1、Microenvironmental autophagy promotes tumour growthNadja S.Katheder1,2,Rojyar Khezri1,2,*,Fergal OFarrell1,2,*,Sebastian W.Schultz1,2,Ashish Jain1,2,3,Mohammed M.Rahman1,2,Kay O.Schink1,2,Theodossis A.Theodossiou4,Terje Johansen3,Gbor Juhsz5,6,David Bilder7,Andreas Brech1,2,Harald Stenmark1,2,and To
2、r Erik Rusten1,21Department of Molecular Cell Biology,Institute for Cancer Research,Oslo University Hospital,Montebello,N-0379 Oslo,Norway2Centre for Cancer Biomedicine,Faculty of Medicine,University of Oslo,Montebello,N-0379 Oslo,Norway3Molecular Cancer Research Group,Institute of Medical Biology,U
3、iT-The Arctic University of Norway,9037 Troms,Norway4Department of Radiation Biology,Institute for Cancer Research,Oslo University Hospital,Montebello,N-0379 Oslo,Norway5Institute of Genetics,Biological Research Centre,Hungarian Academy of Sciences,Szeged,H-6726 Hungary6Department of Anatomy,Cell an
4、d Developmental Biology,Etvs Lornd University,Budapest H-1117,Hungary7Department of Molecular and Cell Biology,University of California,Berkeley,Berkeley,California 94720-3200,USAAbstractAs malignant tumours develop,they interact intimately with their microenvironment and can activate autophagy1,a c
5、atabolic process which provides nutrients during starvation.How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial2.Here we demonstrate,using a well characterized Drosophila melanogaster malignant tumour model3,4,that non-cell-autonomous autophagy is indu
6、ced both in the tumour microenvironment and systemically in distant tissues.Tumour growth can be pharmacologically restrained using Reprints and permissions information is available at and requests for materials should be addressed to T.E.R.(tor.erik.rustenrr-research.no).*These authors contributed
7、equally to this work.Supplementary Information is available in the online version of the paper.Author Contributions N.S.K.,H.S.,D.B.,T.J.and T.E.R.designed the research;N.S.K.,R.K.,F.O.F,A.J.,S.W.S.,M.M.R.,K.O.S.,T.A.T.and T.E.R.,performed experiments and analysed the data;G.J.developed transgenic a
8、utophagy reporter animals;and N.S.K.,H.S.,D.B.,and T.E.R.wrote the manuscript.The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper.Reviewer Information Nature thanks T.Igaki,H.Zhang and the other anonymous reviewer(s)for their contrib
9、ution to the peer review of this work.HHS Public AccessAuthor manuscriptNature.Author manuscript;available in PMC 2017 September 25.Published in final edited form as:Nature.2017 January 19;541(7637):417420.doi:10.1038/nature20815.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptau
10、tophagy inhibitors,and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment.Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells,whereas tum
11、our growth depends on active amino acid transport.We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts.We conclude that transformed cells engage surrounding normal cells as active and essential micr
12、oenvironmental contributors to early tumour growth through nutrient-generating autophagy.To assess autophagy during Drosophila tumour growth,we generated green fluorescent protein(GFP)-labelled malignant RasV12scrib/eye imaginal disc(EAD)tumours in animals carrying the autophagosome marker Atg8a tag
13、ged with mCherry(ChAtg8a)under the control of its endogenous promoter5,6.These tumours grow and invade the central nervous system,eventually killing the host3,4,7.In contrast to RasV12-expressing clones,ChAtg8a puncta accumulated strongly in epithelial cells surrounding RasV12scrib/tumour cells,with
14、 only marginal accumulation in the tumour itself(Fig.1a,b and Extended Data Fig.1a,b).ChAtg8a structures were readily assigned to autophagic structures by correlative light and electron microscopy(Extended Data Fig.1c)and were not observed in cells deficient in the essential autophagy gene atg13,and
15、 lysosomal processing of ChAtg8a increased in the presence of tumour cells.Together,these data demonstrate that the presence of ChAtg8a reflects autophagy activity in this system(Fig.1c,d;for gel source data,see Supplementary Data).As adult Drosophila tumours have recently been found to induce a cac
16、hexia-like response in the animal8,9,we assessed whether peripheral organs responded to eye-specific tumours in larvae.Upregulation of autophagy was observed in the muscle,fat body and midgut of RasV12scrib/but not RasV12 tumour-bearing larvae and was lost altogether in atg13-deficient animals8,9(Fi
17、g.1eg and Extended Data Fig.2a).Taken together,these data show that malignant RasV12scrib/,but not benign RasV12 tumours lead to local and systemic non-cell-autonomous autophagy(NAA).Oral administration of the autophagy flux inhibitor chloroquine10 led to a significant reduction in tumour growth and
18、 invasion(Fig.1h and Extended Data Fig.2b).Therefore,we investigated whether NAA in the microenvironment,distal organs or both may contribute to tumour growth.We generated recombinant chromosomes that allowed autophagy to be ablated through the induction of atg13 or atg14 deficiency in specific comp
19、artments during tumour growth(Extended Data Figs 3,4).RasV12scrib/tumours were generated with the simultaneous prevention of autophagy:(i)within the tumour only;(ii)in the surrounding cell population only;(iii)in both cell populations within the tumour-bearing organ;or(iv)in the entire animal.RasV12
20、scrib/tumours overgrew,with most invading into the ventral nerve cord(VNC)by day 8(refs 3,4,7)(Fig.2a,f and Extended Data Fig.5a).Consistent with previous work,we found that preventing autophagy within the tumour caused a moderate,albeit significant,Katheder et al.Page 2Nature.Author manuscript;avai
21、lable in PMC 2017 September 25.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptreduction in tumour volume11,12,but did not reduce invasion(Fig.2b,f and Extended Data Fig.5a).By contrast,ablation of autophagy in cells surrounding RasV12scrib/clones strongly reduced tumour growth a
22、nd invasion(Fig.2c,f and Extended Data Fig.5a).Removal of autophagy in both cell populations through the creation of a mutant animal with complete loss of function in atg13 reduced tumour volume and invasiveness even further(Fig.2d,f and Extended Data Fig.5a).In both contexts,tumour growth was rescu
23、ed by complementation with a genomic atg13 construct(Fig.2e,f and Extended Data Fig.5bf).Each of these manipulations reduced the elevated cell cycle entry and progression displayed by tumour cells(Extended Data Fig.6ap),with no marked increase in tumour apoptosis(Extended Data Fig.6qu).Finally,we in
24、duced defective autophagy specifically in the tumour-bearing organ through mitotic recombination between the atg14 and atg13,scrib-carrying chromosome arms.Notably,tumour growth was reduced by a similar extent in this organ-specific mutant as in fully mutant atg13 animals,suggesting that the role au
25、tophagy plays in support of tumour growth is local(Fig.2gj).To test this,we rescued atg13 expression specifically in eye discs(ey3.5-atg13)13 and found that this restored autophagic activity and significantly rescued tumour growth in tumour-bearing atg13 mutant animals(Fig.2k and Extended Data Fig.5
26、g,h).Taken together,these results show that RasV12scrib/tumours require local NAA for initial tumour growth and expansive invasion into neighbouring tissue.Earlier studies have established that the growth and invasion of RasV12scrib/tumours involve the TNFJNKFos and IL-6JAKSTAT cytokine signalling p
27、athways3,1419.RasV12scrib/tumours failed to induce NAA in TNF-deficient(egr/)animals or when JNK activity was blocked in the tumour(Fig.3ad and Extended Data Fig.7b,k),indicating that autophagy requires tumour-specific TNF/JNK signalling.Notably,systemic autophagy activation remained when JNK signal
28、ling was inhibited,suggesting that local and systemic control of autophagy are achieved by different mechanisms(Extended Data Fig.7a).JNK cooperates with Hippo(encoded by hpo)/Yorkie(encoded by yki)/Scalloped(encoded by sd)signalling to drive tumour growth in RasV12scrib/animals18.To evaluate the ro
29、le of Hippo signalling,we knocked down the Scrib polarity module component discs large(encoded by dlg1)while simultaneously overexpressing RasV12 in half of the eye-antennal disc15,20.These tumours phenocopied RasV12scrib/tumours,rapidly out-growing the neighbouring microenvironmental cells that exh
30、ibited robust induction of NAA(Fig.3e).However,JNK inhibition reversed NAA,validating the system for epistasis analysis(Extended Data Fig.7c,l).The overexpression of Ykiact alone recapitulated induction of NAA(Extended Data Fig.7d,k),whereas silencing of either yki or sd in RasV12dlg1RNAi tumours re
31、versed overgrowth and NAA(Fig.3f and Extended Data Fig.7e,l).This does not appear to be a consequence of reduced tumour growth,as discs in which tumour burden was reduced by expression of dominant negative PI3K(dp110DN)still showed robust NAA(Extended Data Fig.7f,l).The data suggest that the transcr
32、iptional target(s)that are required for NAA respond both to Yki/Sd and to Fos.Targets of Yki/Sd and Fos include the insulin-binding antagonist ImpL2,a mediator of organ wasting8,9,and the IL-6-like inflammatory cytokines unpaired 1,unpaired 2 and unpaired 3(ref.19).Although ImpL2 was neither necessa
33、ry nor sufficient for the induction of NAA,the overexpression of either upd or upd3 Katheder et al.Page 3Nature.Author manuscript;available in PMC 2017 September 25.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptby RasV12 animals induced NAA,suggesting that these cytokines can m
34、ediate NAA(Fig.3g and Extended Data Fig.7gi,l,m).To investigate how cytokine signalling may regulate NAA,we inactivated downstream JAKSTAT signalling in either tumours or the microenvironment specifically.We observed a robust NAA response in discs in which microenvironmental signalling was disrupted
35、,but this was absent when signalling was inactivated in the tumour(Fig.3h and Extended Data Fig.7j,m),suggesting that NAA induction involves autocrine signalling.Assays revealed that RasV12scrib/tumours have damaged mitochondria,increased mitochondrial mass and production of reactive oxygen species(
36、ROS),and altered respiration(Fig.3i,j and Extended Data Fig.8af).Experimental mitochondrial ROS generation induced by ND75 knockdown was sufficient to trigger autophagy21(Extended Data Fig.8g,h).Ykiact-induced NAA did not involve increased ROS and the growth of these benign-like tumours does not dep
37、end on NAA(Extended Data Fig.8i,j).Inhibition of either JNK or JAKSTAT in RasV12scrib/tumours reversed ROS accumulation18(Fig.3k,l).Thus,high levels of ROS and NAA are co-regulated in RasV12scrib/tumours and rely on co-transcriptional changes in Fos-,Yki/Sd-and STAT-mediated mitogenic signalling.The
38、 direct inducer of NAA in this context has still to be defined,but it may not be a single factor.Efforts to scavenge ROS by pharmaceutical or genetic means failed to reverse RasV12scrib induced NAA,although scavenger efficacy could not be confirmed.Nevertheless,these data suggest that highly prolife
39、rative RasV12scrib/Drosophila tumours are metabolically stressed,produce high levels of ROS and potentially rely on increased import of nutrientsas in humans.Indeed,we observed an increase in the import of the fluorescently labelled glucose analogue(2-NBDG)in RasV12scrib tumour cells(Extended Data F
40、ig.8k,l)Microenvironmental autophagy has been proposed to provide recycled nutrients,including amino acids,locally in order to support tumour growth22.If this is the case,then tumour growth should be hypersensitive to reduced amino acid import.To test this,we silenced the cationic and neutral amino
41、acid transporter slimfast(encoded by slif),the loss of which has previously been shown to have minimal effect on proliferation of wild-type cells23.Silencing of slif markedly reduced growth of RasV12dlgRNAi tumours,indicating that the metabolic demand of tumour cells to sustain growth and proliferat
42、ion is met,in part,by the increased import of amino acids(Fig.4a and Extended Data Fig.9a).To investigate whether the requirement of autophagy for tumour growth is permanent and reliant on local factors,we performed allograft experiments24.RasV12scrib/allografts grew into large tumours,with a concom
43、itant increase in control host ChAtg8a processing(Extended Data Fig.9b,c).Tumour growth could be pharmacologically restricted by administration of autophagy inhibitors;notably,tumours also grew more poorly in atg145.2/atg14EY14568 hypomorphic hosts than in control hosts(Fig.4b and Extended Data Fig.
44、9d,e).This suggests that the autophagy capacity of the host,and not tumour location or original tumour environment,determines growth.To investigate whether the lack of tumour growth in autophagy-deficient animals is reversible,we transplanted poorly growing Katheder et al.Page 4Nature.Author manuscr
45、ipt;available in PMC 2017 September 25.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptRasV12atg13/scrib/atg13/tumours into control or atg145.2/atg14EY14568 hosts.Strikingly,this autophagy-deficient tumour tissue,which grew very poorly in atg13 mutant larvae(Fig.2d),was capable o
46、f regrowth and formed large tumours when transplanted into a control host(Fig.4c).By contrast,RasV12atg13/scrib/atg13/tumours transplanted into atg145.2/atg14EY14568 or atg8 hosts remained smaller(Fig.4c and Extended Data Fig.9f).Thus,autophagy-deficient tumours are dormant but can resume growth in
47、an autophagy-proficient ectopic environment.Malignant tumour growth induces autophagy,both in the microenvironment and distal tissues.Locally,normal epithelial cells are engaged as an active part of the microenvironment,supporting tumour proliferation by autophagy.Systemically,autophagy in distant o
48、rgans may support the growth of allografted tumour tissue.Most previous studies have focused on the cell-autonomous role of autophagy in Ras-driven malignancies2,11,25.In line with these studies,we find that cell-autonomous autophagy supports tumour growth,although our experimental model shows that
49、the non-cell-autonomous effects of autophagy are much more striking.Our studies are consistent with a model in which early tumours concomitantly upregulate the import of nutrients and engage the microenvironment to recycle nutrients to support their own growth through autophagy(Fig.4d).Given the con
50、served pathways studied,similar tumourmicroenvironment co-operation may occur in human cancers.METHODSFly husbandryStocks and crosses were kept at 25 C on standard potato mash fly food containing 32.7 g dried potato powder,60 g sucrose,27.3 g dry yeast,7.3 g agar,4.55 ml propionic acid,and 2 g nipag
