by G9a methyl- that since the role of autophagy is different for each cancer type, it is crucial to identify transferase in autophagy-related genes [89]. However, a decrease in G9a recruitment and the detailed function of autophagy according to cancer type and select an appropriate subsequent H3K9 dimethylation is observed by starvation. EZH2, a methyltransferase of therapeuticH3 K27, regulated agent.the Inexpression addition, ofepigenetic mTOR pathway-related regulators can genes control through not H3K27 only methy- histone modificationlation and knockdown but also non-hi of EZH2-inducedstone modification. autophagy If [HDAC90]. In addition,inhibitors EZH2 modulate inhibitors histone acetylation,such as GSK126 overall and transcription EPZ-011989 may induced be activated, autophagy but [90 it,91 remains] (Table 3difficult). Interestingly, to predict the how transcriptionepigenetic inhibitor will alter induces if they autophagy modulate by an altering unknown the transcription non-histone of target. the target Since genes. target specificityMoreover, canin vivo varystudies depending revealed on that the these type EZH2 of drug inhibitors as well showed as the significant concentration tumor and treatmentgrowth inhibition time of inthe lymphoma drug, a fundamental and myeloma st [92udy,93 ].on However, how and studies which on target how autophagyis controlled beforeinduction application by EZH2 to inhibitors cancer patients affected is overall required. tumor growth inhibition remain lacking.

FigureFigure 4. 4.HistoneHistone H3 H3 modifications modiﬁcations associated associated with autophagy regulation.Histoneregulation.Histone H3 H3 tail tail is is methylated methylated by by G9a, G9a, EZH2 EZH2 or or CARM1.CARM1. The The epigenetic epigenetic modulators modulators can can induce induce autophagy (inhibitor(inhibitor of of H3K27 H3K27 me) me) or or block block induced induced autophagy autophagy (inhibitor (inhibitor of ofH3R17 H3R17 me). me).

Table 3. Autophagy modulator associated with epigenetics.

Clinical Trials in Progress or Compound Mechanism/Use Structure Completed for Cancer Treatment (NCT Number)

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Life 2021, 11, x FOR PEER REVIEW Table 3. Autophagy modulator associated with epigenetics. 15 of 20 Life 2021, 11, x FOR PEER REVIEW 15 of 20

Clinical Trials in Progress or Compound Mechanism/Use Structure Completed for Cancer Treatment (NCT Number) EZH2 EZH2 Discontinued in phase 1 (solid GSK126 inhibition/autophagy Discontinued in phase 1 (solid GSK126 inhibition/autophagyEZH2 tumors and lymphoma) inducer Discontinuedtumors and in lymphoma) phase 1 (solid GSK126 inhibition/autophagyinducerEZH2 EZH2 Discontinuedtumors and in lymphoma) phase 1 (solid GSK126 inhibition/autophagyinducer Discontinued in phase 1 (solid GSK126 EZH2inhibition/autophagy inhibition/autophagy Discontinuedtumors and in phase lymphoma) 1 (solid GSK126 inducer tumors and lymphoma) inducerinducer tumors and lymphoma)

EZH2 EPZ-011989 inhibition/autophagyEZH2 Preclinical EPZ-011989 inhibition/autophagyinducerEZH2 Preclinical EPZ-011989 inhibition/autophagyinducerEZH2 Preclinical EZH2 inhibition/autophagyEZH2 EPZ-011989EPZ-011989 inhibition/autophagyinducer PreclinicalPreclinical EPZ-011989 inhibition/autophagyinducerinducer Preclinical inducer

CARM1

Ellagic acid inhibition/blockingCARM1 Preclinical Ellagic acid autophagyinhibition/blockingCARM1 induction Preclinical CARM1 inhibition/blockingCARM1 EllagicEllagic acid acid autophagyinhibition/blocking induction PreclinicalPreclinical Ellagic acid autophagyautophagyinhibition/blockingCARM1 induction induction Preclinical Ellagic acid autophagyinhibition/blocking induction Preclinical autophagyHDAC induction Approved for the treatment of T- SAHA/vorinostat inhibition/autophagyHDAC Approved for the treatment of T- SAHA/vorinostatHDAC inhibition/autophagy inhibition/autophagyHDAC Approvedcell lymphoma for the treatment (CTCL) of SAHA/vorinostat inducer Approvedcell lymphoma for the treatment (CTCL) of T- SAHA/vorinostat inhibition/autophagyinducerinducerHDAC T-cell lymphoma (CTCL) HDAC Approvedcell lymphoma for the treatment (CTCL) of T- SAHA/vorinostat inhibition/autophagyinducer Approved for the treatment of T- SAHA/vorinostat inhibition/autophagy cell lymphoma (CTCL) inducer cell lymphoma (CTCL) inducer Phase 2 in lung cancer

PhasePhase(NCT02954991 2 in2 in lung lung cancer cancer Phase(NCT02954991)(NCT02954991 2 in lung cancer HDAC PhasePhase 2 in 2urothelial in urothelial carcinoma MGCD0103/MGCD0103/mocetinoHDAC inhibition/autophagyHDAC PhasePhase 2 in(NCT02954991 urothelial2 in lung cancer carcinoma MGCD0103/mocetino inhibition/autophagy carcinoma(NCT02236195) (NCT02236195) mocetinostatstat inhibition/autophagyinhibitorHDAC PhasePhase 2 (NCT02236195)(NCT02954991in 2urothelial in lung cancer carcinoma MGCD0103/mocetinostat inhibitor PhasePhase 2 in2 in lymphoma lymphoma inhibition/autophagyinhibitorHDAC Phase(NCT02282358,Phase 2 in(NCT02954991(NCT02236195) urothelial 2 in NCT02429375, lymphoma carcinoma MGCD0103/mocetino (NCT02282358, NCT02429375, stat HDAC Phase 2NCT00359086) in urothelial carcinoma MGCD0103/mocetino inhibition/autophagyinhibitor (NCT02282358,Phase(NCT02236195)NCT00359086) 2 in lymphoma NCT02429375, stat inhibition/autophagy (NCT02236195) stat inhibitor (NCT02282358,PhaseNCT00359086) 2 in lymphoma NCT02429375,

inhibitor (NCT02282358,PhaseNCT00359086) 2 in lymphoma NCT02429375,

(NCT02282358,NCT00359086) NCT02429375,

7.Histone Conclusions H3R17 and methylation Future Perspective is associated with autophagy inductionNCT00359086) (Figure4). Un- 7. Conclusions and Future Perspective der nutrientClever starvation cancer conditions,cells use autophagy stabilized CARM1in a direction methylated favorable H3R17 to and cancer activated survival, autophagyadaptation7. ConclusionsClever and lysosomalandcancer and rapid Futurecells genesproliferation. use Perspective [ 94autophagy]. As CARM1Therefore, in methyltransferasea direction targeting favorable autophagy activity to iscancer on no H3R17 doubtsurvival, is an inhibited7.excellentadaptation ConclusionsClever by ellagicstrategy andcancer and acidrapid for Futurecells cancer [95 proliferation.], use which Perspective therapy. autophagy is a However, naturallyTherefore, in a the occurringdirection targeting controversial phenolicfavorable autophagy role acid ofto autophagy abundantiscancer no doubtsurvival, should in an 7. Conclusions and Future Perspective fruitsbeexcellentadaptation and keptClever vegetables, in strategy mind. andcancer Autophagyrapid for treatment cells cancer proliferation. use therapy. withactivation autophagy ellagic However, Therefore,can acid inbe wasana thedirection excellent targeting ablecontroversial to blockstrategyfavorable autophagy autophagy role in of treatingto autophagy canceris inductionno early doubtsurvival, should stages an adaptationofbeexcellent keptcancer.Clever in strategy mind. Toxic andcancer Autophagyrapidmutagen for cells cancer proliferation. use or therapy. activation geneticautophagy However, defectsTherefore, can in be aanare thedirection excellent targetingeliminated controversial strategyfavorable autophagy by role autophagy in of totreating autophagycanceris no toearly doubtsurvival,maintain should stages an adaptationexcellentcellularofbe keptcancer. inhomeostasis. strategy mind. Toxicand Autophagyrapid mutagenfor cancer However, proliferation. or therapy. activation genetic autophagy However, Therefore,defects can becan anare the also excellent targeting eliminatedcontroversial support strategy autophagythe by rolegrowth autophagy in of treating autophagy isof cancerno toearly doubt maintain shouldcells stages an as excellentbecellularof keptcancer. inhomeostasis. strategy mind.Toxic Autophagy mutagenfor cancer However, ortherapy. activation genetic autophagy However, defectscan be can an arethe also excellent controversialeliminated support strategy the by rolegrowth autophagy in of treating autophagy of cancer earlyto maintain shouldcells stages as beofcellular keptcancer. in homeostasis. mind.Toxic Autophagymutagen However, or activation genetic autophagy defects can be can anare also excellent eliminated support strategy the by growthautophagy in treating of cancer toearly maintain cells stages as ofcellular cancer. homeostasis. Toxic mutagen However, or genetic autophagy defects can are also eliminated support the by growthautophagy of cancer to maintain cells as cellular homeostasis. However, autophagy can also support the growth of cancer cells as

Life 2021, 11, 839 14 of 19

even in starvation conditions (Table3). Furthermore, ellagic acid inhibited pancreatic cancer growth in the xenograft model [96]. There are many reports that histone deacetylase (HDAC) inhibitors regulate autophagy [97], but the effects are a little complex and controversial (Table3). It has been reported that SAHA/vorinostat, one of the HDAC inhibitors, inhibits mTOR and induces autophagy [97]. Additionally, treatment with SAHA improved autophagy flux by enhanc- ing α-tubulin acetylation and alleviated Cockayne syndrome (CS), a condition of premature aging mainly caused by mutations in the csb gene [98]. Currently, SAHA is approved to treat T-cell lymphoma (CTCL), malignant mesothelioma and multiple myeloma. In the case of cancer in which reduced autophagy promotes tumorigenesis, treatment with SAHA can be effective. Another HDAC inhibitor, that is, MGCD0103/mocetinostat, has been reported to inhibit autophagy [99]. MGCD0103 decreased the autophagic flux in primary lymphocytic leukemia cells by activating the mTOR pathway and downregulating autophagy gene transcription [99]. This suggests that since the role of autophagy is different for each cancer type, it is crucial to identify the detailed function of autophagy according to cancer type and select an appropriate therapeutic agent. In addition, epigenetic regulators can control not only histone modification but also non-histone modification. If HDAC inhibitors modulate histone acetylation, overall transcription may be activated, but it remains difficult to predict how transcription will alter if they modulate an unknown non-histone target. Since target specificity can vary depending on the type of drug as well as the concentration and treatment time of the drug, a fundamental study on how and which target is controlled before application to cancer patients is required.

7. Conclusions and Future Perspective Clever cancer cells use autophagy in a direction favorable to cancer survival, adaptation and rapid proliferation. Therefore, targeting autophagy is no doubt an excellent strategy for cancer therapy. However, the controversial role of autophagy should be kept in mind. Autophagy activation can be an excellent strategy in treating early stages of cancer. Toxic mutagen or genetic defects are eliminated by autophagy to maintain cellular homeostasis. However, autophagy can also support the growth of cancer cells as tumors develop. Additionally, cancer cells use autophagy to meet their increased metabolic demands by recycling macromolecules and supplying building blocks. Accordingly, autophagy modulators should be applied to consider various cancer characteristics such as cancer development stage, cancer types, cancer microenvironments and oncogenic mutations. Of course, it is necessary to determine the state of autophagy of cancer before applying activators or inhibitors. Many clinical trials using autophagy modulators are combinatorial therapy with standard chemotherapy or radiotherapy. Since enhanced or impaired autophagy in cancer is one of the various characteristics of cancers, it will be difficult to show dramatic anticancer effects with autophagy modulator alone. However, the effect of disappearing chemotherapy resistance caused by prolonged standard treatments is positive by inducing an imbalance of autophagy. These effects may be due to the induction of apoptosis, alterations in the tumor microenvironment or a still unknown mechanism. More detailed mechanistic studies of how autophagy modulators overcome chemotherapy resistance are needed for the stronger efficacy of autophagy modulators. It is interesting that epigenetic drugs such as EZH2 inhibitors or HDAC inhibitors regulate autophagy. However, the specific mechanism by which epigenetic drugs affect autophagy remains largely unknown. Since the effect of autophagy in cancer depends on cancer type and tumor stage, it is challenging to determine whether autophagy regulation by epigenetic drugs will have a positive/negative effect on cancer treatment. Therefore, it is necessary to study the autophagy mechanisms and treatment outcomes of epigenetic drugs in a wider range of cancers. Metformin has been used for a long time in patients with type 2 diabetes and has been clinically proven to be safe. Moreover, HCQ and CQ have been used in patients with Life 2021, 11, 839 15 of 19

malaria. Repurposing drugs with potential antitumor properties might have the advantage of improving survival while saving time and money. These drugs have successfully passed phase I clinical trials for cancer patients and are currently undergoing phases II/III. It is expected for use in the treatment of cancer patients after its effectiveness and safety have been conﬁrmed. Recently, immunotherapy is attracting attention as a new cancer treatment. So far, the synergistic effect of autophagy and antibody-targeted therapy has frequently been reported [100]. However, autophagy also enhances or attenuates the effectiveness of immunotherapy, depending on the cancer type [93]. Currently, phase I/II clinical trials on HCQ in combination with nivolumab/ipilimumab in advanced melanoma are recruiting patients (NCT04464759). If positive clinical results are obtained, it is expected that the effective range of autophagy modulators as cancer immunotherapeutics will be further expanded.

Author Contributions: H.J.N. wrote and edited the article. The author has read and agreed to the published version of the manuscript. Funding: This research was funded by the Bio & Medical Technology Development Program of the National Research Foundation, grant number 2020M3A9I4036072. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work was supported by a grant from the Bio & Medical Technology Development Program of the National Research Foundation (NRF), which is funded by the Ministry of Science & ICT (2020M3A9I4036072). Conﬂicts of Interest: The author declares no conﬂict of interest.

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