1
Channeling o newly syn hesized a y acids o choles e ol es e i ica ion
limi s iglyce ide syn hesis in SND1-o e exp essing hepa oma cells
Hia Na a o-Imaz, Yolanda Chico, Yu i Rueda and Ola z F esnedo
Lipids & Li e Resea ch G oup, Depa men o Physiology, Facul y o Medicine and Nu sing,
Uni e si y o he Basque Coun y UPV/EHU. Bº Sa iena s/n, 48940 Leioa, Spain.
Hia Na a o-Imaz: hia .na a [email p o ec ed]om
Yolanda Chico: [email protected]
Yu i Rueda: yu i. [email protected]
Co esponding au ho : Ola z F esnedo, Depa men o Physiology, Facul y o Medicine and
Nu sing, Uni e si y o he Basque Coun y UPV/EHU, 48940 Leioa, Spain. Tel: +34 946015667;
Fax: +34 946015662. E-mail add ess: ola z. [email protected]
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Biochimica e Biophysica Ac a - Molecula and Cell Biology o
Lipids 1864(2) : 137-146 (2019), which has been published in inal o m a h ps://doi.o g/10.1016/j.bbalip.2018.11.004. © 2018 Else ie
unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
2
Abs ac
SND1 is a pu a i e oncop o ein whose molecula unc ion emains unclea . I s o e exp ession
in hepa ocellula ca cinoma impai s choles e ol homeos asis due o he al e ed ac i a ion o he
s e ol egula o y elemen -binding p o ein (SREBP) 2, which esul s in he accumula ion o
cellula choles e yl es e s (CE). In his wo k, we explo ed whe he high choles e ol syn hesis and
es e i ica ion o igina es changes in glyce olipid me abolism ha migh a ec cell g ow h, gi en
ha ace yl-coenzyme A is equi ed o choles e ogenesis and a y acids (FA) a e he subs a es
o acyl-coenzyme A:choles e ol acyl ans e ase (ACAT). SND1-o e exp essing hepa oma cells
show low iglyce ide (TG) syn hesis, bu phospholipid biosyn hesis o cell g ow h a e no
a ec ed. Limi ed TG syn hesis is no due o low ace yl-coenzyme A o NADPH a ailabili y. We
demons a e ha he main ac o limi ing TG syn hesis is he u iliza ion o FAs o choles e ol
es e i ica ion. These me abolic adap a ions a e linked o high Scd1 exp ession, needed o he
de no o p oduc ion o oleic acid, he main FA used by ACAT. We conclude ha high
choles e ogenesis due o SND1 o e exp ession migh de e mine he channeling o FAs o CEs.
Key wo ds: SND1, choles e ol homeos asis, iglyce ide accumula ion, cance lipid me abolism,
hepa ocellula ca cinoma
Highligh s
SND1-o e exp essing hepa oma cells (McA-S) show low iglyce ide (TG) syn hesis
Limi ed TG syn hesis in McA-S cells is no due o low subs a e o NADPH a ailabili y
Fa y acid (FA) u iliza ion o choles e ol es e i ica ion limi s TG syn hesis
High choles e ogenesis migh de e mine he channeling o FAs o choles e yl es e s
Abb e ia ions
ACAT, acyl-coenzyme A:choles e ol acyl ans e ase; ACC, ace yl-coenzyme A ca boxylase; ACLY,
ATP-ci a e lyase; CE, choles e yl es e ; CoA, coenzyme A; EMEM, Eagle’s minimum essen ial
medium; FA, a y acid; HCC, hepa ocellula ca cinoma; OA, oleic acid; PC, phospha idylcholine;
PE, phospha idyle hanolamine; PL, phospholipid; PPAR, pe oxisome p oli e a o -ac i a ed
ecep o ; S-58035, Sandoz-58035; SCD1, s ea oyl-coenzyme A desa u ase-1; SN, s aphylococcal
nuclease; SND1, s aphylococcal nuclease domain-con aining p o ein 1; SREBP, s e ol egula o y
elemen -binding p o ein; TG, iglyce ide.
3
1. In oduc ion
S aphylococcal nuclease domain-con aining p o ein 1 (SND1), also called Tudo -SN, TSN, SND
p102 o p100, is a highly conse ed p o ein. The s uc u al pa e n o ou s aphylococcal
nuclease (SN) domains, a Tudo domain and a 5 h incomple e SN domain has been ound in he
homologues o di e en species and di e en cell ypes [1-6].
Mul iple unc ions ha e been disco e ed o SND1, which e eals a complex biology. I was i s
desc ibed as a ansc ip ional coac i a o (p100 coac i a o [7]) and since hen he egula ion o
gene exp ession is he mos epo ed unc ion [8-11]. Func ions based on he in e ac ion o
SND1 wi h nucleic acids include egula ion o splicing ac i i y [12], deg ada ion o mRNA linked
o RNA-induced silencing complex [13] and egula ion o ansla ion [11].
In he ield o pa hophysiology i is conside ed an oncogenic p o ein based on a a ie y o e ec s
o he di e en ial exp ession o SND1. Loss o con ac -media ed g ow h inhibi ion [14],
modula ion o he esponse agains induced apop osis [15] and o he iabili y and deg ada ion
o umo supp esso genes [16], p omo ion o he exp ession o angiogenic ac o s [17] and
induc ion o epi helial-mesenchymal ansi ion [3,18], among o he s, a e p ocesses o
ca cinogenesis in which SND1 o e exp ession seems o be in ol ed. Se e al wo ks also show
ha he p omo e ac i i y o Snd1 gene is associa ed o cell ac o s linked o ca cinogenesis [19-
21]. I has been shown ha SND1 exp ession is al e ed in se e al cance ypes, o example
b eas cance [22,23], colon cance [14,24,25] p os a e cance [26,27], lung cance [28,29] and
gliomas [30].
The e is also some e idence ha ela es SND1 pa hophysiology wi h lipid me abolism.
Rajaseka an e al. desc ibed ha SND1 p omo es he deg ada ion o monoglyce ide lipase, ac
ha has been associa ed o a lowe endocannabinoid deg ada ion a e in hepa ocellula
ca cinoma (HCC) [31]. The e o e, SND1 migh ha e an i umo igenic e ec s h ough he
modula ion o apop osis, angiogenesis and cellula mig a ion a ibu ed o endocannabinoids
[32]. O he s udies ound ha SND1 associa es o lipidic s uc u es in di e en s ea ogenic
condi ions [33,34]. I also seems ha SND1 plays an essen ial ole in adipogenesis as a
coac i a o o a y acid (FA) and glucose me abolism egula o pe oxisome p oli e a o -
ac i a ed ecep o (PPAR) γ [35]. PPARγ ac i a ion has been desc ibed o ac as a possible
ini ia o o oxici y pa hways in hepa ocy es, by inducing FA up ake and syn hesis in he li e
( e iewed in [36]). Inc ease o FA le els can lead o lipo oxici y and in lamma ion, he e o e
some o he dele e ious e ec s o SND1 in li e ca cinogenesis migh be ela ed o he al e a ion
o FA me abolism.
4
In a p e ious wo k, we showed ha SND1 o e exp ession in HCC impai s cellula choles e ol
dis ibu ion and homeos asis [37]. S e ol egula o y elemen -binding p o ein (SREBP) 2 is
o e ac i a ed due o low choles e ol con en o endoplasmic e iculum memb anes, which
igge s an exace ba ed choles e ogenesis linked o enhanced choles e ol es e i ica ion. Those
al e a ions esul in he accumula ion o choles e yl es e s (CE), a dis inc i e ai o SND1-
o e exp essing hepa oma cells. Choles e ol es e i ica ion is ca alyzed by acyl-coenzyme
A:choles e ol acyl ans e ase (ACAT) and equi es FAs, which can be ei he newly syn hesized o
exogenous FAs. In his wo k, we explo ed whe he he al e a ions in choles e ol egula o y
sys ems caused by SND1 o e exp ession o igina e changes in glyce olipid me abolism ha migh
a ec cell g ow h.
2. Ma e ials and Me hods
2.1. Cell lines, cul u e and ea men s
The cell lines used in his s udy we e ob ained in a p e ious wo k [37]. McA-RH7777 a
hepa oma cells we e ans ec ed wi h linea ized pcDNA6 (In i ogen) con aining Snd1 (McA-S
cells) o LacZ cDNA (McA-L cells). The cell line wi h he highes SND1 exp ession le el (McA-S2)
was used in his wo k; ele an cha ac e is ics o McA-S1 cell line, which has a lowe SND1
exp ession le el [37] a e also shown in Figu e S1. Cells we e cul u ed in gela in-coa ed pla es
wi h Eagle’s minimum essen ial medium (EMEM) supplemen ed wi h 9% e al bo ine se um
(ATTC), 1.675 mM L-glu amine, 85 U/ml penicillin, 85 μg/ml s ep omycin and 2 μg/ml
blas icidin. Analyses and expe imen s we e pe o med 72 h a e cell seeding. A o as a in (0.1
μM; Sigma) o ehicle (0.6% DMSO), Sandoz-58035 (S-58035; ACAT inhibi o ) (10 μM; Sigma) o
ehicle (0.03% DMSO) and s ea oyl-CoA desa u ase (SCD1) inhibi o A939572 (1 μM; Toc is) o
ehicle (0.001% DMSO) ea men s we e done by adding hem o cul u e medium.
2.2. MTT and B dU inco po a ion assays
The quan i ica ion o he cellula me abolic a e was pe o med using he “Cell p oli e a ion ki
(MTT)” comme cial ki om Roche ollowing he manu ac u e s’ ins uc ions. Fo B dU
inco po a ion assays he “Cell p oli e a ion ELISA, B dU” comme cial ki om Roche was used.
Fo bo h assays, 104 cells we e seeded in gela in-coa ed 96-well pla es.
5
2.3. Cell ac iona ion
Fo he isola ion o he cy osolic ac ion McA-L and McA-S cells (5-7·106) we e lysed by a eeze-
haw cycle as desc ibed elsewhe e [38]. The supe na an o 20,000 xg (10 min, 4 °C)
cen i uga ion was eco e ed (cy osolic ac ion) and samples we e dilu ed o adjus p o ein
con en o a inal concen a ion o 3-6 mg/ml.
Isola ion o he memb ane ac ion was ca ied ou basically as desc ibed in [39]. B ie ly, McA-L
and McA-S cells (5-7·106) we e ha es ed and passed h ough a 27 gauge needle 30 imes.
Supe na an ob ained by a 7 min 1,000 xg cen i uga ion was cen i uged a 100,000 xg o 30
min in a TLA-55 Beckman o o . The pelle ed memb ane ac ion was esuspended in he
adequa e bu e .
2.4. Quan i ica ion o me aboli es and dehyd ogenase assays
The quan i ica ion o cy osolic ace yl-coenzyme A (CoA) was pe o med using he “Coenzyme A
Colo ime ic/Fluo ome ic Assay” ki om BioVision. Ci a e and uma a e we e quan i ied using
comme cially a ailable ki s om Sigma.
Dehyd ogenase assays we e pe o med as desc ibed in [38]. Kine ic assays we e done using 30-
100 µg o cy osolic p o ein in a 96-well pla e and ac i i ies o all enzymes we e exp essed as
nmol o NADPH p oduced/min/mg o p o ein.
2.5. Lipid ex ac ion and quan i ica ion
Lipids we e exhaus i ely ex ac ed ollowing he me hod desc ibed by Bligh and Dye [40]. Lipid
quan i ica ion was pe o med by hin laye ch oma og aphy and image analysis as desc ibed
elsewhe e [41].
2.6. Radioac i e labeling
Quan i ica ion o adioac i e p ecu so inco po a ion in o lipids was done as desc ibed by
Aspichue a e al. [39] using hepa oma cells cul u ed in 60 mm diame e pla es (3.5·105 cells
seeded). In he ace a e and olea e inco po a ion expe imen s, a e 68 h o cul u e, medium was
changed o se um- ee EMEM medium supplemen ed wi h a ace o [3H]ace a e (20 µCi pe
pla e; Pe kin Elme ) o [3H]olea e (2 µCi pe pla e; Pe kin Elme ). In some cases, se um- ee
medium was addi ionally supplemen ed wi h 0.02 o 0.4 mM oleic acid (OA) (conjuga ed wi h
6
0.025 o 0.5 % bo ine se um albumin, espec i ely) 4 h be o e he inco po a ion assays. When
included, 0.05 and 0.2 mM ace a e we e added o cul u e medium wi h he adioac i e ace. In
he glucose inco po a ion assay a ace o [3H]glucose (20 µCi pe pla e; Pe kin Elme ) was added
a e 68 h o cul u e and in he case o [14C]glyce ol (2 µCi pe pla e; Pe kin Elme ) a e 72 h.
Cells we e incuba ed o 4 h wi h labeled ace a e, olea e and glucose and o 10 min wi h
glyce ol.
2.7. Glucose quan i ica ion and up ake assay
Glucose con en o he cul u e medium was measu ed using a comme cially a ailable enzyma ic
ki (Mena ini). The glucose up ake assay was pe o med in 35 mm diame e pla es (1.2·105 cells
seeded) by adding 10 µCi o [3H]glucose pe pla e. A e 2 minu es, cells we e washed wice wi h
PBS and ha es ed o measu e he o al adioac i i y inco po a ed by liquid scin illa ion.
2.8. P o ein measu emen and wes e n blo ing
P o ein concen a ion was de e mined by he bicinchoninic acid me hod (The mo Fishe
Scien i ic) including 2% SDS in all samples o a oid e oneous measu es due o he p esence o
lipid. Fo wes e n blo ing analysis, p o ein ac iona ion by SDS-PAGE, immunode ec ion and
quan i ica ion by image analysis we e pe o med as desc ibed elsewhe e [42]. P ima y
an ibodies and dilu ions we e he ollowing: an i ace yl-CoA ca boxylase (ACC) (Cell Signalling
3662; 1:1000), an i phospho yla ed ACC (P-ACC) (Cell Signalling 3661S; 1:1000), an i SCD1 (Cell
Signalling 2794; 1:1000) and an i glyce aldehyde 3-phospha e dehyd ogenase (Abcam ab8245;
1:20,000).
2.9. Rela i e mRNA quan i ica ion by eal ime RT-PCR
To al RNA was ex ac ed using T izol Reagen (Li e Technologies) and cDNAs we e ob ained by
e o ansc ip ion (Supe Sc ip III RT; Li e Technologies) ollowing he manu ac u e s’
ins uc ions. Quan i ica ion o mRNA by eal ime PCR was pe o med using he SYBR G een
de ec ion sys em (Applied Biosys ems) in an Applied Biosys ems 7000 Real Time PCR Sys em.
Samples we e analyzed in iplica e and Gapdh mRNA was used o no maliza ion.
P ime sequences we e he ollowing:
7
Acaca : gg gcagagg accgaag g, : g cg ag ggccg c gaaag; Acly : gc acggacagagagccacac; :
c c gaaa gcc ggc gac; Agpa 9 : g gaagc cc c ccacc g; : c ccaagg caccagga; Dga 1 :
cc accggga g caa c, : acag g c gggcagcag; Fasn : g ggaca gg cacagacga g, :
g ggaccccaaaaaaggagg; Gapdh : g gccagcc cg c ga agac, : aaggcagccc gg aaccag; Gpam :
ac ggg gac g ggc c, : cg gca gaa agcaacacc; Pnpla2 : c gac cgag cgga gga, :
caaaca ggcc gga aagc c; Scd1 : gcc aa ca cccaagaacc c, : cagcg g cc cc gagc; S eb 1 :
cgcagacgagga ca cca, : cacgaggc gcac ga.
2.10. S a is ics
Da a a e p esen ed as he mean ± s anda d de ia ion (SD). S a is ical signi icance o esul s was
assessed by he unpai ed S uden ’s - es using P ism so wa e (G aphPad).
3. Resul s
3.1. McA-S hepa oma cells ha e low s o ed iglyce ide le els
Seeing ha SND1 o e exp ession in hepa oma cells p omo es an o e ac i a ion o choles e ol
syn hesis [37] we wonde ed whe he his me abolic al e a ion a ec s o he pa hways o lipid
biosyn hesis. Fi s , we cha ac e ized he glyce olipid p o ile o McA-S cells compa ing i wi h
McA-L con ol cells.
Figu es 1A and S1A show ha SND1 o e exp ession is associa ed o low le els o iglyce ide
(TG) and no changes we e obse ed in he summa ion o he phospholipids (PL) de ec ed
(phospha idylcholine, PC; phospha idyle hanolamine, PE; phospha idylinosi ol,
phospha idylse ine and ca diolipin) o in he pe cen ages o he majo PLs, PC and PE (Figu e
1B). To assess he con ibu ion o gene exp ession modi ica ions in main aining low TG le els,
we quan i ied mRNA le els o se e al ele an p o eins in ol ed in he de no o TG biosyn hesis
p ocess (Figu es 2A and 2B). Figu es 2C and S1B show ha mRNA le els o he majo enzymes
o he FA biosyn hesis pa hway a e enhanced in SND1-o e exp essing cells, pa icula ly hose o
Scd1, esponsible o he o ma ion o he double bond o OA. O e exp ession o SCD1 p o ein
was also con i med by wes e n blo ing analysis (Figu e 2E). In addi ion, Agpa and Dga 1, he
main enzymes in ol ed in FA es e i ica ion in o TGs, and TG hyd olase Pnpla2 a e all
o e exp essed (Figu es 2D and S1B), which sugges s ha he abili y o s o e and mobilize TG is
no unbalanced in McA-S cells.
8
Figu e 1. Low iglyce ide (TG) con en o SND1-o e exp essing McA-S2 cells. (A) Lipids
we e ex ac ed and quan i ied by image analysis a e hin laye ch oma og aphy
sepa a ion. TGs and he summa ion o all iden i ied phospholipid (PL) species a e shown.
(B) Pe cen age o PLs co esponding o he majo species, phospha idyle hanolamine (PE)
and phospha idylcholine (PC). Values ep esen he mean ± SD o N ≥ 26 ob ained in 6
independen expe imen s. S uden 's - es : **P ≤ 0.01.
I is well known ha sho - e m egula o s ha a ec enzyme ac i i ies also egula e he
lipogenic lux. One o he majo limi ing me abolic s eps is he ca alyzed by ACC (Acaca gene).
No only i s exp ession bu also i s enzyma ic ac i i y can be egula ed. Ci a e, which is a dono
o cy osolic ace yl-CoA (Figu e 2A), is an allos e ic egula o o ACC ac i i y, and he
phospho yla ion s a e o ACC is especially impo an . Figu e 2E depic s ha al hough ACC
p o ein shows a 2- old o e exp ession in McA-S2 cells, P-ACC/ACC a io is no signi ican ly
al e ed. We also quan i ied he cy osolic ci a e concen a ion and no signi ican di e ences
we e de ec ed be ween McA-S2 and McA-L cells (Figu e 2F). Howe e , he e was a endency o
ci a e le els o be lowe in McA-S2 cells, and so we also quan i ied he cy osolic ace yl-CoA, he
p ecu so o lipogenesis and choles e ogenesis ob ained om ci a e by cy osolic ATP-ci a e
lyase (ACLY). The concen a ion o cy osolic ace yl-CoA in McA-S2 was only sligh ly highe
(S uden ’s es , P = 0.057) han in con ol cells and he le els o cy osolic uma ic acid (cy osolic
dica boxylic acids a e indica i e o ci a e expo om mi ochond ia and me abolizing) we e
highe (Figu e 2F). Al oge he , hese esul s sugges ha McA-S cells ha e he capaci y o
p o ide p ecu so s and syn hesize FAs, and o es e i y hem o glyce olipid p oduc ion.
TG
0
30
60
90
120
**
McA-L McA-S2
nmol/mg p o ein
PL
0
100
200
300
400
500
McA-L McA-S2
nmol/mg p o ein
%PE
0
10
20
30
40
50
McA-L McA-S2
% o o al PL
%PC
0
15
30
45
60
McA-L McA-S2
% o o al PL
9
Figu e 2. The lipogenic p og am is o e exp essed in McA-S2 hepa oma cells. (A) Diag am
wi h he main s eps o he a y acid syn hesis pa hway and impo an p ocesses in he
lipogenic p og am. (B) Diag am o he iglyce ide biosyn he ic pa hway. (C) Rela i e mRNA
le els o lipogenic enzymes analyzed by RT-qPCR. Gapdh exp ession was used o
no maliza ion. (D) Rela i e mRNA le els o iglyce ide biosyn he ic enzymes and
iglyce ide hyd olase Pnpla2 analyzed by RT-qPCR. Gapdh exp ession was used o
no maliza ion. (E) P o ein le el o ace yl-CoA ca boxylase (ACC), phospho yla ed ACC (P-
ACC) and s ea oyl-CoA desa u ase (SCD1) analyzed by wes e n blo ing. P o ein samples
om 3 independen McA-L and McA-S2 cul u e pla es we e used. The g aphs show he
quan i ica ion pe o med by image analysis using he loading con ol, GAPDH, as
no malize . (F) Cy osolic ci a e, ace yl-CoA and uma a e le els we e measu ed using
comme cially a ailable ki s. Values ep esen he mean ± SD o N ≥ 5. S uden 's - es : *P ≤
0.05, **P ≤ 0.01, ***P ≤ 0.001.
A
Acaca Acly Fasn Scd1 S eb 1
0
3
6
9McA-L
McA-S2
**** ***
***
***
mRNA (a bi a y uni s)
C
0
50
100
150
200 Ace yl-CoA
p=0.057
McA-L McA-S2
% o con ol
E
0
50
100
150
200
250 Fuma a e
McA-L McA-S2
***
% o con ol
F
B
Gpam
Agpa
Dga 1
0
1
2
3
****
*
mRNA (a bi a y uni s)
D
Pnpla2
0.0
0.5
1.0
1.5 ***
mRNA (a bi a y uni s)
0
30
60
90
120 Ci a e
McA-L McA-S2
% o con ol
McA-L
McA-S2
ACC
P-ACC
GAPDH
SCD1
GAPDH
McA-L
McA-S2
0
200
400
600 **
McA-L McA-S2
SCD1
0
1
2
McA-L McA-S2
PACC/ACC
16
agg essi eness o he umo s [46,47]. In a p e ious s udy, we showed ha hepa oma cells
o e exp essing SND1 p o ein o e accumula e CEs due o he impai men o he homeos a ic
mechanism based on he ac i a ion o SREBP2 [37]. He e we wan ed o analyze whe he he
accele a ion o CE syn hesis caused by o e exp ession o SND1 in hepa oma cells migh ha e an
e ec on he me abolism o glyce olipids. Since McA-S cells ha e a lowe TG con en han con ol
cells (Figu es 1A and S1A) linked o a lowe inco po a ion o lipogenic subs a es in o TGs
(Figu es 3B, 5C, 5D, 6B and S1C), we wan ed o sea ch o he me abolic ac o s linking CE and
TG anabolic pa hways.
In ecen yea s, many wo ks ha e unde lined he ole ha adap a ions o FA me abolism play in
umo de elopmen . Se e al enzymes o he FA biosyn he ic pa hway such as ACLY, ACC and
a y acid syn hase a e o e exp essed in mos cance ypes, ensu ing an enhanced cy osolic
ace yl-CoA a ailabili y and high FA syn hesis a e [43,48,49]. Thus, low TG biosyn he ic abili y
could be a consequence o some kind o an i umo igenic e ec o SND1 o e exp ession.
Ne e heless, mRNA le el analysis showed ha limi a ion in TG syn hesis in McA-S cells is no
due o modi ica ions in he lipogenic p og am (Figu es 2B, 2D and S1B). The e o e, da a indica e
ha low TG accumula ion capaci y is linked o changes in he me abolic channeling o some
me aboli e in ol ed in TG anabolism. Fi s , o analyze whe he SND1 o e exp ession-induced
excess choles e ogenesis could ha e a di ec consequence on TG syn hesis, we inhibi ed
choles e ol syn hesis bu TG syn hesis was una ec ed (Figu e 3A).
Gi en ha lowe ing choles e ogenesis does no induce TG accumula ion, he a ailabili y o
lipogenic subs a e ace yl-CoA and educing powe seem unlikely o be accoun able o he low
TG syn hesis a e. We con i med his idea by measu emen s o cy osolic ace yl-CoA and NADPH-
gene a ing enzyme ac i i ies (Figu e 2F and Table 1). Addi ionally, cells we e cul u ed in
p esence o me abolically ele an ace a e concen a ions ha should enhance in acellula
ace yl-CoA a ailabili y. This ea men p omo ed mino changes in TG con en in McA-S2 cells
compa ed o con ol cells (Figu e 3B). Acco ding o ou esul s, sca ci y o lipogenic subs a es
can be ejec ed as an explana ion o he low TG syn hesis a e o McA-S cells.
One o he main al e a ions con ibu ing o choles e ol homeos asis impai men in McA-S cells
is he high es e i ica ion a e o choles e ol [37]. In ac , inhibi ion o he ACAT ac i i y using he
compound S-58035 e e s he o e ac i a ion o SREBP2 by ising ee choles e ol le els in he
mic osomal memb anes [37]. The induc ion o choles e ol es e i ica ion migh be a p o ec i e
cell s a egy o espond o he high choles e ogenesis, bu channeling FAs o ha pa hway migh
limi hei a ailabili y o p ocesses like TG syn hesis. A e incuba ion wi h he ACAT ac i i y
inhibi o , a apid inc ease in TG syn hesis a e and he accumula ion o TG in he memb ane
17
ac ion we e obse ed in McA-S2 cells bu no in con ol cells. These esul s place, o he i s
ime in ou knowledge, he es e i ica ion o choles e ol as a limi ing me abolic ac o o TG
accumula ion, p obably linked in o he enhanced choles e ogenic capaci y o McA-S cells in his
case. The ac ha PL le els and hei biosyn he ic a e we e no a ec ed by SND1
o e exp ession o by he ea men s ha modi y me aboli e luxes, s ongly sugges s ha he
egula o y e en s ha channel FAs o CE o TG mus occu in he las s ep o he lipogenic
pa hway, he es e i ica ion o diacylglyce ol. Equaliza ion o TG syn hesis in McA-L and McA-S2
cells by ex acellula OA a ailabili y (Figu e 6B) poin s o apid subs a e channeling
modi ica ions as a po en ial egula o y mechanism.
Due o he po en ial oxici y o ee choles e ol, i s es e i ica ion has o be gua an eed by a
su icien FA supply; hus, a pa allel inc ease o bo h he choles e ogenic capaci y and he
lipogenic p og am makes sense. In he choles e ol es e i ica ion eac ion ACAT ac i i y
p e e en ially uses oleoyl-CoA as he acyl dono [50]. Miyazaki e al. [51] showed ha SCD1
de iciency abolishes CE accumula ion in mouse li e , hus, he choles e ol syn hesis pa hway
and OA p oduc ion seem o be linked. In ou SND1-o e exp essing hepa oma cell model, he
enhanced lipogenic p og am includes he nea ly 5- old inc eased le els o SCD-1 p o ein (Figu e
2E). By inhibi ing SCD1 ac i i y, TG le els d opped in McA-S2 cells bu no in con ol ones
indica ing highe dependence on OA p oduc ion (Figu e 7). The ac ha he simul aneous
inhibi ion o ACAT ac i i y eco e ed a leas pa ially he TG le els in SND1-o e exp essing
hepa oma (Figu e 7) ag ees wi h he idea o channeling o newly syn hesized OA o choles e ol
es e i ica ion as a limi ing ac o o TG syn hesis.
Figu e 8 summa izes he p ocesses o lipid me abolism ha cha ac e ize he me abolic luxes in
SND1-o e exp essing hepa oma. This wo k shows a me abolic link be ween wo al e a ions
dis inc i e o cance de elopmen and agg essi eness, i.e. CE accumula ion and al e a ion o FA
me abolism. Using s ably ans ec ed hepa oma cells, we ha e shown ha o e exp ession o
SND1, a pu a i e oncop o ein ha induces choles e ogenesis and CE accumula ion, induced a
educ ion o he use o FA o TG syn hesis bu no a educ ion o FA syn hesis. The lipogenic
p ocess leading o oleoyl-CoA syn hesis seems o be linked o choles e ol syn hesis. These
esul s emphasize he pa hophysiological impo ance o SND1 in lipid homeos asis and
ep esen a no el disco e y in he link be ween choles e ol and acylglyce ol me abolism in
cance cells and he e o e a new ield o po en ial he apeu ic in e es .
18
Figu e 8. P oposed model o he glyce olipid me abolism al e a ion induced by SND1
o e exp ession. (A) SND1 induces choles e ogenesis and o e exp ession o s ea oyl-CoA
desa u ase-1 (SCD1), which p oduces oleic acid o choles e ol es e i ica ion. Al oge he ,
ha educes a y acid a ailabili y o iglyce ide (TG) syn hesis bu no o phospholipid
(PL) syn hesis. (B) When oleic acid is exogenously added, pa o he a y acid pool can be
di e ed o TG syn hesis. CE, choles e yl es e ; DG, diglyce ide.
Funding
This esea ch was suppo ed by he Basque Go e nmen g an IT-971-16 and he Spanish
Minis y o Economy and Compe i i eness g an SAF2015-64352-R. H.N.I. was ecipien o a
esea ch aining ellowship om he Basque Go e nmen .
Re e ences
[1] Po a A, Colonna-Romano S, Callebau I, F anco A, Ma zullo L, Kobayashi GS, e al. An
homologue o he human 100-kDa p o ein (p100) is di e en ially exp essed by His oplasma
capsula um du ing in ec ion o mu ine mac ophages. Biochem Biophys Res Commun 1999 Jan
27;254(3):605-13.
[2] Sami-Subbu R, Choi SB, Wu Y, Wang C, Oki a TW. Iden i ica ion o a cy oskele on-associa ed
120 kDa RNA-binding p o ein in de eloping ice seeds. Plan Mol Biol 2001 May;46(1):79-88.
19
[3] Abe S, Sakai M, Yagi K, Hagino T, Ochi K, Shiba a K, e al. A Tudo p o ein wi h mul iple SNc
domains om pea seedlings: cellula localiza ion, pa ial cha ac e iza ion, sequence analysis,
and phylogene ic ela ionships. J Exp Bo 2003 Ma ;54(384):971-83.
[4] Zhao CT, Shi KH, Su Y, Liang LY, Yan Y, Pos le hwai J, e al. Two a ian s o zeb a ish p100 a e
exp essed du ing emb yogenesis and egula ed by Nodal signaling. FEBS Le 2003 May
22;543(1-3):190-5.
[5] Abe S, Wang PL, Takahashi F, Sasaki E. S uc u al analysis o cDNAs coding o 4SNc-Tudo
domain p o ein om ish and hei exp ession in yellow ail o gans. Ma Bio echnol (NY) 2005
No -Dec;7(6):677-86.
[6] Hossain MJ, Ko de R, Singh S, Mohmmed A, Dasa adhi PV, Chauhan VS, e al. Tudo domain
p o eins in p o ozoan pa asi es and cha ac e iza ion o Plasmodium alcipa um udo
s aphylococcal nuclease. In J Pa asi ol 2008 Ap ;38(5):513-26.
[7] Tong X, D apkin R, Yalamanchili R, Mosialos G, Kie E. The Eps ein-Ba i us nuclea p o ein
2 acidic domain o ms a complex wi h a no el cellula coac i a o ha can in e ac wi h TFIIE.
Mol Cell Biol 1995 Sep;15(9):4735-44.
[8] Le e son JD, Koskinen PJ, O ico FC, Rainio EM, Jalkanen KJ, Dash AB, e al. Pim-1 kinase and
p100 coope a e o enhance c-Myb ac i i y. Mol Cell 1998 Oc ;2(4):417-25.
[9] Yang J, Ai omaki S, Pesu M, Ca e K, Saa inen J, Kalkkinen N, e al. Iden i ica ion o p100 as
a coac i a o o STAT6 ha b idges STAT6 wi h RNA polyme ase II. EMBO J 2002 Sep
16;21(18):4950-8.
[10] Low SH, Vasan h S, La son CH, Mukhe jee S, Sha ma N, Kin e MT, e al. Polycys in-1, STAT6,
and P100 unc ion in a pa hway ha ansduces cilia y mechanosensa ion and is ac i a ed in
polycys ic kidney disease. De Cell 2006 Jan;10(1):57-69.
[11] Paukku K, Kalkkinen N, Sil ennoinen O, Kon ula KK, Leh onen JY. p100 inc eases AT1R
exp ession h ough in e ac ion wi h AT1R 3'-UTR. Nucleic Acids Res 2008 Aug;36(13):4474-87.
[12] Yang J, Valine a T, Hong J, Bu T, Yao Z, Jensen ON, e al. T ansc ip ional co-ac i a o p o ein
p100 in e ac s wi h snRNP p o eins and acili a es he assembly o he spliceosome. Nucleic
Acids Res 2007;35(13):4485-94.
[13] Caudy AA, Ke ing RF, Hammond SM, Denli AM, Ba hoo n AM, Tops BB, e al. A mic ococcal
nuclease homologue in RNAi e ec o complexes. Na u e 2003 Sep 25;425(6956):411-4.
[14] Tsuchiya N, Ochiai M, Nakashima K, Ubagai T, Sugimu a T, Nakagama H. SND1, a componen
o RNA-induced silencing complex, is up- egula ed in human colon cance s and implica ed in
ea ly s age colon ca cinogenesis. Cance Res 2007 Oc 1;67(19):9568-76.
[15] Sunds om JF, Vaculo a A, Sme enko AP, Sa enko EI, Golo ko A, Minina E, e al. Tudo
s aphylococcal nuclease is an e olu iona ily conse ed componen o he p og ammed cell
dea h deg adome. Na Cell Biol 2009 No ;11(11):1347-54.
20
[16] Yoo BK, San hekadu PK, G edle R, Chen D, Emdad L, Bhu ia S, e al. Inc eased RNA-induced
silencing complex (RISC) ac i i y con ibu es o hepa ocellula ca cinoma. Hepa ology 2011
May;53(5):1538-48.
[17] San hekadu PK, Das SK, G edle R, Chen D, S i as a a J, Robe son C, e al. Mul i unc ion
p o ein s aphylococcal nuclease domain con aining 1 (SND1) p omo es umo angiogenesis in
human hepa ocellula ca cinoma h ough no el pa hway ha in ol es nuclea ac o kappaB
and miR-221. J Biol Chem 2012 Ap 20;287(17):13952-8.
[18] San hekadu PK, Akiel M, Emdad L, G edle R, S i as a a J, Rajaseka an D, e al.
S aphylococcal nuclease domain con aining-1 (SND1) p omo es mig a ion and in asion ia
angio ensin II ype 1 ecep o (AT1R) and TGFbe a signaling. FEBS Open Bio 2014 Ap 1;4:353-
61.
[19] A mengol S, A e xe E, Rod iguez L, Ochoa B, Chico Y, Ma inez MJ. NF-kappaB, Sp1 and NF-
Y as ansc ip ional egula o s o human SND1 gene. Biochimie 2013 Ap ;95(4):735-42.
[20] A mengol S, A e xe E, Enzunza L, Mula S, Ochoa B, Chico Y, e al. The p omo e o cell
g ow h- and RNA p o ec ion-associa ed SND1 gene is ac i a ed by endoplasmic e iculum s ess
in human hepa oma cells. BMC Biochem 2014 Dec 11;15:25,014-0025-2.
[21] A mengol S, A e xe E, Enzunza L, Llo en e I, Mendibil U, Na a o-Imaz H, e al. SREBP-2-
d i en ansc ip ional ac i a ion o human SND1 oncogene. Onco a ge 2017 No
21;8(64):108181-94.
[22] Ho J, Kong JW, Choong LY, Loh MC, Toy W, Chong PK, e al. No el b eas cance me as asis-
associa ed p o eins. J P o eome Res 2009 Feb;8(2):583-94.
[23] Yu L, Di Y, Xin L, Ren Y, Liu X, Sun X, e al. SND1 ac s as a no el gene ansc ip ion ac i a o
ecognizing he conse ed Mo i domains o Smad p omo e s, inducing TGFbe a1 esponse and
b eas cance me as asis. Oncogene 2017 Jul 6;36(27):3903-14.
[24] Tsuchiya N, Nakagama H. Mic oRNA, SND1, and al e a ions in ansla ional egula ion in
colon ca cinogenesis. Mu a Res 2010 No 10;693(1-2):94-100.
[25] Wang N, Du X, Zang L, Song N, Yang T, Dong R, e al. P ognos ic impac o Me adhe in-SND1
in e ac ion in colon cance . Mol Biol Rep 2012 Dec;39(12):10497-504.
[26] Ku uma H, Kama a Y, Takahashi H, Iga ashi K, Kimu a T, Miki K, e al. S aphylococcal
nuclease domain-con aining p o ein 1 as a po en ial issue ma ke o p os a e cance . Am J
Pa hol 2009 Jun;174(6):2044-50.
[27] Cappella i M, Bielli P, Pa one o MP, Ciccosan i F, Fimia GM, Saa ike u J, e al. The
ansc ip ional co-ac i a o SND1 is a no el egula o o al e na i e splicing in p os a e cance
cells. Oncogene 2014 Jul 17;33(29):3794-802.
[28] Zag yazhskaya A, Su o a O, Akba NS, Alla ena G, Gyu aszo a K, Zbo o skaya IB, e al. Tudo
s aphylococcal nuclease d i es chemo esis ance o non-small cell lung ca cinoma cells by
egula ing S100A11. Onco a ge 2015 May 20;6(14):12156-73.
21
[29] Xing A, Pan L, Gao J. p100 unc ions as a me as asis ac i a o and is a ge ed by umo
supp essing mic oRNA-320a in lung cance . Tho ac Cance 2018 Jan;9(1):152-8.
[30] Tong L, Wang C, Hu X, Pang B, Yang Z, He Z, e al. Co ela ed o e exp ession o me adhe in
and SND1 in glioma cells. Biol Chem 2016 Jan;397(1):57-65.
[31] Rajaseka an D, Ja iwala N, Mendoza RG, Robe son CL, Akiel MA, Dozmo o M, e al.
S aphylococcal Nuclease and Tudo Domain Con aining 1 (SND1 P o ein) P omo es
Hepa oca cinogenesis by Inhibi ing Monoglyce ide Lipase (MGLL). J Biol Chem 2016 May
13;291(20):10736-46.
[32] He manson DJ, Ma ne LJ. Cannabinoids, endocannabinoids, and cance . Cance
Me as asis Re 2011 Dec;30(3-4):599-612.
[33] Keenan TW, Win e S, Rackwi z HR, Heid HW. Nuclea coac i a o p o ein p100 is p esen
in endoplasmic e iculum and lipid d ople s o milk sec e ing cells. Biochim Biophys Ac a 2000
Sep 1;1523(1):84-90.
[34] Ga cia-A cos I, Rueda Y, Gonzalez-Ko he P, Palacios L, Ochoa B, F esnedo O. Associa ion o
SND1 p o ein o low densi y lipid d ople s in li e s ea osis. J Physiol Biochem 2010
Ma ;66(1):73-83.
[35] Duan Z, Zhao X, Fu X, Su C, Xin L, Saa ike u J, e al. Tudo -SN, a no el coac i a o o
pe oxisome p oli e a o -ac i a ed ecep o gamma p o ein, is essen ial o adipogenesis. J Biol
Chem 2014 Ma 21;289(12):8364-74.
[36] Al Sha i M, Alo P, Vi che a V, Paje a I, Tsako ska I. Modes-o -Ac ion Rela ed o Repea ed
Dose Toxici y: Tissue-Speci ic Biological Roles o PPAR gamma Ligand-Dependen Dys egula ion
in Nonalcoholic Fa y Li e Disease. PPAR Res 2014;2014:432647.
[37] Na a o-Imaz H, Rueda Y, F esnedo O. SND1 o e exp ession de egula es choles e ol
homeos asis in hepa ocellula ca cinoma. Biochim Biophys Ac a 2016 Sep;1861(9 P A):988-96.
[38] B own LJ, Longac e MJ, Hasan NM, Kend ick MA, S oke SW, Macdonald MJ. Ch onic
educ ion o he cy osolic o mi ochond ial NAD(P)-malic enzyme does no a ec insulin
sec e ion in a a insulinoma cell line. J Biol Chem 2009 Dec 18;284(51):35359-67.
[39] Gong Y, Lee JN, Lee PC, Golds ein JL, B own MS, Ye J. S e ol- egula ed ubiqui ina ion and
deg ada ion o Insig-1 c ea es a con e gen mechanism o eedback con ol o choles e ol
syn hesis and up ake. Cell Me ab 2006 Jan;3(1):15-24.
[40] Bligh EG, Dye WJ. A apid me hod o o al lipid ex ac ion and pu i ica ion. Can J Biochem
Physiol 1959 Aug;37(8):911-7.
[41] Ruiz JI, Ochoa B. Quan i ica ion in he subnanomola ange o phospholipids and neu al
lipids by monodimensional hin-laye ch oma og aphy and image analysis. J Lipid Res 1997
Jul;38(7):1482-9.
[42] Palacios L, Ochoa B, Gomez-Lechon MJ, Cas ell JV, F esnedo O. O e exp ession o SND p102,
a a homologue o p100 coac i a o , p omo es he sec e ion o lipop o ein phospholipids in
p ima y hepa ocy es. Biochim Biophys Ac a 2006 Jul;1761(7):698-708.
22
[43] San os CR, Schulze A. Lipid me abolism in cance . FEBS J 2012 Aug;279(15):2610-23.
[44] Rao S, Po e DC, Chen X, He liczek T, Lowe M, Keyoma si K. Lo as a in-media ed G1 a es
is h ough inhibi ion o he p o easome, independen o hyd oxyme hyl glu a yl-CoA educ ase.
P oc Na l Acad Sci U S A 1999 Jul 6;96(14):7797-802.
[45] Clendening JW, Pandy a A, Bou os PC, El Gham asni S, Khos a i F, T en in GA, e al.
Dys egula ion o he me alona e pa hway p omo es ans o ma ion. P oc Na l Acad Sci U S A
2010 Aug 24;107(34):15051-6.
[46] Yue S, Li J, Lee SY, Lee HJ, Shao T, Song B, e al. Choles e yl es e accumula ion induced by
PTEN loss and PI3K/AKT ac i a ion unde lies human p os a e cance agg essi eness. Cell Me ab
2014 Ma 4;19(3):393-406.
[47] de Gonzalo-Cal o D, Lopez-Vila o L, Nasa e L, Pe ez-Olaba ia M, Vazquez T, Escuin D, e
al. In a umo choles e yl es e accumula ion is associa ed wi h human b eas cance
p oli e a ion and agg essi e po en ial: a molecula and clinicopa hological s udy. BMC Cance
2015 Jun 9;15:460,015-1469-5.
[48] Baenke F, Peck B, Miess H, Schulze A. Hooked on a : he ole o lipid syn hesis in cance
me abolism and umou de elopmen . Dis Model Mech 2013 No ;6(6):1353-63.
[49] Belo ibi-Dje a lia S, Vasseu S, Guillaumond F. Lipid me abolic ep og amming in cance
cells. Oncogenesis 2016 Jan 25;5:e189.
[50] Chang TY, Chang CC, Cheng D. Acyl-coenzyme A:choles e ol acyl ans e ase. Annu Re
Biochem 1997;66:613-38.
[51] Miyazaki M, Kim YC, G ay-Kelle MP, A ie AD, N ambi JM. The biosyn hesis o hepa ic
choles e ol es e s and iglyce ides is impai ed in mice wi h a dis up ion o he gene o s ea oyl-
CoA desa u ase 1. J Biol Chem 2000 Sep 29;275(39):30132-8.
23
Supplemen a y ma e ial
Figu e S1. Cha ac e iza ion o McA-S1 cells. (A) Lipid con en s. Lipids we e ex ac ed and quan i ied by
image analysis a e hin laye ch oma og aphy sepa a ion. T iglyce ides (TG), choles e yl es e s (CE) and
he summa ion o all iden i ied phospholipid (PL) species a e shown. Values ep esen he mean ± SD o
N = 7. (B) Rela i e mRNA le els o lipogenic enzymes, iglyce ide biosyn he ic enzymes and iglyce ide
hyd olase Pnpla2 analyzed by RT-qPCR. Gapdh exp ession was used o no maliza ion. P esen ed da a a e
ela i e o cellula p o ein. (C) Inco po a ion o adioac i e ace ic acid (AA) o oleic acid (OA) in o TGs.
Cells cul u ed o 68 h we e ea ed wi h a 20 µCi/pla e ace o [3H]ace a e o wi h 20 µM OA wi h a 2
µCi/pla e ace o [3H]olea e o 4 h. Cells we e ha es ed, lipids ex ac ed and sepa a ed by hin laye
ch oma og aphy and adioac i i y associa ed o TG measu ed by liquid scin illa ion. Values ep esen he
mean ± SD o N = 5. Da a o ganiza ion in g aphs is he same as in Fig. 2. Values ep esen he mean ± SD
o N = 4. S uden 's - es : *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
TG CE PL
0
50
150
250
McA-L
McA-S1
**
***
nmol/mg p o
Acaca Acly Fasn Scd1 S eb 1
0
3
6
9
12
*
*** ***
***
***
mRNA (a bi a y uni s)
Gpam Agpa Dga 1
0
1
2
3
4
***
***
**
mRNA (a bi a y uni s)
Pnpla2
0.0
0.5
1.0
1.5 ***
mRNA (a bi a y uni s)
A Lipid con en
C Inco po a ion o lipogenic
subs a es in o cellula TG
B mRNA
le els
0
5
10
15
20
AA OA
*** ***
% o o al lipids
