Autophagy and cancer: Dynamism of the ... - ResearchGate

ARTICLE IN PRESS2 K. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxxFinally, maturated autophagosomes fuse with the lysosome.The engulfed components, as well as the inner membraneof the autophagosome, are degraded by lysosomalhydrolases such as cathepsins. Autophagic processes havebeen well characterized in yeast, and more than 30autophagy-related genes (ATG) that encode the proteinsexecuting autophagy have been identified in the field ofyeast genetics [6]. Similar autophagic machineries havebeen observed in mammalian cells, and mammalian orthologsfor ATGs and other autophagy regulating moleculeshave also been identified [7]. The detailed molecularevents involved in these processes are described in anothercomprehensive review [8].Compared with the ubiquitin–proteasome system,which recognizes specific targets for degradation, autophagyhas been thought to engulf cytoplasmic constituentsnon-selectively. Recently, selective sequestration targetingspecific organelles or invasive microbes has also attractedattention [9].A very concise description of the function of autophagyis recycling; reducing waste and yielding resources. Damagedorganelles and misfolded proteins accumulate insenescent cells or cells under various stresses, such as oxidativestress and infection. The autophagic machinery degradesand reduces this cellular ‘‘garbage”. This functionis particularly effective in postmitotic cells, like neuronsand cardiac myocytes, since these cells cannot dilute suchsuperfluous components by undergoing cell division. Basalautophagy is constitutively observed in these postmitoticcells and may have an important function for the qualitycontrol of cellular components [10]. On the other hand,self-digested components provide nutrients and materialsfor newly constructed cellular structures. The amino acidsand fatty acids generated by autophagic degradation areused by the tricarboxylic acid (TCA) cycle to produce ATP,a main energy source for various cellular events. Methylpyruvate,a membrane-permeable derivative of pyruvatethat serves as a substrate for the TCA cycle, restored ATPproduction in autophagy-deficient cells. Furthermore, supplementationwith methylpyruvate rescued these cellsfrom metabolic stress-induced cell death [11].Observations of clinical samples and animal and cellularmodels have suggested a variety of physiological and pathologicalroles of autophagy, such as development, aging,host defense system, neurodegenerative diseases, muscleand cardiac diseases and cancer [12,13]. However, the relevanceof autophagy in tumor formation and progression isstill controversial. Although previous findings stronglysuggest that autophagy contributes to sustainable cell survival,anti-tumorigenic roles of autophagy have been alsomentioned. Here, we will summarize and discuss recentstudies of autophagy in cancer biology with the goal ofclarifying this issue.2. Autophagy protects cancer cells from starvationCancer cells in solid tumors obtain their necessarynutrients from blood flow. Aberrantly proliferating cancercells may have high bioenergetic demands and requiremore nutrients than non-cancerous cells. Tumor angiogenesisis a reasonable way to increase blood flow. During theinitial phase of tumorigenesis, tumor vessels have not yetbeen induced and the nutritional demands of tumor cellsare likely to surpass the supply from normal vasculature.Moreover, even after tumor vessels have been established,the oxygen tension and glucose concentration in locallyadvanced tumors remain at a low level. This suggests thatthe tumor microvasculature is functionally and structurallyimmature to support sufficient blood flow [14–16].Or, even once functionally and structurally adequate tumorvessels were established, soon or later, the balance of supplyand demand would be ruined by disorganized tumorcell proliferation resulting in the disastrous dysfunctionof the tumor vessels. The reduction of functional bloodflow is significant in clinically hypovascular tumors suchas pancreatic cancers [17]. Under these conditions, cancercells are likely to encounter chronic ischemia leading to ashortage of nutrients. Cancer cells might adapt themselvesto such a harsh microenvironment. In experimental cellculture systems, several cancer cells were resistant tonutrient-deprived conditions. For example, several pancreaticcancer- and colorectal cancer-derived cell linesshowed a survival rate of more than 50% after 48 h of culturein a medium completely lacking carbon and nitrogensources. Contrastingly, non-transformed fibroblasts werecompletely abolished within a day under the same conditions[18]. These findings suggest that cancer cells usealternative metabolic processes for their survival understarved conditions.Metabolic stresses reportedly induce apoptosis [19]. Inmany tumor cells, apoptosis is suppressed by the overexpressionof anti-apoptotic molecules or by the lack ofpro-apoptotic molecules. Less apoptosis may explain thelimited death of cancer cells under metabolic stresses,but the manner in which cancer cells obtain their necessarynutrients remains a mystery. One possible solutionis that the cells digest their own components and obtainamino acids as an alternative energy source. Autophagyseems ideal for cancer cells to maintain energy homeostasisin such an autonomous fashion. Autophagy is known tobe induced by different forms of metabolic stress, includingnutrient deprivation, growth factor deprivation, andhypoxia [13,20]. Since these conditions are often observedin physiological tumor microenvironments, autophagy islikely activated in cancer cells.We should also remind the apparent discrepancy betweenthe tumor ‘‘cell” doubling time and the tumor ‘‘volume”doubling time. The potential tumor cell doublingtime estimated by in vivo measurement of S-phase durationwas remarkably rapid in many solid tumors, a medianvalue of the order of 5 days. However, tumor volume doublingtime determined by radiological measurement of thesize of these tumors was much slower, months or years[21,22]. It suggests that about half of the proliferating cancercells are subsequently lost under tumor microenvironmentthough many of them have already acquired antiapoptoticfunction. Is there any physiological relevance ofsuch dynamism of cell kinetics? A potential scenario is thatthe vanishing cells serve nutritional sources for surroundingsurviving cells. In this sense, inducing cell death andsupporting cell survival under metabolic stresses are notPlease cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

ARTICLE IN PRESSK. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxx 3contradicting. Autophagy is also known to induce celldeath and possibly sacrifice some part of individual tumorcell for supporting a prolonged survival and continuousgrowth of other cells in the tumor mass.Several experimental methods have been used to identifythe activation of autophagy (Table 1) [23]. Measuringthe turnover of long-lived proteins provides a biochemicalclue to autophagy. Transmission electron microscopy revealsthe ultrastructure characteristics of the autophagosome.Autophagosome formation is also convenientlymonitored by following a phosphatidylethanolamine (PE)-conjugated form of yeast Atg8 or mammalian LC3, one ofthe mammalian orthologs of Atg8. During autophagy, LC3shifts from a soluble form to a membrane-bound form(LC3-II) and is incorporated into the autophagosomal innermembrane. The existence of autophagosome-incorporatedLC3-II can be detected biochemically (immunoblottingagainst LC3) or microscopically (immunocytochemistryagainst LC3 or exogenously expressed, fluorescently taggedLC3).The ultrastructure of autophagosomes in tumor cellshas been observed in several experimental systems,including a rat pancreatic cancer model [24]. A high potentialfor autophagic protein degradation was observed in anundifferentiated colon cancer cell line, HT29, and othertransformed cells [25]. Nutrient deprivation-induced LC3-II turnover was observed in colorectal cancer-derived celllines that showed resistance to starvation [26]. In additionto these findings in cultured cancer cells, the increasedexpression of autophagy-related proteins, including BNIP3and LC3, was observed specifically in colorectal and gastriccancer epithelia in surgically-resected specimens [26,27].Increasing evidence implies that autophagy has a cytoprotectiverole in cancer cells under metabolic stress.Transplanted epithelial tumors in which the Beclin 1 orAtg5 alleles were deleted showed a reduction in autophagyand an increase in cell death in regions exposed to metabolicstress [28–30]. Similarly, a cytoprotective functionof autophagy was observed in cultured cancer cells. The geneticinactivation of autophagy by the suppression of ATGexpression using RNA interference or the constitutive activationof PI3K induced cell death in response to metabolicstresses [28–30]. RNA interference of ATG5, Beclin 1 andATG7 enhanced tamoxifen-induced apoptosis in tamoxifen-resistantbreast cancer cell lines [31]. ATG5 knockdownalso enhanced the effect of alkylating drug-inducedcell death [32]. The pharmacological inhibition of autophagyalso induced nutrient deprivation-induced cell death[26]. Chloroquine, which interferes with lysosomal function,inhibited autophagy and suppressed Myc-inducedlymphomagenesis in a transgenic mouse model [32].Though the above findings suggest that autophagy maycontribute to tumor cell survival and tumor formation, themolecular mechanisms underlying the acquisition of vigorousautophagic activity in cancer cells have remained unclear.Recently, Kroemer and his colleagues reported apotential anti-autophagic function of cytoplasm-localizingp53 [33]. They observed the degradation of p53 under metabolicstresses followed by the induction of autophagy.Contrastingly, the loss of p53 resulted in the consistentactivation of autophagy in a series of cell lines. While overexpressionof wild-type p53 reduced the aberrant autophagosomeformation, a mutant p53 protein whichharbors a codon 175 mutation (R175H), which is frequentlyidentified in clinical human cancers, did not affectthe higher basal autophagic activity of p53-null cells.Though most of their observation was limited in the culturecell system, further studies which clarify its relevanceto tumor formation and progression are awaited.3. Autophagy and tumor suppressionAn opposite perspective was presented by a studyexamining a Bcl-2-binding protein, Beclin 1. Levine andTable 1Recommanded methods for monitoring autophagy in higher eukaryotes [23].CriteriaMethodsMonitoring phagophore and autophagosome formation by steady state methods1. Electron microscopy (increase in autophagosome quantity) Quantitative electron microscopy, immunoelectron microscopy2. Atg8/LC3 Western blotting and ubiquitin-like protein conjugation Western blotsystems(increase in the amount of LC3-II, and Atg12–Atg5 conjugation)3. Fluorescence microscopy (increase in punctate LC3 (or Atg18)) Fluorescence, immunofluorescence and immunoelectron microscopy4. TOR and Atg1 kinase activity Western blot, immunoprecipitation or kinase assays5. Transcriptional regulation Northern blot or qRT-PCRMonitoring autophagy by flux measurements1. Autophagic protein degradation Turnover of long-lived proteins2. Turnover of LC3-II Western blot +/ lysosomal fusion or degradation inhibitors3. GFP-Atg8/LC3 lysosomal delivery, and proteolysisFluorescence microscopy, FACS Western blot +/ lysosomal fusion or degradation(to generate free GFP)inhibitors4. p62 Western blot Western blot with qRT-PCR or Northern blot to assess transcription5. Autophagic sequestration assays Lysosomal accumulation by biochemical or multilabel fluorescence techniques6. Turnover of autophagic compartments Electron microscopy morphometry/stereology7. Autophagosome-lysosome colocalization and dequenching assay Fluorescence microscopy8. Sequestration and processing assays in plants Chimeric RFP fluorescence and processing, light and electron microscopy9. Tandem mRFP-GFP fluorescence microscopy Fluorescence microscopy of tandem mRFP-GFP-LC310. Tissue fractionation Centrifugation, Western blot and electron microscopy11. Analyses in vivo Fluorescence microscopy and immunohistochemistryPlease cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

ARTICLE IN PRESS4 K. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxxher colleagues first identified Beclin 1, a mammalian orthologof ATG6, as a candidate tumor suppressor. Beclin 1interacts with class III PI3-kinase, Vps34, and this interactionwas crucial for the induction of autophagy and suppressionof the growth of the xenografted breast cancercell lines [34–36]. Vps34 is also required for normal proteintrafficking pathways such as the delivery of proteasesfrom the trans-Golgi network to the lysosomes. Atg6(Vsp30) was reportedly involved in the regulation of bothautophagy and endosomal membrane trafficking in yeast.Whether mammalian Beclin 1 takes part in the membranetrafficking is still controversial [34,35].The hemiallelic loss of the Beclin 1 coding gene was observedin 40–75% of sporadic human cancers in the breast,ovary and prostate [37]. A gene-targeted mouse model ofBeclin 1 provided another clue [38,39]. The homozygousdeletion of Beclin 1 led to embryonic lethality. Meanwhile,Beclin 1 heterozygous mutant mice showed decreasedautophagy and increased spontaneous tumors, includinglung and liver cancers and lymphomas. In these mice, noloss of heterozygosity (LOH) was observed and the remainingwild-type allele of Beclin 1 was intact. Furthermore,Beclin 1 protein expression was reduced but not completelydiminished in mouse tumors as well as human clinicalsamples [36]. These findings suggest that Beclin 1 is ahaplo-insufficient tumor-suppressor gene. The tumor-suppressivefunction of Beclin 1 was reinforced by the relevanceof Beclin 1-associated proteins to tumorsuppression. UVRAG was initially identified from the cDNAlibrary, which partially rescued the UV sensitivity of axeroderma pigmentosum (XP) cell line. The screening ofBeclin 1-binding proteins revealed that UVRAG was recruitedto the Beclin 1–class III PI3K complex. UVRAG activatedBeclin 1 and induced autophagosome formation [40].In addition, UVRAG is involved in autophagosome maturation.UVRAG interacts with class C tethering proteins(Vps11, Vps16, Vps18 and Vps33) resulting in activationof a small GTPase, Rab7 which enhances the fusion of autophagosomesto endosomes [41]. As seen with Beclin 1, UV-RAG is monoallelically mutated in human colorectalcancers [40]. Bif-1 (also known as Endophilin B1) interactswith Beclin 1 through UVRAG. Bif-1 activated class III PI3Kand induced autophagy. Spontaneous tumor formationwas increased in Bif-1 deficient mice [42].During investigations of autophagy in mammalian cells,several signaling pathways have been revealed to regulateautophagy. Interestingly, correlations between pro-autophagicmolecules and tumor suppressors and betweenanti-autophagic molecules and oncogene products can bepointed out (Fig. 1). The mammalian target of rapamycin(mTOR) is a key molecule for regulating cancer cell proliferation.Rapamycin inhibits mTOR function followed byautophagy induction [11,43]. Molecules known to suppressmTOR, including PTEN and TSC, both of which are regardedas tumor-suppressor gene products, induce autophagy[44,45]. Meanwhile, mTOR-activating molecules like classI PI3K and Akt, which are frequently activated in variouscancer cells, inhibit autophagy [11,46]. The involvement ofp53 in the activation of autophagy has also been suggested.In contrast to the inhibitory effect of cytoplasmic p53 men-Fig. 1. Cellular events during autophagy (macroautophagy). In the first step of autophagy, the isolation membrane (phagophore) is elongated andsequestrates the cytoplasmic constituents. Fusion of the edges of the isolation membrane forms an autophagosome, which contains the sequestratedcomponents. A lysosome then fuses with the autophagosome, and lysosomal hydrolases degrade the engulfed components along with the inner membraneof the autophagosome. Mammalian homologues of ATG (autophagy-related genes) have been identified. Class I PI3K and mTOR signaling inhibits autophagyactivation, whereas tumor-suppressor proteins like p53 and PTEN induce autophagy. Rapamycin inhibits mTOR and induces autophagy. 3-Methyladenine,chloroquine, bafilomycin A1, E64d and pepstatin A inhibit autophagy at different points. These small molecules are used to inhibit autophagy inexperimental model systems.Please cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

ARTICLE IN PRESSK. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxx 5tioned above, the transactivating function of nuclear p53takes a role in the autophagy induction. DRAM is a directtranscriptional target of p53, and its product localizes inthe lysosomal membrane. The activation of p53 inducedan increase in autophagy in a DRAM-dependent manner[47]. p53 was also reported to affect autophagy via the suppressionof mTOR [45]. DAPk, death-associated protein kinase,induces autophagy as well as apoptosis. The tumorsuppressivefunction of DAPk has also been described, andpromoter hypermethylation of the DAPk locus has been reportedin several human cancers [48].These findings suggest the likely relevance of autophagyto tumor suppression. However, the above-mentionedpro- and anti-autophagic molecules, like p53 and mTOR,are pluripotent proteins that regulate cell proliferationand death via various molecular events. The net contributionof autophagy to cell fate decisions during tumorigenesisshould be carefully estimated, but this objective hasnot yet been successfully accomplished.4. Autophagy and cell deathAutophagy has attracted much attention in connectionwith cell death. ‘‘Type II” or ‘‘autophagic” non-apoptoticprogrammed cell death is morphologically defined by theexistence of autophagosomes [49]. Autophagic death hasbeen reported in cancer cells, especially those that havebeen treated with chemotherapeutic or radiotherapeuticagents [50]. This cytotoxic effect has been supposed to bethe main reason for the tumor-suppressive function ofautophagy. Self-cannibalism, that is, the situation in whichexcess autophagic catabolism exceeds the capacity for cellularanabolism, is frequently argued as a potential mechanismfor autophagic death. Though autophagy has beenregarded to degrade cytoplasmic constituents non-specifically,the specific autophagic degradation of target proteinsand organelles has also been reported. The specific degradationof cytoprotective factors is another undeniablemechanism of autophagic death [51]. However, experimentalevidence supporting these ideas has not yet beenobtained. Moreover, cytotoxic stimuli, such as oxidativestress, induces both autophagy and cell death, but whetherautophagy is an active death-inducing mechanism (celldeath by autophagy) or a result of an unsuccessful effortto prolong the survival of damaged cells (cell death withautophagy) is difficult to distinguish.Recent findings have raised a question about the relevanceof autophagic death to tumor suppression. The allelicloss of Beclin 1 inhibited both basal and stress-inducedautophagy in immortalized baby mouse kidney epithelialcells. Metabolic stress-induced cell death was apparentlyincreased, suggesting loss of autophagy led impaired cytoprotectivefunction. Despite impairment in autophagymediatedcell survival, Beclin 1+/ cells were more tumorigenicthan the wild-type control cells [28]. To make aplausible explanation for these contradictory findings, theloss of cytoprotection induces tumorigenesis, is stillchallenging.5. Potential mechanisms for the tumor-suppressivefunction of autophagyRecently, White and colleagues have proposed interestinghypotheses about the above issues (Fig. 2). Under meta-Fig. 2. Both the gain and loss of autophagy are possibly involved in tumor formation and growth. The activation of autophagy supplies nutrients to tumorcells under metabolic stress. On the other hand, the eradication of tumor cells by autophagy-related cell death is assumed to occur, and the loss ofautophagy may lead to tumor cell survival. A recent mouse model using transplanted autophagy-deficient tumor cells revealed that the loss of autophagyinduced DNA damage and inflammation, enhancing tumor formation and growth.Please cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

ARTICLE IN PRESS6 K. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxxbolic stress, Beclin 1+/ epithelial cells overexpressing antiapoptoticBcl-2 showed an increase in DNA double strandbreaks, gene amplification, and chromosomal number disorder.They assumed that the loss of autophagy inducesDNA damage and chromosomal instability, followed by increasedtumor susceptibility [30]. The exact mechanismsfor how autophagy maintains genome stability remain unclear.One possibility is that cells lacking autophagy are unableto reduce damaged mitochondria and peroxisomes.These damaged organelles are potential sources of reactiveoxygen species that induce genotoxic stress.When the same autophagy-deficient epithelial cellswere exposed to starvation, apparent necrotic cell deathwas observed when apoptosis was inhibited. Furthermore,significant inflammation was induced in the tumor tissuesin which autophagy-deficient cells were transplanted [28].Necrosis is often associated with macrophage infiltrationin vivo, and tumor-associated macrophages enhance tumorprogression [52]. It is an interesting hypothesis that theinflammation induced by a lack of autophagy may be correlatedwith tumor progression in vivo.The above scenarios are not incompatible with the ideathat autophagy protects cancer cells from metabolic stressinduceddeath, and these potential mechanisms are worthyof further discussion to elucidate the physiological roles ofautophagy in tumorigenesis.6. Perspective – remaining tasksThe relevance of autophagy to cancer remains a frustratingtopic to discuss. It is necessary to understand theconsequences of the loss or gain of autophagy in the contextof the tumor microenvironment. Previously proposedhypotheses for how autophagy contributes to tumor biology,either pro- or anti-tumorigenic, are possible and interesting,but most of them have arisen from artificialexperimental systems and no direct clinico-pathologicalevidence supports these ideas. Evaluating autophagy inclinical tumor samples has been difficult, mainly becauseof the lack of appropriate markers for detecting activeautophagy. Autophagosome formation in tumor tissueshas been morphologically confirmed using electronmicroscopy, but this method is not suitable for handlinga large number of specimens. Recently, anti-LC3 antibodiesfor immunostaining have become available [26,53,54].LC3-II proteins incorporated into autophagosome membranesexhibit a punctuate cytoplasmic staining patternduring active autophagy. LC3 immunostaining is an easiermethod of evaluating autophagosome formation in conventionalformaldehyde-fixed surgically-resected specimens.We applied polyclonal anti-LC3 antibody for theimmunostaining of colorectal cancer specimens. In over90% of the cases, LC3 accumulated specifically in cancerousepithelia but not in adjacent non-cancerous mucosa [26].Similar cancer-specific accumulation of LC3 was observedin pancreatic cancer. Interestingly, strong LC3 expressionin the peripheral area of cancer tissue was correlated witha poor prognosis (Fig. 3) [55]. As we mentioned earlier,both pancreatic and colorectal cancer-derived cell linesare often resistant to the nutrient-starvation. It seemsmore than coincidence that specific LC3 accumulationwas observed in the clinical samples of these tumors. Furtherinvestigation using various cancer tissue samples mayreveal the contribution of the gain or loss of autophagy intumor formation and progression more clearly.Autophagy is executed by dynamic and multiple cellularprocesses, including the formation of the autophagosome,the delivery of cytoplasmic constituents to thelysosome, and the digestion and recycling of these targetmolecules and organelles. For the precise evaluation ofautophagic activity, the ‘‘autophagic flux” must be estimated.For example, the accumulation of autophagosomesreflects either an increase in autophagosome formation(activation of autophagy) or a reduction in the degradationof autophagosomes (inhibition of autophagy). To determinethe flux, the rate of long-lived protein degradationmust be measured or the changes in appropriate autophagymarkers, such as the LC3-II protein level, must be assessedwith or without the arrest of autophagic flux at agiven point of blockage. Recently, Klionsky and 231 otherscientists published guidelines for the use and interpretationof assays for monitoring autophagy in higher eukaryotes(Table 1) [23]. In these guidelines, the authors stronglyrecommended the measurement of autophagic flux formonitoring autophagy. Detecting autophagosome formationusing steady state methods, including electronmicroscopy or LC3 immunostaining, should be combinedwith flux measurements. However, applying such dynamicassays to clinical samples is often technically difficult.The above-mentioned guidelines do not provide a fullresolution of the above difficulties. Further discussionmight be needed to establish standard methods for evaluatingautophagy in clinical samples. To assess autophagicactivity using steady state methods, not only one parameter,but multiple autophagy-related molecules should beapplied. For example, while the loss of Beclin 1 implies areduction in autophagosome formation, this outcomeshould be confirmed by a decrease in autophagosome-specificmarkers, such as LC3-II. Mimicking in vivo events onex vivo systems may support the findings obtained in clinicalsamples using steady state methods. In such cases, itshould be remembered that autophagy is induced undercomplex microenvironments in cancer tissues. As mentionedpreviously, cancer cells are often exposed to chronicischemia. Under this situation, not only nutrients but alsooxygen, growth factors and other components providedby blood flow might be decreased. Meanwhile, metabolitesand other components excreted from cancer cells may notbe properly removed from the cancer tissues. Cancer cellsare also directly and indirectly influenced by surroundingstromal cells and extracellular matrices. Reconstitutingall these factors in an experimental cell culture system ispractically unfeasible, but the above restrictions should alwaysbe taken into account when evaluating the resultsobtained from any model system.To analyze autophagy function under more physiologicalconditions, appropriate animal models are eagerlyneeded. Xenografted and/or isografted animals in whichthe transplanted transformed cells lacking autophagy-relatedgenes are useful, but not perfect. Transplanted tumorsdo not always reproduce the microenvironmentalPlease cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

ARTICLE IN PRESSK. Tsuchihara et al. / Cancer Letters xxx (2008) xxx–xxx 7Fig. 3. Autophagosome formation in the surgically-resected specimens. (A) Autophagosome-incorporated LC3 protein was detected using GFP-LC3 fusionprotein in a cultured colorectal cancer cell line, SW480, during amino acid-deprivation. (B) Tumor cell-specific LC3 accumulation in colorectal cancer tissuewas detected using immunohistochemistry with anti-LC3 antibody and hematoxylin and eosin staining. (C) LC3 accumulation at the peripheral area of thepancreatic cancer tissue. Nv: nerve cells in which LC3 is constitutively expressed. (inlet) Higher magnification of (B) and (C). LC3 proteins were localized inthe cytoplasm with irregular condensation. (D) The group with strongly positive expression of LC3 protein in the peripheral area of the pancreatic cancertissue had a significantly shorter survival time. (A) and (B) are quoted from Ref. [26], (C) and (D) are quoted from Ref. [55].conditions of clinical tumors. Furthermore, such models donot provide information regarding the initial steps oftumorigenesis, in which autophagy might have criticalroles for pampering aberrantly proliferating cancer cellswithout extra blood supply. Examining the effect of variousautophagy inhibitors in tumorigenic animals is anotherway to evaluate the roles of autophagy in vivo. But it hasthe drawback that these reagents are not completelyautophagy-specific [32]. Recently, several genetically engineeredmouse models targeting autophagy-related geneshave been reported [12]. However, these mice often showembryonic or neonatal lethality, hampering the long-termobservation of tumorigenesis in adult animals. Tissue-specificor inducible conditional knockout systems may beuseful for this purpose.Whether the control of autophagy is useful for cancertherapy and prevention is another important issue. Severalsmall molecules are known to activate or inhibit autophagy(Fig. 1) [56]. Inhibiting the class I PI3K-mTOR axis byrapamycin induces autophagy. The inhibition of autophagycan be achieved by targeting several points. A class III PI3Kinhibitor (3-methyladenine), lysosomotropic alkalines(chloroquine and 3-hydroxychloroquine), a lysosomal protonpump inhibitor (bafilomycin A1) and lysosomal enzymeinhibitors (E64d and pepstatin A) are available forexperimental use. Since the contribution of autophagy totumorigenesis is still controversial, we should carefullyconsider the application of autophagy inhibitors or activatorsfor therapeutic use. Increasing evidence emphasizesthe importance of basal autophagy for cellular quality control[57]. The disturbance of autophagy might result inunexpected adverse effects, especially in postmitotic cellsin the central nervous system and cardiovascular system.The specificity of such activators and inhibitors is anotherissue to consider. The above-mentioned chemicals are notautophagy-specific. Also, the targeted kinases (PI3K,mTOR) or organelles (lysosome) have broad functionsother than autophagy regulation.In the last decade, autophagy has appeared in the centerstage of cancer biology and is now attracting much attentionin the development of new cancer therapeutics. Asmentioned above, a considerable number of issues remainto be clarified. However, similar to its molecular process,the research field of autophagy is very dynamic. Novelstrategies for monitoring autophagy and experimentalmodels are appearing, and various small molecules regulatingautophagic process will be available. Further investigationwill reveal the dynamism of metabolism in cancercells and tissues that is still behind the scene now. And itwill likely open up a new field of cancer biology.Conflicts of interest statementNone declared.AcknowledgementsThis work was supported by Grants for the Third-TermComprehensive 10-Year Strategy for Cancer Control, and aGrant-in-Aid for Cancer Research from the Ministry ofHealth, Labour and Welfare and a Grant-in-Aid for ScientificResearch on Priority Areas from the Ministry of Education,Culture, Sports, Science and Technology – Japan.References[1] D.J. Klionsky, The molecular machinery of autophagy: unansweredquestions, J. Cell Sci. 118 (2005) 7–18.Please cite this article in press as: K. Tsuchihara et al., Autophagy and cancer: Dynamism of the metabolism of tumor cells..., Cancer Lett. (2008), doi:10.1016/j.canlet.2008.09.040

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