WORKING HYPOTHESIS

The Significance of Autophagy in Cancer

Grace Ng1* and Jingxiang Huang2

1Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, United Kingdom2Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts

Autophagy, a vacuolar process of cytoplasmic degradation, is implicated in a form of programmed cell death distinct

from apoptosis. The regulation of autophagy is complex and signalling pathways such as the target of rapamycin (Tor)kinase pathway, play important roles in tumourigenesis. Beclin 1, an autophagic protein, has been found to be atumour suppressor. Conversely, cancer cells may exploit autophagy as a means to adapt to hypoxic and nutrient-

limiting environments. The relative importance of autophagic cell death and apoptosis in carcinogenesis remains to beestablished, and the Bcl-2 family of proteins may be instrumental in coordinating the two pathways of programmedcell death. � 2005 Wiley-Liss, Inc.

Key words: Beclin 1; Tor; phosphatidylinositol 3-kinase; Bcl-2; programmed cell death

INTRODUCTION

Evading death is an important facet of carcinogen-esis as cancer cells are under tremendous survivalpressures—from intrinsic triggers such as deregu-lated proliferation and DNA damage, to extrinsicfactors such as nutrient deprivation and immuneresponse. In the 1970s, apoptosis was initially identi-fied as a form of controlled or programmed celldeath, in contrast to necrosis, which was thought tobe ‘passive’ [1]. For some time, it has been recognisedthat a form of ‘apoptosis’ exists that is caspase-independent.

Further understanding of cell death resultedin dichotomisation of programmed cell death—apoptotic and autophagic cell death. Apoptosis ischaracterised by chromatin condensation, cellshrinkage, DNA degradation and fragmentation ofthe cell into apoptotic bodies. The remnants of thecell are then removed by phagocytosis. On the otherhand, autophagic cell death is defined by theappearance of double- or multi-membrane vesicleswhich engulf bulk cytoplasm and organelles. Auto-phagic vesicles and their contents are then destroyedby endogenous lysosomes.

WHAT IS AUTOPHAGY?

The term autophagy comes from Greek wordsmeaning ‘self’ and ‘eating’, and was coined todescribe how a cell, facing starvation, degrades intra-cellular components to obtain nutrients for survival.It is one of the two major eukaryotic pathways ofprotein degradation that helps in the normal turn-over of cellular constituents and organelles. Whereasproteasomal degradation is confined to cytosolic

proteins, autophagic degradation includes the degra-dation of entire organelles.

There are three identifiable types of autophagy—macroautophagy, microautophagy and chaperone-mediated autophagy.

Macroautophagy begins with the formation of ahalf circle of double- or multi-membrane-boundstructure in the cytosol that eventually surroundsbulk cytoplasm and/or cytoplasmic organelles suchas mitochondria, and fuses with a lysosome, re-leasing inside a single membrane-enclosed vesiclecalled an autophagic body. Both the membrane andcontent of the autophagic body are then degraded bythe proteases in the lysosome.

In microautophagy, cytoplasm is engulfed directlyby the lysosomal membrane via invagination of themembrane. The invaginations of the lysosomal mem-brane extend deep into the lysosome until they finallypinch off as vesicles into the lumen of the lysosome.

Dice et al. have characterised a chaperone-mediated route for selective transport of cytosolicproteins into lysosomes, in which a receptor belong-ing to the family of heat shock cognate proteins

MOLECULAR CARCINOGENESIS 43:183–187 (2005)

� 2005 WILEY-LISS, INC.

Abbreviations: Tor, target of rapamycin; PI3K, phosphatidylinosi-tol 3-kinase; PTEN, phosphatase and tensin homolog deleted onchromosome 10; GDP, guanosine 50-diphosphate; DAPk, death-associated protein kinase.

*Correspondence to: Medical Research Council Cancer Cell Unit,Hutchison/MRC Research Centre, Box 197, Hills Road, CambridgeCB2 2XZ, United Kingdom.

Received 19 December 2004; Revised 20 January 2005; Accepted26 January 2005

DOI 10.1002/mc.20097

(Hsc73) binds to soluble proteins containing thepeptide recognition signal KFERQ, initiating theirtransport into a prelysosomal (e.g. an endosome orautophagosome) or lysosomal compartment, ulti-mately resulting in the complete degradation of theprotein [2].

By far, macroautophagy appears to be the beststudied form of autophagy in relation to humanpathophysiology [3].

WHAT REGULATES AUTOPHAGY?

Target of rapamycin (Tor), a kinase that is involvedin the response to changes in intracellular aminoacid and ATP levels, acts as the gatekeeper for theinitiation of the autophagic pathway (Figure 1). Themechanisms by which the Tor protein regulatesautophagy have been studied in yeast. Undernutrient-rich conditions, Tor phosphorylates Apg13.When exposed to conditions of starvation, Toractivity is inhibited; Apg13 is dephosphorylated andtightly associates with Apg1 kinase, resulting in theactivation of Apg1 kinase which leads to the induc-tion of autophagy [4].

Tor was discovered in yeast as the target of anti-fungal drug rapamycin, which inhibits Tor activityand induces autophagy in yeast and mammaliancells. Conversely, the phosphorylation of proteins inthe Tor pathway, such as S6K and its target ribosomalprotein S6, leads to the inhibition of autophagy [5].

The class I phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt/PKB) signalling pathway pro-motes cell growth in response to mitogenic signals.Mutations in several proteins in this pathway havebeen found in human malignancies [6]. Class I PI3Kgenerates PI(3,4)P2 and PI(3,4,5)P3, which bind toAkt. Activation of the Akt pathway reduces autopha-gic degradation [7]. This phenomenon may be partlyexplained by the fact that Akt activates Tor, whichinhibits autophagy. The tumour suppressor phos-phatase and tensin homolog deleted on chromo-some 10 (PTEN) antagonizes the PI3K/Akt pathway,and positively regulates autophagy. Mutations inPTEN result in the activation of the Akt pathway andinactivation of autophagy [8]. In cells treated withPI3K inhibitors autophagosome precursors are notgenerated, indicating that PI3K activity is essential inthe early stages of autophagic vesicle formation.

Class III PI3K and its product phosphatidylinositol3-phosphate are also shown to be involved in auto-phagy signalling. In yeast, a PI3K complex consistingof Apg6, Apg14 and Vps15 and a PI3K, Vps34 isinvolved in autophagic vesicle formation. Beclin 1 inmammals is the orthologue of Apg6 in yeast. Uponamino acid starvation, Beclin 1 expression in MCF7breast cancer cells increased the number of autopha-gic vesicles. In addition, overexpression of Beclin 1 inApg6 disrupted yeast complemented the defectiveautophagic activity. The addition of 3-methylade-

Figure 1. Pathways involved in the regulation of autophagy.

184 NG AND HUANG

nine (3-MA), a PI3K inhibitor, prevented autophagyinduction by Beclin 1 in MCF7 cells [9].

In the colon cancer cell line HT-29, autophagy isregulated by heterotrimeric G proteins localised tothe Golgi/endoplasmic reticulum membranes [10],the guanosine 50-diphosphate (GDP) bound formof the Gai3 subunit being the autophagy-inducingform of the system. The hydrolysis of guanosine 50-triphosphate to GDP by a GTPase-activating protein,G-alpha interacting protein stimulates autophagy, asdoes the expression of activator of G-protein signal-ling 3, a protein that stabilizes the GDP bound formof the Gai3. G-alpha interacting protein is in turnregulated by the Ras/Raf-1/ERK1/2 signalling path-way, and activation of this pathway induces autop-hagy in HT-29 cells. This pathway is sensitive tochanges in amino acid availability. In the presence ofamino acids, activated Raf-1 fails to induce autop-hagy. There is further ‘crosstalk’ with the PI3K/Aktpathway as Akt phosphorylates and inactivatesRaf-1, thereby inhibiting autophagy.

In mammalian cells, phosphorylation of theeukaryotic initiation factor 2a (eIF2a), which occursin response to nutrient starvation, reduces proteintranslation and induces autophagy.

Death-associated protein kinase (DAPk) andDAPk-related protein-1 belong to a family of death-associated kinases. Overexpression of each incarcinoma cell lines induces autophagy [11]. InMCF-7 breast cancer cells, a dominant-negativeconstruct of DAPk-related protein-1 reduces the levelof starvation and tamoxifen-induced autophagy,while reduction of DAPk expression in HeLa cellsreduced interferon-g-induced autophagy.

DOES AUTOPHAGY HAVE A ROLEIN TUMOUR SUPPRESSION?

In the 1980s, an inverse relationship betweenautophagic activity and malignant transformationwas established, when it was found that the autop-hagic capacity of cancer cell lines was often lowerthan their normal counterparts. In animal models ofcarcinogenesis, a decrease in autophagic capacitywas also observed [12]. It was proposed that thedownregulation of autophagy provided a survivaladvantage resulting from the improved proteinbalance obtained by preventing excessive proteinloss upon starvation. In vivo, the maintenance of apositive protein balance may offer a clonal advan-tage to preneoplastic cells. Paradoxically, studies oncarcinogen-induced pancreatic cancer in rats show-ed that cells from premalignant nodules had anincreased autophagic capacity. However, cells frompancreatic adenocarcinoma had decreased autopha-gic activity [13]. These observations suggest thatautophagic capacity first increases during premalig-nant stages of pancreatic carcinogenesis, and thendecreases during the adenoma to adenocarcinomatransition. It may be deduced that the breakdown of

the autophagic process contributes to the develop-ment of cancer, although direct causal relationshipshave yet to be demonstrated.

The autophagic capacity of cells may correlateinversely with their ability to proliferate. Autophagyis the major degradative pathway of long-lived pro-teins, and an increase in the rate of their degradationdecreases the protein mass of the cell. As proliferat-ing cells including cancer cells need to reach aminimal size prior to cell division, increased auto-phagic activity may serve to inhibit proliferation.

Autophagy also plays a role in the elimination ofdamaged organelles. A defective autophagic systemleads to the accumulation of damaged organelles andan increase in genotoxic substances, which mayinduce mutations.

Additionally, autophagy may be a means to inhibittumour vascularisation. Characteristics of autopha-gic cell death have been observed in endothelial cellsin the presence of the angiogenesis inhibitor endo-statin [14]. As angiogenesis in solid tumours facil-itates metastasis and mass increase, the inhibition ofangiogenesis by autophagic cell death may inhibittumour progression.

Some previously known oncogenes and tumoursuppressor genes affect autophagic pathways, andthe deregulation of the autophagy-related aspects oftheir mode of action may contribute to malignanttransformation. These genes include PTEN, class IPI3K, Akt, Ras and Myc. In contrast, genes, such asBeclin 1, have been identified by their direct roles inthe execution of autophagy, before their tumoursuppressive functions were recognised [9].

Nevertheless, it may also be argued that thesuppression or activation of autophagy may beincidental for the regulators of autophagy found tobe involved in tumour progression, because theseregulators have alternative carcinogenic or tumoursuppressive roles. For example, Akt is involved in theregulation of a range of substrates that participate incell growth and survival, and tumourigenesis arisingfrom the activation of Akt may result from thedisturbance of a pathway other than autophagy.

CAN AUTOPHAGY PROMOTE TUMOUR FORMATION?

Although autophagy has been linked to oncogen-esis, whether autophagy causes, protects or plays adual role in the oncogenic process remains unclear.While its involvement in cancer cell death, and itsrole in limiting cell size and removing damagedorganelles which could generate free radicals andpromote mutations, support its function as a tumoursuppressor, there exists possible paradoxical roles ofautophagy in tumour promotion. In the later stagesof tumour growth, autophagy may be a means bywhich cancer cells survive nutrient-limiting and low-oxygen conditions in the internal region of thetumour, which is poorly vascularised. Autophagymay also protect against some cancer treatments, such

AUTOPHAGY IN CANCER 185

as ionising radiation, by removing damaged organ-elles, hence protecting the transformed cell againstapoptosis, and allowing its continued survival.

IS AUTOPHAGY LINKED TO APOPTOSIS?

As autophagic cell death and apoptosis are path-ways to the same end, a functional connectionbetween both forms of cell death is likely to beoperative. It may be envisioned that autophagyprecedes apoptosis by scavenging ‘leaky’ mitochon-dria, thereby delaying apoptosis. Alternatively, bothforms of cell death can act as backup mechanisms ofeach other, under conditions where cell death isimperative. For example, death by autophagy occurr-ed when the apoptotic pathway of HeLa cells wasblocked [15]. Correspondingly, apoptotic deathoccurred in malignant glioma cells exposed tobafilomycin A1—an inhibitor of autophagy [16].

The close interrelationship between the two path-ways is reflected in the overlapping effects of theirchemical suppressors. 3-Methyladenine, an inhibi-tor of autophagy, can also inhibit cytochrome crelease and caspase activation in neurons, therebydelaying apoptosis [17]. Similarly, the commonlyused pan-caspase inhibitor benzyloxycarbonyl-Valyl-Alanyl-Aspartyl-fluoromethylketone, also inhibitsthe lysosomal protease cathepsin, thus affectingautophagic degradation [18].

Recently, it was demonstrated that although Bcl-2and Bcl-XL inhibit apoptosis, they facilitate non-apoptotic death through interaction with Beclin 1[19]. Thus, it may be hypothesised that there exists acentral signalling pathway, possibly centred aroundthe mitochondria, that coordinates the fate of thedying cell.

CONCLUSION

Autophagic cell death may represent an alterna-tive form of programmed cell death, by which cellsthat have accumulated irreversible organelle damageand are at risk of malignant transformation aredestroyed. Recent research on the role of the Bcl-2family of proteins in autophagic cell death hint athow both pathways may be coordinated. However,the conditions which lead to the preferential execu-tion of one pathway over the other are still notentirely clear, and their significance in carcinogen-esis remains to be elucidated. There exist practicaldifficulties in parallel studies of the two pathways,especially for investigations involving tissue mate-rial. Whilst terminal deoxynucleotidyl transferase-mediated, dUTP-biotin nick end-labelling (TUNEL)may be used to determine the apoptotic index ofparaffin-embedded tissue sections, there is no corre-sponding method of quantifying the prevalence ofautophagic cell death in human cancer samples [20].

Not only does it remain to be established thatdeficient autophagy plays a causative role in tumour-igenesis, there exists the paradoxical possibility that

autophagy actually promotes tumour growth inlow-nutrient and hypoxic conditions, in the samemanner by which it supports survival in normal cells.Perhaps, partial inhibition of autophagy results inincreased cell death, whilst more complete abroga-tion of autophagy results in tumourigenesis. Specificinhibitors of autophagy that can precisely modulateautophagic activity in cell or animal models can helpresolve this issue.

Further answers to this apparent paradox may liein a greater understanding of the biological role ofBeclin 1 and other markers of autophagy. Unravel-ling the molecular mechanisms of autophagy mayprovide new insights into the relationship betweenautophagy and oncogenesis.

ACKNOWLEDGMENTS

We thank Dr. Z.-M. Yuan and J. Stuart (HarvardSchool of Public Health) for their invaluable com-ments on this manuscript.

REFERENCES

1. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biologicalphenomenon with wide-ranging implications in tissuekinetics. Br J Cancer 1972;26:239–257.

2. Chiang HL, Terlecky SR, Plant CP, Dice JF. A role for a 70-kilodalton heat shock protein in lysosomal degradation ofintracellular proteins. Science 1989;246:382–385.

3. Shintani T, Klionsky DJ. Autophagy in health and disease: Adouble edged sword. Science 2004;306:990–995.

4. Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M,Ohsumi Y. Tor-mediated induction of autophagy via an Apg1protein kinase complex. J Cell Biol 2000;150:1507–1513.

5. Scott RC, Schuldiner O, Neufeld TP. Role and regulation ofstarvation-induced autophagy in the Drosophila fat body.Dev Cell 2004;7:167–178.

6. Blume-Jensen P, Hunter T. Oncogenic kinase signaling.Nature 2001;411:355–365.

7. Meijer AJ, Codogno P. Regulation and role of autophagy inmammalian cells. Int J Biochem Cell Biol 2004;36:2445–2462.

8. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTENpositively regulates macroautophagy by inhibiting the phos-phatidylinositol 3-kinase/protein kinase B pathway. J BiolChem 2001;276:35243–35246.

9. Liang XH, Jackson S, Seaman M, et al. Induction of auto-phagy and inhibition of tumorigenesis by beclin 1. Nature1999;402:672–676.

10. Ogier-Denis E, Couvineau A, Maoret JJ, et al. A hetero-trimeric Gi3-protein controls autophagic sequestration in thehuman colon cancer cell line HT-29. J Biol Chem 1995;270:13–16.

11. Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A. DAP kinase andDRP-1 mediate membrane blebbing and the formation ofautophagic vesicles during programmed cell death. J Cell Biol2002;157:455–468.

12. Schwarze PE, Seglen PO. Reduced autophagic activity,improved protein balance and enhanced in vitro survival ofhepatocytes isolated from carcinogen-treated rats. Exp CellRes 1985;157:15–28.

13. Toth S, Nagy K, Palfia Z, Rez G. Cellular autophagic capacitychanges during azaserine-induced tumour progression in therat pancreas. Up-regulation in all premalignant stages anddown-regulation with loss of cycloheximide sensitivity ofsegregation along with malignant transformation. CellTissue Res 2002;309:409–416.

186 NG AND HUANG

14. Chau YP, Lin SY, Chen JH, Tai MH. Endostatin inducesautophagic cell death in EAhy926 human endothelial cells.Histol Histopathol 2003;18:715–726.

15. Xue L, Fletcher GC, Tolkovky AM. Mitochondria areselectively eliminated from eukaryotic cells after blockadeof caspases during apoptosis. Curr Biol 2001;11:361–365.

16. KanzawaT, Kondo Y, Ito H, Kondo S, Germano I. Induction ofautophagic cell death in malignant glioma cells by arsenictrioxide. Cancer Res 2003;63:2103–2108.

17. Xue L, Fletcher GC, Tolkovsky AM. Autophagy is activated byapoptotic signalling in sympathetic neurons: An alternative

mechanism of death execution. Mol Cell Neurosci 1999;14:180–198.

18. Foghsgaard L, Wissing D, Mauch D, et al. Cathepsin B acts asa dominant execution protease in tumour cell apoptosisinduced by tumour necrosis factor. J Cell Biol 2001;153:999–1010.

19. Shimizu S, Kanaseki T, Mizushima N, et al. Role of Bcl-2 familyproteins in a non-apoptotic programmed cell death depen-dent on autophagy genes. Nat Cell Biol 2004;6:1221–1228.

20. Mizushima N. Methods for monitoring autophagy. Int JBiochem Cell Biol 2004;36:2491–2502.

AUTOPHAGY IN CANCER 187