Review Article Autophagy, Warburg, and Warburg Reverse ...downloads.hindawi.com/journals/bmri/2014/926729.pdf · Review Article Autophagy, Warburg, and Warburg Reverse ... homeostasis

Review ArticleAutophagy Warburg and Warburg Reverse Effects inHuman Cancer

Claudio D Gonzalez12 Silvia Alvarez1 Alejandro Ropolo1 Carla Rosenzvit2

Maria F Gonzalez Bagnes2 and Maria I Vaccaro1

1 Institute of Biochemistry and Molecular Medicine National Council for Scientific and Technological ResearchSchool of Pharmacy and Biochemistry University of Buenos Aires Junin 956 p5 1113 Buenos Aires Argentina

2Department of Pharmacology CEMIC University Institute 1113 Buenos Aires Argentina

Correspondence should be addressed to Maria I Vaccaro mvaccaroffybubaar

Received 23 April 2014 Accepted 24 July 2014 Published 12 August 2014

Academic Editor Genichiro Ishii

Copyright copy 2014 Claudio D Gonzalez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Autophagy is a highly regulated-cell pathway for degrading long-lived proteins as well as for clearing cytoplasmic organellesAutophagy is a key contributor to cellular homeostasis and metabolism Warburg hypothesized that cancer growth is frequentlyassociated with a deviation of a set of energy generation mechanisms to a nonoxidative breakdown of glucose This cellularphenomenon seems to rely on a respiratory impairment linked to mitochondrial dysfunction This mitochondrial dysfunctionresults in a switch to anaerobic glycolysis It has been recently suggested that epithelial cancer cells may induce the Warburgeffect in neighboring stromal fibroblasts in which autophagy was activated These series of observations drove to the proposal of aputative reverse Warburg effect of pathophysiological relevance for at least some tumor phenotypes In this review we introducethe autophagy process and its regulation and its selective pathways and role in cancer cell metabolism We define and describe theWarburg effect and the newly suggested ldquoreverserdquo hypothesis We also discuss the potential value of modulating autophagy withseveral pharmacological agents able to modify the Warburg effect The association of the Warburg effect in cancer and stromalcells to tumor-related autophagy may be of relevance for further development of experimental therapeutics as well as for cancerprevention

1 Introduction

Autophagy is an evolutionarily conserved and highly reg-ulated lysosomal pathway involved in the degradation ofmacromolecules and cytoplasmic organelles [1ndash3] Auto-phagy is a crucial contributor to maintain cellular home-ostasis The quality control of mitochondria structure andfunction for instance is important in the maintenance of cellenergy and this process seems to involve autophagy

By 1920 Otto Warburg hypothesized that tumor cellsmainly generate energy by nonoxidative breakdown of glu-cose making cancer growth feasible This phenomenonis known as ldquoWarburg effectrdquo This cellular event relieson mitochondrial dysfunction characterized by respiratoryimpairment resulting in a switch to glycolysis

Originally the Warburg effect was thought to occuronly in cancer cells Nevertheless in 2008 Vincent et aldemonstrated that human skin keloid fibroblasts displaysimilar bioenergetic changes as cancer cells in generatingATP mainly from glycolysis The hypoxic microenvironmentis a common fact in solid tumors and keloids which may bethe explanation for this thermodynamic phenomenon [4] Inline with these findings Pavlides and col suggested in 2009a novel hypothesis for understanding the Warburg effect intumors [5] they proposed that epithelial cancer cells inducethe Warburg effect in neighboring stromal fibroblasts

A clear association among mitochondrial function War-burg effect the reverse Warburg effect and autophagy canbe established The objective of this review is to discuss theautophagy process its regulation the selective pathways and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 926729 10 pageshttpdxdoiorg1011552014926729

2 BioMed Research International

its role in cancer cell metabolism We define Warburg effectand the ldquoreverserdquo hypothesis and we discuss the potentialvalue of modulating autophagy The relevance of these inter-actions on cancer cell biology will be also discussed as well astheir potential impact on disease prevention and treatment

2 Autophagy and Cancer

Autophagy is a highly regulated-cellular pathway for degrad-ing long-lived proteins and is the only known pathway forclearing cytoplasmic organelles This process is involvedin the turnover of long-lived proteins and other cellularmacromolecules andwhennormal itmight play a protectiverole in development aging cell death and defense againstintracellular pathogens [6 7] Autophagy has been associatedwith a variety of pathological processes such as degenerativediseases (diabetes neurodegenerative processes etc) andcarcinogenesis with highlights of biomedical relevance [8 9]

Autophagy consists of several sequential steps induc-tion autophagosome formation autophagosome-lysosomefusion and degradationThreemajor types of autophagy existin eukaryotes (1) chaperonemediated autophagy (CMA) (2)microautophagy and (3) macroautophagy hereafter referredto as autophagy [10] CMA allows the direct lysosomalimport of unfolded soluble proteins that contain a particularpentapeptidemotif Inmicroautophagy cytoplasmicmaterialis directly engulfed into the lysosome at the surface of thelysosome by membrane rearrangement Finally autophagyinvolves the sequestration of cytoplasm into a double-membrane cytosolic vesicle referred to as an autophagosomethat subsequently fuses with a lysosome to form an autolyso-some for the degradation by lysosomal hydrolases [11]

Autophagy is characterized by sequestration of bulkcytoplasm and organelles in double-membrane vesicles calledautophagosomes which eventually acquire lysosomal-likefeatures [11 12]

Autophagy is mediated by a set of evolutionarily con-served gene products (termed the ATG proteins) originallydiscovered in yeast [13] In mammalian cells BECN1 [2 14ndash16] promotes autophagosome formation when it functionsas part of a complex with the class III phosphatidylinos-itol 3-kinase (PI3K) mediating the localization of otherautophagic proteins to the autophagosomal membrane [17]However despite the advances in understanding autophagyautophagosome formation in mammalian cells is a complexprocess and neither the molecular mechanisms nor all theimplicated genes involved in its formation are fully eluci-dated

More than 30 highly conserved genes that are involvedin autophagy have been identified so far [18] A core mo-lecular machinery has been defined and is composed offour subgroups first the ATG1unc-51-like kinase (ULK)complex second the class III phosphatidylinositol 3 kinase(PtdIns3K)Vps34 complex I third two ubiquitin-like pro-tein (ATG12 and ATG8 (LC3) conjugation systems and fourtwo transmembrane proteins ATG9mATG9 (and associatedproteins involved in its movement such as ATG18WIPI-1)and VMP1 (whose expression triggers autophagy) [19ndash21]

Basal autophagy in unstressed cells is kept down by the actionof the mammalian target of rapamycin complex 1 (mTORC1)Key upstream regulators of mTORC1 include the class Iphosphoinositide 3-kinase (PI3K)-Akt pathway which keepsmTORC1 active in cells with sufficient growth factors and theAMP-activated protein kinase (AMPK) pathway that inhibitsmTORC1 upon starvation and calcium signals [22 23]

Autophagy is strongly induced in many types of culturedcells under stress conditions These stress conditions includeamino acid starvation The effects of individual amino acidsdiffer in their abilities to regulate autophagy It has beensuggested that amino acid starvation is followed by anactivation of serinethreonine kinase mammalian target ofrapamycin (mTOR) and the subsequent regulation of theclass III PI3K The mTOR is involved in the control ofmultiple cell processes in response to changes in nutrientconditions [24] Particularly mTOR complex 1 (mTORC1)requires Rag GTPase Rheb and Vps34 for its activation andthe corresponding inhibition of autophagy in response toamino acids [25 26] AMP activated protein kinase (AMPK)senses energy levels and constitutes a key factor for cellularenergy homeostasis When energy levels are low AMPK isactivated The activated AMPK then inactivates mTORC1through TSC1TSC2 and Rheb protein [27]This inactivationofmTORC1 is an essential step for the induction of autophagyand plays a central role in autophagy In addition to aminoacid signaling it has also been reported that other factorscan regulate autophagy such as hormones growth factorsand many other factors including bcl-2 [28] reactive oxygenspecies (ROS) [29] calcium [30] BNIP3 [31] p19ARF [32]DRAM [33] calpain [34] TRAIL [35] FADD [36] andmyo-inositol-145-triphosphate (IP3) [37] But it is importantto point out that not all autophagy signals are transducedthrough mTOR signaling A recent study showed that small-molecule enhancers of the cytostatic effects of rapamycin(called SMERs) induce autophagy independently of mTOR[38]

Depending on nutrient conditions the activities of theULK1 kinase complex can be regulated by mTOR Undergrowing and high-nutrient conditions the active mTORC1interacts with the ULK1 kinase complex (ULK1-mATG13-FIP200-ATG101) and phosphorylatesULK1 andmATG13 andtherefore inhibits themembrane targeting of theULK1 kinasecomplex On the other hand during starvation conditionthe inactivated mTORC1 dissociates from the ULK1 kinasecomplex This dissociation results in the ULK1 kinase com-plex free to phosphorylate components such as mATG13 andFIP200 in the ULK1 kinase complex leading to autophagyinduction [39]

The vacuole membrane protein 1 (VMP1) a pancreatitis-associated protein is a transmembrane protein with noknown homologues in yeast Its expression induces autopha-gosome formation even under nutrient-replete conditionswhile remaining an integrated autophagosomal membraneprotein in mammalian cells [40] Hyperstimulation of Gq-coupled CCK receptor in pancreatic acinar cells during acutepancreatitis [41] and mutated KRas in pancreatic cancer cells[42] induce VMP1 expression In addition VMP1 interactswith Beclin 1ATG6 through its hydrophilic C-terminal

BioMed Research International 3

region (VMP1-ATG domain) which is necessary for earlysteps of autophagosome formation [40 43] Besides EPG-3VMP1 is one of three essential autophagy genes conservedfrom worms to mammals EPG-3VMP1 regulates early stepsof the autophagic pathway in Caenorhabditis elegans [44]VMP1 along with ULK1 and ATG14 localizes in the endo-plasmic reticulum-associated autophagosome formationsites in a PI3K activity-independent manner confirmingthe key role of VMP1 in the formation of autophagosomes[19] Interestingly an accumulation of huge ubiquitin-positive protein aggregates containing the autophagy markerATG8LC3 was seen and p62 homolog [45] in Dictyosteliumcells lacking Vmp1 gene showed Moreover the knockdownof VMP1 expression abolishes starvation and rapamycin-induced autophagosome formation [40] It also abolishesautophagy induced by hyperstimulation of Gq-coupled CCKreceptor in pancreatic acinar cells [41] or by chemotherapyin pancreatic tumor cells [46] Furthermore VMP1 is theonly human disease-inducible ATG-protein described so far

It has been shown that both downregulated and excessiveautophagy have been implicated in the pathogenesis ofdiverse diseases These diseases include a certain type ofneuronal degeneration diabetes and its complications andcancer [47] Autophagy has also been implicated in celldeath called autophagic or type II programmed cell deathwhich was originally described on the basis of morphologicalstudies detecting autophagic vesicles during tissue involution[48]

In general cancer cells tend to undergo less autophagythan their normal counterparts at least for some tumors[49 50] There is a monoallelic deletion of beclin1 autophagygene in 40ndash74 of cases of human sporadic breast ovar-ian and prostate cancer [50] Heterozygous disruption ofbeclin1 increases the frequency of spontaneous malignanciesand accelerates the development of virus-induced premalig-nant lesions [50] This suggests that defective regulation ofautophagy promotes tumor genesis It has been proposed thatautophagy can suppress carcinogenesis by a cell-autonomousmechanism that involves the protection of genome integrityand stability and a nonautonomous mechanism that involvesthe suppression of inflammation and necrosis On the otherhand autophagymay support the survival of rapidly growingcancer cells that have outgrown their vascular supply andare exposed to a hostile environment with an inadequateoxygen supply ormetabolic stress In contrast excessive levelsof autophagy promote cell death [51] Accordingly it hasbeen proposed that autophagy can play an important roleboth in tumor progression and in promotion of cancer celldeath [52] For instance in pancreatic ductal adenocarcinoma(PDAC) the cellular response to ROS initiates a survival orcell death pathway dependent on the severity of the oxidativedamage [53] ROS such as H

2O2can induce autophagy The

deregulation of the AGER ligand HMGB1 is expressed inmany cancer cells including pancreatic cancer cells ROScan increase the release of HMGB1 from necrotic cells andthen activates Beclin-1-dependent autophagy by binding toAGER in pancreatic cancer cells [53 54] In addition ROScan promote cytosolic translocation of HMGB1 to bind toBeclin-1 and then enhance autophagy [55] Recent studies

have demonstrated that oxidative stress increases the activityof NF-120581B which upregulates the expression of AGER inpancreatic cancer [56] This in turn promotes autophagyflux by upregulation of LC3-II levels and protects pancreaticcancer cells from oxidative injury On the other handascorbate leads to cell death in PDAC through a unique ROS-mediated caspase-independent autophagy pathway [57] andgemcitabine and cannabinoids combination induces ROS-mediated autophagic cell death in pancreatic tumor cells [58]

There are suggestions that autophagy may be a cancercell survival response to tumor-associated hypoxia Tumorhypoxia has been used as a marker of poor prognosis [59]in any case how cancer cells become more malignant orsurvive with an extremely poor blood supply is poorlyunderstood When cancer cells are exposed to hypoxiaanaerobic glycolysis increases and provides energy for cellsurvival but as the glucose supply is also insufficient dueto the poor blood supply there must be an alternativemetabolic pathway that provides energy when both oxygenand glucose are depleted [60 61] In pancreatic cancerthere have been reports of hypoxia increasing the malignantpotential of the tumor [59] Proliferating cancer cells requiremore nutrients than surrounding noncancerous cells doNutrition of these proliferating cancer cells is supplied viafunctionally structurally immature neovessels Autophagymay react to the cancer microenvironment to favor thesurvival of rapidly growing cancer cells This is becauseautophagy-specific genes promote the survival of normal cellsduring nutrient starvation in all eukaryotic organisms LC3expression has been seen in surgically resected pancreaticcancer tissue that shows activated autophagy in the peripheralarea which included the invasive border and concomitantlyexhibits enhanced expression of carbonic anhydrase [62]This suggests that autophagy may promote cell viability inhypovascularized cancer tissue

Another proposal is that autophagy is a survival cancercell response to tumor-associated inflammation [63] Thepromotion of carcinogenesis and resistance to therapy aretwo results of cancer-associated inflammation Several phe-notypic alterations observed in cancer cells are a result ofinflammatory signals found within the tumor microenviron-ment [63] The receptor for advanced glycation end products(RAGE) is an induced inflammatory receptor It is consti-tutively expressed on many murine and human epithelialtumor cell lines [64 65] Murine and human pancreatic ade-nocarcinoma tumors have shown the highest levels of RAGEexpression Genotoxic andormetabolic stress lead tomodestbut reproducible increases in overall expression of RAGEon epithelial cell lines There is a direct correlation betweenRAGE expression and the ability of both murine and humanpancreatic tumor cell lines to survive cytotoxic aggressionTargeted knockdown of RAGE significantly increases celldeath whereas forced overexpression promotes survival Itwas recently reported that the enhanced sensitivity to celldeath in the setting of RAGE knockdown is associated withincreased apoptosis and decreased autophagy In contrastoverexpression of RAGE is associated with an increasedautophagy but diminished apoptosis and enhances cancer


cell viability Knockdown of RAGE enhances mTOR phos-phorylation in response to chemotherapy therefore there is aprevention of an induction of a survival response Inhibitionof autophagy by means of silencing beclin1 expression inpancreatic cancer cells enhances apoptosis and cell death[66] These findings suggest that RAGE expression in cancercells has a role in tumor cell response to environmentallyinduced stress through the enhancement of autophagy How-ever increased sensitivity to chemotherapeutic agents inRAGE-knockdown pancreatic cancer cells is dependent onATG5 expression but independent of BECN1 expression [66]These last findings suggested that the role of autophagy in theresistance to microenvironment insult or in the sensitivity tochemotherapeutic agent is the result of complex molecularpathways in the tumor cell

On the other hand inhibition of autophagy has beensuggested as a tumor cell response to prolonged hypoxic con-ditions Pancreatic cancer cell response to prolonged hypoxiamay consist in inhibition of autophagic cell death Amemberof the basic helix-loop-helix family of transcriptional regu-lators [67] the short isoform of single-minded 2 (SIM2s)is upregulated in pancreatic cancer The procell death geneBNIP3 has been identified by microarray studies as a targetof SIM2s repression Prolonged hypoxia induces cell deathvia an autophagic pathway involving the HIF1alfa-mediatedupregulation of BNIP3 [31 68] There is an associationbetween the deregulation of both SIM2s andBNIP3with poorprognostic outcomes [69] Decreased BNIP3 levels and poorprognosis correlate with elevated SIM2s expression in pan-creatic cancerThe loss of BNIP3 either by hypermethylationor by transcriptional repression correlates with inhibition ofcell death [70 71] On the contrary upregulation of BNIP3sensitizes pancreatic carcinoma cells to hypoxia-induced celldeath [72] SIM2s expression concomitant with its repressionof BNIP3 enhances tumor cell survival under prolongedhypoxic conditions Recent data linked the increased SIM2sexpression with enhanced cell survival during hypoxia-stressassociated with BNIP3 repression and the attenuation ofhypoxia-induced autophagic processes Therefore inhibitionof autophagic cell death by BNIP3 repression enhances tumorcell survival under prolonged hypoxic conditions

In some cancer cells a relation between a decreasedautophagy and malignant stages of the disease has beenfoundCancer cells present a general tendency to undergo lessautophagy than their normal counterparts this supports theidea that defective autophagic cell death plays an importantrole in the tumor progression process Pancreatic adenocarci-noma cells have lower autophagic capacity than premalignantcells This has been proved by studies of carcinogen-inducedpancreatic cancer in animal models [73] The WIPI proteinfamily which includes ATG18 the WIPI-1 homolog in Scerevisiae was genetically identified as a gene contributingto autophagy [73] Human WIPI-1a a member of a highlyconservedWD-repeats protein family is linked to starvation-induced autophagic processes in the mammalian systemThe deprivation of amino acids triggers an accumulation ofendogenous hWIPI-1 protein They are contained in largevesicular and cup-shaped structures where colocalize withLC3 The starvation-induced hWIPI-1 formation is blocked

by wortmannin a principal inhibitor of PI-3 kinase-inducedautophagosome formation [74] An interesting fact is thatWIPI proteins are linked pathologically to cellular transfor-mation This is because all human WIPI genes are reportedaberrantly expressed in a variety of matched human cancersamples Strikingly hWIPI-2 and hWIPI-4 mRNA expres-sion is substantially decreased in 70 of matched kidney(10 patients) and 100 of pancreatic (seven patients) tumorsamples Most of these samples were derived from tumorsin an advanced stage such as pancreatic adenocarcinomasstages IndashIV Therefore cancer-associated downregulation ofhWIPI-2 andhWIPI-4 supports the possibility that decreasedautophagic activity is necessary for the malignant stages ofpancreatic cancer

3 Warburg Effect and Cancer Cell Biology

Otto Warburg and colleagues performed studies measuringlactate production and oxygen consumption on liver ratcarcinoma tissue and were able to propose that cancer cellsdisplay some very relevant differences when compared withnormal tissues with regard to their glucose metabolismglycolysis is favored despite oxygen availability The hypoth-esis of Warburg was that cancer growth is caused by thefact that tumor cells mainly generate energy (in the formof ATP) by nonoxidative breakdown of glucose This viewcontrasts with the observation that normal cells produce ATPduring oxidative phosphorylation obtaining ldquofuelrdquo throughthe oxidative breakdown of glucose [75]

Glycolysis under anaerobic condition produces 2ATP permolecule of glucose This yield of ATP is much lower thanthe production of ATP by means of a complete oxidation ofglucose to CO

2under aerobic conditions (30 or 32 ATP per

molecule of glucose) [76] In other words about a 15 timeshigher amount of glucose is consumed anaerobically whencompared to the aerobic pathway to yield the same amountof ATP As consequence glucose uptake takes place about tentimes faster in most solid tumors than in normal tissues [77]Commonly cancer cells depend on anaerobic glycolysis fortheir ATP production due to their exposure to a limited O

2

supply (hypoxia)The ldquoWarburg effectrdquo was the denomination given to this

phenomenon of preferred aerobic glycolysis which resultsin an increased lactate production even in presence of ade-quate pO

2 It was suggested that this cellular behavior relies

on mitochondrial dysfunction characterized by respiratoryimpairment resulting into a switch to glycolysis It was alsosuggested that the high glycolytic rate might also result froma decreased mitochondrial mass in tumor cells [78]

This effect first described in cancer tissues was furtheridentified in many other rapidly dividing normal cells [79]Several mechanisms have been proposed to explain theWarburg effect in cancer tissues and they may be involvedin transcriptional and posttranslational related metabolicchanges

A reduced expression of the tumor suppressor proteinp53 in cancer cells might be linked to the Warburg effectP53 is known to reduce the glycolysis rate by increasing the


activity of a fructose-26-bisphosphatase This mechanism isalso involved in the regulatory pathways of apoptosis [80 81]In addition this mechanism seems to increase the oxida-tive phosphorylation process Other transcription regulatorssuch as the alpha estrogen-related receptor (of potentialrelevance in breast cancer) might be linked to the Warburgeffect in the same way an increased expression of oncogeneslike MYC also seems to be associated with an increasedglycolytic rate and might be involved in the pathophysiologyof themetabolic modifications found in tumors [82] Besidesglycolytic enzymes and glucose transmembrane transport areactivated by MYC overexpression

As mentioned before the posttranslational regulation ofthe Warburg effect was also under scrutiny As a relevantexample the activation of the PI3KAKTdownstreamderivesinto an increased glucose influx and the phosphorylation ofsome enzymes like hexokinase and phosphofructokinase-2with an upregulation of the glycolytic pathway [80] Severalposttranslational modifications of the M2 isoform pyruvatekinase result in a change in its activity modulating theglycolytic pathway in several tissues The K305 acetylation ofthis M2 isoform reduces its enzymatic activity and increasesthe enzyme degradation via chaperone-mediated autophagy[80]The posttranscriptional modification of the M2 isoformof the pyruvate kinase has been shown to influence glycolysisat various models and experimental conditions by oxidationacetylation phosphorylation and so forth A recent linkwas described among tumor overexpression of endogenousmicroRNA (miRNA) metabolic regulation of cancer cellsand the ldquoWarburg effectrdquo [80] Even when attractive thebiological impact of this association remains to be clarified

4 The Reverse Warburg Effect in CancerPharmacological Modulation of Warburgand Reverse Warburg Effects

As stated before it was thought that the Warburg effectonly occurred in cancer cells Recently it has been shownthat human skin keloid fibroblasts were able to generateenergy (ATP) mainly from glycolysis this phenomenon wasexplained through the existence of similar hypoxic microen-vironments in tumors and keloids [4 5]This observation ledto suggest a new hypothesis where epithelial cancer cells areable to induce the Warburg effect in stromal fibroblasts Thisprocess was termed ldquoreverseWarburg effectrdquo and it is based instudies performed in cocultures systems (eg stromal fibrob-lasts and human breast cancer cells) [4] This hypothesis isconsistent with the original view and is important to pointout that in this situation the Warburg effect is not occurringin cancer cells but in the stroma For a clearer understandingthe reverse Warburg effect can be explained as occurring intwo steps

As a first step cancer-associated fibroblasts undergomyo-fibroblastic differentiation and secrete lactate and pyruvatethrough the glycolytic pathway As stated before this processis induced in cancer cells by amechanism involving oxidativestress in association with loss of Caveolin-1 mitophagy

andor mitochondrial dysfunction and increased productionof NO [83]

Following these changes epithelial cancer cells take upthe energy-rich metabolites which in turn enter in thetricarboxylic acid (TCA) pathway This leads to productionof ATP by oxidative phosphorylation The mitochondrialmass of these cells expands to satisfy the increased metabolicdemand In addition antioxidant enzymes are upregulated inorder to cope with the oxidative stress generated and increasetumor aggressive behavior [84]

It is conceivable that different variants of similar types ofcancer may differ with regard to their metabolic behaviorBreast cancer seems to be heterogeneous in its metabolicstatus and therefore it can be classified into variousmetabolicphenotypes ldquoWarburgrdquo and mixed variants had been identi-fied closely associated with the triple negative breast cancerphenotype whereas the reverse Warburg and null typeswere frequently identified within the luminal type of breasttumors suggesting a correlation between metabolic pheno-type and the biology of breast cancer [85]

The Warburg effect might be modified and reversed bysome pharmacological interventions Even though severalmechanisms for such actions were reported in general theclinical relevance of these findings is still on the way of beingclarified

One of the most studied agents in this area is a well-known antidiabetic agent metformin This drug has beenproposed as a potential multifaceted agent for cancer pre-vention Metformin acts as an indirect activator of AMPKand is able to reduce mitochondrial complex I activity Thesehave been proposed as mechanisms for reducing hepaticglucose output in patients with type 2 diabetes Metformintreatment was associated with an increased cell death in P53-deficient cancer cells In normal cells there is an increase inglycolysis rates as an alternative ATP-producing mechanismthat follows metformin treatment In fact one very rare butstill possible adverse effect of metformin is lactic acidosis Itseems that P53-deficient cells experience problems in switch-ing their metabolic pattern This is followed by an enhancedcell death rate Metformin diminishes ROS generation atmitochondria [86] This is mainly achieved by reducing theactivity of the respiratory chain complex I Acknowledgingthis is important because the role of ROS in tumorigenesisand in cancer growth has been widely recognizedMetforminexhibits a mild to moderate antiangiogenic effect this is aneffect that it shares with thrombospondin and endostatinThis effect on angiogenesis may be on the basis of its potentialactions on cancer cells andor its stroma [86]

In addition as mentioned before metformin activatesthe ATMLKB1AMPK axis A very well-characterized tumorsuppressor in the pathophysiology of melanoma and pan-creatic and lung cancer the tumor suppressor LKB1 mightparticipate at the mechanism of action of metformin It isthought that part of the preventive effects of metforminmight be mediated by this suppressing factor Metforminmay inhibit the mTOR pathway by activating AMPK thiseffect has been proposed as an explanation for the potentialantineoplastic effects ofmetformin in breast and renal tumors[87] Metforminrsquos effects on the Warburg effect may be


explained by many of the mentioned mechanisms This drughas been suggested to reduce glycolysis and to increasemitochondrial respiration in tumors and both effects havebeen associated with growth arrest [87] It has been proposedthat pyruvate kinase expression in fibroblast of tumoralstroma is linked to cancer growth Cancer cells produce ROSthat promote oxidative stress in fibroblasts This results inthe activation of HIF1 and NF-120581B NF-120581B increases proin-flammatory cytokines and HIF1 alpha promotes autophagyand anaerobic glycolysis Pyruvate kinase activity resultsin an increase in ketones and lactate and these nutrientsare transferred to cancer cells where they are used formitochondrial oxidative metabolism As it has been saidbefore metformin reduces the mitochondrial chain activityby inhibiting complex I activity In this manner metforminmay alter some of the mechanisms involved into the reverseWarburg effect [88] It may also affect cell reprogrammingbymodifying the lipogenic enzymes acetyl-Co A carboxylaseand fatty acid synthase [89] These changes may also affectthe metabolic behavior of both stroma and tumor cells Asmentioned before the clinical impact of these modificationsis still uncertain

There are other drugs that exhibit potential for themodification of Warburg effect and authophagy rates Mildautophagy induction by hypoxia or starvation seems toprotect the cells but rapamycin or sulforaphane leads toits elimination [90] In contrast an excessive autophagyrate may induce cell death Elimination of highly aggres-sive pancreatic adenocarcinoma cells can be achieved byinhibition of autophagy by monensin or 3-methyladenine[90] This is possible because these drugs may totally blockcontinuous recycling of cellular components necessary fornew synthesis and survival This information suggests thatboth inhibitors and activators of autophagy may have utilityin the treatment of patients with pancreatic ductal ade-nocarcinoma since strong overactivation as well as stronginhibition of autophagy induces death in highly aggres-sive adenocarcinoma cells and sensitizes them to hypoxia-starvation [90] Both autophagy activating (eg rapamycinmdashderivates sirolimus and temsirolimus or sulforaphane-a nat-urally occurring dietary substance enriched in broccoli) orinhibiting drugs (eg antibiotic monensin antimalarial drugchloroquine) are available and generally tolerated well bypatients

Autophagy may be necessary for the maintenance of thetumor in advanced cancer This is why multiple clinical trialsare on their way to test this as a therapeutic approach inhuman patients using hydroxychloroquine (HCQ) [91 92]

Autophagy can be affected in different manner andseveral ways by standard cancer chemotherapies Gemc-itabine monotherapy or its combination with other agentshas become the standard chemotherapy for the treatmentof advanced pancreatic cancer Gemcitabine is a relativelyeffective chemotherapeutic agent acting by competition withdCTP for the incorporation intoDNAcausing chain termina-tion On the other hand gemcitabine serves as an inhibitoryalternative substrate for ribonucleotide reductase and leads toa reduction of deoxynucleotide pools [93 94] This moleculeinhibits cells that are insensitive to classic anticancer drugs

including other nucleoside analogs with similar structuresIt has been recently suggested that gemcitabine also inducesautophagy in pancreatic cancer cells [46] even though gemc-itabine seems to exert its toxicity at least in part by activationof apoptosis [93] It has been proposed that the early induc-tion of autophagy with gemcitabine may be mediated by anincreased expression of VMP1 [46] Capecitabine which is apyrimidine analog induces apoptosis in several cancer linesand shows a modest efficacy in locally advanced pancreaticductal adenocarcinoma when associated with limited fieldradiotherapy [95] It has been proposed that capecitabinemodulates autophagy by displaying a Src kinase modulatoryeffect [96] but the results on this area are still contradictoryIrinotecan is a topoisomerase I inhibitorwhich preventsDNAfrom unwinding In a phase III trial the combination of5-fluouracil leucovorin oxaliplatin and irinotecan resultedin better responses progression-free survival and overallsurvival when compared with the standard single drugtherapy with gemcitabine for metastatic pancreatic ductaladenocarcinoma [97] In small-cell prostatic carcinomairinotecan promoted an increase in autophagy of treatedtumors as indicated by an increase in LC3B expression [9899] Nevertheless authors of this research state that the roleof autophagy is complex This can be said because there isevidence that autophagy supports both promotion and sup-pression of cancer growth In general as mentioned beforea considerable amount of caution should be exercised for theinterpretation of the consequences of cancer chemotherapyon autophagy Other chemotherapeutic agents like the glyco-side oleandrin some platinum compounds the multikinaseinhibitor sorafenib and some histone-deacetylase inhibitorshave demonstrated effects on the autophagy rate in pancre-atic carcinoma cell lines [98 99] As proposed autophagymay be involved in carcinogenesis tumor progression anddissemination and may be associated at least in part withthe actions of some chemotherapy for pancreatic ductaladenocarcinoma as well All these modifications may alterWarburg and reverse Warburg effects but it is importantto remember that the real contribution of these metabolicchanges to tumor cell survival and clinical prognosis remainsunclear

5 Conclusions

Autophagy regulation involves a set of key processes neededfor a normal cell survival and turnover The associationbetween abnormal or defective autophagy and cancer devel-opment has been strongly suggested by several authors Thisassociation is currently under an intense scrutiny aimed tocontribute to a better understanding of the tumor cell biol-ogy Figure 1 summarizes the link between abnormalities inautophagy and the Warburg and the reverse Warburg effectscritical to understand several tumor adaptive behaviors Abetter knowledge of these metabolic interactions may be ofimportance in the development of new therapeutic agents inoncology as well as for the development of more efficientpreventive strategies for some cancer phenotypes


Stroma

Oxidativestress Cancer

cellsIncreased tumorAutophagy

(mitophagy)

Warburg effectsAerobic glycolysis

ATP + lactate + pyruvate

Warburg reverse effects

aggressiveness

Figure 1 AutophagyWarburg andWarburg reverse effects in human cancerThe link between abnormalities in autophagy and theWarburgand the reverse Warburg effects seems to be critical to understand several tumor adaptive behaviors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authorsrsquo work is supported by grants from the NationalAgency for the Scientific and Technologic Promotion(ANPCyT) the National Council for Scientific and TechnicalResearch (CONICET) and the University of Buenos Aires(UBA-UBACyT) Buenos Aires Argentina

References

[1] A M Cuervo ldquoAutophagy in sickness and in healthrdquo Trends inCell Biology vol 14 no 2 pp 70ndash77 2004

[2] B Levine and D J Klionsky ldquoDevelopment by self-digestionmolecular mechanisms and biological functions of autophagyrdquoDevelopmental Cell vol 6 no 4 pp 463ndash477 2004

[3] Z Yang and D J Klionsky ldquoAn overview of the molecularmechanism of autophagyrdquo Current Topics in Microbiology andImmunology vol 335 no 1 pp 1ndash32 2009

[4] A S Vincent T T Phan A Mukhopadhyay H Y Lim BHalliwell and K P Wong ldquoHuman skin keloid fibroblastsdisplay bioenergetics of cancer cellsrdquo Journal of InvestigativeDermatology vol 128 no 3 pp 702ndash709 2008

[5] S Pavlides D Whitaker-Menezes R Castello-Cros et al ldquoThereverse Warburg effect aerobic glycolysis in cancer associatedfibroblasts and the tumor stromardquo Cell Cycle vol 8 no 23 pp3984ndash4001 2009

[6] T Hara K Nakamura M Matsui et al ldquoSuppression of basalautophagy in neural cells causes neurodegenerative disease inmicerdquo Nature vol 441 no 7095 pp 885ndash889 2006

[7] X Qu Z Zou Q Sun et al ldquoAutophagy gene-dependent clear-ance of apoptotic cells during embryonic developmentrdquo Cellvol 128 no 5 pp 931ndash946 2007

[8] S Pattingre and B Levine ldquoBcl-2 inhibition of autophagy a newroute to cancerrdquoCancer Research vol 66 no 6 pp 2885ndash28882006

[9] T Shintani andD J Klionsky ldquoAutophagy in health and diseasea double-edged swordrdquo Science vol 306 no 5698 pp 990ndash9952004

[10] D J Klionsky ldquoAutophagyrdquo Current Biology vol 15 no 8 pp282ndash283 2005

[11] D J Klionsky and S D Emr ldquoAutophagy as a regulated pathwayof cellular degradationrdquo Science vol 290 no 5497 pp 1717ndash17212000

[12] NMizushima ldquoThe pleiotropic role of autophagy from proteinmetabolism to bactericiderdquoCell Death ampDifferentiation vol 12no 2 pp 1535ndash1541 2005

[13] D J Klionsky J M Cregg W A Dunn Jr et al ldquoA unifiednomenclature for yeast autophagy-related genesrdquo Developmen-tal Cell vol 5 no 4 pp 539ndash545 2003

[14] X H Liang S Jackson M Seaman et al ldquoInduction ofautophagy and inhibition of tumorigenesis by beclin 1rdquo Naturevol 402 no 6762 pp 672ndash676 1999

[15] S Pattingre A Tassa X Qu et al ldquoBcl-2 antiapoptotic proteinsinhibit Beclin 1-dependent autophagyrdquo Cell vol 122 no 6 pp927ndash939 2005

[16] C Liang P Feng B Ku et al ldquoAutophagic and tumour suppres-sor activity of a novel Beclin1-binding protein UVRAGrdquoNatureCell Biology vol 8 no 7 pp 688ndash698 2006

[17] A Kihara T Noda N Ishihara and Y Ohsumi ldquoTwo distinctVps34 phosphatidylinositol 3-kinase complexes function inautophagy and carboxypeptidase y sorting in Saccharomycescerevisiaerdquo Journal of Cell Biology vol 153 no 3 pp 519ndash5302001

[18] CHe andD J Klionsky ldquoRegulationmechanisms and signalingpathways of autophagyrdquo Annual Review of Genetics vol 43 pp67ndash93 2009

[19] E Itakura and N Mizushima ldquoCharacterization of autophago-some formation site by a hierarchical analysis of mammalianAtg proteinsrdquo Autophagy vol 6 no 6 pp 764ndash776 2010

[20] M I Vaccaro A Ropolo D Grasso and J L Iovanna ldquoA novelmammalian trans-membrane protein reveals an alternative


initiation pathway for autophagyrdquo Autophagy vol 4 no 3 pp388ndash390 2008

[21] Z Yang andD J Klionsky ldquoMammalian autophagy coremolec-ular machinery and signaling regulationrdquo Current Opinion inCell Biology vol 22 no 2 pp 124ndash131 2010

[22] M Hoslashyer-Hansen and M Jaattela ldquoAMP-activated proteinkinase a universal regulator of autophagyrdquo Autophagy vol 3pp 381ndash383 2007

[23] M Zheng Y Wang X Wu et al ldquoInactivation of Rheb byPRAK-mediated phosphorylation is essential for energy-deple-tion-induced suppression of mTORC1rdquoNature Cell Biology vol13 no 3 pp 263ndash272 2011

[24] T Nobukuni M Joaquin M Roccio et al ldquoAmino acidsmediate mTORraptor signaling through activation of class 3phosphatidylinositol 3OH-kinaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no40 pp 14238ndash14243 2005

[25] S Wullschleger R Loewith and M N Hall ldquoTOR signaling ingrowth andmetabolismrdquoCell vol 124 no 3 pp 471ndash484 2006

[26] Y Sancak L Bar-Peled R Zoncu A L Markhard S Nada andD M Sabatini ldquoRagulator-rag complex targets mTORC1 to thelysosomal surface and is necessary for its activation by aminoacidsrdquo Cell vol 141 no 2 pp 290ndash303 2010

[27] D M Gwinn D B Shackelford D F Egan et al ldquoAMPKphosphorylation of raptor mediates a metabolic checkpointrdquoMolecular Cell vol 30 no 2 pp 214ndash226 2008

[28] B Levine S Sinha and G Kroemer ldquoBcl-2 family membersdual regulators of apoptosis and autophagyrdquo Autophagy vol 4no 5 pp 600ndash606 2008

[29] J Botti M Djavaheri-Mergny Y Pilatte and P CodognoldquoAutophagy signaling and the cogwheels of cancerrdquo Autophagyvol 2 no 2 pp 67ndash73 2006

[30] D R Green and R Wang ldquoCalcium and energy making thecake and eating it toordquo Cell vol 142 no 2 pp 200ndash202 2010

[31] K Tracy B C Dibling B T Spike J R Knabb P SchumackerandK FMacleod ldquoBNIP3 is anRBE2F target gene required forhypoxia-induced autophagyrdquo Molecular and Cellular Biologyvol 27 no 17 pp 6229ndash6242 2007

[32] C J Sherr ldquoAutophagy by ARF a Short Storyrdquo Molecular Cellvol 22 no 4 pp 436ndash437 2006

[33] D Crighton S Wilkinson and K M Ryan ldquoDRAM linksautophagy to p53 and programmed cell deathrdquo Autophagy vol3 no 1 pp 72ndash74 2007

[34] H G Xia L Zhang G Chen et al ldquoControl of basal autophagyby calpain1 mediated cleavage of ATG5rdquo Autophagy vol 6 no1 pp 61ndash66 2010

[35] K R Mills M Reginato J Debnath B Queenan and JS Brugge ldquoTumor necrosis factor-related apoptosis-inducingligand (TRAIL) is required for induction of autophagy duringlumen formation in vitrordquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 10 pp3438ndash3443 2004

[36] J O Pyo M H Jang Y K Kwon et al ldquoEssential roles of Atg5and FADD in autophagic cell death dissection of autophagiccell death into vacuole formation and cell deathrdquoThe Journal ofBiological Chemistry vol 280 no 21 pp 20722ndash20729 2005

[37] S Sarkar andD C Rubinsztein ldquoInositol and IP3 levels regulateautophagy biology and therapeutic speculationsrdquo Autophagyvol 2 no 2 pp 132ndash134 2006

[38] S Sarkar E O Perlstein S Imarisio et al ldquoSmall moleculesenhance autophagy and reduce toxicity in Huntingtonrsquos disease

modelsrdquo Nature Chemical Biology vol 3 no 6 pp 331ndash3382007

[39] N Mizushima ldquoThe role of the Atg1ULK1 complex in auto-phagy regulationrdquo Current Opinion in Cell Biology vol 22 no2 pp 132ndash139 2010

[40] A Ropolo D Grasso R Pardo et al ldquoThe pancreatitis-inducedvacuole membrane protein 1 triggers autophagy in mammaliancellsrdquo Journal of Biological Chemistry vol 282 no 51 pp 37124ndash37133 2007

[41] D Grasso A Ropolo A Lo Re et al ldquoZymophagy a novelselective autophagy pathway mediated by VMP1-USP9x-p62prevents pancreatic cell deathrdquo Journal of Biological Chemistryvol 286 no 10 pp 8308ndash8324 2011

[42] A E Lo Re M G Fernandez-Barrena L L Almada et alldquoNovel AKT1-GLI3-VMP1 pathway mediates KRAS oncogene-induced autophagy in cancer cellsrdquo The Journal of BiologicalChemistry vol 287 no 30 pp 25325ndash25334 2012

[43] M IMolejon A Ropolo A L Re V Boggio andM I VaccaroldquoTheVMP1-Beclin 1 interaction regulates autophagy inductionrdquoScientific Reports vol 3 article 1055 2013

[44] Y Tian Z Li W Hu et al ldquoC elegans screen identifiesautophagy genes specific to multicellular organismsrdquo Cell vol141 no 6 pp 1042ndash1055 2010

[45] J Calvo-Garrido and R Escalante ldquoAutophagy dysfunctionand ubiquitin-positive protein aggregates in Dictyostelium cellslacking Vmp1rdquo Autophagy vol 6 no 1 pp 100ndash109 2010

[46] R Pardo A Lo Re C Archange et al ldquoGemcitabine inducesthe VMP1-mediated autophagy pathway to promote apoptoticdeath in human pancreatic cancer cellsrdquo Pancreatology vol 10no 1 pp 19ndash26 2010

[47] N Mizushima B Levine A M Cuervo and D J Klion-sky ldquoAutophagy fights disease through cellular self-digestionrdquoNature vol 451 no 7182 pp 1069ndash1075 2008

[48] M Hoslashyer-Hansen and M Jaattela ldquoAutophagy an emergingtarget for cancer therapyrdquo Autophagy vol 4 pp 574ndash580 2008

[49] S Toth K Nagy Z Palfia and G Rez ldquoCellular autophagiccapacity changes during azaserine-induced tumour progressionin the rat pancreas up-regulation in all premalignant stagesand down-regulation with loss of cycloheximide sensitivity ofsegregation along with malignant transformationrdquo Cell andTissue Research vol 309 no 3 pp 409ndash416 2002

[50] X Qu J Yu G Bhagat et al ldquoPromotion of tumorigenesis byheterozygous disruption of the beclin 1 autophagy generdquo Journalof Clinical Investigation vol 112 no 12 pp 1809ndash1820 2003

[51] B Levine ldquoCell biology autophagy and cancerrdquo Nature vol446 pp 745ndash747 2007

[52] R Mathew V Karantza-Wadsworth and E White ldquoRole ofautophagy in cancerrdquo Nature Reviews Cancer vol 7 no 12 pp961ndash967 2007

[53] R Kiffin U Bandyopadhyay and A M Cuervo ldquoOxidativestress and autophagyrdquo Antioxidants and Redox Signaling vol 8no 1-2 pp 152ndash162 2006

[54] D Tang R Kang K M Livesey H J Zeh III and M T LotzeldquoHigh mobility group box 1 (HMGB1) activates an autophagicresponse to oxidative stressrdquo Antioxidants amp Redox Signalingvol 15 no 8 pp 2185ndash2195 2011

[55] D Tang R Kang K M Livesey et al ldquoEndogenous HMGB1regulates autophagyrdquo Journal of Cell Biology vol 190 no 5 pp881ndash892 2010

[56] R Kang D Tang K M Livesey N E Schapiro M TLotze and H J Zeh ldquoThe receptor for advanced glycation


end-products (RAGE) protects pancreatic tumor cells againstoxidative injuryrdquo Antioxidants and Redox Signaling vol 15 no8 pp 2175ndash2184 2011

[57] J Du S MMartin M Levine et al ldquoMechanisms of ascorbate-induced cytotoxicity in pancreatic cancerrdquo Clinical CancerResearch vol 16 no 2 pp 509ndash520 2010

[58] MDonadelli I Dando T Zaniboni et al ldquoGemcitabinecanna-binoid combination triggers autophagy in pancreatic cancercells through a ROS-mediated mechanismrdquo Cell Death andDisease vol 2 no 4 article e152 2011

[59] P Buchler H A Reber R S Lavey et al ldquoTumor hypoxiacorrelates with metastatic tumor growth of pancreatic cancer inan orthotopic murine modelrdquo Journal of Surgical Research vol120 no 2 pp 295ndash303 2004

[60] K Izuishi K Kato T Ogura T Kinoshita and H EsumildquoRemarkable tolerance of tumor cells to nutrient deprivationpossible new biochemical target for cancer therapyrdquo CancerResearch vol 60 no 21 pp 6201ndash6207 2000

[61] H Esumi K Izuishi K Kato et al ldquoHypoxia and nitricoxide treatment confer tolerance to glucose starvation in a 51015840-AMP-activated protein kinase-dependent mannerrdquo Journal ofBiological Chemistry vol 277 no 36 pp 32791ndash32798 2002

[62] S Fujii S Mitsunaga M Yamazaki et al ldquoAutophagy isactivated in pancreatic cancer cells and correlates with poorpatient outcomerdquo Cancer Science vol 99 no 9 pp 1813ndash18192008

[63] D G DeNardo M Johansson and L M Coussens ldquoInflaminggastrointestinal oncogenic programmingrdquo Cancer Cell vol 14no 1 pp 7ndash9 2008

[64] R Abe and S Yamagishi ldquoAGE-RAGE system and carcinogene-sisrdquoCurrent Pharmaceutical Design vol 14 no 10 pp 940ndash9452008

[65] T Arumugam D M Simeone K Van Golen and C D Logs-don ldquoS100P promotes pancreatic cancer growth survival andinvasionrdquo Clinical Cancer Research vol 11 no 15 pp 5356ndash5364 2005

[66] R Kang D Tang N E Schapiro et al ldquoThe receptor foradvanced glycation end products (RAGE) sustains autophagyand limits apoptosis promoting pancreatic tumor cell survivalrdquoCell Death and Differentiation vol 17 no 4 pp 666ndash676 2010

[67] R J Kewley M L Whitelaw and A Chapman-Smith ldquoThemammalian basic helix-loop-helixPAS family of transcrip-tional regulatorsrdquo The International Journal of Biochemistry ampCell Biology vol 36 no 2 pp 189ndash204 2004

[68] M B Azad Y Chen E S Henson et al ldquoHypoxia inducesautophagic cell death in apoptosis-competent cells through amechanism involving BNIP3rdquo Autophagy vol 4 no 2 pp 195ndash204 2008

[69] T R Burton and S B Gibson ldquoThe role of Bcl-2 familymemberBNIP3 in cell death and disease NIPping at the heels of celldeathrdquoCell Death and Differentiation vol 16 no 4 pp 515ndash5232009

[70] J Okami D M Simeone and C D Logsdon ldquoSilencing ofthe hypoxia-inducible cell death protein BNIP3 in pancreaticcancerrdquo Cancer Research vol 64 no 15 pp 5338ndash5346 2004

[71] P C Mahon P Baril V Bhakta et al ldquoS100A4 contributesto the suppression of BNIP3 expression chemoresistance andinhibition of apoptosis in pancreatic cancerrdquo Cancer Researchvol 67 no 14 pp 6786ndash6795 2007

[72] T Abe M Toyota H Suzuki et al ldquoUpregulation of BNIP3by 5-aza-21015840-deoxycytidine sensitizes pancreatic cancer cells to

hypoxia-mediated cell deathrdquo Journal of Gastroenterology vol40 no 5 pp 504ndash510 2005

[73] J Guan P E Stromhaug M D George et al ldquoCvt18Gsa12 isrequired for cytoplasm-to-vacuole transport pexophagy andautophagy in Saccharomyces cerevisiae and Pichia pastorisrdquoMolecular Biology of the Cell vol 12 no 12 pp 3821ndash3838 2001

[74] T Proikas-Cezanne S Waddell A Gaugel T Frickey ALupas and A Nordheim ldquoWIPI-1120572 (WIPI49) a member of thenovel 7-bladed WIPI protein family is aberrantly expressed inhuman cancer and is linked to starvation-induced autophagyrdquoOncogene vol 23 no 58 pp 9314ndash9325 2004

[75] OWarburg ldquoOn the origin of cancer cellsrdquo Science vol 123 no3191 pp 309ndash314 1956

[76] D Nelson and D Cox Lehninger Principles of Biochemistrychapter 14 WH Freeman and Co New York NY USA 2008

[77] R Bartrons and J Caro ldquoHypoxia glucose metabolism and theWarburgrsquos effectrdquo Journal of Bioenergetics and Biomembranesvol 39 no 3 pp 223ndash229 2007

[78] V Gogvadze B Zhivotovsky and S Orrenius ldquoThe Warburgeffect and mitochondrial stability in cancer cellsrdquo MolecularAspects of Medicine vol 31 no 1 pp 60ndash74 2010

[79] M Vincent ldquoCancer a de-repression of a default survivalprogram common to all cellsrdquo BioEssays vol 34 no 1 pp 72ndash82 2012

[80] S J Bensinger andHR Christofk ldquoNew aspects of theWarburgeffect in cancer cell biologyrdquo Seminars inCell andDevelopmentalBiology vol 23 no 4 pp 352ndash361 2012

[81] K Bensaad A Tsuruta M A Selak et al ldquoTIGAR a p53-indu-cible regulator of glycolysis and apoptosisrdquo Cell vol 126 no 1pp 107ndash120 2006

[82] L M Nilsson T Z Plym Forshell S Rimpi et al ldquoMousegenetics suggests cell-context dependency for myc-regulatedmetabolic enzymes during tumorigenesisrdquo PLoS Genetics vol8 no 3 Article ID e1002573 2012

[83] U E Martinez-Outschoorn S Pavlides D Whitaker-Menezeset al ldquoTumor cells induce the cancer associated fibroblastphenotype via caveolin-1 degradation implications for breastcancer andDCIS therapy with autophagy inhibitorsrdquoCell Cyclevol 9 no 12 pp 2423ndash2433 2010

[84] M P Lisanti U E Martinez-Outschoorn B Chiavarina etal ldquoUnderstanding the ldquolethalrdquo drivers of tumor-stroma co-evolution emerging role(s) for hypoxia oxidative stress andautophagymitophagy in the tumor micro-environmentrdquo Can-cer Biology andTherapy vol 10 no 6 pp 537ndash542 2010

[85] T Smith-Vikos ldquoA report of the James Watson lecture at YaleUniversityrdquo The Yale Journal of Biology and Medicine vol 85no 3 pp 417ndash419 2012

[86] S del Barco A Vazquez-Martin S Cufı et al ldquoMetforminmulti-faceted protection against cancerrdquo Oncotarget vol 2 no12 pp 896ndash917 2011

[87] B Chiavarina D Whitaker-Menezes U E Martinez-Out-schoorn et al ldquoPyruvate kinase expression (PKM1 and PKM2)in cancer associated fibroblasts drives stromal nutrient produc-tion and tumor growthrdquoCancer Biology andTherapy vol 12 no12 pp 1101ndash1113 2011

[88] A Vazquez-Martin B Corominas-Faja S Cufi et al ldquoThemitochondrial H+-ATP synthase and the lipogenic switch newcore components of metabolic reprogramming in inducedpluripotent stem (iPS) cellsrdquo Cell Cycle vol 12 no 2 pp 207ndash218 2013


[89] V Rausch L Liu A Apel et al ldquoAutophagy mediates survivalof pancreatic tumour-initiating cells in a hypoxicmicroenviron-mentrdquoThe Journal of Pathology vol 227 no 3 pp 325ndash335 2012

[90] R K Amaravadi J Lippincott-Schwartz X Yin et al ldquoPrinci-ples and current strategies for targeting autophagy for cancertreatmentrdquo Clinical Cancer Research vol 17 no 4 pp 654ndash6662011

[91] J Choi H Kim do W H Jung and J S Koo ldquoMetabolic inter-action between cancer cells and stromal cells according to breastcancer molecular subtyperdquo Breast Cancer Research vol 15 no5 article R78 2013

[92] J D Mancias and A C Kimmelman ldquoTargeting autophagy ad-diction in cancerrdquoOncotarget vol 2 no 12 pp 1302ndash1306 2011

[93] B Ewald D Sampath and W Plunkett ldquoNucleoside analogsmolecular mechanisms signaling cell deathrdquo Oncogene vol 27no 50 pp 6522ndash6537 2008

[94] I Vivanco and C L Sawyers ldquoThe phosphatidylinositol 3-kinase-AKT pathway in human cancerrdquoNature Reviews Cancervol 2 no 7 pp 489ndash501 2002

[95] A SN Jackson P Jain G RWatkins et al ldquoEfficacy and tolera-bility of limited field radiotherapy with concurrent capecitabinein local advanced pancreatic cancerrdquo Clinical Oncology vol 22no 7 pp 570ndash577 2010

[96] R Sheikh N Walsh M Clynes R Orsquoconnor and R McDer-mott ldquoChallenges of drug resistance in the management ofpancreatic cancerrdquo Expert Review of AnticancerTherapy vol 10no 10 pp 1647ndash1661 2010

[97] T Conroy F Desseigne M Ychoy et al ldquoRandomized phaseIII trial comparing FOLFIRINOX (F 5FUleucovorin [LV]irinotecan [I] and oxaliplatin [O]) versus gemcitabine (G) asfirst-line treatment for metastatic pancreatic adenocarcinoma(MPA) preplanned interim analysis results of the PRODIGE4ACCORD 11 trialrdquo Journal of Clinical Oncology vol 28supplement 15s abstract 4010 2010

[98] W Tung YWang PW Gout DM Liu andM Gleave ldquoUse ofirinotecan for treatmentof small cell carcinoma of the prostaterdquoProstate vol 71 no 7 pp 675ndash681 2011

[99] M I Vaccaro C D Gonzalez S Alvarez and A RopololdquoModulating autophagy and the lsquoreverse warburg effectrsquordquo inTumor Metabolome Targeting and Drug Development CancerDrug Discovery and Development S Kanner Ed Springer NewYork NY USA 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


its role in cancer cell metabolism We define Warburg effectand the ldquoreverserdquo hypothesis and we discuss the potentialvalue of modulating autophagy The relevance of these inter-actions on cancer cell biology will be also discussed as well astheir potential impact on disease prevention and treatment

2 Autophagy and Cancer

Autophagy is a highly regulated-cellular pathway for degrad-ing long-lived proteins and is the only known pathway forclearing cytoplasmic organelles This process is involvedin the turnover of long-lived proteins and other cellularmacromolecules andwhennormal itmight play a protectiverole in development aging cell death and defense againstintracellular pathogens [6 7] Autophagy has been associatedwith a variety of pathological processes such as degenerativediseases (diabetes neurodegenerative processes etc) andcarcinogenesis with highlights of biomedical relevance [8 9]

Autophagy consists of several sequential steps induc-tion autophagosome formation autophagosome-lysosomefusion and degradationThreemajor types of autophagy existin eukaryotes (1) chaperonemediated autophagy (CMA) (2)microautophagy and (3) macroautophagy hereafter referredto as autophagy [10] CMA allows the direct lysosomalimport of unfolded soluble proteins that contain a particularpentapeptidemotif Inmicroautophagy cytoplasmicmaterialis directly engulfed into the lysosome at the surface of thelysosome by membrane rearrangement Finally autophagyinvolves the sequestration of cytoplasm into a double-membrane cytosolic vesicle referred to as an autophagosomethat subsequently fuses with a lysosome to form an autolyso-some for the degradation by lysosomal hydrolases [11]

Autophagy is characterized by sequestration of bulkcytoplasm and organelles in double-membrane vesicles calledautophagosomes which eventually acquire lysosomal-likefeatures [11 12]

Autophagy is mediated by a set of evolutionarily con-served gene products (termed the ATG proteins) originallydiscovered in yeast [13] In mammalian cells BECN1 [2 14ndash16] promotes autophagosome formation when it functionsas part of a complex with the class III phosphatidylinos-itol 3-kinase (PI3K) mediating the localization of otherautophagic proteins to the autophagosomal membrane [17]However despite the advances in understanding autophagyautophagosome formation in mammalian cells is a complexprocess and neither the molecular mechanisms nor all theimplicated genes involved in its formation are fully eluci-dated

More than 30 highly conserved genes that are involvedin autophagy have been identified so far [18] A core mo-lecular machinery has been defined and is composed offour subgroups first the ATG1unc-51-like kinase (ULK)complex second the class III phosphatidylinositol 3 kinase(PtdIns3K)Vps34 complex I third two ubiquitin-like pro-tein (ATG12 and ATG8 (LC3) conjugation systems and fourtwo transmembrane proteins ATG9mATG9 (and associatedproteins involved in its movement such as ATG18WIPI-1)and VMP1 (whose expression triggers autophagy) [19ndash21]

Basal autophagy in unstressed cells is kept down by the actionof the mammalian target of rapamycin complex 1 (mTORC1)Key upstream regulators of mTORC1 include the class Iphosphoinositide 3-kinase (PI3K)-Akt pathway which keepsmTORC1 active in cells with sufficient growth factors and theAMP-activated protein kinase (AMPK) pathway that inhibitsmTORC1 upon starvation and calcium signals [22 23]

Autophagy is strongly induced in many types of culturedcells under stress conditions These stress conditions includeamino acid starvation The effects of individual amino acidsdiffer in their abilities to regulate autophagy It has beensuggested that amino acid starvation is followed by anactivation of serinethreonine kinase mammalian target ofrapamycin (mTOR) and the subsequent regulation of theclass III PI3K The mTOR is involved in the control ofmultiple cell processes in response to changes in nutrientconditions [24] Particularly mTOR complex 1 (mTORC1)requires Rag GTPase Rheb and Vps34 for its activation andthe corresponding inhibition of autophagy in response toamino acids [25 26] AMP activated protein kinase (AMPK)senses energy levels and constitutes a key factor for cellularenergy homeostasis When energy levels are low AMPK isactivated The activated AMPK then inactivates mTORC1through TSC1TSC2 and Rheb protein [27]This inactivationofmTORC1 is an essential step for the induction of autophagyand plays a central role in autophagy In addition to aminoacid signaling it has also been reported that other factorscan regulate autophagy such as hormones growth factorsand many other factors including bcl-2 [28] reactive oxygenspecies (ROS) [29] calcium [30] BNIP3 [31] p19ARF [32]DRAM [33] calpain [34] TRAIL [35] FADD [36] andmyo-inositol-145-triphosphate (IP3) [37] But it is importantto point out that not all autophagy signals are transducedthrough mTOR signaling A recent study showed that small-molecule enhancers of the cytostatic effects of rapamycin(called SMERs) induce autophagy independently of mTOR[38]

Depending on nutrient conditions the activities of theULK1 kinase complex can be regulated by mTOR Undergrowing and high-nutrient conditions the active mTORC1interacts with the ULK1 kinase complex (ULK1-mATG13-FIP200-ATG101) and phosphorylatesULK1 andmATG13 andtherefore inhibits themembrane targeting of theULK1 kinasecomplex On the other hand during starvation conditionthe inactivated mTORC1 dissociates from the ULK1 kinasecomplex This dissociation results in the ULK1 kinase com-plex free to phosphorylate components such as mATG13 andFIP200 in the ULK1 kinase complex leading to autophagyinduction [39]

The vacuole membrane protein 1 (VMP1) a pancreatitis-associated protein is a transmembrane protein with noknown homologues in yeast Its expression induces autopha-gosome formation even under nutrient-replete conditionswhile remaining an integrated autophagosomal membraneprotein in mammalian cells [40] Hyperstimulation of Gq-coupled CCK receptor in pancreatic acinar cells during acutepancreatitis [41] and mutated KRas in pancreatic cancer cells[42] induce VMP1 expression In addition VMP1 interactswith Beclin 1ATG6 through its hydrophilic C-terminal




















2

























5 Conclusions



Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































2

























5 Conclusions



Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2

























5 Conclusions



Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































5 Conclusions



Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions



Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Stroma



(mitophagy)




aggressiveness




Acknowledgments


References
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article Autophagy, Warburg, and Warburg Reverse ...downloads.hindawi.com/journals/bmri/2014/926729.pdf · Review Article Autophagy, Warburg, and Warburg Reverse ... homeostasis