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