SUMOyla ion egula es LKB1 localiza ion and i s oncogenic ac i i y
in li e cance
Imanol Zubie e-F anco
a,1
,Juan L. Ga cía-Rod íguez
a,1
,Fe nando Lopi z-O soa
a,1
, Ma ina Se ano-Macia
a
,
Jo ge Simon
a
, Pablo Fe nández-Tussy
a
, Lucía Ba bie -To es
a
, Da id Fe nández-Ramos
a
,
Vi ginia Gu ié ez-de-Juan
a
,Se gio López de Da alillo
a
, Onin za Ca le a is
b
, Adol o Begui is ain Gómez
c
,
E ica Villa
d
, Diego Cal isi
e
,Césa Ma ín
, Edu ne Be a
b
, Pa icia Aspichue a
g,k
, Naia a Be aza
a
,
Ma a Va ela-Rey
a
, Ma ias Á ila
h
, Manuel S. Rod íguez
i
,José M. Ma o
a
,I ene Díaz-Mo eno
j
,
An onio Díaz-Quin ana
j
, Te esa C. Delgado
a,
⁎,Ma ía L. Ma ínez-Chan a
a,
⁎
a
Li e Disease and Li e Me abolism Lab, CIC bioGUNE, Cen o de In es igación Biomédica en Red de En e medades Hepá icas y Diges i as (CIBERehd), 48160 De io, Bizkaia, Spain
b
Physiopa hology o he Hypoxia-Signalling Pa hway Lab, CIC bioGUNE, 48160 De io, Bizkaia, Spain
c
Uni e si y Hospi al o Donos ia, 20014 Donos ia, Gipuzkoa, Spain
d
Depa men o Gas oen e ology, Azienda Ospedalie o-Uni e si a ia & Uni e si y o Modena and Reggio Emilia, 41124 Modena, I aly
e
Ins i u e o Pa hology, Uni e si y Klinic o Regensbu g, 93053 Regensbu g, Ge many
Ins i u o Biofisika (CSIC, UPV/EHU) and Depa amen o de Bioquímica y Biología Molecula , UPV/EHU, 48940 Leioa, Spain
g
Depa men o Physiology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y, 48940 Leioa, Bizkaia, Spain
h
Hepa ology Depa men , Cen o de In es igación Médica Aplicada (CIMA), Uni e sidad de Na a a, 31008 Pamplona, Spain
i
UbiCARE, Ad anced Technology Ins i u e in Li e Sciences (ITAV)-CNRS-IPBS, 31106 Toulouse, F ance
j
Ins i u o de In es igaciones Químicas (IIQ) –Cen o de In es igaciones Cien íficas Isla de la Ca uja (cicCa uja), Uni e sidad de Se illa –Consejo Supe io de In es igaciones Cien íficas (CSIC),
41092 Se illa, Spain
k
Bioc uces Heal h Resea ch Ins i u e, 48093 Ba akaldo, Bizkaia, Spain
abs ac a icle in o
A icle his o y:
Recei ed 13 June 2018
Recei ed in e ised o m 13 Decembe 2018
Accep ed 14 Decembe 2018
A ailable online xxxx
Backg ound: E en hough li e kinase B1 (LKB1) is usually desc ibed as a umo supp esso in a wide a ie y o
issues, i has been shown ha LKB1 abe an exp ession is associa ed wi h bad p ognosis in Hepa ocellula
Ca cinoma (HCC).
Me hods: He ein we ha e o e exp essed LKB1 in human hepa oma cells and by using his idine pull-down assay
we ha e in es iga ed he ole o he hypoxia- ela ed pos - ansla ional modifica ion o Small Ubiqui in- ela ed
Modifie (SUMO)yla ion in he egula ion o LKB1 oncogenic ole. Molecula modelling be ween LKB1 and i s
in e ac o s, in ol ed in egula ion o LKB1 nucleocy oplasmic shu ling and LKB1 ac i i y, was pe o med. Finally,
high a fini y SUMO binding en i ies-based echnology we e used o alida e ou findings in a p e-clinical mouse
model and in clinical HCC.
Findings: We ound ha in human hepa oma cells unde hypoxic s ess, LKB1 o e exp ession inc eases cell ia-
bili y and agg essi eness in associa ion wi h changes in LKB1 cellula localiza ion. Mo eo e , by using si e-
di ec ed mu agenesis, we ha e shown ha LKB1 is SUMOyla ed by SUMO-2 a Lys178 hampe ing LKB1
nucleocy oplasmicshu lingand uelinghepa oma cellg ow h.Molecula modellingo SUMOmodifiedLKB1 u -
he confi med s e ic impedance be ween SUMOyla ed LKB1 and he STe20-Rela edADap o co ac o (STRADα),
in ol ed in LKB1 expo om he nucleus. Finally, we p o ide e idence ha endogenous LKB1 is modified by
SUMO in p e-clinical mouse models o HCC and clinical HCC, whe e LKB1 SUMOyla ion is highe in as g owing
umo s.
In e p e a ion: O e all, SUMO-2 modifica ion o LKB1 a Lys178 media es LKB1 cellula localiza ion and i s onco-
genic ole in li e cance .
Fund: This wo k was suppo ed by g an s om NIH (US Depa men o Heal h and Human se ices)-
R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobie no Vasco-Depa amen o de Salud 2013111114 ( o M.L.M.-
C), ELKARTEK 2016, Depa amen o de Indus ia del Gobie no Vasco ( o M.L.M.-C), MINECO: SAF2017–87301-R
and SAF2014–52097-R in eg ado en el Plan Es a al de In es igación Cien ifica y Técnica y Inno ación
2013–2016 cofinanciado con Fondos FEDER ( o M.L.M.-C and J.M.M., espec i ely), BFU2015–71017/BMC
Keywo ds:
LKB1
SUMO
HCC
SIRT1
STRADα
EBioMedicine xxx (2018) xxx
⁎Co esponding au ho s a : CIC bioGUNE, Ed. 801A Pa que Tecnológico de Bizkaia, 48160 De io, Bizkaia, Spain.
E-mail add esses: ca [email protected] (T.C. Delgado), mlma [email protected] (M.L. Ma ínez-Chan a ).
1
Join fi s au ho s.
EBIOM-01832; No o Pages 16
h ps://doi.o g/10.1016/j.ebiom.2018.12.031
2352-3964/© 2018 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Con en s lis s a ailable a ScienceDi ec
EBioMedicine
jou nal homepage: www.ebiomedicine.com
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
MINECO/FEDER, EU ( o A.D.Q. and I.D.M.), BIOEF (Basque Founda ion o Inno a ion and Heal h Resea ch): EITB
Ma a oia BIO15/CA/014; Ins i u o de Salud Ca los III:PIE14/00031, in eg ado en el Plan Es a al de In es igación
Cien ifica y Técnica y Inno acion 2013–2016cofinanciado con Fondos FEDER ( o M.L.M.-C and J.M.M), Asociación
Española con a el Cánce (T.C.D, P·F-T and M.L.M-C), Daniel Alagille awa d om EASL ( o T.C.D), Fundación
Cien ífica de la Asociación Española Con a el Cance (AECC Scien ific Founda ion) Ra e Tumo Calls 2017 ( o
M.L.M and M.A), La Caixa Founda ion P og am ( o M.L.M), P og amma di Rice ca Regione-Uni e si à
2007–2009 and 2011–2012, Regione Emilia-Romagna ( o E.V.), Ramón A eces Founda ion and he Andalusian
Go e nmen (BIO-198) (A.D.Q. and I.D.M.), ayudas pa a apoya g upos de in es igación del sis ema Uni e si a io
Vasco IT971–16 (P.A.), MINECO:SAF2015–64352-R (P.A.), Ins i u Na ional du Cance , FRANCE, INCa
g an PLBIO16–251 (M.S.R.), MINECO - BFU2016–76872-R o (E.B.). Wo k p oduced wi h he suppo o a
2017 Leona do G an o Resea che s and Cul u al C ea o s, BBVA Founda ion (M.V-R). Finally,
Cibe ehd_ISCIII_MINECO is unded by he Ins i u o de Salud Ca los III. We hank MINECO o he Se e o Ochoa
Excellence Acc edi a ion o CIC bioGUNE (SEV-2016-0644). Fundingsou ces had no in ol emen in s udy design;
in he collec ion, analysis, and in e p e a ion o da a; in he w i ing o he epo ; and in he decision o submi
he pape o publica ion.
© 2018 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://
c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
Li e Kinase B1 (LKB1) is a 50 kDa se ine/ h eonine kinase ubiqui-
ously exp essed in adul and e al issues, pa icula ly in panc eas,
li e , es es and skele al muscle [1]. LKB1 is an ups eam ac i a o o
14 kinases om he ARK (AMP-ac i a ed p o ein kinase- ela ed kinase)
amily including AMP-ac i a ed p o ein kinase (AMPK). In ac , he
de ec i e ac i a ion o AMPK in LKB1-null cells can be escued by e-
exp ession o LKB1 [2,3]. AMPK unc ions as a cellula ene gy senso o
p o ide me abolic adap a ions unde ATP-dep i ed condi ions such as
s a a ion and oxygen dep i a ion (hypoxia) [4]. Unde hese ci cum-
s ances, AMPK ac i a ion esul s in s imula ion o bioene ge ic pa h-
ways and inhibi ion o ATP- and NADPH-consuming p ocesses such as
biosyn hesis and p oli e a ion. I is pa icula ly ele an in his espec
ha AMPK inhibi s he mammalian a ge o apamycin complex 1
(mTORC1) pa hway [5] wi h concomi an down egula ion o he glyco-
ly ic pa hway [6,7]. Indeed, silencing LKB1 in mouse emb yonic fib o-
blas s p omo es a me abolic swi ch o ae obic glycolysis, also called
he Wa bu g e ec , a hallma k o highly p oli e a i e umo cells,
suppo ed by mTOR ac i i y and d i en by hypoxia inducible ac o
(HIF)-1α[8]. Mo eo e , AMPK can egula e ene gy expendi u e by
modula ing mi ochond ial biogenesis and con olling he exp ession
o oxida i e enzymes [9–11]. Al e na i ely, LKB1-induced ac i a ion o
AMPK signaling can o e a p o ec i e e ec by allowing he cell ime
o a emp o e e se he abe an ly high a io o AMP/ATP, ha o he -
wise can cause cell dea h [12].
Ge mlinemu a ionso dele ions in he LKB1 genea e esponsible o
hePeu z-Jeghe s Synd ome (PTS), a cance -p oneau osomaldominan
inhe i ed diso de [1,13]. Likewise, soma ic mu a ions o LKB1 gene a e
in ol ed in he de elopmen o spo adic cance s, such as ce ical, p os-
a e and lung cance s, among o he s [14–16]. Al hough gene ic e idence
suppo s he umo -supp essi e ole o LKB1, o he e idence e ealed
ha LKB1 may also exhibi p o-oncogenic unc ions. In he con ex o
li e disease, a con olled balance in hepa ic LKB1 le els has been de-
sc ibed as a ga ekeepe o hepa ocy e p oli e a ion du ing egene a ion
[17,18]. Fu he mo e, LKB1 exp ession o ac i i y ha e been p e iously
shown o be augmen ed in Hepa ocellula Ca cinoma (HCC), he mos
common ype o li e cance , especially ela ed o bad p ognosis and
la e s age HCC [19,20]. The mechanisms unde lying he oncogenic ole
o LKB1 in HCC emain a he unexplo ed.
LKB1 localiza ion wi hin he cell is c i ical o he egula ion o i s ac-
i i y. LKB1 has a N- e minal egula o y domain in he mos N- e minal
egiona e he kinase domain and a C- e minal egula o y domain [21].
LKB1 has also wo specific ecogni ion sequences called nuclea localiza-
ion sequences (NLS) in i s N- e minal egion [22], ha allow he shu -
ling be ween cy oplasm and nucleus o LKB1 in mammalian cells. LKB1
nucleocy oplasmic shu ling is media ed by co ac o s, such as he
STe20-Rela ed ADap o (STRADα) and mouse p o ein 25 (MO25) [2].
B iefly, STRADαinduces elocaliza ion o LKB1 om he nucleus o he
cy oplasm whe eas MO25 s abilizes he STRADα-LKB1 in e ac ion
[23,24]. On he o he hand, STRADαinhibi s nuclea impo o LKB1
by compe ing wi h ka yophe in impo in-α o binding o LKB1 [25].
To da e, se e al egula ing mechanisms ha e been p oposed o explain
LKB1 cellula localiza ion: i) abe an exp ession o STRADαwas associ-
a ed wi h nuclea LKB1 du ing co icogenesis [26]; ii) he o phan nu-
clea ecep o Nu 77 can bind and seques e LKB1 in he nucleus [27];
and finally iii) e e sible pos - ansla ional modifica ions o LKB1 ha e
been shown o egula e i s s abili y and ac i i y [19,28,29].
Pos - ansla ional modifica ion is a c i icale en in he dynamic eg-
ula ion o p o ein s abili y, loca ion, s uc u e, unc ion, ac i i y and
Resea ch in con ex
E idence be o e his s udy
LKB1 is a se ine h eonine kinase p o ein wi h an ambiguous ole in
cance p og ession. LKB1 exp ession o ac i i y is augmen ed in
bad p ognosis and la e s age li e cance . LKB1 cellula localiza-
ion is ele an o i s ac i i y. LKB1 has been shown o be a a ge
o SUMO pos - ansla ional modi ica ions. SUMO-media ed mod-
i ica ions a e known o play an impo an ole in p o ein a ge sub-
cellula localiza ion du ing cance p og ession.
Added alue o his s udy
In his s udy, we show ha LKB1 o e exp ession in human hepa-
oma cells du ing hypoxic s ess p omo es umo cell g ow h and
su i al. Mo eo e , we p o ide e idence ha endogenous LKB1
is modi ied by SUMO in p e-clinical mouse models o HCC as
well as in li e biopsies o HCC pa ien s. Finally, ou da a sugges
ha LKB1 SUMOyla ion is abe an in li e cance being essen ial
o he egula ion o i s subcellula localiza ion and oncogenic ole.
Implica ions o all he a ailable e idence
Ou esul s suppo a po en ial ole o LKB1 SUMOyla ion as a
no el oncogenic mechanism in HCC and he eby he pu a i e he -
apeu ic po en ial o p o ein-based, pep idyl and small molecule in-
hibi o s o a ious SUMO speci ic p o eases iso o ms.
2I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
in e ac ion wi h o he p o eins. Recen ly, Ri ho and colleagues ha e de-
sc ibed o he fi s ime LKB1 Small Ubiqui in- ela ed MOdifie
(SUMO)-media ed modifica ions and i s implica ion unde me abolic
s ess ci cums ances whe e SUMO-media ed modifica ion o LKB1 is es-
sen ial in p omo ing i s in e ac ion wi h i s downs eam a ge AMPK
ia a SUMO-in e ac ing mo i (SIM) essen ial o AMPK ac i a ion [29].
In addi ion, he SUMO p o ein modifica ion has an ex ensi e and c i ical
ole in he adap i e cellula esponse o hypoxia [30–34]. Ch onic hyp-
oxia is an impo an mic o-en i onmen al ac o o es ablishing he ag-
g essi eness o HCC by p omo ing umo in asion and me as asis
[35,36]. Thus, SUMOyla ion appea s o be up egula ed in many ypes
o cance , including HCC [37–39]. P e iously, exp ession o bo h he
SUMO E2 conjuga ing enzyme (Ubc9) and he E3 SUMO-p o ein ligase
CBX4 we e ound o be up egula ed and ela ed o poo p ognosis in
HCC [38,39].
In his s udy, we aimed o explo e he unc ional ole o LKB1 in
hepa oca cinogenesis and p og ession wi h special ocus on he ole
playedby SUMOyla ed LKB1 in hemodula ion o i s cellula localiza ion
in hepa oma cells du ing hypoxic s ess and in li e cance .
2. Ma e ials and me hods
2.1. Cell lines
Huh-7, Hep G2, and PLC/PRF/5, human hepa oma cell lines; and
MLP-29, mouse li e p ogeni o cells we e used. All cells we e g own
a 37 °C in a humidified a mosphe e o 5% CO
2
–95% ai and cul u ed in
Dulbecco's Modified Eagle Medium (DMEM) supplemen ed wi h 10%
FBS (Gibco, The mo Fishe Scien ific, Spain), unless o he wise s a ed.
2.2. Cell ans ec ion
Cell lines we e ansien ly ans ec ed using Lipo ec amine 2000
(In i ogen, USA), acco ding o he manu ac u e 's ins uc ions. B iefly,
Lipo ec amine 2000 (2.5 μl/1 μg DNA) was dilu ed in OPTI-MEM (Gibco)
medium o ans ec ions and incuba ed o 5 min. A e incuba ion, he
mix u e was added o OPTI-MEM con aining plasmid DNA (1 o 6 μgde-
pending on he expe imen ), and incuba ed o a leas 20 min a oom
empe a u e (RT) o allow o he o ma ion o he DNA-Lipo ec amine
complexes. DNA-lipo ec amine complexes p e iously o med we e
added o he cul u e pla es con aining he co esponding cul u e me-
dium wi h 10% FBS bu wi hou an ibio ics and wi h he cells in suspen-
sion. Plasmid DNA used a e lis ed in Supplemen al Table 1. Pu a i e
SUMO sequence binding si es on LKB1 we e analyzed using he
SUMOplo Analysis P og am (h p://www.abgen .com/SUMOplo ,
Abgen , USA). The 4 highes anking si es p edic ed by SUMOplo
we e used o c ea e he mu an s (lysines 96, 97, 178 and 235). Lysines
we e mu a ed o a ginines (R). An addi ional ace yla ion LKB1 mu an
(K48R) was also c ea ed. The mu an LKB1 plasmid cons uc s we e c e-
a ed using he QuickChange ki o di ec ed mu agenesis (S a agene,
USA), acco ding o he manu ac u e 's ins uc ions, wi h wo comple-
men a y oligonucleo ides and wi h he pcDNA3-FLAG LKB1 plasmid as
a empla e. The p oduc s we e sequenced (STAB ida, Po ugal).
2.3. Cell ea men s
The selec i e inhibi o o si uin 1 (SIRT1), Ex-527 (Sigma-Ald ich)
in DMSO, was gi en o cells o 24 h a 30 μM. Cycloheximide, a euka y-
o e p o ein syn hesis inhibi o was added o cells a 50 μg/ml in H
2
O.
2.4. Hypoxia in i o
Fo hypoxia expe imen s, cells we e incuba ed in an In i o
2
400
hypoxia Wo ks a ion (Bake Ruskinn, USA) a 1% O
2
o un il 72 h and
lysed/fixed in he hypoxia chambe .
2.5. Se um dep i a ion in i o
Cells we e g own wi hou FBS o a 24-hou pe iod and compa ed o
cells main ained on 10%FBS.
2.6. Mouse models
All animal expe imen s we e pe o med acco ding o he ARRIVE
guidelines and ca ied ou in acco dance wi h he Na ional Ins i u es
o Heal h guide o heca e and use o Labo a o y animals (NIH Publica-
ions N0.8023, e ised 1978) and he guidelines o Eu opean Resea ch
Council o animal ca e and use. Adul male C57BL/6 mice and mouse
models ha de elop HCC a 8-mon hs old, such as he glycine N-
me hyl ans e ase (Gnm ) deficien (Gnm
−/−
)and wild ype
(Gnm
+/+
)mice, we e b ed and housed in he animal uni o CIC
bioGUNE, which is an AAALAC-acc edi ed acili y. Animals we e housed
unde con olled empe a u e (22 °C) and humidi y condi ions in a 12 h
ligh /da k cycle wi h ad libi um access o ood and wa e .
2.7. In i o hypoxia ea men
Th ee-mon h-old C57BL/6 mice males we e exposed o sys emic
hypoxia in a sealed wo ks a ion (Bake -Ruskinn In i O
2
400, Cul ek).
The final 10% O
2
en i onmen was eached a e a 2 h30 pe iod in
which oxygen le els we e g adually educed. Feeding and ligh cycles
we e kep uni o m in he hypoxia wo ks a ion. Con ol mice we e also
exposed o he same chambe bu unde a 21% O
2
en i onmen . Mice
we e eu hanized by ce ical disloca ion inside he chambe , and he dis-
sec ed o gans we e di ec ly fixed.
2.8. Human HCC samples
The wo k desc ibed has been ca ied ou in acco dance wi h he
code o e hics o he Wo ld Medical Associa ion (Decla a ion o Hel-
sinki) o expe imen s in ol ing humans a e ob aining in o med con-
sen . Su gically esec ed li e specimens o 22 pa ien s wi h HCC (10
Hepa i is C, 10 ASH and 2 NASH) we e examined. The Basque Biobank
(h p://www.biobanco asco.o g) p o ided he da a and ype o
biospecimen. In addi ion, we ha e used ozen pai ed HCC umo and
su ounding issue li e biopsies ob ained du ing umo esec ion
(n = 6) as well as HCC li e biopsies om a se o samples p e iously
classified as as g owing (n = 3) o slow g owing (n = 3). The la e
samples we e included in a p e iously published la ge s udy, which in-
cluded wo coho s o pa ien s wi h ci hosis o any e iology on ul a-
sound su eillance ( aining se = 78 and alida ion se = 54). A fi s
iden ifica ion o HCC, hey unde wen wo CT scans 6 weeks apa
wi h no ea men in-be ween. Fas g owing pa ien s has a median
umo doubling ime o 42 days while o he slow g owing pa ien 's
median umo doubling ime was 97 days [40]. US-guided li e biopsies
we e ini ially ob ained and used o gene a e a mic oa ay (Agilen
Whole Human Genome Oligo) acco ding o he MIAME guidelines.
Gene exp ession da a is a ailable a he Gene Exp ession Omnibus
websi e (h p://www.ncbi.nlm.nih.go /geo) unde he accession num-
be : GSE54236.
2.9. P o ein ex ac ion and wes e n blo ing analysis
P o ein ex ac ion and Wes e n blo ing analysis was pe o med as
p e iously desc ibed [41]. P ima y an ibodies and hei op imal incuba-
ion condi ions a e de ailed in Supplemen al Table 2.
2.10. C ys al iole iabili y assay
Cell iabili y was assessed using c ys al iole s aining (Sigma-
Ald ich).
3I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
2.11. Sc a ching wound-healing assay
Cells we e seeded o confluence o e 12 mm co e slip a e an o e -
nigh ans ec ion. A pipe e ip (200 μl) was used o sc a ch a s aigh
line h ough all he wells. Media was changed wice o emo e dead
and una ached cells. The wells we e hen placed in hypoxia du ing
72 h. Pic u es o he sc a ch we e aken using an Eclipse TS100 mic o-
scope (Nikon, Japan).
2.12. Subcellula p o eome ex ac ion ki
P o eoEx ac ® Subcellula P o eome Ex ac ion Ki (S-PEK) om
Me ck (Spain) was used.
2.13. Immunocy ofluo escence
Cells seeded on 12 mm co e slips we e fixed in PBS 4% pa a o mal-
dehyde (San a C uz Bio echnology, USA). Co e slips we e hen blocked
and pe meabilized wi h PBS con aining 0.1% BSA, 10% goa se um and
0·2% T i on X-100 o 30 min a RT. A e blocking, he co e slips
we e washed in PBS and incuba ed o e nigh in a humid chambe
wi h he p ima y an ibody (FLAG 1:100) in PBS. Co e slips we e
washed in PBS and incuba ed du ing 1 h a RT in blocking solu ion
wi h DAPI bu no T i on X-100 wi h seconda y an ibody (dilu ion
1:200, Cy3 conjuga ed an i-mouse o FITC-conjuga ed an i- abbi , Jack-
son ImmunoResea ch labo a o ies, USA). Co e slips we e moun ed in
Dako fluo escence moun ing medium (Dako, Denma k). Fi e Images
om each expe imen al condi ion we e aken using an Axioimage D1
(Zeiss). Blind quan ifica ion o LKB1 posi i e nuclea cells e sus o al
numbe o s ained cells was pe o med manually by an immunohis o-
chemis y echnician.
2.14. Nickel-His idine a fini y pu ifica ion using nickel-ni iolo iace ic acid
(Ni
2+
-NTA) beads
Cells we e ans ec ed wi h he espec i e cons uc s and His
6
-
SUMO as desc ibed. A e ea men s, His
6
-SUMOyla ed p o eins we e
pu ifiedas p e iously desc ibedby usinglowdensi y Ni
2+
-NTA-aga ose
beads (ABT, Spain) [19,42].
2.15. P o ein immunop ecipi a ion assays
To al p o ein ex ac s we e immunop ecipi a ed wi h 5 μg o IgG1
(BD Pha migen), an i-LKB1 o an i-STRADαan ibodies by using A/G
PLUS-Aga ose Beads (San a C uz).
2.16. SUMO binding en i ies (SUBEs)
SUBEs, ea lie desc ibed and alida ed [43], we e used as a cap u ing
sys em o endogenous SUMOyla ed LKB1. Li e issue (75 mg) om
Gnm
−/−
and Gnm
+/+
as well as omHCC pa ien s we e used. B iefly,
li e s we e lysed in lysis bu e [50 mM T is pH 8.5; 150 mMNaCl, 5 mM
EDTA, 1% Igepal, supplemen ed wi h 1× p o ease inhibi o cock ail
(Roche) and 50 μM o PR-619 (ubiqui in and ubiqui in-like
isopep idases inhibi o , Li eSenso s)]. Lysa es we e cen i uged a
14000 ×gand he supe na an was incuba ed wi h 50 μlo GST-
aga ose beads con aining 50 μg o SUBEs o GST and 1 mM DTT (Di hio-
h ei ol) o 2 h, a 4 °C. Beads we e hen pulled down by cen i uga ion,
1000 ×g o 5 min, and 1/10 o he unbound ac ion was sa ed o wes -
e n blo analysis (flow h ough-FT). Washes we e ca ied ou using 30
column olumes o wash bu e (50 mM T is pH 8.5; 50 mM NaCl,
5 mMEDTA and 1% Igepal).Elu ionswe epe o med in onecolumn ol-
ume o 2 Laemmli Bu e . Fo Wes e n blo analysis, samples we e sep-
a a ed in NUPAGE 4–12% BT Gels, 1.5 mm, 15Well.
2.17. Modelling
Compu a ional analysis o he a ious LKB1 species used he X- ay
di ac ion coo dina es o he LKB1-STRADα-MO25αcomplex by
Zequi aj e al. (2009; pdb 2WTK). Highly mobile egions o LKB1 we e
absen and, he e o e, needed modelling. Fo his pu pose, 250 s uc-
u es we e gene a ed using Modelle 9 7 [44] and classified acco ding
o hei Disc e e Op imized P o ein Ene gy (zDOPE) sco es o selec
he model wi h i s lowes alue. Molecula Dynamics ajec o ies we e
compu ed wi h he AMBER 16 package [45], using he 14SB o ce field
[46]. Fo he ace yla ed lysine esidue, Papamokos' pa ame e iza ions
[47] we e used. Simula ions un unde pe iodic bounda y condi ions
in o ho hombic boxes. Ini ially, he minimum dis ance be ween p o-
ein and cell aces was 10 Å. PME elec os a ics we e se wi h he
Ewald summa ion cu -o a 9 Å. Sodium coun e -ions neu alized he
cha ges o he sys em. The s uc u es we e sol a ed wi h SPC wa e
molecules [48]. P o ein side-chains we e ene gy-minimized (100
s eepes descen and 1400 conjuga e g adien s eps)down o a RMS en-
e gy g adien o 0·01 kJ mol-1 Å-1. A e wa ds, sol en was subjec ed o
1000 s eps o s eepes descen minimiza ion ollowed by 500 ps NPT-
MD compu a ions using iso opic molecule posi ion scaling and a p es-
su e elaxa ion ime o 2 ps a 298 K. Tempe a u e was egula ed wi h
Be endsen's hea ba h algo i hm [49], wi h a coupling ime cons an
equal o 0·5 ps. The densi y o he sys em eached a pla eau a e ca.
150 ps simula ion. Then, o each p o ein, he whole sys em was ene gy
minimized and submi ed o NVT-MD a 298 K, using 2·0 s in eg a ion
ime s eps. Snapsho s we e sa ed e e y 100 ps. SHAKE algo i hm
(Ryckae e al., 1977) was used o cons ain bonds in ol ing hyd ogen
a oms. Coo dina e files we e p ocessed using CPPTRAJ [50]. Fu he p o-
cessing was made in O igin 16 (O iginlab) and g aphic displays we e
buil in UCSF Chime a [51].
2.18. Immunohis ochemis y
Pa a fin-embedded sec ions (5 μm hick)o o malin-fixed li e
samples we e ini ially depa afinized in xylene o xylene-subs i u e
and ehyd a ed h ough g aded alcohol solu ions. Specific an ibodies
and expe imen al condi ions used in immunohis ochemis y can be
ound in supplemen al Table 3. Fo he analysis, images we e aken
wi h an up igh ligh mic oscope (Zeiss, Ge many). The a e age sum
o in ensi ies and s aineda ea pe cen age o each sample wascalcula ed
using FRIDA so wa e (h p://bui3.win.ad.jhu.edu/ ida/,JohnHopkins
Uni e si y).
2.19. S a is ical analysis
Da a is exp essed as mean ± SEM (s anda d e o o he mean). S a-
is ical significance was es ima ed using he Mann-Whi ney U es . A p
alue o b0·05 was conside ed significan .
3. Resul s
3.1. LKB1 o e s su i al and in asi eness ad an age o human hepa oma
cells du ing hypoxic s ess
LKB1 is endogenously exp essed in human hepa oma cells (Suppl.
Fig. 1a). These esul s a e in ag eemen wi h ea lie e idence showing
augmen ed exp ession o LKB1 in oden hepa oma cells [52–54]. In
o de o u he explo e he unc ional ole o LKB1 in HCC p og ession,
we ansien ly o e exp essed LKB1 in Huh-7 human hepa oma cells
bo h a e se um dep i a ion (0% FBS) o unde hypoxic (1% O
2
) condi-
ions in compa ison wi h cells unde no moxia (21% O
2
) and cul u ed
wi h 10% FBS. LKB1 ec opic ansien o e exp ession in Huh-7 hepa-
oma cells esul s in compa able le els o LKB1 a e 24 h o hypoxic,
se um dep i a ion o no moxic s imuli (Fig. 1a, Suppl. Fig. 1b).
4I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
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ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
Fig. 1. Li e Kinase B1(LKB1) o e s su i al andin asi eness ad an age o humanhepa oma cells du ing hypoxic s ess. LKB1 o e exp ession was induced in human hepa oma Huh-7 cell
line by using he pcDNA3-FLAG-LKB1 WildType plasmid (LKB1)and compa ed o con ol o e nigh ans ec ion wi h he pcDNA™3.3-TOPO® plasmid (C l), ollowed by 24 h ea men
unde con ol condi ions o no moxia and comple e media (21%oxygen,10% se um), se um dep i a ion (SD) and hypoxia (1% oxygen, 10% se um). a. Rep esen a i e Wes e n blo o LKB1,
i s downs eam a ge AMP-ac i a ed p o ein (AMPK) and phospho yla ed AMPK a Th 172 and hypoxia inducible ac o (HIF1α), a hypoxic ma ke , a e shown. [β-ac in was used as
loading con ol]. Quan ifica ions a e shown in Suppl. Fig. 1b; b. Cell iabili y as de ec ed by s aining o a ached cells wi h c ys al iole dye; c. Time-cou se o cell iabili y and cell
mig a ion using a wound-healing sc a ch assay a e LKB1 o e exp ession unde hypoxia; d. Rep esen a i e immunofluo escence s aining o LKB1 (FLAG) in Huh-7 hepa oma cells
and quan ifica ion o he pe cen age o LKB1 nuclea posi i e s aining cells. Scale ba co esponds o 50 μm; and e. Wes e n blo o LKB1 le els in cy oplasmic and nuclea ac ions
[Glyce aldehyde 3-phospha e (GAPDH) was used as loading con ol o cy oplasmic ac ions and His one H3 o nuclea ac ions]. Quan ifica ions a e shown in Suppl. Fig. 1b. A leas
iplica es we e used pe expe imen al condi ion. Da a is shown as mean ± SEM. *p b0·05 and **p b0·01 a e indica ed (Mann-Whi ney U es ). (Fo in e p e a ion o he e e ences
o colou in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
5I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031

Fig. 2. Li e KinaseB1 (LKB1) is SUMOyla ed by SUMO-2in human hepa oma cells. a. Ni
2+
-NTA aga ose bead pulldown in Huh-7 human hepa oma cells a e ans ec ion wi h His-SUMO-
1, 2, o 3, wi h he pcDNA3-FLAG-LKB1 Wild ype plasmid (LKB1 WT) in he p esence and absence o ubiqui in conjuga ing enzyme 9 (UBC9). b. Ni
2+
-NTA aga ose bead pulldown in Huh-7
human hepa oma cells a e ans ec ion wi h His-SUMO-2 wi h he LKB1 WT plasmid in he p esence o he di e en SUMO E3 ligases PIAS 1, 2α,2β,and4.c.Ni
2+
-NTA aga ose bead
pulldown in Huh-7 human hepa oma cells a e ans ec ion wi h His-SUMO-2 and wi h he LKB1 WT plasmid in he p esence o he di e en SUMO-specific p o eases, SENPs 1–7. d.
Ni
2+
-NTA aga ose bead pulldown in Huh-7 human hepa oma cells a e ans ec ion wi h His-SUMO-2 and wi h he LKB1 WT plasmid o he pcDNA3-FLAG-LKB1 Kinase Dead K78I
plasmid. e. Immunop ecipi a ion assay be ween LKB1 and he STe20-Rela ed ADap o (STRADα) co ac o a e STRADαo e exp ession in Huh-7 human hepa oma cells. No malized
quan ifica ions ela i e o inpu s a e shown below he panel.
6I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
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ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
LKB1 is an ups eam ac i a o o AMPK p omo ing he phospho yla ion
o AMPK Th -172 in he ac i a ion loop o i s αsubuni in esponse o
me abolic s ess in o de o inhibi biosyn hesis and p oli e a ion [2,3].
He ein, LKB1 o e exp ession inc eased phospho yla ion o AMPK a
Th -172 excep du ing hypoxic s ess, a condi ion cha ac e ized by
high HIF-1αle els (Fig. 1b, Suppl. Fig. 1b). In e es ingly, a e 24 h o
hypoxia, LKB1 up egula ion was associa ed wi h inc eased cell iabili y
in compa ison wi h con ol and se um dep i ed cells, bo h in Huh-7
(Fig. 1b) and ano he cell line o mouse li e p ogeni o cells, he
MLP-29 cells (Suppl. Fig. 2a). A ime cou se indica es ha whe eas hep-
a oma cells g ow h is hampe ed du ing hypoxia, LKB1 o e exp ession is
able o induce cell g ow h unde hese condi ions (Fig. 1c). Fu he -
mo e, hypoxia is known o unleash he in asi e po en ial o umo
cells. Sc a ch wound-healing assay e ealed ha LKB1 o e exp ession
also p o ides and in asi eness ad an age o umo cells unde hypoxia
(Fig. 1c).
LKB1 cellula localiza ion plays an impo an ole i s ac i i y. Du ing
se um dep i a ion and no moxia condi ions LKB1 ac i ely shu les be-
ween he nucleus and cy oplasm whe eas unde hypoxia, he LKB1
nucleocy oplasmic shu ling is hampe ed and LKB1 is mo e p esen in
he nucleus (Fig. 1d, e, Suppl. Fig. 1c).
O e all, LKB1 o e exp ession p o ides g ow h su i al and in a-
si eness ad an age o hepa oma cells du ing hypoxic s ess which
ag ees wi h p e ious e idence om ou labo a o y and o he s showing
ha LKB1 exp ession is induced in HCC umo s [19,20], umo s cha ac-
e ized by a highly hypoxic en i onmen .
3.2. Inc eased LKB1 SUMOyla ion in human hepa oma cells
As p e iously men ioned, SUMOyla ion pos - ansla ional modifica-
ions a e c i ical du ing hypoxia and he eby ele an in HCC [38,39]. In
mammals, he e a e fi e SUMO pa alogues, being SUMO-1, -2 and -3
Fig. 3. Li e Kinase B1 (LKB1) is SUMOyla ed by SUMO-2 a Lys178 in human hepa oma cells. a. Schema ic ep esen a ion o LKB1 showing he Nuclea (NLS) localiza ion and he Kinase
domain (KDN). SUMOyla ion LKB1 mu an s used a e also shown and desc ibed. b. Ni
2+
-NTA aga ose bead pulldown in Huh-7 human hepa oma cells a e ans ec ion wi h His-SUMO-2
and he LKB1 SUMO mu an s, LKB1 K96R, LKB1 K97R, LKB1 K178R and LKB1 K235R. No malized quan ifica ions ela i e o inpu s a e shown below each panel.
7I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
mo e s udied, compa ed o SUMO-4 and -5 [55–57]. Hepa ic SUMO-1
p o ein le els a e low, on he con a y o o he issues such as lung,
u e us and p os a e, whe eas SUMO-2/3 le els in he li e a e high
[58]. On his ega d, we ha e ound ha mice exposed o hypoxia
show inc eased li e LKB1 nuclea exp ession and SUMO-2/3 le els
(Suppl. Fig. 3a). Fu he mo e, we show ha endogenous LKB1
SUMOyla ion by SUMO-2/3 is induced in Huh-7 hepa oma cells a e
24 h o hypoxia in compa ison wi h cells g own unde no moxic condi-
ions (Suppl. Fig. 3b). To u he explo e he ole o SUMO-media ed
modifica ions o LKB1 in hepa oma cells, Ni
2+
-NTA aga ose bead-
pulldowns we e pe o med in Huh-7 human hepa oma cells a e co-
ans ec ion o pcDNA3-FLAG-LKB1 wild ype (WT) and pcDNA3-His
6
-
SUMO1, pcDNA3-His
6
-SUMO2 o pcDNA3-His
6
-SUMO3 plasmids. Ou
esul s show ha LKB1 is mos ly modified by SUMO-2, in a SUMO-
conjuga ing enzyme UBC9 dependen p ocess, bo h in Huh-7 human
hepa oma cells (Fig. 2a) and in MLP-29 cells (Suppl. Fig. 4a). Co-
ans ec ion wi h he E3 SUMO-p o ein ligases PIAS, especially PIAS 1,
u he inc eased LKB1 SUMOyla ion by SUMO-2 in Hu7–7 cells
whe eas co- ans ec ion wi h SUMO-specific p o eases, pa icula ly
SENP2, and SENP-1 and -3, dec eased LKB1 SUMOyla ion (Fig. 2b,c). In
Fig. 4. Li e Kinase B1 (LKB1) SUMOyla ion a Lys178 by SUMO-2 egula es human hepa oma cell su i al by hampe ing LKB1 nucleocy oplasmic shu ling. LKB1 o e exp ession was
induced in Huh-7 cells wi h pcDNA3-FLAG-LKB1 Wild Type plasmid (LKB1 WT) o he pcDNA3-FLAG-LKB1 K178R plasmid (LKB1 K178R) in he p esence o His-SUMO-2. a. Wes e n
blo analysis o LKB1, [Glyce aldehyde 3-phospha e (GAPDH) was used as loading con ol]. Quan ifica ions a e shown in Suppl. Fig. 4a; b and c. Cell iabili y as de ec ed by s aining o
a ached cells wi h c ys al iole dye and numbe o cells; d. Immunop ecipi a ion assay be ween LKB1 WT and LKB1 K178R and he STe20-Rela ed ADap o (STRADα) co ac o a e
STRADαo e exp ession in Huh-7 human hepa oma cells. No malized quan ifica ions ela i e o inpu s a e shown below he panel; e. Rep esen a i e immunofluo escence s aining o
LKB1 (FLAG) in Huh-7 cells and quan ifica ion o he pe cen age o LKB1 nuclea posi i e s aining cells. Scale ba co esponds o 50 μm. . LKB1 le els in cy oplasmic (Cy o) and nuclea
ac ions (Nuc) [Glyce aldehyde 3-phospha e (GAPDH) was used as loading con ol o cy oplasmic ac ions and His one H3 o nuclea ac ions]. Quan ifica ions a e shown in Suppl.
Fig. 4b. A leas iplica es we e used pe expe imen al condi ion. Da a is shown as mean ± SEM. *p b0·05 is indica ed (Mann-Whi ney U es ). (Fo in e p e a ion o he e e ences o
colou in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
8I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
ag eemen , SENP2 has been p e iously epo ed o play a c i ical ole in
he con ol o HCC cell g ow h [59]. The SUMOyla ion o LKB1 does no
depend on LKB1 kinase ac i i y domain as a kinase dead (KD) mu an ,
pcDNA3-FLAG-LKB1 K78I, was equally modified by SUMO-2
(Fig. 2de
). Finally, SUMO-2-media ed modifica ion o LKB1 educed
he in e ac ion be ween LKB1 and STRADα, a co- ac o in ol ed in
LKB1 nuclea expo , as shown by immunop ecipi a ion assay, du ing
STRADαo e exp ession (Fig. 2e).
Fig. 5. Li e Kinase B1 (LKB1) is modified by SUMO-2 in Lys178 a e i s ace yla ion a Lys48 in human hepa oma cells. a. Schema ic ep esen a ion o LKB1 showing he Nuclea (NLS)
localiza ion, ace yla ion domain (AD) and he Kinase domain (KDN). Ace yla ion LKB1 mu an used is shown. b. Ni
2+
-NTA aga ose bead pulldown in Huh-7 human hepa oma cells
a e ans ec ion wi h he pcDNA3-FLAG-LKB1 Wild ype plasmid (LKB1 WT), His-SUMO-2 and ea men wi h si uin 1 (SIRT1). c. Ni
2+
-NTA aga ose bead pulldown in Huh-7 human
hepa oma cells a e ans ec ion he LKB1 WT, LKB1 ace yla ion mu an , LKB1 K48R o he LKB1 SUMOyla ion mu an , LKB1 K178R, wi h His-SUMO-2, in he p esence and absence o
he Ex-527, he SIRT1 inhibi o . No malized quan ifica ions ela i e o inpu s a e shown below each panel; d. Rep esen a i e immunofluo escence s aining o FLAG and
quan ifica ions in Huh-7 hepa oma cells a e ans ec ion wi h he LKB1 WT o he LKB1 SUMOyla ion mu an LKB1 K178R and SUMO-2 in he p esence and absence o Ex-527. Scale
ba co esponds o 50 μm. A leas iplica es we e used pe expe imen al condi ion. Da a is shown as mean ± SEM. *p b0·05 and **p b0·01 a e indica ed (Mann-Whi ney U es ).
9I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031
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16 I. Zubie e-F anco e al. / EBioMedicine xxx (2018) xxx
Please ci e his a icle as: I. Zubie e-F anco, J.L.Ga cía-Rod íguez, F. Lopi z-O soa, e al., SUMOyla ion egula es LKB1 localiza ion and i s oncogenic
ac i i y in li e cance , EBioMedicine, h ps://doi.o g/10.1016/j.ebiom.2018.12.031