cance s
A icle
Se um Response Fac o (SRF) D i es he T ansc ip ional
Up egula ion o he MDM4 Oncogene in HCC
Rossella Pelleg ino 1,* , Abhishek Tha amani 2, Diego F. Cal isi 3, Jan Budczies 1, A iane Neumann 1,
Robe Ge e s 4, Jasmin K oeme 1, Dama is G eule 1, Pe e Schi mache 1, Al ed No dheim 2and
Thomas Longe ich 1


Ci a ion: Pelleg ino, R.;
Tha amani, A.; Cal isi, D.F.;
Budczies, J.; Neumann, A.; Ge e s, R.;
K oeme , J.; G eule, D.;
Schi mache , P.; No dheim, A.; e al.
Se um Response Fac o (SRF) D i es
he T ansc ip ional Up egula ion o
he MDM4 Oncogene in HCC.
Cance s 2021,13, 199.
h ps://doi.o g/10.3390/
cance s13020199
Recei ed: 3 Oc obe 2020
Accep ed: 4 Janua y 2021
Published: 8 Janua y 2021
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional clai-
ms in published maps and ins i u io-
nal a ilia ions.
Copy igh : © 2021 by he au ho s. Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Ins i u e o Pa hology, Uni e si y Hospi al Heidelbe g, 69120 Heidelbe g, Ge many;
[email p o ec ed] (J.B.); [email p o ec ed] (A.N.);
[email p o ec ed]g.de (J.K.); [email p o ec ed]g.de (D.G.);
Pe e [email p o ec ed] (P.S.); [email p o ec ed] (T.L.)
2Depa men o Molecula Biology, In e acul y Ins i u e o Cell Biology, Uni e si y o Tuebingen,
72074 Tuebingen, Ge many; [email p o ec ed] (A.T.);
al ed.no [email p o ec ed] (A.N.)
3Ins i u e o Pa hology, Uni e si y Hospi al Regensbu g, 93053 Regensbu g, Ge many;
[email p o ec ed]g.de
4Genome Analy ics, Helmhol z Cen e o In ec ion Resea ch, 38124 B aunschweig, Ge many;
obe .ge [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +49-(0)6221-56-34094
Simple Summa y:
Hepa ocellula ca cinoma (HCC) ep esen s he mos common ype o li e cance
and has a poo p ognosis. The e o e, he e is an u gen need o he iden i ica ion o new he apeu ic
op ions. The mouse double minu e homolog 4 (MDM4) gene, a known p53 inhibi o , is up egula ed
in mos HCCs. He e, we aimed o in es iga e he mechanisms leading o MDM4 ansc ip ional
up egula ion and o e alua e whe he he apeu ic a ge ing o hese mechanisms migh ep esen
a sui able app oach o u u e he apy. Using human HCC cell lines, a mouse model, and human
HCC coho s, we ha e iden i ied se um esponse ac o (SRF), ETS ansc ip ion ac o s ELK1 and
ELK4 as ansc ip ion ac o s (TFs) d i ing MDM4 exp ession. T ea men o HCC cell lines wi h
XI-011, a pha maceu ical inhibi o o MDM4 ansc ip ion, educed he exp ession o bo h he TFs
and MDM4 and impai ed umo g ow h, sugges ing ha a ge ing he MDM4 ansc ip ion may
p o ide a a ionale o u u e a ge ed he apy o HCC.
Abs ac :
Di e en molecula mechanisms suppo he o e exp ession o he mouse double minu e
homolog 4 (MDM4), a unc ional p53 inhibi o , in human hepa ocellula ca cinoma (HCC). Howe e ,
he ansc ip ion ac o s (TFs) leading o i s ansc ip ional up egula ion emain unknown. Following
p omo e and gene exp ession analyses, pu a i e TFs we e in es iga ed using gene-speci ic siRNAs,
cDNAs, luci e ase epo e assays, ch oma in immunop ecipi a ion, and XI-011 d ug ea men
in i o
. Addi ionally, MDM4 exp ession was in es iga ed in SRF-VP16
iHep
ansgenic mice. We
obse ed a copy-numbe -independen up egula ion o MDM4 in human HCCs. Se um esponse
ac o (SRF), ELK1 and ELK4 we e iden i ied as TFs ac i a ing MDM4 ansc ip ion. While SRF
was cons i u i ely de ec ed in TF complexes a he MDM4 p omo e , p esence o ELK1 and ELK4
was cell- ype dependen . Fu he mo e, MDM4 was up egula ed in SRF-VP16-d i en mu ine li e
umo s. The pha macological inhibi o XI-011 exhibi ed an i-MDM4 ac i i y by down egula ing he
TFs d i ing MDM4 ansc ip ion, which dec eased HCC cell iabili y and inc eased apop osis. In
conclusion, SRF d i es ansc ip ional MDM4 up egula ion in HCC, ac ing in conce wi h ei he
ELK1 o ELK4. The ansc ip ional egula ion o MDM4 may be a p omising a ge o p ecision
oncology o human HCC, as XI-011 ea men exe s an i-MDM4 ac i i y independen om he
MDM4 copy numbe and he p53 s a us.
Keywo ds:
HCC; MDM4; ELK1; ELK4; ETS ansc ip ion ac o s; ERK; umo p o ein p53; SRF;
MDM4 ansc ip ional egula ion; XI-011
Cance s 2021,13, 199. h ps://doi.o g/10.3390/cance s13020199 h ps://www.mdpi.com/jou nal/cance s
Cance s 2021,13, 199 2 o 17
1. In oduc ion
Li e cance is he i h mos common ype o cance in he wo ld and he second
mos equen cause o cance - ela ed dea h, wi h limi ed he apeu ic op ions o pa ien s
wi h ad anced s ages o he disease [
1
,
2
]. Hepa ocellula ca cinoma (HCC) accoun s o
he majo i y (80%) o li e cance cases, and he unde lying e iological ac o s a e well
known [
1
]. Du ing he las ew yea s, he molecula landscape o human HCC has been
comp ehensi ely cha ac e ized a genomic, ansc ip omic, epigenomic, and p o eomic
le els [
3
–
7
]. The umo supp esso p53 ep esen s he second mos equen ly mu a ed gene
in HCC. I s mu a ion equency shows a s ong geog aphical a ia ion, which pa allels
wi h he exposu e o a la oxin B1 and he p e alence o ch onic HBV in ec ion. In endemic
egions, such as sub-Saha an A ica o Eas Asia, a mu a ion a e o up o 50% has been
epo ed [
8
], while he p53 mu a ion equency is much lowe in wes e n coun ies, anging
om <10% o 25% [
5
,
9
,
10
]. P53 is egula ed by a ne wo k o in e ac ing ac o s, mos p omi-
nen ly by genes o he mouse double minu e (MDM) amily [
11
]. Bo h MDM2 and MDM4
a e inhibi o s o p53. They a e able o bind o he N- e minal ansac i a ion domain o p53.
While MDM4 is a po en inhibi o o p53 ansc ip ional ac i i y [
12
,
13
], MDM2 mainly
unc ions as a nega i e eedback egula o o p53 signaling. T ansc ip ionally ac i a ed
by p53, MDM2 ac s as an E3 ligase a ge ing p53 o p o easomal deg ada ion [
13
,
14
]; o
no e, he o ma ion o MDM2-MDM4 he e odime complexes is essen ial o p53 polyu-
biqui ina ion, while MDM2 alone ma ks p53 o monoubiqui ina ion and hus does no
p omo e comple e p53 deg ada ion [
15
].
In i o
and
in i o
expe imen s highligh ed
oncogenic p ope ies o bo h MDM amily genes, bu mouse models sugges ed MDM4
o be a mo e po en p53 inhibi o han MDM2 [
16
,
17
]. Dys egula ion o MDM4 has been
de ec ed in di e en cance ypes and a ious mechanisms may p omo e i s up egula-
ion [
18
,
19
]. We p e iously epo ed ecu en ampli ica ion o he MDM4 gene locus in
human HCC [
3
], which was ecen ly alida ed by a la ge in e na ional conso ium [
7
].
Fu he mo e, we p oposed a pos - ansc ip ional mechanism, by which ac i a ion o he
EEF1A2/PI3K/AKT/mTOR signaling axis os e s he p o umo igenic unc ion o MDM4
in human HCC and showed ha he MDM4 p o ein le el is associa ed wi h he su i al
p obabili y o HCC pa ien s ollowing li e esec ion [20].
The obse a ion ha inc eased MDM4 exp ession le els can also be de ec ed in human
HCC samples wi h balanced MDM4 gene locus, led us o hypo hesize ha ansc ip ional
dys egula ion may lead o an up egula ion o MDM4 in hese cases. As he ansc ip ional
egula ion o MDM4 emains la gely elusi e, we sc eened he pu a i e basal MDM4
p omo e o ansc ip ion ac o (TF) binding si es in silico, pe o med alida ion o he
candida e TFs
in i o
, and explo ed whe he ansc ip ional dys egula ion o MDM4 migh
be a d ug a ge o u u e ansla ional s udies. Mo eo e , we used an SRF ansgenic
mouse model ha spon aneously de elops HCC [
21
] o u he alida e ou hypo hesis
in i o
. The da a p esen ed he e highligh he ole o SRF in d i ing MDM4 ansc ip ion,
which equi es in e ac ion wi h ELK1 o ELK4 in a cell con ex -dependen manne .
2. Resul s
2.1. T ansc ip ional Ac i a ion Con ibu es o MDM4 Up egula ion in Human HCC
We ha e p e iously epo ed ha genomic gains occu a he MDM4 gene locus
(1q32.1) and MDM4 mRNA and p o ein le els a e up egula ed in human HCCs compa ed
o no mal li e s (NLs) [
3
,
4
]. Howe e , mic oa ay exp ession p o iling da a om 37 human
HCCs e ealed no signi ican di e ence in MDM4 mRNA le els be ween HCCs wi h
balanced (n= 13) o gained (n= 24) MDM4 gene locus, espec i ely (balanced:
2.3 ±0.4
s.
gained: 3.7
±
0.4, p> 0.05; Figu e 1A), sugges ing ha abe an ansc ip ional ac i a ion
may con ibu e o MDM4 o e exp ession in HCC. Pu a i e TFs in ol ed in he egula ion
o MDM4 ansc ip ion we e iden i ied by an in silico analysis o he MDM4 p omo e
egion using he MAPPER [
22
] and Jaspa [
23
] da abases. As shown in Figu e 1B, a high
a ini y SRF binding si e (Jaspa sco e: 10.1), as well as binding si es o ELK1 and ELK4
(Jaspa sco e: 9.8 and 8.6, espec i ely), we e de ec ed in he 5
0
-UTR o he MDM4 gene
Cance s 2021,13, 199 3 o 17
p edic ed o con ain he basal p omo e . O no e, he sequence iden i ied as an ELK1
binding si e (CCGGAAG) di e ed om he complemen a y e e se ELK4 ecogni ion
sequence (TTTCCGG) by only one nucleo ide (G); he e o e, his sequence was conside ed
a pu a i e ELK1/ELK4 binding si e. ELK1 and ELK4 a e ETS amily p o eins o he e na y
complex ac o (TCF) sub amily, which o m e na y complexes wi h DNA-bound SRF [
24
].
Cance s 2021, 13, x 3 o 17
may con ibu e o MDM4 o e exp ession in HCC. Pu a i e TFs in ol ed in he egula ion
o MDM4 ansc ip ion we e iden i ied by an in silico analysis o he MDM4 p omo e
egion using he MAPPER [22] and Jaspa [23] da abases. As shown in Figu e 1B, a high
a ini y SRF binding si e (Jaspa sco e: 10.1), as well as binding si es o ELK1 and ELK4
(Jaspa sco e: 9.8 and 8.6, espec i ely), we e de ec ed in he 5′-UTR o he MDM4 gene
p edic ed o con ain he basal p omo e . O no e, he sequence iden i ied as an ELK1 bind-
ing si e (CCGGAAG) di e ed om he complemen a y e e se ELK4 ecogni ion se-
quence (TTTCCGG) by only one nucleo ide (G); he e o e, his sequence was conside ed
a pu a i e ELK1/ELK4 binding si e. ELK1 and ELK4 a e ETS amily p o eins o he e na y
complex ac o (TCF) sub amily, which o m e na y complexes wi h DNA-bound SRF
[24].
Figu e 1. Abe an ansc ip ional ac i a ion may be in ol ed in up egula ion o mouse double minu e homolog 4
(MDM4) in human HCC. (A) Rela i e MDM4 mRNA exp ession in human hepa ocellula ca cinomas (HCCs) wi h bal-
anced (n = 13) and gained MDM4 (n = 24) gene loci, espec i ely. Mann–Whi ney U es : p > 0.05. (B) An in silico analysis
o he basal MDM4 p omo e egion iden i ied pu a i e ansc ip ion ac o binding si es o se um esponse ac o (SRF),
ELK1, and ELK4. (C) Exp ession p o iling e ealed a posi i e associa ion be ween MDM4 mRNA and he exp ession le el
o he pu a i e ansc ip ion ac o s SRF, ELK1, and ELK4 in human HCC samples (n = 37). (D) MDM4, SRF, and ELK4
mRNA le els we e associa ed wi h he su i al p obabili y o HCC pa ien s in a second coho (n = 32). Each median
exp ession le el was used o s a i ica ion. Abb e ia ion: n.s., no s a is ically signi ican .
Gene exp ession p o iling e ealed a signi ican posi i e associa ion be ween he
mRNA exp ession o all h ee pu a i e TFs and MDM4 in a human HCC coho (n = 37;
Figu e 1C). These indings we e co obo a ed in a second se ies o human HCC (n = 32;
(Figu e S1), which e ealed signi ican ly inc eased MDM4, SRF, ELK1, and ELK4 mRNA
Figu e 1.
Abe an ansc ip ional ac i a ion may be in ol ed in up egula ion o mouse double minu e homolog 4 (MDM4)
in human HCC. (
A
) Rela i e MDM4 mRNA exp ession in human hepa ocellula ca cinomas (HCCs) wi h balanced (n= 13)
and gained MDM4 (n= 24) gene loci, espec i ely. Mann–Whi ney U es : p> 0.05. (
B
) An in silico analysis o he basal
MDM4 p omo e egion iden i ied pu a i e ansc ip ion ac o binding si es o se um esponse ac o (SRF), ELK1, and
ELK4. (
C
) Exp ession p o iling e ealed a posi i e associa ion be ween MDM4 mRNA and he exp ession le el o he
pu a i e ansc ip ion ac o s SRF,ELK1, and ELK4 in human HCC samples (n= 37). (
D
)MDM4,SRF, and ELK4 mRNA
le els we e associa ed wi h he su i al p obabili y o HCC pa ien s in a second coho (n= 32). Each median exp ession
le el was used o s a i ica ion. Abb e ia ion: n.s., no s a is ically signi ican .
Gene exp ession p o iling e ealed a signi ican posi i e associa ion be ween he
mRNA exp ession o all h ee pu a i e TFs and MDM4 in a human HCC coho (
n= 37
;
Figu e 1C). These indings we e co obo a ed in a second se ies o human HCC (n= 32;
(Figu e S1), which e ealed signi ican ly inc eased MDM4,SRF,ELK1, and ELK4 mRNA
le els in human HCCs compa ed o pai ed su ounding non-neoplas ic li e issues (SL)
(Figu e S2A–D). Fu he mo e, su i al analysis o he la e coho showed ha high
mRNA le els o MDM4,SRF, and ELK4 we e associa ed wi h a lowe su i al p obabili y
o HCC pa ien s ollowing li e esec ion (p= 0.0003, p= 0.0386, and p= 0.0151, espec i ely;
Figu e 1D), while ELK1 exp ession le els we e o no p ognos ic alue (p> 0.05; pe sonal
obse a ion, 2020). Addi ionally, su i al was analyzed using he TCGA da a se (LIHC)
Cance s 2021,13, 199 4 o 17
ollowing s a i ica ion o pa ien s based on he p53 gene s a us [
7
]. As shown in Figu e
S2E, he o e all su i al o pa ien s whose HCC showed a wild- ype p53 gene sequence
was lowe , bu no signi ican ly di e en when compa ed o p53-mu a ed cases. In addi ion,
u he s a i ica ion o HCC cases ega ding he gene exp ession le el o MDM4,SRF,
and ELK4 did no e eal signi ican di e ences in e ms o su i al p obabili y be ween
he indi idual g oups (Figu e S2F). No ably, he posi i e associa ion o he MDM4 mRNA
le el wi h he gene exp ession o SRF and ELK4 could be again alida ed in he la ge LIHC
coho (p< 0.001, pe sonal obse a ion, 2020).
2.2. SRF, ELK1, and ELK4 Regula e MDM4 Exp ession in HCC Cell Lines
As SRF is conside ed he cen al media o o he immedia e cellula se um esponse [
25
],
e al cal se um (FCS) was used o s imula e HCC cells. Indeed, bo h HepG2 and HLE cells
ha had been s a ed o e nigh showed signi ican ly highe MDM4 mRNA and p o ein
le els upon s imula ion wi h FCS compa ed o he nons imula ed con ol cells (Figu e 2A
and Figu e S3A). This e ec co ela ed wi h an inc eased phospho yla ion o ex acellula
signal- ela ed kinase 1/2 (ERK 1/2), indica ing an ac i e se um esponse (Figu e S3A).
To u he co obo a e ou indings, MDM4 gene exp ession was assessed in se um-
s a ed HepG2 cells s imula ed wi h FCS in combina ion wi h he PI3K inhibi o LY294002
o he ERK inhibi o LY3214996, espec i ely. The e icacy o PI3K and ERK inhibi ion was
con i med by de ec ion o dec eased AKT and ERK phospho yla ion in ea ed cells compa ed
o con ols, espec i ely (Figu e S3C,F). As expec ed, he inhibi ion o he indi idual pa hways
p e en ed FCS-induced up egula ion o MDM4 exp ession (Figu e S3B–E). Also, knockdown
o SRF exp ession by wo di e en gene-speci ic siRNAs (siSRF_1/2) signi ican ly educed
MDM4 mRNA and p o ein le els in HepG2 and HLE cells compa ed o ans ec ion o a
sc ambled, nonsense siRNA (siNS) (Figu e 2B,C). Simila esul s we e ob ained when ELK1
o ELK4 we e a ge ed by gene-speci ic siRNAs in HCC cell lines (Figu e 2D–G).
To alida e he essen ial ole o SRF in he up egula ion o MDM4 ollowing FCS
s imula ion, MDM4 p o ein le els we e analyzed upon FCS s imula ion in o e nigh
se um-s a ed cells p e iously ans ec ed wi h an SRF-speci ic siRNA and compa ed o
siNS- ans ec ed cells. As o ERK and PI3K pa hway inhibi ion, FCS could no induce
MDM4 exp ession in SRF-deple ed HCC cells, while con ol cells we e s ill FCS esponsi e
(Figu e 2H). Fu he mo e, MDM4 mRNA and p o ein le els we e signi ican ly inc eased
in HuH7 cells, which showed low basal SRF le els (pe sonal obse a ion, 2020) when
ansien ly ans ec ed wi h an SRF-VP16 plasmid, which encodes o a ull-leng h SRF
cDNA used o he ansc ip ional ac i a ion domain o he he pes simplex i us p o ein
VP16 (Figu e 2I). SRF-VP16 is able o bind o SRF a ge si es in p omo e egions and
ac i a e ansc ip ion wi hou addi ional co- ac o s ha a e physiologically equi ed o
SRF-media ed ansc ip ional ac i a ion (see below) [26].
Cance s 2021,13, 199 5 o 17
Cance s 2021, 13, x 5 o 17
Figu e 2. SRF, ELK1, and ELK4 egula e MDM4 exp ession in HCC cell lines. (A) Inc eased MDM4 mRNA le els in HepG2
and HLE cell lines ollowing e al cal se um (FCS) s imula ion compa ed o s a ed con ol cells. (B) MDM4 mRNA and
(C) p o ein le els ollowing siRNA-media ed knockdown o SRF compa ed o con ol cells ans ec ed wi h a sc ambled,
nonsense siRNA (siNS) in HepG2 and HLE cells. (D) MDM4 mRNA and (E) p o ein le els ollowing siRNA-media ed
knockdown o ELK1 compa ed o con ol cells ans ec ed wi h a sc ambled, nonsense siRNA (siNS) in HepG2 and HLE
cells. (F) MDM4 mRNA and (G) p o ein le els ollowing siRNA-media ed knockdown o ELK4 compa ed o con ol cells
ans ec ed wi h a sc ambled, nonsense siRNA (siNS) in HepG2 and HLE cells. (H) siRNA-media ed knockdown o SRF
(siSRF_2) p e en s FCS-s imula ed MDM4 p o ein up egula ion. (I) MDM4 mRNA and p o ein exp ession 48 h ollowing
ans ec ion o HuH7 cells wi h an SRF-VP16 exp ession ec o compa ed o mock ans ec ed con ol cells. O iginal wes -
e n blo s a e shown in Figu es S7 and S8. Da a a e p esen ed as mean ± SEM. Mann–Whi ney U es : * p < 0.05, ** p < 0.01,
*** p < 0.001. Abb e ia ions: siNS—sc ambled, nonsense siRNA; siSRF_1/_2, siELK1_1/_2, siELK4_1/_2—siRNA 1 and 2
speci ically a ge ing SRF, ELK1, and ELK4, espec i ely.
Figu e 2.
SRF, ELK1, and ELK4 egula e MDM4 exp ession in HCC cell lines. (
A
) Inc eased MDM4 mRNA le els in HepG2
and HLE cell lines ollowing e al cal se um (FCS) s imula ion compa ed o s a ed con ol cells. (
B
) MDM4 mRNA and
(
C
) p o ein le els ollowing siRNA-media ed knockdown o SRF compa ed o con ol cells ans ec ed wi h a sc ambled,
nonsense siRNA (siNS) in HepG2 and HLE cells. (
D
) MDM4 mRNA and (
E
) p o ein le els ollowing siRNA-media ed
knockdown o ELK1 compa ed o con ol cells ans ec ed wi h a sc ambled, nonsense siRNA (siNS) in HepG2 and HLE
cells. (
F
) MDM4 mRNA and (
G
) p o ein le els ollowing siRNA-media ed knockdown o ELK4 compa ed o con ol cells
ans ec ed wi h a sc ambled, nonsense siRNA (siNS) in HepG2 and HLE cells. (
H
) siRNA-media ed knockdown o SRF
(siSRF_2) p e en s FCS-s imula ed MDM4 p o ein up egula ion. (
I
) MDM4 mRNA and p o ein exp ession 48 h ollowing
ans ec ion o HuH7 cells wi h an SRF-VP16 exp ession ec o compa ed o mock ans ec ed con ol cells. O iginal wes e n
blo s a e shown in Figu es S7 and S8. Da a a e p esen ed as mean
±
SEM. Mann–Whi ney U es : * p< 0.05, ** p< 0.01, ***
p< 0.001. Abb e ia ions: siNS—sc ambled, nonsense siRNA; siSRF_1/_2, siELK1_1/_2, siELK4_1/_2—siRNA 1 and 2
speci ically a ge ing SRF, ELK1, and ELK4, espec i ely.

Cance s 2021,13, 199 6 o 17
2.3. ELK1 and ELK4 A e Co-Fac o s o SRF-Media ed T ansc ip ional Regula ion o MDM4 in
HCC Cells
To demons a e ha SRF, ELK1, and ELK4 indeed e ec i ely induce he ansc ip ion
o he MDM4 gene, he speci ic siRNAs a ge ing SRF, ELK1, and ELK4, oge he wi h
an MDM4 p omo e cons uc ca ying a Gaussia luci e ase as epo e , we e ansien ly
ans ec ed in HCC cells. E icien deple ion o SRF, ELK1, and ELK4 by gene-speci ic
siRNAs (Figu e S3F) esul ed in a signi ican dec ease o luci e ase ac i i y compa ed o
con ols (Figu e 3A–C).
Cance s 2021, 13, x 6 o 17
2.3. ELK1 and ELK4 A e Co-Fac o s o SRF-Media ed T ansc ip ional Regula ion o MDM4 in
HCC Cells
To demons a e ha SRF, ELK1, and ELK4 indeed e ec i ely induce he ansc ip ion
o he MDM4 gene, he speci ic siRNAs a ge ing SRF, ELK1, and ELK4, oge he wi h an
MDM4 p omo e cons uc ca ying a Gaussia luci e ase as epo e , we e ansien ly
ans ec ed in HCC cells. E icien deple ion o SRF, ELK1, and ELK4 by gene-speci ic siR-
NAs (Figu e S3F) esul ed in a signi ican dec ease o luci e ase ac i i y compa ed o con-
ols (Figu e 3A–C).
Figu e 3. ELK1 and ELK4 a e essen ial co- ac o s o SRF-media ed ansc ip ional egula ion o MDM4 in HCC. Luci e ase
ac i i y o a MDM4 p omo e epo e upon siRNA-media ed knockdown o (A) SRF, (B) ELK1, and (C) ELK4 in HepG2
and HLE cells compa ed o con ols. (D) MDM4 mRNA le els a e co- ans ec ion o an ELK1 cDNA wi h siNS o siSRF.
T ans ec ion e icacy was con i med by de ec ion o ELK1 and SRF mRNA le els. (E) MDM4 mRNA le els ollowing
ans ec ion o he indica ed cDNA plasmids. ELK1 S383A ep esen s an inac i e a ian , which canno be ac i a ed by
phospho yla ion o S383 and is hus unable o ini ia e a ge gene ansc ip ion. T ans ec ion e icacy was con i med by
de ec ion o ELK1 and SRF mRNA le els. Da a a e p esen ed as mean ± SEM. Mann-Whi ney U es : * p < 0.05, ** p < 0.01,
*** p < 0.001. Abb e ia ions: siNS, sc ambled, nonsense; siSRF_1/_2, siELK1_1/_2 siELK4_1/_2, siRNA 1 and 2 speci ically
a ge ing SRF, ELK1 and ELK4, espec i ely; ELK1, ELK1 cDNA; ELK1 S383A, ELK1 S383A cDNA; GLuc, Gaussia luci e -
ase; SEAP, Sec e ed Alkaline Phospha ase; no m., no malized agains con ol.
While ELK1 is capable o ini ia ing a ge gene ansc ip ion on i s own [27], SRF e-
qui es addi ional co ac o s o ac i a e he ansc ip ion o i s a ge genes. These include
ei he membe s o he e na y complex ac o (TCF) amily o ETS domain p o eins (ELK1,
ELK4, NET) o he myoca din- ela ed ansc ip ion ac o (MRTF) amily (MKL1 and
MKL2) [24,28]. Modula ion o MRTF gene exp ession did no a ec MDM4 mRNA ex-
p ession (pe sonal obse a ion, 2020). In con as , ansien ELK1 ans ec ion signi ican ly
inc eased MDM4 and SRF mRNA le els in HLE cells, which we e blocked by p e ious
siRNA-media ed knockdown o SRF exp ession (Figu e 3D). Addi ionally, co- ans ec ion
Figu e 3.
ELK1 and ELK4 a e essen ial co- ac o s o SRF-media ed ansc ip ional egula ion o MDM4 in HCC. Luci e ase
ac i i y o a MDM4 p omo e epo e upon siRNA-media ed knockdown o (
A
) SRF, (
B
) ELK1, and (
C
) ELK4 in HepG2
and HLE cells compa ed o con ols. (
D
)MDM4 mRNA le els a e co- ans ec ion o an ELK1 cDNA wi h siNS o siSRF.
T ans ec ion e icacy was con i med by de ec ion o ELK1 and SRF mRNA le els. (
E
)MDM4 mRNA le els ollowing
ans ec ion o he indica ed cDNA plasmids. ELK1 S383A ep esen s an inac i e a ian , which canno be ac i a ed by
phospho yla ion o S383 and is hus unable o ini ia e a ge gene ansc ip ion. T ans ec ion e icacy was con i med by
de ec ion o ELK1 and SRF mRNA le els. Da a a e p esen ed as mean
±
SEM. Mann-Whi ney U es : * p< 0.05, ** p< 0.01,
*** p< 0.001. Abb e ia ions: siNS, sc ambled, nonsense; siSRF_1/_2, siELK1_1/_2 siELK4_1/_2, siRNA 1 and 2 speci ically
a ge ing SRF, ELK1 and ELK4, espec i ely; ELK1, ELK1 cDNA; ELK1 S383A, ELK1 S383A cDNA; GLuc, Gaussia luci e ase;
SEAP, Sec e ed Alkaline Phospha ase; no m., no malized agains con ol.
While ELK1 is capable o ini ia ing a ge gene ansc ip ion on i s own [
27
], SRF
equi es addi ional co ac o s o ac i a e he ansc ip ion o i s a ge genes. These in-
clude ei he membe s o he e na y complex ac o (TCF) amily o ETS domain p o eins
(ELK1, ELK4, NET) o he myoca din- ela ed ansc ip ion ac o (MRTF) amily (MKL1
and MKL2) [
24
,
28
]. Modula ion o MRTF gene exp ession did no a ec MDM4 mRNA ex-
p ession (pe sonal obse a ion, 2020). In con as , ansien ELK1 ans ec ion signi ican ly
inc eased MDM4 and SRF mRNA le els in HLE cells, which we e blocked by p e ious
Cance s 2021,13, 199 7 o 17
siRNA-media ed knockdown o SRF exp ession (Figu e 3D). Addi ionally, co- ans ec ion
o SRF and an inac i e ELK1 mu an (ELK1 S383A) signi ican ly lowe ed he induc ion
o MDM4 mRNA compa ed o a wild ype ELK1 cDNA in HuH7 cells (Figu e 3E). Taken
oge he , hese da a demons a e ha SRF d i es he up egula ion o MDM4 in HCC cells,
mos likely in combina ion wi h ei he ELK1 o ELK4.
2.4. SRF and TCF Family Membe s Con ol he Ac i i y o he MDM4 P omo e in HCC Cell Lines
To es o he physical in e ac ion be ween he h ee TF candida es and he MDM4
p omo e sequence in HCC cell lines, ChIP expe imen s we e pe o med as ou lined in
Figu e 4A. Speci ic binding o SRF, ELK1, and ELK4 we e de ec ed a hei espec i e TF
binding si es in he MDM4 p omo e in bo h HepG2 and HLE cells (Figu e 4B). Compa ing
he en ichmen o ELK1 and ELK4 a he MDM4 p omo e , ELK1 was mo e en iched in
HLE cells, while ELK4 was he mos p e alen TCF amily membe a he MDM4 p omo e
in HepG2 cells (ELK1: 17.0
±
1.93 (HLE) s. 1.5
±
0.04 (HepG2), p< 0.05; ELK4:
5.1 ±0.92
(HLE) s. 5.6
±
0.27 (HepG2), p> 0.05). The speci ici y o TF binding o hei cogna e
sequences was e i ied using con ol p ime s ups eam (2.1 Kb) and downs eam (1.3 Kb)
om he basal p omo e egion (p< 0.05 o all he TFs analyzed).
Cance s 2021, 13, x 7 o 17
o SRF and an inac i e ELK1 mu an (ELK1 S383A) signi ican ly lowe ed he induc ion o
MDM4 mRNA compa ed o a wild ype ELK1 cDNA in HuH7 cells (Figu e 3E). Taken
oge he , hese da a demons a e ha SRF d i es he up egula ion o MDM4 in HCC cells,
mos likely in combina ion wi h ei he ELK1 o ELK4.
2.4. SRF and TCF Family Membe s Con ol he Ac i i y o he MDM4 P omo e in HCC Cell
Lines
To es o he physical in e ac ion be ween he h ee TF candida es and he MDM4
p omo e sequence in HCC cell lines, ChIP expe imen s we e pe o med as ou lined in
Figu e 4A. Speci ic binding o SRF, ELK1, and ELK4 we e de ec ed a hei espec i e TF
binding si es in he MDM4 p omo e in bo h HepG2 and HLE cells (Figu e 4B). Compa -
ing he en ichmen o ELK1 and ELK4 a he MDM4 p omo e , ELK1 was mo e en iched
in HLE cells, while ELK4 was he mos p e alen TCF amily membe a he MDM4 p o-
mo e in HepG2 cells (ELK1: 17.0 ± 1.93 (HLE) s. 1.5 ± 0.04 (HepG2), p < 0.05; ELK4: 5.1 ±
0.92 (HLE) s. 5.6 ± 0.27 (HepG2), p > 0.05). The speci ici y o TF binding o hei cogna e
sequences was e i ied using con ol p ime s ups eam (2.1 Kb) and downs eam (1.3 Kb)
om he basal p omo e egion (p < 0.05 o all he TFs analyzed).
Figu e 4. MDM4 gene p omo e is ac i a ed by an SRF-ETS amily ansc ip ion ac o complex in HCC cell lines. (A)
Schema ic ep esen a ion o he posi ioning o p ime s used o ChIP analyses o he MDM4 p omo e egion. (B) Speci ic
binding o SRF, ELK1, and ELK4 a hei cogna e binding si es in he MDM4 p omo e compa ed o con ol p ime s loca ed
ei he down- o ups eam o he p edic ed basal MDM4 p omo e as de ec ed by quan i a i e eal- ime PCR o immuno-
p ecipi a ed ch oma in. (C) Rela i e en ichmen o SRF-immunop ecipi a ed DNA in HuH7 cells ans ec ed wi h SRF-
VP16 exp ession plasmid compa ed o mock ans ec ed con ol cells. Da a a e p esen ed as mean ± SEM. Mann–Whi ney
U es : ** p < 0.01, *** p < 0.001. Abb e ia ions: ups . con ol, con ol p ime ampli ying a egion ups eam o he MDM4
p omo e ; downs . con ol, con ol p ime ampli ying a egion downs eam o he MDM4 p omo e .
Figu e 4.
MDM4 gene p omo e is ac i a ed by an SRF-ETS amily ansc ip ion ac o complex in HCC cell lines.
(
A
) Schema ic ep esen a ion o he posi ioning o p ime s used o ChIP analyses o he MDM4 p omo e egion. (
B
)
Speci ic binding o SRF, ELK1, and ELK4 a hei cogna e binding si es in he MDM4 p omo e compa ed o con ol p ime s
loca ed ei he down- o ups eam o he p edic ed basal MDM4 p omo e as de ec ed by quan i a i e eal- ime PCR o
immunop ecipi a ed ch oma in. (
C
) Rela i e en ichmen o SRF-immunop ecipi a ed DNA in HuH7 cells ans ec ed
wi h SRF-VP16 exp ession plasmid compa ed o mock ans ec ed con ol cells. Da a a e p esen ed as mean
±
SEM.
Mann–Whi ney U es : ** p< 0.01, *** p< 0.001. Abb e ia ions: ups . con ol, con ol p ime ampli ying a egion ups eam
o he MDM4 p omo e ; downs . con ol, con ol p ime ampli ying a egion downs eam o he MDM4 p omo e .
Cance s 2021,13, 199 8 o 17
O no e, ELK1 exp ession was lowe in HepG2 compa ed o HLE cells, while bo h cell
lines exp essed simila ELK4 le els, likely explaining he di e en ial pa e n obse ed in
he immunop ecipi a ion expe imen s (Figu e S4). Fu he mo e, he binding o SRF o he
MDM4 co e p omo e was alida ed by o e exp ession o SRF-VP16, which esul ed in a
signi ican en ichmen o SRF a he MDM4 p omo e in HuH7 cells compa ed o con ol
cells (Figu e 4C). To u he in es iga e he ele ance o SRF o MDM4 gene exp ession
in i o
, SRF-VP16 ansgenic mice (SRF-VP16
iHep
), which exp ess a cons i u i ely ac i e
SRF-VP16 usion p o ein in hepa ocy es, we e analyzed. These mice de elop HCC ia
a p emalignan nodula s age [
21
]. Immunohis ochemis y e ealed up egula ion o he
MDM4 p o ein in HCCs o SRF-VP16
iHep
mice compa ed o con ol mice (Figu e 5). In
line wi h he
in i o
da a, inc eased nuclea pELK1 and ELK4 p o ein exp ession we e
de ec ed in SRF-VP16-induced HCCs compa ed o wild ype li e ma es (Figu e 5).
Cance s 2021, 13, x 8 o 17
O no e, ELK1 exp ession was lowe in HepG2 compa ed o HLE cells, while bo h cell
lines exp essed simila ELK4 le els, likely explaining he di e en ial pa e n obse ed in
he immunop ecipi a ion expe imen s (Figu e S4). Fu he mo e, he binding o SRF o he
MDM4 co e p omo e was alida ed by o e exp ession o SRF-VP16, which esul ed in a
signi ican en ichmen o SRF a he MDM4 p omo e in HuH7 cells compa ed o con ol
cells (Figu e 4C). To u he in es iga e he ele ance o SRF o MDM4 gene exp ession
in i o, SRF-VP16 ansgenic mice (SRF-VP16iHep), which exp ess a cons i u i ely ac i e
SRF-VP16 usion p o ein in hepa ocy es, we e analyzed. These mice de elop HCC ia a
p emalignan nodula s age [21]. Immunohis ochemis y e ealed up egula ion o he
MDM4 p o ein in HCCs o SRF-VP16iHep mice compa ed o con ol mice (Figu e 5). In line
wi h he in i o da a, inc eased nuclea pELK1 and ELK4 p o ein exp ession we e de-
ec ed in SRF-VP16-induced HCCs compa ed o wild ype li e ma es (Figu e 5).
Figu e 5. MDM4 is up egula ed in SRF-VP16iHep ansgenic mice. (A) No mal li e pa enchyma in
con ol mice (HE s aining). (B) Well-di e en ia ed HCC in a 30-week-old SRF-VP16iHep mouse
showing abecula disa ay and pseudogland o ma ion. MDM4 immunos aining is nega i e in
con ol mice (C), while a di use, p edominan ly nuclea s aining is seen in SRF-VP16iHep mice (D).
Indi idual hepa ocy e nuclei (a ow) a e posi i e o phospho yla ed-ELK1 in he con ol li e (E),
whe eas he numbe o p-ELK1 posi i e nuclei is signi ican ly inc eased in SRF-VP16iHep mice (F).
Figu e 5.
MDM4 is up egula ed in SRF-VP16
iHep
ansgenic mice. (
A
) No mal li e pa enchyma
in con ol mice (HE s aining). (
B
) Well-di e en ia ed HCC in a 30-week-old SRF-VP16
iHep
mouse
showing abecula disa ay and pseudogland o ma ion. MDM4 immunos aining is nega i e in
con ol mice (
C
), while a di use, p edominan ly nuclea s aining is seen in SRF-VP16
iHep
mice (
D
).
Indi idual hepa ocy e nuclei (a ow) a e posi i e o phospho yla ed-ELK1 in he con ol li e (
E
),
whe eas he numbe o p-ELK1 posi i e nuclei is signi ican ly inc eased in SRF-VP16
iHep
mice (
F
).
The e is no ELK4 immunosignal in con ol mice (
G
). In con as , he SRF-VP16-induced HCC e eals
weak o mode a e nuclea ELK4 s aining (H). Scale ba : 20 µM.
Cance s 2021,13, 199 9 o 17
Fu he mo e, MDM4 mRNA exp ession was signi ican ly up egula ed in neoplas ic
lesions om SRF-VP16
iHep
mice compa ed o hei co esponding con ols (no mal li e
0.13
±
0.07 s. HCC 1.51
±
0.66 (mean
±
SEM), Wilcoxon es p< 0.01), con i ming ha
SRF ac i a es MDM4 ansc ip ion and esul s in an up egula ion o MDM4 in i o.
2.5. XI-011 Inhibi s MDM4 T ansc ip ion by TF Down egula ion
Recen ly, a d ug sc eening s udy showed ha XI-011, a pseudou ea de i a i e, is
a po en p53 ac i a o and educes MDM4 p o ein le els in b eas as well as head and
neck cance cells [
29
]. Simila ly, XI-011 ea men signi ican ly educed MDM4 mRNA
le els in a dose-dependen manne in HepG2 and HLE cells (Figu e 6A). The s onges
e ec was eco ded a concen a ions o 0.5 and 1
µ
M XI-011 in bo h cell lines, espec i ely.
Down egula ion o MDM4 p o ein was consis en ly de ec ed a e 16 h o XI-011 ea men
in hese cell lines (Figu e 6B) and simila esul s we e ob ained a e e y ime poin es ed
(Figu e S5A). O no e, MDM4 mRNA and p o ein le els we e also dec eased ollowing
XI-011 ea men in Hep3B cells wi h dele ed p53 alleles (Figu e S5B,C). In line wi h his,
XI-011 signi ican ly educed MDM4 p omo e ac i i y in HepG2 and HLE cells (Figu e
6C). O no e, MDM2 p o ein le els we e no a ec ed by XI-011 ea men in HCC cell lines
(Figu e S5D). XI-011-media ed educ ion o MDM4 exp ession es o ed he p53 unc ion in
HepG2 cells, as indica ed by he up egula ion o p53 p o ein and induc ion o apop osis
(PARP clea age) (Figu e 6D). Addi ionally, eac i a ion o he p53-media ed ansc ip ion
was con i med by up egula ion o p21 mRNA in HepG2 cells (Figu e 6E). O no e, XI-
011-induced PARP clea age was dec eased ollowing siRNA-media ed MDM4 deple ion
in HepG2 cells compa ed wi h siNS- ans ec ed con ol cells, indica ing ha apop osis
induc ion by XI-011 equi es MDM4 exp ession (Figu e S5E). Al hough HLE cells ha bo a
mu an p53 gene (p.R249S), XI-011 ea men also esul ed in up egula ion o p21 mRNA
and induc ion o PARP clea age in hese cells (Figu e 6D,E). A e inhibi ion o p o ein
biosyn hesis by cycloheximide (CHX) ea men , he hal -li e ime o he wild- ype p53
p o ein was inc eased in HepG2 cells ollowing XI-011 ea men compa ed o dime hylsul-
oxide (DMSO)- ea ed con ol cells (Figu e 6F). In con as , he hal -li e ime o mu an p53
in HLE cells was no a ec ed by XI-011 (Figu e 6F), con i ming ha mu an p53 may escape
om p o easomal deg ada ion induced by MDM2-MDM4 he e odime s, as p e iously
epo ed om o he cance en i ies [
30
]. Howe e , p53 mRNA le els we e no a ec ed by
XI-011 ea men in HepG2 cells, whe eas inc eased p53 gene exp ession was obse ed
in HLE cells ea ed wi h 0.2 and 0.5
µ
M XI-011, which con as s wi h he unal e ed p53
p o ein le els o his cell line (Figu e S5I), sugges ing he possibili y ha he p53 a ian
R249S has no comple ely los i s unc ion o ansc ip ionally ac i a e (some) p53 a ge
genes. Addi ionally, XI-011 ea men signi ican ly dec eased he iabili y o HepG2 and
HLE cells compa ed o DMSO- ea ed con ols in a dose- and ime-dependen manne
(Figu e 6G), simila ly o educed cell g ow h obse ed ollowing MDM4 deple ion
in i o
and
in i o
[
3
,
20
]. O no e, siRNA-media ed p53 inhibi ion in combina ion wi h XI-011
ea men lowe ed he MDM4 gene exp ession and consequen ly educed he iabili y o
HepG2 and HLE cells compa ed o he co esponding con ols, excluding he possibili y
ha he obse ed XI-011-d i en biological e ec s we e media ed by p53 (
Figu e S5F–H
).
Since we obse ed ha XI-011 educed MDM4 ansc ip ion, we hypo hesized ha XI-011
may a ec he machine y d i ing MDM4 ansc ip ion. Indeed, SRF, ELK1, and ELK4
p o ein le els we e signi ican ly educed upon XI-011 ea men (0.5 and 1
µ
M) in bo h
HCC cell lines (Figu e 6H). Thus, XI-011 educed MDM4 exp ession by a ge ing he cen-
al TFs equi ed o ac i a e MDM4 ansc ip ion. Impo an ly, XI-011 did no a ec he
p o ein hal -li e ime o he TFs (Figu e S6A), sugges ing ha he obse ed down egula-
ion o ansc ip ion ac o s was no due o inc eased p o easomal deg ada ion. In line
wi h his, he exp ession o a selec ion o canonical SRF a ge s (VCL1,VIM,BCL2) [
31
]
was educed ollowing XI-011 ea men in HLE cells compa ed o DMSO- ea ed con ol
cells (Figu e S6B), while ACTB mRNA le els we e no signi ican ly a ec ed by he same
ea men (Figu e S6B). Addi ionally, c-MYC exp ession, which was ound up egula ed
Cance s 2021,13, 199 16 o 17
Acknowledgmen s:
The au ho s would like o hank Ve onika Geissle and he Cen e o Model
Sys em and Compa a i e Pa hology (CMCP) a he Ins i u e o Pa hology in Heidelbe g o hei
excellen echnical suppo in pe o ming he immunohis ochemis y s aining and he NCT Tissue
Bank Heidelbe g o suppo wi h issue banking.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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