Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer

In e na ional Jou nal o Molecula Sciences Re iew Zeb a ish as a Model O ganism o he De elopmen o D ugs o Skin Cance Fa emeh Boo o abi 1, Hamed Manoucheh i 2, Reza Changizi 2, Ha lan Ba ke 3, Elisabe a Palazzo 4ID , Annalisa Sal a i 4, Ma aleena Pa ikka 5, Ca lo Pincelli 4 and Ashok Aspa wa 3,*ID 1Biosenso Resea ch Cen e , Endoc inology and Me abolism Molecula -Cellula Sciences Ins i u e, Teh an Uni e si y o Medical Sciences, 14114 Teh an, I an; [email p o ec ed] 2Depa men o Aquacul u e, Babol B anch, Islamic Azad Uni e si y, 47134 Babol, I an; [email p o ec ed] (H.M.); [email p o ec ed] (R.C.) 3 Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, 33014 Tampe e, Finland; [email p o ec ed] 4Labo a o y o Cu aneous Biology, Depa men o Su gical, Medical, Den al and Mo phological Sciences, Uni e si y o Modena and Reggio Emilia, 41100 Modena, I aly; [email p o ec ed] (E.P.); [email p o ec ed] (A.S.); [email p o ec ed] (C.P.) 5Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, O al and Maxillo acial Uni , Tampe e Uni e si y Hospi al, 33014 Tampe e, Finland; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +358-465962117 Academic Edi o : Julie e Legle Recei ed: 12 Ap il 2017; Accep ed: 11 July 2017; Published: 18 July 2017 Abs ac : Skin cance , which includes melanoma and squamous cell ca cinoma, ep esen s he mos common ype o cu aneous malignancy wo ldwide, and i s incidence is expec ed o ise in he nea u u e. This condi ion de i es om acqui ed gene ic dys egula ion o signaling pa hways in ol ed in he p oli e a ion and apop osis o skin cells. The de elopmen o animal models has allowed a be e unde s anding o hese pa homechanisms, wi h he possibili y o ca ying ou oxicological sc eening and d ug de elopmen . In pa icula , he zeb a ish (Danio e io) has been es ablished as one o he mos impo an model o ganisms o cance esea ch. This model is pa icula ly sui able o li e cell imaging and high- h oughpu d ug sc eening in a la ge-scale ashion. Thanks o he ecen ad ances in genome edi ing, such as he clus e ed egula ly-in e spaced sho palind omic epea s (CRISPR)/CRISPR-associa ed p o ein 9 (Cas9) me hodologies, he mechanisms associa ed wi h cance de elopmen and p og ession, as well as d ug esis ance can be in es iga ed and comp ehended. Wi h hese unique ools, he zeb a ish ep esen s a powe ul pla o m o skin cance esea ch in he de elopmen o a ge he apies. He e, we will e iew he ad an ages o using he zeb a ish model o d ug disco e y and oxicological and pheno ypical sc eening. We will ocus in de ail on he mos ecen p og ess in he ield o zeb a ish model gene a ion o he s udy o melanoma and squamous cell ca cinoma (SCC), including cance cell injec ion and ansgenic animal de elopmen . Mo eo e , we will epo he la es compounds and small molecules unde in es iga ion in melanoma zeb a ish models. Keywo ds: melanoma; squamous cell ca cinoma; inhibi o sc eening; ansgenic zeb a ish; skin cance ; d ug de elopmen 1. In oduc ion Zeb a ish is a small e eb a e opical ish ha has ecen ly eme ged as one o he mos use ul models o s udying human diseases, including cance s. In ac , zeb a ish displays high ecundi y and is sui able o gene ic manipula ion and bo h e e se and o wa d gene ic s udies. Thanks o In . J. Mol. Sci. 2017,18, 1550; doi:10.3390/ijms18071550 www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2017,18, 1550 2 o 15 i s ela i ely low cos o use, he zeb a ish is ideal o la ge-scale sc eening app oaches and allows bo h chemical and gene ic sc eening o iden i y genes and pa hways unde lying diseases, as well as pheno ypic sc eening o he disco e y o new d ugs [ 1 , 2 ]. The compounds, d ugs o small molecules, can be added di ec ly o he wa e en i onmen o he zeb a ish [ 3 ]. Mo eo e , gi en he high gene ic and physiological simila i ies wi h humans, zeb a ish can be used as a use ul and cos -e ec i e ehicle o high- h oughpu sc eening (HTS) [2,4–6]. The g owing in e es in his model is de i ed om he op ical anspa ency o zeb a ish emb yos and la ae, as well as hei as de elopmen ex u e o. In ac , zeb a ish a e capable o e ilizing 200–300 eggs e e y 5–7 days and ha e an equi alen longe i y and gene a ion ime o mice (3–5 mon hs). Zeb a ish emb yos apidly de elop ex u e o, and less han one week is equi ed o he de elopmen o he diges i e, ne ous and ca dio ascula o gan sys ems [ 7 – 12 ]. This p ope y allows o as e s udy o he physiological and pa hological mechanisms by a di ec li e-cell imaging in i o . Fo hese easons, zeb a ish has become an impo an model o human diseases. Mo eo e , he high le el o simila i y be ween human and zeb a ish la ae in e ms o he gene ics and physiology o he inna e immune sys em p omo es special in es iga ion on cance esea ch [ 1 ]. Zeb a ish is a sui able model o umo induc ion by he use o se e al me hods, such chemical ea men s [ 13 ], gene ic knockou [ 14 ], gene o e exp ession [ 15 ] and xeno ansplan a ion [ 16 ]. Zeb a ish has been used o he s udy o di e en ype o cance s, such as skin cance [ 14 , 15 ] panc ea ic cance [ 16 ], b eas cance [ 17 ], leukemia [18], glioma [19] and lung cance [20]. Skin cance , which includes melanoma and squamous cell ca cinoma (SCC), ep esen s he mos common ype o cu aneous malignancy wo ldwide, and i s incidence is expec ed o ise in he nea u u e [ 21 ]. Melanoma is he deadlies o m o skin cance , wi h espec o all skin cance - ela ed dea hs, and has a mo ali y a e o 80% [ 22 , 23 ]. Melanoma a ises om melanocy es, he pigmen ed epide mal cells esponsible o he p oduc ion o melanin. In he ea ly s ages, melanoma is con ined o he epide mis ( adial g ow h phase (RGP) melanoma) and can be emo ed by su gical excision [ 24 ]. These ypes o melanomas a e usually associa ed wi h a good p ognosis. On he con a y, in he nex s ages o umo p og ession, melanoma cells in ade he subcu aneous issues ( e ical g ow h phase (VGP) melanoma) and e en ually p og ess owa d he me as a ic phase. A his s age, ew he apeu ic op ions a e a ailable, and melanoma equen ly elapses and becomes incu able [6,24]. Cu aneous SCC (cSCC) mos ly de i es om al e a ions wi hin he epide mal homeos asis caused by ul a iole exposu e and elies on uncon olled p oli e a ion o he epide mal cells, namely ke a inocy es [ 25 ]. cSCC is he second mos equen ype o non-melanoma skin cance and ep esen s 20% o all skin malignancies [ 25 , 26 ]. Al hough in si u SCCs a e cu able by su gical excision, me as a ic SCCs a e esponsible o he majo i y o dea hs due o non-melanoma skin cance s [ 21 ]. As a ke a inocy e-de i ed epi helial umo , head and neck squamous cell ca cinoma (HNSCC) a ises in se e al places in he head and neck egion, such as o opha ynx and la yngopha ynx, and is e y common wo ldwide, ep esen ing 4% o all cance s in he Uni ed S a es [ 23 ]. In pa icula , o al squamous cell ca cinoma (OSCC) accoun s o 24% o HNSCC wi h a mo ali y a e o 200,000 cases each yea [ 26 , 27 ]. Despi e di e en he apeu ic op ions, such as adio o chemo he apy o su ge y, he i e-yea su i al a e is app oxima ely 50% [26,28,29]. Gi en he signi ican impac o melanoma and SCC, on a wo ld-wide scale, he de elopmen o new ools o s udy hese pa hologies and hei ea men ep esen s an impo an ask. In he p esen e iew, we desc ibe he zeb a ish as a powe ul in i o model o skin cance (melanoma and SCC) and summa ize he ad an ages o d ug sc eening in skin cance esea ch. We epo in de ail on he use o ansgenic zeb a ish o s udy melanoma de elopmen , p og ession and ea men [ 30 , 31 ] and he use o zeb a ish emb yos o e alua e po en ial a ge s and compounds o SCC [22,25]. 1.1. Zeb a ish as a Model O ganism o Pha macological and Toxicological Sc eening In ecen yea s, esea ch has begun o de elop an unde s anding o how zeb a ish can be used as an in i o model o human diseases, in e ms o a ge s o he apy, physiological simila i y, In . J. Mol. Sci. 2017,18, 1550 3 o 15 d ug me abolism, d ug sa e y and oxici y and pha macology [ 2 ] (Figu e 1). The zeb a ish genome sha es o e 70% simila i y wi h he human genome, and o e 80% o known human disease genes, including oncogenes and umo supp esso genes, ha e o hologues in zeb a ish [ 32 ]. Se e al pa hways a e also well conse ed be ween human and zeb a ish allowing a ge ing o a speci ic biological p ocess [ 32 ]. The e o e, he esul s ob ained by using zeb a ish models o d ug sc eening can be pa icula ly ele an o human disease and cance he apy. In . J. Mol. Sci. 2017, 18, 1550 3 o 15 also well conse ed be ween human and zeb a ish allowing a ge ing o a speci ic biological p ocess [32]. The e o e, he esul s ob ained by using zeb a ish models o d ug sc eening can be pa icula ly ele an o human disease and cance he apy. Figu e 1. Zeb a ish as a ele an model o human disease and cance he apy. The zeb a ish genome shows up o 80% simila i y wi h human disease-associa ed genes. Mo eo e , hanks o a well- conse ed physiology, he pha macological beha io and me abolism o se e al d ugs ha e been sc eened in zeb a ish wi h e ec s simila o humans. Cell-based assays mos ly p o ide limi ed esul s and in o ma ion abou he abso p ion, dis ibu ion, me abolism and oxici y o he sc eened molecules, compounds and d ugs. By con as , sc eening in zeb a ish o e comes his p oblem; mo eo e , he e a e physiological simila i ies in he blood b ain ba ie [10], endo helial cells [33], endo helial cells and immunological esponses [34], gi ing as da a ela ed o he pha macological cha ac e is ics [35–37]. When injec ed wi h a compound, ei he a d ug o small molecule, zeb a ish and mammals p esen o e lapping physiologic esponses, such as he induc ion o me aboli es, enzyme ac i i y agains di e en an igens and upon oxida i e s ess condi ions [4,9]. A numbe o he biological p ope ies o zeb a ish p o ides ad an ages o d ug sc eening. Ea ly-s age zeb a ish emb yos ha e a anspa en body, making i ela i ely easy o collec nume ous da a using a high-quali y imaging a e ea men . Mo eo e , gi en ha a single emale can lay 10,000 eggs pe annum [38], i is possible o each high h oughpu (1000–10,000) o mo e assays pe day, as only space becomes a limi ing ac o [39] (Figu e 2). Fo his eason, zeb a ish emb yos ha e been p oposed as he in i o animal model ha could b idge he gap be ween cell-based assays and biological alida ion o a compound. Mo eo e , he p esence o he cho ion, an acellula en elope made o h ee in e c ossing laye s ha su ound he emb yo du ing de elopmen , allows some ma e ials o easily pass h ough o he emb yo ia passi e di usion [40]. This aspec is ele an in e ms o physical-chemical p ope ies o he compounds ha could be es ed by zeb a ish models, by a ec ing he e icacy o he ea men s o he le el o dose-speci ic oxici y. Because o he anspa ency o zeb a ish emb yos and la ae and he possibili y o conduc mo phological obse a ion o a ious o gans and ana omic s uc u es du ing de elopmen [8,10– 12,41], zeb a ish has assumed an impo an ole in oxicology esea ch, including eco oxicology and de elopmen al s udies. The g ea es ad an age o zeb a ish in oxicology s udies is he low cos wi h espec o oden s and he possibili y o pe o m analysis wi h a la ge numbe o compounds a he same ime. In his espec , zeb a ish can be u ilized o la ge-scale sc eening o compounds/lib a ies and d ugs, as i may gi e as e esul s. In ac , hund eds o di e en hi s om a p ima y HTS can be Figu e 1. Zeb a ish as a ele an model o human disease and cance he apy. The zeb a ish genome shows up o 80% simila i y wi h human disease-associa ed genes. Mo eo e , hanks o a well-conse ed physiology, he pha macological beha io and me abolism o se e al d ugs ha e been sc eened in zeb a ish wi h e ec s simila o humans. Cell-based assays mos ly p o ide limi ed esul s and in o ma ion abou he abso p ion, dis ibu ion, me abolism and oxici y o he sc eened molecules, compounds and d ugs. By con as , sc eening in zeb a ish o e comes his p oblem; mo eo e , he e a e physiological simila i ies in he blood b ain ba ie [ 10 ], endo helial cells [ 33 ], endo helial cells and immunological esponses [ 34 ], gi ing as da a ela ed o he pha macological cha ac e is ics [ 35 – 37 ]. When injec ed wi h a compound, ei he a d ug o small molecule, zeb a ish and mammals p esen o e lapping physiologic esponses, such as he induc ion o me aboli es, enzyme ac i i y agains di e en an igens and upon oxida i e s ess condi ions [4,9]. A numbe o he biological p ope ies o zeb a ish p o ides ad an ages o d ug sc eening. Ea ly-s age zeb a ish emb yos ha e a anspa en body, making i ela i ely easy o collec nume ous da a using a high-quali y imaging a e ea men . Mo eo e , gi en ha a single emale can lay 10,000 eggs pe annum [ 38 ], i is possible o each high h oughpu (1000–10,000) o mo e assays pe day, as only space becomes a limi ing ac o [ 39 ] (Figu e 2). Fo his eason, zeb a ish emb yos ha e been p oposed as he in i o animal model ha could b idge he gap be ween cell-based assays and biological alida ion o a compound. Mo eo e , he p esence o he cho ion, an acellula en elope made o h ee in e c ossing laye s ha su ound he emb yo du ing de elopmen , allows some ma e ials o easily pass h ough o he emb yo ia passi e di usion [ 40 ]. This aspec is ele an in e ms o physical-chemical p ope ies o he compounds ha could be es ed by zeb a ish models, by a ec ing he e icacy o he ea men s o he le el o dose-speci ic oxici y. Because o he anspa ency o zeb a ish emb yos and la ae and he possibili y o conduc mo phological obse a ion o a ious o gans and ana omic s uc u es du ing de elopmen [8,10–12,41] , zeb a ish has assumed an impo an ole in oxicology esea ch, including eco oxicology and de elopmen al s udies. The g ea es ad an age o zeb a ish in oxicology s udies In . J. Mol. Sci. 2017,18, 1550 4 o 15 is he low cos wi h espec o oden s and he possibili y o pe o m analysis wi h a la ge numbe o compounds a he same ime. In his espec , zeb a ish can be u ilized o la ge-scale sc eening o compounds/lib a ies and d ugs, as i may gi e as e esul s. In ac , hund eds o di e en hi s om a p ima y HTS can be es ed o elimina e he oxic compounds and hen p io i izing hose ha will be u ilized in u he assays and d ug de elopmen . This implici coun e -sc een o oxici y is an in eg al pa o disease supp esso sc eens in he o ganism [2]. In . J. Mol. Sci. 2017, 18, 1550 4 o 15 es ed o elimina e he oxic compounds and hen p io i izing hose ha will be u ilized in u he assays and d ug de elopmen . This implici coun e -sc een o oxici y is an in eg al pa o disease supp esso sc eens in he o ganism [2]. Figu e 2. Zeb a ish model o high- h oughpu d ug sc eening. Zeb a ish is a aluable ool o high- h oughpu sc eening assays. In e ms o oxici y, zeb a ish ha e been used o e alua e a speci ic o gan o beha io wi h espec o oxici y. In d ug sc eening, zeb a ish is also es ablished as p ecious pla o m o mul iple sys emic pheno ype s udies simul aneously wi h me abolic p o iles and oxici y epo e lines. In addi ion, he ansplan a ion o human cance cells in o zeb a ish o gene a e xenog a models ep esen s a powe ul ool o moni o cance p oli e a ion, umo angiogenesis, me as asis and d ug esponse in eal ime. The use o luo escen p obes o epo e genes du ing oxicological o cance - ela ed s udies gi es he oppo uni y o pe o m li e-imaging o bo h ansgenic and xeno ansplan a ion models. Fo example, melanoma cells can be s ained wi h Vyb an Cell-Labeling Solu ion o (5(6)- Ca boxy luo escein N-hyd oxysuccinimidyl es e (CFSE) dye be o e being mic oinjec ed in he yolk [42]. To iden i y he ole o Ra and phosphoinosi ide 3-kinase (P13K) signaling pa hways in melanoma cells, a G een Fluo escen p o ein (GFP) epo e gene was used unde he con ol o he p omo e o he melanoma cell speci ic gene, mic oph halmia-associa ed ansc ip ion ac o (mi ) [24]. Simila ly, in o de o in es iga e he implica ion o Ex acellula signal-Regula ed kinases (ERK1/2)-Mi ogen-Ac i a ed P o ein kinases (MAPK) signaling pa hways in he de elopmen o u eal melanoma, ansgenic emb yos we e labeled wi h GFP and coupled o he GNAQQ209P ec o in o de o exp ess his oncogene in melanocy es [30]. Mo eo e , in e -segmen al blood essel cells we e labeled wi h GFP in ansgenic emb yos o he s udy o melanoma cell adhesion [15]. Ne e heless, as a consequence o he complexi y and inhe en di e ences o mammals, i is impo an o highligh ha zeb a ish can ne e eplace oden s in he la e phases o d ug disco e y, bu may be complemen a y o oden o cell-based assays a ea lie s ages [43]. Indeed, clinical ials using se e al mu ine models o human in lamma o y pa hologies ail o show a signi ican success a e in humans [44]. Hence, unde s anding he ad an ages, disad an ages and limi a ions o mammalian models helps o choose he bes model o pha macological s udies acco ding o he speci ic a ge . 1.2. Pheno ype-Guided D ug Disco e y Despi e he achie emen o signi ican p og ess in d ug disco e y, he iden i ica ion o new he apeu ic a ge s o he ea men o human diseases emains a big challenge in science [45]. Figu e 2. Zeb a ish model o high- h oughpu d ug sc eening. Zeb a ish is a aluable ool o high- h oughpu sc eening assays. In e ms o oxici y, zeb a ish ha e been used o e alua e a speci ic o gan o beha io wi h espec o oxici y. In d ug sc eening, zeb a ish is also es ablished as p ecious pla o m o mul iple sys emic pheno ype s udies simul aneously wi h me abolic p o iles and oxici y epo e lines. In addi ion, he ansplan a ion o human cance cells in o zeb a ish o gene a e xenog a models ep esen s a powe ul ool o moni o cance p oli e a ion, umo angiogenesis, me as asis and d ug esponse in eal ime. The use o luo escen p obes o epo e genes du ing oxicological o cance - ela ed s udies gi es he oppo uni y o pe o m li e-imaging o bo h ansgenic and xeno ansplan a ion models. Fo example, melanoma cells can be s ained wi h Vyb an Cell-Labeling Solu ion o (5(6)-Ca boxy luo escein N-hyd oxysuccinimidyl es e (CFSE) dye be o e being mic oinjec ed in he yolk [ 42 ]. To iden i y he ole o Ra and phosphoinosi ide 3-kinase (P13K) signaling pa hways in melanoma cells, a G een Fluo escen p o ein (GFP) epo e gene was used unde he con ol o he p omo e o he melanoma cell speci ic gene, mic oph halmia-associa ed ansc ip ion ac o (mi ) [ 24 ]. Simila ly, in o de o in es iga e he implica ion o Ex acellula signal-Regula ed kinases (ERK1/2)-Mi ogen-Ac i a ed P o ein kinases (MAPK) signaling pa hways in he de elopmen o u eal melanoma, ansgenic emb yos we e labeled wi h GFP and coupled o he GNAQQ209P ec o in o de o exp ess his oncogene in melanocy es [ 30 ]. Mo eo e , in e -segmen al blood essel cells we e labeled wi h GFP in ansgenic emb yos o he s udy o melanoma cell adhesion [15]. Ne e heless, as a consequence o he complexi y and inhe en di e ences o mammals, i is impo an o highligh ha zeb a ish can ne e eplace oden s in he la e phases o d ug disco e y, bu may be complemen a y o oden o cell-based assays a ea lie s ages [ 43 ]. Indeed, clinical ials using se e al mu ine models o human in lamma o y pa hologies ail o show a signi ican success a e in humans [ 44 ]. Hence, unde s anding he ad an ages, disad an ages and limi a ions o mammalian models helps o choose he bes model o pha macological s udies acco ding o he speci ic a ge . 1.2. Pheno ype-Guided D ug Disco e y Despi e he achie emen o signi ican p og ess in d ug disco e y, he iden i ica ion o new he apeu ic a ge s o he ea men o human diseases emains a big challenge in science [45]. In . J. Mol. Sci. 2017,18, 1550 5 o 15 While p e ious s udies we e es ic ed o in e eb a e model o ganisms, such as Caeno habdi is elegans, o o in i o cell-based assays, because o ex ensi e labo and cos equi emen s, he use o zeb a ish has opened a new a enue [ 45 ]. Thanks o he andom mu agenesis s a egy, a g ea e ange o pheno ypes esembling human diseases can be in es iga ed in compa ison o o he e eb a es such as mice and a s. In con as o a ge -based app oaches, pheno ype-guided d ug disco e y aims o ca y ou a la ge chemical sc eening by combining HTS wi h animal models o a human disease. Hence, using he pheno ype-guided d ug disco e y, i is possible o iden i y an e ec i e compound by looking a pheno ype al e a ion in he whole o ganism, independen ly o he speci ic a ge . The e o e, his app oach imp o es he de elopmen o new d ugs and, a he same ime, allows he iden i ica ion o he pa hways ha a e implica ed in he de elopmen o p og ession o he speci ic disease [ 6 ]. Two examples o he use o zeb a ish o pheno ype-based d ug disco e y a e gi en by he sc eening o chemical molecules as supp esso s o wo speci ic gene ic mu a ions: (1) g idlock and (2) C umbs homolog (c b) mu a ions [ 45 ]. G idlock mu an s, which p esen a ascula de ec due o a mu a ion in he hey2 gene, ha e been ea ed wi h a lib a y o 5000 small molecules o e e he disease pheno ype. F om he sc eening, wo compounds, named GS4012 and GS3999, ha e been iden i ied. These molecules ac i a e he ascula endo helial g ow h ac o (VEGF) pa hway o escue he pa hological pheno ype. The second mu an ha bo s a mu a ion in he Myb p o o-oncogene p o ein also known as ansc ip ional ac i a o Myb (myb) gene, which leads o genome ins abili y and de ec s in he cell-cycle. A lib a y o 16,320 compounds was sc eened in c b emb yos, allowing he disco e y o h ee di e en classes o supp esso s o he c b mu an pheno ype. The possibili y o epo human disease alleles in zeb a ish u he suppo s he high impac o his me hod in d ug disco e y [ 46 – 49 ]. 2. Zeb a ish as a Model O ganism o S udy SCC The zeb a ish model has been ecen ly used o iden i y key molecules in cSCC and HNSCC [23], as well as compounds o SCC a ge he apy [50]. Cichon and co-wo ke s ound ha he y osine kinase ecep o Axl, which is highly exp essed in SCC whe e i p omo es cell su i al, is implica ed in umo o ma ion in he cSCC xenog a model in zeb a ish [ 51 ]. Indeed, Axl dele ion in cSCC cells educed umo mass, a e cell injec ion in he yolk sac o one-day-old emb yos, hus indica ing ha Axl is a po en ial he apeu ic a ge o SCC. Using a simila app oach, he ole o ype VII collagen (Col7) in cSCC umo g ow h and angiogenesis was also e alua ed. Mu a ion in he COL7A1 gene is esponsible o dys ophic epide molysis bullosa (RDEB), an inhe i ed blis e ing diso de ha p edisposes o he de elopmen o an unexplained agg essi e SCC [ 25 ]. Recombinan ype VII collagen (h Col7) was able o e e se SCC angiogenesis in he xenog a model [25]. The y osine kinase discoidin domain ecep o 2 (DDR2), which has a ole in cell p oli e a ion, adhesion, di e en ia ion and in asion, is implica ed in HNSCC [ 21 ]. DDR2 has been epo ed o be inhibi ed by dasa inib, a Food and D ug Adminis a ion (FDA)-app o ed inhibi o o Abelson mu ine leukemia i al oncogene homolog, P o o-oncogene y osine-p o ein kinase (ABL, SRC) and c-Ki [ 21 ]. Von Massenhausen and collabo a o s analyzed he unc ional ole o DDR2 in an in i o xenog a model wi h o wi hou dasa inib ea men and demons a ed ha DDR2 inhibi ion blocked HNSCC cell mig a ion and in asion [ 21 ]. Thei esea ch indica es ha dasa inib can be po en ially used as a y osine kinase inhibi o in DDR2-posi i e HNSCC pa ien s. Zeb a ish emb yos ha e also been used o e alua e he e ec o Flo illin-1 o e exp ession in KB cells (a subline o he KERATIN- o ming umo cell line HeLa), which a e an OSCC cell line [ 26 ]. Flo illin-1 is a componen o he lipid a s and plays an essen ial ole in cell adhesion, cell mo phology and p o ein sec e ion [ 26 ]. Zeb a ish emb yos we e injec ed wi h luo escen -labeled umo cells o e exp essing Flo illin-1 o analyze umo me as asis. The esul s demons a ed ha he exp ession o Flo illin-1 inc eases he cell g ow h and mo ili y o KB cells [26]. The mo ili y o OSCC was also a ge ed by he use o he neu alizing monoclonal an ibody NZ-1 and lec in (MASL) agains podoplanin (PDPN)-exp essing OSCC cells in zeb a ish. This molecule In . J. Mol. Sci. 2017,18, 1550 6 o 15 is a ansmemb ane ecep o ha p omo es umo cell mo ili y in OSCC, and i migh be used as a chemo he apeu ic a ge o p ima y and me as a ic cance s [6]. Among he compounds being e alua ed in zeb a ish o SCC, iazine compound S06 educes OSCC in asion. I s mechanism o ac ion is a ge ing o he chape on hea -shock p o ein 90 (Hsp90); hus, inhibi ing ca cinoma-associa ed ib oblas (CAF)-de i ed p oin asi e chemokinases by S06 is capable o inhibi ing umo cell mig a ion in a zeb a ish xenog a model a 48 h pos - e iliza ion [ 50 ]. The ma ine mic obial ex ac luminacin was e alua ed in zeb a ish emb yos in e ms o an i- umo ac i i y in HNSCC [ 23 ]. Luminacin ea men o cance cells was able o inhibi g ow h and cance p og ession by p omo ing au ophagy o HNSCC cell lines. Taken oge he , hese wo ks well suppo he use o zeb a ish as a model o ganism o he analysis o he key playe s in SCC de elopmen and p og ession, as well as o d ug sc eening and oxici y assays. 3. Zeb a ish as Model O ganism o Melanoma Resea ch While he impo ance o he ac i a ion o oncogenes and inac i a ion o umo supp esso genes in umo o ma ion is well app ecia ed, ou unde s anding o he ea ly e en s o cance ini ia ion emains limi ed. The mechanisms ha enable a subpopula ion o umo cells o comple e he con e sion o a malignan s a e among a la ge g oup o cance -p one cells (desc ibed as a “cance ized ield”) is s ill unclea [ 52 ]. To be e unde s and he mechanisms unde lying umo ini ia ion, in melanoma esea ch, zeb a ish can be used as an excellen ool, h ough he use o xenog a [ 42 , 53 ] and ansgenic models [30,54]. The main ad an ages o xeno ansplan a ion a e li e cell imaging and he lack o he adul immune sys em, which is comple ely unc ional only a 28 days o de elopmen . The injec ion o luo escen -labelled melanoma cells in o zeb a ish la ae enables he s udy o cance angiogenesis and umo cell sp ead. The ea ly phases o melanoma p og ession and he pheno ype swi ching owa d me as a ic beha io ha e been ecen ly in es iga ed in zeb a ish h ough he s udy o he neu o ophin ecep o CD271 (p75NTR) [ 42 ]. CD271 is a iably exp essed in melanoma cells, showing highe le els in p ima y umo s compa ed o he me as a ic umo s de i ed om he same pa ien . The injec ion o melanoma cells a e CD271 silencing o o e exp ession in anspa en la ae e ealed ha CD271 absence is associa ed wi h a highe numbe o me as ases in zeb a ish [42]. Since adul zeb a ish a e no longe anspa en , in o de o isualize melanoma cells by luo escen gene exp ession, a anspa en adul ish, named caspe , has been c ea ed by Whi e e al. [55]. Al e na i ely, he s udy o melanoma de elopmen can be pe o med in ansgenic zeb a ish de i ed om he exp ession o oncogenes o om mu a ions in umo supp esso genes induced wi h N-e hyl-N-ni osou ea (ENU). ENU is a powe ul mu agen wi h alkyla ing ac i i y used as a highly e icien s a egy o induce andom mu a ions in he genome o zeb a ish. This app oach consis s o exposing zeb a ish o epea ed ea men s wi h ENU o gene a e poin mu a ions in p e-meio ic ge m cells [ 53 ]. This ea men causes all possible base pai changes, such as nonsense, splicing and missense mu a ions. ENU has been success ully used o c ea e a se ies o knockou zeb a ish [ 56 ]. Gi en ha he pheno ype associa ed wi h al e a ions in he zeb a ish de elopmen was easily no iceable, ENU was used o in es iga e he ole o mu a ions in emb yonic pa e ning and de elopmen . Howe e , some disad an ages limi ed he use o his andom mu agenesis app oach, such as he gene a ion o he e ozygous mu an s ha hampe he iden i ica ion o ecessi e inhe i ed pheno ypes. By using his app oach, h ee mu an s named “young” (Yng), “pe plexed” (plx) and “con used” (c s) a ec ed by de ec s in e inal lamina ion ha e been iden i ied [ 57 ]. Mo e ecen ly, he clus e ed egula ly-in e spaced sho palind omic epea s (CRISPR)/CRISPR-associa ed p o ein 9 (Cas9) sys em has eme ged as a new genome edi ing ool o induce mu a ions in speci ic genes wi hin o ganisms [ 58 ]. CRISPR comp ises segmen s o DNA con aining sho , epe i i e base sequences. These sequences play a ole in a bac e ial immune sys em. The CRISPR/Cas9 s a egy is based on deli e ing he Cas9 DNA nuclease wi h a guide RNA (gRNA) in o a cell o cu he genome a a speci ic loca ion. A se ies o success ul s udies In . J. Mol. Sci. 2017,18, 1550 7 o 15 used his echnology o e alua e he ole o speci ic gene ic pe u ba ions on he pheno ype o zeb a ish. Fo example, i has been used o s udy he e ec o MMP21 knock-ou , e ealing a ole in ca diac de ec s and al e a ion o No ch signaling [ 59 ]. Mo eo e , his genome edi ing sys em has a g ea impac in cance esea ch, as i pe mi s issue-speci ic modelling o human cance , such as melanoma [ 60 – 62 ]. Melanoma equen ly o igina es due o mu a ion in he BRAF and NRAS genes (43% and 30% o cases, espec i ely) [ 63 ]. Because BRAF ac s as a se ine/ h eonine kinase and becomes ac i a ed by a soma ic poin mu a ion, i plays an impo an ole as a a ge o d ug de elopmen in malignan melanoma [ 64 ]. The i s melanoma zeb a ish model ca ying he V600E mu a ion was ob ained by placing he human BRAF V600E gene unde he con ol o he mi (mic oph halmia-associa ed ansc ip ion ac o ) p omo e , a ansc ip ion ac o in ol ed in he lineage-speci ic pa hway o melanocy es [ 65 ]. The BRAF V600E mu a ion is associa ed wi h melanoma de elopmen ; howe e , he concomi an loss o unc ion o he p53 gene is needed o make melanocy ic nae i p og ess o cance [ 64 ]. E en a e he gene a ion o an ex ensi e “cance ized ield”, wi h all melanocy es ha bo ing bo h BRAF V600E and p53-/- mu a ions in zeb a ish, only one o h ee melanomas will de elop and only a e se e al mon hs [ 65 ]. This lesse numbe and ime ame indica e ha o he molecula al e a ions and pa hways play an impo an ole in umo o ma ion. By sc eening he genes implica ed in he apid onse o melanoma, Zon and colleagues ound ha he o e exp ession o his one-lysine N-me hyl ans e ase (SETDB1) can accele a e he onse and in asion o melanoma [ 4 ]. High le els o SETDB1 a e common in human melanoma, indica ing ha ch oma in emodeling may be c i ical in melanoma p og ession due o changes in gene egula ion, such as subse o homeo ic genes (hox) genes [66]. T ansgenic zeb a ish ca ying RAS mu a ions o he s udy o melanoma ha e also been c ea ed [ 24 ]. Exp ession o he HRAS G12V p o ein, unde he mi a p omo e , in melanocy e p ogeni o s induces a hype -pigmen ed pheno ype and an abno mal g ow h o melanocy es in he la ae; ul ima ely d i ing melanoma o ma ion in he adul ish [ 67 ]. Mo eo e , ele a ed ac i i y o RAC, a sub amily o Rho GTPases, o en associa ed wi h melanoma in humans, can accele a e he p og ession o HRAS V12 -d i en malignan melanoma [ 68 ]. Zeb a ish BRAF V600E models ha e some limi a ions, such as he ime needed o spon aneous umo o ma ion and he di e en numbe o mu a ions as compa ed o human melanomas [ 69 ]. On he o he hand, HRAS V12 mu a ions accele a e melanoma o ma ion and enable melanoma o be isualized a he ea lies s ages in zeb a ish [7,24,70]. Finally, he zeb a ish model allows he s udy o genes in ol ed in he lineage-speci ic pa hway o melanocy e de elopmen and how hei mis egula ion is esponsible o melanoma ini ia ion [ 71 ]. Fo his pu pose, a empe a u e-sensi i e zeb a ish model ha bo ing a mu a ion in he mi gene (mi a c7) has ecen ly been c ea ed [71–73]. 4. Compounds and Inhibi o s o Melanoma T ea men P e ious s udies ha e shown ha mi is a po en ial he apeu ic a ge o melanoma ea men [67,74,75] . MITF plays a c i ical ole in he de elopmen o he melanocy ic lineage [76,77] and egula es genes associa ed wi h melanogenesis, cell di e en ia ion, p oli e a ion and su i al [78–80] by a ge ing BCL-2 (B-cell leukemia/lymphoma 2) [ 81 ], ML-IAP (melanoma inhibi o o apop osis) [ 82 , 83 ], HIF1a (hypoxia-inducible ac o 1a) [ 84 ] and MET [ 85 ]. Mo eo e , MITF can egula e he exp ession o CDKN1A [ 86 , 87 ], which encodes o p21, an inhibi o o he cell-cycle. Th ough sc eening o a small molecule lib a y con aining 2000 compounds, SKLB226 has been iden i ied as a speci ic supp esso o mi exp ession, subsequen ly able o inhibi he iabili y and mig a ion o melanoma cells [ 88 ]. In addi ion, he e alua ion o SKLB226 ac ion in zeb a ish showed i s abili y o block pigmen a ion and cell mig a ion in i o [ 89 ]. Le i e al. used zeb a ish emb yos o es a lib a y o 6000 compounds iden i ying wo molecules (12G9 and 36E9) able o inhibi melanocy e de elopmen by a ge ing mi [ 90 ]. 12G9 and 36E9 inhibi ed pigmen a ion o bo h skin and e inal pigmen epi helium [ 91 ] and induced apop osis o melanocy es [ 76 ]. In pa icula , he compounds impai ed he p oli e a ion and di e en ia ion o melanoblas s and induced a educed numbe o mi a and In . J. Mol. Sci. 2017,18, 1550 8 o 15 dc (dopach ome au ome ase, a p o ein equi ed o he syn hesis o eumelanin) posi i e cells [ 91 ]. In addi ion, among he sul u -con aining molecules, 4 (phenylsul anyl) bu an-2 educes he exp ession o melanin syn hesis- ela ed p o eins and exhibi s a good sa e y p o ile. This compound induces a ema kable supp ession o melanogenesis in zeb a ish a e 48 h. In addi ion, i s abili y o ep ess in i o pigmen a ion could ha e signi ican applica ions in he cosme ic ield [ 91 ]. Fu he mo e, his whi ening subs ance is sa e and e ec i e, and i could be used o p e en hype -pigmen a ion [ 91 ]. Zeb a ish emb yos ca ying BRAFV 600E exp ession ha e been used o he s udy o he neu al c es -de i ed cell lineage [ 92 ]. In pa icula , ansgenic mi -BRAF V600E ; p53/zeb a ish emb yos ha e been c ea ed o he e alua ion o ea ly ansc ip ional ac i i y wi hin melanoma pa hogenesis and o p o ide a model o chemical gene ic sc eening in he con ex o melanoma he apy. Inhibi o s o dihyd oo o a e dehyd ogenase (DHODH), such as le lunomide (le ), a e able o educe he abili y o sel - enewal o neu al c es cells and a ge he ac i i y o hose genes equi ed o neu al c es de elopmen and melanoma umo g ow h [ 93 ], including mi and myc a ge s [ 92 ]. Resea ch da a sugges ha le would be mo e e ec i e in combina ion wi h a BRAFV 600E inhibi o and may help o o e come esis ance o BRAFV 600E inhibi o s. Cu en goals in ol e he iden i ica ion o mo e lineage-speci ic supp esso s in zeb a ish emb yos ha can be gene alized o o he cell ypes, wi h less side e ec s and oxici y and di ec ele ance o human cance . The inhibi ion o DHODH by le abla es zeb a ish neu al c es de elopmen and supp esses melanoma g ow h by blocking he ansc ip ional elonga ion o key genes, such as c es in and mi a [ 92 ]. HEXIM1 (hexame hylene bisace amide inducible 1) wo ks as a gene al RNA polyme ase II ansc ip ion inhibi o , and i s exp ession egula es gene exp ession du ing di e en ia ion. HEXIM1 supp esses melanoma umo s in esponse o nucleo ide s ess, and i s o e exp ession in a zeb a ish model supp esses melanoma o ma ion [ 94 ]. HEXIM1 knockdown by mo pholinos pa ially escued c es in and mi a exp ession in le ea ed zeb a ish emb yos [94]. This mechanism in ol es con ol o he elonga ion phase o RNA ansc ip ion and egula es gene exp ession du ing di e en ia ion [ 93 ]. The unc ion o HEXIM1 in melanoma was in es iga ed a e i s o e exp ession and dele ion in a zeb a ish model, e ealing i s ole as a umo supp esso gene. O he cell ypes may be suscep ible o di e en cellula s esses and espond in a simila manne by ac i a ing HEXIM1 o help he cell epai o induce cell dea h. Finding such dependencies in o he cance s could lead o he de elopmen o new cance he apies using he zeb a ish model (Figu e 3) [62,95]. In . J. Mol. Sci. 2017, 18, 1550 8 o 15 educes he exp ession o melanin syn hesis- ela ed p o eins and exhibi s a good sa e y p o ile. This compound induces a ema kable supp ession o melanogenesis in zeb a ish a e 48 h. In addi ion, i s abili y o ep ess in i o pigmen a ion could ha e signi ican applica ions in he cosme ic ield [91]. Fu he mo e, his whi ening subs ance is sa e and e ec i e, and i could be used o p e en hype -pigmen a ion [91]. Zeb a ish emb yos ca ying BRAFV600E exp ession ha e been used o he s udy o he neu al c es -de i ed cell lineage [92]. In pa icula , ansgenic mi -BRAFV600E; p53/zeb a ish emb yos ha e been c ea ed o he e alua ion o ea ly ansc ip ional ac i i y wi hin melanoma pa hogenesis and o p o ide a model o chemical gene ic sc eening in he con ex o melanoma he apy. Inhibi o s o dihyd oo o a e dehyd ogenase (DHODH), such as le lunomide (le ), a e able o educe he abili y o sel - enewal o neu al c es cells and a ge he ac i i y o hose genes equi ed o neu al c es de elopmen and melanoma umo g ow h [93], including mi and myc a ge s [92]. Resea ch da a sugges ha le would be mo e e ec i e in combina ion wi h a BRAFV600E inhibi o and may help o o e come esis ance o BRAFV600E inhibi o s. Cu en goals in ol e he iden i ica ion o mo e lineage-speci ic supp esso s in zeb a ish emb yos ha can be gene alized o o he cell ypes, wi h less side e ec s and oxici y and di ec ele ance o human cance . The inhibi ion o DHODH by le abla es zeb a ish neu al c es de elopmen and supp esses melanoma g ow h by blocking he ansc ip ional elonga ion o key genes, such as c es in and mi a [92]. HEXIM1 (hexame hylene bisace amide inducible 1) wo ks as a gene al RNA polyme ase II ansc ip ion inhibi o , and i s exp ession egula es gene exp ession du ing di e en ia ion. HEXIM1 supp esses melanoma umo s in esponse o nucleo ide s ess, and i s o e exp ession in a zeb a ish model supp esses melanoma o ma ion [94]. HEXIM1 knockdown by mo pholinos pa ially escued c es in and mi a exp ession in le ea ed zeb a ish emb yos [94]. This mechanism in ol es con ol o he elonga ion phase o RNA ansc ip ion and egula es gene exp ession du ing di e en ia ion [93]. The unc ion o HEXIM1 in melanoma was in es iga ed a e i s o e exp ession and dele ion in a zeb a ish model, e ealing i s ole as a umo supp esso gene. O he cell ypes may be suscep ible o di e en cellula s esses and espond in a simila manne by ac i a ing HEXIM1 o help he cell epai o induce cell dea h. Finding such dependencies in o he cance s could lead o he de elopmen o new cance he apies using he zeb a ish model (Figu e 3) [62,95]. Figu e 3. The umo supp esso hexame hylene bisace amide inducible 1 (HEXIM1) gene inhibi s melanoma in he zeb a ish model. HEXIM1 plays an impo an ole as a melanoma umo supp esso in esponse o nucleo ide s ess. HEXIM1 o ms a complex wi h posi i e ansc ip ion elonga ion ac o (P-TEFb) in o de o inhibi he kinase o ini ia e ansc ip ion elonga ion a umo igenic genes. Al e a ion o gene exp ession, in pa allel wi h an i- umo igenic RNAs binding o HEXIM1, a o s he “an i-cance ” gene exp ession. Pol II: DNA polyme ase II; CDK9: cyclin-dependen kinase; CCNT1: Cyclin-T. Figu e 3. The umo supp esso hexame hylene bisace amide inducible 1 (HEXIM1) gene inhibi s melanoma in he zeb a ish model. HEXIM1 plays an impo an ole as a melanoma umo supp esso in esponse o nucleo ide s ess. HEXIM1 o ms a complex wi h posi i e ansc ip ion elonga ion ac o (P-TEFb) in o de o inhibi he kinase o ini ia e ansc ip ion elonga ion a umo igenic genes. Al e a ion o gene exp ession, in pa allel wi h an i- umo igenic RNAs binding o HEXIM1, a o s he “an i-cance ” gene exp ession. Pol II: DNA polyme ase II; CDK9: cyclin-dependen kinase; CCNT1: Cyclin-T. In . J. Mol. Sci. 2017,18, 1550 9 o 15 A ansgenic zeb a ish melanoma model was de eloped by coupling he zeb a ish mi a p omo e wi h he human oncogenic HRASG12V [ 96 ]. These ansgenic emb yos de eloped melanocy e hype plasia wi h he induc ion o RAS-RAF-MEK-ERK and RAS-PI3K-AKT-mTOR signaling pa hways. In his con ex , he zeb a ish model was use ul o he sc eening o compounds di ec ed agains mi ogen-ac i a ed p o ein kinases, ex acellula signal- egula ed kinases (MEK/ERK) and PI3K/mTORi pa hways, alone o in combina ion. Rapamycin, a well-known mTOR inhibi o , is conside ed as one o he s onges d ugs in he ield [ 97 ]. I was sugges ed ha apamycin analogs migh also imp o e access o he kinase ac i e si e o ATP compe i i e inhibi o s. The abili y o apamycin analogs o co-ope a e wi h MEKi o supp ess melanoma cell g ow h was p e iously obse ed. Because o he e icacy o cock ails o inhibi o s obse ed wi h HTS assays in i o , clinical ials using apamycin analogs combined wi h MEKi o PIK3K/mTORi a e cu en ly unde way [ 98 ] (Figu e 4). In . J. Mol. Sci. 2017, 18, 1550 9 o 15 A ansgenic zeb a ish melanoma model was de eloped by coupling he zeb a ish mi a p omo e wi h he human oncogenic HRASG12V [96]. These ansgenic emb yos de eloped melanocy e hype plasia wi h he induc ion o RAS-RAF-MEK-ERK and RAS-PI3K-AKT-mTOR signaling pa hways. In his con ex , he zeb a ish model was use ul o he sc eening o compounds di ec ed agains mi ogen-ac i a ed p o ein kinases, ex acellula signal- egula ed kinases (MEK/ERK) and PI3K/mTORi pa hways, alone o in combina ion. Rapamycin, a well-known mTOR inhibi o , is conside ed as one o he s onges d ugs in he ield [97]. I was sugges ed ha apamycin analogs migh also imp o e access o he kinase ac i e si e o ATP compe i i e inhibi o s. The abili y o apamycin analogs o co-ope a e wi h MEKi o supp ess melanoma cell g ow h was p e iously obse ed. Because o he e icacy o cock ails o inhibi o s obse ed wi h HTS assays in i o, clinical ials using apamycin analogs combined wi h MEKi o PIK3K/mTORi a e cu en ly unde way [98] (Figu e 4). Figu e 4. D ug de elopmen and inhibi o sc eening using selec ed MEKi and PI3K/mTOR inhibi o s. Zeb a ish plays an impo an ole o he sc eening o compounds a ge ing MEK/ERK and PI3K/mTOR pa hways, bo h alone and in combina ion. (A) Example o sc eening o he FDA lib a y molecules using zeb a ish emb yos; (B) s eps showing hi selec ion a e he sc eening p ocedu e wi h di e en d ugs and d ug dose esponse using he melanin assay. A he end o his p ocess, 11 hi s we e de ec ed o be u he e alua ed in cell cul u e. MEKi: mi ogen-ac i a ed p o ein kinases inhibi o ; PI3K: phosphoinosi ide 3-kinase; mTOR: mechanis ic a ge o apamycin. 5. Conclusions and Fu u e Di ec ions As zeb a ish esea ch de elops u he , new knowledge and ools a e becoming a ailable ha will signi ican ly impac he ange and quali y o in i o chemical sc eening. Recen and u u e ad ances in zeb a ish esea ch will ce ainly ha e an impac ha will include imp o emen s in da a acquisi ion (pa icula ly imaging), inc eased au oma ion capabili y, ad ances in gene ic manipula ion and he de elopmen o new pheno ypic endpoin s. A new e a o combined he apy o me as a ic melanoma is now on he way. To u he add ess he de elopmen o esis ance, clinical ials in ol ing iple combina ion he apies a e ongoing. In his espec , he combina ion o BRAF and MEK inhibi o s wi h an i-E bB3 an ibodies, a leas based on p eclinical da a, p edic s a u u e clinical use. Replacing he a ge -based d ug disco e y wi h he newly-in oduced pheno ype-guided disco e y has opened an impo an ga e o chemical and gene ics sc eening. HTS in combina ion wi h zeb a ish as an in i o disease model ep esen s a aluable ool o oxici y s udies and small molecules sc eening. A he same ime, a la ge numbe o molecules and compounds can be sc eened wi h he use o zeb a ish in o de o iden i y e ec i e d ugs and obse e disease pheno ypes. The incidence o skin cance is showing a apid inc ease wo ldwide o se e al easons, om unp o ec ed exposu e o he Sun o he aging o he popula ion [68]. SCC and melanoma a e malignan condi ions o he skin o which he need o d ug de elopmen is c ucial, because o he numbe o pa ien s wi h me as ases and he poo ou come o ad anced s ages. In his con ex , zeb a ish models gi e he oppo uni y o bo h explo e new pa hways and es new compounds o small molecules ha could be used in clinical ials. Simul aneously, he e ec on emb yonic Figu e 4. D ug de elopmen and inhibi o sc eening using selec ed MEKi and PI3K/mTOR inhibi o s. Zeb a ish plays an impo an ole o he sc eening o compounds a ge ing MEK/ERK and PI3K/mTOR pa hways, bo h alone and in combina ion. ( A ) Example o sc eening o he FDA lib a y molecules using zeb a ish emb yos; ( B ) s eps showing hi selec ion a e he sc eening p ocedu e wi h di e en d ugs and d ug dose esponse using he melanin assay. A he end o his p ocess, 11 hi s we e de ec ed o be u he e alua ed in cell cul u e. MEKi: mi ogen-ac i a ed p o ein kinases inhibi o ; PI3K: phosphoinosi ide 3-kinase; mTOR: mechanis ic a ge o apamycin. 5. Conclusions and Fu u e Di ec ions As zeb a ish esea ch de elops u he , new knowledge and ools a e becoming a ailable ha will signi ican ly impac he ange and quali y o in i o chemical sc eening. Recen and u u e ad ances in zeb a ish esea ch will ce ainly ha e an impac ha will include imp o emen s in da a acquisi ion (pa icula ly imaging), inc eased au oma ion capabili y, ad ances in gene ic manipula ion and he de elopmen o new pheno ypic endpoin s. A new e a o combined he apy o me as a ic melanoma is now on he way. To u he add ess he de elopmen o esis ance, clinical ials in ol ing iple combina ion he apies a e ongoing. In his espec , he combina ion o BRAF and MEK inhibi o s wi h an i-E bB3 an ibodies, a leas based on p eclinical da a, p edic s a u u e clinical use. Replacing he a ge -based d ug disco e y wi h he newly-in oduced pheno ype-guided disco e y has opened an impo an ga e o chemical and gene ics sc eening. HTS in combina ion wi h zeb a ish as an in i o disease model ep esen s a aluable ool o oxici y s udies and small molecules sc eening. A he same ime, a la ge numbe o molecules and compounds can be sc eened wi h he use o zeb a ish in o de o iden i y e ec i e d ugs and obse e disease pheno ypes. The incidence o skin cance is showing a apid inc ease wo ldwide o se e al easons, om unp o ec ed exposu e o he Sun o he aging o he popula ion [ 68 ]. SCC and melanoma a e malignan condi ions o he skin o which he need o d ug de elopmen is c ucial, because o he numbe o pa ien s wi h me as ases and he poo ou come o ad anced s ages. In his con ex , zeb a ish models gi e he oppo uni y o bo h explo e new pa hways and es new compounds o small