cancer cells are like the spoiled siblings of normal cells , running rampant with 
 unrestricted growth . to deal with them , it is important to understand two questions : 
 what serves as a critical barrier to prevent normal cells from turning into cancer cells 
 and what provides the crucial ( and addictive ) support for cancer cells allowing them to 
 maintain their high demand for metabolism and growth ?
current studies suggest autophagy 
 as one solution to both of these questions . in this review ,
we summarize results from 
 recent autophagy studies about the mutational landscape of autophagy genes in human 
 cancers , the mouse genetic evidence for the roles of autophagy in tumor promotion or 
 inhibition , current anti - autophagy drugs in clinical trials for cancer treatment , and 
 future directions in cancer therapy targeting autophagy .
autophagy is first linked to human cancer through the identification of beclin 
 1 ( becn1 hereafter , italic for the gene , nonitalic for the protein ) , an 
 autophagy essential gene , as a haploid - insufficient tumor suppressor . 

the becn1 gene has been mapped to a tumor - susceptibility locus on human 
 chromosome 17q21 that is monoallelically deleted in up to 4075% of ovarian 
 cancers , breast cancers , and prostate cancers , raising the possibility that 
 it may be a tumor suppressor . however , in the deleted chromosome region , 
 becn1 is adjacent to brca1 ( breast cancer 1 , early 
 onset ) , which is a breast cancer tumor suppressor gene frequently mutated in familial 
 breast and ovarian cancers .
passenger  which is coincidently deleted with 
 brca1 , rather than the deletion itself being a cause of breast or 
 ovarian cancers , because the statistical 
 analysis of the mutation profiles of brca1 and becn1 
 in breast and ovarian cancers from the cancer genome atlas ( tcga ) showed that large 
 deletions in human breast and ovarian cancers often encompass both 
 brca1 and becn1 ( the majority ) , or 
 brca1 alone ( a small proportion ) , but rarely becn1 
 alone .
interestingly , brca1 is not a haploid - insufficient tumor suppressor 
 gene ; therefore monoallelic deletion may not affect its function or affect tumorigenesis 
 of those breast cancers bearing deletions .
brca1 is a classical tumor suppressor and it 
 is inactivated only when both gene copies / alleles are mutated ( one by germ - line mutation 
 and the other by somatic mutation ) . together ,
brca1 and 
 brca2 germ - line mutations account for about 2025% of 
 hereditary breast cancers , while they rarely occur in sporadic breast cancers . 

mouse genetic studies indicated that one copy of brca1 is sufficient to maintain its 
 tumor suppressor function .
about ten different conventional brca1 mouse 
 mutants have been generated , each carrying a mutation in different regions of the gene . 

in contrast , mouse genetic studies showed that one copy of becn1 is 
 insufficient to support its full function in autophagy ( haploinsufficiency ) , which 
 therefore may cause harmful effects and promote tumorigenesis .
unlike 
 brca1 , heterozygous disruption of becn1 in mice 
 increased the frequency of spontaneous malignancies , although over a long latent period 
 ( 13 - 18 months ) .
these observations support the notion that 
 becn1 is a haploid - insufficient tumor suppressor gene and 
 distinguish it from other classic tumor suppressors that require both alleles be 
 inactivated in promoting tumorigenesis .
the haploid - insufficient properties of 
 becn1 correlate well with the frequent monoallelic deletion of the 
 becn1 containing region in breast and ovarian cancers , suggesting a 
 critical role of becn1 and likely autophagy in tumor suppression .
if combining 
 becn1 monoallelic deletion with other cancer promoting models , 
 becn1 monoallelic deletion has a mixed effect in different cancer 
 models ( table 1 ) .
abbreviations : atm , ataxia - telangiectasia mutated ; wt , wild type ; palb2 , 
 partner and localizer of brca2 ; erbb2 , v - erb - b2 avian erythoblastic leukemia 
 viral oncogene homolog 2 ; pymt , polyoma middle t ; panin , pancreatic 
 intraepithelial neoplasia ; pdac , pancreatic ductal adenocarcinoma .
while a large body of studies is focused on the becn1 gene relevance to 
 human cancers , a number of studies showed that other atg genes are also 
 oncogenically associated .
single amino 
 acid mutations in mtor are found in several types of human cancers , 
 which confer constitutive activation of this autophagy negative regulator .
somatic point mutations of atg5 are also identified in 135 
 patient samples of gastric cancer , colorectal cancer , and hepatocellular carcinoma , 
 which are associated with decreased cellular expression of atg5 protein .
furthermore , frameshift mutations have been 
 found in atg2b , atg5 , and atg9b 
 ( alone or in combination ) in gastric cancer and colorectal cancer samples .
given the number of identified autophagy 
 genes in humans ( over 30 ) , more extensive genome sequencing efforts should be targeted 
 towards autophagy genes in cancer patients . in this way ,
the mutational landscape of 
 autophagy genes in human cancers can be more precisely defined .
if autophagy plays an important role in tumor suppression , as suggested in 
 becn1 heterozygous mutant mice , one would expect other autophagy 
 essential genes would also have a function in tumor suppression .
this seemingly 
 straightforward statement is brought into question by the absolute requirement of 
 autophagy genes in embryonic , neonatal and adult development .
inducible knockout of atg7 in adult mice ( 810 
 weeks ) killed the animals within 37 months with hyperglycemia and 
 neurodegeneration .
the acute lethality 
 of autophagy - deficient mice compromises their use in studying tumorigenesis , as that 
 probably requires a longer time window .
mizushima 's group found their atg5 ; 
 cag - cre mutant mice displayed a mosaic phenotype for an unknown 
 reason even with the ubiquitous expression of the cre recombinase .
although , in most cases , 60 - 90% of cells in various organs 
 of atg5 ; cag - cr are 
 atg5 null , it appears that the remaining atg5 is 
 enough to execute the minimum function of autophagy .
consequently , 
 atg5 ; cag - cre mice were viable and can 
 survive more than 19 months .
this mosaic atg5 mutant mouse strain 
 allows researchers to study the contribution of atg5 to tumorigenesis . 

premalignant liver tumors were detected in all 17 
 atg5 ; cag - cre mice after 9 
 months .
consistently , premalignant live 
 tumors were also observed in liver - specific atg7-deficient mice with nearly 100% 
 penetrance after 12 months .
although no 
 malignant cancer was detected in this setting , the high penetrance to premalignant tumor 
 suggests that autophagy likely functions as a tumor suppression mechanism .
it is 
 currently unknown why autophagy deficiency can only lead to premalignant tumors but not 
 malignant cancers , which are often more invasive and metastatic .
it is not clear , at 
 least from these studies , if these premalignant tumors in autophagy - deficient mice are 
 in a  benign
 state that requires additional genetic alterations to 
 promote malignant transformation ( e.g. p53 mutation ) , or if autophagy inhibition can 
 only promote premalignant / benign tumor formation but somehow protects cells from 
 advancing to malignant cancers ( figure 1 ) .
it 
 is also possible that the premalignant tumor formation is secondary to other cancer 
 prone responses , like inflammation caused by autophagy deficiency . in normal cells
, autophagy inhibits their transformation to premalignant tumor 
 cells by reducing reactive oxygen species ( ros ) , dna damage , protein aggregation , 
 and mitochondrial abnormality etc . in established tumor cells
recent evidence indicates 
 that autophagy inhibits the regression of malignant tumor cells to benign tumors . 

it has yet to be determined if autophagy promotes the progression of premalignant 
 tumor cells to malignant tumor cells .
several factors have been suggested as candidates for blocking autophagy - deficient 
 animals from developing malignant cancer .
ryan 's group proposed that growth 
 arrest , senescence , and cell death mediated by p53 might act as barriers to the 
 progression of premalignant cancer lesions to malignant cancer in a humanized 
 genetically - modified mouse model of pancreatic ductal adenocarcinoma ( pdac ) .
they showed that mice null for atg7 or 
 atg5 , but not wild - type mice , prevent progression to malignant pdac driven by 
 kras . 

however , if p53 is concomitantly deleted from the mouse genome , atg7 deletion 
 accelerates rather than inhibits progression to pdac , probably because autophagy loss increases glucose uptake . 

this study raised an interesting point , at least in the 
 kras - driven pdac mouse model : malignant tumor 
 development is highly dependent on autophagy and p53 status .
in addition to these 
 studies , evidence supporting the role of autophagy in tumor suppression is summarized in 
 table 1 .
the current consensus is that autophagy has a dual role in cancer ( figure 1 ) . in 
 normal cells , autophagy functions as a surveillance mechanism to remove damaged 
 organelles and aggregated proteins . in doing so
, autophagy reduces dna damage , reactive 
 oxygen species ( ros ) , and mitochondrial abnormality , which likely protects normal cells 
 from transforming to tumor cells . on the other hand , in established tumor cells
, 
 autophagy is hijacked to serve as a cell survival mechanism that plays a vital role in 
 facilitating tumor cell growth .
several potential mechanisms have been suggested : 
 autophagy promotes metabolite turnover and absorption in tumor cells to meet their high 
 demand of metabolism and growth ; autophagy inhibits apoptosis caused by mitochondrial 
 malfunctions and ros production ; or autophagy increases drug resistance . of note
, certain types of cancer cell , 
 including pancreatic and lung cancer cells , are heavily reliant on growth benefits 
 brought by autophagy activation , therefore becoming  addicted  to 
 autophagy .
inhibition of autophagy in these autophagy  addicted  cancer 
 cells might remarkably change the  life and death  balance of tumor cells 
 and consequently suppresses tumorigenesis . in the kras and 
 braf driven lung cancer models , autophagy 
 ablation altered the tumor fate from adenoma and carcinoma ( malignant ) to oncocytoma 
 ( benign ) , indicating that autophagy inhibition promotes the 
 regression of malignant tumors to benign tumors ( figure 1 ) .
possibly , autophagy maintains the survival of malignant tumor cells by 
 removing defective mitochondria and reducing ros .
autophagy inhibition removes this 
 protection and exposes malignant tumor cells to ros cytotoxicity . in a recent study ,
white 's group utilized a novel strategy to dissect the 
 function of autophagy in tumorigenesis globally rather than in a tissue - specific manner 
 .
this strategy was used to bypass the 
 requirement of autophagy to embryogenesis : atg7 was inducibly deleted from 810 
 weeks adult mice with floxed alleles of atg7 .
the expression of the cre 
 recombinase in this study is under the control of a ubiquitously expressed promoter that 
 leads to a nearly complete and sustained loss of atg7 protein in all examined tissues 
 ( different from the mosaic atg5 ko mice from mizushima 's lab as 
 we discussed above ) . a week after atg7 was deleted ; lung tumorigenesis was initiated by 
 kras activation and p53 depletion by adenoviral expression of the flp recombinase .
three 
 weeks later , there was no detectable difference in tumor frequency or burden between 
 atg7-intact or atg7-deleted mice , suggesting that autophagy may not be required for 
 tumor initiation in this case .
however , this experimental condition is clearly different 
 from the experiment using the atg5 mosaic mutant mice described by mizushima 's 
 group . in such a short period after atg7 
 deletion ( 4 weeks )
, it is unlikely that premalignant or benign tumors can form ( it takes 
 9 months for the atg5 mosaic mutant mice to develop premalignant liver 
 tumors ) to prime malignant progression . 
 nevertheless , at least in this
experimental setting , loss of autophagy seems to have no 
 dramatic effect in an acute tumorigenesis mouse model . on the other hand ,
white 's group observed a very robust effect of autophagy 
 ablation in reversing malignant cancers to benign tumors : in a converse experiment , lung 
 tumors were generated before inducible atg7 deletion , after only 5 weeks , both tumor 
 size and burden were significantly reduced .
these observations are consistent with a previous report from the same 
 group showing that lung - specific deletion of atg7 , concurrently with tumor initiation by 
 kras activation and p53 deletion , also reduces tumor burden and generates benign 
 oncocytomas . in addition , deletion of 
 fip200 suppressed pymt - driven mammary tumor initiation and progression , 
 which represents another example of an autophagy gene that , when deleted , impairs 
 tumorigenesis . in all
, these studies 
 further consolidate the concept that autophagy inhibition can be exploited to treat 
 human cancers .
thus , pharmaceutical inhibition of autophagy emerges as a promising 
 strategy in cancer therapy .
currently , there are about 32 ongoing clinical trials assessing the effect of autophagy 
 manipulation on human cancer treatment , half of which are using chloroquine and 
 hydroxychloroquine ( the chloroquine derivative ) ( refer to reviews in [ 26 - 28 ] ) .
a number of the latest phase i / ii clinical trial reports evaluated the 
 maximum tolerated dose , safety , preliminary activity , pharmacokinetics , and 
 pharmacodynamics of hydroxychloroquine when combined with other therapies in treating 
 glioblastoma , advanced solid tumor , and myeloma [ 29 - 33 ] . in one study ,
the 
 combination of temsirolimus ( mtor inhibitor ) and hydroxychloroquine improved the median 
 progression - free survival in 13 melanoma patients to 3.5 months and increased the rate 
 of stable disease in patients .
the 
 beneficial effect of this combination was also observed in another phase i trial study 
 conducted by the same group . in another 
 study , the combination of bortezomib ( proteasome inhibitor ) and hydroxychloroquine 
 showed a high rate of partial response and stable disease in patients with 
 relapsed / refractory myeloma . whereas , a 
 phase i / ii trial of hydroxychloroquine in conjunction with radiation therapy and 
 concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma 
 multiforme led to no significant improvement in overall survival , which could be due to 
 inconsistent autophagy inhibition in patients treated with this regimen .
these clinical trial studies indicate that 
 autophagy inhibition with chloroquine / hydroxychloroquine treatment is achievable in 
 patients and represents a potentially useful strategy for improving outcomes in cancer 
 therapy . on the other hand
, they also suggest the need to develop other compounds that 
 can induce more consistent autophagy inhibition than chloroquine / hydroxychloroquine in 
 certain tumors .
hydroxychloroquine / chloroquine inhibits autophagy by entering the lysosomes 
 ( lysosomotropic ) and changing the acidity through chelating protons . as a result , the 
 finishing step in autophagy that requires acidic lysosomes for degradation is blocked . 
 in preclinical trials ,
the effect of chloroquine and hydroxychloroquine on tumorigenesis 
 has been extensively investigated in mouse cancer models , including genetic and 
 xenograft models ( summarized in table 2 ) .
as 
 mentioned above , pancreatic cancers and kras - driven lung cancers are highly dependent on 
 autophagy for tumorigenic development , whereas other types of cancers might only use 
 autophagy as a cell survival mechanism when stressed .
this notion suggests a promising 
 avenue in treating certain pancreatic and lung cancers that are notoriously refractory 
 to pharmaceutical intervention .
two studies from kimmelman 's group demonstrated 
 that chloroquine was able to suppress pdac tumor growth in xenograft mouse models , 
 irrespective of p53 status .
this is in contrast to studies from 
 ryan 's group that showed that chloroquine accelerated pdac development in p53 
 null mice instead of suppressing it , 
 suggesting that the anti - tumor effects of chloroquine are p53 dependent .
ryan 's group 
 used a mouse model of embryonic homologous deletion of p53 , while kimmelman 's 
 group used a loss of heterozygosity mouse model of p53 ( closer to human tumors ) .
abbreviations : pdac , pancreatic ductal adenocarcinoma ; atm , 
 ataxia - telangiectasia mutated ; sclc , small cell lung cancer ; tlr9 , toll like 
 receptor 9 ; nsclc , non - small cell lung cancer ; 5-fu , 5-fluorouracil . in most cases of preclinical and clinical trials ,
chloroquine is used as a 
 sensitizer / enhancer to chemotherapy and immunotherapy [ 35 - 47 ] .
the rationale 
 is that chemotherapy and immunotherapy can induce autophagy in tumor cells , which in 
 turn is used by tumor cells as a survival mechanism to resist pharmaceutical invention . 
 as such , blocking of autophagy with chloroquine produces a synergistic effect and 
 increases drug cytotoxicity .
this combined treatment shows a significant preclinical 
 effect on several types of cancers , but marginal effects on many others ( table 2 ) , suggesting that the 
 chloroquine effect is likely tissue- or tumor - type - dependent .
inconsistent 
 inhibition of autophagy in humans is a major issue for its clinical trials .
what 's more , 
 prolonged chloroquine treatment might promote autophagosome biogenesis . from the perspective of cell biology
, it is believed that chloroquine 
 achieves most of its pharmaceutical effects through inhibiting lysosomal functions . 

lysosomes are involved not only in autophagy , but also in endosomal trafficking and 
 antibody presentation . as a result
in addition , chloroquine has non - lysosomal targets . for 
 example , chloroquine acts on endosomal notch1 trafficking and signaling to normalize 
 tumor vasculature in an autophagy - independent manner .
such 
 promiscuous properties of chloroquine may account for its adverse effects during 
 therapy , including retinopathy and immunosuppression , which 
 highlights the need for the development of more specific inhibitors targeting only 
 autophagy .
it is commonly speculated that new therapeutic approaches might be developed by 
 targeting the autophagic pathway , which consists of a number of steps including 
 initiation , membrane nucleation , membrane elongation , membrane closure , membrane fusion , 
 and lysosomal degradation ( figure 2 ) .
currently , the drugs targeting autophagy initiation , membrane nucleation , and 
 lysosomal degradation are under experimental or clinical investigation . other 
 regulatory steps of autophagy , including the membrane fusion mechanism , could also 
 serve as drug targets to block autophagy with high potency and selectivity .
torc1 , 
 target of rapamycin complex 1 ; pi3k , phosphoinositide 3-kinase ; snare , soluble 
 n - ethylmaleimide - sensitive factor attachment protein receptors .
as discussed above , current autophagy inhibitors , such as chloroquine , 
 hydroxychloroquine , and bafilomycin a1 , mainly inhibit lysosomal functions .
recently the 
 lipid kinase vps34 , which is essential for autophagy , also emerged as a target of 
 pharmaceutical interest .
while a number of inhibitors are known to effectively inhibit 
 vps34 activity , including 3-methyladenine and wortmannin , their clinical application has 
 limitations , because they also inhibit class i and ii lipid kinases and other protein 
 kinases .
most recently , however , two 
 structurally distinct vps34 inhibitors , which possess higher selectivity compared with 
 existing inhibitors , have been reported .
pik - iii and sar405 were 
 discovered and developed in large - scale screens of chemicals that inhibit vps34 activity 
 in vitro and pi3p production in vivo , respectively . 

interestingly , both inhibitors are bound within the atp binding cleft but adopt 
 different binding modes to interact with surrounding residues .
these unique contacts 
 confer high specificity to new inhibitors , because they are not present in other similar 
 kinases , including class i pi3k and mtor .
while these findings represent a step forward 
 towards the development of more specific autophagy inhibitors , it should be noted that 
 vps34 is also involved in the endosomal signaling pathway by forming the endosomal class 
 iii pi3k complex ( vps34/p150/becn1/uvrag / rubicon ) in mammals [ 64 - 70 ] . inhibition of 
 vps34 activity will affect both autophagy and endosomal trafficking .
vps34 activity in 
 autophagy is tightly controlled by assembling the autophagy specific class iii pi3k 
 subcomplex ( vps34/p150/becn1/atg14 ) [ 66,67,71 - 74 ] . specific inhibition of 
 vps34 activity in the complex form might differentiate the effect on autophagy or 
 endocytosis . in this regard , a large - scale screen of chemicals targeting activity of 
 vps34/p150/becn1/atg14 instead of vps34 alone might be more appropriate .
new inhibitors 
 might be identified through inhibiting vps34 activity in an allosteric manner and thus 
 guarantee high selectivity .
the membrane fusion process in autophagy can also be exploited to develop more autophagy 
 specific inhibitors .
the snare proteins ( soluble n - ethylmaleimide - sensitive factor 
 attachment protein receptors ) mediate vesicular fusion events inside the cells .
a unique 
 set of autophagic snare proteins syntaxin17 , snap29 , and vamp8 are essential for the 
 fusion between autophagosomes and lysosomes .
recently , we have been 
 able to reconstitute their fusogenic activity in intro ( unpublished 
 data , zhong et al . ) , and this fusion activity is likely precisely 
 regulated by multiple autophagy specific protein factors .
even if highly specific autophagy inhibitors are developed , one should be cautious about 
 their potential  side - effects  when used as a cancer therapy .
acute 
 ablation of autophagy leads to animal death caused by infection , diabetes , and 
 neurodegeneration , as well as muscle dystrophy and reduced fat storage .
on the other hand , there is 
 considerable interest in developing autophagy activators to battle against infectious 
 diseases and neurodegeneration , in which autophagy activation should alleviate the 
 symptoms . in closing ,
given the broad involvement of autophagy in defense against infection , 
 neurodegenerative disorders , diabetes , cancer and aging , reagents that specifically 
 regulate autophagy will have wide therapeutic applications .
most importantly , our 
 understanding of autophagy in cancer will finally give us an advantage in the war with 
 cancer .
 autophagy is a catabolic degradation process in which cellular proteins and 
 organelles are engulfed by double - membrane autophagosomes and degraded in lysosomes . 
 
 autophagy has emerged as a critical pathway in tumor development and cancer therapy , 
 although its precise function remains a conundrum . 
 the current consensus is that 
 autophagy has a dual role in cancer . 
 on the one hand , autophagy functions as a tumor 
 suppressor mechanism by preventing the accumulation of damaged organelles and 
 aggregated proteins . on the other hand , 
 autophagy is a key cell survival mechanism 
 for established tumors ; therefore autophagy inhibition suppresses tumor progression . 
 here 
 , we summarize recent progress on the role of autophagy in tumorigenesis and 
 cancer therapy . 
Introduction
Are autophagy genes mutated or altered in human cancers?
Does autophagy suppress tumorigenesis?
Does autophagy inhibition suppress tumorigenesis?
Current autophagy inhibitors in clinical trials for cancer treatment
Future directions
Disclosures