Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis

In eg ins Coope a e Wi h he EGFR/ Ras Pa hway o P ese e Epi helia Su i al and A chi ec u e in De elopmen and Oncogenesis And ea Valencia-Expósi o 1 † , M. Jesús Gómez-Lama ca 1 , 2 † , Thomas J. Widmann 3 and Ma ía D. Ma ín-Be mudo 1 * 1 Cen o Andaluz de Biología del Desa ollo CSIC-Uni e sidad Pablo de Ola ide, Se illa, Spain, 2 Depa amen o de Biología Celula , Uni e sidad de Se illa, Se illa, Spain, 3 GENYO, G anada, Spain Adhesion o he ex acellula ma ix (ECM) is equi ed o no mal epi helial cell su i al. Dis up ion o his in e ac ion leads o a specific ype o apop osis known as anoikis. Ye , he e a e physiological and pa hological si ua ions in which cells no connec ed o he ECM a e p o ec ed om anoikis, such as du ing cell mig a ion o me as asis. The main ecep o s ansmi ing signals om he ECM a e membe s o he in eg in amily. Howe e , al hough in eg in-media ed cell-ECM ancho age has been long ecognized as c ucial o epi helial cell su i al, he in i o significance o his in e ac ion emains o be weighed. In his wo k, we ha e used he D osophila wing imaginal disc epi helium o analyze he impo ance o in eg ins as su i al ac o s du ing epi helia mo phogenesis. We show ha educing in eg in exp ession in he wing disc induces caspase-dependen cell dea h and basal ex usion o he dead cells. In his case, anoikis is media ed by he ac i a ion o he JNK pa hway, which in u n igge s exp ession o he p oapop o ic p o ein Hid. In addi ion, ou esul s s ongly sugges ha , du ing wing disc mo phogenesis, he EGFR pa hway p o ec s cells unde going cell shape changes upon ECM de achmen om anoikis. Fu he mo e, we show ha oncogenic ac i a ion o he EGFR/Ras pa hway in in eg in mu an cells escues hem om apop osis while p omo ing hei ex usion om he epi helium. Al oge he , ou esul s suppo he idea ha in eg ins p omo e cell su i al du ing no mal issue mo phogenesis and p e en he ex usion o ans o med cells. Keywo ds: in eg ins, su i al, JNK, EGFR, oncogenesis INTRODUCTION To su i e o die is a cellula decision con olled by en i onmen al cues and physical s imuli. This li e and dea h decision is deeply influenced by he ex acellula ma ix (ECM) (Me edi h e al., 1993). When cells lose hei no mal in e ac ions wi h he ECM, he cell cycle is a es ed and a specific o m o apop osis, knownasanoikis,isini ia ed(F isch and Ruoslah i, 1997). Du ing de elopmen , abe an anoikis al e s issue a chi ec u e and unc ion, comp omising emb yonic iabili y (Mole e al., 2021). Con e sely, p og ammed anoikis con ibu es o physiological de elopmen al p ocesses and issue enewal (Gilmo e, 2005;Chia ugi and Giannoni, 2008). Anoikis is also an impo an su eillance mechanism, ensu ing ha any cell ha loses i s app op ia e posi ion wi hin a issue is a ge ed o dea h. Fo his eason, impai ed anoikis con ibu es o he malignancy o many cance cells, allowing hem o su i e wi hou ECM Edi ed by: Claudia Tanja Mie ke, Leipzig Uni e si y, Ge many Re iewed by: Susan LaFlamme, Albany Medical College, Uni ed S a es Dan Be gs alh, Uni e si y o Roches e , Uni ed S a es *Co espondence: Ma ía D. Ma ín-Be mudo [email p o ec ed] † These au ho s ha e con ibu ed equally o his wo k Special y sec ion: This a icle was submi ed o Cell Adhesion and Mig a ion, a sec ion o he jou nal F on ie s in Cell and De elopmen al Biology Recei ed: 09 Ma ch 2022 Accep ed: 09 May 2022 Published: 13 June 2022 Ci a ion: Valencia-Expósi o A, Gómez-Lama ca MJ, Widmann TJ and Ma ín-Be mudo MDD (2022) In eg ins Coope a e Wi h he EGFR/Ras Pa hway o P ese e Epi helia Su i al and A chi ec u e in De elopmen and Oncogenesis. F on . Cell De . Biol. 10:892691. doi: 10.3389/ cell.2022.892691 F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926911 ORIGINAL RESEARCH published: 13 June 2022 doi: 10.3389/ cell.2022.892691 ancho age and acili a ing hei dispe sion [ e iewed in (Zhong and Resco la, 2012)]. Un a eling he mechanisms egula ing anoikis is he e o e c ucial o unde s and bo h no mal mo phogenesis and cance p og ession. Al hough he concep o ancho age-dependen cell su i al has been ecognized o many yea s, i was in he ea ly 1990’swheni was demons a ed ha cells dep i ed o ECM a achmen unde go anoikis (Me edi h e al., 1993;F isch and F ancis, 1994). Anoikis can be conside ed a mul is ep p ocess [ e iewed in (Zhong and Resco la, 2012)].Fi s o all,losso ancho age esul sininac i a iono ocal complex signalling molecules, including FAK and S c (Gianco i, 2000;Pa sons, 2003). As a consequence, se e al p o- su i al pa hways such as PI3K/Ak and Ra /MEK/ERK a e dis up ed [ e iewed in (Vachon, 2011)].Theseconds epin anoikis is he simul aneous disassembly o ocal adhesions and he des abiliza ion o he cy oskele on, which leads o he elease o p o- apop o ic Bcl-2 p o eins (Ma in and Vuo i, 2004). The final s ep is he ac i a ion o apop o ic kinases, including JNK, which esul s in an inc ease in he exp ession and phospho yla ion o he p o-apop o ic BH3-only p o eins BIM an BMF (Gi nius and Da is, 2017). Howe e , he oleo JNKinanoikis emainscon o e sial,as, depending on he cell ype, kinase iso o m and s imulus, i can pe o m p o- o an i-apop o ic unc ions (Liu and Lin, 2005). Analysing JNK unc ion in di e en cellula con ex s shall help esol ing i s deba ed ole in anoikis. The majo ansmi e s o su i al signals om he ECM a e membe s o he in eg in amily (F isch and Ruoslah i, 1997;Me edi h e al., 1993;Vachon, 2011). In eg ins a e a widely exp essed amily o he e odime ic ansmemb ane glycop o eins, composed o an aand a βsubuni , ha link he ECM o he ac in cy oskele on (Hynes, 1992). The e eb a e in eg in amily is comp ised o 18 aand 8 β ansmemb ane subuni s enabling abou 24 di e en he e odime ic ecep o s o a di e se a ay o ECM p o eins. Howe e , only some in eg ins a e capable o egula ing cell iabili y and hese include he β1in eg insub amily(S upack and Che esh, 2002). The egula ion o cell su i al media ed by in eg ins implica es an inc easing complexi y o playe s depending on he issue, cell ype, specie and he cell di e en ia ion s a e, emphasizing he need o s udy anoikis in a gi en issue wi hin i s physiological con ex [ e iewed in (Igno z and Massague, 1987;F isch and Sc ea on, 2001;Vachon, 2011)]. None heless, he ea ly emb yonic le hali y obse ed in some e eb a e models lacking β1 in eg ins has long p ecluded a di ec e idence o a ole o β1 in eg ins as su i al ac o s in i o (S ephens e al., 1995). The use o cell lineage-specific gene dele ion app oaches and ansplan a ion expe imen s o ci cum en ea ly emb yonic le hali y has e ealed ha he ole o in eg in unc ion in cell su i al in i o is mul i ace ed and some imes con adic o y. Thus, while deple ion o β1 in eg ins om ei he he de eloping mouse lens, he epide mis o he endo helia esul s in apop osis (DiPe sio e al., 2000;Simi skii e al., 2007;Ca lson e al., 2008), condi ional dele ion in he in es inal epi helium causes anoikis esis ance, inc eased cell p oli e a ion and de ec i e di e en ia ion (Jones e al., 2006). Fu he mo e, in some si ua ions, cells a e p o ec ed om anoikis, such as du ing cell mig a ion o me as asis. In hese cases, cells escape dea h using a a ie y o s a egies, including hype ac i a ion o ecep o y osine kinases (Guadamillas e al., 2011;Paolie al.,2013). Since he main componen s o he cell dea h machine y a e e olu iona y conse ed,D osophila, due o i s unique gene ic and cell biological ad an ages, has become an ideal model sys em o s udy apop osis in he con ex o a de eloping o ganism. As in mammals, he caspase amily o cys eine p o eases is essen ial o he egula ion o apop osis also in D osophila. These p o eins a e cons i u i ely exp essed as p ocaspases ha ha e low bu significan p o ease ac i i y. To p e en unwan ed consequences o basal p ocaspase ac i i y, li ing cells exp ess high le els o he Inhibi o o Apop osis (IAP) p o eins, Diap1 and Diap2, which ac as E3- ubiqui in ligases and a ge ac i a o and e ec o caspases o p o easome deg ada ion [ e iewed in (Hay, 2000)]. Upon an apop o ic s imulus, Diap1 an agonis s, including hid, eape ( p ), g im,sickle (skl), ja ac2,anddOmi/H A2 a e ansc ip ionally ac i a ed and physically in e ac wi h Diap1 [ e iewed in (Xu e al., 2009)]. This leads o he blockage o i s caspase inhibi o y unc ion and he deg ada ion o Diap1 by au o-inhibi o y ubiqui iniza ion. As a consequence, he caspase cascade is ac i a ed and he apop osome is o med. Fi s o all, ini ia o p o- caspases, including DRONC, he casp9 o holog, DREDD and DCP- 2, a e ac i a ed by au o-clea age. Then, ini ia o caspases ca alyse he ac i a ion o he e ec o p o-caspases d ICE and DCP-1, and his in u n clea e hei subs a es, including cy oskele on p o eins and DNA me abolic p o eins (Coope e al., 2009). Howe e , despi e he conse a ion o he cell dea h esponse be ween flies and e eb a es, li le is known abou he p ocess o anoikis in D osophila. Using he p imo dium o he D osophila wing, he wing imaginal disc, as model sys em, we ha e s udied he implica ion o in eg ins in he egula ion o epi helial cell su i al in he con ex o a de eloping issue. The o ma ion o he wing s a s du ing ea ly emb yonic de elopmen when abou 30 cells a e alloca ed o o m he wing imaginal disc p imo dium. Du ing la al li e, disc cells di ide and o m an epi helial sac comp ised o a olded columna epi helium on one side and a squamous epi helium on he o he (Ga cia-Bellido and Me ian, 1971).Thema u ediscwillgi e ise o headul wing and no um. A he onse o me amo phosis, local cell shape changes p omo e he olding o he single-laye ed columna epi helium in o a bi-laye ed shee in which epi helial cells ace each o he along hei basal sides (F is om e al., 1993). In eg ins pe o m wo dis inc unc ions in he wing disc, o hold he wo laye s oge he and o egula e he cell shape changes unde lying olding (B aban e al., 1996;Dominguez-Gimenez e al., 2007). Howe e , despi e he known ole o in eg ins as su i al ac o s in bo h benign and malignan epi helia [ e iewed in (Gilc ease, 2007)], in eg ins ha e no ye been implica ed in cell su i al in wing disc epi helial cells. He e, we show ha low in eg in le els induces caspase-dependen wing disc cell dea h. This anoikis p ocess is media ed by he ac i a ion o he JNK pa hway, which in u n induces exp ession o he p oapop o ic gene hid.Wealsofind ha in eg ins coope a e wi h he EGFR pa hway o main ain wing disc epi helial cell su i al. Finally, ou esul s demons a e ha an oncogenic e sion o Ras, Ras V12 , blocks anoikis in he wing disc and ha elimina ion o in eg ins om ans o med Ras V12 cells s imula es hei basal ex usion om he wing disc epi helium. Ou esul s un a el new oles o in eg ins in epi helial cells in i o, p omo ing cell su i al du ing issue mo phogenesis and p e en ing he ex usion o ans o med cells. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926912 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s MATERIALS AND METHODS Fly S ains The ollowing s ocks we e used: UAS-Ras V12 (Lee e al., 1996); nub- Gal4 (Calleja e al., 1996); hid 5′F-GFP (Tanaka-Ma aka su e al., 2009); UAS-DER DN ,UAS-Diap1,UAS-puc,ap-Gal4,puc-LacZ, p - LacZ,andb k-LacZ (Blooming on D osophila S ock Cen e ); mys RNAi ,UAS-diβ(Ma in-Be mudo and B own, 1999)and hid RNAi (Vienna D osophila RNA-i Cen e ). To gene a e mu an clonesin hewingdiscweused heFRT/FLP echnique(Chou and Pe imon, 1992). Mu an clones we e ma ked by he absence o GFP. The ollowing mu an alleles and ch omosomes we e used: mys 11 [also known as mys XG43 (Bunch e al., 1992)] and e22c-Gal4 UAS- flipase (Du y e al., 1998). The e22c-Gal4 d i e is exp essed in some pos e io wing disc cells and was he e o e used in combina ion wi h UAS- flp o gene a e la ge mys clones. Flies we e aised a 25°C. Immunohis ochemis y and Imaging Wing imaginal discs we e s ained using s anda d p ocedu es and moun ed in Vec ashield (Vec o Labo a o ies, Bu lingame, CA, Uni ed S a es). The ollowing p ima y an ibodies we e used: goa an i-GFP FICT (Abcam, 1:500), abbi an i-caspase Dcp1 (Cell Signaling; 1:100), abbi an i-pJNK (P omega, 1:200), mouse an i- βGal (P omega, 1:1000), mouse an i-βPS (De elopmen al S udies Hyb idoma Bank, DSHB, Uni e si y o Iowa, Uni ed S a es, 1:50) and mouse an i-myc (P omega, 1:100). The seconda y an ibodies used we e Alexa fluo 488,(Molecula P obes TM ) and Cy3 and Cy5 (Jackson ImmunoReseach Labo a o ies, Inc.) a 1: 200. DNA was labelled using Hoechs (Molecula P obes, 1:1000). Con ocal images we e ob ained using a Leica SP5-MP-AOBS, equipped wi h a Plan- Apoch oma 40X oil objec i e (NA 1.4). Quan ifica ion o Fluo escence In ensi y To quan i y fluo escen in ensi y o he di e en ma ke s, fluo escen signaling was measu ed om maximum p ojec ions o 30 Z-con ocal sec ions aken wi h 1 μm o in e al pe wing disc and geno ype. Quan ifica ions we e made in he wing pouch egion, which was manually selec ed using he FIJI-Image J line selec ion ool. Mic oscope se ings we e main ained be ween imaging sessions in each expe imen . The backg ound alue aken om cell- ee egion was sub ac ed om all da a se ies. Measu emen s o whole fluo escence in ensi y we e done di iding he mean o all included pixels in ensi y by he ou lined cell a ea, using he FIJI-Image J measu e ool. S uden ’s es s we e used o s a is ical compa isons o fluo escence in ensi y alues. RESULTS In eg ins a e Requi ed o he Su i al o Wing Disc Epi helial Cells To deepen ou unde s anding o he ole o in eg ins as su i al ac o s du ing epi helia mo phogenesis, we es ed whe he emo ing in eg ins in D osophila wing disc epi helial cells a ec ed hei su i al. The D osophila genome con ains wo in eg in βsubuni s, βPS and βν [ e iewed in (B own, 1993;Yee and Hynes, 1993). βPS, encoded by he gene myosphe oid (mys), is he only βchain p esen in he wing imaginal disc (B aban e al., 1996); Supplemen a y Figu e S1]. Fi s , we gene a ed mosaic wing disc epi helia con aining clones o cells homozygous o he null allele mys XG43 ( om now on mys cells, whi e a ow in Figu es 1A–B”). To de ec apop osis, we used an an ibody o clea ed e ec o caspase Dcp-1 (Yu e al., 2002). In con ol wing imaginal discs, Dcp-1 ac i i y is e y low, being de ec ed only occasionally in a ew cells sca e ed h oughou he disc (Figu e 1A,n= 20, whe e n ep esen s he numbe o wing discs analyzed). In con as , all wing discs con aining mysclonesshowed apop o ic cells wi hin he clone a ea (Figu e 1B,n=18).Second,we educed in eg in le els in la ge a eas o he disc using he nubbin (nub) Gal4 line o exp ess a mys RNAi (nub>mys RNAi)in hewing pouch (inse in Figu e 1C) egion in he cen e o he disc ha gi es ise o he adul wing, indica ed wi h a do ed whi e ci cle in all figu es (B and and Pe imon, 1993;Calleja e al., 1996). Immunos aining o con ol (n= 30) and expe imen al nub>mys RNAi wing imaginal discs (n= 36) wi h an ibodies agains he βPS p o ein and clea ed Dcp-1 showed ha he exp ession o he mys specificRNAiin hewing pouch caused a s ong educ ion in βPS p o ein le els (Figu es 1C,C’) and a obus inc ease in apop osis (Figu es 1C,C’’,E). Thus, in eg in exp ession in wing disc epi helial cells p e en hem om unde going cell dea h. I is known ha emb yonic epi helial cells unde going apop osis a e emo ed om he epi helium by ex usion a he han phagocy osis (Rosenbla e al., 2001). Simila ly, in eg in-de ec i e cells we e ex uded (Figu es 1C–C’’) and accumula ed be ween he basal su ace o he disc epi helium and he ECM, as seen wi h an an ibody agains he ECM componen Pe lecan (Supplemen a y Figu e S2). In eg ins can media e bo h signaling and adhesion [ e iewed in (Adams and Wa , 1993)]. To add ess which in eg in unc ion is equi ed o acili a e cell su i al in he wing imaginal disc, we used a chime ic in eg in (diβ) ha lacks in eg in-adhesi e unc ion bu main ains i s abili y o signal (Ma in-Be mudo and B own, 1999;Dominguez-Gimenez e al., 2007). We had p e iously shown ha exp ession o diβin he wing disc inhibi ed he basal localiza ion o endogenous in eg in [(Supplemen a y Figu es S3B,B’;(Dominguez-Gimenez e al., 2007)]. He e, we ound ha i caused an inc ease in apop osis (Supplemen a y Figu es S3B,B”), s ongly sugges ing ha in eg in signaling is no su ficien o p e en cell dea h in de ached wing disc cells. Al oge he , hese esul s p opose ha in eg in-media ed adhesion o he ECM p omo es cell su i al in he wing disc epi helium by egula ing caspase ac i i y. To u he suppo his hypo hesis, we es ed whe he o e exp ession o one o he D osophila IAP (inhibi o o apop osis) p o eins, Diap1, supp essed he cell dea h caused by exp ession o mys RNAi and ound ha i did (Figu es 1D-D’’,E,n= 35). In eg ins DownRegula e Caspase Ac i i y by Inhibi ing Hid Exp ession P e ious expe imen s ha e shown ha o e exp ession o hid o p induces down egula ion o Diap1 le els and apop osis in D osophila wing imaginal discs (Milan e al., 1997;Be gan inos e al., 2010;Yoo e al., 2002). As Diap1 o e exp ession escues cell dea h due o in eg in down egula ion, in eg ins could p omo e cell su i al h ough he egula ion o hid and/o p exp ession. To es his, we analysed hid and p exp ession in con ol and F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926913 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s nub>mys RNAi wing discs, using he hid 5′F-GFP [ om now on hidGFP,(Tanaka-Ma aka su e al., 2009)] and p LacZ (No ds om e al., 1996) ansc ip ional epo e s. hidGFP le els in con ol discs (hidGFP;nubGal4) a e ha dly de ec able, consis en wi h a low occu ence o cell dea h in his issue (Tanaka-Ma aka su e al., 2009); Figu es 2A-A’’’,E]. This exp ession was d ama ically up egula ed in nub >mys RNAi discs (n= 25, Figu es 2B-B’’’). In con as , he no mal exp ession pa e n o p LacZ, confined o a small egion o he no um and wo s ipes in he D-V and A-P bounda ies [(No ds om e al., 1996), Supplemen a y Figu es S4A,A’], was no al e ed in nub >mys RNAi discs (n= 28, Supplemen a y Figu es S4B,B’). These esul s sugges ha in eg ins block hid, bu no p exp ession in no mal wing disc epi helial cells o p omo e cell su i al. Fu he mo e, consis en wi h he idea ha D osophila hid ac s ups eam o Diap1 (Wang e al., 1999;Goyal e al., 2000), we ound ha exp ession o Diap1 was no able o escue he inc ease in hidGFP le els obse ed in mys RNAi cells (n= 24, Figu es 2C–C’’’). To u he confi m hid in ol emen in in eg in loss o unc ion- dependen cell dea h, we es ed whe he educing hid le els could escue apop osis in nub >mys RNAi discs. We ound ha exp ession FIGURE 1 | In eg ins p omo e cell su i al in he wing imaginal disc. (A–D99 )Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d-ins a la ae s ained wi h an i-βPS [g een in (A–D), whi e in (A9–D9)], an i-Dcp1 [ ed in (A–D), whi e in (A99 –D99 )] and he nuclea ma ke Hoechs [DNA, blue in (A–D)]. (A) Con ol nubGal4 wing disc. (B) Wing disc ca ying mys mu an clones (whi e a ow), ma ked by he absence o βPS (g een). (C) Wing disc exp essing GFP o a mys RNAi unde he con ol o nubGal4,nub >GFP (inse ) and nub >mys RNAi , espec i ely. (yz) Con ocal yz sec ion along he whi e do ed line shown in (C).(D) Wing disc co- exp essing a mys RNAi and Diap-1 unde he con ol o nubGal4,nub >mys RNAi ;Diap1.(E) Quan ifica ion o Dcp1 le els in wing discs o he designa ed geno ypes. The do ed whi e ci cles indica e he wing pouch a ea in all figu es. The s a is ical significance o di e ences was assessed wi h a - es , ***, ** and * p alues <0.001, <0.01, and <0.05, espec i ely. Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926914 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s o an RNAi agains hid (hidGFP;nub >mys RNAi ;hid RNAi ) esul ed in a significan educ ion o mys RNAi-induced apop osis (n=20, Figu es 2A-A’’’,B-B’’’,D-D’’’,E). These findings demons a e ha in eg ins p omo e cell su i al in wing imaginal discs h ough he nega i e egula ion o hid exp ession. Al e ing he le els o Decapen aplegic (Dpp), a D osophila ans o ming g ow h ac o βhomologue), in wing discs esul s in up egula ion o b inke (b k), a ansc ip ional ep esso ha p e en s apop osis (Adachi-Yamada e al., 1999;Mo eno e al., 2002). Howe e , educing in eg in unc ion does no seem o a ec Dpp signalling, as he exp ession pa e n o he b k epo e b kLacZ was no al e ed in nub >mys RNAi wing discs (n=26,Supplemen a y Figu e S5B). Down egula ion o In eg in Exp ession in Wing Discs Induces JNK Ac i a ion As men ioned in he in oduc ion, he ole o JNK in anoikis is con o e sial, as, depending on he cell ype and cellula con ex , i FIGURE 2 | In eg ins s imula e cell su i al h ough inhibi ion o hid exp ession. (A–D-)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs s ained wi h an i-βPS [blue in (A–D), whi e in (A9–D9)], an i-Dcp1 [ ed in (A–D), whi e in (A99 –D99 )] and an i-GFP [g een in (A–D), whi e in (A-–D-)].(A–A-)Con ol nubGal4 wing disc ca ying a epo e o hid exp ession, hidGFP,hidGFP; nubGal4.(B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4, hidGFP;nub >mys RNAi .(C–D-)hidGFP wing disc co-exp essing a mys RNAi unde he con ol o nubGal4 wi h ei he Diap1, hidGFP; nub >mys RNAi ;Diap1 (C–C-), o a hid RNAi, hidGFP;nub >mys RNAi ;hid RNAi (D–D-).(E) Quan ifica ion o Dcp1 le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , ***, ** and * p alues <0.001, <0.01, and <0.05, espec i ely. NS, non-s a is ically significan . Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926915 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s can be ei he essen ial (F isch e al., 1996)o dispensable(Khwaja and Downwa d, 1997). In D osophila wing discs, JNK ac i a ion igge s apop osis in esponse o se e al s imuli, including dis o ion o posi ional in o ma ion (Adachi-Yamada e al., 1999), TNF signalling (Igakie al.,2002;Mo eno e al., 2002)o i adia ion (Luo e al., 2007). To s udy he ole o he JNK pa hway in apop osis induced by in eg in loss o unc ion, we analyzed JNK ac i a ion using an an ibody agains phospho yla ed JNK (pJNK, Figu e 3). Compa ed o con ols (n=16,Figu es 3A,A’,D), pJNK le els we e up egula ed in nub >mys RNAi discs (n=18,Figu es 3B,B’,D), sugges ing ha in eg ins con ol JNK ac i i y in he wing disc. This was u he confi med using a epo e ha moni o s he ansc ip ion o a a ge o he JNK pa hway, he JNK inhibi o pucke ed (pucLacZ)(Adachi-Yamada e al., 1999). We ound ha while in wild ype wing discs, pucLacZ exp ession was es ic ed o a small p oximal egion (Adachi-Yamada e al., 1999), Supplemen a y Figu es S6A,A’), in nub >mys RNAi wing discs, pucLacZ le els we e s ongly up egula ed in he pouch (n= 28,Supplemen a y Figu es S6B,B’). In some con ex s, JNK signalling can be ac i a ed as a consequence o apop osis (Ku anaga e al., 2002). To es whe he ac i a ion o JNK lies ups eam o downs eam o loss o in eg in unc ion, JNK ac i a ion was assessed in nub >mys RNAi ;Diap1 wing discs (Figu e 3). We ound ha pJNK (n=20,Figu es 3C,D)and pucLacZ (n= 25, Supplemen a y Figu es S6C,C’) le els we e s ill up egula ed despi e escue o apop osis (Figu e 1D), sugges ing ha JNK ac i a ion is ups eam o cell dea h due o in eg in loss o unc ion in wing disc cells. S ess induced cell dea h in he wing disc ac i a es JNK ups eam o caspase ac i a o p o eins, which, in u n, ein o ce JNK ac i a ion in a posi i e eedback loop o ampli y he apop o ic esponse (Shle ko and Mo a a, 2012). He e, we ound ha , in ac , he inc ease in JNK ac i i y o nub >mys RNAi ;Diap1 FIGURE 3 | Elimina ion o in eg ins ac i a es he JNK pa hway. (A–C9)Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d-ins a la ae s ained wi h an i-pJNK [g een in (A–C), whi e in (A9–C9)] and he nuclea ma ke Hoechs [DNA, blue in (A–C)]. (A,A9)Con ol nubGal4 wing disc. (B,B9)Wing disc exp essing a mys RNAi unde he con ol o nubGal4,nub >mys RNAi .(C,C9)Wing disc co-exp essing RNAi agains mys and Diap1 unde he con ol o nubGal4,nub >mys RNAi ; Diap1.(D) Quan ifica ion o pJNK le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and ** p alues <0.001 and <0.01, espec i ely. Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926916 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s discs was lowe han he one obse ed in nub >mys RNAi discs (Figu es 3B–D). This esul sugges s ha he eedback loop be ween JNK ac i i y and apop osis obse ed in s ess-induced apop osis may also ope a e in in eg in loss o unc ion-dependen apop osis. JNK Media es Apop osis due o Loss o In eg in Func ion in Wing Disc Cells To es whe he he JNK pa hway media es cell dea h due o in eg in loss o unc ion in wing imaginal discs, we analyzed i o e exp ession o he JNK inhibi o puc supp essed he cell dea h caused by mys RNAi (nub >mys RNAi ;puc) and ound ha i did (Figu es 4A–D,n= 24). The ela ionship be ween JNK signalling, p o-apop o ic p o eins and caspase ac i a ion is con ex -dependen . Thus, he JNK pa hway can ac ups eam o downs eam o p o-apop o ic genes such as p o hid. Fo ins ance, expe imen s in D osophila S2 cells sugges ed ha Rp could s imula e JNK ac i a ion h ough DIAP1 deg ada ion (Ku anaga e al., 2002). In con as , JNK signalling p omo es hid exp ession (and apop osis) in eye discs, and i is equi ed o p - epo e induc ion in esponse o adia ion in wing discs (Ku anaga e al., 2002;Mo eno e al., 2002). In ou sys em, inhibi ion o JNK signalling ia puc o e exp ession was able o supp ess hid up egula ion in nub > mys RNAi wing discs o a la ge ex en (n=17,Figu es 4C-C’’’,E). Al oge he , hese esul s allow us o conclude ha educ ion o in eg in unc ion in wing discs leads o apop osis by s imula ing hid ansc ip ion h ough JNK ac i a ion. In eg ins Coope a e Wi h he EGFR Pa hway o P omo e Cell Su i al The EGFR ac i a es a ne wo k o signaling pa hways p omo ing su i al in many di e en cellula con ex s [ e iewed in (Henson FIGURE 4 | In eg ins p omo e cell su i al by inhibi ing he JNK pa hway. (A–C-)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs ca ying he epo e hidGFP, s ained wi h an i-βPS [blue in (A–C), whi e in (A9–C9)], an i-Dcp1 [ ed in (A–C), whi e in (A99 –C99 )] and an i-GFP [g een in (A–C), whi e in (A-–C-)]. (A–A-)Con ol hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,hidGFP;nub >mys RNAi .(C–C-)hidGFP wing disc co-exp essing a mys RNAi and he nega i e egula o o he JNK pa hway puc, unde he con ol o nubGal4 hidGFP;nub >mys RNAi ;puc.(D,E) Quan ifica ion o Dcp1 (D) and hidGFP (E) le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and * p alues < 0.001, <0.01, and <0.05, espec i ely. NS, non-s a is ically significan . Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926917 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s and Gibson, 2006)]. In addi ion, expe imen s mainly om cell cul u e ha e e ealed syne gis ic coope a ion be ween g ow h ac o s and in eg ins in cell su i al (Regina o e al., 2003;Mi an i and B ugge, 2002).Fo ins ance, EGFR p omo es su i al h ough egula ion o he Ras/MAPK pa hway and consequen inhibi ion o he p oapop o ic gene hid in he eye disc (Be gmann e al., 1998;Ku ada and Whi e, 1998;Yu e al., 2002). Mo eo e , wing disc mu an clones o EGFR o o some o hei dowms eam e ec o s, such as Ras, a e smalle han hei win spo , sugges ing a ole o he EGFR pa hway in cell su i al (Diaz-Benjumea and Ga cia-Bellido, 1990; Diaz-Benjumea and Ha en, 1994; Zecca and S uhl, 2002). To di ec ly add ess a ole o EGFR in cell su i al in he wing disc, we exp essed an EGFR dominan nega i e cons uc (UAS-DER DN ) unde he con ol o he nub-Gal4 line and ound i led o inc ease cell dea h in he pouch egion (nub >DER DN ,n= 21, Figu es 5A, A’,C,C’,E). Howe e , in con as o wha happens in nub >mys RNAi wing discs (Figu es 5B, B’,E), whe e cell dea h was ound dis ibu ed all o e he pouch, exp ession o DER DN es ic ed apop osis o egions o high EGFR ac i i y (Gabay e al., 1997), such as he wing ma gin and he p esump i e ein e i o ies (yellow a ow and as e isks in Figu es 5A, A’,C,C, espec i ely). In addi ion, and con a y o wha happens in he eye, EGFR did no seem o exe i s p o-su i al unc ion h ough egula ion o hid o p ansc ip ion, as hidGFP o p -lacZ exp ession we e no al e ed in nub >DER DN wing discs (Figu es 5A, A’,C,C”;Supplemen a y Figu e S7). We hus conclude ha he EGFR pa hway has a limi ed ole in he con ol o cell su i al in he wing disc ha is independen o he ansc ip ional egula ion o he p o- apop o ic genes hid and p . Finally, we examined a possible coope a ion be ween in eg ins and he EGFR pa hway o media e cell su i al. We ound ha he simul aneous co- exp ession o mys RNAi and DER DN in wing discs (nub > mys RNAi ;DER DN ) enhanced he cell dea h pheno ype caused by he exp ession o any o hem on hei own (Figu es 5B–E), s ongly sugges ing ha in eg ins and he EGFR pa hway ac in pa alell o p omo e cell su i al in wing imaginal discs. Oncogenic Ras Supp esses Anoikis in Wing Discs Supp ession o anoikis a e de achmen o cance cells om he ECM is a key s ep in umo me as asis [ e iewed in (Simpson e al., 2008)]. In ac , he abili y o oncogenic Ras o supp ess anoikis has long been conside ed c i ical o Ras ans o ma ion. Howe e , ecen e idence sugges s ha Ras and o he oncogenes can induce bo h p o- and an i-apop o ic signals depending on he cell ype and con ex [ e iewed in (Cox and De , 2003)]. In D osophila, Ras o e ac i a ion ende s cells e ac o y o s ess- and i adia ion-induced apop osis (Be gmann e al., 1998; Ku ada and Whi e, 1998;Pinal e al., 2018). To es whe he oncogenic Ras could p o ec cells om anoikis in he wing imaginal disc, we o e exp essed an ac i a ed o m o DRas, Ras V12 (Ka im and Rubin, 1998) and ound i subs an ially supp essed he cell dea h pheno ype caused by in eg in deple ion (nub >mys RNAi , as V12 ;Figu es 6A-A’’,6B–B’’, C–C’’,D,n= 18). These esul s demons a e ha oncogenic Ras is able o supp ess anoikis in wing imaginal discs. In epi helial cell lines, oncogenic Ras con e s esis ance o anoikis pa ly by down egula ing he exp ession o p oapop o ic Bcl-2 membe s FIGURE 5 | In eg ins and EGFR coope a e o p omo e cell su i al. (A–D”)Maximal p ojec ion o con ocal iews o wing imaginal discs om hi d- ins a la ae ca ying he epo e hidGFP, s ained wi h an i-Dcp1 [ ed in (A–D), whi e in (A9–D9)], an i-GFP [g een in (A–D), whi e in (A’’–D’’)] and he nuclea ma ke Hoechs [DNA, blue in (A–D)]. (A–D’’)Con ol hidGFP; nubGal4 wing disc. (B–B’’)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,hidGFP;nub >mys RNAi .(C–C’’)hidGFP wing disc exp essing a dominan nega i e o m o he EGFR, DER DN , unde he con ol o nubGal4,hidGFP;nub >DER DN .(C–D’’)hidGFP wing disc co-exp essing a mys RNAi and DER DN unde he con ol o nubGal4 hidGFP;nub >mys RNAi ; DER DN .(E) Quan ifica ion o Dcp1 le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and ** p alues <0.001 and <0.01, espec i ely. NS, non-s a is ically significan . Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926918 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s (Rak e al., 1995) and by p e en ing down egula ion o an iapop o ic p o eins (Rosen e al., 2000). In D osophila,i appea s ha he Ras pa hway egula es Hid ac i i y a he le el o bo h, p o ein phospho yla ion and gene exp ession, in hid- induced apop osis (Be gmann e al., 1998;Ku ada and Whi e, 1998). Howe e , we ound ha , in he D osophila wing disc epi helium, he abili y o oncogenic Ras o con e esis ance o anoikis does no in ol e ansc ip ional egula ion o hid,as hidGFP le els we e no al e ed in nub >mys RNAi ; Ras V12 (n= 21, Figu es 6A’’’,C’’’) discs compa ed o nub >mys RNAi (n= 20, Figu e 6B’’’). Ec opic exp ession o Ras V12 in he do sal compa men o wing imaginal discs, by means o he ap e ous Gal4 (ap) Gal4 line, causes issue o e g ow h and cell shape changes, which esul s in he o ma ion o ec opic olds [ap >Ras V12 ,n= 20, Figu es 7A,A’,C,C’,(P obe and Edga , 2000;Sole Bea y e al., 2021). Reducing in eg in unc ion also al e s cell shape causing a mild olding pheno ype (Dominguez-Gimenez e al., 2007), Figu es 7B, B’E, E’]. He e, we ound ha co-exp ession o mys RNAi and Ras V12 enhanced he cell shape and olding pheno ype caused by he sole exp ession o any o hem (ap >mys RNAi ; Ras V12 ,n= 20, Figu es 7D,D’). In his wo k, we show ha educing in eg in exp ession ensued basal ex usion o he dead cells (Figu es 1C–C’’, Figu es 7F,G). Howe e , e en hough oncogenic K-Ras MCDK cells su i e and ex ude basally, a mechanism p oposed o ini ia e in asion in o su ounding issues (Sla um e al., 2014), we ound ha exp ession o Ras V12 on i s own in wing disc cells (ap >Ras V12 ) did no esul in cell ex usion (Figu e 7H). In s iking con as , he exp ession o Ras V12 in FIGURE 6 | Ec opic ac i a ion o he Ras pa hway escues anoikis. (A–C’’’)Maximal p ojec ion o con ocal iews o hi d-ins a wing imaginal discs ca ying he epo e hidGFP, s ained wi h an i-βPS [blue in (A–C), whi e in (A9–C9)], an i-Dcp1 [ ed in (A–C), whi e in (A’’–C’’)] and an i-GFP [g een in (A–C), whi e in (A-–C-)]. (A–A-)Con ol hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc exp essing a mys RNAi unde he con ol o nubGal4,hidGFP;nub >mys RNAi .(C–C-)hidGFP wing disc co-exp essing a mys RNAi and an ac i a ed o m o Ras, Ras V12 , unde he con ol o nubGal4 hidGFP;nub >mys RNAi ; Ras V12 .(D) Quan ifica ion o Dcp1 le els in wing discs o he indica ed geno ypes. The s a is ical significance o di e ences was assessed wi h a - es , *** and * p alues <0.001 and <0.05, espec i ely. Scale ba in all panels, 30 μm. F on ie s in Cell and De elopmen al Biology | www. on ie sin.o g June 2022 | Volume 10 | A icle 8926919 Valencia-Expósi o e al. In eg ins: Essen ial De elopmen al Su i al Fac o s