ANA INÊS LOURENÇO DE ALMEIDA
AN APPROACH TO MOLECULAR GENETICS OF THYROID
CANCER: FROM NOVEL MUTATIONS TO A ZEBRAFISH MODEL
Tese de candida u a ao g au de
Dou o em Pa ologia e Gené ica Molecula
subme ida ao Ins i u o de Ciências Biomédicas Abel Salaza
da Uni e sidade do Po o
O ien ado – Dou o a Ana Paula Soa es Dias
Fe ei a
Ca ego ia – P o esso a Auxilia , Faculdade de
Medicina da Uni e sidade do Po o e
Coo denado a do G upo Cance Signaling &
Me abolism, IPATIMUP/Ins i u o de
In es igação e Ino ação em Saúde
A iliação – Faculdade de Medicina da
Uni e sidade do Po o e IPATIMUP/Ins i u o
de In es igação e Ino ação em Saúde
Coo ien ado – Miguel Godinho Fe ei a
Ca ego ia – Coo denado do G upo Telome e
and Genome S abili y, Ins i u o Gulbenkian
de Ciência
A iliação – Ins i u o Gulbenkian de Ciência
Financial Suppo
The candida e was suppo ed by a PhD ellowship (SFRH/BD/79135/2011) om
Fundação pa a a Ciência e Tecnologia (FCT).
The e a e many people in my li e
And hen he e’s you,
To my Dad, o my Mum
Acknowledgmen s
Thank you o e e y one o you ha came along on my i e-yea jou ney.
Belie e me, I ha en’ o go en each one o you. Yes, you!
Thank you Paula.
Thank you Miguel.
Thank you P o esso Sob inho Simões.
A BIG THANK YOU o my Dad and my Mum.

P e ácio
Eu ac edi o que cada pessoa em a sua p óp ia iloso ia. Na minha iloso ia há uma
g ande disposição pa a abso e a expe iência da ida e com ela ap ecia o mundo.
G ande pa e da minha sa is ação pessoal p o ém das minhas opções p o issionais.
Há cinco anos a ás lancei um desa io. Candida ei-me a um p og ama dou o al. E
acei o meu caminho. Ao longo dele encon ei aquezas, encon ei espe anças. E
ui gua dando udo o que i, udo o que iz. A minha ese e lec e o meu pe cu so
des es cinco anos.
Comecei o meu p ojec o de dou o amen o no g upo Cance Biology no IPATIMUP,
o qual epo ou uma ele ada p e alência de mu ações BRAF em ca cinomas
papila es da i óide espo ádicos e linhas celula es de i adas des es ca cinomas. A
p ocu a de ac o es adicionais que explicassem a umo igénese do canc o da
i óide po encialmen e elacionadas com as mu ações do BRAF e a descobe a de
mu ações no p omo o da elome ase pe mi i am ao g upo explo a es as úl imas
mu ações nas á ias sé ies exis en es no banco de umo es. En e an o, enquan o
ou os elemen os do g upo p ocu a am pe cebe mecanis icamen e o e ei o das
mu ações no p omo o da elome ase, eu oquei-me no desen ol imen o de um
modelo animal que pe mi isse inicialmen e comp eende o e ei o de al e ações
equen emen e encon adas no canc o da i óide (mu ações nos genes BRAF e p53)
e u u amen e es uda ac o es adicionais que ago a se sabem e em um papel
ele an e na umo igénese ais como as mu ações no p omo o da elome ase. Po
es a al u a oi es abelecida uma colabo ação com o g upo Telome es and Genome
S abili y no IGC passando de uma ciência anslacional pa a uma ciência básica. O
desen ol imen o de um modelo pa a o es udo do e ei o das mu ações nos genes
BRAF e p53 em peixe-zeb a oi o u o dessa colabo ação. Deixa ei um legado de
e amen as que mui os pode ão usu ui .
Publica ions
Ao ab igo do dispos o do nº 2, alínea a) do a igo 31º do Dec e o-Lei n.º 115/2013
de 7 de Agos o azem pa e in eg an e des a ese de dou o amen o os seguin es
abalhos já publicados ou subme idos pa a publicação:
A igo I - Vinag e J, Almeida A, Pópulo H, Ba is a R, Ly a J, Pin o V, Coelho R,
Celes ino R, P aze es H, Lima L, Melo M, da Rocha AG, P e o A, Cas o P, Cas o L,
Pa dal F, Lopes JM, San os LL, Reis RM, Cameselle-Teijei o J, Sob inho-Simões M,
Lima J, Máximo V, Soa es P. 2013. F equency o TERT p omo e mu a ions in human
cance s. Na Commun. 4:2185.
A igo II – Almeida A, Sob inho-Simões M, Fe ei a MG, Soa es P. (Subme ido).
O seguin e capí ulo de li o não az pa e do co po p incipal de esul ados des a
ese, mas é pa e in eg an e da mesma, endo sido u ilizado na sua In odução e
Discussão.
Appendix I - Almeida AL, Boa en u a P, Soa es P, Clinical Managemen o Thy oid
Cance : E iopa hogenic ac o s o hy oid cance , Pages 46-62, Fu u e Medicine.
2013
i
Table o con en s
Abb e ia ions
i
Abs ac
ix
Resumo
xi
Chap e I
–
In oduc ion
1
I.1 Thy oid gland
2
I.1.1 Thy oid physiology
2
I.1.2 Thy oid
diso de s
3
I.1.2.1 Goi e
4
I.1.2.2 Neoplasias
5
Papilla y hy oid cance
7
Molecula gene ics o papilla y hy oid cance
9
Signaling pa hways al e ed in papilla y hy oid ca cinomas
19
I.2 Zeb a ish as a model sys em
23
I.2.1 Zeb a ish in
Cance Resea ch
23
I.2.2 S udies on zeb a ish hy oid physiology and unc ion
25
I.3 Aims
28
Chap e II
–
Ma e ial & Me hods
30
II.1 Plasmid cloning
31
II.1.1 p5E- g p omo e plasmid
31
II.1.2 pME
-
mChe y and p3E
-
polyA plasmids
31
II.1.3 pME
-
mChe y
-
T2A
-
BRAF
WT
and pME
-
mChe y
-
T2A
-
BRAF
V600E
plasmids
32
II.1.4 pTol2A2- g:mChe y–pA and pTol2CG2- g:mChe y–pA
plasmids
32
II.1.5 pTol2CG2- g:mChe y-T2A- BRAF
V600E
–pA plasmids
33
II.1.6 pTol2CG2- g:loxP-CFP-loxP-mChe y–pA and pTol2CG2-
g:loxP-CFP-loxP-mChe y-T2A-BRAF
V600E
–pA plasmids
34
II.1.7 pCS2-CMV:mChe y-pA plasmid
36
ii
II.1.8 pCS2-CMV:mChe y-T2A-BRAF
WT
-pA and pCS2-
CMV:mChe y-T2A-BRAF
V600E
-pA plasmids
36
II.1.9 Summa y o he plasmids gene a ed
36
II.2 Cloning
-
auxilia y echniques
38
II
.2.1 PCR
38
II.2.2 DNA sequencing
38
II.2.3 DNA quan i ica ion
38
II.2.4 Res ic ion diges ion and
liga ion
39
II.2.5 Isola ion o DNA by aga ose gel elec opho esis
39
II.2.6 Plasmid ans o ma ion in compe en E. Coli
39
II.2.7 Plasmid g ow h in solid and liquid cul u es
40
II.2.8 Plasmid pu i ica ion
40
II.3 Capped mRNA syn hesis
40
II.3.1 Capped ansposase mRNA syn hesis
40
II.3.2 Capped mChe y mRNA, mChe y
-
T2A
-
BRAF
WT
mRNA and
mChe y-T2A-BRAF
V600E
mRNA syn hesis
41
II.4 Mic oinjec ions
41
II.4.1 DNA plasmid mic oinjec ions
41
II.4.2 Capped mRNA mic oinjec ions
42
II.5 T ansgenesis
42
II.5.1 WT lines
43
II.5.2 p53
M214K
lines
44
II.6 Fish s ains and husband y
45
II.7 Sc eening and Imaging
46
II.8 Fin clip and gDNA ex ac ion
46
II.9 Geno yping
46
II.10
His opa hology
47
II.11 Measu emen o s anda d leng h in la ae
47
II.12 Measu emen o body mass index (BMI)
48
II.13 Measu emen o hy oid olume in adul ish
48
II.14 P epa a ion o emb yo lysa es
49
II.15 Dissec ion o hy oid issue in adul
zeb a ish
49
II.16 P epa a ion o issue lysa es
49
II.17 Immunoblo ing
50
II.18 Hea shock and d ug ea men
51
II.19 S a is ical analysis
51
ix
Abs ac
Thy oid diseases a e ex emely equen and a e mos o en o benign na u e.
Thy oid cance is he mos common endoc ine malignancy in humans and he
majo i y o umo s ha bo gene ic al e a ions such as he BRAF mu a ion. Mos o
his mu a ion is an ac i a ing mu a ion in he kinase domain o he BRAF. In
spo adic papilla y hy oid ca cinomas (PTCs), BRAF gene mu a ions a e ound in
29%-83% o all cases and almos ne e co-exis wi h RAS mu a ions o RET
(RET/PTC) and NTRK1 ea angemen s. As a esul o BRAF mu a ion, he MAPK
pa hway is ac i a ed and cellula p ocesses such as p oli e a ion, su i al, mo ili y
and in asion a e p omo ed. Recen ly, mu a ions in he elome ase e e se
ansc ip ase (TERT) p omo e ha e been desc ibed in hy oid cance and
conside ed one o he possible mechanisms ha unde lies TERT eexp ession in
se e al ypes o human umo s including hose o he hy oid.
In he i s pa o he hesis, I desc ibe he s udy ega ding TERT p omo e
mu a ions in which I was deeply in ol ed. Ou s udy highligh ed he p esence o
ecu en soma ic mu a ions in he TERT p omo e in cance s o he cen al ne ous
sys em (43%), bladde (59%), hy oid ( ollicula cell-de i ed, 10%) and skin
(melanoma, 29%). Conce ning hy oid cance , he p esence o TERT p omo e
mu a ions was ound o be signi ican ly associa ed wi h highe TERT mRNA
exp ession and wi h olde age o he pa ien s. We concluded ha TERT p omo e
mu a ions a e ela i ely equen in se e al speci ic ypes o human cance and ha
such mu a ions may enhance exp ession o elome ase.
O e he yea s, hy oid cance has been s udied using mice models. These models
ha e p o ided e idence showing ha hy oid-speci ic exp ession o BRAF
V600E
induced goi e as well as in asi e PTCs which p og ess o poo ly di e en ia ed
ca cinoma closely ecapi ula ing some human hy oid umo pheno ypes. Ve y
success ul, mice models o hy oid cance a e being used o explo e molecula
mechanisms in ol ed in hy oid umo igenesis. Mice models can also be used o
moni o umo s and o pe o m d ug sc eening in he se ing o hy oid cance bu
such asks emain ime-consuming.
In he second pa o he hesis, I de eloped a hy oid- a ge ed BRAF
V600E
-
exp essing ansgenic zeb a ish and e alua ed he hy oid issue pheno ypes

x
du ing all s ages o de elopmen up o 12 mon hs o age. I obse ed ha hy oid-
speci ic exp ession o BRAF
V600E
induced abno mal hy oid mo phogenesis ea ly in
li e ha de eloped la e on in o hype plasia by ~2-3 mon hs o age and colloid
goi e by 12 mon hs o age. BRAF
V600E
–exp essing cells disclosed up egula ion o
p oli e a ion, concomi an wi h MAPK pa hway ac i a ion, and he e was p omo ion
o apop osis. High le els o p53 sugges ed ha his p o ein may be es aining
p og ession o malignancy. Loss o WT p53 using a p53
M214K
zeb a ish p e en ed
impai men o hy oid mo phogenesis induced by BRAF
V600E
and su p isingly no
e idence o hy oid hype plasia, goi e and/o neoplasia was de ec ed in hose
animals up o 12 mon hs o age. p53
M214K
BRAF
V600E
–exp essing cells we e low
p oli e a i e, consis en wi h down egula ion o he MAPK pa hway; supp ession o
apop osis was also obse ed.
In conclusion, my wo k showed ha TERT p omo e mu a ions a e ela i ely
equen in speci ic ypes o human cance , including hose o he hy oid, and ha
may enhance elome ase exp ession. Also, hy oid-speci ic exp ession o BRAF
V600E
induces hype plasia and colloid goi e in zeb a ish and oge he wi h he absence
o WT p53, BRAF
V600E
was no able o de elop hy oid cance . This da a p o ides
e idence ha BRAF ac i a ion is su icien o hy oid cell ans o ma ion and ha
BRAF and p53 pa hways mus in e ac gene ically in zeb a ish hy oid.
xi
Resumo
As doenças na i óide são ex emamen e equen es e são ge almen e de na u eza
benigna. O canc o da i óide é o umo endóc ino maligno mais comum em
humanos e a maio ia des es umo es possui al e ações gené icas ais como a
mu ação no gene BRAF. A maio ia das mu ações do BRAF ac i am o domínio de
cinase da p o eína BRAF. Em ca cinomas papila es da i óide (CPT) espo ádicos, as
mu ações no gene BRAF são encon adas em 29% a 83% do o al de casos e quase
nunca co-exis em com as mu ações do RAS e ea anjos do RET (RET/PTC) e
NTRK1. Como esul ado das mu ações do BRAF, a ia de sinalização das MAP
cinases é ac i ada e p ocessos celula es, ais como p oli e ação, sob e i ência,
mo ilidade e in asão, são p omo idos. Recen emen e, o am desc i as mu ações
no p omo o da elome ase ansc ip ase e e sa (TERT) e es as são conside adas
um dos possí el mecanismos de eexp essão da elome ase em á ios ipos de
canc o humano incluindo os da i óide.
Na p imei a pa e da ese eu desc e o o es udo ela i o às mu ações no p omo o
da TERT no qual eu es i e en ol ida. O nosso es udo ealçou a p esença de
mu ações somá icas eco en es no p omo o da TERT em umo es como os do
sis ema ne oso cen al (43%), da bexiga (59%), da i óide com o igem nas células
olicula es (10%) e da pele (melanoma) (29%). Rela i amen e ao canc o da i óide,
oi encon ada uma associação signi ica i a en e a p esença de mu ações no
p omo o da TERT e ní eis ele ados de exp essão de mRNA e ambém uma
associação com pacien es mais elhos. Concluímos que as mu ações no p omo o
da TERT são ela i amen e equen es em de e minados ipos de canc o humanos
e que es as mu ações podem aumen a a exp essão da TERT.
Ao longo dos anos, o canc o da i óide em sido es udado usando modelos de
a inho. Es es modelos de am e idências que demons am que a exp essão
especí ica de BRAF
V600E
induziu bócio bem como CPT in asi os que p og edi am
pa a ca cinomas pouco di e enciados ecapi ulando alguns dos enó ipos dos
umo es de i óide humanos. Os modelos de a inho ainda são usados pa a
explo a mecanismos molecula es en ol idos na umo igénese da i óide. Es es
modelos podem ambém se usados pa a moni o iza umo es e pa a ealiza
ensaios de d ogas no con ex o do canc o da i óide mas es as a e as são mo osas.
xii
Na segunda pa e do ese eu desen ol i uma linha ansgénica com exp essão
especí ica de BRAF
V600E
na i óide de peixe-zeb a e a aliei os enó ipos na i óide
du an e odos os es ádios de desen ol imen o e a é aos doze meses de idade.
Obse ei que a exp essão especí ica de BRAF
V600E
na i óide induziu uma
mo ogénese ano mal des e ecido em es ádios iniciais que se desen ol em em
hipe plasia aos 2-3 meses de idade e bócio colóide ao im de 12 meses. Células
da i óide que exp essa am BRAF
V600E
inham uma sob e egulação da p oli e ação,
concomi an e com a ac i ação da ia de sinalização das MAP cinases, e oi
obse ada indução da apop ose. Ní el ele ados de p53 suge em que es a p o eína
pode e con ido a p og essão pa a malignidade. Pe da da p o eína sel agem de
p53, usando uma linha homozigo a pa a a mu ação M214K do p53, p e eniu
mo ogénese ano mal da i óide induzida pelo BRAF
V600E
e su p eenden emen e não
o am encon adas e idências de hipe plasia, bócio e/ou ca cinomas em peixes a é
doze meses de idade. Células da i óide que exp essa am BRAF
V600E
e am pouco
p oli e a i as, consis en e com des egulação da ia de sinalização das MAP cinases,
e oi obse ada sup essão da apop ose.
Em conclusão, o meu abalho demons ou que as mu ações no p omo o da TERT
são ela i amen e equen es em de e minados ipos de canc o humanos, incluindo
os da i óide, e podem aumen a a exp essão da TERT. A exp essão especí ica de
BRAF
V600E
induziu hipe plasia e bócio colóide em peixe-zeb a e que em conjun o
com a ausência da p o eína p53, o BRAF
V600E
não oi capaz de induzi canc o na
i óide. Es as obse ações demons am e idências de que a ac i ação do BRAF é
su icien e pa a a ans o mação de células da i óide e que as ias de sinalização
da qual azem pa e o BRAF e o p53 de em in e agi gene icamen e na i óide do
peixe-zeb a.
xiii
1
Chap e I
In oduc ion

2
THE HUMAN ENDOCRINE SYSTEM comp ises pa o he body’s communica ion
sys em connec ing he b ain o he o gans which in u n con ol he me abolism,
g ow h and ep oduc ion. Tigh con ol o he sys em is possible h ough complex
eedback mechanisms ha main ains homeos asis. Any dis up ion o an endoc ine
gland o o he eedback mechanisms may esul in endoc ine dis u bance.
Ul ima ely, cance may ei he con ibu e o be he ou come o such dis u bance.
I.1 Thy oid gland
The human hy oid gland is a bu e ly-shaped gland loca ed on he achea and
comp ises wo lobes connec ed by an is hmus (VanPu e e al., 2010). The gland is
highly ascula ized and is one o he la ges endoc ine glands in he human body
(VanPu e e al., 2010). The hy oid gland comp ises nume ous and a ying sized
ollicles consis ing o a hin-laye o cuboidal epi helial cells and a cen al lumen.
The lumen is illed wi h a homogeneous p o ein- ich colloid named hy oglobulin
which is essen ial o hy oid ho mone (TH) syn hesis (Manson e al., 1973; VanPu e
e al., 2010). Two ho mones a e p oduced in he hy oid gland by he ollicula cells
in esponse o hy oid-s imula ing ho mone (TSH) eleased om he pi ui a y: i-
iodo hy onine (T3) and hy oxine (T4). These ho mones ha e an e ec on all body
sys ems a all s ages o li e egula ing he basal me abolic a e and issue g ow h
and ma u a ion (Manson e al., 1973; Kuma e al., 2005; VanPu e e al., 2010).
Pa a ollicula cells sec e ing calci onin a e also ound in clus e s su ounding he
ollicles and in he connec i e issue (VanPu e e al., 2010).
I.1.1 Thy oid physiology
Thy oid ollicles a e he ac o y and he s o age o THs. The p esence o TSH is
indispensable o he syn hesis and sec e ion o THs as well as an adequa e iodide
nu i ion.
The i s s ep in he syn hesis o THs is he up ake o iodide by sodium-iodine
sympo e (NIS) which is con e ed o iodine and hen condensed on o y osine
esidues om he backbone o hy oglobulin, a p o ein p oduced inside he hy oid
cells. The newly o med iodo hy oglobulin can be ei he mono-iodina ed o di-
iodina ed. When coupled, wo di-iodo y osine molecules esul in he o ma ion o
3
T4 whe eas a di-iodo y osine coupled wi h a mono-iodo y osine esul s in T3.
Al hough he T3 is mo e biologically ac i e han he T4, he p oduc ion o T3 occu s
p e e en ially ou side he hy oid gland by pe iphe ical con e sion om T4. THs
a e s o ed inside he hy oid ollicles composing he majo i y o he colloid
ma e ial. T3 and T4 a e eleased by p o eolysis om he hy oid o he bloods eam
whe e hey bind o TH binding p o eins namely he hy oxin binding globulin (TBG)
(Figu e 1) (Kuma e al., 2005; B ix e al., 2011).
Figu e 1.
TH syn hesis. TSH signaling ia he TSH ecep o con ols TH syn hesis. NIS a
he basola e al memb ane akes up iodide om he blood. Iodide is o gani ied in he y osyl
esidues o g in a eac ion ca alyzed by hy oid pe oxidase (TPO). T
3
, and T
4
a e s o ed in
colloid un il hey a e eleased in o he blood.
I.1.2 Thy oid diso de s
Thy oid diso de s can ange om an enla ged hy oid gland ha does no need
ea men o hy oid cance . The mos common hy oid p oblems include goi e
and benign hy oid nodules. Also ela i ely equen , and clinically mo e e iden , is
he abno mal p oduc ion o TH ha can be classi ied in o wo g oups:
hype hy oidism and hypo hy oidism (Manson e al., 1973; VanPu e e al., 2010).
4
In hype hy oidism, he hy oid gland is o e ac i e p oducing high le els o hy oid
ho mones and speeding up he me abolism. G a es’ disease also known as oxic
di use goi e is he mos common cause o hype hy oidism (Manson e al., 1973;
VanPu e e al., 2010). In hypo hy oidism, he hy oid gland is unde ac i e
p oducing inadequa e le els o hy oid ho mones and slowing down he
me abolism. Hashimo o’s hy oidi is, congeni al hypo hy oidism and i adia ion
a e some o he causes o hypo hy oidism (Manson e al., 1973; VanPu e e al.,
2010).
The p ope ea men o hype hy oidism and hypo hy oidism depends on he
symp oms o he disease and he ae iology. In hype hy oidism, ea men s include
hiou acils o hioamides, adioiodine he apy, hy oidec omy, adioac i e iodine
and/o be a blocke s. In hypo hy oidism, le o hy oxine is a ho mone eplacemen
used o ea men (VanPu e e al., 2010).
I.1.2.1 Goi e
A goi e is an enla ged hy oid gland, i can be ei he di use o nodula and i may
ex end in o he e os e nal space wi h o wi hou subs an ial an e io enla gemen
(Kuma e al., 2005; Lam e al., 2014).
A de iciency in iodine in ake o in TH syn hesis leads o an inc eased TSH
p oduc ion which in u n sus ains inc eased cellula i y and hype plasia o he
hy oid gland as an a emp o no malize he le els o he TH. TH de iciency can be
due o de ec s on ho mone syn hesis, iodine de iciency and goi ogens (Figu e 2).
Also, a goi e may appea as a esul o he s imula ion o he hy oid gland by
hy oid s imula ing ho mone ecep o (TshR) agonis s such as TSH ecep o
an ibodies, pi ui a y esis ance o hy oid ho mone, adenomas o he
hypo halamus o pi ui a y gland and human cho ionic gonado opin-p oducing
umou s (Kuma e al., 2005; Lam e al., 2014).
5
Figu e 2.
Rela ion be ween hypo halamus-pi ui a y- hy oid (HPT) axis and human
goi e de elopmen . Iodine de iciency and/o goi ogens dis up he TH syn hesis in many
ways. Upon insu icien TH le els, he nega i e eedback inhibi ion is los esul ing in
inc eased sec e ion o opic ho mones (TRH and TSH). High TSH le els s imula e hy oid
cells p omo ing goi e . Also, TSH ecep o agonis s can ilici ly mimick he biological ac i i y
o TSH.
Small benign eu hy oid goi e s do no equi e any ea men , howe e hei size
may be educed wi h le o hy oxine supp essi e he apy. La ge and complica ed
goi e s usually equi e su gical and/o adia ion ea men ollowed by TH
eplacemen (Kuma e al., 2005; Lam e al., 2014).
I.1.2.2 Neoplasias
Thy oid cance is he 16
h
mos common cance wo ldwide and accoun s o
app oxima ely 2% o o al o human malignancies wi h a ound 298.000 new cases
diagnosed in 2012 wo ldwide (la es da a epo ed) (Niki o o , 2012; Fe lay e al.,
2013). Incidence a es o hy oid cance a e highes in No he n Ame ica and o he
de eloped coun ies and lowes in wes e n A ica bu his is pa ly due o da a
quali y and e lec s di e en p e alence o isk ac o s and sc eening and
12
mice de eloped PTC ha closely ecapi ula ed he pheno ype in humans. S ikingly,
ea men o hese mice wi h a MEK inhibi o educed hy oid size, es o ed he
p oduc ion o THs and inhibi ed umo igenesis (Cha les e al., 2011).
Mo e e idence o BRAF
V600E
in ol emen in mice hy oid umo igenesis was obse ed
when BRAF
V600E
exp ession was induced in ollicula hy oid cells in a doxycycline-
inducible manne . As ea ly as one week a e doxycycline ea men , de elopmen
o highly pene an and high-g ade PTCs wi h poo ly di e en ia ed ea u es and a
e e sible ac i a ion o he MAPK pa hway we e obse ed. Upon doxycycline
wi hd awal, ollicula a chi ec u e was ees ablished bu a second induc ion no
only esul ed in hypo hy oidism bu also educed hy oid-speci ic genes
exp ession (Chak a a y e al., 2011).
I is easonable o assume ha BRAF
V600E
is an ea ly e en in hy oid umo igenesis
due o a high p e alence o BRAF
V600E
in papilla y mic oca cinomas and de elopmen
o umo s wi h his ological ea u es o human PTCs induced by exp ession o
BRAF
V600E
in ansgenic mice in he absence o any o he gene ic al e a ions (Pa k e
al., 2010).
In iguingly, i has been showed ha some human PTCs ha e in a umo
he e ogenei y o he BRAF geno ype as he e a e wo dis inc cell popula ions ei he
wi h he wild- ype (WT) o he BRAF
V600E
. This may sugges ha he clonal occu ence
o BRAF mu a ion is a a e e en , occu ing only in a subpopula ion o cells, and
BRAF mu a ions a e a he a la e subclonal e en in PTCs (Gue a e al., 2012).
Also, genome-wide allelo yping and BRAF mu a ion analysis o oci in mul i ocal
human PTCs showed ha BRAF
V600E
mu a ion is an ea ly e en du ing clonal
e olu ion in mos bu no all cases. In ac , BRAF
V600E
is no always p esen in all
umo oci which sugges s ha o he gene ic ac o s in he p ima y umo clone
may ha e igge ed neoplas ic ans o ma ion (Jo ano ic e al., 2008).
None heless, in mos mice s udies BRAF
V600E
is exp essed in all hy oid cells e y
ea ly in li e ( e al o du ing he i s mon h) and BRAF-induced supp ession o
hy oid unc ion led o TSH ele a ion which in u n p omo ed hy oid
umo igenesis. One pa icula s udy gene a ed a model in which BRAF
V600E
exp ession was empo ally and spa ially es ic ed so ha i can ecapi ula es he
human spo adic PTC ha usually a ises pos na ally om ollicula cells unde
physiological se um TSH concen a ions. Wi h his app oach, hy oid ca cinomas
unde no mal TSH le els we e no ound (Shimamu a e al., 2013). This shows ha

13
he iming o BRAF ac i a ion may be he key o de e mine cell ans o ma ion as
he induc ion o an oncogene in poo ly di iding cells, such as he ollicula hy oid
cells, du ing adul hood may no igge umo igenesis (Shimamu a e al., 2013).
These da a ques ions whe he BRAF
V600E
ini ia es hy oid umo igenesis o BRAF
V600E
is a consequence o umo de elopmen and no a d i e mu a ion. An al e na i e
scena io is ha BRAF
V600E
does ini ia e he o ma ion o a PTC howe e as seconda y
gene ic al e a ions and/o epigene ic changes ake o e o main ain umo
sus ainabili y, BRAF
V600E
is no longe selec ed and/o impo an o umo
main enance (Xing, 2012).
RET/PTC and NTRK1 ea angemen s
The RET gene is a membe o he cadhe in
supe amily and encodes one o he i s TK ecep o s ha we e ound o ha e a
ole in human cance (Phay e al., 2010). RET ligands belong o he glial cell-de i ed
neu o ophic ac o amily and, when bound o RET co- ecep o s (GFRα-1), b ings
oge he wo RET molecules leading o he au ophospho yla ion o he in acellula
y osine po ion (Manie e al., 2001; Ai aksinen e al., 2002; T o isco e al., 2007).
In u n, he e is he ec ui men and binding o adap o p o eins and subsequen
ac i a ion o signaling pa hways such as he MAPK pa hway which a e able o
con ol cell p oli e a ion, di e en ia ion, mo ili y and su i al (Manie e al., 2001;
Ai aksinen e al., 2002; T o isco e al., 2007).
NTRK1 gene encodes a membe o he neu o ophic TK ecep o amily. Upon
neu o ophin binding, his memb ane-bound ecep o au o-phospho yla es and
ac i a es o he membe s o he MAPK signaling pa hway leading o cell
di e en ia ion (Teng e al., 2004; T o isco e al., 2007).
Rea angemen s o RET and NTRK1 usually in ol e he usion wi h he e ologous
genes esul ing in RET/PTC and NTRK1 chime ic ansc ip s. The chime ic p o eins
ha e an abe an and pe sis en ac i a ion o hei TK domains (T o isco e al.,
2007).
Soma ic ea angemen s o RET gene a e ound in 3% and up o 60% o spo adic
PTCs and lead o a de no o exp ession o he TK on RET domain in he cy oplasm
o ollicula hy oid cells (Niki o o , 2002; San o o e al., 2002; F asca e al., 2008).
The mos common RET ea angemen s a e he RET/PTC1 and RET/PTC3.
RET/PTC1 is by a he mos p e alen ype comp ising 60-70% o all he
14
ea angemen s howe e RET/PTC3 is he mos equen ea angemen ound
ea ly a e adia ion exposu e. RET/PTC1 and RET/PTC3 a e pa acen ic
ea angemen s wi h CCDC6 (coiled-coil domain con aining 6) and NCOA4 (nuclea
ecep o coac i a o 4) genes, espec i ely (T o isco e al., 2007). Ano he
ea angemen , RET/PTC2, in ol es ecip ocal ansloca ions wi h he p o ein
kinase, cAMP-dependen , egula o y, ype I, alpha (PRKAR1A) gene. RET gene can
s ill be in ol ed in o he ea angemen s bu i is mainly associa ed wi h adia ion
(T o isco e al., 2007). RET/PTC usions lea e in ac he TK domain o he RET
ecep o enabling he p o ein o induce ac i a ion o signaling cascades including
MAPK and phospha idylinosi ol-4,5-bisphospha e 3-kinase (PI3K)-V-Ak mu ine
hymoma i al oncogene homolog (AKT) pa hways (Ku oda e al., 2003; Knau e
al., 2009).
Rea angemen s o NTRK1 gene a e a e and a e ound in less han 10% o spo adic
PTCs (T o isco e al., 2007).
RAS mu a ions
The RAS gene encodes o a amily o ela ed p o eins ha s ay a
he cen e o a cascade o molecula in e ac ions. Mos p o eins a e ac i a ed by
RAS upon phospho yla ion as Ras swi ches be ween i s “on” and “o ” s a e. Usually,
RAS binds o guanosine-5'-diphospha e (GDP) bu upon a ecep o ac i a ion, GDP
is expelled allowing guanosine-5'- iphospha e (GTP) o bind. In u n, GTP causes
a sub le ea angemen o he RAS p o ein ul ima ely leading o he ac i a ion o
MAPK signaling pa hway. As GTP is hyd olyzed o GDP, RAS u ns i sel “o ”, sel -
limi ing i s ac i i y (Lodish e al., 2000).
RAS mu a ions lead o a loss o he GTPase ac i i y in RAS p o ein in such a way
ha i locks RAS in a cons i u i ely ac i e GTP-bound s a e which po en ia e
uncon olled p oli e a i e signals (Lodish e al., 2000).
In PTCs, RAS gene mu a ions a e mo e equen ly ound in he ollicula a ian o
PTC (Zhu e al., 2003, Gio dano e al., 2005, F asca e al., 2008). The p e alence o
RAS mu a ions in PTCs anges om 0% o 16% and neu oblas oma RAS i al ( - as)
oncogene homolog (NRAS) gene is he mos p edominan ly mu a ed namely on
codon 61 (Zhu e al., 2003; T o isco e al., 2007).
PAX8-PPAR gamma ea angemen s
The pai ed box 8 (PAX8) gene encodes o
a membe o he pai ed box amily o ansc ip ion ac o s in ol ed in ollicula
15
hy oid cell de elopmen and exp ession o hy oid-speci ic genes (Kimu a, 2011).
Pe oxisome p oli e a o -ac i a ed ecep o gamma (PPARG) gene encodes o a
pe oxisome p oli e a o -ac i a ed ecep o ha egula es he exp ession o a ge
genes in ol ed in cell p oli e a ion, di e en ia ion and immune and in lamma o y
esponses (K oll e al., 2000).
PAX8-PPAR gamma ea angemen s a e ipically ound in ollicula hy oid
adenomas, FTCs and on he ollicula a ian o PTCs, in he la e wi h a p e alence
up o 38% (Cas o e al., 2006).
PTEN and PIK3CA mu a ions
The phospha ase and ensin homolog (PTEN) gene
encodes o a phospha idylinosi ol-3,4,5- iphospha e 3-phospha ase which
egula es dephospho yla ion o phosphoinosi ide subs a es he eby nega i ely
egula ing he PI3K-AKT signaling pa hway (Sun e al., 1999; Hou e al., 2007).
PTEN mu a ions a e ound in 1-2% o PTCs and mu a ions in his umo supp esso
gene ac i a e he PI3K-AKT pa hway (Hou e al., 2007).
Phospha idylinosi ol-4,5-bisphospha e 3-kinase, ca aly ic subuni alpha (PIK3CA)
encodes o a ca aly ic subuni ha uses ATP o phospho yla e
phospha idylinosi ol-4-monophospha e and phospha idylinosi ol-4,5-
diphospha e (Samuels e al., 2004). PIK3CA mu a ions ha e also a e y low
p e alence in PTCs (1-3%) and mos a e ound in he helical and kinase domains o
he p110α p o ein p oducing a ian s ha a e independen o he egula o y
subuni s and a e capable o inducing cell p oli e a ion, in asi eness and esis ance
o apop osis (Gymnopoulos e al., 2007; San a pia e al., 2010). PIK3CA copy gain
p e alence goes up o 14% (Wang e al., 2007; Hou e al., 2007). Ei he mu a ions
in he PIK3CA gene o inc ease in copy numbe esul in he gain o unc ion
e lec ed by high o cons i u i e ac i a ion o he PI3K ac i i y and p omo ion o
umo igenesis (San a pia e al., 2010).
p53 mu a ions
The TP53 gene encodes o a p o ein ha main ains genome
in eg i y by binding speci ically o a DNA consensus sequence o induce g ow h
inhibi o y genes o nonspeci ically o damaged si es leading o DNA epai o
apop osis (Liu e al., 2001). The le els o p53 in no mal cells a e e y low bu upon
p53 ac i a ion in esponse o en i onmen al challenges such as cellula s ess, p53
p o ein is accumula ed and s abilized (Liu e al., 2001). p53 is capable o a es ing
he cell cycle a G1, G2 o in S-phase by inducing p21 which in u n blocks he
16
cycling-dependen kinases (CDKs) esponsible o checkpoin egula ion and
p og ession o he cell cycle (Bai e al., 2006). This allows ime o epai damaged
DNA o induce cell dea h (Bai e al., 2006). p53 is also an ac i a o o he MDM2
p o o-oncogene, E3 ubiqui in p o ein ligase (MDM2) gene which nega i ely au o-
egula es p53 main aining low le els o he p53 p o ein in no mal cells (Bai e al.,
2006).
A a iance wi h o he human umo s, TP53 mu a ions a e no equen in hy oid
cance (only 10%) and mos ha e been documen ed in anaplas ic ca cinomas.
Indeed, well-di e en ia ed hy oid cance s do no ha bo mu a ions in TP53
sugges ing a ole on hy oid cance p og ession o poo ly di e en ia ed and
agg essi e pheno ypes (Mo i a e al., 2008). O no e, s udies on hy oid umo
samples e ealed an accumula ion o p53 in poo ly di e en ia ed and anaplas ic
o ms bu also well-di e en ia ed umo s in he absence o any p53 mu a ion
sugges ing ha p53 inac i a ion may esul om loss o in e ac ion wi h MDM2
(Soa es e al., 1994). In hy oid cance , TP53 mu a ions a e commonly ound a
codons 213 and 238 and in anaplas ic ca cinomas a e also ound a codons 248
and 273 (Bai e al., 2006).
Modeling hy oid cance in mice ha e shown ha acqui ed mu a ions d i e umo
p og ession and in gene al BRAF
V600E
is su icien o ini ia e PTCs. Al eady
demons a ed is e idence ha p53 cons ains p og ession om papilla y o
anaplas ic hy oid ca cinoma (P e o e al., 2004). By gene a ing a hy oid-speci ic
C e ecombinase-es ogen ecep o (C eER) ansgenic mouse and using a C e-
egula ed BRAF
V600E
and a condi ional T p53, i was ound ha p53 loss does enable
p og ession o agg essi e anaplas ic hy oid cance bu addi ional e en s may be
equi ed o ull anaplas ic con e sion (McFadden e al., 2014).
Telome ase p omo e mu a ions
Telome ase is a ibonucleop o ein complex ha
adds elome es epea s sequences o he ends o elome ic DNA. The p o ein
componen has e e se ansc ip ase (TERT) ac i i y while he RNA componen
se es as a empla e o he elome e epea (Capezzone e al., 2009; Hanahan e
al., 2011).
Telome ase is ac i e in he as majo i y o human cance cells (80-90%) enabling
hei eplica i e immo ali y (Figu e 4) (Hanahan e al., 2011) and spo adic hy oid
ca cinomas a e no excep ion. In PTCs, elome ase ac i i y measu ed by TRAP assay
17
anged om 20% o 87.5% (Capezzone e al., 2009) bu he high le els epo ed in
some s udies may be due o he p esence o lymphocy ic in il a ion coexis ing wi h
he neoplasia (B ousse e al., 1997; Umb ich e al., 1997; Saji e al., 1999). O
no e, elome ase ac i i y in no mal hy oid issue is almos absen (Capezzone e
al., 2009).
Figu e 4.
Regula ion o elome e leng h by elome ase. (A) In no mal soma ic cells,
elome ase is absen and he elome e epea sequence (ligh blue boxes) is los e e y ime
he cell di ides. A e many cell di isions, elome es each a c i ically sho leng h igge ing
senescence and cessa ion o p oli e a ion. (B) In cance cells, eexp ession o he
elome ase exp ession bypasses senescence and elome e leng h is compa ible wi h
p oli e a ion. Telome e elonga ion sus ains cance .
Telome ase ac i i y is conside ed he esul o clonal selec ion as elome es become
c i ically sho ened (Sk o zo e al., 2009). I was shown ha gene ic mechanisms
ha p omo e elome ase eac i a ion in human umo s include TERT al e na i e
splicing, TERT gene ampli ica ion and TERT p omo e mu a ions (Figu e 5)
(Sk o zo e al., 2009). As ecen ly epo ed wo ecu en , non-o e lapping
soma ic mu a ions on ch omosome 5, -124C>T (C228T) and -146C>T (C250T)
(whe e -1 is he base jus ups eam o he ATG ansla ion s a si e o he TERT

18
gene), in he TERT p omo e a e e y equen in spo adic melanoma (Huang e al.,
2013, Ho n e al., 2013). These mu a ions we e subsequen ly ound in se e al
umo s including hy oid cance lines and ei he well-di e en ia ed o poo ly and
anaplas ic hy oid ca cinomas (Vinag e e al., 2014).
TERT p omo e mu a ions c ea e an ele en-base nucleo ide s e ch 5’-
CCCCTTCCGGG3’ which con ains a new consensus binding si e GGAA, gene a ing
de no o consensus binding mo i s o E- wen y-six (e y h oblas ans o ma ion-
speci ic –ETS- ansc ip ion ac o s). I was shown ha i inc eases he
ansc ip ional ac i i y o he elome ase p omo e by wo- o-six- old in human
cance cell lines (Huang e al., 2013; Ho n e al., 2013; Liu e al., 2013). This is
consis en wi h a mildly enhanced TERT exp ession wi h espec o no mal issues
(Muzza e al., 2015). One o he ansc ip ion ac o s ound o be ec ui ed
speci ically o he mu an p omo e is he mul ime ic GA-binding p o ein (GABP)
(Bell e al., 2015).
TERT p omo e mu a ions a e ound in 8% o 22% o PTCs, being he -124C>T
mu a ion mo e p e alen (Liu e al., 2013; Landa e al., 2013; Vinag e e al., 2013;
Melo e al., 2014; Liu e al., 2014; Wang e al., 2014; Muzza e al., 2015).
No iceably, 33% o PTCs wi h dis an me as asis ha bo ed TERT p omo e mu a ions
(Melo e al., 2014; Xing e al, 2014; Gandol i e al., 2015).
I was p oposed ha TERT p omo e mu a ions may be mo e common in cance s
de i ed om e minally di e en ia ed cells which ha e a low sel - enewing
capaci y. Indeed, ollicula hy oid cells ha e a e y low mi o ic a e pos na ally
(p oli e a i e a e lowe han 0.1 in adul s). Also, well-di e en ia ed PTCs a e
usually indolen lesions wi h low a e o g ow h. A signi ican a e o TERT p omo e
mu a ions in hose ca cinomas sugges s ha hy oid cance cells may bene i om
his mechanism o main ain elome ase leng hening (Saad e al., 2006; Killela e
al., 2013).
Mo eo e , i was ound a signi ican o e ep esen a ion o TERT p omo e
mu a ions in hy oid umo s ha bo ing al e a ions in BRAF o RAS genes. TERT
p omo e mu a ions seemed o be mo e equen in BRAF
V600E
ha in BRAF
WT
-PTCs
(Liu e al., 2013; Melo e al., 2014; Xing e al., 2014).
In di e en ia ed hy oid ca cinomas, TERT p omo e mu a ions a e associa ed wi h
olde age a diagnosis, umo size and male gende and a e co ela ed wi h a
19
educed p og ession ee su i al and o e all su i al (Vinag e e al., 2013; Xing e
al., 2014; Melo e al., 2014).
This comes as no su p ise ha TERT p omo e mu a ions a e associa ed wi h olde
age a diagnosis which is consis en wi h a p og essi e sho ening o elome es in
ollicula cells du ing li e ime. When hese cells acqui e gene ic al e a ions, such as
he BRAF
V600E
mu a ion, and begin o eply o he oncogenic s imuli by inducing
p oli e a ion, e y sho elome es also igge elome e dys unc ion which may be
compensa ed by elome ase eac i a ion ia TERT p omo e mu a ions (Londoño-
Vallejo, 2008; Muzza e al., 2015). Fu he mo e, BRAF mu a ions leading o MAPK
ac i a ion can be concei ed as induce s o exp ession o membe s o he ETS
ansc ip ion ac o amily. Ha ing de no o consensus binding si es o ETS ac o s
p omo ed by TERT p omo e mu a ions, he li espan o BRAF-d i en clones is
ex ended. Fu he accumula ion o addi ional gene ic de ec s is p omo ed which in
u n allows p og ession o ad anced umo s ages (P a ilas e al., 2009; Huang e
al., 2013; Ho n e al., 2013; Liu e al., 2013).
These hypo hesis could explain why well di e en ia ed PTCs ha bo ing BRAF
mu a ions a e mo e likely o ha bo TERT p omo e mu a ions han hose PTCs
wi hou BRAF mu a ions. I is also consis en wi h an en ichmen o TERT p omo e
mu a ions in poo ly di e en ia ed hy oid ca cinomas and anaplas ic hy oid
ca cinomas which ha e pa ially and comple ely los di e en ia ion, espec i ely,
and a e he mos agg essi e hy oid ca cinomas (Landa e al., 2013; Liu e al.,
2013). O no e, TERT p omo e mu a ions mus be only one o he se e al
mechanisms ha illegi ima ely ac i a e elome ase in human cance .
Signaling pa hways al e ed in papilla y hy oid ca cinomas
MAPK signaling pa hway
The MAPK pa hway comp ises e olu iona ily conse ed
kinase modules ha join ex acellula signals o he machine y esponsible o cell
g ow h, p oli e a ion, di e en ia ion, mig a ion and apop osis. One o he g oups
o MAPK cha ac e ized in mammals is he ex acellula signal- egula ed kinase
(ERK)1/2 (Dhillon e al., 2007).
Cons i u i e ac i a ion o he MAPK signaling pa hway is a equen e en in human
cance s pa icula ly in PTCs and melanomas (Xing, 2013).
20
Fo mos cance s, cons i u i e ac i a ion o ERK signaling is es ablished by
sus ained au oc ine o pa ac ine p oduc ion o ac i a ing ligands, o e exp ession
and ac i a ing mu a ions o he TK ecep o and ac i a ing mu a ions in RAS and
BRAF (Dhillon e al., 2007; Knau e al., 2009). In hy oid cance s, besides RAS and
BRAF mu a ions, RET-PTC, NTRK1 and ALK mu a ions also media e umo igenesis
ia he MAPK pa hway (Xing, 2013).
In his pa hway, ligand-media ed ac i a ion o TK ecep o p omo e RAS GTPase
con e sion which ec ui s and ac i a es RAF kinases o he plasma memb ane. ERK1
and ERK2 a e ac i a ed upon phospho yla ion by MEK1 and MEK2 which a e
hemsel es ac i a ed when phospho yla ed by RAF p o eins. Ac i a ed ERKs
phospho yla e cy oplasmic and nuclea a ge s including kinases, phospha ases,
ansc ip ion ac o s and cy oskele al p o eins (Figu e 5) (Dhillon e al., 2007).
The e ec o ERK signaling ac i a ion is consis en o he cellula p ocesses ha
i sel egula es. Sus ained ERK signaling p omo es phospho yla ion and
s abiliza ion o FBJ mu ine os eosa coma i al oncogene homolog (Fos), Jun p o o-
oncogene (Jun), V-Myc a ian myelocy oma osis i al oncogene homolog (Myc) and
ETS-Rela ed (E g-1) genes and also cyclin D1 he eby p omo ing cell-cycle en y
and can ep ess genes esponsible o inhibi ing p oli e a ion (Yamamo o e al.,
2006). On he o he hand, high le els o ERKs can induce cell-cycle a es by
exp ession o CDK-inhibi o p o eins such as p21 and p27 ha mus be
coun e ac ed by ele a ed RhO signaling o ac i a ion o AKT so ha cells con inue
o p oli e a e (Dhillon e al., 2007).
PI3K-AKT signaling pa hway
PI3Ks ep esen a amily o kinases ha
phospho yla e he 3’-hyd oxyl g oup o phospha idylinosi ol inosi ides ha a e
ac i a ed by many TK ecep o s. Class I o PI3Ks consis s o he e odime s o
egula o y (p85) and ca aly ic (p110) subuni s. p110α and p110β subuni s ha e an
impo an ole in umo igenesis as RAS unc ion is media ed by i s in e ac ion wi h
he RAS-binding si e p esen in hose subuni s. Also, ac i a ion o TK ecep o by
ex acellula signals is i sel su icien o ac i a e p110 subuni s which in u n
phospho yla es phospha idylinosi ol (P dIns)-3,4-P2 p oducing P dIns-3,4,5-P3
leading o he ec ui men o AKT o he cy osolic memb ane. AKT is hen
phospho yla ed and ac i a ed by PDK1 esul ing in he phospho yla ion o
downs eam e ec o such as he mammalian a ge o apamycin (mTOR) (Figu e
21
5). This esul in a b oad cascade o signaling esponsible o cell g ow h and
p oli e a ion, glucose up ake, mig a ion and apop osis esis ance (Saji e al., 2010).
Cons i u i e ac i a ion o he PI3K- egula ed signaling pa hway is ele an in a wide
a ie y o human umo s including hy oid cance . This is pa icula ly alid in
Cowden’s synd ome, ha p esen hy oid ca cinomas, which is cha ac e ized by
mu a ions in he PTEN gene ha encodes a phospha ase ha dephospho yla e
P dIns-3,4,5-P3 and he eby nega i ely egula es he PI3K-AKT pa hway.
Addi ionally, RAS mu a ions, RET/PTC ea angemen s and PIK3CA and AKT1
mu a ions a e u he e idences ha PI3K signaling pa hway has a undamen al ole
in hy oid umo igenesis (Saji e al., 2010, Xing, 2010).
mTOR signaling pa hway
mTOR is a se ine/ h eonine kinase ha belongs o he
phosphoinosi ide 3-kinase (PI3K)- ela ed kinase amily and unc ions as a
egula o o cell g ow h- ela ed p ocesses. mTOR can o m wo dis inc complexes
wi h o he p o eins, mTOR complex 1 (mTORC1) and complex 2 (mTORC2), ha
ha e di e en ups eam inpu s and downs eam ou pu s. Regula ion o mTOR by
g ow h ac o s occu s h ough he PI3K/AKT pa hway which is coun e ac ed by
PTEN. The e is e idence o o e ac i a ion o AKT/mTOR pa hway in PTCs when
compa ed o o he di e en ia ed hy oid ca cinomas and co ela ion wi h BRAF
V600E
mu a ion which could be explained by BRAF-induced phospho yla ion o umo
supp esso li e kinase B1 (LKB1) Se 428, a main ups eam kinase o AMP-ac i a ed
p o ein kinase (Faus ino e al., 2012).
28
I.3 Aims
No so long ago cance was seen as a ising owing o he accumula ion o mu a ions
in c i ical genes ha al e ed he no mal p og amme o cell p oli e a ion,
di e en ia ion and dea h. Bu he impac o such mu a ions, including hose in he
BRAF gene, is only pa o he cance pa adigm, indeed many o he mechanisms
media e umo igenesis.
One o he mos no el mechanism is ela ed o TERT p omo e mu a ions. TERT
p omo e mu a ions we e ini ially ound a high equency in human melanoma bu
no in ne i and a e belie ed o illici ly con ibu e o elome ase eac i a ion in
melanoma as in o he cance s. I was in ol ed in a s udy o in es iga e whe he TERT
p omo e mu a ions we e p esen in cance ypes o he han melanomas: hy oid,
kidney, bladde , gas oin es inal s omal umo s (GIST), ad enal medulla and
cen al ne ous sys em (CNS) umo s. The equency o TERT p omo e mu a ions
was assessed in he abo e-men ioned cance s and con ibu ed o u he s udies
o unde s and why hose mu a ions a e impo an in some cance s bu no in o he s
and how do hey ela e wi h addi ional gene ic al e a ions.
Vi ually e e y b eak h ough in he s udy o human diseases has been he di ec
esul o using animal models. Al hough many al e na i e animal- eplacing
me hods a e also used, li ing sys ems emain a necessi y in esea ch.
In hy oid cance s udies, mice models ha e p o ided enough e idence ha
hy oid-speci ic exp ession o BRAF
V600E
induced goi e and in asi e PTCs which
p og ess o poo ly di e en ia ed ca cinomas ha closely ecapi ula ed he human
PTC pheno ypes. Those mice models a e s ill used nowadays o disclose new
mechanisms o hy oid umo igenesis, howe e umo imaging and ansla ional
s udies namely d ug sc eening a e di icul and ime-consuming.
In he las yea s, zeb a ish has p o ed o be a good addi ion o animal models o
human cance no only because hey de elop cance spon aneously, a e mu agen
exposu e o h ough ansgenesis bu also because umo s ha a ise esemble
hose o he humans a he his ological, gene ic and genomic le els. Taking
ad an age o his e sa ile animal model, I de eloped a no el ansgenic line ha
exp essed BRAF
V600E
speci ically in hy oid cells o zeb a ish o unde s and he e ec
o BRAF
V600E
in his issue and alida e a model use ul o s udy hy oid disease.
Up egula ion o p53 was ound o play an impo an ole in he BRAF
V600E
-induced
pheno ype in ha line. Also, human and mice da a showed ha p53 loss is essen ial

29
in umo p og ession. The e o e, I de eloped a ansgenic line ha exp essed
BRAF
V600E
speci ically in hy oid cells o p53
M214K
zeb a ish o unde s and whe he
he absence o WT p53 would p omo e cance o a leas exace ba e he BRAF
V600E
e ec on hy oid. Finally, o closely ecapi ula e he iming o BRAF ac i a ion in
spo adic hy oid ca cinomas, I de eloped a ansgenic model ha allowed
empo ally es ic ed exp ession o BRAF
V600E
in hy oid cells o zeb a ish.
30
Chap e II
Ma e ial &
Me hods
31
The ma e ial and me hods om he “F equency o TERT p omo e mu a ions in
human cance s” wo k a e exclusi ely desc ibed in chap e III.1. The ma e ial and
me hods om he s udies in zeb a ish a e desc ibed in de ail in his chap e and
b ie ly in chap e III.2
II.1 Plasmid cloning
II.1.1 p5E-
g
p omo e plasmid
The (-2041; -1) egion, being -1 he posi ion immedia ely ups eam o he ATG
sequence o he zeb a ish hy oglobulin ( g) gene, was ampli ied by polyme ase
chain eac ion (PCR) om bac e ial a i icial ch omosome (BAC) genomic clone
DKEY-97I18 BAC, GenBank CR855311.15 (Sou ceBioScience) using he ollowing
p ime s: o wa d p ime 5’-CAGCTGGTACTCTAAATGTGAGAAA-3’ and e e se
p ime 5’-TGTTTAAAAGGGACGATGTAGC-3’. The agmen was ini ially cloned
in o a pCR™-Blun II-TOPO® plasmid (The mo Fishe Scien i ic Inc.). The inse was
hen excised using es ic ion si es om he backbone plasmid and cloned in o he
KpnI (The mo Fishe Scien i ic Inc.) and XhoI (The mo Fishe Scien i ic Inc.)
es ic ion si es on he mul icloning si e (MCS) o a p5E-MCS plasmid, con aining
a L4 and a R1 ecombina ion si es and p o ided by he Tol2Ki (Kwan e al., 2007).
Liga ion p oduc was ans o med in o One Sho ® TOP10 Chemically Compe en
Cells (The mo Fishe Scien i ic Inc.). The ull-leng h cons uc was sequenced o
accu acy.
II.1.2 pME-mChe y and p3E-polyA plasmids
pME-mChe y is a middle en y (ME) clone con aining a L1 and a L2
ecombina ion si es. p3E-polyA is a 3’ en y clone con aining simian acuola ing
i us 40 (SV40) la e polyA signal and a R2 and a L3 ecombina ion si es. Bo h
en y clones we e p o ided by he Tol2Ki (Kwan e al., 2007). pME-mChe y and
p3E-polyA plasmids we e ans o med in o One Sho ® TOP10 Chemically
Compe en Cells (The mo Fishe Scien i ic Inc.). The ull-leng h cons uc s we e
sequenced o accu acy.
32
II.1.3 pME-mChe y-T2A-BRAF
WT
and pME-mChe y-T2A-BRAF
V600E
plasmids
Full-leng h human B- ype Ra kinase (BRAF)
WT
and BRAF
V600E
coding sequences we e
ampli ied by PCR om exp ession ec o s p e iously gene a ed in my lab: pCMV-
BRAF
WT
and pCMV-BRAF
V600E
, espec i ely (Faus ino e al., 2012) using he ollowing
p ime s: o wa d p ime 5’-CCGGCCCTATGGCGGCGCTGAGC-3’ and e e se
p ime 5’- GTTTCCTGTCCACTGATGATATCG-3’. mChe y coding sequence was
ampli ied by PCR om pME-mChe y (Kwan e al., 2007) using he ollowing
p ime s: o wa d p ime 5’-GGGCCCCCCCTCGAGGGCCGCCACCATGGTG-3’ and
e e se p ime 5’-GAGAATCCCGGCCCTATGGCGGC-3’. T2A sequence (5’-
GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT-3’)
was syn he ized as an oligome o 54bp and ampli ied by PCR. All PCR p oduc s
gene a ed we e pu i ied indi idually and assembled in ame in o a p e iously
linea ized pME-MCS (con aining a L1 and a L2 ecombina ion si es) using he
GeneA ® Seamless Assembly Ki (The mo Fishe Scien i ic) and acco ding o he
ollowing o de : (1
s
) mChe y - (2
nd
) T2A – (3
d
) BRAF
WT
o BRAF
V600E
. Liga ion p oduc
was ans o med in o One Sho ® TOP10 Chemically Compe en Cells (The mo
Fishe Scien i ic Inc.). The ull-leng h cons uc s we e sequenced o accu acy.
II.1.4 pTol2A2-
g
:mChe y–pA and pTol2CG2-
g
:mChe y–pA plasmids
p5E- g p omo e , pME-mChe y and p3E-polyA we e assembled in o a
pDes Tol2pA2, con aining a R4 and a R3 ecombina ion si es and p o ided by he
Tol2Ki (Kwan e al., 2007), using a Ga eway LR Clonase II Enzyme Mix (The mo
Fishe Scien i ic Inc.) and acco ding o he manu ac u e ’s ins uc ions. Liga ion
p oduc was ans o med in o One Sho ® TOP10 Chemically Compe en Cells
(The mo Fishe Scien i ic Inc.). The ull-leng h pTol2A2- g:mChe y–pA cons uc
was sequenced o accu acy. Same p o ocol was pe o med o gene a e he
pTol2CG2- g:mChe y–pA, howe e he h ee en y clones we e assembled in o a
pDes Tol2CG2, con aining he cmlc2:EGFP casse e and he a R4 and a R3
ecombina ion si es, p o ided by he Tol2Ki (Kwan e al., 2007) (Figu e 1). The
ull-leng h pTol2CG2- g:mChe y–pA cons uc was sequenced o accu acy.
33
Figu e 1.
Ga eway cloning s a egy o gene a e he pTol2CG2-
g
:mChe y-pA plasmid.
Schema ic o he h ee-pa LR ecombina ion eac ion used o gene a e pTol2CG2-
g:mChe y-pA. En y clones (p5E- g, pME-mCh and p3E-pA) con aining a L si es and
pDes Tol2CG2 des ina ion plasmid con aining a LR si es ecombined when LR clonase was
added o he eac ion.
II.1.5 pTol2CG2-
g
:mChe y-T2A- BRAF
V600E
–pA plasmids
p5E- g p omo e , pME-mChe y-T2A-BRAF
V600E
and p3E-polyA we e assembled in o
a pDes Tol2CG2, con aining he ca diac myosin ligh chain (cmlc2): enhanced
g een luo escen p o ein (EGFP) casse e and con aining a R4 and a R3
ecombina ion si es, p o ided by he Tol2Ki (Kwan e al., 2007) using a Ga eway
LR Clonase II Enzyme Mix (Li e Technologies) and acco ding o he manu ac u e ’s
ins uc ions (Figu e 2). Liga ion p oduc was ans o med in o One Sho ® TOP10
Chemically Compe en Cells (The mo Fishe Scien i ic Inc.). The ull-leng h
pTol2CG2- g:mChe y-T2A-BRAF
V600E
–pA cons uc was sequenced o accu acy.

34
Figu e 2.
Ga eway cloning s a egy o gene a e he pTol2CG2-
g
:mChe y-T2A-
BRAF
V600E
-pA plasmid. Schema ic o he h ee-pa LR ecombina ion eac ion used o
gene a e pTol2CG2- g:mChe y-T2A-BRAF
V600E
–pA. En y clones (p5E- g, pME-mCh-T2A-
BRAF
V600E
and p3E-pA) con aining a L si es and pDes Tol2CG2 des ina ion plasmid
con aining a LR si es ecombined when LR clonase was p esen in he eac ion.
II.1.6 pTol2CG2-
g
:loxP-CFP-loxP-mChe y–pA and pTol2CG2-
g
:loxP-
CFP-loxP-mChe y-T2A-BRAF
V600E
–pA plasmids
pTol2CG2- g:mChe y-pA was cu wi h SalI (The mo Fishe Scien i ic Inc.), gel
pu i ied and dephospho yla ed wi h Sh imp Alkaline Phospha ase (SAP) (The mo
Fishe Scien i ic Inc.). loxP-cyan luo escen p o ein (CFP)-loxP was ampli ied om
a dono plasmid gene a ed in he lab (unpublished plasmid) using he ollowing
p ime s: o wa d p ime 5’-
CAAGCTTATCGATACCGCCCGGGATAACTTCGTATAATGTATGCTATACGAAGTTATC
TTG-3’ and e e se p ime 5’-CTAGAGAAGCTGAGGACAGGGATCCGGCCGGCC-3’.
These p ime s we e enginee ed o ecombina ion deoxy ibonucleic acid (DNA)
assembly and he PCR p oduc was gel pu i ied. Plasmid and inse we e liga ed
using he Gibson Assembly Cloning Ki (New England Biolabs) and liga ion p oduc
35
was ans o med in o One Sho ® TOP10 Chemically Compe en Cells (The mo
Fishe Scien i ic Inc.). The ull-leng h pTol2CG2- g:loxP-CFP-loxP-mChe y-pA
cons uc was sequenced o accu acy. Same p o ocol was pe o med o gene a e
he pTol2CG2- g:loxP-CFP-loxP-mChe y-T2A-BRAF
V600E
–pA howe e pTol2CG2-
g:mChe y-T2A-BRAF
V600E
–pA was used as a backbone. The ull-leng h pTol2CG2-
g:loxP-CFP-loxP-mChe y-T2A-BRAF
V600E
–pA cons uc was sequenced o
accu acy.
Figu e 3.
Cloning s a egy o gene a e condi ional cons uc s. (A) pTol2CG2- g:loxP-
CFP-loxP-mChe y–pA and (B) pTol2CG2- g:loxP-CFP-loxP-mChe y-T2A-BRAF
V600E
–pA
plasmids. The loxP-CFP-loxP casse e was inse ed in o p e iously gene a ed cons uc s.
Scisso ep esen SalI cu .
36
II.1.7 pCS2-
CMV
:mChe y-pA plasmid
pTol2- g:mChe y–pA and pCS2-CMV plasmid we e excised wi h BamI (The mo
Fishe Scien i ic Inc.). mChe y inse was gel pu i ied and linea ized pCS2-CMV
was dephospho yla ed wi h SAP (The mo Fishe Scien i ic Inc.). Plasmid and inse
we e liga ed using T4 ligase (The mo Fishe Scien i ic Inc.) and liga ion p oduc was
ans o med in o One Sho ® TOP10 Chemically Compe en Cells (The mo Fishe
Scien i ic Inc.) (Figu e 3). The ull-leng h pCS2-CMV:mChe y-pA cons uc was
sequenced o accu acy.
II.1.8 pCS2-
CMV
:mChe y-T2A-BRAF
WT
-pA and pCS2-
CMV
:mChe y-
T2A-BRAF
V600E
-pA plasmids
pME-mChe y-T2A-BRAF
WT
and pCS2-CMV plasmid we e excised wi h XbaI
(The mo Fishe Scien i ic Inc.) and XhoI (The mo Fishe Scien i ic Inc.). mChe y-
T2A-BRAF
WT
inse was gel pu i ied and linea ized pCS2-CMV was
dephospho yla ed wi h SAP (The mo Fishe Scien i ic Inc.). Plasmid and inse we e
liga ed using T4 ligase (Li e Technologies) and liga ion p oduc was ans o med
in o One Sho ® TOP10 Chemically Compe en Cells (The mo Fishe Scien i ic Inc.).
The ull-leng h pCS2-CMV:mChe y-T2A-BRAF
WT
-pA cons uc was sequenced o
accu acy. Same p o ocol was pe o med o gene a e he pCS2-CMV:mChe y-T2A-
BRAF
V600E
-pA howe e pME-mChe y-T2A-BRAF
V600E
was used o excise he
mChe y-T2A-BRAF
V600E
inse (Figu e 4). The ull-leng h pCS2-CMV:mChe y-T2A-
BRAF
V600E
-pA cons uc was sequenced o accu acy.
II.1.9 Summa y o he plasmids gene a ed
Table 1 summa izes he plasmids gene a ed in his wo k.
37
Name
Inse
Size
5’ en y clones
(kan
amycin
esis an )
p5E- g p omo e
2.0 kb
g
p om
o e
4843
Middle en y clones (
kan
amycin
esis an )
pME
-
mChe y
mCh
3261
pME
-
mChe y
-
T2A
-
BRAF
WT
mCh
and BRAF
WT
sepa a ed by T2A sequence
5818
pME
-
mChe y
-
T2A
-
BRAF
V600E
mCh
and BRAF
V600E
sepa a ed by T2A sequence
5818
3’ en y clone (
kan
amycin
esis an )
p3E
-
polyA
SV40 la e polyA signal
2838
Des ina ion ec o (amp
icillin
esis an )
pTol2- g:mChe y-pA
g
p omo e and mCh
epo e *
†
7385
pTol2CG2
-
g:mChe y-pA
g
p omo e and mCh
epo e
*
†
9298
pTol2CG2
-
g:mChe y-T2A-
BRAF
V600E
-pA
g
p omo e , mCh
epo e
and
BRAF
V600E
sepa a ed by T2A sequence*
†
11855
pTol2CG2- g:loxP-
CFP-loxP-mChe y–pA
g
p omo e , CFP lanked by lox
P in e ed
sequences and mCh epo e *
†
10604
pTol2CG2- g:loxP-
CFP-loxP-mChe y-
T2A-BRAF
V600E
–pA
g
p omo e , CFP lanked by lox
P in e ed
sequences and mCh epo e and BRAF
V600E
sepa a ed by T2A sequence*
†
13161
Plasmids o in i o RNA ansc ip ion
pCS2-CMV: mChe y-
pA
CMV/SP6 casse
e wi h mCh
epo e
*
4934
pCS2-CMV: mChe y-
T2A-BRAF
WT
-pA
CMV/SP6 casse e wi h mCh
epo e and
and
BRAF
WT
sepa a ed by T2A sequence*
7203
pCS2-CMV: mChe y-
T2A-BRAF
V600E
-pA
CMV/SP6 casse e wi h mCh
epo e and
and
BRAF
V600E
sepa a ed by T2A sequence*
7203
Table 1.
Lis o plasmids gene a ed o ansgenesis o in i o RNA ansc ip ion.
* wi h SV 40 polyA;
†
lanked by Tol2 in e ed epea s
44
g(
g
:mCh-T2A-BRAF
V600E
;
cmlc2
:EGFP) line
Emb yos om AB s ain we e injec ed
wi h pTol2CG2- g:mChe y-T2A-BRAF
V600E
-pA plasmid o gene a e mosaic
g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) ish. Founde ish we e c ossed wi h WT ish
o gene a e he g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) line. This line was selec ed
o mCh and EGFP epo e s.
g(
g
:mCh
; g
:mCh-T2A-BRAF
V600E
;
cmlc2
:EGFP) line g:mCh ish we e c ossed
wi h g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) ish o p oduce he g( g:mCh; g:mCh-
T2A-BRAF
V600E
;cmlc2:EGFP) line. This line was selec ed o mCh and EGFP epo e s.
g(
hsp70l
:mCh-T2A-C eER
T2
;
g
:loxP-CFP-loxP-mCh;
cmlc2
:EGFP) line
g(hsp70l:mCh-T2A-C eER
T2
) ish we e used o p o ide emb yos o injec he
pTol2CG2- g:loxP-CFP-loxP-mChe y–pA plasmid and gene a e mosaic
g(hsp70l:mCh-T2A-C eER
T2
; g:loxP-CFP-loxP-mCh; cmlc2:EGFP) ish. Founde
ish we e c ossed wi h WT ish i hey ca ied he hsp70l:mCh-T2A-C eER
T2
allele
o g(hsp70i:mCh-T2A-C eER
T2
) ish i hey did no . This line was selec ed o CFP
and EGFP epo e s and he hsp70l:mCh-T2A-C eER
T2
allele.
g(
hsp70l
:mCh-T2A-C eER
T2
;
g
:loxP-CFP-loxP-mCh T2A-BRAF
V600E
;
cmlc2
:EGFP) line
g(hsp70l:mCh-T2A-C eER
T2
) ish we e used o p o ide emb yos
o injec he pTol2CG2- g:loxP-CFP-loxP-mChe y-T2A-BRAF
V600E
–pA plasmid and
gene a e mosaic g(hsp70l:mCh-T2A-C eER
T2
; g:loxP-CFP-loxP-mCh T2A-
BRAF
V600E
; cmlc2:EGFP) ish. Founde ish we e c ossed wi h WT ish i hey ca ied
hsp70l:mCh-T2A-C eER
T2
allele o g(hsp70i:mCh-T2A-C eER
T2
) ish i hey did no .
This line was selec ed o CFP and EGFP epo e s and he hsp70l:mCh-T2A-C eER
T2
allele.
II.5.2
p53
M214K
lines
p53
M214K
g(
g
:mCh) line
p53
M214K
ish we e c ossed wi h g( g:mCh) ish o
p oduce he e ozygous p53
M214K
g( g:mCh). This line was c ossed again wi h
p53
M214K
ish o p oduce homozygous p53
M214K
g( g:mCh) ish. All lines we e

45
selec ed o mCh epo e . p53
M214K
mu a ion was con i med by geno yping he in
o selec ed ish.
p53
M214K
g(
g
:mCh;
cmlc2
:EGFP) line
p53
M214K
ish we e c ossed wi h
g( g:mCh;cmlc2:EGFP) ish o p oduce he e ozygous p53
M214K
g( g:mCh;cmlc2:EGFP). This line was c ossed again wi h p53
M214K
ish o p oduce
homozygous p53
M214K
g( g:mCh;cmlc2:EGFP) ish. All lines we e selec ed o mCh
and EGFP epo e s. p53
M214K
mu a ion was con i med by geno yping he in o
selec ed ish.
p53
M214K
g(
g
:mCh-T2A-BRAF
V600E
;
cmlc2
:EGFP) line
p53
M214K
ish we e c ossed
wi h g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) ish o p oduce a he e ozygous p53
M214K
g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) line. This line was c ossed again wi h
p53
M214K
ish o p oduce a homozygous p53
M214K
g( g:mCh-T2A-
BRAF
V600E
;cmlc2:EGFP) line. All lines we e selec ed o mCh and EGFP epo e s.
p53
M214K
mu a ion was con i med by geno yping he in o selec ed ish.
p53
M214K
g(
g
:mCh
; g
:mCh-T2A-BRAF
V600E
;
cmlc2
:EGFP) line p53
M214K
g( g:mCh) ish we e c ossed wi h p53
M214K
g( g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP)
ish o p oduce he p53
M214K
g( g:mCh; g:mCh-T2A-BRAF
V600E
;cmlc2:EGFP) line.
This line was selec ed o mCh and EGFP epo e s. p53
M214K
mu a ion was
con i med by geno yping he in o selec ed ish.
II.6 Fish s ains and husband y
AB wild- ype ish was he zeb a ish s ain used h oughou he wo k. Emb yos and
la ae <7dp /dpi we e collec ed om pai ma ing o ou c osses and kep in emb yo
medium (5.0mM NaCl, 0.17mM KCl, 0.33mM CaCl, 0.33mM MgSO
4
, 0.05%
me hylene blue, pH 7.4) a 28ºC on a 14 hou ligh /10 hou da k cycle. Zeb a ish
≥7dp /7dpi we e kep in a eci cula ing sys em a 28ºC on a 14 hou ligh /10 hou
da k cycle acco ding o Wes e ield e al., 2000. Fish we e kep in s anda d
densi ies (10-12 ish/li e o wa e ).
46
II.7 Sc eening and Imaging
The ansgenic lines gene a ed exp essed mChe y, EGFP and/o CFP epo e s.
Sc eening o emb yos, la ae and adul ish was pe o med on a Zeiss S e eo LUMAR
s e eoscope. Images we e acqui ed on he Zeiss S e eo Luma .V12 s e eoscope
equipped wi h a Hamama sy O ca-ER CCD Came a, con olled wi h he
Mic oManage 1.14 so wa e. B igh ield (BF) images we e acqui ed using he
Luma il e LP420 and luo escence images we e acqui ed wi h Luma il e s o
Texas Red®, EGFP and CFP.
II.8 Fin clip and gDNA ex ac ion
Fish we e as en, p io o anes hesia, in icaine me hane sul ona e
(MS222) (168ug/L, Sigma). The in was clipped wi h a sha p scalpel a a poin no
g ea e han hal way be ween he ip o he in and he poin whe e he scales end.
Fish we e hen immedia ely ans e ed o a con aine wi h esh sys em wa e and
moni o ed un il hey eco e ed and egained swimming abili y. Fin issue was
placed in an eppendo ube con aining 100μL o 50mM NaOH. The sample was
hea ed a 95ºC o 15 minu es o un il he issue was no iceably iable. The sample
was cooled o 4ºC and 1/10
h
olume o 1M T is-HCl, pH 8.0 was added o
neu alize he basic solu ion. A cen i uga ion o pelle he deb is was pe o med
a maximum speed o 10 minu es a oom empe a u e. The supe na an
con aining he genomic DNA was collec ed o a new ube and s o ed a 4ºC o
longe a -20ºC.
II.9 Geno yping
p53
M214K
mu a ion was geno yped using p ime s o ampli y he loss-o - unc ion
(LOF) poin mu a ion on exon 7 o he zeb a ish p53 gene (homozygous ish we e
selec ed i T>A was in bo h alleles; he e ozygous ish we e selec ed i T>A was in
one allele) as desc ibed by Be ghmans e al., 2005. The ollowing p ime s we e
used: o wa d p ime 5’-CACAAGTGTCCTGTTATCGAT-3’ and e e se p ime 5’-
CATGGCAAGGCAACTGAACTGT-3’.
Condi ional lines we e also geno yped o he hsp70l:mCh-T2A-C eER
T2
allele using
p ime s o ampli y he C e ecombinase (C e) coding sequence. The
47
g(hsp70l:mChe y-T2A-C eER
T2
) line (Hans e al., 2011) does no ha e a
ansgenesis ma ke and p ogeny om his line can esul in WT o ansgenic ish.
I was only possible o geno ype adul s. The ollowing p ime s we e used: o wa d
p ime 5’-GCATTTCTGGGGATTGCTTA-3’ and e e se p ime 5’-
CCCGGCAAAACAGGTAGTTA-3’.
II.10 His opa hology
7dp la ae we e eu hanized by subme sion in ice wa e (5 pa s o ice/1 pa o
wa e , 0-4ºC) o a leas wen y minu es o ensu e dea h by hypoxia. La ae ≥8dp
we e eu hanized by an o e dose o MS222 (200mg/L, Sigma) by p olonged
imme sion. La ae 7-14dp we e ixed in o maldehyde (Sigma-Ald ich) o one
day and la ae ≥15dp we e ixed in o maldehyde (Sigma-Ald ich) o wo days
and hen decalci ied in 0.5M e hylenediamine e aace ic acid (EDTA) o one o wo
mo e days be o e pa a in embedding and sec ioning. 3μm longi udinal sec ions
ep esen a i e o he whole la ae o ep esen a i e o he adul head we e
pe o med. Hema oxylin-Eosin (HE) s aining we e pe o med acco ding o s anda d
echniques by he His opa hUni a Ins i u o Gulbenkian de Ciência (IGC).
Images we e acqui ed on a Leica DM LB2 up igh mic oscope equipped wi h an IDS
colo CCD came a using he ollowing objec i es: 10x 0.25NA and 40x 0.75NA and
using he uEye Cockpi so wa e (Imaging De elopmen Sys ems GmbH, Ge many).
II.11 Measu emen o s anda d leng h in la ae
Pa ichy e al., 2009 p oposed s anda d leng h (SL), de ined by he dis ance om
he snou o he caudal peduncle, as a eadou o de elopmen al s age (Pa ichy e
al., 2009). SL was measu ed a 7dp in la ae anes he ized in icaine me hane
sul ona e (MS222 168ug/L, Sigma). Images we e acqui ed on he Zeiss S e eo
LUMAR s e eoscope equipped wi h a Hamama sy O ca-ER CCD Came a and EGFP
and Texas Red® il e se s, con olled wi h he Mic oManage 1.14 so wa e.
Measu emen s we e pe o med using he LAS AF Li e so wa e (Leica
Mic osys ems).
48
II.12 Measu emen o body mass index (BMI)
Body weigh (kg) and body leng h (m) o adul ish we e measu ed a 3 mon hs pos
e iliza ion (mp ) and 12mp . Fish we e as en p io o anes hesia in
MS222 (168ug/L, Sigma). Body leng h was measu ed om he head o he end o
he caudal in. Body weigh and body leng h we e used o calcula e he BMI
acco ding o he o mula: BMI = (body weigh kg)/(body leng h m
2
).
II.13 Measu emen o hy oid olume in adul ish
Pa a in blocks we e exhaus i ely sec ioned in o 3-μm- hick sec ions co e ing he
whole hy oid o he adul ish as p e iously desc ibed. Thy oid middle sec ions
we e de e mined acco ding o he dis ance be ween he i s and he las sec ions
displaying hy oid issue and he en al ao a posi ion. 5 longi udinal sec ions o
each ish we e chosen (middle sec ion, 1/3 and 2/3 o o al hy oid sec ions
s a ing om he middle sec ion) (Figu e 5). Images we e acqui ed on a Leica DM
LB2 up igh mic oscope equipped wi h an IDS colo CCD came a using he ollowing
objec i es: 10x 0.25NA and 20x 0.5NA and using he uEye Cockpi so wa e
(Imaging De elopmen Sys ems GmbH, Ge many). To measu e he ollicle a ea,
measu emen s we e pe o med on ImageJ2 (Na ional Ins i u es o Heal h, USA) and
o each ish o al ollicle a ea was calcula ed by he sum o all indi idual a eas. To
calcula e a ep esen a i e o al hy oid olume, o al ollicle a ea and hickness
we e mul iplied.
Figu e 5.
Measu emen o hy oid olume in adul ish using i e 3-μm- hick sec ions
ha would be ep esen a i e o he olume. Pink ci cles ou lined by pu ple dashes
ep esen hy oid ollicles and g ey boxes ep esen 3μm- issue sec ions.
49
II.14 P epa a ion o emb yo lysa es
Emb yos om mRNA expe imen s we e manually decho iona ed and deyolked a
4hp /4hpi (n=50) as desc ibed by Link e al., 2006. Emb yo lysa es we e p epa ed
by adding 100μL o adioimmunop ecipi a ion assay bu e (RIPA) Bu e (10 mM
T is-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% T i on X-100, 0.1% sodium
deoxychola e, 0.1% SDS, 140 mM NaCl and 1 mM PhenylMe hylSul onyl Fluo ide-
PMSF) supplemen ed wi h 1x phospha ase and 1x p o ease inhibi o s
(The moFishe Scien i ic). Emb yos we e mashed wi h a cell pes le o 30 seconds
on ice. P o ein ex ac s we e incuba ed o 30 minu es on ice and cen i uged a
13.000 pm o 10 minu es a 4ºC. The supe na an was collec ed o a new
eppendo ube and s o ed a -80ºC p io o use.
II.15 Dissec ion o hy oid issue in adul zeb a ish
Adul zeb a ish was eu hanized by an o e dose o MS222 (200mg/L, Sigma, MI,
USA) by p olonged imme sion, inse in wa e and hen d ied on a pape owel.
Thy oid issue was dissec ed wi h he help o a Zeiss S e eo LUMAR s e eoscope
and immedia ely ans e ed o an eppendo placed on d y ice un il p epa a ion o
issue lysa es.
II.16 P epa a ion o issue lysa es
Tissue lysa es we e p epa ed by adding 100μL o RIPA Bu e (10 mM T is-Cl pH
8.0, 1 mM EDTA, 0.5 mM EGTA, 1% T i on X-100, 0.1% sodium deoxychola e, 0.1%
SDS, 140 mM NaCl and 1 mM PMSF) supplemen ed wi h 1x phospha ase and 1x
p o ease inhibi o s (The moFishe Scien i ic). Dissec ed issue was mashed wi h a
cell pes le o 45 seconds on ice. P o ein ex ac s we e incuba ed on ice o 30
minu es and cen i uged a 13000 pm o 10 minu es a 4ºC. The supe na an was
collec ed o a new eppendo ube and s o ed a -80ºC p io o use.

50
II.17 Immunoblo ing
To al p o ein ex ac s om issue o emb yo lysa es we e hawed and p epa ed by
adding an equal olume o 2x Laemmli sample bu e (100 mM T is-Cl pH 6.8, 2%
SDS and 20% glyce ol supplemen ed wi h 1/10 olume o B-me cap oe hanol).
Samples we e hea ed a 95ºC o 5 minu es and immedia ely un in 10% sodium
dodecyl sul a e polyac ylamide gel elec opho esis (SDS PAGE) gels. A e p ope
esolu ion, samples we e ans e ed o HyBond-P PVDF memb anes (GE
Heal hca e) p e iously ac i a ed wi h me hanol. Following he ans e , he
memb anes we e s ained wi h Ponceau Red o con i m p o ein loading. Memb anes
we e blocked wi h 5% non- a milk in 1x is-bu e ed saline (TBS)- 20 o 60
minu es in slow shaking. Then, memb anes we e p obed o e nigh a 4ºC wi h
p ima y an ibodies. The ollowing p ima y an ibodies and dilu ions we e used:
Li ing Colo s® DsRed Polyclonal An ibody (dilu ion 1:1000 in 5% non- a milk in 1x
TBS- 20, Clon ech Lab), Ra -B An ibody - C19 sc:166, (dilu ion 1:500 in 5% non-
a milk in 1x TBS- 20, San a C uz Bio echnology), phospho p44/42 MAPK (ERK1/2)
(Th 202/Ty 204) An ibody (dilu ion 1:1000 in 5% non- a milk in 1x TBS- 20, Cell
Signaling), PCNA An ibody FL-261 (dilu ion 1:1000 in 5% non- a milk in 1x TBS-
20, San a C uz Bio echnology), CDKN2a/p16 (F12) An ibody (dilu ion 1:1000 in 5%
non- a milk in 1x TBS- 20, San a C uz Bio echnology), p53 An ibody (dilu ion
1:500 in 5% non- a milk in 1x TBS- 20, Anaspec), An i-ac i e caspase 3 An ibody
(dilu ion 1:500 in 5% non- a milk in 1x TBS- 20, Abcam), An i-pan-Ak (Phospho
T308) An ibody (dilu ion 1:1000 in 5% non- a milk in 1x TBS- 20, Abcam),
Phospho-Ak (Se 473) (D9E) XP® An ibody (dilu ion 1:1000 in 5% non- a milk in
1x TBS- 20, Cell Signaling) and Phospho-p38 MAPKinase (Th 180/Ty 182)
An ibody (dilu ion 1:1000 in 5% non- a milk in 1x TBS- 20, Cell Signaling).
O e nigh p obing was ollowed by washes in 1x TBS- 20 and by incuba ion o 1-
3 hou s a oom empe a u e wi h species-speci ic ho se adish pe oxidase (HRP)-
conjuga ed an ibodies. The ollowing seconda y an ibodies and dilu ions we e
used: goa an i-mouse IgG-HRP An ibody (dilu ion 1:1000 o 1:2000 in 5% non-
a milk in 1x TBS- 20, San a C uz Bio echnology) and goa an i- abbi IgG-HRP
An ibody (dilu ion 1:1000/1:2000 in 5% non- a milk in 1x TBS- 20, San a C uz
Bio echnology, USA). Memb anes we e washed in 1x TBS- 20 and de eloped
manually using he Pie ce™ ECL Plus Wes e n Blo ing Subs a e de ec ion sys em
(The moFishe Scien i ic). P o ein exp ession le els om wes e n blo band
densi ies we e quan i ied on ImageJ2 and no malized o ubulin.
51
II.18 Hea shock and d ug ea men
2 ½ mp ish we e hea -shocked in a 37ºC wa e ba h o 30 minu es and
immedia ely a d ug ea men was ca ied ou . 4-hyd oxyl- amoxi en (4-OHT)
(H7904, Sigma) was dissol ed a 10mM in 100% e hanol and s o ed a -20ºC
(p o ec ed om ligh ). Subsequen dilu ions o p epa e wo king solu ions we e
made immedia ely be o e use, in emb yo medium, o 2.5μM. Con ols we e ea ed
wi h an equi alen amoun o 100% e hanol dilu ed in emb yo medium. Fish
ecei ed ea men a 28ºC o 5 hou s in he da k ollowed by a eco e y pe iod in
esh wa e o 12 hou s.
II.19 S a is ical analysis
S a is ical analysis was pe o med using G aphPad P ism e sion 6.0 (G aphPad
So wa e Inc, San Diego, CA) using T-S uden es and Mann-Whi ney U es . Resul s
we e conside ed s a is ically signi ican when p<0.05 (*p<0.05; **p<0.01;
***p<0.001 and ****p<0.0001) and no s a is ically signi ican when p≥0.05.
52
Chap e III
Resul s
53
III.1 F equency o
TERT
p omo e mu a ions in human cance s
SUPPLEMENTARY INFORMATION FOR JVINAGRE - NCOMMS-13-03296-A
Supplemen a y Figu e S1 - The panel illus a es he h ee mu a ions iden i ied in his
s udy and hei loca ion in ch omosome 5 in ela ion o he TERT ATG ansla ion s a
si e.
Supplemen a y Figu e S2 - Ba plo indica ing he numbe o cases wi h TERT
mu a ions in BRAF-mu a ed and BRAF-wild- ype con en ional papilla y hy oid
ca cinomas (cPTC) and melanomas. Fo bo h umou ypes, he p esence o TERT
mu a ions is signi ican ly associa ed wi h he p esence o BRAF mu a ions (as e isks,
Fishe ’s exac es wo-sided).

Supplemen a y Table S1 - Gene ic al e a ions in hy oid and melanoma cell lines
O igin
Cell line
BRAF
RAS
RET
GNAQ
TERT
Thy oid
XTC-1
WT
WT
ND
-
WT
HTH74
V600E
WT
ND
-
-124G>A
K1
V600E
WT
ND
-
-124G>A
BCPAP
V600E
WT
ND
-
-124/-125GG>AA
C643
WT
HRASG13E
ND
-
-124G>A
8505C
V600E
WT
ND
-
-146G>A
TPC-1
WT
WT
RET/PTC1
-
-124G>A
T238
WT
TT
C634W
WT
MZ-CRC-1
M918T
WT
Skin
melanoma
A375
V600E
WT
WT
-146G>A
BLM
WT
WT
WT
-146G>A
G361
V600E
WT
WT
-138/-139GG>AA
Mewo
WT
WT
WT
-146G>A
U eal
melanoma
92.1
WT
WT
Q209L
WT
OMM1
WT
WT
WT
WT
OMM2.3
WT
WT
Q209L
WT
Mel 202
WT
WT
Q209L
WT
Mel 270
WT
WT
Q209P
WT
Mel 285
WT
WT
WT
WT
*All he cell lines a e deposi ed in he cell line bank om he IPATIMUP and we e au hen ica ed
using DNA p o ile analysis, ob ained wi h he Powe Plex 16 sys em (P omega, Madison, USA),
acco ding o ATCC and HSRRB a ailable DNA p o iles.
Supplemen a y Table S2 - Gene ic al e a ions in cell lines
O igin
De i a ion
Cell line
TERT
Colo ec al
Colon adenoca cinoma
COLO-205
WT
Colon adenoca cinoma
Caco-2
WT
Colon adenoca cinoma
SW 480
WT
Colon adenoca cinoma
HT29
WT
Colon adenoca cinoma
HCT-15
WT
Colon adenoca cinoma
HCT-116
WT
Colon adenoca cinoma
SW48
WT
Colon adenoca cinoma
Co-115
WT
Gas ic
Gas ic adenoca cinoma
AGS
WT
Gas ic adenoca cinoma
MKN28
WT
Well-di e en ia ed gas ic ca cinoma
NCI-N87
WT
Poo ly-di e en ia ed gas ic ca cinoma
SNU-638
WT
Poo ly-di e en ia ed gas ic ca cinoma
MKN-45
WT
Poo ly-di e en ia ed gas ic ca cinoma
Me as asis
KATO III
WT
Di use Gas ic Ca cinoma
IPA220A
WT
Di use Gas ic Ca cinoma
GP202
WT
B eas
B eas adenoca cinoma
MDA-MB-231
-124G>A
B eas adenoca cinoma
MCF-7AZ
WT
B eas adenoca cinoma
SKBR3
WT
B eas adenoca cinoma
MCF-7/6
WT
B eas adenoca cinoma
BT-549
-146G>A
B eas adenoca cinoma
BT-20
WT
B eas adenoca cinoma
MDA-MB-468
WT
B eas adenoca cinoma
SUM149
WT
Kidney
CCRCC
Caki-2
-124G>A
CCRCC
786-O
-124G>A
Leukemia
Acu e p omyelocy ic leukemia
NB-4
WT
Acu e p omyelocy ic leukemia
HL-60
WT
Pe iphe al B
Lymphocy es
Lymphoblas oid cell lines
GM20770
WT
Lymphoblas oid cell lines
GM20890
WT
Lymphoblas oid cell lines
GM11840
WT
Lymphoblas oid cell lines
GM19782 B
WT
Lymphoblas oid cell lines
GM20515
WT
Lymphoblas oid cell lines
GM19777
WT
Lymphoblas oid cell lines
GM20797
WT
Lymphoblas oid cell lines
GM10847 B
WT
Lung
Lung Adenoca cinoma
A549
WT
CNS
Glioblas oma
SF-767
WT
*All he cell lines a e deposi ed in he cell line bank om he IPATIMUP and we e
au hen ica ed using DNA p o ile analysis, ob ained wi h he Powe Plex 16 sys em
(P omega, Madison, USA), acco ding o ATCC and HSRRB a ailable DNA p o iles.
Supplemen a y Table S3 – Summa y o clinico-pa hological and gene ic da a in 56
skin melanoma
Case Code
Melanoma sub ype
Age
Gende
Thickness (mm)
BRAF
NRAS
TERT
MEL1
Supe icial sp eading
74
M
1.20
Pos
MEL2
Supe icial sp eading
36
F
0.95
MEL3
Supe icial sp eading
NA
F
0.50
V600E
MEL4
Supe icial sp eading
NA
M
1.40
-146 G>A
MEL5
Supe icial sp eading
46
F
3.10
MEL6
Nodula
52
F
0.90
MEL7
Nodula
59
M
3.20
V600E
MEL8
Len igo maligna
67
F
0.50
V600E
MEL9
Nodula
54
F
16.00
MEL10
Nodula
33
M
19.50
V600E
MEL11
Supe icial sp eading
57
F
3.65
-146 G>A
MEL12
Len igo maligna
79
F
0
V600E
-146 G>A
MEL13
Ac al len iginous
94
F
2.10
MEL14
Ac al len iginous
50
F
1.00
MEL15
Supe icial sp eading
49
M
10.00
Pos
-124 G>A
MEL16
Supe icial sp eading
55
M
2.30
MEL17
Nodula
82
F
3.50
V600E
MEL18
Nodula
35
F
2.50
V600E
-124 G>A
MEL19
Supe icial sp eading
58
M
7.70
MEL20
Nodula
42
F
1.70
V600E
-124 G>A
MEL21
Nodula
61
F
4.40
-124 G>A
MEL22
Ac al len iginous
69
M
4.00
MEL23
Supe icial sp eading
41
M
0
V600E
MEL24
Ac al len iginous
72
F
0.70
MEL25
Supe icial sp eading
.
M
0.50
MEL26
Nodula
76
M
6.00
MEL27
Supe icial sp eading
66
M
0
-124 G>A
MEL28
Supe icial sp eading
55
F
1.78
MEL29
Ac al len iginous
67
F
4.00
MEL30
Nodula
63
M
5.00
MEL31
Ac al len iginous
60
M
5.40
V600E
Pos
-124 G>A
MEL32
Supe icial sp eading
NA
M
0.50
V600E
-146 G>A
MEL33
Ac al len iginous
71
M
5.20
MEL34
Supe icial sp eading
75
F
70.00
V600E
-124 G>A
MEL35
Nodula
69
F
7.00
V600E
-146 G>A
MEL36
Ac al len iginous
9
F
3.25
MEL37
Supe icial sp eading
39
F
2.30
MEL38
Ac al len iginous
68
M
4.50
MEL39
Ac al len iginous
NA
M
2.70
MEL40
Supe icial sp eading
NA
F
1.20
V600E
-146 G>A
MEL41
Supe icial sp eading
59
F
3.30
V600E
MEL42
Ac al len iginous
NA
F
4.00
MEL43
Len igo maligna
NA
M
5.90
Pos
Supplemen a y Table S4 - Summa y o clinico-pa hological and gene ic da a in 118
gliomas
Case Code
Age
Gende
Diagnosis (G ade)
TERT
GLI1
4
F
Pilocy ic As ocy oma (I)
GLI2
27
M
Pilocy ic As ocy oma (I)
GLI3
22
M
Pilocy ic As ocy oma (I)
GLI4
31
F
Pilocy ic As ocy oma (I)
GLI5
27
M
Pilocy ic As ocy oma (I)
-124 G>A
GLI6
18
M
Pilocy ic As ocy oma (I)
GLI7
30
F
Pilocy ic As ocy oma (I)
GLI8
12
F
Pilocy ic As ocy oma (I)
GLI9
NA
NA
Pilocy ic As ocy oma (I)
GLI10
NA
NA
Pilocy ic As ocy oma (I)
GLI11
NA
NA
Pilocy ic As ocy oma (I)
GLI12
NA
NA
Pilocy ic As ocy oma (I)
GLI13
NA
NA
Pilocy ic As ocy oma (I)
GLI14
18
F
Di use As ocy oma (II)
GLI15
NA
NA
Di use As ocy oma (II)
-124 G>A
GLI16
38
M
Di use As ocy oma (II)
GLI17
22
M
Di use As ocy oma (II)
GLI18
50
M
Di use As ocy oma (II)
GLI19
31
F
Di use As ocy oma (II)
GLI20
30
M
Di use As ocy oma (II)
GLI21
59
M
Di use As ocy oma (II)
GLI22
NA
NA
Di use As ocy oma (II)
GLI23
53
F
Di use As ocy oma (II)
GLI24
20
M
Di use As ocy oma (II)
GLI25
69
M
Di use As ocy oma (II)
-146 G>A
GLI26
42
F
Di use As ocy oma (II)
MEL44
Len igo maligna
NA
F
0.50
MEL45
Ac al len iginous
68
F
2.20
V600E
MEL46
Nodula
71
F
7.80
MEL47
Supe icial sp eading
59
M
1.20
MEL48
Supe icial sp eading
NA
M
0.35
MEL49
Melanoma nodula
42
M
20.00
V600E
-124 G>A
MEL50
Supe icial sp eading
61
F
4.30
V600E
MEL51
Nodula
79
M
22.00
Pos
-146 G>A
MEL52
Len igo maligna
61
F
3.40
MEL53
Ac al len iginous
75
M
6.10
MEL54
Supe icial sp eading
49
F
2.90
V600E
-146 G>A
MEL55
Ac al len iginous
NA
M
4.40
MEL56
Len igo maligna
75
M
0.30
GLI27
27
F
Di use As ocy oma (II)
GLI28
60
F
Di use As ocy oma (II)
-124 G>A
GLI29
NA
NA
Di use As ocy oma (II)
GLI30
NA
NA
Di use As ocy oma (II)
GLI31
NA
NA
Di use As ocy oma (II)
GLI32
NA
NA
Di use As ocy oma (II)
GLI33
NA
NA
Di use As ocy oma (II)
GLI34
72
F
Oligodend oglioma (II)
-124 G>A
GLI35
9
M
Oligodend oglioma (II)
GLI36
33
M
Oligodend oglioma (II)
GLI37
42
F
Oligodend oglioma (II)
-124 G>A
GLI38
46
F
Oligodend oglioma (II)
GLI39
45
F
Oligodend oglioma (II)
-124 G>A
GLI40
43
M
Oligodend oglioma (II)
GLI41
53
M
Oligodend oglioma (II)
-146 G>A
GLI42
53
F
Oligodend oglioma (II)
-124 G>A
GLI43
54
F
Oligodend oglioma (II)
-124 G>A
GLI44
43
F
Oligodend oglioma (II)
-124 G>A
GLI45
34
M
Oligodend oglioma (II)
-146 G>A
GLI46
40
M
Oligodend oglioma (II)
GLI47
45
F
Oligodend oglioma (II)
-124 G>A
GLI48
NA
NA
Oligodend oglioma (II)
GLI49
NA
NA
Oligodend oglioma (II)
GLI50
NA
NA
Oligodend oglioma (II)
GLI51
NA
NA
Oligodend oglioma (II)
GLI52
NA
NA
Oligodend oglioma (II)
GLI53
NA
NA
Oligodend oglioma (II)
GLI54
NA
NA
Oligodend oglioma (II)
GLI55
NA
NA
Oligodend oglioma (II)
-124 G>A
GLI56
65
M
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI57
47
F
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI58
65
F
Anaplas ic Oligodend oglioma (III)
GLI59
50
M
Anaplas ic Oligodend oglioma (III)
-146 G>A
GLI60
36
F
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI61
53
M
Anaplas ic Oligodend oglioma (III)
-146 G>A
GLI62
65
M
Anaplas ic Oligodend oglioma (III)
-146 G>A
GLI63
47
M
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI64
50
M
Anaplas ic Oligodend oglioma (III)
GLI65
70
F
Anaplas ic Oligodend oglioma (III)
GLI66
40
M
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI67
67
F
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI68
64
F
Anaplas ic Oligodend oglioma (III)
-146 G>A
GLI69
44
M
Anaplas ic Oligodend oglioma (III)
GLI70
42
F
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI71
68
F
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI72
NA
NA
Anaplas ic Oligodend oglioma (III)

GLI73
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI74
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI75
NA
NA
Anaplas ic Oligodend oglioma (III)
-124 G>A
GLI76
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI77
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI78
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI79
NA
NA
Anaplas ic Oligodend oglioma (III)
GLI80
56
M
Glioblas oma (IV)
-146 G>A
GLI81
27
F
Glioblas oma (IV)
GLI82
54
F
Glioblas oma (IV)
-124 G>A
GLI83
46
F
Glioblas oma (IV)
GLI84
77
M
Glioblas oma (IV)
-124 G>A
GLI85
67
F
Glioblas oma (IV)
-146 G>A
GLI86
50
F
Glioblas oma (IV)
-146 G>A
GLI87
73
M
Glioblas oma (IV)
-146 G>A
GLI88
57
M
Glioblas oma (IV)
-124 G>A
GLI89
28
M
Glioblas oma (IV)
GLI90
51
F
Glioblas oma (IV)
-124 G>A
GLI91
69
M
Glioblas oma (IV)
-146 G>A
GLI92
53
M
Glioblas oma (IV)
GLI93
56
F
Glioblas oma (IV)
-124 G>A
GLI94
66
M
Glioblas oma (IV)
-124 G>A
GLI95
68
F
Glioblas oma (IV)
GLI96
58
F
Glioblas oma (IV)
GLI97
73
M
Glioblas oma (IV)
-124 G>A
GLI98
61
F
Glioblas oma (IV)
GLI99
66
F
Glioblas oma (IV)
-124 G>A
GLI100
60
F
Glioblas oma (IV)
-124 G>A
GLI101
54
M
Glioblas oma (IV)
-124 G>A
GLI102
66
F
Glioblas oma (IV)
-124 G>A
GLI103
79
M
Glioblas oma (IV)
-124 G>A
GLI104
31
M
Glioblas oma (IV)
GLI105
NA
NA
Glioblas oma (IV)
-124 G>A
GLI106
NA
NA
Glioblas oma (IV)
GLI107
NA
NA
Glioblas oma (IV)
-124 G>A
GLI108
NA
NA
Glioblas oma (IV)
-124 G>A
GLI109
NA
NA
Glioblas oma (IV)
-124 G>A
GLI110
NA
NA
Glioblas oma (IV)
-124 G>A
GLI111
NA
NA
Glioblas oma (IV)
GLI112
NA
NA
Glioblas oma (IV)
GLI113
NA
NA
Glioblas oma (IV)
-124 G>A
GLI114
NA
NA
Glioblas oma (IV)
GLI115
NA
NA
Glioblas oma (IV)
-146 G>A
GLI116
NA
NA
Glioblas oma (IV)
GLI117
NA
NA
Glioblas oma (IV)
GLI118
NA
NA
Glioblas oma (IV)
Supplemen a y Table S5 – Compa ison o he clinico-pa hological ea u es o umou s
wi h and wi hou TERT mu a ions
Tumou
ype
Clinical ea u es
All
pa ien s
TERT wild
ype
TERT
mu a ed
p
alue
Thy oid*
(n=263)
Mean age a
diagnosis, y (±SD)
Gende
Male
Female
Mean umou size,
cm
48±17
61 (24%)
189 (76%)
3.5±3.4
46±16
50 (22%)
174 (78%)
3.4±3.4
62±13
11(42%)
15 (58%)
4.6±2.7
<0,0001
0,03
0,008
CNS
(n=118)
Mean age a
diagnosis, y (±SD)
Gende
Male
Female
47±18
42 (49%)
43 (51%)
38±17
21 (51%)
20 (49%)
57±12
20 (47%)
23 (53%)
<0,0001
NS
Skin
melanoma
(n=56)
Mean age a
diagnosis, y (±SD)
Gende
Male
Female
60±16
26 (46%)
30 (54%)
60±14
19 (48%)
21 (52%)
63±12
7 (44%)
9 (56%)
NS
NS
Bladde
(n=82)
Mean age a
diagnosis, y (±SD)
Gende
Male
Female
63±10
66 (80%)
16 (20%)
65±12
24 (70%)
10 (30%)
63±12
42 (88%)
6 (12%)
NS
NS
The numbe s in pa en heses indica e pe cen ages. In some cases in o ma ion ega ding age,
gende o umou size was missing.
*Only ollicula cell-de i ed hy oid ca cinomas: FTC, PTC, PDTC and ATC.
Supplemen a y Table S6 – Clinico-pa hological cha ac e is ics and TERT p omo e mu a ion in he 399 hy oid samples analysed
His o ypes
No. O cases
Gende a io (♀:♂)
Mean age (yea s±SE)
Tumou size (cm±SE)
BRAFV600E mu a ion
TERT n (%)
No mal Thy oid
27
3,5:1
47,0 ± 14,8
NA
0
0 (0,0)
Lymphocy ic
Thy oidi is
9
9:0
44,6 ± 15,7
NA
0
0 (0,0)
Nodula Goi e
12
1,75:1
56,2 ± 13,4
NA
0
0 (0,0)
Follicula Thy oid Adenoma
Con en ional
52
7:1
43,8 ± 12,1
3,5 ± 1,6
0
0 (0,0)
Oncocy ic Va ian
8
7:0
47,0 ± 14,0
2,7 ± 1,2
0
0 (0,0)
Follicula Thy oid Ca cinoma
Con en ional
36
2,5:1
53,7 ± 14,7
4,5 ± 2,8
0
9 (25,0)
Oncocy ic Va ian
28
5,5:1
51,4 ± 20,2
4,6 ± 3,6
0
0 (0,0)
Papilla y hy oid ca cinoma
Con en ional
110
3,1:1
43,1 ± 16,2
2,6 ± 1,6
61
12 (10,9)
Follicula Va ian
39
3,9:1
45,8 ±14,7
2,6 ± 1,2
2
0 (0,0)
Mic oca cinoma
5
5:0
46,0 ±14,4
0,4 ± 0,4
1
0 (0,0)
Mucoepide moid
Ca cinoma
4
2:0
43,5 ± 33,2
NA
0
1 (25,0)
Oncocy ic Va ian o PTC
Con en ional
Pa e n
4
4:0
45,3 ±11,1
2,8 ± 1,8
4
0 (0,0)
Follicula Pa e n
3
2:1
45,3 ±22,5
5,2 ± 3,9
1
0 (0,0)
Wa hin-like PTC
4
4:0
55,5 ±11,2
2,0 ± 0,8
4
0 (0,0)
PDTC
14
1:1
57,0 ± 17,4
6,7 ± 2,8
1
2 (14,2)
Anaplas ic
16
1,3:1
62,1 ± 12,8
6,9 ± 3,1
5
2 (12,5)
MTC
28
2,3:1
46,4 ± 16,7
3,0 ± 0,9
0
0 (0,0)
Supplemen a y Table S7 - Summa y o clinico-pa hological and gene ic da a in 351
umou and umou like hy oid lesions
Case Code
Age
Gende
Diagnosis
RAS
BRAF
TERT
THY1
50
F
FTA
THY2
50
F
FTA
THY3
59
M
FTA
THY4
28
F
FTA
THY5
45
F
FTA
THY6
51
F
FTA
THY7
37
F
FTA
THY8
53
F
FTA
THY9
57
F
FTA
THY10
66
F
FTA
THY11
63
F
FTA
THY12
40
F
FTA
THY13
34
F
FTA
THY14
65
F
FTA
THY15
35
F
FTA
THY16
34
F
FTA
THY17
18
F
FTA
THY18
37
F
FTA
THY19
43
F
FTA
THY20
53
F
FTA
THY21
45
F
FTA
THY22
33
F
FTA
THY23
34
F
FTA
THY24
46
M
FTA
THY25
53
F
FTA
THY26
65
F
FTA
THY27
29
F
FTA
THY28
34
F
FTA
THY29
34
F
FTA
THY30
NA
NA
FTA
THY31
NA
NA
FTA
THY32
56
F
FTA
THY33
33
F
FTA
THY34
47
M
FTA
THY35
30
F
FTA
THY36
55
M
FTA
THY37
NA
F
FTA
THY308
60
M
Anaplas ic
V600E
-124 G>A
THY309
72
M
Anaplas ic
THY310
NA
NA
Anaplas ic
V600E
THY311
74
F
Anaplas ic
V600E
THY312
52
F
Anaplas ic
THY313
55
M
Anaplas ic
THY314
NA
NA
Anaplas ic
THY315
NA
F
Anaplas ic
THY316
56
F
Anaplas ic
THY317
59
F
Anaplas ic
THY318
75
F
Anaplas ic
V600E
THY319
82
F
Anaplas ic
THY320
69
M
Anaplas ic
V600E
THY321
39
M
Anaplas ic
THY322
70
M
Anaplas ic
THY323
44
F
Anaplas ic
-146 G>A
THY324
63
M
MTC
THY325
NA
NA
MTC
THY326
NA
NA
MTC
THY327
32
F
MTC
THY328
NA
NA
MTC
THY329
NA
NA
MTC
THY330
NA
NA
MTC
THY331
NA
NA
MTC
THY332
NA
NA
MTC
THY333
NA
NA
MTC
THY334
68
F
MTC
THY335
NA
NA
MTC
THY336
37
F
MTC
THY337
NA
NA
MTC
THY338
71
F
MTC
THY339
40
M
MTC
THY340
NA
NA
MTC
THY341
56
F
MTC
THY342
NA
NA
MTC
THY343
38
F
MTC
THY344
NA
NA
MTC
THY345
NA
NA
MTC
THY346
23
M
MTC
THY347
NA
NA
MTC
THY348
36
F
MTC
THY349
NA
NA
MTC
THY350
NA
NA
MTC
THY351
NA
NA
MTC

Supplemen a y Table S8 – Compa ison o he clinico-pa hological ea u es o
con en ional PTC wi h and wi hou TERT mu a ions
Tumou
ype
Clinical ea u es
All
pa ien s
TERT wild
ype
TERT
mu a ed
p
alue
Con en ional
Papilla y
Thy oid
Ca cinoma
(n=110)
Mean age a
diagnosis, y (±SD)
Gende
Male
Female
Mean umou size,
cm
LN me as asis
(posi i e/ o al)
BRAF mu a ion
(posi i e/ o al)
NRAS mu a ion
(posi i e/ o al)
43±16
26 (25%)
80 (75%)
2,6±1,6
67 (73%)
61 (57%)
4(7%)
41±15
18 (19%)
76 (81%)
2,4±1,4
56 (68%)
49 (50%)
4 (10%)
63±14
8 (67%)
4 (33%)
4,3±2,2
11 (100%)
12 (100%)
0 (0%)
<0,0001
0,0012
0,0004
0,03
0,001
NS
Supplemen a y Table S9 - Summa y o clinico-pa hological and gene ic da a in 82
bladde cance s
Case Code
Age
Gende
S age
G ade
TERT
BC1
66
M
Ta
Low G ade
-146 G>A
BC2
NA
M
T1
Low G ade
BC3
80
M
Ta
Low G ade
-146 G>A
BC4
70
M
Ta
Low G ade
BC5
67
M
Ta
Low G ade
-124 G>A
BC6
80
M
Ta
Low G ade
-124 G>A
BC7
65
M
Ta
Low G ade
-124 G>A
BC8
40
M
Ta
Low G ade
-124 G>A
BC9
36
M
T1
Low G ade
-124 G>A
BC10
64
M
Ta
Low G ade
BC11
65
M
Ta
Low G ade
-124 G>A
BC12
47
F
Ta
Low G ade
BC13
54
M
Ta
Low G ade
-124 G>A
BC14
41
M
T1
Low G ade
BC15
50
M
T1
Low G ade
-124 G>A
BC16
67
M
Ta
Low G ade
-124 G>A
BC17
97
M
Ta
Low G ade
-146 G>A
BC18
40
F
Ta
Low G ade
-124 G>A
BC19
60
M
Ta
Low G ade
BC20
70
M
T1
Low G ade
-146 G>A
BC21
46
F
Ta
Low G ade
BC22
61
F
Ta
High G ade
-146 G>A
BC23
NA
M
T1
High G ade
-124 G>A
BC24
59
M
T1
High G ade
-124 G>A
BC25
77
M
T1
High G ade
-146 G>A
BC26
65
M
T1
High G ade
-124 G>A
BC27
83
F
T1
High G ade
-124 G>A
BC28
64
M
T1
High G ade
BC29
NA
M
NA
High G ade
-124 G>A
BC30
NA
M
NA
High G ade
-146 G>A
BC31
67
M
T1
High G ade
-124 G>A
BC32
NA
M
Ta
High G ade
BC33
NA
M
T1
High G ade
BC34
NA
M
T1
High G ade
BC35
NA
M
T1
High G ade
-146 G>A
BC36
74
M
T1
High G ade
-124 G>A
BC37
75
M
Ta
High G ade
BC38
76
F
T1
High G ade
BC39
45
M
Ta
High G ade
BC40
NA
M
T1
High G ade
BC41
76
F
T1
High G ade
BC42
NA
M
T1
High G ade
-124 G>A
BC43
72
F
Ta
High G ade
BC44
NA
F
T1
High G ade
BC45
67
M
T1
High G ade
-124 G>A
BC46
NA
M
T1
High G ade
-146 G>A
BC47
NA
M
T1
High G ade
-146 G>A
BC48
51
M
T1
High G ade
-146 G>A
BC49
50
M
Ta
High G ade
-124 G>A
BC50
74
M
T1
High G ade
-124 G>A
BC51
70
F
T1
High G ade
-124 G>A
BC52
50
M
T1
High G ade
-146 G>A
BC53
68
M
T1
High G ade
-124 G>A
BC54
56
M
T1
High G ade
BC55
53
M
T1
High G ade
BC56
60
F
T1
High G ade
BC57
74
M
T1
High G ade
BC58
73
M
T1
High G ade
BC59
60
F
T1
High G ade
BC60
64
F
T1
High G ade
BC61
74
M
T1
High G ade
BC62
51
M
T1
High G ade
BC63
63
M
T1
High G ade
-124 G>A
BC64
57
M
Ta
High G ade
-146 G>A
BC65
67
F
Ta
High G ade
-146 G>A
BC66
58
M
Ta
High G ade
-124 G>A
BC67
66
M
T1
High G ade
-124 G>A
BC68
64
M
T1
High G ade
BC69
70
M
T1
High G ade
-146 G>A
BC70
68
M
Ta
High G ade
-146 G>A
BC71
64
M
T1
High G ade
BC72
59
F
T1
High G ade
-124 G>A
BC73
63
M
T1
High G ade
-124 G>A
BC74
66
M
Ta
High G ade
BC75
76
M
T1
High G ade
-124 G>A
BC76
71
M
T1
High G ade
BC77
61
M
T1
High G ade
-124 G>A
BC78
76
M
T1
High G ade
-124 G>A
BC79
61
M
T1
High G ade
BC80
68
F
T1
High G ade
BC81
72
M
T1
High G ade
BC82
71
M
T1
High G ade
-146 G>A
Supplemen a y Table S10 - Summa y o clinico-pa hological and gene ic da a in 26
kidney cance s
CCRC – Clea cell enal ca cinoma; C omRCC – ch omophobe enal cell cance ; PRCC – Papilla y
enal cell ca cinoma;
Case code
Diagnosis
Age
Gende
Nuclea g ade
S aging
TERT
RCC1
CCRCC
68
M
RCC2
CCRCC
44
M
RCC3
CCRCC
49
M
RCC4
CCRCC
49
M
3
pT1a Nx Mx
RCC5
CCRCC
57
M
2
pT1a Nx Mx
RCC6
CCRCC
48
F
2
pT1a Nx Mx
RCC7
CCRCC
48
F
2
pT1a Nx Mx
RCC8
CCRCC
68
M
2
pT3a Nx Mx
RCC9
CCRCC
50
M
4
pT3b Nx M0
RCC10
CCRCC
50
M
4
pT3b Nx M1
RCC11
CCRCC
62
M
3
pT2 Nx Mx
RCC12
CCRCC
62
M
3
pT2 Nx Mx
RCC13
C omRCC
59
M
RCC14
C omRCC
77
F
2
pT2 Nx Mx
RCC15
C omRCC
77
F
2
pT2 Nx Mx
RCC16
C omRCC
72
M
4
pT2 Nx Mx
RCC17
PRCC
NA
M
RCC18
PRCC
61
M
RCC19
PRCC
26
F
2
pT2 N2 MX
RCC20
PRCC
78
M
2
pT1b Nx Mx
RCC21
PRCC
34
M
1
pT1a Nx Mx
RCC22
PRCC
60
M
pT1b Nx Mx
RCC23
PRCC
60
M
pT1b Nx Mx
RCC24
PRCC
71
M
RCC25
PRCC
71
M
RCC26
PRCC
75
M
3
pT1a Nx Mx

Supplemen a y Table S11 - Summa y o clinico-pa hological and gene ic da a in 17
phaeoch omocy oma
Case code
Age
Gende
Tumo size (cm)
MEN/NF/VHL
TERT
PHEO1
51
M
4,5
NA
PHEO2
56
M
7
NA
PHEO3
54
F
4
NA
PHEO4
46
F
7
NA
PHEO5
53
F
2,5
NA
PHEO6
42
M
8,5
NA
PHEO7
20
F
1,5
MEN2
PHEO8
55
F
3,5
NA
PHEO9
19
F
7
NA
PHEO10
56
M
4,5
NA
PHEO11
62
M
6
NA
PHEO12
45
M
7
NA
PHEO13
73
F
5
NA
PHEO14
57
M
3,5
NA
PHEO15
61
M
1,7
NA
PHEO16
20
F
6
MEN2
PHEO17
46
F
10
NF1
Supplemen a y Table S12 - Summa y o clinico-pa hological and gene ic da a in 36
gas oin es inal s omal umou
Case code
Age
Sex
Tumo Size (cm)
KIT
PDGFRA
TERT
GIST1
80
F
exon 11 N567K, L576V, 568del575
GIST2
59
M
1,2
exon 11 V560D
GIST3
52
M
9,5
exon 11 69997:T>A
GIST4
67
M
17
exon 11 W557F delK558
GIST5
58
F
3,5
exon 11 556del557
GIST6
54
M
19
exon 11 V559D
GIST7
79
F
7
exon 11 del557-558
GIST8
46
M
5,5
exon 11 del 550-557, ins550-551
GIST9
54
M
3,1
exon 11 552del557, K550Q, P551R
GIST10
76
F
6,7
exon 18 D842V
GIST11
55
F
6,5
exon 11 del560V
GIST12
43
M
14,5
exon 11 564del578
GIST13
73
F
0,8
exon 11 delV559
GIST14
62
M
2,5
exon 11 del_K558
GIST15
38
M
3,5
exon 11 V560E
GIST16
88
F
23
exon 11 L576P
GIST17
81
F
exon 11 L576P
GIST18
78
F
exon 12 583del586
GIST19
40
F
4
exon 9 502-503dup
GIST20
63
F
9
exon 9 502-503dup
GIST21
75
M
8
exon 11 Q575H, P577T, delL576
GIST22
59
F
exon 11 552del570
GIST23
78
F
24
exon 11 551del553
GIST24
58
M
13,5
exon 11 557del558
GIST25
76
M
14
exon 9 502-503dup
GIST26
20
F
2
GIST27
77
F
6
GIST28
74
M
3
GIST29
58
F
3,5
GIST30
72
F
8
GIST31
62
M
6,5
GIST32
73
M
6
GIST33
66
F
14
GIST34
63
F
6,5
GIST35
55
M
4
GIST36
82
F
7
83
III.2 Ta ge ed exp ession o BRAF
V600E
in hy oid cells o
ansgenic zeb a ish induces hype plasia e e ed by loss o
WT p53
Abs ac
The BRAF
V600E
mu a ion is he mos common gene ic al e a ion in papilla y hy oid
ca cinomas (PTCs) and a e ound in 29%-83% o all cases. PTCs ha bo ing B- ype
Ra kinase (BRAF)
V600E
a e o en in asi e and his mu a ion is also p esen in mo e
ad anced s ages o he disease such as in poo ly di e en ia ed and anaplas ic
ca cinomas a ising om PTCs. BRAF
V600E
kinase ac i a e he mi ogen-ac i a ed
p o ein kinases (MAPK) pa hway, p omo ing cellula p ocesses such as
p oli e a ion, su i al, mo ili y and in asion.
To explo e he ole o BRAF
V600E
in hy oid cance pa hogenesis, I a ge ed i s
exp ession o hy oid cells o ansgenic zeb a ish unde a hy oid-speci ic
p omo e . Fish showed impai men o no mal hy oid mo phogenesis a ea ly
s ages o de elopmen and hey seemed o compensa e o BRAF
V600E
-induced
hy oid dys unc ion by de eloping hype plasia and goi e bu no neoplasia. Also,
ac i a ion o BRAF
V600E
exp ession in hy oid cells o ansgenic zeb a ish du ing
adul hood esul ed in a hype plasic pheno ype simila o when BRAF
V600E
was
exp essed sho ly a e bi h. In BRAF
V600E
-exp essing animals, up egula ion o p53
was obse ed sugges ing a p o ec i e mechanism o p e en cance . Thus, I
a ge ed BRAF
V600E
exp ession o hy oid cells o p53
M214K
zeb a ish in o de o
o e come p53 blockage and p og ession o cance . Sup isingly, BRAF
V600E
-
exp essing p53
M214K
ish de eloped a no mal hy oid un il adul hood.
In summa y, hy oid-speci ic exp ession o BRAF
V600E
induced goi e bu no
neoplasia which was p e en ed by loss o WT p53 p o ein.
In oduc ion
To c ea e unc ional ollicles, hy oid cells mus agg ega e, pola ize and es ablish
selec i e pe meabili y ba ie s be ween he lumen and he ou e compa men s.
Cellula adhesion, in acellula a icking, specialized cell-cell junc ion assembly
84
and p ecise egula ed mo phogene ic cell mo emen s mus all be coo dina ed (Yap
e al., 1997).
Dis u bances in hese cellula p ocesses can be media ed by ex e nal ac o s such
as goi ogens o in e nal ac o s such as ho monal imbalance o gene ic al e a ions.
Ul ima ely, hese ac o s ha e impo an implica ions in hy oid diseases, mos ly
cha ac e ized by dis u bed ollicula a chi ec u e.
PTCs a e cha ac e ized by nono e lapping gene ic al e a ions in mo e han 70% o
he cases. These al e a ions include ea angemen s in he y osine kinase (TK)
ecep o s Rea anged-du ing- ans ec ion (RET), Neu o ophic Ty osine Kinase,
Recep o , Type 1 (NTRK1), Anaplas ic Lymphoma Recep o Ty osine Kinase (ALK)
and A Kinase (PRKA) Ancho P o ein 9/B- ype Ra kinase (AKAP9/BRAF) and also
poin mu a ions in Ra Sa coma Vi al Oncogene Homolog (RAS) and BRAF genes.
Ul ima ely, mos cases will lead o an abe an ac i a ion o he RAS-RAF-MEK-ERK
kinase pa hway (DeLellis e al., 2004; Kuma e al., 2005; Niki o o , 2012). Up o
90% o he BRAF gene mu a ions consis in he BRAF
V600E
(Ga ne e al., 2004; F asca
e al., 2008).
The ini ial e idence ha BRAF
V600E
is equi ed o he cell p oli e a ion,
ans o ma ion and umo igenici y o ollicula hy oid cells was demons a ed in a
xenog a mice model (Liu e al., 2007) e en hough many s udies ha e suppo ed
he ole o BRAF mu a ion in umo ini ia ion.
Ta ge ed exp ession o he BRAF
V600E
in hy oid cells was induced in ansgenic
FVB/N mice using a bo ine hy oglobulin p omo e . This s udy showed ha BRAF
induced hy oid dys unc ion which was compensa ed by inc eased le els o
hy oid-s imula ing ho mone (TSH) and goi e de elopmen . Also, mul i ocal
umo s in ol ing bo h lobes o he hy oid gland wi h mixed papilla y and ollicula
g ow h pa e n we e obse ed in 12 and 22-week-old mice. PTCs p esen ed he
classical a chi ec u e, all-cell ea u es and a high po en ial o in asi eness.
Tumo s om one o he ansgenic lines p og essed in o poo ly di e en ia ed
ca cinomas (Knau e al., 2005). PTCs we e also obse ed in a hy ocy e-speci ic
knock-in o BRAF
V600E
in mice bu wi h a e y sho la ency and comple e pene ance
by 3 weeks. When his model was c ossed wi h a hy oid s imula ing ho mone
ecep o (TshR) knockou mice o gene ically abla e TSH signaling, hy oid g ow h
was educed and low-g ade PTCs we e obse ed bu only a 9 weeks o age (F anco
e al., 2011). Mo e e idence o BRAF
V600E
in ol emen in mice hy oid umo igenesis
was pe cei ed when BRAF
V600E
exp ession was induced in ollicula hy oid cells in a
91
Figu e 3
. g(
g
:mCh) line. (A) 14dp g( g:mCh) la ae e ealed (B) mChe y p o ein
exp ession speci ically in hy oid cells. La ae is o ien ed wi h an e io o he le . Scale ba :
250μM.
A his poin , EGFP epo e exp ession in he hea was used o selec animals om
he BRAF
V600E
-exp essing line. By c ossing he g( g:mCh) wi h he g( g:mCh-T2A-
BRAF
V600E
) line, a g( g:mCh; g:mCh-T2A- BRAF
V600E
) line was gene a ed in o de o
ha e a hy oid-speci ic epo e in he BRAF
V600E
-exp essing line. Emb yos showing
mChe y exp ession in he hy oid gi en by he Tol2 g:mCh ansgene and EGFP
exp ession in he hea gi en by he Tol2CG2 g:mCh-T2A-BRAF
V600E
ansgene we e
g own o adul hood.
Because he g( g:mCh-T2A- BRAF
V600E
) line was no exp essing mChe y, an
expe imen o de e mine he p o ein ansla ion e iciency om mCh-T2A-BRAF
V600E
was pe o med in o de o exclude possible p oblems wi h he 2A clea age.
A pCS2+ exp ession plasmid was chosen because i con ains a s ong
enhance /p omo e (simian CMV) ollowed by a polylinke and a SV40 la e
polyadenyla ion si e. Also, a SP6 p omo e is p esen in he 5’ un ansla ed egion
o he mRNA om he sCMV p omo e which allows in i o RNA syn hesis o
sequences cloned in o he polylinke . mChe y coding sequence was used as a
con ol and mCh-T2A-BRAF
WT
was used as a con ol o T2A e iciency and BRAF
oxici y, because BRAF
w
p o eins do no ha e an e ec on he de elopmen o
zeb a ish emb yos (Anas asaki e al., 2012). Capped mRNA was in i o ansc ibed
om he cons uc s gene a ed: pCS2-CMV:mCh, pCS2- CMV:mCh-T2A-BRAF
WT
and
pCS2-CMV:mCh-T2A-BRAF
V600E
(Figu e 4A).

92
A e injec ing capped mCh-T2A-BRAF
V600E
mRNA in WT emb yos, a se e e
de elopmen al a es was ound a a ound 4hpi p e en ing emb yos o u he
de elop beyond he blas ula s age (Figu e 4B). Mal o ma ions we e ound in he
majo i y o injec ed emb yos (90.43%) (Figu e 4C) and su i al a 24 hou s pos
injec ion (hpi) was e y low (34.8%) (Figu e 4D). By luo escence mic oscopy, a
polka-do pa e n was obse ed con i ming mChe y exp ession in he cell (Figu e
4B). The pheno ype p oduced was belie ed o be due o exp ession o mChe y and
BRAF
V600E
because no pheno ypes we e obse ed in non-injec ed WT emb yos
(Figu e 4B). WT emb yos injec ed wi h capped mCh o mCh-T2A- BRAF
WT
mRNAs
showed no de elopmen al a es (Figu e 4B), a e y low equency o mal o ma ions
(0.76% and 0.64%, espec i ely) (Figu e 4C) and su i al a 24hpi (85.8% and 75.5%,
espec i ely) no signi ican ly di e en om non-injec ed WT emb yos (Figu e 4D).
By luo escence mic oscopy, i was obse ed an ubiqui ous exp ession o mChe y
in WT emb yos injec ed wi h capped mCh o mCh-T2A-BRAF
WT
mRNAs (Figu e 4B)
which was consis en wi h he obse a ions epo ed by Anas asaki e al., 2012. In
o de o con i m i BRAF
V600E
was indeed inducing a pheno ype, a wes e n blo o
emb yo lysa es was pe o med by p obing wi h an ibodies o mChe y and human
BRAF. Blo s con i med he p esence o bo h mChe y and BRAF p o eins in emb yos
injec ed wi h mCh-T2A-BRAF
V600E
mRNA and BRAF p obing was simila o hose
injec ed wi h he WT o m o BRAF (Figu e 4E; E’ and E’’).
93
94
95
Figu e 4.
mRNA expe imen in WT emb yos. (A) Schema ic ep esen a ion o he pCS2-
CMV:mCh, pCS2-CMV:mCh-T2A-BRAF
WT
and pCS2-CMV:mCh-T2A-BRAF
V600E
cons uc s
( op o bo om) con aining he SP6 p omo e o d i e in i o RNA syn hesis. (B)
Rep esen a i e images o 24hp non-injec ed and 24hpi injec ed WT emb yos wi h one o
he h ee mRNAs: mCh, mCh-T2A-BRAF
WT
o mCh-T2A- BRAF
V600E
. On he le a e shown BF
images; on he igh images acqui ed wi h a Texas Red® il e . Scale ba : 300μm. (C)
Pe cen age o de elopmen al de ec s a 24hp o 24hpi (s a is ical analysis only shown o
ele an compa isons; p<0.001). (D) Su i al a 24hp o 24hpi (s a is ical analysis only
shown o ele an compa isons; p<0.001). (E) Wes e n blo analysis o lysa es om
emb yos a 4hp o 4hpi. 50 emb yos we e pooled o each condi ion. Memb anes we e
blo ed wi h mCh and BRAF an ibodies. Ponceau S s ain showed equal amoun o p o ein in
all lanes. (E’) mChe y and (E’’) BRAF p o ein le els we e quan i ied and no malized o he
amoun o p o ein. Values a e shown in ba g aphs and ep esen h ee independen
expe imen s (s a is ical analysis only shown o ele an compa isons; p<0.001).
To ule ou a possible ac i a ion o p53-media ed apop osis due o o - a ge ing
e ec o he mRNA injec ed (Robu e al., 2007), an mRNA expe imen was
conduc ed in p53
M214K
emb yos. p53
M214K
emb yos lack apop osis and cell-cycle
a es esponses o deoxy ibonucleic acid (DNA) damage because he p53-
media ed cell-cycle con ol is absen (Be ghmans e al., 2005).
96
A e injec ing capped mCh-T2A-BRAF
V600E
mRNA in p53
M214K
emb yos, a lowe
de elopmen al a es was ound in emb yos a a ound 4hpi (Figu e 5A) when
compa ed o p53
M214K
emb yos. Mal o ma ions we e ound in injec ed emb yos
(39.40%) (Figu e 5B) and su i al was low (33.00%) (Figu e 5C). By luo escence
mic oscopy, ubiqui ous exp ession o mChe y was obse ed (Figu e 5A). In
con as , p53
M214K
emb yos injec ed wi h capped mCh o mCh-T2A- BRAF
WT
mRNAs
showed no de elopmen al a es (Figu e 5A), e y low equency o mal o ma ions
(1.00% and 0.28%, espec i ely) (Figu e 5B) and su i al a 24hpi (92.60% and
85.50%, espec i ely) (Figu e 5C) simila o non-injec ed p53
M214K
emb yos (0.00%
and 98% o mal o ma ion equency and su i al, espec i ely) (Figu e 5B and 5C).
Because a less se e e pheno ype was obse ed in BRAF
V600E
-exp essing p53
M214K
emb yos, a wes e n blo o emb yo lysa es was conduc ed o de e mine whe he
he pheno ype would be due o a dec ease o BRAF exp ession. Blo s con i med he
p esence o bo h mChe y and BRAF p o eins in p53
M214K
emb yos injec ed wi h
mCh-T2A-BRAF
V600E
mRNA bu BRAF p o ein le els we e signi ican ly lowe han
hose ound o BRAF
WT
-exp essing p53
M214K
emb yos (Figu e 5D; D’ and D’’)
(p<0.001) o e en BRAF
V600E
-exp essing WT emb yos (Figu e 4E’’).

97
98
Figu e 5.
mRNA expe imen in
p53
M214K
emb yos. (A) Rep esen a i e images o 24hp
non-injec ed and 24hpi p53
M214K
emb yos injec ed wi h one o he h ee mRNAs: mCh,
mCh-T2A-BRAF
WT
o mCh-T2A-BRAF
V600E
. On he le a e shown BF images; on he igh
99
images acqui ed wi h a Texas Red® il e . Scale ba : 300μm. (B) Pe cen age o
de elopmen al de ec s a 24hp o 24hpi (s a is ical analysis only shown o ele an
compa isons; p<0.001). (C) Su i al a 24hp o 24hpi (s a is ical analysis only shown o
ele an compa isons; p<0.001). (D) Wes e n blo analysis o lysa es om emb yos a 4hp
o 4hpi. 50 emb yos we e pooled o each condi ion. Memb anes we e blo ed wi h mCh
and BRAF an ibodies. Ponceau S s ain showed equal amoun o p o ein in all lanes. (D’)
mChe y and (D’’) BRAF p o ein le els we e quan i ied and no malized o he amoun o
p o ein. Values a e shown in ba g aphs and ep esen h ee independen expe imen s
(s a is ical analysis only shown o ele an compa isons; p<0.001).
Taken oge he , mRNA expe imen s showed ha BRAF
V600E
-induced pheno ype in
zeb a ish emb yos was condi ioned by p53 and he p esence o a WT p53 p o ein
igge ed p o ec i e mechanisms (possibly apop osis and/o cell cycle a es )
agains he oncogene.
The mRNA expe imen s also con i med an e icien ansla ion o wo independen
p o eins om mCh-T2A-BRAF
V600E
mRNA. This sugges ed ha , independen ly o he
absence o mChe y exp ession in he g( g:mCh-T2A-BRAF
V600E
) ish, he mu an
o m o BRAF was able o be ansla ed. To con i m BRAF exp ession in he s able
g( g:mCh-T2A-BRAF
V600E
) line, wes e n blo s o p o ein ex ac s om hy oid
issues o h ee mon h-old ish (Figu e 6A) using a BRAF an ibody aised agains a
pep ide o human o igin we e pe o med. I was con i med an inc ease in he le els
o BRAF when compa ed o g( g:mCh) (Figu e 6B). Also, a obus inc ease in he
le els o phospho-
Ex acellula Regula ed Kinase (pERK), a downs eam o BRAF,
con i med ac i a ion o he MAPK pa hway by BRAF
V600E
(Figu e 6B). This sugges ed
ha he BRAF p o ein was indeed unc ional in hy oid cells o he ansgenic line.
100
Figu e 6.
BRAF and pERK exp ession in g(
g
:mCh-T2A-BRAF
V600E
) ish. (A) Schema ic
ep esen a ion o he hy oid issue dissec ed om mCh-exp essing ansgenic zeb a ish
o p oduce issue lysa es. All ollicles we e dissec ed and sepa a ed om non-mCh
exp essing issues. (B) Wes e n blo analysis o lysa es om hy oid issues o g( g:mCh)
( i s lane) and g( g:mCh-T2A-BRAF
V600E
) (second lane) ish a 3 mon hs o age. Th ee ish
we e pooled o each line. Memb anes we e blo ed wi h BRAF and pERKs an ibodies.
Ponceau S showed equal amoun o p o ein in bo h lanes.
107
Pa adoxically, BRAF
V600E
was also ound o induce senescence and apop osis by
blocking p oli e a ion (Wajapeyee e al., 2008). In BRAF
V600E
-exp essing ansgenic
animals i was be expec ed he opposi e obse a ion as p oli e a ion was being
induced.
p16, an impo an media o in blocking cell p og ession om G1 o S phase, was
e alua ed o exclude BRAF
V600E
-induced senescence as a es aining ac o o
malignancy. p16 p o ein le els we e down egula ed in g( g:mCh-T2A-BRAF
V600E
)
ish (p>0.05) (Figu e 8F and F’) sugges ing ha BRAF
V600E
did no induce senescence.
Ac i a ed caspase 3, an impo an p o ein in he execu ion-phase o cell apop osis,
was also e alua ed o exclude BRAF
V600E
-induced apop osis as a mechanism o clea
gene ically uns able cells. Sup isingly, ac i a ed caspase 3 p o ein le els we e
inc eased in g( g:mCh-T2A-BRAF
V600E
) ish (p<0.0001) (Figu e 8F and F’).
To con i m i caspase ac i a ion was induced by p53 as a esponse o oncogenic
s ess, p53 p o ein le els we e assessed. Inc eased le els o p53 p o ein we e
ound in g( g:mCh-T2A-BRAF
V600E
) ish (p<0.0001) (Figu e 8F and F’) con i ming
ha apop osis was igge ed in BRAF
V600E
-exp essing hy oid cells.
To de e mine whe he p53 ac i a ion was media ed indi ec ly by BRAF
V600E
h ough
BRAF
V600E
-media ed p38 ac i a ion, le els o p38 we e assessed. p38 p o ein le els
in g( g:mCh-T2A-BRAF
V600E
) ish we e no di e en om he con ols (p>0.05)
(Figu e 8F and F’).

108
109
110
Figu e 8.
Cha ac e iza ion o g(
g
:mCh-T2A-BRAF
V600E
) ju eniles. (A) Rep esen a i e
pheno ypes o g( g:mCh) and g( g:mCh-T2A-BRAF
V600E
) a 3 mon hs o age. Scale ba :
5mm. (B) BMI measu ed om g( g:mCh) (n=39) and g( g:mCh-T2A-BRAF
V600E
) (n=39)
(p<0.0001). (C) HE s ain o longi udinal sec ions ep esen a i e o he lowe jaw a 2- and
3mp . Scale ba : 250μm. C1’’ and C2’’ a e highe magni ica ions o he ollicles. Scale ba :
62μm. (D) Incidence o hype plasia a 3mp . Values co espond o he pe cen age o ish
wi h hype plasia con i med by his ology ( en ish we e s udied om each line) (p<0.0001).
(E) Rep esen a i e hy oid olume assessed in issue sec ions. Values co espond o he
olume o each ish s udied; en in o al o each line (p<0.05). (F) Wes e n blo analysis o
lysa es om hy oid issue a 3mp . Th ee ish we e pooled o each line. Memb anes we e
blo ed wi h ele an an ibodies. Ponceau S is shown in Figu e 6. (F’) P o ein le els we e
quan i ied and no malized o he amoun o p o ein. Values a e shown in ba g aphs and
ep esen h ee independen expe imen s (s a is ical analysis only shown o ele an
compa isons; p<0.05, p<0.01 and p<0.0001).
g( g:mCh-T2A-BRAF
V600E
)
ish we e moni o ed un il hey each 12 mon hs o age.
A his s age, he di e ences in body weigh and body leng h p o ed no o be
ansien (Figu e 9A) as e lec ed by a 11% dec ease in body mass when compa ed
o g( g:mCh) ish (p<0.001) (Figu e 9B).
A his s age, i was obse ed a pa e n o mChe y exp ession ha sugges ed
enla ged ollicles so his ological examina ion was pe o med o con i m such
hypo hesis. g( g:mCh) ish displayed hy oid ollicles sca e ed p edominan ly in
he connec i e issue nea he en al ao a (Figu e 9C: C1 and C1’). Each ollicle
was ound o o al and was lined by a single-laye ed epi helium; he cen e o he
ollicle was illed wi h homogenous colloid (Figu e 9C: C1 and C1’). g( g:mCh-
T2A-BRAF
V600E
)
ish displayed signs indica ing colloid goi e which in ol ed he
lowe jaw. Follicles we e p edominan ly la ge o e y la ge close o each o he wi h
a ying shapes (Figu e 9C: C2 and C2’). Small ollicles we e also obse ed. Follicles
we e lined by basophilic and cuboidal epi helial cells and ollicles wi h excessi e
colloid lined by a la ened laye o epi helial cells (Figu e 9C: C2 and C2’). These
ollicles we e well-di e en ia ed and in some cases due o hei size, ollicles e en
cons ic ed he en al ao a. Taken oge he , none o he g( g:mCh) (0/20) bu
mos o he g( g:mCh-T2A-BRAF
V600E
)
ish (18/20) displayed colloid goi e as
con i med by his ology (goi e incidence a 12mp : p<0.0001) (Figu e 9D).
111
When examining issue sec ions o g( g:mCh-T2A-BRAF
V600E
)
ish, many exhibi ed
ollicles ha we e ma kedly la ge (Figu e 9C: C2 and C2’). In o de o de e mine
he ex ension o he goi e ous hy oid, 3-μm- hick sec ions co e ing he whole
hy oid o he adul ish we e selec ed o de e mine hy oid olume. On a e age,
g( g:mCh-T2A-BRAF
V600E
)
ish displayed a 6- old inc ease in hy oid olume when
compa ed o con ols (p<0.001) (Figu e 9E).
Al hough colloid goi e has a good p ognosis and su i al in humans, hy oid
dys unc ion wi hou a p ope ollow-up can esul in complica ions. In g( g:mCh-
T2A-BRAF
V600E
)
ish he e was ne e an expe imen al in e e ence in he endoc ine
homeos asis besides he exp ession o BRAF oncogene by hy oid cells and ish
we e subjec ed o he same husband y condi ions as age-ma ched con ols. To
e i y whe he a goi e ous s a e could ha e in luenced he li espan, all ish om
his wo k ha we e no used o his ological examina ion we e included o
de e mine su i al a es. Su i al up o 12mp was simila o ha obse ed o he
g( g:mCh) ish (p>0.05) (Figu e 9F).
112

113
Figu e 9.
Cha ac e iza ion o g(
g
:mCh-T2A-BRAF
V600E
) adul s. (A) Rep esen a i e
pheno ypes o g( g:mCh) and g( g:mCh-T2A-BRAF
V600E
) a 12 mon hs o age. Scale ba :
7.5mm. (B) BMI measu ed om g( g:mCh) (n=25) and g( g:mCh-T2A-BRAF
V600E
) (n=28)
(p<0.001). (C) HE s ain o longi udinal sec ions ep esen a i e o he lowe jaw a 12mp .
Scale ba : 250μm. C1’ and C2’ a e highe magni ica ions o he ollicles. Scale ba : 62μm.
(D) Incidence o goi e a 12mp . Values co espond o he pe cen age o ish wi h goi e
con i med by his ology ( wen y ish we e s udied om each line) (p<0.0001). (E)
Rep esen a i e hy oid olume assessed in issue sec ions. Values co espond o he olume
o each ish s udied; wen y in o al o each line (p<0.001). (F) Su i al cu es o g( g:mCh)
(n=25) and g( g:mCh-T2A-BRAF
V600E
) (n=25) up o 12mp (p>0.05). Values a e pe cen ages
o ish ali e o a ce ain imepoin .
114
Mu a ing p53 p e en s BRAF
V600E
-induced hy oid dys unc ion in ansgenic ish
g( g:mCh-T2A-BRAF
V600E
) ish displayed an inc ease in he p o ein le els o WT p53
by 3 mon hs o age concomi an wi h hy oid hype plasia sugges ing ha p53 was
es aining umo p og ession. In o de o acili a e cance de elopmen , a p53
M214K
zeb a ish line was used.
p53
M214K
-homozygous zeb a ish a e iable and de elop umo s by 16 mon hs o
age ha his ologically esemble malignan pe iphe al neu al sheal h umo s no
seen in humans and mice wi h ge mline mu a ions o TP53 (Be ghmans e al., 2005;
S o e e al., 2010). O no e, zeb a ish p53 is highly simila o mammalian p53 in
bo h s uc u e and unc ion sha ing 48% homology in amino acid sequence o
human p53 (Cheng e al., 1997). p53 is ubiqui ously exp essed ea ly in zeb a ish
de elopmen (1hp ), a he pha yngula s age (24-48hp ) is p edominan ly
exp essed in he head and by 48hp he p o ein le els a e ba ely de ec able (Cheng
e al., 1997; Lee e al., 2008).
p53
M214K
missense mu a ion a ec s he p53 p o ein a an aminoacid posi ion ha
is o hologous o TP53 mu a ions ound in human cance cells. The o hologous
human codon o he p53
M214K
mu a ion is in exon 7, me hionine-246, which was
ound o be mu a ed in 124 di e en human umo s, 8 o which exhibi he same
amino acid change (Oli ie e al., 2002; Be ghmans e al., 2005). This codon is also
posi ioned be ween o he known mu a ion ho spo s in he DNA-binding domain
(DBD) o he human TP53 gene a codons 245, 248, and 249 o exon 7 (Oli ie e
al., 2002; Be ghmans e al., 2005).
p53
M214K
mu a ed p o ein is no able o ac i a e ansc ip ion h ough he p21
esponse elemen and beha es in a dominan -nega i e manne , inhibi ing he
abili y o he WT p53 o ac i a e downs eam e ec o s (Be ghmans e al., 2005).
Homozygous p53
M214K
ish we e iden i ied om an inc oss o he e ozygous ish.
The o med we e c ossed wi h g( g:mCh) ish o p oduce he e ozygous p53
M214K
g( g:mCh) o wi h g( g:mCh-T2A-BRAF
V600E
) o p oduce he e ozygous p53
M214K
g( g:mCh-T2A-BRAF
V600E
) ish. He e ozygous ish we e inc ossed and he
homozygous p ogeny also ha bo ing he ansgene we e used o all u he
expe imen s. These clu ches exhibi ed Mendelian seg ega ion o he p53
M214K
mu a ion and emb yonic de elopmen o he mu an was no a ec ed as desc ibed
by Be ghmans e al., 2005.
115
When analyzing li e p53
M214K
g( g:mCh-T2A-BRAF
V600E
) emb yos, a obus hy oid-
speci ic epo e signal became de ec able a a ound 34hp and i was main ained
h oughou hy oid mo phogenesis simila o p53
M214K
g( g:mCh) emb yos.
To s udy whe he BRAF
V600E
would in e e e wi h hy oid mo phogenesis in he
absence o WT p53 p o ein in p53
M214K
g( g:mCh-T2A-BRAF
V600E
), mChe y
exp ession was e alua ed om 60-72hp and up o 7dp . In ha line i was
obse ed he o ganiza ion o ollicula -like s uc u es in he pha yngeal egion
consis en wi h no mal hy oid mo phogenesis simila ly o p53
M214K
g( g:mCh).
Th oughou he sc eening i was ob ious ha homozygous BRAF
V600E
-exp essing
la ae de eloped no mally and we e indis inguishable om homozygous con ols
in e ms o iabili y and e ili y (Figu e 10A). When assessing he pe cen age o
la ae displaying no mal hy oid mo phogenesis, 84% (137/163) o p53
M214K
g( g:mCh; g:mCh-T2A-BRAF
V600E
) and 90% (87/96) o con ols showed signs
indica ing no impai men o hy oid mo phogenesis (p>0.05) (Figu e 10B). The
numbe o ollicles we e quan i ied in o de o con i m ha hy oid mo phogenesis
was indeed no mal. In p53
M214K
g( g:mCh; g:mCh-T2A-BRAF
V600E
) la ae he
numbe o ollicles (2-5 ollicles) was simila o he numbe ound in p53
M214K
g( g:mCh) (2 o 6 ollicles) (p>0.05) (Figu e 10C).
A simila g ow h be ween bo h lines was e i ied when measu ing SL a 7dp
(p>0.05) (Figu e 10D).
By con en ional luo escence mic oscopy, i was e i ied ha hy oid was
de eloping no mally in p53
M214K
g( g:mCh; g:mCh-T2A-BRAF
V600E
) la ae. Howe e ,
i was impo an o cla i y whe he hy oid cells we e o ganized simila ly o p53
M214K
con ols so his ological examina ion o he lowe jaw was pe o med om 7-, 14-
, 21- and 28-days-old la ae. p53
M214K
g( g:mCh) e ealed hy oid ollicles loca ed
p edominan ly in he connec i e issue o he lowe jaw. The indi idual ollicles
we e ound o o al and we e lined by a single-laye ed cuboidal epi helium; he
cen e o he ollicle was illed wi h colloid and esicles we e seen (Figu e 10E).
p53
M214K
g( g:mCh-T2A-BRAF
V600E
) la ae displayed he same his ology ea u es
desc ibed abo e (Figu e 10E).
The su i al in p53
M214K
g( g:mCh-T2A-BRAF
V600E
) was simila o he con ols which
was consis en wi h no mal hy oid g ow h du ing he la al s age (p>0.05) (Figu e
10F).
116
123
olume when compa ed o con ols (p<0.001) (Figu e 12E), s ill lowe ha he
olume measu ed in g( g:mCh-T2A-BRAF
V600E
) o he same age.
To e alua e su i al, all ish om his wo k ha we e no used o his ological
examina ion we e included. Su i al up o 12mp was simila o ha obse ed o
con ols (p>0.05) (Figu e 12F).

124
Figu e 12.
Cha ac e iza ion o
p53
M214K
g(
g
:mCh-T2A-BRAF
V600E
) adul s. (A)
Rep esen a i e pheno ypes o p53
M214K
g( g:mCh) and p53
M214K
g( g:mCh-T2A-BRAF
V600E
) a
12 mon hs o age. Scale ba : 7.5mm. (B) BMI measu ed om p53
M214K
g( g:mCh) (n=25)
and p53
M214K
g( g:mCh-T2A-BRAF
V600E
) (n=28) (p>0.05). (C) HE s ain o longi udinal
sec ions ep esen a i e o he lowe jaw a 12mp . Scale ba : 250μm. C1’ and C2’ a e highe
magni ica ions o he ollicles. Scale ba : 62μm. (D) Incidence o goi e a 12mp . Values
a e pe cen ages o ish wi h goi e con i med by his ology (ele en and nine ish we e
s udied om BRAF
V600E
and con ol line, espec i ely) (p>0.05). (E) Rep esen a i e hy oid
olume assessed by issue sec ions. Values co espond o he olume o each ish s udied;
ele en and nine ish we e s udied om BRAF
V600E
and con ol line, espec i ely (p<0.001). (F)
125
Su i al cu es o p53
M214K
g( g:mCh) (n=25) and p53
M214K
g( g:mCh-T2A-BRAF
V600E
)
(n=25) up o 12mp (p>0.05). Values a e pe cen ages o ish ali e o a ce ain imepoin .
Reduced BRAF
V600E
exp ession in p53
M214K
ish may explain absence o BRAF-induced
pheno ype
I was obse ed an abno mal hy oid mo phogenesis in g( g:mCh-T2A-BRAF
V600E
)
la ae by 7dp and accele a ed g ow h ha was no seen in age-ma ched p53
M214K
g( g:mCh-T2A-BRAF
V600E
) ish (p<0.0001) (Figu e 13A). g( g:mCh-T2A-BRAF
V600E
)
ish de eloped hy oid hype plasia by 3 mon hs o age and colloid goi e was
obse ed a 12 mon hs o age as well as a delay in g ow h (BMI a 3mp : p<0.01
and a 12mp : p<0.01), concomi an wi h BRAF
V600E
exp ession in hy oid cells
(Figu e 13B). Hype plasia and educed g ow h we e no obse ed in age-ma ched
p53
M214K
g( g:mCh-T2A-BRAF
V600E
) ish (Figu e 13B). This sugges ed ha in he
absence o WT p53, he BRAF
V600E
-induced pheno ypes in ish we e no sus ained.
In one hand, BRAF
V600E
exp ession a 3 mon hs o age in g( g:mCh-T2A-BRAF
V600E
)
ish is consis en wi h ac i a ion o MAPK and PI3K-AKT pa hways h ough pERKs
and pATKs, espec i ely (Figu e 13D). This sugges ed a ole o hese pa hways in
he hype plasia obse ed. On he o he hand, up egula ion o p53 and ac i a ed
caspase 3 (Figu e 13D) sugges ed ha p53 pu a b ake in BRAF-exp essing cells.
When compa ed o he p53
M214K
g( g:mCh-T2A-BRAF
V600E
) ish, BRAF
V600E
exp ession
is educed concomi an wi h low le els o pERKs and pATKs (Figu e 13D). This
sugges ed ha e en in he absence o he b ake ha would a o p og ession o
malignancy, he down egula ion o impo an pa hways ha e con ibu ed o
p oduce no e idences o hype plasia o cance un il 12 mon hs o age in he
BRAF
V600E
-exp essing p53
M214K
line.
126
127
Figu e 13.
Compa ison be ween BRAF
V600E
-exp essing p53
w
and p53
M214K
lines. (A)
SL measu ed a 7dp om p53
w
g( g:mCh-T2A-BRAF
V600E
) (n=31) and p53
M214K
g( g:mCh-
T2A-BRAF
V600E
) (n=29) (p<0.0001). (B) BMI measu ed a 3 mon hs o age om p53
w
g( g:mCh-T2A-BRAF
V600E
) (n=39) and p53
M214K
g( g:mCh-T2A-BRAF
V600E
) (n=30) (p<0.01).
(C) BMI measu ed a 12 mon hs o age om p53
w
g( g:mCh-T2A-BRAF
V600E
) (n=27) and
p53
M214K
g( g:mCh-T2A-BRAF
V600E
) (n=18) (p<0.01). (D) Wes e n blo analysis o lysa es
om hy oid issue a 3mp . Values a e shown in ba g aphs and ep esen h ee
independen expe imen s, each co esponding o a pool o h ee ish (s a is ical analysis
only shown o ele an compa isons; p<0.001 and p<0.0001).
Condi ional a ge ed exp ession o BRAF
V600E
in hy oid cells o ansgenic zeb a ish
(p elimina y da a)
Exp essing BRAF
V600E
in hy oid cell lines and in he hy oid o mice models ha e
shown con adic o y da a. Mi su ake e al., 2005 showed ha in i o condi ional
BRAF
V600E
exp ession ailed o ans o m a di e en ia ed hy oid PCCL3 cells and
induce apop osis in pa allel wi h inc eased DNA syn hesis, dedi e en ia ion and
ch omosomal ins abili y (Mi su ake e al., 2005). Mo eo e , Vizioli e al., 2011
demons a ed ha p ima y cul u es o hy oid ans ec ed wi h BRAF
V600E
p omo e
oncogene-induced senescence bu no oncogenic ans o ma ion (Vizioli e al.,
2011). In mice, Shimamu a e al., 2013 desc ibed ha condi ional BRAF
V600E
exp ession did no induce umo o ma ion and enginee ed pos na al exp ession
o BRAF
V600E
in a small numbe o hy oid cells did no ini ia e umo igenesis in
hy oid (Shimamu a e al., 2013) despi e o mice modeled o hy oid-speci ic
exp ession o BRAF
V600E
de eloped goi e and in asi e PTCs ea ly in li e (Knau e al.,
2005; Me ce e al., 2005; F anco e al., 2011).
In g( g:mCh-T2A-BRAF
V600E
) ish, a e y ea ly impai men o hy oid mo phogenesis
and de elopmen o hype plasia was obse ed by 2 mon hs o age. In o de o
model spo adic hy oid cance , a ansgenic model was de eloped in which BRAF
V600E
was exp essed la e in li e and con olled in a e y e icien manne unde
physiological se um TSH concen a ions.
128
In he pas decade, he C e-loxP echnology, when combined wi h inducible
sys ems, has allowed a con olled spa ial and/o empo al exp ession o mu a ions
om umo supp esso genes and oncogenes.
The g(hsp70l:mChe y-T2A-C eER
T2
) ansgenic line is a non-leaky condi ional line
ha has a bicis onic mRNA coding o mChe y and C eER
T2
sepa a ed by a i al
T2A pep ide sequence unde he con ol o he zeb a ish hea shock cogna e 70-
kd p o ein, like (hsp70l) p omo e (Hans e al., 2011). In he absence o amoxi en
(TAM), C e-loxP-si e-speci ic ecombina ion does no occu whe eas in he
p esence o TAM and a e hea shock ull ecombina ion can be achie ed. A
cons uc , in which BRAF
V600E
exp ession would be no mally supp essed by he
p esence o a loxed gene bu induced when his gene was ea anged by C e, was
de eloped.
To acili a e cloning, he ollowing p e ious cons uc s we e used: Tol2CG2 g:mCh
and Tol2CG2 g:mCh-T2A-BRAF
V600E
. The loxP-CFP-loxP casse e was cloned
downs eam o he g p omo e and ups eam o he mCh o mCh-T2A-BRAF
V600E
(Figu e 14A and 14B, espec i ely). CFP epo e was chosen because i would allow
o moni o he ecombina ion p ocess in hy oid cells upon hea shock and TAM
ea men .
Tol2CG2 g:loxP-CFP-loxP-mCh o Tol2CG2 g:loxP-CFP-loxP-mCh-T2A-BRAF
V600E
was injec ed wi h capped ansposase mRNA in o one-cell-s age
g(hsp70l:mChe y-T2A-C eER
T2
) emb yos. By luo escence mic oscopy, mosaic
CFP exp ession was obse ed in hy oid issue o F0 animals injec ed wi h
Tol2CG2 g:loxP-CFP-loxP-mCh o Tol2CG2 g:loxP-CFP-loxP-mCh-T2A-BRAF
V600E
.
This was he con i ma ion ha he cons uc was unc ional and g p omo e was
su icien o d i e exp ession o CFP speci ically o he hy oid cells.
Emb yos p esen ing mosaic CFP o EGFP exp ession in he hy oid o he hea ,
espec i ely we e g own o adul hood and F0 ounde s we e iden i ied by a
speci ic- epo e signal exclusi ely in hose issue o hei p ogeny. S able
ansgenic lines we e es ablished using F0 ounde s. A obus CFP exp ession was
con i med in hy oid cells o bo h F0-hsp70l:mChe y-T2A-C eER
T2
; g:loxP-CFP-
loxP-mCh and F0-hsp70l:mChe y-T2A-C eER
T2
; g:loxP-CFP-loxP-mCh-T2A-
BRAF
V600E
lines.

129
When analyzing li e emb yos om he lines abo e-men ioned, a obus hy oid
speci ic epo e signal became de ec able a a ound 34hp and i was main ained
h oughou hy oid g ow h simila o g( g:mCh) emb yos.
A p elimina y expe imen was conduc ed by inducing C e-loxP-media ed
ecombina ion in g(hsp70l:mChe y-T2A-C eER
T2
; g:loxP-CFP-loxP-mCh-T2A-
BRAF
V600E
) ish a 2 ½ mon hs o age. This age was chosen no only because
ecombina ion du ing la al s age would no po ayed he spo adic cance bu also
because ish would handle be e he anes hesia, pe o med in o de o moni o
he epo e exp ession.
The ecombina ion was induced by hea shock ollowed by exposu e o he ac i e
me aboli e o TAM, 4-Hyd oxyl-Tamoxi en (4-OHT). Recombina ion e iciency was
assessed by a g adual loss o CFP exp ession and g adual gain o mChe y
exp ession, which was ubiqui ous in he ini ial hou s a e he hea shock and
speci ically o he hy oid cells in he days ha ollowed (Figu e 14C).
130
Figu e 14.
Condi ional cons uc s (A) Schema ic ep esen a ion o he (A) Tol2 g:loxP-
CFP-loxP-mCh and (B) Tol2 g:loxP-CFP-loxP-mCh-T2A-BRAF
V600E
cons uc s con aining
Tol2 elemen s and he cmlc2:EGFP-pA casse e. (C) Schema ic ep esen a ion o he C e-
media ed ecombina ion o he hsp70l:mCh-T2A-C eER
T
allele in he blue- o- ed epo e
g:loxP-CFP-loxP-mCh/mCh-T2A-BRAF
V600E
line in he p esence o hea and 4-OHT
exposu e and inal ansgenes kep in he genome.
T ansgenic emb yos ca ying he CFP epo e and he g(hsp70l:mCh-T2A-
C eER
T2
) allele displayed a s ong CFP signal un il 2 ½ mon hs o age. Hea shock
and exposu e o 4-OHT led sho ly a e o a s ong ubiqui ous mChe y exp ession
131
indica ing success ul induc ion o C eER
T2
exp ession. Fish we e moni o ed and CFP
exp ession was comple ely los du ing he i s weeks a e induc ion. This
con i med he success ul ecombina ion e en in mos o all hy oid cells. mChe y
exp ession was ne e de ec ed; obse a ion ha co obo a ed wi h he absen
mChe y exp ession in g( g:mCh-T2A-BRAF
V600E
) ish.
Wi hou ha ing an exp essing epo e ha would help o moni o hy oid
mo phology, ish we e g own un il hey eached 4 mon hs o age and hen
his ological examina ion was pe o med. g(hsp70l:mChe y-T2A-C eER
T2
; g:loxP-
CFP-loxP-mCh-T2A-BRAF
V600E
) ish, in which ecombina ion was no induced,
displayed hy oid ollicles sca e ed p edominan ly in he connec i e issue nea
he en al ao a (Figu e 15 A). Follicles we e ound o o al and we e lined by a
single-laye ed epi helium con ining a e y homogenous colloid (Figu e 15 A’). Fish
in which ecombina ion was induced showed ollicula hype plasia in he lowe jaw
(Figu e 15B). The ollicles we e ei he big o e y small and we e e y close. The
epi helium lining he ollicles was a iably basophilic cuboidal o columna (Figu e
15B’). The e was a ma ked in lamma o y in il a e likely o be o lymphocy ic o igin
su ounding he ollicles and inside he blood essels nea by (Figu e 15: B’’ and
B’’’). Because he hype plasia desc ibed esembled ha ound in g( g:mCh-T2A-
BRAF
V600E
) ish, i sugges ed ha exp ession o BRAF
V600E
was induced success ully.
132
Figu e 15
. C e-media ed ecombina ion o he
hsp70l
:mCh-T2A-C eER
T2
allele in he
blue- o- ed epo e g(
g:
loxP-CFP-loxP-mCh-T2A-BRAF
V600E
) line in he absence
and p esence o hea and 4-OHT exposu e. (A) Scheme o he ansgene in he absence
o ecombina ion and HE s ain o longi udinal sec ions ep esen a i e o he lowe jaw. Scale
ba : 250μm. (B) Highe magni ica ion o he ollicles. Scale ba : 62μm. B: Scheme o he