At
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Journal Pre-proofAt a Glance:A History of Autophagy and CancerXin Wen,Daniel J.KlionskyPII:S1044-579X(19)30167-1DOI:https:/doi.org/10.1016/j.semcancer.2019.11.005Reference:YSCBI 1718To appear in:Seminars in Cancer BiologyReceived Date:4 June 2019Revised Date:10 October 2019Accepted Date:5 November 2019Please cite this article as:Wen X,Klionsky DJ,At a Glance:A History of Autophagy andCancer,Seminars in Cancer Biology(2019),doi:https:/doi.org/10.1016/j.semcancer.2019.11.005This is a PDF file of an article that has undergone enhancements after acceptance,such asthe addition of a cover page and metadata,and formatting for readability,but it is not yet thedefinitive version of record.This version will undergo additional copyediting,typesetting andreview before it is published in its final form,but we are providing this version to give earlyvisibility of the article.Please note that,during the production process,errors may bediscovered which could affect the content,and all legal disclaimers that apply to the journalpertain.2019 Published by Elsevier.1 At a Glance:A History of Autophagy and Cancer Xin Wen1,Daniel J.Klionsky1*klionskyumich.edu 1Life Sciences Institute and Department of Molecular,Cellular and Developmental Biology,University of Michigan,Ann Arbor,Michigan 48109,USA *Correspondence to:Daniel J.Klionsky(e-mail:)Keywords:ATG,autophagy,cancer,lysosome,stress,tumor Abstract Since the first discovery of the lysosome and the definition of autophagy by Christian de Duve more than 60 years ago,research on autophagy,a process targeting cytoplasmic materials for lysosomal degradation and recycling,has expanded dramatically.This research has extended our understanding of the basic mechanism of autophagy as well as its role in pathophysiology.Autophagy deficiency has been reported to be involved in numerous diseases,among which cancer has been extensively studied,in part because autophagy appears to play a dual role,depending on the stage of tumorigenesis.In this review,we will briefly revisit the intriguing history of autophagy and cancer,underscoring the importance of harnessing this pathway for the benefit of human health.1.Introduction Autophagy,meaning“self-eating”,is a highly regulated cellular primarily catabolic process,conserved from yeast to more complex eukaryotes,including humans 1.During autophagy,cytoplasmic materials are delivered into the lysosome(or the analogous organelle,the vacuole,in yeast)for degradation by resident hydrolases;the resulting breakdown products are recycled back to the cytoplasm to maintain cellular homeostasis 2.Under normal nutrient conditions,autophagy Journal Pre-proof 2 occurs at a basal level,which can play a critical role in cell physiology by inhibiting the otherwise gradual accumulation of damaged protein aggregates and organelles;thus autophagy functions in part as a quality control system 3.When cells face particular stimuli or stress from the intracellular or extracellular microenvironment,autophagy can be massively upregulated to handle the increased demand.The normal functions of autophagy are also fundamental in maintaining other aspects of cell physiology,such as efficient cell metabolism and genomic integrity 4.In mammalian cells,there are various types of autophagy,which can be differentiated based on the mechanisms of cytoplasmic sequestration as well as the form of cargo delivered to the lysosome 5.Among these types,macroautophagy is the best characterized 6.Accordingly,we primarily focus on macroautophagy(simply referred to as autophagy hereafter)in this review.The malfunction of autophagy is associated with various diseases,including cancer,aging,metabolic and neurodegenerative diseases 7.Compared to other pathophysiologies,the role of autophagy is more complicated in cancer because autophagy can either inhibit or promote tumorigenesis 8.Some of the pioneering work connecting autophagy and cancer can be traced back to the 1970s and 1980s.For example,in 1976,researchers found that deprivation of serum and amino acids can induce autophagy in HeLa cells 9.Later,a 1977 study from Gunn and colleagues compared the proteolysis rate between non-growing rat liver hepatocytes and chemically transformed hepatoma,as well as mouse fibroblasts and virus-transformed fibroblasts.The transformed cells exhibit a lower rate of protein degradation,which became an initial indicator for analyzing autophagy in cancer cells 10.In this review,we will briefly summarize the mechanism of autophagy,and its involvement in cancer based on a timeline of the past 20 years,coinciding with the identification of the molecular machinery involved in autophagy.The potential of autophagy as a therapeutic target for cancer treatment will be further discussed.Journal Pre-proof 3 2.Molecular Mechanism of Autophagy The key morphological feature of autophagy is the formation of a double-membrane vesicle termed the autophagosome,which is derived from a phagophore 11.The mammalian phagophore is initially generated near the endoplasmic reticulum(ER),and different membrane sources can contribute to the formation of this transient double-membrane structure 12,13.The phagophore is the active sequestering compartment of autophagy;this structure expands and ultimately seals to form the autophagosome 14.The completed autophagosome subsequently fuses with a lysosome;in many cell types the autophagosome may first fuse with an endosome,and the resulting amphisome then fuses with the lysosome.The fusion of the autophagosome or amphisome with the lysosome generates an autolysosome.In this compartment,the cytoplasmic cargo is exposed to the lumenal contents of the lysosome and subsequently degraded by resident hydrolases 15,16.Although autophagy had been studied morphologically and pharmacologically since the 1950s,detailed studies into the molecular mechanism of autophagy only started in the late 1990s,following the analysis of this pathway in yeast,and the identification of the autophagy-related(ATG)genes 17.At present,over 40 ATG genes have been identified in yeast,and,because this process is evolutionarily conserved,many of them have homologs in all other eukaryotes.The ATG proteins participate in different stages of autophagy including induction,nucleation of the phagophore,expansion of the phagophore,autophagosome completion and fusion with the lysosome,and the final steps of degradation and efflux of the breakdown products;those components that are required to form an autophagosome are referred to as the core autophagy machinery 18-21.Based on their roles in these different stages,the core ATG proteins can be roughly categorized into the following major groups(Fig.1):A.The ULK kinase complex Journal Pre-proof 4(including ULK1 or ULK2,RB1CC1/FIP200,ATG13 and ATG101)can phosphorylate downstream factors for the induction of autophagy;B.The class III phosphatidylinositol 3-kinase(PtdIns3K)complex 1,mainly composed of BECN1/beclin 1,PIK3C3/VPS34,PIK3R4/VPS15,ATG14 and NRBF2,acts in phagophore nucleation;C.The only transmembrane protein,ATG9A works together with ATG2A/ATG2B and WDR45/WIPI4 to function in phagophore membrane elongation;D.Two ubiquitin-like systems function in the completion of the autophagosome,utilizing ATG12(along with components ATG5,ATG7,ATG10,and ATG16L1)and LC3/GABARAP(along with ATG3,ATG4A to ATG4D,ATG7 and WIPI2);there are seven isoforms of LC3/GABARAP,referred to as Atg8-family proteins because of their homology to yeast Atg8.These core ATG proteins may not only work at one stage.For example,when binding to UVRAG(UV irradiation resistance associated)instead of ATG14,BECN1 functions as part of the PtdIns3K complex 2 to help in autophagosome formation and fusion with the lysosome 22.In addition,RB1CC1/FIP200 may interact with ATG16L1 and regulate its targeting to the phagophore 23.ATG13,a component of the ULK kinase complex,can also interact with ATG9A for the recruitment of the latter to the site of autophagosome formation 24.In addition to the above-mentioned core ATG proteins,the success of autophagy also relies on other key factors.The autophagy cargo receptor SQSTM1/p62 can recognize ubiquitin on cargo and deliver it to the phagophore by interacting with LC3 on the concave side of the membrane 25.Various receptors function in different types of selective autophagy,connecting the cargo with the phagophore via binding to an Atg8-family protein.The RAB GTPase RAB7 and the HOPS complex,along with SNARE proteins(STX17,SNAP29 and VAMP8),modulate various processes related to autophagy,such as autophagosome maturation,lysosomal biogenesis and maintenance,as well as autophagosome-lysosome fusion 26(Fig.1).Journal Pre-proof 5 As alluded to above,autophagy can either be non-selective or selective:the non-selective mode is thought to involve degradation of random portions of cytoplasm,whereas the selective mode is highly specific for certain components 1,27.Mitophagy is one of the best-studied types of selective autophagy,which involves the sequestration of damaged or superfluous mitochondria and prevents the generation of reactive oxygen species(ROS)28.This process is essential for maintaining the integrity of mitochondria and oxygen homeostasis for cellular and organismal survival.PINK1(PTEN induced kinase 1)and the E3 ubiquitin ligase PRKN/Parkin are two well-characterized factors that mediate mitophagy;however,mitophagy can also be independent of PINK1-PRKN,and not all mammalian cells express PRKN 29.Mitophagy receptors include SQSTM1 and OPTN,which can bind to LC3 and recognize the polyubiquitinated mitochondria 30,31.Other receptors include LIR-containing outer mitochondrial membrane proteins BNIP3L/Nix and FUNDC1,as well as the inner mitochondrial membrane protein PHB2 32(Fig.2).Different studies have reported selective autophagic degradation of other components including,but not limited to,protein aggregates(aggrephagy),peroxisomes(pexophagy),lysosomes(lysophagy),endoplasmic reticulum(reticulophagy),pathogen invasion(xenophagy),ferritin(ferritinophagy)and glycogen(glycophagy)5.3.A Brief History of Autophagy and Cancer As made clear by various studies,autophagy is important for multiple aspects of cancer biology,and its role in tumorigenesis is context dependent 33.That is,autophagy can be tumor-suppressive,tumor-promoting or even neutral,depending on the dynamic progression of the cancer 34.During tumor development,cytoprotective autophagy plays an anticancer role by maintaining cellular and genomic integrity.In 2007,Eileen Whites group showed that autophagy can suppress tumor progression by restricting chromosomal instability,and a similar result has been further Journal Pre-proof 6 validated in a recently published paper from Jan Karlseders group 35,36.Alternatively,a 2015 study suggested that defective autophagy may interfere with cellular senescence,a process that can limit the proliferation of damaged cells;thereby leading to aberrant proliferation of cancer progenitor cells 37.Once a tumor is formed,autophagy may have an opposite outcome,enabling tumor progression and proliferation 38.In addition,the functional consequences of autophagy can expand to endothelial,stromal and immune cells of the tumor microenvironment,further reinforcing the complicated role of autophagy in cancer.For instance,the upregulation of angiogenesis,a mechanism by which tumors gain nutrients and oxygen,has been regarded as a hallmark of tumorigenesis 39.The balance between pro-and anti-angiogenic factors governs the angiogenic switch in cancer cells.Several studies have shown that autophagy defects can either cause cerebrovascular disorders 40,or show increased angiogenic potential 41,42;therefore,autophagy has both both pro-and anti-angiogenic effects.The cancer-associated fibroblasts and cancer-associated adipocytes are examples of cancer-related stromal cells that can exhibit upregulation of autophagy to drive cancer progression 43,44.In these cells,autophagy participates in several intricate signaling pathways to influence tumor progression,and may represent a future target for tumor treatment 8.Furthermore,the relation between autophagy and immunity under the context of cancer is also complicated.Autophagy can play a bona fide role in both innate and adaptive immunity by controlling antigen presentation and modulating lymphocyte homeostasis,while the immune system can modulate autophagy in turn 45-47.Overall,the controversial role of autophagy in cancer leads to another question for cancer therapeutics:is it better to inhibit or to enhance autophagy?The last decade has witnessed a surge in research into the role of autophagy in cancer,and several comprehensive reviews have been Journal Pre-proof 7 published on the connection of autophagy and tumorigenesis 8,33,48,49.Here,instead of explaining the complicated relation between cancer and autophagy,we will briefly review the history of autophagy and cancer,with a focus on key events that occurred in the past 20 years(Fig.3).3.1 Core ATGs in Cancer The first landmark study to link autophagy and cancer was from Beth Levines group in 1999,which identified BECN1,the mammalian homolog of yeast Vps30/Atg6(an essential component for autophagy activity),as a tumor suppressor 50.Later,a 2003 study from the Levine group used the targeted mutant mouse model to further demonstrate that heterozygous disruption of BECN1 can promote tumorigenesis 51.That same year,Zhenyu Yue,Shengkan Jin,Arnold Levine and Nathaniel Heintz published a paper further supporting the view that BECN1 is a haploinsufficient tumor suppressor 52.Subsequent studies have confirmed that BECN1 is monoallelically lost in several cancer cell lines while Becn1 heterozygous mutant mice are prone to develop various malignancies 53.Downregulation of BECN1 mRNA expression is also associated with poor prognosis in human breast cancers 54.The importance of BECN1-mediated autophagy in tumor suppression is further strengthened by its association with UVRAG.The latter is a coiled-coil protein,which is monoallelically mutated at high frequency in human colon and breast cancers 55.In 2006,Jae Jungs lab found that UVRAG can interact with BECN1,activate autophagy and suppress colon cancer tumorigenicity 56.Through UVRAG,SH3GLB1/Bif-1(SH3 domain containing GRB2 like,endophilin B1)forms a complex with BECN1 and regulates autophagosome formation.Furthermore,SH3GLB1 ablation significantly enhances the development of spontaneous tumors in a BECN1-dependent manner in mice 57.Journal Pre-proof 8 In addition,several studies have been carried out with the goal of revealing the direct link between other ATG proteins and cancer.For example,a 2009 study from Sug Hyung Lees lab observed frameshift mutations of ATG2,ATG5,ATG9 and ATG12 in gastric and colorectal cancers with microsatellite instability 58.In 2011,Noboru Mizushimas group generated autophagy-deficient mice with a systemic mosaic deletion of ATG5 or the liver-specific k