scieee Science in your language
[en] (orig)

Unravelling the role of the spinocerebellar ataxia type 3-associated protein ATXN3 in glioblastoma

Sousa, Ana Margarida Machado · 2022

Abstract

O glioblastoma (GBM) é o tipo de glioma mais comum e maligno em adultos. Apesar dos esforços para investigar diversos tratamentos, os pacientes com GBM apresentam uma evolução clínica rápida e desfavorável, com uma sobrevida mediana de apenas 15 meses após o diagnóstico. Para além disso, a elevada atividade proliferativa e a natureza heterogénea e complexa do GBM estão associadas a um resultado clínico imprevisível e diverso. Assim, a identificação de marcadores moleculares de prognóstico que permitam a categorização de subgrupos de pacientes com GBM é fundamental para contribuir para a melhoria do seu resultado clínico. A ataxina-3 (ATXN3), a proteína envolvida na doença neurodegenerativa Ataxia espinocerebelosa tipo 3 (SCA3), é uma proteína conservada evolutivamente e expressa de forma ubíqua, que foi proposta como sendo uma enzima desubiquitinase. Além do seu envolvimento em SCA3, foi sugerido que a ATXN3 desempenha um papel em cancro, desempenhando funções oncogénicas ou supressoras tumorais dependendo do tipo tumoral. Uma vez que nenhum estudo, até ao momento, explorou o potencial envolvimento da ATXN3 em gliomas, e, particularmente em GBM, neste trabalho pretendemos, pela primeira vez, avaliar o papel funcional da ATXN3 em GBM e a sua relevância clínica nesta doença. Observámos que a expressão da ATXN3 diminui significativamente nos graus mais elevados de glioma, sendo menos expressa em GBM quando comparada com gliomas de baixo grau. Adicionalmente demonstrámos que a expressão da ATXN3 está associada à mutação de IDH e à codeleção 1p/19q. Em células de GBM, observámos que a ATXN3 é expressa em todos os modelos testados, ao nível de RNAm e ao nível da proteína. Funcionalmente, a sobre-expressão da ATXN3 foi associada a uma diminuição significativa da viabilidade celular e da invasão em modelos in vitro de GBM. No entanto, em relação à proliferação e à migração celular não foi detetado um efeito estatisticamente significativo nos mesmos modelos. Estes dados sugerem que a ATXN3 pode ter funções de gene supressor tumoral em GBM, diminuindo a agressividade destes gliomas in vitro. Em pacientes com GBM, descobrimos que a ATXN3 tem valor de prognóstico clínico, estando associada a uma sobrevida global mais longa, independentemente de outros potenciais fatores de prognóstico. Em suma, este trabalho permitiu que se compreendesse o papel da ATXN3 em GBM ao identificá la como um gene supressor tumoral, e como um novo biomarcador de prognóstico favorável, trazendo novos conhecimentos sobre os mecanismos moleculares subjacentes a esta doença mortal.

Read accessible full text

Unravelling the role of the spinocerebellar ataxia type 3-associated protein ATXN3 in glioblastoma

Author: Sousa, Ana Margarida Machado
Year: 2022
Source: https://repositorium.uminho.pt/bitstreams/e9c8db68-ec2d-4ab4-a5be-7004ea525f77/download
Uni e sidade do Minho
Escola de Medicina
Ana Ma ga ida Machado Sousa
Un a elling he ole o he spinoce ebella
a axia ype 3-associa ed p o ein ATXN3 in
glioblas oma
dezemb o de 2021
UMinho | 2021 Ma ga ida Sousa Un a elling he ole o he spinoce ebella a axia
ype 3-associa ed p o ein ATXN3 in glioblas oma
Ana Ma ga ida Machado Sousa
Un a elling he ole o he spinoce ebella
a axia ype 3-associa ed p o ein ATXN3 in
glioblas oma
Disse ação de Mes ado
Mes ado em Ciências da Saúde
T abalho e e uado sob a o ien ação da
Dou o a And eia Alexand a Ne es de Ca alho
e da
Dou o a Céline Sa ai a Gonçal es
Uni e sidade do Minho
Escola de Medicina
dezemb o de 2021
ii
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS
Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as
e boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos.
Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada.
Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não
p e is as no licenciamen o indicado, de e á con ac a o au o , a a és do Reposi ó iUM da Uni e sidade
do Minho.
Licença concedida aos u ilizado es des e abalho
A ibuição-NãoCome cial-SemDe i ações
CC BY-NC-ND
h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
iii
AGRADECIMENTOS
Ao inaliza es es 2 anos de dedicação e abalho, gos a ia de exp essa o meu ag adecimen o a
odas as pessoas que me apoia am e con ibuí am pa a a ealização des e abalho.
Começo po ag adece às minhas o ien ado as Dou o a And eia e Dou o a Céline po me e em
dado a opo unidade de ap ende e e olui como in es igado a e po oda a disponibilidade e
ensinamen os. Espe o que es e abalho os aça ica o gulhosas. Ag adeço ambém ao Dou o B uno
Cos a e à P o esso a Pa ícia Maciel po me e em dado a opo unidade de in eg a os seus g upos de
in es igação. Ob igada ambém a odos os colegas de g upo e de labo a ó io. Em especial que o
ag adece à Edua da, pela disponibilidade, pela ajuda cons an e e c ucial, pela paciência e pelos
conhecimen os ansmi idos. Fos e, sem somb a de dú idas, uma g ande ajuda nes e ano. Ag adeço
ambém à Liliana e à Dou o a Sa a Sil a, que i e am ambém um papel impo an e na minha
ap endizagem, po es a em semp e disponí eis pa a me ajuda em e i a em dú idas semp e que
possí el. Es ou- os g a a!
Ag adeço aos meus colegas de mes ado, em especial à Ad iana, Ma cela, Bea iz e Joana po
odas as pala as de con o o quando necessi ei e po es a em semp e disponí eis. A odos os meus
amigos, especialmen e ao Cenas, po odos as con e sas alea ó ias, po odo o apoio nos maus
momen os e po es a em semp e lá.
Po úl imo, gos a ia de ag adece à minha amília (a melho do mundo) e sob e udo aos meus
pais e ao Rúben, pelo amo , po me apoia em incondicionalmen e, pela o ça a con inua e po
ac edi a em semp e em mim, mui as das ezes mais do que eu p óp ia. Não há pala as pa a exp imi
udo o que sin o. Sem ocês nada dis o se ia possí el, de o- os udo!
FUNDING
The wo k p esen ed in his hesis was pe o med in he Li e and Heal h Sciences Resea ch Ins i u e
(ICVS), Uni e si y o Minho. Financial suppo was p o ided by Fundação Calous e Gulbenkian and Liga
Po uguesa Con a o Canc o ( o B uno M. Cos a); by g an s om he ICVS Scien i ic Mic oscopy Pla o m,
membe o he na ional in as uc u e PPBI - Po uguese Pla o m o Bioimaging (PPBI-POCI-01-0145-
FEDER-022122; by he p ojec NORTE-01-0145-FEDER-000055, suppo ed by No e Po ugal Regional
Ope a ional P og amme (NORTE 2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough he
Eu opean Regional De elopmen Fund (ERDF) and by Na ional unds, h ough he Founda ion o Science
and Technology (FCT) - p ojec UIDB/50026/2020 and UIDP/50026/2020.

i
STATEMENT OF INTEGRITY
I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used
plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o
i s elabo a ion.
I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o
Minho.
RESUMO
Des endando o papel da p o eína associada à a axia espinoce ebelosa ipo 3 ATXN3 em glioblas oma
O glioblas oma (GBM) é o ipo de glioma mais comum e maligno em adul os. Apesa dos es o ços
pa a in es iga di e sos a amen os, os pacien es com GBM ap esen am uma e olução clínica ápida e
des a o á el, com uma sob e ida mediana de apenas 15 meses após o diagnós ico. Pa a além disso, a
ele ada a i idade p oli e a i a e a na u eza he e ogénea e complexa do GBM es ão associadas a um
esul ado clínico imp e isí el e di e so. Assim, a iden i icação de ma cado es molecula es de p ognós ico
que pe mi am a ca ego ização de subg upos de pacien es com GBM é undamen al pa a con ibui pa a
a melho ia do seu esul ado clínico. A a axina-3 (ATXN3), a p o eína en ol ida na doença
neu odegene a i a A axia espinoce ebelosa ipo 3 (SCA3), é uma p o eína conse ada e olu i amen e e
exp essa de o ma ubíqua, que oi p opos a como sendo uma enzima desubiqui inase. Além do seu
en ol imen o em SCA3, oi suge ido que a ATXN3 desempenha um papel em canc o, desempenhando
unções oncogénicas ou sup esso as umo ais dependendo do ipo umo al. Uma ez que nenhum
es udo, a é ao momen o, explo ou o po encial en ol imen o da ATXN3 em gliomas, e, pa icula men e
em GBM, nes e abalho p e endemos, pela p imei a ez, a alia o papel uncional da ATXN3 em GBM e
a sua ele ância clínica nes a doença.
Obse ámos que a exp essão da
ATXN3
diminui signi ica i amen e nos g aus mais ele ados de
glioma, sendo menos exp essa em GBM quando compa ada com gliomas de baixo g au. Adicionalmen e
demons ámos que a exp essão da
ATXN3
es á associada à mu ação de IDH e à codeleção 1p/19q. Em
células de GBM, obse ámos que a ATXN3 é exp essa em odos os modelos es ados, ao ní el de RNAm
e ao ní el da p o eína. Funcionalmen e, a sob e-exp essão da ATXN3 oi associada a uma diminuição
signi ica i a da iabilidade celula e da in asão em modelos
in i o
de GBM. No en an o, em elação à
p oli e ação e à mig ação celula não oi de e ado um e ei o es a is icamen e signi ica i o nos mesmos
modelos. Es es dados suge em que a ATXN3 pode e unções de gene sup esso umo al em GBM,
diminuindo a ag essi idade des es gliomas
in i o
. Em pacien es com GBM, descob imos que a ATXN3
em alo de p ognós ico clínico, es ando associada a uma sob e ida global mais longa,
independen emen e de ou os po enciais a o es de p ognós ico.
Em suma, es e abalho pe mi iu que se comp eendesse o papel da ATXN3 em GBM ao iden i icá-
la como um gene sup esso umo al, e como um no o bioma cado de p ognós ico a o á el, azendo
no os conhecimen os sob e os mecanismos molecula es subjacen es a es a doença mo al.
Pala as-cha e:
A axina-3; Bioma cado de P ognós ico; Gene Sup esso Tumo al; Glioblas oma;
Glioma
i
ABSTRACT
Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
Glioblas oma (GBM) is he mos common and malignan ype o glioma in adul s. Despi e he e o s
o in es iga e a ious ea men s, GBM pa ien s exhibi a apid and un a o able clinical e olu ion, wi h a
median o e all su i al o only 15 mon hs a e diagnosis. Fu he mo e, he high p oli e a i e ac i i y, and
he e ogeneous and complex na u e o GBM is associa ed wi h an unp edic able and dis inc clinical
ou come. Thus, he iden i ica ion o molecula p ognos ic ma ke s ha allow he ca ego iza ion o
subg oups o pa ien s wi h GBM is c i ical o con ibu e o he imp o emen o hei clinical ou come.
A axin-3 (ATXN3), he p o ein in ol ed in he neu odegene a i e disease Spinoce ebella A axia Type 3
(SCA3), is an e olu iona ily conse ed and ubiqui ously exp essed p o ein, which has been p oposed o
ac as a deubiqui ina ing enzyme. In addi ion o i s in ol emen in SCA3, ATXN3 was sugges ed o play a
ole in cance , pe o ming oncogenic o umo supp essi e unc ions depending on he umo ype. Since
no s udy o da e has explo ed he po en ial in ol emen o ATXN3 in gliomas, and pa icula ly in GBM, in
his wo k we in end, o he i s ime, o e alua e he unc ional ole o ATXN3 in GBM and he clinical
ele ance o his p o ein in his deadly disease.
We obse ed ha
ATXN3
exp ession dec eases signi ican ly wi h glioma g ade, being less
exp essed in GBM when compa ed o lowe -g ade gliomas, and ha i is associa ed wi h IDH mu a ion
and 1p/19q codele ion. Speci ically in GBM cells, we obse ed ha ATXN3 is exp essed in all cell models
es ed, bo h a he mRNA and p o ein le el. Func ionally, ATXN3 o e exp ession was associa ed wi h a
signi ican dec ease in he cell iabili y and in asion o GBM
in i o
models, al hough no s a is ically
signi ican e ec was obse ed ega ding cell p oli e a ion and mig a ion. This da a sugges s ha ATXN3
may ha e umo supp essi e unc ions in GBM, dec easing i s agg essi eness
in i o
. In GBM pa ien s,
we ound ha
ATXN3
has clinical p ognos ic alue, being associa ed wi h longe o e all su i al,
independen ly o o he po en ial p ognos ic ac o s.
In summa y, his wo k allowed he unde s anding o he ole o ATXN3 in GBM by iden i ying i as
a umo supp esso gene, and as a new p ognos ic bioma ke o a o able ou come, b inging new
knowledge abou he molecula mechanisms unde lying his deadly disease.
Keywo ds:
A axin-3, Glioblas oma, Glioma, P ognos ic bioma ke , Tumo supp esso gene
ii
CONTENTS
DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS ........................ ii
AGRADECIMENTOS ............................................................................................................................ iii
FUNDING ........................................................................................................................................... iii
STATEMENT OF INTEGRITY ................................................................................................................ i
RESUMO .............................................................................................................................................
ABSTRACT ......................................................................................................................................... i
CONTENTS ....................................................................................................................................... ii
ABBREVIATIONS ................................................................................................................................ ix
FIGURE LIST ..................................................................................................................................... xii
TABLE LIST ....................................................................................................................................... xii
1. | INTRODUCTION .......................................................................................................................... 1
1.1 Cance O e iew ................................................................................................................ 2
1.2 P ima y B ain Tumo s ........................................................................................................ 3
1.3 Glioma .............................................................................................................................. 4
1.4 Glioblas oma (GBM) ........................................................................................................... 6
1.4.1 GBM T ea men : a mul imodal app oach ........................................................................ 8
1.4.2 Molecula p ognos ic ac o s o GBMs ............................................................................ 9
1.5 The ATXN3 p o ein .......................................................................................................... 10
1.5.1 ATXN3 po en ial unc ion(s) .......................................................................................... 12
1.5.2 ATXN3 in cance .......................................................................................................... 13
2. | OBJECTIVES ............................................................................................................................. 16
3. | MATERIALS AND METHODS ...................................................................................................... 18
3.1 Glioma da ase s ............................................................................................................... 19
3.2 Cell lines and cul u e condi ions ....................................................................................... 19
3.3 Plasmid ans o ma ion in o
Esche ichia coli
(
E. coli
) ........................................................ 20
3.4 Len i i us p oduc ion ....................................................................................................... 20
3.5 ATXN3 o e exp ession in GBM cells ................................................................................. 21
3.6 RNA ex ac ion ................................................................................................................. 21
3.7 DNA ex ac ion ................................................................................................................. 22
3.8 cDNA syn hesis ................................................................................................................ 22
3.9 Quan i a i e Polyme ase Chain Reac ion (qPCR) ............................................................... 23
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
2
1.
INTRODUCTION
1.1
Cance O e iew
Cance is a mul i ace ed public heal h p oblem and he second mos common cause o dea h
a ound he wo ld (B ay e al., 2021), despi e he e o s obse ed o e he pas ew decades o imp o e
di e en po en ial ea men s. A s agge ing numbe o people a e a ec ed by cance – acco ding o he
Wo ld Heal h O ganiza ion (WHO), i is es ima ed ha abou 19.3 million o new cance cases we e
diagnosed and 9.96 million cance dea hs we e accoun ed in 2020 wo ldwide (Fe lay, E ik, e al., 2020;
Sung e al., 2021). The cance bu den is p ojec ed o inc ease, wi h a p edic ed 24.6 million new cance
cases and 12.9 million cance - ela ed dea hs occu ing annually by 2030 (Fe lay, La e sanne, e al.,
2020), a consequence o popula ion g ow h and aging (Fidle e al., 2018).
Cance is usually a disease associa ed wi h apid p oli e a ion and uncon olled cell g ow h.
Howe e , he de elopmen o malignancy is a much mo e complex and highly dynamic p ocess, in ol ing
mul iple s eps (G izzi & Chi i a-In e na i, 2006). Di e en ypes o no mal cells p og essi ely e ol e in o
a malignan s a e h ough he acquisi ion o mul iple biological cha ac e is ics (
Figu e 1
). These dis inc
cells pa icipa e in he e o ypic in e ac ions wi h one ano he o ming a complex issue, called a umo .
These biological cha ac e is ics, esponsible o he malignancy o he umo , a e designa ed “hallma ks
o cance ” and we e p oposed by Hanahan & Weinbe g, 2011: (i) e ading g ow h supp esso s, (ii)
a oiding immune des uc ion, (iii) enabling eplica i e immo ali y, (i ) umo -p omo ion in lamma ion, ( )
ac i a ing in asion and me as asis, ( i) induc ing angiogenesis, ( ii) genome ins abili y and mu a ion, ( iii)
esis ing cell dea h, (ix) de egula ing cellula ene ge ics, and (x) sus aining p oli e a i e signaling.
The e olu ion o hese al e a ions leading o umo p og ession and associa ed he e ogenei y
in ol es he g adual accumula ion o gene ic and epigene ic cance -p omo ing changes, a ec ing many
o he cell’s egula o y mechanisms and unc ions – DNA mu a ions, copy numbe a ia ions (CNV -
dele ions and ampli ica ions), ch omosomal ea angemen s (dele ions, in e sions and ansloca ion) and
epigene ic modi ica ions (DNA me hyla ion and his one modi ica ions) (B ai & Sid ansky, 2011; Hanahan
& Weinbe g, 2011). Gene ally, hese gene ic and epigene ic al e a ions lead o he ac i a ion o oncogenes
and inac i a ion o umo supp esso genes (Vogels ein & Kinzle , 2004).

1. | In oduc ion
3
Figu e 1
.
The eigh hallma ks and wo enabling cha ac e is ics o cance p oposed by Hanahan and Weinbe g.
Cance cells
p esen biological cha ac e is ics ha con ibu e o umo g ow h and p og ession (Adap ed om Hanahan & Weinbe g, 2011).
1.2
P ima y B ain Tumo s
P ima y b ain umo s o igina e om abno mal b ain cells, in con as o me as a ic b ain umo s
ha appea elsewhe e in he body and sp ead o he b ain egion, usually h ough he bloods eam (Fayed,
2020). Despi e hei low incidence, accoun ing only o app oxima ely 2% o all cance s, p ima y b ain
umo s ha e a high mo bidi y and mo ali y a e (Buckne e al., 2007). Fu he mo e, hey ank i s
ega ding a e age o yea s o li e los among all umo ypes (Bu ne e al., 2005), and a e also he leading
cause o cance - ela ed dea h in people unde 40 in Eu ope (Fe lay, E ik, e al., 2020). Indeed, hese
ypes o umo s a e one o he mos ea ed o ms o cance no only because o hei poo p ognosis, bu
also because o he di ec epe cussions on pa ien s’ quali y o li e and cogni i e unc ion. Acco ding o
WHO, in 2020 he wo ld es ima ed incidence o b ain and ne ous sys em umo s was app oxima ely
308000 new cases, being he 19 h mos common cance ype. Rega ding mo ali y, in he same yea ,
app oxima ely 251300 dea hs we e es ima ed wo ldwide, being he 12 h mos deadly cance ype. In
Po ugal, 1105 new cases and 933 dea hs we e epo ed in 2020 (Fe lay, E ik, e al., 2020).
Geog aphically, No he n Eu ope, he USA whi e popula ion and Is ael a e he egions ha ha e
he highes a es o epo ed cases and dea h due o p ima y malignan b ain umo s (11-20 pe 100000
inhabi an s), while India and he Philippines ha e he lowes a es (2–4 pe 100,000 inhabi an s) (Os om
e al., 2017; Walsh e al., 2016). Howe e , hese di e ences in b ain umo s’ incidence by geog aphic
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
4
egion a e equen ly due o wo ldwide di e ences in medical ca e access and diagnos ic impossibili y
(Os om e al., 2017).
1.3
Glioma
Glioma is he mos common ype o malignan b ain umo , accoun ing o abou 80% o all p ima y
malignan b ain umo s, and is cha ac e ized by being he e ogeneous and equen ly le hal (Os om e
al., 2020). Gliomas a e di ided in o di use and ci cumsc ibed gliomas; howe e , he la e a e no he
ocus o his disse a ion and will no be explo ed. Since 2016, acco ding o he WHO, umo s o he
cen al ne ous sys em (CNS) a e classi ied acco ding o hei localiza ion, mo phological simila i ies wi h
di e en ypes o neu oglial cells (his ologic ea u es), molecula pa ame e s and g ades o malignan
beha io (
Figu e 2
). This ecen classi ica ion was a s ep o wa d in imp o ing he molecula
cha ac e iza ion o gliomas, highligh ing key molecula al e a ions such as isoci a e dehyd ogenase (IDH)
mu a ion and 1p/19q codele ion (Louis e al., 2016). This app oach combining his ological and molecula
classi ica ions esul s in g ea e clinical/diagnos ic accu acy and a be e de e mina ion o he apeu ic
s a egies, which e lec s in be e pa ien managemen .
IDH mu a ions
The inding o soma ic mu a ions in he
IDH1
and
IDH2
genes, in a subg oup o glioblas omas
(GBM), in genomic s udies conduc ed by Pa sons e al. in 2008 was pe haps he mos impo an
b eak h ough in he molecula unde s anding and diagnosis o gliomas (Pa sons e al., 2008).
IDH is an NADP+-dependen enzyme ha ca alyzes he oxida i e deca boxyla ion o isoci a e o α-
ke oglu a a e wi h simul aneous p oduc ion o NADPH (Rei man & Yan, 2010). The mu a ion a ec s he
amino acid a ginine a posi ion 132 – c i ical o isoci a e binding – which is usually eplaced by his idine
(R132H) (Yan e al., 2009). This mu a ion causes he ac i e si e esidues o be shi ed, esul ing in
s uc u al al e a ions ha p e en IDH om pe o ming i s usual enzyma ic unc ion. As a esul , he
mu an IDH enzyme has he abili y o con e α-ke oglu a a e o R-2-hyd oxyglu a a e, excessi e
accumula ion o which con ibu es o umo igenesis (Dang e al., 2009). IDH mu a ions a e highly
equen in WHO g ade II and III gliomas (60-90%), bu a ely occu in GBM (5-10%) (Cohen e al., 2013;
Tu kalp e al., 2014). Fu he mo e, IDH mu a ions occu mainly in younge pa ien s and p edic longe
pa ien su i al. In ac , GBM pa ien s p esen ing IDH mu a ion ha e a median o e all su i al (OS) o 31
mon hs, compa ed o 15 mon hs o pa ien s who did no ha e he mu a ion (Yan e al., 2009).
1. | In oduc ion
5
1p/19q codele ion
The 1p/19q codele ion is an e en o gene ic loss, being associa ed wi h umo s o he
oligodend oglial lineage. I is es ima ed ha 80-90% o g ade II oligodend ogliomas and 50-70% o g ade
III oligodend ogliomas ha e he 1p/19q codele ion (Cai nc oss & Jenkins, 2008; Jansen e al., 2010).
The 1p/19q codele ion in ol es he dele ion o he sho a m o ch omosome 1 and he dele ion o he
long a m o ch omosome 19, esul ing in an unbalanced ansloca ion in ol ing he cen ome ic egions
(Jenkins e al., 2006). So a , he ole his codele ion plays in ca cinogenesis is no clea . Howe e ,
pa ien s wi h his gene ic e en ha e been shown o ha e a signi ican ly be e OS and p ognosis (J. S.
Smi h e al., 2000).
His ologically, gliomas a e di ided, based on mic oscopic simila i ies wi h glial cells o o igin, in
as ocy omas and oligodend ogliomas (Louis e al., 2016, 2021). Fu he mo e, conside ing he WHO
classi ica ion, gliomas a e g ouped in o ou g ades (I o IV) acco ding o he his ological
p esence/absence o cy ological a ypia, anaplasia, mi o ic ac i i y, mic o ascula p oli e a ion, and
nec osis. G ade I gliomas a e conside ed benign, ha e low p oli e a i e po en ial and well-di e en ia ed
cells. G ade II gliomas, despi e ha ing low agg essi eness, a e classi ied as malignan umo s since hey
ha e di use in il a ion capaci y, which makes su gical emo al o he umo di icul . G ade III gliomas
a e cha ac e ized by a highe cellula densi y and p esen e idence o malignancy, such as nuclea a ypia
and high mi o ic ac i i y. G ade II and III gliomas end o p og ess o highe -g ade gliomas (HGG). Finally,
g ade IV gliomas ep esen he mos malignan glioma, exhibi ascula p oli e a ion and nec osis, and
p esen a apid p og ession o he disease wi h a le hal ou come, despi e agg essi e mul imodal ea men
(Louis e al., 2007; Riemenschneide & Rei enbe ge , 2009; S ien & Mabon, 1949; Welle e al., 2015).
Thus, gliomas include (i) as ocy omas (g ade II and III), IDH-mu an o IDH-wild ype; (ii)
oligodend ogliomas (g ade II and III), IDH-mu an , 1p/19q codele ed; and (iii) GBM (g ade IV), IDH-mu an
o IDH-wild ype (
Figu e 2
) (Louis e al., 2016). In 2021, his classi ica ion was upda ed, emphasizing he
ole o molecula diagnosis. The majo al e a ion o he glioma classi ica ion is ha GBM IDH-mu an
umo s a e now classi ied as as ocy oma, IDH-mu an g ade IV (Louis e al., 2021).
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
6
Figu e 2
.
Classi ica ion o di use gliomas acco ding o he 2016 WHO classi ica ion o CNS umo s.
In addi ion o he
mo phological appea ances al eady de ined and he loca ion o he umo , he 2016 classi ica ion began o include impo an
molecula al e a ions such as he IDH mu a ion and he 1p/19q codele ion s a us.
E o s o es ablish possible glioma p edisposi ion isk ac o s ha e been made. Howe e , mos o
he esul s we e inconsis en and a de ini i e link is ye o be disco e ed. In e ms o en i onmen al isk
ac o s, ionizing adia ion (exposu e o he apeu ic o diagnos ic doses, o high-dose adia ion) is he only
linked o an inc eased isk o b ain umo s, mo e speci ically, glioma (B aganza e al., 2012; Elsamadicy
e al., 2015; P es on e al., 2007). Indeed, di e en s udies ha e shown ha su i o s o a omic bomb
explosions in Nagasaki and Hi oshima exposed o high doses adia ion ha e an inc eased incidence a e
o b ain umo s, including gliomas (P es on e al., 2007). Addi ionally, i has been epo ed ha he
he apeu ic use o ionizing adia ion o ea
Tinea capi is
and skin hemangioma in child en and in an s
is associa ed wi h an inc eased ela i e isk o de eloping glioma (Sade zki e al., 2005). O he s udies
ha e e alua ed he po en ial e ec o die (Chen e al., 2002; Qin e al., 2014), smoking (Shao e al.,
2016), elec omagne ic ields (Coble e al., 2009), cell phone exposu e (Ahlbom e al., 2009; Benson e
al., 2013), among o he ac o s, on he isk o glioma incidence. Howe e , he esul s we e inconsis en ,
and no conclusi e co ela ion was obse ed. G owing e idence has shown ha pa ien s wi h alle gies o
au oimmune diseases a e linked o a lowe isk o glioma (Schoemake e al., 2006; Schwa zbaum e
al., 2012; Wiemels e al., 2002).
1.4
Glioblas oma (GBM)
Glioblas oma (GBM), classi ied by he WHO as g ade IV, is he mos common and malignan ype
o glioma in adul s, accoun ing o mo e han 50% o all gliomas, and wi h a global annual incidence a e
o 3.23 pe 100000 popula ion (Os om e al., 2020). Unde s anda d-o -ca e ea men hese pa ien s
p esen a median su i al o app oxima ely 15 mon hs a e diagnosis (S upp e al., 2005). Al hough
1. | In oduc ion
7
GBMs can occu in all age g oups, he incidence peak usually occu s be ween 45 and 70 yea s. Males
a e mo e a ec ed han emales (1.6:1) and Whi es mo e han Blacks (2:1) (Walsh e al., 2016).
His ologically, GBM p esen s cellula polymo phism, nuclea a ypia, equen mi o ic ac i i y,
ascula h ombosis, mic o ascula p oli e a ion in he umo ma gin and nec osis obse ed in he cen al
po ion o he umo . The umo mass is cha ac e ized by ex ensi e he e ogenei y a he cellula and
molecula le el and a weak delimi a ion wi hou capsule, which makes GBMs highly agg essi e, in il a ing
and di use (So e al., 2021) These cha ac e is ics esul in an ex ensi e sp ead o umo cells wi hin he
b ain, which makes comple e su gical esec ion di icul and ecu ences almos ce ain (B andes e al.,
2008; Louis e al., 2007; C. Smi h & I onside, 2007; Welle e al., 2015).
As p e iously men ioned, in he 2016 WHO classi ica ion, GBM we e subdi ided in o wo sub ypes,
aking in o accoun he s a us o IDH mu a ion: (i) GBM, IDH-wild ype and (ii) GBM, IDH-mu an (Louis e
al., 2016). IDH-wild ype GBMs a e he mos common, accoun ing o app oxima ely 90% o cases, and
appea as a
de no o
p ocess, ha is, wi hou p e-exis ing clinical o his ological e idence o a lowe -g ade
p ecu so . These umo s end o be e y agg essi e and o de elop quickly, occu ing p e e en ially in
elde ly pa ien s (incidence peak is 62 yea s) (Masui e al., 2016; Ohgaki & Kleihues, 2013). In con as ,
IDH-mu an GBMs de elop p og essi ely om a p e-exis ing di use o anaplas ic as ocy oma, gene ally
o e a pe iod o 5 o 10 yea s. The incidence peak occu s in younge pa ien s, a ound 44 yea s old, and
hey ha e a be e p ognosis (Louis e al., 2016; Ohgaki & Kleihues, 2013). The molecula p o ile o hese
umo s is simila o ha o IDH-mu an as ocy omas (Masui e al., 2016). As a esul , in he new 2021
classi ica ion o CNS umo s, GBM IDH-mu an we e ca ego ized as as ocy omas, IDH-mu an , g ade IV.
Thus, GBM IDH-wild ype became he unique ype o GBM (Louis e al., 2021).
GBMs occu exclusi ely in he b ain and a e commonly loca ed in he sup a en o ial egion,
occu ing in he ou lobes: on al (26.8%), empo al (20.2%), pa ie al (11.6%) and occipi al (2.8%).
Howe e , al hough ela i ely a e, GBMs can also appea in he b ains em (4.3%) and ce ebellum (2.8%).
(Os om e al., 2020). These umo s a e highly in il a ing, and app oxima ely hal in il a e mo e han one
lobe and app oxima ely 5% g ow mul i ocally, in adul s (Djalilian e al., 1999; Wi sching e al., 2016). The
clinical ou come o GBM pa ien s has been demons a ed o be in luenced by umo si e. Indeed, a s udy
demons a ed ha pa ien s wi h on al lobe GBM had a longe su i al compa ed o pa ien s wi h
empo al o pa ie al lobe GBM (11.4 mon hs
s
. 9.1 and 9.6 mon hs, espec i ely) (Simpson e al., 1993).
While GBM me as ases o dis an o gans a e ex emely a e, me as ases o bones, lungs, pleu a, li e ,
mesen e y, lymph nodes, li e and neck ha e been epo ed (Cunha & Maldaun, 2019; Pasquie e al.,
1980; Rosen e al., 2018; Schwei ze e al., 2001; Seo e al., 2012).

| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
8
Depending on he size and loca ion o he umo , he clinical p esen a ion o a pa ien wi h a newly
diagnosed GBM can a y widely. The inc eased in ac anial p essu e, which is a consequence o he
g adual inc ease in umo size and he edema su ounding he umo , o en causes headache and ocal
o p og essi e neu ological de ici s. Seizu es mani es in abou 20% o 40% o pa ien s and gai imbalance
and incon inence can also be mani es ed, usually in la ge umo s. Non-speci ic complain s include
headaches, dizziness, nausea, le ha gy, hemipa esis, isual loss, speech di icul ies, s oke-like
symp oms, memo y p oblems o pe sonali y changes, he las o en con used wi h psychia ic diso de s
o demen ia, especially in he elde ly indi iduals (Hani e al., 2017; Iacob & Dinca, 2009; Omu o &
DeAngelis, 2013).
1.4.1
GBM T ea men : a mul imodal app oach
Despi e he e o s o in es iga e di e en success ul ea men s and ecen ad ances in he
unde s anding o GBM molecula mechanisms, e ec i ely ea ing GBM pa ien s p esen ing a apid and
un a o able clinical e olu ion emains di icul . GBM has an unp edic able esponse o mos he apies
mainly because o i s high p oli e a i e ac i i y, in il a ion o su ounding issues, and he e ogeneous and
complex na u e. In addi ion, he blood-b ain ba ie (BBB) makes ea men e en mo e di icul as i limi s
he e ec i eness o a ge ed-si e he apies (Iacob & Dinca, 2009; Taylo e al., 2019).
A mul imodal app oach is needed o he ea men o GBM. The cu en s anda d he apy is based
on maximal su gical esec ion, ollowed by adio he apy and chemo he apy wi h adminis a ion o
alkyla ing agen (Cla ke e al., 2010; S upp e al., 2005, 2006, 2009; Wilson e al., 2014). Su gical
esec ion is pe o med wi h maximum sa e y in o de o educe he umo load and a oid pu ing he
pa ien 's neu ological unc ion a isk (Lukas e al., 2019). Howe e , owing o he in asi e na u e o GBM,
his he apy is no cu a i e and a ely elimina es esidual umo cells which may lead o disease
p og ession o ecu ence in he u u e (B andes e al., 2008; Lukas e al., 2019). Thus, pos -su gical
ea men – concomi an and adju an adio he apy and chemo he apy wi h alkyla ing agen – is needed
o p e en ecu ence (Hani e al., 2017; Lukas e al., 2019). The alkyla ing agen s cause DNA damage
and selec i e cy o oxici y, which leads o apop osis and cell dea h, by adding me hyl g oups a di e en
posi ions in he DNA (S obel e al., 2019). Se e al alkyla ing agen s ha e been es ed o hei
e ec i eness in ea ing GBM, wi h emozolomide (TMZ) eme ging as he gold s anda d chemo he apeu ic
agen (S upp e al., 2005). TMZ ac s by adding a me hyl g oup a he N7 posi ion o guanine, O3 posi ion
o adenine and O6 posi ion o guanine. Alkyla ion o he O6 si e on guanine is he main eason ha leads
o a cy o oxic e ec in umo cells, esul ing in double-s and b eaks and base mispai ing in DNA, esul ing
in cell cycle a es and apop osis (S obel e al., 2019; J. Zhang e al., 2012). TMZ was disco e ed in he
1. | In oduc ion
9
1970s and app o ed by he Food and D ug Adminis a ion in 2005 a e a la ge in e na ional clinical ial
by S upp e al. ha e demons a ed ha TMZ adminis a ion esul ed in p olonged su i al o GBM pa ien s.
This s udy showed ha when pa ien s we e ea ed wi h concomi an and adju an adio he apy wi h TMZ
compa ed o adio he apy alone, hei OS imp o ed by 2.5 mon hs (12.1 mon hs
s
14.6 mon hs) (S upp
e al., 2005). Mo eo e , TMZ is able o c oss he BBB and o each he apeu ically ele an concen a ions
in he b ain (S obel e al., 2019; Taylo e al., 2019). Thus, he s anda d o ca e o he ea men o
GBM consis s o pos ope a i e adio he apy (a o al o 60 Gy in 30 ac ions – 2 Gy pe daily ac ion) wi h
concomi an daily TMZ adminis a ion, ollowed by 6 cycles o adju an TMZ (S upp e al., 2005, 2009;
Wilson e al., 2014).
1.4.2
Molecula p ognos ic ac o s o GBMs
Un o una ely, he s anda d he apy cu en ly a ailable o GBM pa ien s is unable o change he
le hali y o his disease. Fu he mo e, he molecula and gene ic he e ogenei y o GBMs con ibu es o a
a ied esponse o ea men s. Thus, he iden i ica ion o molecula ma ke s o p ognosis allowing he
ca ego iza ion o subg oups o GBM pa ien s is c i ical o imp o e hei ea men ou comes and
a emp ing o pe sonalize hei clinical managemen . Pa ien ’s age, Ka no sky pe o mance s a us (KPS),
and su gical esec ion’s ex en a e he mos consis en and well-es ablished p ognos ic a iables in GBM.
Pa ien ’s age is a p edic o o poo p ognosis, as olde pa ien s end o p esen a sho e OS han younge
pa ien s. Fu he mo e, pa ien s wi h a highe KPS ha e a longe OS. Also, mo e comple e esec ions a e
associa ed wi h be e OS esul s (Ahmadloo e al., 2013; Xa ie -Magalhães e al., 2013). Mu a ions in
he
IDH
gene ( o me ly men ioned) (Cohen e al., 2013) and he me hyla ion o he O-6-me hylguanine-
DNA me hyl ans e ase (
MGMT
) gene p omo e
(Hegi e al., 2004) a e he only ones cu en ly being used
in he clinical con ex o GBM pa ien s a i ica ion.
MGMT
p omo e me hyla ion s a us
The mos p omising bioma ke o esponse o he apy so a is, undoub edly, he p omo e
me hyla ion o he
MGMT
gene. This gene encodes a ubiqui ously exp essed DNA epai enzyme ha
emo es alkyl g oups om he O6 posi ion o guanine (Wick e al., 2014). This DNA alkyla ion si e is he
a ge o he TMZ alkyla ing agen in ea ing umo cells (S obel e al., 2019). Thus, he ac i i y o he
MGMT enzyme in e e es wi h he e ec o TMZ, coun e ac ing i s he apeu ic e icacy, which ep esen s
a po en ial mechanism o esis ance o he apy (Feldheim e al., 2019; Wick e al., 2014).
Hype me hyla ion o he
MGMT
p omo e esul s in i s epigene ic silencing, hus inducing loss o
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
10
exp ession. Consequen ly, DNA epai ac i i y is educed leading o inc eased sensi i i y o alkyla ing
agen s (Es elle e al., 2000). O no e,
MGMT
exp ession is educed in a ound 50% o GBMs. S udies ha e
shown ha his silencing o he
MGMT
p omo e egion is associa ed wi h a highe OS o GBM pa ien s.
Indeed, Hegi and colleagues obse ed ha he median su i al was 21.7 mon hs o pa ien s whose
umo con ained he me hyla ed
MGMT
p omo e compa ed o 12.7 mon hs o pa ien s wi h
unme hyla ed
MGMT
(Hegi e al., 2004, 2005).
While he cu en ly es ablished bioma ke s hold p omise in helping o imp o e he ea men o
GBM, pa ien s s ill p esen a poo p ognosis (Taylo e al., 2019). The e o e, scien is s a e s ill wo king
ying o ind o he use ul bioma ke s. Wi hin he scope o his hesis, we will s udy he po en ial ole o
he a axin-3 (ATXN3) p o ein in he con ex o GBM. This p o ein has al eady been shown o play a ole in
se e al o he ypes o cance , which will also be discussed la e in his hesis.
1.5
The ATXN3 p o ein
ATXN3 is a p o ein encoded by he
ATXN3
gene wi h an app oxima e molecula weigh o 42 kDa.
This p o ein is known o be in ol ed in Spinoce ebella a axia ype 3 (SCA3), a neu odegene a i e disease
caused by he uns able expansion o a cy osine-adenine-guanidine (CAG) inucleo ide wi hin he coding
egion o he
ATXN3
gene.
ATXN3 is ubiqui ously exp essed in neu onal and pe iphe al issues al hough wi h some cellula
di e ences in he exp ession pa e n (T o ie e al., 1998). In e ms o subcellula localiza ion, ATXN3 is
p edominan ly ound in he cy oplasm (Paulson e al., 1997), al hough i has been epo ed o be able o
ansloca e om he cy oplasm o he nucleus and ice e sa (Macedo-Ribei o e al., 2009), and o be
associa ed wi h he nuclea ma ix (Tai e al., 1998). This nucleocy oplasmic shu ling is media ed by a
nuclea -localiza ion signal (NLS) and wo po en ial nuclea expo signals (NES), which a e p esen in he
ATXN3
sequence (An ony e al., 2009; Macedo-Ribei o e al., 2009). Fu he mo e, ATXN3 is an
e olu iona ily conse ed p o ein, wi h o hologs in se e al o ganisms – mice (Do Ca mo Cos a e al.,
2004), a (Schmi e al., 1997), chicken (Linha o á e al., 1999),
C. elegans
(A.J. Rod igues e al.,
2007), among o he s. These homologous p o eins sha e unc ional domains, such as he Josephin
domain (JD) and he ubiqui in-in e ac ing mo i s (UIM), ne e heless, he long polyglu amine (polyQ) ac
appea s o be human-speci ic, since i is nea ly absen in o he species, such as mouse and
C. elegans
,
which only con ain six and one glu amine, espec i ely (Do Ca mo Cos a e al., 2004; A.J. Rod igues e
al., 2007).
1. | In oduc ion
11
As a esul o a ia ions in he ca boxyl e minal o he
ATXN3
gene p oduc caused by al e na i e
splicing and a s op codon polymo phism, se e al ATXN3 iso o ms can be ansla ed, di e ing in he
numbe o UIMs and in hei C- e minal sequence (Be encou e al., 2010; Go o e al., 1997; Weishäupl
e al., 2019). The e a e wo main iso o ms: he o iginal ATXN3 iso o m isola ed om he SCA3 human
b ain in 1994 con ains 2 UIMs and a C- e minus o hyd ophobic na u e (Kawaguchi e al., 1994); la e ,
ano he iso o m was iden i ied ha con ains 3 UIMs and a hyd ophilic C- e minal egion, being p oposed
as he mos abundan iso o m in he b ain (Ha is e al., 2010; Ichikawa e al., 2001).
ATXN3 belongs o he cys eine p o eases amily and, s uc u ally, i is composed by: a s uc u ed
and ex emely conse ed globula N- e minal JD (1-1998 aa), ollowed by a lexible and uns uc u ed C-
e minal ha con ains he polymo phic polyQ ac o a iable leng h and wo o h ee UIMs depending
on he iso o m - wo UIMs be o e and one a e he polyQ ac (
Figu e 3A
) (Masino e al., 2003; Nicas o
e al., 2005). The JD and UIMs a e he main unc ional uni s o ATXN3 ha syne gis ically con ol i s
ac i i y as a deubiqui ina ing (DUB) enzyme, playing a ole in cellula p o ein quali y con ol, h ough
Ubiqui in-p o easome sys em (UPS), and in egula ion o he quali y and s abili y o di e en subs a es
(B. Bu ne e al., 2003; Cos a e al., 2010; Ne es-Ca alho e al., 2015; Winbo n e al., 2008). Nuclea
magne ic esonance analysis e ealed ha he JD is mainly composed by wo subdomains – a helical
hai pin and a globula ca aly ic subdomain comp ising a ca aly ic si e composed o a iad o cys eine
(C14), his idine (H119) and aspa agine (N134), cha ac e is ic o cys eine p o eases (
Figu e 3B
), and wo
binding si es o ubiqui in (Ub) (Chow e al., 2004; Nicas o e al., 2005; Scheel e al., 2003). E idence
has shown ha he Q9 esidue is equally impo an o ATXN3’s ca aly ic ac i i y (Mao e al., 2005;
Nicas o e al., 2005). The JD has g ea e a ini y o , and clea es, poly-ubiqui yla ed p o eins con aining
ou o mo e Ub molecules (B. Bu ne e al., 2003). The UIMs a e 15-aa mo i s ha media e he speci ic
ecogni ion and posi ioning o Ub chains ela i ely o he ca aly ic si e o p o eoly ic clea age by ATXN3
(Be ke e al., 2005; Chai e al., 2004). Howe e , e idence has shown ha he UIMs may be dispensable
o clea age (Todi e al., 2009).
ATXN3 is subjec o pos - ansla ional modi ica ions, such as phospho yla ion (Ma os e al., 2016;
Muelle e al., 2009), ubiqui yla ion (Todi e al., 2009, 2010) and SUMOyla ion (Almeida e al., 2015)
(
Figu e 3A
). These modi ica ions may in luence i s unc ion, subcellula localiza ion, and in e ac ion wi h
o he molecules (Ca alho e al., 2018; Ma os e al., 2019).
3. | MATERIALS AND METHODS

3. | Ma e ials and Me hods
19
3.
MATERIALS AND METHODS
3.1
Glioma da ase s
ATXN3
gene exp ession and clinical da a we e ob ained om The Cance Genome A las (TCGA)
(The Cance Genome A las Resea ch Ne wo k, 2008), a e e ence da ase in cance esea ch, a ailable
o download a h ps://po al.gdc.cance .go /. Mic oa ay and RNAseq da a e alua ed by Agilen
G4502A 244K and Illumina HiSeq 2000 Sequencing Sys em, espec i ely, we e collec ed. Mic oa ay
da a include 573 GBM, 27 lowe -g ade gliomas (LGG; 7 g ade II and 20 g ade III gliomas), and 10 non-
umo al samples, while RNAseq da a include 161 GBM, 466 LGG (226 g ade II and 240 g ade III
gliomas), and 5 non- umo al samples. When mo e han one po ion was a ailable pe pa ien , he median
exp ession alue was used o a oid epea ed en ies om he same pa ien , as p e iously desc ibed
(Gonçal es e al., 2020). Clinical da a used o each pa ien includes in o ma ion on age a diagnosis,
gende , ea men ecei ed, OS, Ka no sky pe o mance (KPS) and i al s a us.
To assess he exp ession o
ATXN3
by glioma g ade he ollowing da ase s (mic oa ay da a),
a ailable o download a GlioVis websi e (h p://glio is.bioin o.cnio.es/) (Bowman e al., 2017), we e
also used: Remb and (100 g ade I/II, 85 g ade III and 130 g ade IV gliomas) (Madha an e al., 2009),
G a endeel (32 g ade I/II, 85 g ade III and 159 g ade IV gliomas) (G a endeel e al., 2009), F eije (26
g ade III and 59 g ade IV gliomas) (F eije e al., 2004), Phillips (24 g ade III and 76 g ade IV gliomas)
(Phillips e al., 2006), Vi al (12 LGG – 3 g ade I, 3 g ade II and 6 g ade III – and 28 g ade IV gliomas)
(Vi al e al., 2010) and Kamoun (46 g ade II, 102 g ade III and 21 g ade IV gliomas) (Kamoun e al.,
2016) da ase s. Fo su i al analyses, only GBM pa ien s da a was used, om he ollowing da ase s:
Remb and , G a endeel, F eije, Phillips, Vi al, Joo (Joo e al., 2013), LeeY (Y. Lee e al., 2008) and Nu
(Nu e al., 2003) da ase s. Fo su i al analysis, he op imal cu -o de e mined by GlioVis (calcula ed
using he maximum selec ed ank s a is ics) was used o de ine
ATXN3
-high and
ATXN3
-low glioma
pa ien s (Bowman e al., 2017). Clinical da a included OS and i al s a us o he pa ien s.
3.2
Cell lines and cul u e condi ions
The comme cially a ailable human GBM cell lines U87MG and U373MG we e kindly p o ided by
D . Joseph Cos ello, Uni e si y o Cali o nia San F ancisco. The comme cially a ailable human GBM cell
lines U251MG, A172 e LN229 we e pu chased om Ame ican Type Cul u e Collec ion (ATCC). The
comme cially a ailable human GBM cell line SNB19 was pu chased om DSMZ, Ge many. Immo alized
human as ocy es (hTERT/E6/E7) we e p e iously es ablished (Tsu uga e al., 2008). The cells we e
cul u ed in Dulbecco's Modi ied Eagle Medium (DMEM; Sigma-Ald ich) supplemen ed wi h 10% Fe al
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
20
Bo ine Se um (FBS; Sigma-Ald ich). HEK293T cells we e cul u ed in Op i-MEM (Gibco) supplemen ed
wi h 10% FBS, 1% glu aMAX (Gibco), penicillin 100 U/ml and s ep omycin 100 mg/ml (Gibco). All he
cells we e incuba ed in a humidi ied a mosphe e a 37°C and 5% ( / ) CO2 h oughou he s udies.
Tes ing o mycoplasma con amina ion was pe o med egula ly. A neu oblas oma cell line (SH-SY5Y),
pu chased om ATCC, was used as a posi i e con ol.
3.3
Plasmid ans o ma ion in o
Esche ichia coli
(
E. coli)
The bac e ial ans o ma ion was pe o med in o de o p opaga e he cons uc ed plasmids. The
ans o ma ion was pe o med in o
E. coli DH5 alpha
compe en cells, using he hea shock
ans o ma ion me hod. B ie ly,
E. coli
compe en cells and plasmids we e hawed on ice o 30 minu es
and hen 1 µl o he DNA was added o 50 µL o
E. coli
. The mix u e was incuba ed on ice o 30 minu es.
A e incuba ion, he mix u e was hea shocked a 42ºC o 45 seconds and hen placed back on ice o
30 minu es. Then, Lu ia Be ani (LB) medium wi hou an ibio ic was added o he cells and incuba ed a
37ºC wi h shaking o 2 hou s. O e nigh a 37ºC, 100 µL o he cul u e was g own on LB aga pla es
wi h he app op ia e an ibio ic. The nex day, a colony was inocula ed in LB medium wi h ampicillin (100
mg/mL; Sigma) a 37ºC, o e nigh wi h agi a ion. Plasmid DNA was isola ed using he GeneJET Plasmid
Midip ep ki (The mo Scien i ic) acco ding o manu ac u e ’ p o ocol. The concen a ion and pu i y o DNA
we e assessed using Nanod op (The mo Scien i ic NanoD op 1000 Spec opho ome e ).
3.4
Len i i us p oduc ion
Len i i al pa icles we e p oduced using HEK293T cells. The cells we e pla ed in a 12-well pla e a
a densi y o 250000 cells/well in Op i-MEM supplemen ed wi h 10% FBS. On he ollowing day, he a ge
gene ec o s (pLen i +
ATXN3
– ec o con aining he ull-leng h o
ATXN3 –
and he espec i e emp y
ec o ) and he len i i al ec o s (psPax2 and pMD2.G) we e co- ans ec ed in o HEK293T cells using
Fugene eagen (P omega), acco ding o he manu ac u e 's ecommenda ions. Fi s , Fugene
eagen :plasmid complex we e dilu ed in Op i-MEM and incuba ed o 5 minu es a oom empe a u e
(RT), and hen, cells we e incuba ed wi h ans ec ion complex du ing app oxima ely 16 hou s. A e he
incuba ion, he medium was enewed. Th ee days a e ans ec ion, he supe na an was collec ed and
il e ed h ough a 0.45 µm il e in o de o emo e cell deb is.
3. | Ma e ials and Me hods
21
3.5
ATXN3 o e exp ession in GBM cells
The A172 cell line was pla ed a a cell densi y o 40000 cells/well in a 12-well pla e in DMEM
supplemen ed wi h 10% FBS. On he ollowing day, cells we e in ec ed wi h he len i i al pa icles
con aining he o e exp ession ec o (A172-ATXN3;
Figu e 5
) o he espec i e emp y ec o (A172-C l;
Addgene, w118-1) in he p esence o polyb ene (8 µg/ml). Hal o he o al olume o i us ob ained was
used. The medium was changed on he nex day and he cells we e allowed o eco e . Subsequen ly,
success ully in ec ed cells we e selec ed wi h pu omycin (0.5 µg/ml; San a C uz Bio ecnologies) since
hese cons uc s p esen a pu omycin esis ance gene. The cell line o u he expe imen s was gene a ed
om he polyclonal expansion o he in ec ed/selec ed cells. The o e exp ession e icacy was con i med
by Polyme ase Chain Reac ion (PCR) and Wes e n blo (WB).
Figu e 5 Cons uc ed ec o used o ATXN3 o e exp ession in GBM cells.
3.6
RNA ex ac ion
To al RNA was ex ac ed om he U251 and A172 cell lines wi h di e en ial le els o
ATXN3
exp ession (A172-C l and A172-ATXN3) using he TRIzol eagen (In i ogen), acco ding o he
manu ac u e 's ecommenda ions. To al RNA om o he human GBM cell lines, p ima y GBM pa ien -
de i ed cul u es and human immo alized as ocy es we e p e iously ob ained by he g oup using he
same me hod. B ie ly, TRIzol was added o he cell pelle s (collec ed om he cell lines by cen i uga ion
a 150 x g o 5 minu es a 4ºC – Mega uge 16 Cen i uge; The moScien i ic). Each sample was
homogenized and incuba ed o allow he cells o lyse. Then, chlo o o m (200 µL/mL o TRIzol) was added
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
22
o each ube and he samples we e incuba ed and cen i uged a 21100 x g (F esco 21 Mic ocen i uge;
The moScien i ic) o 15 minu es a 4ºC o p omo e phase sepa a ion. The uppe clea aqueous phase
(con aining he RNA) was ca e ully collec ed, and isop opanol (500 µL/mL o TRIzol) was added o
p ecipi a e he RNA. A e incuba ion he samples we e cen i uged a 12000 x g o 10 minu es a 4ºC.
The supe na an was emo ed and inally, he p ecipi a ed RNA was washed in 75% e hanol (1mL/mL o
TRIzol) and cen i uged a 7500 x g o 5 minu es a 4ºC. The supe na an was disca ded, and he RNA
was dilu ed in RNase and DNase ee wa e . The quan i y and pu i y o RNA we e assessed using
Nanod op (The mo Scien i ic NanoD op 1000 Spec opho ome e ). RNA in eg i y was con i med by
unning a 1% aga ose gel p epa ed in 1x T is-ace a e-EDTA (TAE) bu e .
3.7
DNA ex ac ion
To de e mine he CAG epea o he
ATXN3
gene in he glioma cell lines, DNA om he A172 cell
line and U87MG was ex ac ed using he Ci ogene DNA Isola ion Ki (Ci omed), acco ding o he
manu ac u e 's ecommenda ions. B ie ly, Cell Lysis solu ion was added o he cells in o de o b eak he
cell memb anes and elease he DNA and p o ein. A e cen i uga ion a 15700 x g (Cen i uge 5415D;
Eppendo ) o 6 minu es, a compac p o ein pelle is o med and he DNA (p esen in he supe na an )
is p ecipi a ed wi h 100% isop opanol. The samples we e cen i uged a 15700 x g o 4 minu es and,
a e wa ds, he DNA, in he o m o a whi e pelle , was washed wi h 70% e hanol. A new cen i uga ion a
15700 x g o 4 minu es was pe o med and inally, DNA hyd a ion solu ion was added o he pelle . The
quan i y and pu i y o DNA we e assessed using Nanod op (The mo Scien i ic NanoD op 1000
Spec opho ome e ).
3.8
cDNA syn hesis
A e RNA quan i ica ion, a ea men wi h DNase was pe o med o emo e possible genomic DNA,
using he DNase I, RNase- ee ki (The mo Scien i ic), acco ding o he manu ac u e 's ecommenda ions.
The eac ion was p epa ed wi h 1 µg o RNA, Reac ion Bu e wi h MgCl2 (1x), DNase I (0.1 U/µL) and
wa e o a olume o 10 µL. The eac ion ook place in he he mo cycle (Bio-Rad T100 The mal Cycle )
a 37ºC o 30 minu es. The eac ion was e mina ed wi h he addi ion o 1 µL o EDTA (5 mM) pe
sample and incuba ion a 65ºC o 10 minu es. Then, he cDNA was syn hesized om 1 µg o RNA using
he iSc ip cDNA Syn hesis Ki (Bio ad). B ie ly, a eac ion mix u e was p epa ed adding nuclease- ee
wa e , 1x iSc ip Reac ion Mix (con ains dNTP’s, p ime s) and iSc ip Re e se T ansc ip ase. The sample
was placed in he he mo cycle (Bio-Rad T100 The mal Cycle ) and p og ammed wi h he ollowing
3. | Ma e ials and Me hods
23
p o ocol: 25ºC o 5 minu es (p ime binding), 46ºC o 20 minu es ( e e se ansc ip ion), 95ºC o 1
minu e (enzyme inac i a ion) and 4ºC in ini ely.
3.9
Quan i a i e Polyme ase Chain Reac ion (qPCR)
The le els o
ATXN3
and
HPRT1
, used as a e e ence gene, we e assessed by qPCR using he
SoFas E a G een RT-PCR eagen ki (Bio-Rad), acco ding o he manu ac u e ’s ecommenda ions. A
eac ion mix was p epa ed by adding DNase and RNase ee wa e , he E aG een enzyme, he o wa d
and e e se p ime s (0.2 µM
HPRT1
and 0.5 µM
ATXN3
;
Table 2
) and he cDNA samples, ob ained as
desc ibed abo e. The samples we e placed in he 7500 Fas Real-Time PCR Sys em (Applied Biosys ems)
and he qRT-PCR cycling condi ions used we e: 95ºC o 30 seconds (enzyme ac i a ion), 95ºC o 5
seconds (DNA dena u a ion) and 60ºC o 30 seconds (p ime s annealing/ex ension). The las 2 s eps
we e epea ed 40 imes, and a e ha a mel ing cu e was pe o med o assess whe he he eac ion
p oduced unique and speci ic p oduc s. In each un, nega i e con ols (mix solu ion, bu wi hou any
cDNA) we e included o sc een o possible con amina ion. The exp ession le els we e no malized o he
ela i e exp ession o he
HPRT1
gene. Resul s we e p esen ed using he ΔΔc me hod (Li ak &
Schmi gen, 2001). The a ia ion be ween he Cq o samples was calcula ed and he ela i e exp ession
was de e mined.
3.10
PCR
A PCR was pe o med o e alua e he
ATXN3
exp ession in he A172-o e exp ession cell line, using
he AmpliTaq Gold 360 DNA Polyme ase ki (Applied Biosysys ems), acco ding o he manu ac u e ’s
ecommenda ions. A eac ion mix was p epa ed by adding DNase and RNase ee wa e , AmpliTaq Gold
360 Bu e (1x), Magnesium Chlo ide (1.5 mM), dNTP mix (200 µM each), AmpliTaq Gold 360 DNA
Polyme ase (2 U), he o wa d and e e se p ime s (0.8 µM;
Table 2
and he cDNA sample. The ollowing
p o ocol was used in he he mo cycle (Bio-Rad T100 The mal Cycle ): 95ºC o 10 minu es (DNA ini ial
dena u a ion), 95ºC o 30 seconds (DNA dena u a ion), 60ºC o 30 seconds (p ime s annealing), 72ºC
o 60 seconds (p ime s ex ension) and 72ºC o 7 minu es ( inal ex ension). The 3 in e media e s eps
we e epea ed 35 imes. The ea e , he PCR p oduc s we e un on a 2% aga ose gel p epa ed in 1x T is-
bo a e-EDTA (TBE) bu e .
The de e mina ion o CAG epea leng h was pe o med in he U87MG and A172 glioma cell lines
by PCR ampli ica ion, using he p e iously ex ac ed DNA. Fo his, a eac ion mix u e was p epa ed by
adding nuclease- ee wa e , MyTaq Reac ion Bu e (1x), MyTaq HS DNA polyme ase (Bioline) and p ime s

| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
24
(0.5 µM;
Table 2
). The ollowing p o ocol was used in he he mo cycle (Bio-Rad T100 The mal Cycle ):
95ºC o 3 minu es (dena u a ion), 95ºC o 15 seconds (annealing), 55ºC o 15 seconds (ex ension),
72ºC o 15 seconds ( inal ex ension). The las 3 s eps we e epea ed 30 imes. The PCR p oduc s we e
analyzed by agmen analysis in compa ison o a size s anda d, as desc ibed in (Sil a-Fe nandes e al.,
2014).
Table 2
.
Sequence o p ime s used in he qRT-PCR, PCR, and in he de e mina ion o CAG leng h.
3.11
Wes e n Blo (WB)
Cells we e washed using PBS 1x and emo ed by sc a ch in he lysis bu e [RIPA bu e : 50 mM
T is-HCl pH 7.4, 250 mM NaCl, 2 mM EDTA, 10% Glyce ol and inhibi o s o p o eases 1x (Roche)]. The
cell lysa e was incuba ed o 15 min and hen cen i uged a 21100 x g (F esco 21 Mic ocen i uge;
The moScien i ic) o 15 min a 4ºC. Using he ob ained supe na an , he o al p o ein concen a ion was
de e mined by he B ad o d me hod, using he ki P o ein Assay Dye Reagen Concen a e (Bio-Rad).
P o ein ex ac s we e dena u ed and educed wi h 2x Laemmli Sample Bu e (Bio-Rad), o which 20 mM
di hio h ei ol (DTT) was added. Then he p o ein (10-20 µg) was sepa a ed in a 10% SDS-polyac ylamide
esol ing gel and a 4% s acking gel by elec opho esis. A molecula weigh ma ke (GRS p o ein ma ke
mul icolou - GRISP) was added. The gel was ans e ed o ni ocellulose memb anes using he T ans-
Blo Tu bo T ans e Sys em (Bio-Rad), and he Ponceau S dye was used o con i m he e iciency o he
ans e . Be o e immunode ec ion, he memb anes we e blocked wi h 5% milk o 1 hou a RT, in o de
o p e en non-speci ic binding o he an ibody. Subsequen ly, an ibodies agains ATXN3 (1H9; Re .
MAB5360; Millipo e), GAPDH (Re : ab9485; Abcam) and b-ac in (Re . 8227; Abcam) we e used o
immunode ec ion, in which he memb anes we e incuba ed o e nigh a 4ºC. A e incuba ion wi h he
p ima y an ibody, he memb anes we e washed wi h washing bu e (2.5% milk dilu ed in 1x TBS and
3. | Ma e ials and Me hods
25
0.1% Tween) o 10 min. Then, he memb anes we e incuba ed o 1 hou a RT wi h pe oxidase-
conjuga ed seconda y an i-mouse (Re . 1706516; Bio-Rad) o an i- abbi IgG an ibodies (Re . 1706515;
Bio-Rad). Blo s we e e ealed using an enhanced chemiluminescence (ECL) solu ion (Cla i y Wes e n ECL
Subs a e; Bio- ad). Chemiluminescence was measu ed using he Sapphi e Biomolecula Image (Azu e
Biosys ems) and wi h Wide Dynamic Range o exposu e. Band quan i ica ion was pe o med using
Azu eSpo , acco ding o he manu ac u e ’s ins uc ions. P o ein exp ession was no malized using b-ac in
(GBM pa en al cells) o GAPDH (A172-o e exp ession model) as a con ol p o ein.
3.12
Cell iabili y assays
3.12.1
T ypan Blue assay
A172 (A172-C l and A172-ATXN3) cells we e pla ed, in iplica e, in 6-well pla es a an ini ial
densi y o 15000 cells/well and allowed o adhe e and g ow o 4 and 6 days. A day 4 and 6, he cells
we e eco e ed by ypsiniza ion and mixed wi h ypan blue dye (1:1 a io; Gibco). Viable cells possess
in ac cell memb anes ha exclude he dye, unlike dead cells, ha ha e comp omised memb ane
in eg i y. The numbe o iable cells om each well was coun ed wi h he help o Neubaue chambe ,
unde he mic oscope in duplica es. The o al numbe o cells was calcula ed using he o mula:
𝑚𝑒𝑎𝑛%𝑜𝑓%𝑣𝑖𝑎𝑏𝑙𝑒%𝑐𝑒𝑙𝑙𝑠% × %𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛%𝑓𝑎𝑐𝑡𝑜𝑟% ×%10!×𝑡𝑜𝑡𝑎𝑙%𝑣𝑜𝑙𝑢𝑚𝑒
.
3.12.2
MTS assay
A172 (A172-C l and A172-ATXN3) cells we e pla ed, in iplica e, in 24-well pla es a an ini ial
densi y o 2000 cells/well and allowed o adhe e and g ow o 6 days. A day 6, he cells we e incuba ed
wi h 10% o he MTS solu ion (CellTi e 96® AQueous One Solu ion Cell P oli e a ion Assay; P omega) in
DMEM supplemen ed wi h 10% FBS, in he da k, in a humidi ied a mosphe e, a 37ºC and 5% CO2 o
app oxima ely 2 hou s. MTS (3-(4,5-Dime hyl hiazol-2-yl)-5-(3-ca boxyme hoxyphenyl)-2-(4-sul ophenyl)-
2H- e azolium) is educed o a pu ple soluble o mazan p oduc in me abolically iable cells by a
NAD(P)H-dependen mi ochond ial dehyd ogenase enzyme, hus p o iding in o ma ion abou he
me abolic iabili y o he cells. A e incuba ion, he o mazan dye p oduced by iable cells was quan i ied
by measu ing he abso bance a 490 nm.
3.13
Cell p oli e a ion assay
Cell p oli e a ion was assessed based on he measu emen o b omodeoxyu idine (B dU), a
syn he ic analogue o hymidine inco po a ed du ing DNA syn hesis, using he Cell P oli e a ion ELISA,
B dU colo ime ic assay ki (Roche), and acco ding o he manu ac u e 's ecommenda ions. The A172
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
26
(A172-C l and A172-ATXN3) cells we e pla ed, in iplica e, in 96-well pla es, a an ini ial densi y o 2000
cells/well and incuba ed o 4 days. Subsequen ly, B dU was added o he cell cul u e, and hey we e e-
incuba ed o 8 hou s. Then, an enzyme-linked immunoso ben assay (ELISA) was pe o med. FixDena
was added o he cells, a solu ion ha ixes he cells and dena u es he DNA o enable an ibody binding.
A e 30 minu es o incuba ion a RT, an i-B dU-POD an ibody was added o he cells and incuba ed o
ano he 90 minu es a RT. Subsequen ly, he an ibody conjuga e was emo ed, and he wells we e
washed 3 imes wi h Washing Solu ion o allow o emo al o unbound an ibodies. Finally, Subs a e
Solu ion was added o allow pho ome ic de ec ion a 370 nm.
3.14
Cell mig a ion assay
Cell mig a ion capaci y was assessed using he wound healing assay. The A172 (A172-C l and
A172-ATXN3) cells we e pla ed, in iplica es, a an ini ial cell densi y o 70000 cells in each side o Ibidi
2-well inse s (Ibidi) and le o adhe e o e nigh . Subsequen ly, he inse s we e emo ed (0 hou s
imepoin ), lea ing a 500 µm cell- ee gap. The c ea ed a i icial wound om each well was pho og aphed
o e ime in he same posi ion using he CKX41 in e ed mic oscope (Olympus), un il he wound was
comple ely closed (app oxima ely 24 hou s). The ela i e cell mig a ion (gap size) was measu ed using
an au oma ed so wa e (beWound - Cell Mig a ion Tool, 1.7, ICVS, Po ugal), and he gap size was e i ied
and co ec ed manually, when necessa y. Ten posi ions equally spaced and pe pendicula o he wound
we e measu ed. The pe cen age o wound closu e was calcula ed by measu ing he wid h o he wound
ela i ely o he ini ial wid h o he wound ( ime 0 h).
3.15
Cell in asion assay
Cell in asion was assessed using he Boyden chambe assay. BD BioCoa ™ Ma igel™ In asion
Chambe s (Co ning®) we e used, acco ding o he manu ac u e 's ecommenda ions. The A172 (A172-
C l and A172-ATXN3) cells we e pla ed in he uppe compa men o he chambe a an ini ial densi y o
20000 cells/well in DMEM supplemen ed wi h 1% o FBS. Epide mal g ow h ac o (EGF; 20 µM;
In i ogen), a chemo ac ic agen , was added o he lowe compa men con aining DMEM supplemen ed
wi h 10% FBS. The cells we e le o incuba e o 22 hou s, and, a e his pe iod, non-in ading cells a
he op o he chambe we e gen ly emo ed by a co on swab and he in ading cells, a ached o he
memb ane, we e ixed wi h 100% cold me hanol and s ained using DAPI wi h moun ing medium
(Vec ashield; Vec o Labo a o ies). Whole memb anes we e scanned using an Olympus Wide ield In e ed
3. | Ma e ials and Me hods
27
IX81 mic oscope (Olympus; objec i e lens magni ica ion: 4x) and he o al numbe o in ading cells was
coun ed using he ImageJ so wa e ( e sion 1.53).
3.16
Cell cycle assay
The cell cycle was assessed by low cy ome y using P opidium Iodide (PI) s aining. The A172
(A172-C l and A172-ATXN3) cells we e pla ed a an ini ial densi y o 200000 cells pe T25 lask in DMEM
supplemen ed wi h 10% FBS. Two days la e , cells we e ypsinized, washed wi h 1x PBS and ixed in
70% cold e hanol. A e wa ds, he cells we e cen i uged a 266 x g (Mega uge 16 Cen i uge;
The moScien i ic) o 5 minu es a 4ºC and we e again washed wi h 1x PBS. Then, ixed cells we e
incuba ed wi h PI s aining solu ion [0.1% i on-X-100, 20 µg/mL PI (The moFishe Scien i ic) and 250
µg/mL RNase (In i ogen) in PBS] in he da k o 1 hou a 50ºC. Flow cy ome y was used o cell cycle
analysis o PI-s ained cells. A leas 10000 single cells e en s pe sample we e acqui ed in a BD LSRII
low cy ome e (BD Biosciences) using he FACS DIVA so wa e (BD Biosciences). The collec ed da a was
analyzed using he FlowJo so wa e ( 10.8.0; T ee S a ). The numbe o cells in each phase o he cell
cycle was quan i ied using he Dean-Je -Fox model.
3.17
Cell dea h assay
Cell dea h was assessed by low cy ome y using annexin V/PI s aining. The A172 (A172-C l and
A172-ATXN3) cells we e pla ed a an ini ial densi y o 40000 cells pe T25 lask. A e 24 hou s, cells
we e ea ed wi h TMZ (800 µM) o wi h DMSO (dime hylsul oxide), used as ehicle. The ea men was
enewed 2 days la e . Cell dea h was assessed a e 6 days o ea men wi h TMZ and DMSO. Cells we e
s ained wi h Annexin V-FITC (BD Bioscience) and PI (5 µg/mL; The moFishe Scien i ic), ollowed by low
cy ome ic analyses. A leas 10000 single cells e en s pe sample we e acqui ed in a BD LSRII low
cy ome e (BD Biosciences) using he FACS DIVA so wa e (BD Biosciences). Resul s we e analyzed using
FlowJo so wa e ( 10.8.0; T ee S a ).
3.18
S a is ical analyses
ATXN3
exp ession in gliomas o di e en g ade was e alua ed using he wo-sided unpai ed
- es
o one-way ANOVA. When no mali y was no e i ied by he Shapi o-Wilk es , he non-pa ame ic Mann-
Whi ney and K us al-Wallis es s we e used. Fo he wound healing, cell dea h and cell cycle assays a wo-
way ANOVA ollowed by he pos -hoc Sidak’s es o mul iple compa ison es ing was used. Fo he o he s
assays, homoscedas ici y was e i ied wi h Le ene’s es and di e ences be ween g oups we e assessed
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
34
Figu e 9
.
ATXN3 exp ession dec eases GBM agg essi eness.
(A)
ATXN3 o e exp ession in he A172 cell line was con i med
by WB (le ) and PCR ( igh ).
(B)
Cell iabili y o A172-ATXN3 and A172-C l cell lines was e alua ed by T ypan Blue assay
(n=3).
(C)
Cell iabili y o A172-ATXN3 and A172-C l cell lines was e alua ed by MTS assay (n=3).
(D)
Cell p oli e a ion o
A172-ATXN3 and A172-C l cell lines was assessed by he B dU assay (n=3).
(E-F)
Cell cycle analysis was pe o med by low
cy ome y a e PI s aining. (E) Rep esen a i e image o cell cycle esul s. The ini ial peak and he las peak ep esen he
G0/G1 and G2/M phases, espec i ely, while be ween he peaks is he S phase. (F) Quan i ica ion o he pe cen age o cells
in each phase o he cycle using an algo i hm (Dean-Je -Fox model; FlowJo so wa e).
(G-H)
The mig a ion capaci y o he
(
con inued on nex page
)

4. | Resul s
35
(
con .)
A172-ATXN3 and A172-C l cell lines we e e alua ed by he wound healing mig a ion assay (0 h-12 h n = 3, and 12 h-24 h n
= 4). (G) Quan i ica ion o he pe cen age o wound closu e o e ime. (H) Rep esen a i e images (4x magni ica ion; scale ba
= 20000 µm)
(I-J)
In asion capaci y o he A172-ATXN3 and A172-C l cell lines was e alua ed by he Ma igel Chambe
in asion assay (n=3). (I) Quan i ica ion o he numbe o in asi e cells. (J) Rep esen a i e imagens wi h cell nuclei s ained wi h
DAPI (scale ba = 100 µm). *,
p
< 0.05 (Unpai ed
- es and wo-way ANOVA wi h pos -hoc Sidak’s es o he wound healing
and cell cycle assays).
he e ec o ATXN3 on GBM cell cycling. Flow cy ome y was used o quan i y he p opo ion o cells in
each s age o he cell cycle s ained wi h PI, a DNA binding dye. The e we e no s a is ically signi ican
di e ences compa ing cells wi h ATXN3 o e exp ession and con ol cells, in wha ega ds he pe cen age
o cells in each cell cycle phase (
p
= 0.9646 o G0/G1,
p
= 0.9919 S,
p
= 0.9857 G2/M;
Figu e 9E and
F
). This esul sugges s ha ATXN3 does no a ec cell cycle p og ession o GBM cells. In o de o assess
whe he ATXN3 modula es he abili y o A172 cells o mig a e, we pe o med a wound healing assay o
24 hou s. We did no obse e s a is ically signi ican di e ences be ween con ol and ATXN3
o e exp essing cell lines, concluding ha ATXN3 appea s o no a ec he mig a ion capaci y o he cells
(
Figu e 9G
and H
). Fu he mo e, as GBMs ha e a g ea capaci y o in ade b ain issue (B andes e al.,
2008; C. Smi h & I onside, 2007), we es ed whe he ATXN3 media es GBM in asion, h ough he Boyden
Chambe assay. In e es ingly, we obse ed ha ATXN3 o e exp ession signi ican ly dec eased he
in asi eness o cells (
p
= 0.0217;
Figu e 9I and J
).
O e all, al hough ATXN3 has no impac on he p oli e a ion, cell cycle o mig a ion capaci y o he
GBM cells, ATXN3 exp ession p esen a unc ional impac on GBM cells, by a ec ing hei iabili y and
capaci y o in ade, which sugges s ha i may ac s as a umo supp esso molecule, educing GBM
agg essi eness
in i o
.
4.4 ATXN3 does no a ec he sensi i i y o GBM cells o TMZ
Al hough TMZ is he i s -line chemo he apeu ic agen used o GBM pa ien s, many pa ien s
de elop esis ance o he d ug, leading o ea men ailu e (S. Y. Lee, 2016). In his con ex , we es ed
whe he ATXN3 exp ession may a ec GBM cell sensi i i y/ esis ance o TMZ. T ea men wi h TMZ o
DMSO (used as ehicle) was applied o 6 days, and cell dea h was assessed by low cy ome y using
Annexin V and PI s aining. T ea men o bo h cells (A172-C l and A172-ATXN3) wi h TMZ led o a
signi ican dec ease in he pe cen age o iable cells (
p
= 0.0078 o A172-C l and
p
= 0.0008 o A172-
ATXN3), as well as a signi ican inc ease in Annexin V + PI posi i e cells (
p
= 0.0244 A172-C l and
p
=
0.0027 A172-ATXN3) and in Annexin V posi i e cells (
p
= 0.0307 A172-C l and
p
= 0.0268 A172-
ATXN3), compa ed o ea men wi h ehicle. Howe e , ATXN3 o e exp ession did no a ec he sensi i i y
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
36
o he GBM cells o TMZ-media ed ea men (
Figu e 10
), since he numbe o iable cells (
p
= 0.9516)
and he numbe o apop o ic cells (
p
= 0.9050) is simila in ATXN3 o e exp essing cells and in con ol
cells.
These esul s sugges ha ATXN3 does no a ec he sensi i i y o TMZ ea men in his GBM
cellula model.
Figu e 10
.
ATXN3 does no a ec he sensi i i y o GBM cells o TMZ. (A-B)
A172-ATXN3 and A172-C l cells we e ea ed wi h
DMSO ( ehicle) o TMZ o 6 days and cell dea h was measu ed by low cy ome y (n=3). (A) Pe cen age o li ing and dead
cells, labeled wi h Annexin V and PI. (B) Rep esen a i e do plo s. *, p < 0.05; **, p < 0.01 and ***, p < 0.001 ( wo-way ANOVA
pos -hoc Tukey es ). DMSO: dime hyl sul oxide; PI: p opidium iodide; TMZ: emozolomide.
4. | Resul s
37
4.5
ATXN3
has p ognos ic alue in GBM pa ien s
Gi en ha ou esul s sugges ha ATXN3 plays a ele an ole in GBM agg essi eness
in i o
(
Figu e 9
), we ques ioned whe he in he clinical con ex he e would be an associa ion be ween
ATXN3
exp ession and he p ognosis o GBM pa ien s. To unde s and ha we assessed he p ognos ic alue o
ATXN3
exp ession in 573 GBM pa ien s wi h su i al da a a ailable in he TCGA da abase. Acco ding o
he esul s, pa ien s whose umo s ha e low
ATXN3
exp ession p esen ed a s a is ically sho e OS (OS
median o 13.9 mon hs) compa ed o pa ien s whose umo s ha e high le els o
ATXN3
exp ession (OS
median o 15.4 mon hs;
p
= 0.0215; Log- ank es ;
Figu e 11A
). Addi ionally, we alida ed he p e ious
esul in 8 addi ional independen da ase s. This obse a ion was consis en among 5 di e en da ase s
(Remb and ,
p
= 0.0074,
Figu e 11B
; Phillips,
p
= 0.0475,
Figu e 11C
; F eije,
p
= 0.0240,
Figu e 11D
;
Vi al,
p
= 0.0002,
Figu e 11E
; and Joo,
p
= 0.0072,
Figu e 11F
), and a simila end was obse ed in he
o he 3 da ase s (G a endeel,
p
= 0.0625,
Figu e 11G
; LeeY,
p
= 0.0673,
Figu e 11H
; and Nu ,
p
=
0.1060,
Figu e 11I
).
In addi ion o uni a ia e analysis, a mul i a ia e Cox analysis was pe o med using da a om he
TCGA da abase. This Cox model allows he use o
ATXN3
exp ession as a con inuous a iable and was
used o ake in o accoun he po en ial con ounding e ec o o he known p ognos ic ac o s such as
pa ien age, KPS and gende . In e es ingly, we obse ed a s a is ically signi ican associa ion be ween
lowe
ATXN3
exp ession alues and sho e OS in GBM pa ien s (
p
= 0.025, Exp(B) = 0.713)
independen ly o o he p ognos ic ac o s (
Table 3
). As expec ed, inc easing pa ien ’s age a diagnosis
was signi ican ly associa ed wi h wo se OS (
p
< 0.0001, Exp(B) = 1.708) and inc eased KPS associa ed
wi h be e p ognosis (
p
< 0.0001, Exp(B) = 0.976). GBM pa ien gende was no signi ican ly associa ed
wi h OS
(p
= 0.085).
We also pe o med a me a-analysis o sys ema ically e alua e all da ase s used and ein o ce he
associa ion be ween
ATXN3
exp ession and he p ognosis o GBM pa ien s. O e all, he esul s
demons a e ha high
ATXN3
exp ession is signi ican ly associa ed wi h a be e p ognosis in GBM
pa ien s (HR = 0.612, 95% CI 0.506 – 0.739;
p
< 0.0001; andom e ec ;
Figu e 11J
).
Al oge he , ou indings show ha
ATXN3
exp ession is associa ed wi h longe o e all su i al,
es ablishing
ATXN3
as a clinically ele an bioma ke o p ognosis in GBM pa ien s.
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
38
Figu e 11.
ATXN3 exp ession has a p ognos ic alue in GBM pa ien s. (A-I)
Kaplan-Meie su i al cu es o GBM pa ien s
de i ed om
(A)
TCGA (n=573),
(B)
Remb and (n=203),
(C)
Phillips (n=56),
(D)
F eije (n=59),
(E)
Vi al (n=26),
(F)
Joo (n=54),
(G)
G a endeel (n=159),
(H)
LeeY (n=191) and
(I)
Nu (n=28) da ase s.
(J)
Me a-analysis wi h he associa ion be ween ATXN3
exp ession and o e all su i al in pa ien s wi h GBM. The size o each squa e ep esen s he weigh o he da ase in he me a-
analysis and iangle is he combined e ec . HR: haza d a io, CI: con idence in e al.
4. | Resul s
39
Table 3
.
Mul i a ia e analysis o he associa ion o
ATXN3
exp ession and su i al o GBM pa ien s, adjus ed o pa ien age,
KPS, and gende .

5. | DISCUSSION
5. | Discussion
41
5.
DISCUSSION
Glioblas oma (GBM) is he mos common and malignan ype o glioma in adul s (Os om e al.,
2020). Du ing he las ew yea s, se e al e o s ha e been made o unde s and he molecula
mechanisms o GBM and de elop new ea men s. None heless, he p ognosis o GBM pa ien s is s ill
ex emely poo , being app oxima ely 15 mon hs a e diagnosis (S upp e al., 2005). Thus, i is c ucial o
iden i y and use new molecula ma ke s allowing o pa ien s a i ica ion and he apy imp o emen .
ATXN3 is a p o ein in ol ed in SCA3 disease, a neu odegene a i e disease caused by a CAG epea
expansion wi hin he coding egion o he
ATXN3
gene (Kawaguchi e al., 1994; Takiyama e al., 1993).
In addi ion o i s in ol emen in he pa hogenesis o SCA3, ATXN3 has been desc ibed o play oncogenic
o umo supp esso unc ions in a a ie y o umo ypes (Aue e al., 2007; E gun e al., 2020; Ge e
al., 2015; Ne es-Ca alho e al., 2015; Sacco e al., 2014; Shi e al., 2018; Song e al., 2021; Zeng e
al., 2014; Zou e al., 2019). Howe e , he impac o ATXN3 in gliomas, pa icula ly in GBM, is comple ely
unknown un il now. Thus, he p esen s udy is he i s a emp o e alua e he exp ession o
ATXN3
in
glioma and i s po en ial ole in his malignan b ain umo .
Ou da a showed ha
ATXN3
exp ession dec eases wi h glioma g ade, being less exp essed in
glioma g ade IV (GBM) when compa ed o less malignan gliomas (g ade I, II and III) and no mal samples
a ailable a he TCGA da abase (
Figu e 6
, le panels), sugges ing ha highe
ATXN3
exp ession is
associa ed wi h lowe glioma malignancy. This was alida ed in 6 addi ional independen coho s om
a ious wo ld egions (
Figu e 7
). Addi ionally, we ound ha
ATXN3
is less exp essed in IDH-wild ype
gliomas (wo se p ognosis) when compa ed o IDH-mu an gliomas and wi h 1p/19q co-dele ion (be e
p ognosis) (
Figu e 6
, igh panels). Thus,
ATXN3
exp ession seems o be associa ed wi h IDH mu a ion
and 1p/19q codele ion – and he e o e wi h a be e ou come – which i s well wi h he esul s ound
ega ding i s dis ibu ion along glioma g ades. Acco dingly, a s udy has shown ha ATXN3 exp ession is
dec eased in gas ic cance , whe e ATXN3 play a ole as umo supp esso gene, when compa ed o non-
cance ous issue (Zeng e al., 2014). In con as , in es icula cance , whe e ATXN3 play a ole as
oncogene, ATXN3 was shown o be signi ican ly o e exp essed in es icula cance issues compa ed o
no mal ones.
Nex , we wan ed o u he explo e he po en ial ole o ATXN3 in GBM. To do so, he exp ession o
ATXN3 was ini ially cha ac e ized in GBM cell lines and pa ien -de i ed cul u es. All GBM cells es ed
p esen ed ATXN3 exp ession a he mRNA and p o ein le el (
Figu e 8
). I should be no ed ha
ATXN3
exp ession is mo e he e ogeneous among GBM cells a he mRNA le el, in con as o ATXN3 exp ession
a he p o ein le el, which is mo e homogeneous. Howe e , he cell line showing he lowes le els o
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
42
ATXN3 exp ession was he same in bo h echniques (A172 cell line), bu he e was no ag eemen be ween
he wo app oaches on which GBM cell p esen s highe exp ession le els o ATXN3. These di e ences
ound be ween mRNA and p o ein le els migh be explained by he ac ha hey ha e di e en egula o y
mechanisms. Indeed, he cen al dogma ha "DNA gene a es RNA ha gene a es p o eins" is no as
simplis ic as ha and mRNA le els do no always co ela e wi h p o ein exp ession, due o pos -
ansc ip ional and pos - ansla ional egula ion (He, 2019; Hewi , 2020). Also, he deg ada ion ime o
p o eins and genes can be di e en (de Sousa Ab eu e al., 2009). Mo eo e , we a e using echniques
wi h comple ely di e en esolu ions, one is quan i a i e ( he qPCR), while he o he is semi-quan i a i e
(WB), which may no allow he de ec ion o smalle di e ences ha migh be iden i iable h ough qPCR.
ATXN3 is he causa i e p o ein o SCA3, a mo o neu odegene a i e disease ha esul s om he
uns able expansion o his p o ein's CAGs (Kawaguchi e al., 1994).
ATXN3
has a polymo phic CAG
inucleo ide epea ac o a iable leng h among indi iduals: he CAG epea leng h a ies om 11 o
37 in heal hy people, whe eas i a ies om 62 o 84 in SCA3 pa ien s (Lindblad e al., 1996; Maciel e
al., 1995; Ranum e al., 1995). Aue e al. demons a ed ha pa ien s wi h amilia and spo adic ch onic
lymphocy ic leukemia, despi e exp essing ATXN3 wi h a numbe o CAG epea s wi hin no mal ange
(non-expanded), had a s a is ically signi ican inc ease in he equency o high-leng h CAG epea s a he
ATXN3
locus compa ed wi h con ol-ma ched popula ion (Aue e al., 2007). Taking his in o accoun , we
decided o de e mine he numbe o CAG epea o he GBM cell line used (A172 cell line) and one o he
cell lines wi h he highes endogenous exp ession o ATXN3 (U87MG cell line). We obse ed ha in bo h
cell lines
ATXN3
has 11 CAG epea s, which is wi hin he no mal ange (Maciel e al., 1995), sugges ing
ha ATXN3 expansion may no be c ucial in GBM. Howe e , his da a should be in e p e ed wi h ca e as
only wo GBM cell lines we e es ed. In he u u e i will be necessa y o de e mine he size o he ATXN3
CAG ac in a la ge numbe o GBM cell lines, as well as in pa ien umo s.
To assess he impac o ATXN3 in GBM we gene ically manipula ed he exp ession o
ATXN3
in he
A172 cell line, o inc ease i s exp ession. Subsequen ly, se e al cance hallma ks we e e alua ed (
Figu e
9
and Figu e 10
). In gene al, ATXN3 had an impac on GBM, dec easing i s agg essi eness and ac ing
mos ly as a umo supp esso gene. We ound ha highe ATXN3 exp ession is associa ed wi h dec eased
GBM cell iabili y in he wo me hods used, in he T ypan Blue a e 6 days o incuba ion and in he MTS
assay (
Figu e 9B and C
). In e es ingly, in he T ypan Blue assay, a e 4 days o incuba ion, no s a is ically
signi ican di e ence was obse ed. In line wi h his, in lung cance , in which ATXN3 is conside ed an
oncogene, ATXN3 was shown o be associa ed wi h an inc ease in cell iabili y (Sacco e al., 2014). In
o al squamous cell ca cinoma, ATXN3 silencing educed he cell iabili y o cispla in- esis an cells (Song
5. | Discussion
43
e al., 2021). The same e ec was seen in es icula cance , whe e ATXN3 p omo ed cell iabili y,
unc ioning as an oncogene. In e es ingly, i was disco e ed ha his abili y o ATXN3 o p omo e es icula
cance cell iabili y was due o he supp ession o PTEN exp ession and o he indi ec ac i a ion o
AKT/mTOR signaling pa hway (Shi e al., 2018). This signaling pa hway cons i u ed an impo an pa hway
in cance , including in GBM, and PTEN is a umo supp esso ha inhibi s his pa hway, helping o p e en
cance . As a esul , he loss o PTEN unc ion allows un es ic ed AKT signaling, esul ing in cell su i al
and umo g ow h (Po a e al., 2014). In GBM, PTEN is equen ly dele ed due o mu a ions o loss o
he e ozygosi y. This loss o PTEN plays a key ole in he umo ’s de elopmen and agg essi e beha io ,
and, u he mo e, i is co ela ed wi h poo su i al in GBM pa ien s (Koul, 2008). Thus, i will be impo an
in he u u e o e alua e whe he and how ATXN3 a ec s he exp ession o his umo supp esso gene in
GBM cells and/o i impac s he ac i a ion o he impo an PI3K/AKT/mTOR pa hway; his can be
achie ed by assessing he exp ession o p o eins ela ed o he pa hway. In ou assays, ATXN3 did no
a ec cell p oli e a ion (
Figu e 9D
), no cell cycle p og ession in GBM (
Figu e 9E and F
), nei he did i
a ec GBM cells’ mig a ion (
Figu e 9G and H
). O no e, silencing ATXN3 in he neu oblas oma cell line
esul ed in a g ea e numbe o cells in S phase and signi ican ly inc eased mig a o y capaci y o he cells
(Ne es-Ca alho e al., 2015). On he o he hand, in b eas cance , whe e ATXN3 was shown o ha e an
oncogenic unc ion, ATXN3 silencing signi ican ly dec eased cance cell mig a ion (Zou e al., 2019).
Al hough GBMs a ely me as asize, hey ha e a highly in il a i e beha io ac oss he b ain issue. This
impo an ea u e o GBM makes comple e su gical esec ion impossible, esul ing in a less e ec i e
ea men (So e al., 2021). He e, we ound ha highe ATXN3 exp ession is associa ed wi h a dec eased
in asion capaci y o he GBM cell model (
Figu e 9I and J
). In he clinical con ex , his associa ion wi h a
dec eased in asion capaci y may pe haps con ibu e o a mo e comple e umo esec ion and,
consequen ly a be e ea men ou come o GBM pa ien s. Thus, his inding is in line wi h he ac ha
we show ha
ATXN3
is a new posi i e bioma ke o GBM, being associa ed wi h a be e p ognosis in
pa ien s wi h he disease (
Figu e 11
). In b eas cance , whe e ATXN3 was conside ed an oncogene, i s
exp ession was associa ed wi h an inc eased in asi e capaci y o he cells, as expec ed, he opposi e o
wha was obse ed by us in GBM (Zou e al., 2019). Al hough he p ecise biological unc ion o ATXN3
emains la gely unknown, i is known ha ATXN3 ac s as a deubiqui ina ing enzyme (DUB), egula ing
he deubiqui ina ion and s abili y o a ious p o eins (B. Bu ne e al., 2003; Winbo n e al., 2008). O
no e, ATXN3 has been iden i ied in b eas cance as he DUB o K üppel-like ac o 4 (KLF4), an impo an
ansc ip ion ac o . Indeed, he impac o ATXN3 on b eas cance cell mig a ion and in asion was shown
o be due o i s egula ion o KLF4 (Zou e al., 2019). In e es ingly, s udies ha e shown ha KLF4 le els
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
50
7.
REFERENCES
Ahlbom, A., Feych ing, M., G een, A., Khei e s, L., Sa i z, D. A., & Swe dlow, A. J. (2009). Epidemiologic
e idence on mobile phones and umo isk: A e iew.
Epidemiology
,
20
(5), 639–652.
h ps://doi.o g/10.1097/EDE.0b013e3181b0927d
Ahmadloo, N., Kani, A. A., Mohammadianpanah, M., Nas olahi, H., Omid a i, S., Mosalaei, A., & Ansa i,
M. (2013). T ea men ou come and p ognos ic ac o s o adul glioblas oma mul i o me.
Jou nal o
he Egyp ian Na ional Cance Ins i u e
,
25
(1), 21–30. h ps://doi.o g/10.1016/j.jnci.2012.11.001
Almeida, B., Ab eu, I. A., Ma os, C. A., F aga, J. S., Fe nandes, S., Macedo, M. G., Gu ié ez-Gallego, R.,
Pe ei a, P. J. B., Ca alho, A. L., & Macedo-Ribei o, S. (2015). SUMOyla ion o he b ain-
p edominan A axin-3 iso o m modula es i s in e ac ion wi h p97.
Biochimica e Biophysica Ac a -
Molecula Basis o Disease
,
1852
(9), 1950–1959.
h ps://doi.o g/10.1016/j.bbadis.2015.06.010
Al es, S., Nascimen o-Fe ei a, I., Au egan, G., Hassig, R., Du ou , N., B ouille , E., Ped oso de Lima, M.
C., Han aye, P., de Almeida, L. P., & Déglon, N. (2008). Allele-speci ic RNA silencing o mu an
a axin-3 media es neu op o ec ion in a a model o Machado-Joseph disease.
PLoS ONE
,
3
(10).
h ps://doi.o g/10.1371/jou nal.pone.0003341
Al es, S., Nascimen o-Fe ei a, I., Du ou , N., Hassig, R., Au egan, G., Nób ega, C., B ouille , E.,
Han aye, P., Ped oso de Lima, M. C., Déglon, N., & de Almeida, L. P. (2010). Silencing a axin-3
mi iga es degene a ion in a a model o Machado-Joseph disease: No ole o wild- ype a axin-3?
Human Molecula Gene ics
,
19
(12), 2380–2394. h ps://doi.o g/10.1093/hmg/ddq111
An ony, P. M. A., Män ele, S., Mollenkop , P., Boy, J., Kehlenbach, R. H., Riess, O., & Schmid , T. (2009).
Iden i ica ion and unc ional dissec ion o localiza ion signals wi hin a axin-3.
Neu obiology o
Disease
,
36
(2), 280–292. h ps://doi.o g/10.1016/j.nbd.2009.07.020
Aue , R. L., Dighie o, G., Goldin, L. R., Synde combe-Cou , D., Jones, C., McElwaine, S., Newland, A. C.,
Fegan, C. D., Capo aso, N., & Co e , F. E. (2007). T inucleo ide epea dynamic mu a ion iden i ying
suscep ibili y in amilial and spo adic ch onic lymphocy ic leukaemia.
B i ish Jou nal o
Haema ology
,
136
(1), 73–79. h ps://doi.o g/10.1111/j.1365-2141.2006.06388.x
Benson, V. S., Pi ie, K., Schüz, J., Ree es, G. K., Be al, V., & G een, J. (2013). Mobile phone use and
isk o b ain neoplasms and o he cance s: p ospec i e s udy.
In e na ional Jou nal o Epidemiology
,
42
(3), 792–802. h ps://doi.o g/10.1093/ije/dy 072
Be ke, S. J. S., Chai, Y., Ma s, G. L., Wen, H., & Paulson, H. L. (2005). De ining he ole o ubiqui in-
in e ac ing mo i s in he polyglu amine disease p o ein, a axin-3.
Jou nal o Biological Chemis y
,
280
(36), 32026–32034. h ps://doi.o g/10.1074/jbc.M506084200
Be encou , C., San os, C., Mon iel, R., Do Ca mo Cos a, M., C uz-Mo ales, P., San os, L. R., Simões,
N., Kay, T., Vasconcelos, J., Maciel, P., & Lima, M. (2010). Inc eased ansc ip di e si y: No el
splicing a ian s o Machado-Joseph Disease gene (ATXN3).
Neu ogene ics
,
11
(2), 193–202.
h ps://doi.o g/10.1007/s10048-009-0216-y
Bowman, R. L., Wang, Q., Ca o, A., Ve haak, R. G. W., & Squa i o, M. (2017). GlioVis da a po al o
isualiza ion and analysis o b ain umo exp ession da ase s.
Neu o-Oncology
,
19
(1), 139–141.
h ps://doi.o g/10.1093/neuonc/now247
B aganza, M. Z., Ki aha a, C. M., Be ing on De González, A., Inskip, P. D., Johnson, K. J., & Raja aman,

7. |
Re e ences
51
P. (2012). Ionizing adia ion and he isk o b ain and cen al ne ous sys em umo s: A sys ema ic
e iew.
Neu o-Oncology
,
14
(11), 1316–1324. h ps://doi.o g/10.1093/neuonc/nos208
B ai , M., & Sid ansky, D. (2011). Cance epigene ics: Abo e and beyond.
Toxicology Mechanisms and
Me hods
,
21
(4), 275–288. h ps://doi.o g/10.3109/15376516.2011.562671
B andes, A. A., Tosoni, A., F anceschi, E., Reni, M., Ga a, G., & Vech , C. (2008). Glioblas oma in adul s.
C i ical Re iews in Oncology/Hema ology
,
67
(2), 139–152.
h ps://doi.o g/10.1016/j.c i e onc.2008.02.005
B ay, F., La e sanne, M., Weide pass, E., & Soe joma a am, I. (2021). The e e -inc easing impo ance
o cance as a leading cause o p ema u e dea h wo ldwide.
Cance
,
127
(16), 3029–3030.
h ps://doi.o g/10.1002/cnc .33587
Buckne , J. C., B own, P. D., O’Neill, B. P., Meye , F. B., We mo e, C. J., & Uhm, J. H. (2007). Cen al
ne ous sys em umo s.
Mayo Clinic P oceedings
,
82
(10), 1271–1286.
h ps://doi.o g/10.4065/82.10.1271
Bu ne , N. G., Je e ies, S. J., Benson, R. J., Hun , D. P., & T easu e, F. P. (2005). Yea s o li e los (YLL)
om cance is an impo an measu e o popula ion bu den - And should be conside ed when
alloca ing esea ch unds.
B i ish Jou nal o Cance
,
92
(2), 241–245.
h ps://doi.o g/10.1038/sj.bjc.6602321
Bu ne , B. G., & Pi man, R. N. (2005). The polyglu amine neu odegene a i e p o ein a axin 3 egula es
agg esome o ma ion.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o
Ame ica
,
102
(12), 4330–4335. h ps://doi.o g/10.1073/pnas.0407252102
Bu ne , B., Li, F., & Pi man, R. N. (2003). The polyglu amine neu odegene a i e p o ein a axin-3 binds
polyubiqui yla ed p o eins and has ubiqui in p o ease ac i i y.
Human Molecula Gene ics
,
12
(23),
3195–3205. h ps://doi.o g/10.1093/hmg/ddg344
Cai nc oss, G., & Jenkins, R. (2008). Gliomas Wi h 1p/19q Codele ion:a.k.a. Oligodend oglioma.
The
Cance Jou nal
,
14
(6), 352–357. h ps://doi.o g/10.1097/PPO.0b013e31818d8178
Ca alho, A. L., Sil a, A., & Macedo-Ribei o, S. (2018).
Polyglu amine-Independen Fea u es in A axin-3
Agg ega ion and Pa hogenesis o Machado-Joseph Disease
(pp. 275–288).
h ps://doi.o g/10.1007/978-3-319-71779-1_14
Chai, Y., Be ke, S. S., Cohen, R. E., & Paulson, H. L. (2004). Poly-ubiqui in Binding by he Polyglu amine
Disease P o ein A axin-3 Links I s No mal Func ion o P o ein Su eillance Pa hways.
Jou nal o
Biological Chemis y
,
279
(5), 3605–3611. h ps://doi.o g/10.1074/jbc.M310939200
Chen, H., Wa d, M. H., Tucke , K. L., G auba d, B. I., McComb, R. D., Po ischman, N. A., Weisenbu ge ,
D. D., & Heineman, E. F. (2002). Die and isk o adul glioma in eas e n Neb aska, Uni ed S a es.
Cance Causes and Con ol
,
13
(7), 647–655. h ps://doi.o g/10.1023/A:1019527225197
Chow, M. K. M., MacKay, J. P., Whiss ock, J. C., Scanlon, M. J., & Bo omley, S. P. (2004). S uc u al
and unc ional analysis o he Josephin domain o he polyglu amine p o ein a axin-3.
Biochemical
and Biophysical Resea ch Communica ions
,
322
(2), 387–394.
h ps://doi.o g/10.1016/j.bb c.2004.07.131
Cla ke, J., Bu owski, N., & Chang, S. (2010). Recen Ad ances in The apy o Glioblas oma.
A chi es o
Neu ology
,
67
(3). h ps://doi.o g/10.1001/a chneu ol.2010.5
Coble, J. B., Dosemeci, M., S ewa , P. A., Blai , A., Bowman, J., Fine, H. A., Shapi o, W. R., Selke , R.
G., Loe le , J. S., Black, P. M., Line , M. S., & Inskip, P. D. (2009). Occupa ional exposu e o
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
52
magne ic ields and he isk o b ain umo s.
Neu o-Oncology
,
11
(3), 242–249.
h ps://doi.o g/10.1215/15228517-2009-002
Cohen, A. L., Holmen, S. L., & Colman, H. (2013). IDH1 and IDH2 Mu a ions in Gliomas.
Cu en
Neu ology and Neu oscience Repo s
,
13
(5), 345. h ps://doi.o g/10.1007/s11910-013-0345-4
Cos a, M. do C., Bajanca, F., Rod igues, A.-J., Tomé, R. J., Co hals, G., Macedo-Ribei o, S., Paulson, H.
L., Loga inho, E., & Maciel, P. (2010). A axin-3 Plays a Role in Mouse Myogenic Di e en ia ion
h ough Regula ion o In eg in Subuni Le els.
PLoS ONE
,
5
(7), e11728.
h ps://doi.o g/10.1371/jou nal.pone.0011728
Cunha, M. L. V. da, & Maldaun, M. V. C. (2019). Me as asis om glioblas oma mul i o me: a me a-
analysis.
Re is a Da Associação Médica B asilei a
,
65
(3), 424–433.
h ps://doi.o g/10.1590/1806-9282.65.3.424
Dang, L., Whi e, D. W., G oss, S., Benne , B. D., Bi inge , M. A., D igge s, E. M., Fan in, V. R., Jang, H.
G., Jin, S., Keenan, M. C., Ma ks, K. M., P ins, R. M., Wa d, P. S., Yen, K. E., Liau, L. M., Rabinowi z,
J. D., Can ley, L. C., Thompson, C. B., Vande Heiden, M. G., … Su, M. (2009). Cance -associa ed
IDH1 mu a ions p oduce 2-hyd oxyglu a a e.
Na u e
,
462
(7274), 739.
h ps://doi.o g/10.1038/na u e08617.Cance -associa ed
de Sousa Ab eu, R., Penal a, L. O., Ma co e, E. M., & Vogel, C. (2009). Global signa u es o p o ein and
mRNA exp ession le els.
Molecula BioSys ems
. h ps://doi.o g/10.1039/b908315d
Djalilian, H. R., Shah, M. V., & Hall, W. A. (1999). Radiog aphic incidence o mul icen ic malignan
gliomas.
Su gical Neu ology
,
51
(5), 554–558. h ps://doi.o g/10.1016/S0090-3019(98)00054-8
Do Ca mo Cos a, M., Gomes-Da-Sil a, J., Mi anda, C. J., Sequei os, J., San os, M. M., & Maciel, P.
(2004). Genomic s uc u e, p omo e ac i i y, and de elopmen al exp ession o he mouse
homologue o he Machado-Joseph disease (MJD) gene.
Genomics
,
84
(2), 361–373.
h ps://doi.o g/10.1016/j.ygeno.2004.02.012
Elsamadicy, A. A., Babu, R., Ki kpa ick, J. P., & Adamson, D. C. (2015). Radia ion-induced malignan
gliomas: A cu en e iew.
Wo ld Neu osu ge y
,
83
(4), 530–542.
h ps://doi.o g/10.1016/j.wneu.2014.12.009
Elsi , T., Edq is , P., Ca lson, J., Ribom, D., Be gq is , M., Ekman, S., Popo a, S. N., Ala uzo , I., Pon en,
F., Nis é , M., & Smi s, A. (2014). A s udy o emb yonic s em cell- ela ed p o eins in human
as ocy omas: Iden i ica ion o Nanog as a p edic o o su i al.
In e na ional Jou nal o Cance
,
134
(5), 1123–1131. h ps://doi.o g/10.1002/ijc.28441
E gun, S., Gunes, S., Buyukalpelli, R., & Aydin, O. (2020). Associa ion o Abl in e ac o 2, ABI2, wi h
pla ele /lymphocy e a io in pa ien s wi h enal cell ca cinoma: A pilo s udy.
In e na ional Jou nal
o Expe imen al Pa hology
,
101
(3–4), 87–95. h ps://doi.o g/10.1111/iep.12349
Es elle , M., Ga cia-Foncillas, J., Andion, E., Goodman, S. N., Hidalgo, O. F., Vanaclocha, V., Baylin, S.
B., & He man, J. G. (2000). Inac i a ion o he DNA-Repai Gene MGMT and he Clinical Response
o Gliomas o Alkyla ing Agen s.
New England Jou nal o Medicine
,
343
(19), 1350–1354.
h ps://doi.o g/10.1056/NEJM200011093431901
E e , B. O., A aujo, J., Viei a-Saecke , A. M., De Vos, R. A. I., Ha endza, S., Klockge he , T., & Wüllne ,
U. (2006). A axin-3 ep esses ansc ip ion ia ch oma in binding, in e ac ion wi h his one
deace ylase 3, and his one deace yla ion.
Jou nal o Neu oscience
,
26
(44), 11474–11486.
h ps://doi.o g/10.1523/JNEUROSCI.2053-06.2006
7. |
Re e ences
53
Fayed, L. (2020).
Di e en ia ing Be ween P ima y and Me as a ic Tumo s
.
h ps://www. e ywellheal h.com/p ima y-and-me as a ic-b ain- umo s-513566
Feldheim, J., Kessle , A. F., Mono anu, C. M., E nes us, R.-I., Löh , M., & Hagemann, C. (2019). Changes
o O6-Me hylguanine DNA Me hyl ans e ase (MGMT) P omo e Me hyla ion in Glioblas oma
Relapse—A Me a-Analysis Type Li e a u e Re iew.
Cance s
,
11
(12), 1837.
h ps://doi.o g/10.3390/cance s11121837
Fe lay, J., E ik, M., Lam, F., Colombe , M., Me y, L., Piñe os, M., Znao , A., Soe joma a am, I., & B ay,
F. (2020).
Global Cance Obse a o y: Cance Today
. Lyon, F ance: In e na ional Agency o
Resea ch on Cance . h ps://gco.ia c. / oday
Fe lay, J., La e sanne, M., E ik, M., Lam, F., Colombe , M., Me y, L., Piñe os, M., Znao , A.,
Soe joma a am, I., & B ay, F. (2020).
Global Cance Obse a o y: Cance Tomo ow
. Lyon, F ance:
In e na ional Agency o Resea ch on Cance .
Fidle , M. M., B ay, F., & Soe joma a am, I. (2018). The global cance bu den and human de elopmen :
A e iew.
Scandina ian Jou nal o Public Heal h
,
46
(1), 27–36.
h ps://doi.o g/10.1177/1403494817715400
F aile, J. M., Quesada, V., Rod íguez, D., F eije, J. M. P., & López-O ín, C. (2012). Deubiqui inases in
cance : New unc ions and he apeu ic op ions.
Oncogene
,
31
(19), 2373–2388.
h ps://doi.o g/10.1038/onc.2011.443
F eije, W. A., Cas o-Va gas, F. E., Fang, Z., Ho a h, S., Cloughesy, T., Liau, L. M., Mischel, P. S., &
Nelson, S. F. (2004). Gene exp ession p o iling o gliomas s ongly p edic s su i al.
Cance
Resea ch
,
64
(18), 6503–6510. h ps://doi.o g/10.1158/0008-5472.CAN-04-0452
Ge, F., Chen, W., Qin, J., Zhou, Z., Liu, R., Liu, L., Tan, J., Zou, T., Li, H., Ren, G., & Chen, C. (2015).
A axin-3 like (ATXN3L), a membe o he Josephin amily o deubiqui ina ing enzymes, p omo es
b eas cance p oli e a ion by deubiqui ina ing K üppel-like ac o 5 (KLF5).
Onco a ge
,
6
(25),
21369–21378. h ps://doi.o g/10.18632/onco a ge .4128
Gonçal es, C. S., Xa ie -Magalhães, A., Ma ins, E. P., Pin o, A. A., Pi es, M. M., Pinhei o, C., Reis, R. M.,
Sousa, N., & Cos a, B. M. (2020). A no el molecula link be ween HOXA9 and WNT6 in glioblas oma
iden i ies a subg oup o pa ien s wi h pa icula poo p ognosis.
Molecula Oncology
,
14
(6), 1224–
1241. h ps://doi.o g/10.1002/1878-0261.12633
Go o, J., Wa anabe, M., Ichikawa, Y., Yee, S. B., Iha a, N., Endo, K., Iga ashi, S., Takiyama, Y., Gaspa ,
C., Maciel, P., Tsuji, S., Rouleau, G. A., & Kanazawa, I. (1997). Machado-Joseph disease gene
p oduc s ca ying di e en ca boxyl e mini.
Neu oscience Resea ch
,
28
(4), 373–377.
h ps://doi.o g/10.1016/S0168-0102(97)00056-4
G a endeel, L. A. M., Kouwenho en, M. C. M., Ge ae , O., De Rooi, J. J., S ubbs, A. P., Duijm, J. E.,
Daemen, A., Bleeke , F. E., B al en, L. B. C., Kloos e ho , N. K., De Moo , B., Eile s, P. H. C., Van
De Spek, P. J., K os, J. M., Sille is Smi , P. A. E., Van Den Ben , M. J., & F ench, P. J. (2009).
In insic gene exp ession p o iles o gliomas a e a be e p edic o o su i al han his ology.
Cance
Resea ch
,
69
(23), 9065–9072. h ps://doi.o g/10.1158/0008-5472.CAN-09-2307
G izzi, F., & Chi i a-In e na i, M. (2006). Cance : Looking o simplici y and inding complexi y.
Cance
Cell In e na ional
,
6
, 1–7. h ps://doi.o g/10.1186/1475-2867-6-4
Hanahan, D., & Weinbe g, R. A. (2011). Hallma ks o cance : The nex gene a ion.
Cell
,
144
(5), 646–
674. h ps://doi.o g/10.1016/j.cell.2011.02.013
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
54
Hani , F., Muza a , K., Pe een, K., Malhi, S. M., & Simjee, S. U. (2017). Glioblas oma mul i o me: A
e iew o i s epidemiology and pa hogenesis h ough clinical p esen a ion and ea men .
Asian
Paci ic Jou nal o Cance P e en ion
,
18
(1), 3–9. h ps://doi.o g/10.22034/APJCP.2017.18.1.3
Ha is, G. M., Dodelzon, K., Gong, L., Gonzalez-Aleg e, P., & Paulson, H. L. (2010). Splice iso o ms o
he polyglu amine disease p o ein a axin-3 exhibi simila enzyma ic ye di e en agg ega ion
p ope ies.
PLoS ONE
,
5
(10). h ps://doi.o g/10.1371/jou nal.pone.0013695
He, C. (2019). Special Issue on Regula ing he Cen al Dogma.
Biochemis y
,
58
(5), 295–296.
h ps://doi.o g/10.1021/acs.biochem.9b00059
Hegde, A. N., & Upadhya, S. C. (2011). Role o ubiqui in–p o easome-media ed p o eolysis in ne ous
sys em disease.
Biochimica e Biophysica Ac a (BBA) - Gene Regula o y Mechanisms
,
1809
(2),
128–140. h ps://doi.o g/10.1016/j.bbag m.2010.07.006
Hegi, M. E., Dise ens, A.-C., Goda d, S., Die ich, P.-Y., Regli, L., Os e mann, S., O en, P., Van Melle, G.,
de T ibole , N., & S upp, R. (2004). Clinical T ial Subs an ia es he P edic i e Value o O-6-
Me hylguanine-DNA Me hyl ans e ase P omo e Me hyla ion in Glioblas oma Pa ien s T ea ed wi h
Temozolomide.
Clinical Cance Resea ch
,
10
(6), 1871–1874. h ps://doi.o g/10.1158/1078-
0432.CCR-03-0384
Hegi, M. E., Dise ens, A.-C., Go lia, T., Hamou, M.-F., de T ibole , N., Welle , M., K os, J. M., Hain ellne ,
J. A., Mason, W., Ma iani, L., B ombe g, J. E. C., Hau, P., Mi imano , R. O., Cai nc oss, J. G.,
Janze , R. C., & S upp, R. (2005). MGMT Gene Silencing and Bene i om Temozolomide in
Glioblas oma .
New England Jou nal o Medicine
,
352
(10), 997–1003.
h ps://doi.o g/10.1056/nejmoa043331
Hei , R., Ablasou, C., Dumon ie , E., Ellio , M., Fago o-Kau mann, C., & Bed o d, F. K. (2006). The UBL
domain o PLIC-1 egula es agg esome o ma ion.
EMBO Repo s
,
7
(12), 1252–1258.
h ps://doi.o g/10.1038/sj.embo .7400823
Hewi , S. M. (2020). Nega i e Consequences o he Cen al Dogma.
Jou nal o His ochemis y and
Cy ochemis y
,
68
(11), 731. h ps://doi.o g/10.1369/0022155420970927
Holmbe g, J., He, X., Pe edo, I., O ego, A., Hesselage , G., E icsson, C., Ho a a, O., Oba-Shinjo, S. M.,
Ma ie, S. K. N., Nis é , M., & Muh , J. (2011). Ac i a ion o Neu al and Plu ipo en S em Cell
Signa u es Co ela es wi h Inc eased Malignancy in Human Glioma.
PLoS ONE
,
6
(3), e18454.
h ps://doi.o g/10.1371/jou nal.pone.0018454
Iacob, G., & Dinca, E. B. (2009). Cu en da a and s a egy in glioblas oma mul i o me.
Jou nal o
Medicine and Li e
,
2
(4), 386–393.
Ichikawa, Y., Go o, J., Ha o i, M., Toyoda, A., Ishii, K., Jeong, S. Y., Hashida, H., Masuda, N., Oga a, K.,
Kasai, F., Hi ai, M., Maciel, P., Rouleau, G. A., Sakaki, Y., & Kanazawa, I. (2001). The genomic
s uc u e and exp ession o MJD, he Machado-Joseph disease gene.
Jou nal o Human Gene ics
,
46
(7), 413–422. h ps://doi.o g/10.1007/s100380170060
Jansen, M., Yip, S., & Louis, D. N. (2010). Molecula pa hology in adul gliomas: diagnos ic, p ognos ic,
and p edic i e ma ke s.
The Lance Neu ology
,
9
(7), 717–726. h ps://doi.o g/10.1016/S1474-
4422(10)70105-8
Jenkins, R. B., Blai , H., Ballman, K. V., Giannini, C., A usell, R. M., Law, M., Flynn, H., Passe, S., Fel en,
S., B own, P. D., Shaw, E. G., & Buckne , J. C. (2006). A (1;19)(q10;p10) Media es he Combined
Dele ions o 1p and 19q and P edic s a Be e P ognosis o Pa ien s wi h Oligodend oglioma.
Cance
7. |
Re e ences
55
Resea ch
,
66
(20), 9852–9861. h ps://doi.o g/10.1158/0008-5472.CAN-06-1796
Joo, K. M., Kim, J., Jin, J., Kim, M., Seol, H. J., Mu ado , J., Yang, H., Choi, Y. La, Pa k, W. Y., Kong, D.
S., Lee, J. Il, Ko, Y. H., Woo, H. G., Lee, J., Kim, S., & Nam, D. H. (2013). Pa ien -Speci ic O ho opic
Glioblas oma Xenog a Models Recapi ula e he His opa hology and Biology o Human
Glioblas omas In Si u.
Cell Repo s
,
3
(1), 260–273.
h ps://doi.o g/10.1016/j.cel ep.2012.12.013
Kamoun, A., Idbaih, A., Dehais, C., Ela ouci, N., Ca pen ie , C., Le ouzé, E., Colin, C., Mokh a i, K.,
Jou e , A., U o-Cos e, E., Ma in-Du e neuil, N., Sanson, M., Dela e, J.-Y., Figa ella-B ange , D.,
de Reyniès, A., & Duc ay, F. (2016). In eg a ed mul i-omics analysis o oligodend oglial umou s
iden i ies h ee subg oups o 1p/19q co-dele ed gliomas.
Na u e Communica ions
,
7
(1), 11263.
h ps://doi.o g/10.1038/ncomms11263
Kawaguchi, Y., Okamo o, T., Taniwaki, M., Aizawa, M., Inoue, M., Ka ayama, S., Kawakami, H.,
Nakamu a, S., Nishimu a, M., Akiguchi, I., Kimu a, J., Na umiya, S., & Kakizuka, A. (1994). CAG
expansions in a no el gene o Machado-Joseph disease a ch omosome 14q32.1.
Na u e Gene ics
,
8
(3), 221–228. h ps://doi.o g/10.1038/ng1194-221
Koul, D. (2008). PTEN Signaling pa hways in glioblas oma.
Cance Biology & The apy
,
7
(9), 1321–1325.
h ps://doi.o g/10.4161/cb .7.9.6954
Lee, S. Y. (2016). Temozolomide esis ance in glioblas oma mul i o me.
Genes & Diseases
,
3
(3), 198–
210. h ps://doi.o g/10.1016/j.gendis.2016.04.007
Lee, Y., Scheck, A. C., Cloughesy, T. F., Lai, A., Dong, J., Fa ooqi, H. K., Liau, L. M., Ho a h, S., Mischel,
P. S., & Nelson, S. F. (2008). Gene exp ession analysis o glioblas omas iden i ies he majo
molecula basis o he p ognos ic bene i o younge age.
BMC Medical Genomics
,
1
(1), 1–12.
h ps://doi.o g/10.1186/1755-8794-1-52
Li, F., Mac a lan, T., Pi man, R. N., & Chak a a i, D. (2002). A axin-3 is a his one-binding p o ein wi h
wo independen ansc ip ional co ep esso ac i i ies.
Jou nal o Biological Chemis y
,
277
(47),
45004–45012. h ps://doi.o g/10.1074/jbc.M205259200
Lindblad, K., Lunkes, A., Maciel, P., S e anin, G., Zande , C., Klockge he , T., Ra zla , T., B ice, A.,
Rouleau, G. A., Hudson, T., Aubu ge , G., & Schalling, M. (1996). Mu a ion de ec ion in Machado-
Joseph disease using epea expansion de ec ion.
Molecula Medicine
,
2
(1), 77–85.
h ps://doi.o g/10.1007/b 03402204
Linha o á, I., Repi z, M., D ábe , P., Nemec, M., Wiche, G., & P ops , F. (1999). Conse ed domains
and lack o e idence o polyglu amine leng h polymo phism in he chicken homolog o he Machado-
Joseph disease gene p oduc a axin-3.
Biochimica e Biophysica Ac a - Gene S uc u e and
Exp ession
,
1444
(2), 299–305. h ps://doi.o g/10.1016/S0167-4781(99)00004-4
Li ak, K. J., & Schmi gen, T. D. (2001). Analysis o Rela i e Gene Exp ession Da a Using Real-Time
Quan i a i e PCR and he 2−ΔΔCT Me hod.
Me hods
,
25
(4), 402–408.
h ps://doi.o g/10.1006/me h.2001.1262
Louis, D. N., Ohgaki, H., Wies le , O. D., Ca enee, W. K., Bu ge , P. C., Jou e , A., Schei haue , B. W., &
Kleihues, P. (2007). The 2007 WHO classi ica ion o umou s o he cen al ne ous sys em.
Ac a
Neu opa hologica
,
114
(2), 97–109. h ps://doi.o g/10.1007/s00401-007-0243-4
Louis, D. N., Pe y, A., Rei enbe ge , G., on Deimling, A., Figa ella-B ange , D., Ca enee, W. K., Ohgaki,
H., Wies le , O. D., Kleihues, P., & Ellison, D. W. (2016). The 2016 Wo ld Heal h O ganiza ion

| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
56
Classi ica ion o Tumo s o he Cen al Ne ous Sys em: a summa y.
Ac a Neu opa hologica
,
131
(6), 803–820. h ps://doi.o g/10.1007/s00401-016-1545-1
Louis, D. N., Pe y, A., Wesseling, P., B a , D. J., C ee, I. A., Figa ella-B ange , D., Hawkins, C., Ng, H.
K., P is e , S. M., Rei enbe ge , G., So ie i, R., Von Deimling, A., & Ellison, D. W. (2021). The 2021
WHO classi ica ion o umo s o he cen al ne ous sys em: A summa y.
Neu o-Oncology
,
23
(8),
1231–1251. h ps://doi.o g/10.1093/neuonc/noab106
Lukas, R. V, Wainw igh , D. A., Ladome sky, E., Sachde , S., Sonabend, A. M., & S upp, R. (2019). Newly
Diagnosed Glioblas oma: A Re iew on Clinical Managemen .
Oncology (Willis on Pa k, N.Y.)
,
33
(3),
91–100. h p://www.ncbi.nlm.nih.go /pubmed/30866031
Macedo-Ribei o, S., Co es, L., Maciel, P., & Ca alho, A. L. (2009). Nucleocy oplasmic Shu ling Ac i i y
o A axin-3.
PLoS ONE
,
4
(6), e5834. h ps://doi.o g/10.1371/jou nal.pone.0005834
Maciel, P., Gaspa , C., DeS e ano, A. L., Sil ei a, I., Cou inho, P., Rad any, J., Dawson, D. M., Suda sky,
L., Guima aes, J., Lou ei o, J. E. L., Neza a i, M. M., Co win, L. I., Lopes-Cendes, I., Rooke, K.,
Rosenbe g, R., MacLeod, P., Fa e , L. A., Sequei os, J., & Rouleau, G. A. (1995). Co ela ion
be ween CAG epea leng h and clinical ea u es in Machado- Joseph disease.
Ame ican Jou nal o
Human Gene ics
,
57
(1), 54–61.
Madha an, S., Zenklusen, J. C., Ko lia o , Y., Sahni, H., Fine, H. A., & Bue ow, K. (2009). Remb and :
Helping pe sonalized medicine become a eali y h ough in eg a i e ansla ional esea ch.
Molecula Cance Resea ch
,
7
(2), 157–167. h ps://doi.o g/10.1158/1541-7786.MCR-08-0435
Mao, Y., Senic-Ma uglia, F., Di Fio e, P. P., Polo, S., Hodsdon, M. E., & De Camilli, M. (2005).
Deubiqui ina ing unc ion o a axin-3: Insigh s om he solu ion s uc u e o he Josephin domain.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica
,
102
(36), 12700–
12705. h ps://doi.o g/10.1073/pnas.0506344102
Masino, L., Musi, V., Menon, R. P., Fusi, P., Kelly, G., F enkiel, T. A., T o ie , Y., & Pas o e, A. (2003).
Domain a chi ec u e o he polyglu amine p o ein a axin-3: A globula domain ollowed by a lexible
ail.
FEBS Le e s
,
549
(1–3), 21–25. h ps://doi.o g/10.1016/S0014-5793(03)00748-8
Masui, K., Mischel, P. S., & Rei enbe ge , G. (2016). Molecula classi ica ion o gliomas. In
Handbook o
Clinical Neu ology
(1s ed., Vol. 134). Else ie B.V. h ps://doi.o g/10.1016/B978-0-12-802997-
8.00006-2
Ma os, C. A., de Almeida, L. P., & Nób ega, C. (2019). Machado–Joseph disease/spinoce ebella a axia
ype 3: lessons om disease pa hogenesis and clues in o he apy.
Jou nal o Neu ochemis y
,
148
(1), 8–28. h ps://doi.o g/10.1111/jnc.14541
Ma os, C. A., de Macedo-Ribei o, S., & Ca alho, A. L. (2011). Polyglu amine diseases: The special case
o a axin-3 and Machado-Joseph disease.
P og ess in Neu obiology
,
95
(1), 26–48.
h ps://doi.o g/10.1016/j.pneu obio.2011.06.007
Ma os, C. A., Nób ega, C., Lou os, S. R., Almeida, B., Fe ei o, E., Vale o, J., De Almeida, L. P., Macedo-
Ribei o, S., & Ca alho, A. L. (2016). A axin-3 phospho yla ion dec eases neu onal de ec s in
spinoce ebella a axia ype 3 models.
Jou nal o Cell Biology
,
212
(4), 465–480.
h ps://doi.o g/10.1083/jcb.201506025
Muelle , T., B eue , P., Schmi , I., Wal e , J., E e , B. O., & Wüllne , U. (2009). CK2-dependen
phospho yla ion de e mines cellula localiza ion and s abili y o a axin-3.
Human Molecula
Gene ics
,
18
(17), 3334–3343. h ps://doi.o g/10.1093/hmg/ddp274
7. |
Re e ences
57
Ne es-Ca alho, A., Dua e-Sil a, S., Sil a, J., Almeida, B., Hee eld, S., So i opoulos, I., Heu ink, P., Li,
K. W., & Maciel, P. (2019). Regula ion o neu onal mRNA splicing and Tau iso o m a io by ATXN3
h ough deubiqui yla ion o splicing ac o s.
BioRxi
, 711424. h ps://doi.o g/10.1101/711424
Ne es-Ca alho, A., Dua e-Sil a, S., Teixei a-Cas o, A., & Maciel, P. (2020). Polyglu amine
spinoce ebella a axias: eme ging he apeu ic a ge s.
Expe Opinion on The apeu ic Ta ge s
,
0
(0).
h ps://doi.o g/10.1080/14728222.2020.1827394
Ne es-Ca alho, A., Loga inho, E., F ei as, A., Dua e-Sil a, S., do Ca mo Cos a, M., Sil a-Fe nandes, A.,
Ma ins, M., Se a, S. C., Lopes, A. T., Paulson, H. L., Heu ink, P., Rel as, J. B., & Maciel, P. (2015).
Dominan nega i e e ec o polyglu amine expansion pe u bs no mal unc ion o a axin-3 in
neu onal cells.
Human Molecula Gene ics
,
24
(1), 100–117.
h ps://doi.o g/10.1093/hmg/ddu422
Nicas o, G., Menon, R. P., Masino, L., Knowles, P. P., McDonald, N. Q., & Pas o e, A. (2005). The
solu ion s uc u e o he Josephin domain o a axin-3: S uc u al de e minan s o molecula
ecogni ion.
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica
,
102
(30), 10493–10498. h ps://doi.o g/10.1073/pnas.0501732102
Nób ega, C., Nascimen o-Fe ei a, I., Ono e, I., Albuque que, D., Hi ai, H., Déglon, N., & de Almeida, L.
P. (2013). Silencing Mu an A axin-3 Rescues Mo o De ici s and Neu opa hology in Machado-
Joseph Disease T ansgenic Mice.
PLoS ONE
,
8
(1).
h ps://doi.o g/10.1371/jou nal.pone.0052396
Nu , C. L., Mani, D. R., Be ensky, R. A., Tamayo, P., Cai nc oss, J. G., Ladd, C., Pohl, U., Ha mann, C.,
McLaughlin, M. E., Ba chelo , T. T., Black, P. M., Von Deimling, A., Pome oy, S. L., Golub, T. R., &
Louis, D. N. (2003). Gene exp ession-based classi ica ion o malignan gliomas co ela es be e
wi h su i al han his ological classi ica ion.
Cance Resea ch
,
63
(7), 1602–1607.
Ohgaki, H., & Kleihues, P. (2013). The de ini ion o p ima y and seconda y glioblas oma.
Clinical Cance
Resea ch
,
19
(4), 764–772. h ps://doi.o g/10.1158/1078-0432.CCR-12-3002
Omu o, A., & DeAngelis, L. M. (2013). Glioblas oma and o he malignan gliomas: A clinical e iew.
JAMA
- Jou nal o he Ame ican Medical Associa ion
,
310
(17), 1842–1850.
h ps://doi.o g/10.1001/jama.2013.280319
Os om, Q. T., Gi leman, H., S e son, L., Vi k, S., & Ba nhol z-Sloan, J. S. (2017). Epidemiology o
In ac anial Gliomas.
P og ess in Neu ological Su ge y
,
30
, 1–11.
h ps://doi.o g/10.1159/000464374
Os om, Q. T., Pa il, N., Cio i, G., Wai e, K., K uchko, C., & Ba nhol z-Sloan, J. S. (2020). CBTRUS
s a is ical epo : P ima y b ain and o he cen al ne ous sys em umo s diagnosed in he Uni ed
S a es in 2013-2017.
Neu o-Oncology
,
22
(Supplemen _1), IV1–IV96.
h ps://doi.o g/10.1093/neuonc/noaa200
Pa sons, D. W., Jones, S., Zhang, X., Lin, J. C.-H., Lea y, R. J., Angenend , P., Mankoo, P., Ca e , H.,
Siu, I.-M., Gallia, G. L., Oli i, A., McLendon, R., Rasheed, B. A., Kei , S., Nikolskaya, T., Nikolsky, Y.,
Busam, D. A., Tekleab, H., Diaz, L. A., … Kinzle , K. W. (2008). An In eg a ed Genomic Analysis o
Human Glioblas oma Mul i o me.
Science
,
321
(5897), 1807–1812.
h ps://doi.o g/10.1126/science.1164382
Pasquie , B., Pasquie , D., N’gole , A., Panh, M. H., & Coude c, P. (1980). Ex aneu al me as ases o
as ocy omas and glioblas omas clinicopa hological s udy o wo cases and e iew o li e a u e.
| Un a elling he ole o he spinoce ebella a axia ype 3-associa ed p o ein ATXN3 in glioblas oma
58
Cance
,
45
(1), 112–125. h ps://doi.o g/10.1002/1097-0142(19800101)45:1<112::AID-
CNCR2820450121>3.0.CO;2-9
Paulson, H. L., Das, S. S., C ino, P. B., Pe ez, M. K., Pa el, S. C., Go sdine , D., Fischbeck, K. H., &
Pi man, R. N. (1997). Machado-Joseph disease gene p oduc is a cy oplasmic p o ein widely
exp essed in b ain.
Annals o Neu ology
,
41
(4), 453–462.
h ps://doi.o g/10.1002/ana.410410408
P oh, R., Lacdao, I. K., & Sa idakis, V. (2015). Deubiqui inases and he new he apeu ic oppo uni ies
o e ed o cance .
Endoc ine-Rela ed Cance
,
22
(1), T35–T54. h ps://doi.o g/10.1530/ERC-14-
0516
Phillips, H. S., Kha banda, S., Chen, R., Fo es , W. F., So iano, R. H., Wu, T. D., Mis a, A., Nig o, J. M.,
Colman, H., So oceanu, L., Williams, P. M., Mod usan, Z., Feue s ein, B. G., & Aldape, K. (2006).
Molecula subclasses o high-g ade glioma p edic p ognosis, delinea e a pa e n o disease
p og ession, and esemble s ages in neu ogenesis.
Cance Cell
,
9
(3), 157–173.
h ps://doi.o g/10.1016/j.cc .2006.02.019
Pojo, M., Gonçal es, C. S., Xa ie -Magalhães, A., Oli ei a, A. I., Gonçal es, T., Co eia, S., Rod igues, A.
J., Cos a, S., Pin o, L., Pin o, A. A., Lopes, J. M., Reis, R. M., Rocha, M., Sousa, N., & Cos a, B. M.
(2015). A ansc ip omic signa u e media ed by HOXA9 p omo es human glioblas oma ini ia ion,
agg essi eness and esis ance o emozolomide.
Onco a ge
,
6
(10), 7657–7674.
h ps://doi.o g/10.18632/onco a ge .3150
Po a, C., Paglino, C., & Mosca, A. (2014). Ta ge ing PI3K/Ak /mTOR Signaling in Cance .
F on ie s in
Oncology
,
4
. h ps://doi.o g/10.3389/ onc.2014.00064
P es on, D. L., Ron, E., Tokuoka, S., Funamo o, S., Nishi, N., Soda, M., Mabuchi, K., & Kodama, K.
(2007). Solid cance incidence in a omic bomb su i o s: 1958-1998.
Radia ion Resea ch
,
168
(1),
1–64. h ps://doi.o g/10.1667/RR0763.1
Qin, S., Wang, M., Zhang, T., & Zhang, S. (2014). Vi amin e in ake is no associa ed wi h glioma isk:
E idence om a me a-analysis.
Neu oepidemiology
,
43
(3–4), 253–258.
h ps://doi.o g/10.1159/000369345
Ranum, L. P. W., Lundg en, J. K., Schu , L. J., Ah ens, M. J., Pe lman, S., Ai a, J., Bi d, T. D., Gomez,
C., & O , H. T. (1995). Spinoce ebella a axia ype 1 and Machado-Joseph disease: Incidence o
CAG expansions among adul -onse a axia pa ien s om 311 amilies wi h dominan , ecessi e, o
spo adic a axia.
Ame ican Jou nal o Human Gene ics
,
57
(3), 603–608.
Ray, S. K. (2016). The T ansc ip ion Regula o K uppel-Like Fac o 4 and I s Dual Roles o Oncogene in
Glioblas oma and Tumo Supp esso in Neu oblas oma.
Fo um on Immunopa hological Diseases
and The apeu ics
,
7
(1–2), 127–139.
h ps://doi.o g/10.1615/Fo umImmunDisThe .2016017227
Rei man, Z. J., & Yan, H. (2010). Isoci a e dehyd ogenase 1 and 2 mu a ions in cance : Al e a ions a a
c oss oads o cellula me abolism.
Jou nal o he Na ional Cance Ins i u e
,
102
(13), 932–941.
h ps://doi.o g/10.1093/jnci/djq187
Riemenschneide , M. J., & Rei enbe ge , G. (2009). Molecula neu opa hology o gliomas.
In e na ional
Jou nal o Molecula Sciences
,
10
(1), 184–212. h ps://doi.o g/10.3390/ijms10010184
Rod igues, A.-J., Coppola, G., San os, C., Cos a, M. do C., Ailion, M., Sequei os, J., Geschwind, D. H., &
Ma iel, P. (2007). Func ional genomics and biochemical cha ac e iza ion o he C. elegans
7. |
Re e ences
59
o hologue o he Machado-Joseph disease p o ein a axin-3 .
The FASEB Jou nal
,
21
(4), 1126–
1136. h ps://doi.o g/10.1096/ j.06-7002com
Rod igues, A. J., do Ca mo Cos a, M., Sil a, T. L., Fe ei a, D., Bajanca, F., Loga inho, E., & Maciel, P.
(2010). Absence o a axin-3 leads o cy oskele al diso ganiza ion and inc eased cell dea h.
Biochimica e Biophysica Ac a - Molecula Cell Resea ch
,
1803
(10), 1154–1163.
h ps://doi.o g/10.1016/j.bbamc .2010.07.004
Rod íguez-Leb ón, E., Cos a, M. D., Luna-Cancalon, K., Pe on, T. M., Fische , S., Boud eau, R. L.,
Da idson, B. L., & Paulson, H. L. (2013). Silencing mu an ATXN3 exp ession esol es molecula
pheno ypes in SCA3 ansgenic mice.
Molecula The apy
,
21
(10), 1909–1918.
h ps://doi.o g/10.1038/m .2013.152
Rosen, J., Blau, T., G au, S. J., Ba be, M. T., Fink, G. R., & Galldiks, N. (2018). Ex ac anial Me as ases
o a Ce eb al Glioblas oma: A Case Repo and Re iew o he Li e a u e.
Case Repo s in Oncology
,
11
(2), 591–600. h ps://doi.o g/10.1159/000492111
Sacco, J. J., Yau, T. Y., Da ling, S., Pa el, V., Liu, H., U bé, S., Clague, M. J., & Coulson, J. M. (2014).
The deubiqui ylase A axin-3 es ic s PTEN ansc ip ion in lung cance cells.
Oncogene
,
33
(33),
4265–4272. h ps://doi.o g/10.1038/onc.2013.512
Sade zki, S., Che i , A., F eedman, L., S o all, M., Modan, B., & No iko , I. (2005). Long- e m ollow-up
o b ain umo de elopmen a e childhood exposu e o ionizing adia ion o inea capi is.
Radia ion Resea ch
,
164
(2), 234. h ps://doi.o g/10.1667/0033-
7587(2005)164[0234:E]2.0.CO;2
Scheel, H., Tomiuk, S., & Ho mann, K. (2003). Elucida ion o a axin-3 and a axin-7 unc ion by in eg a i e
bioin o ma ics.
Human Molecula Gene ics
,
12
(21), 2845–2852.
h ps://doi.o g/10.1093/hmg/ddg297
Schmi , I., B a ig, T., Gossen, M., & Riess, O. (1997). Cha ac e iza ion o he a spinoce ebella a axia
ype 3 gene.
Neu ogene ics
,
1
(2), 103–112. h ps://doi.o g/10.1007/s100480050015
Schoemake , M. J., Swe dlow, A. J., Hepwo h, S. J., McKinney, P. A., Van Tonge en, M., & Mui , K. R.
(2006). His o y o alle gies and isk o glioma in adul s.
In e na ional Jou nal o Cance
,
119
(9),
2165–2172. h ps://doi.o g/10.1002/ijc.22091
Schwa zbaum, J., Ding, B., Johannesen, T. B., Osnes, L. T. N., Ka a odin, L., Ahlbom, A., Feych ing,
M., & G ims ud, T. K. (2012). Associa ion be ween p ediagnos ic IgE le els and isk o glioma.
Jou nal o he Na ional Cance Ins i u e
,
104
(16), 1251–1259.
h ps://doi.o g/10.1093/jnci/djs315
Schwei ze , T., Vince, G., He bold, C., Roosen, K., & Tonn, C. (2001). Ex aneu al me as ases o p ima y
b ain umo s.
J Neu ooncol
,
53
(2), 107–114. h ps://doi.o g/10.1023/a:1012245115209.
Seo, Y. J., Cho, W. H., Kang, D. W., & Cha, S. H. (2012). Ex aneu al me as asis o glioblas oma
mul i o me p esen ing as an unusual neck mass.
Jou nal o Ko ean Neu osu gical Socie y
,
51
(3),
147–150. h ps://doi.o g/10.3340/jkns.2012.51.3.147
Shao, C., Zhao, W., Qi, Z., & He, J. (2016). Smoking and glioma isk: E idence om a me a-analysis o
25 obse a ional s udies.
Medicine (Uni ed S a es)
,
95
(2), 1–9.
h ps://doi.o g/10.1097/MD.0000000000002447
Shi, Z., Chen, J., Zhang, X., Chu, J., Han, Z., Xu, D., Gan, S., Pan, X., Ye, J., & Cui, X. (2018). A axin-3
p omo es es icula cance cell p oli e a ion by inhibi ing an i-oncogene PTEN.
Biochemical and