Cellular Oncology

https://doi.org/10.1007/s13402-021-00595-z

ORIGINAL PAPER

YAP promotes sorafenib resistance in hepatocellular carcinoma

by upregulating survivin
Ting Sun 1 & Wenhao Mao 1 & Hui Peng 1 & Qi Wang 2 & Lin Jiao 3

Accepted: 4 February 2021

# International Society for Cellular Oncology 2021

Abstract
Background Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but its use is hampered
by secondary drug resistance. Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway, which is
crucial for liver tumorigenesis. As yet, however, the mechanism underlying sorafenib resistance and the role of YAP therein is not
fully understood and needs to be explored further.
Methods Western blotting, flow cytometry and CCK-8 assays were used to assess the role of YAP in HCC sorafenib resistance.
Next, qRT-PCR and Western blotting were performed to identify survivin as a YAP downstream effector, and rescue experi-
ments were performed to confirm that YAP induces sorafenib resistance via survivin. Additionally, Western blotting, flow
cytometry and in vivo xenograft models were used to evaluate the effect of verteporfin in combination with sorafenib on HCC.
Results We found that sorafenib enhances YAP nuclear accumulation and activation, thereby promoting sorafenib resistance
through inhibiting apoptosis in HCC cells. In addition, we found that survivin acts as a downstream mediator of YAP to resist
sorafenib-induced apoptosis. Pharmacological inhibition of YAP by verteporfin increased the sensitivity of HCC cells to soraf-
enib and reversed sorafenib resistance. Moreover, verteporfin in combination with sorafenib significantly suppressed in vivo
HCC tumor growth.
Conclusions Our data indicate that YAP promotes sorafenib resistance through upregulation of survivin expression in HCC cells.
Targeting YAP may be a therapeutic strategy to improve the antitumor effects of sorafenib in HCC.

Keywords Hepatocellular carcinoma . Sorafenib resistance . YAP . Survivin

1 Introduction related to viral infection, alcohol consumption or metabolic

Liver cancer is one of the most frequent causes of cancer-
related death worldwide, with a 5-year survival rate of 18 %
[1]. The liver cancer incidence is rising faster than that for any
other cancer in the United States [2]. Hepatocellular carcinoma
(HCC) comprises 90 % of liver cancer cases [3]. China is one
of the most high-risk regions for HCC [4]. HCC usually de-
velops in patients with underlying chronic liver inflammation
* Ting Sun
sunting@zzu.edu.cn
1
Department of Clinical Laboratory, The First Affiliated Hospital of
Zhengzhou University, Zhengzhou 450052, China
2
Department of Pharmacy, Kaifeng Hospital of Traditional Chinese
Medicine, 475000 Kaifeng, China
3
Department of Laboratory Medicine, West China Hospital, Sichuan
University, Chengdu 610041, China
disorders. Despite efforts that have been made to elucidate the
molecular mechanisms underlying HCC development and pro-
gression, our understanding of this disease is still limited and its
therapeutic options are not satisfactory. Sorafenib, a multi-
kinase inhibitor, is the first-line treatment for advanced HCC
patients [5]. Although this treatment has been shown to im-
prove the overall survival of advanced HCC patients, the re-
sponse rate is not satisfactory, and the development of sorafe-
nib resistance often hampers its long-term use [5–9]. Thus, a
systematic understanding of the molecular mechanism under-
lying sorafenib resistance is of critical importance to improve
its antitumor effect in HCC patients.
The transcriptional co-activator YAP is a crucial down-
stream effector of the Hippo signaling pathway, which plays
an important role in organ size control, tissue homeostasis and
cancer [10–12]. YAP can be regulated through phosphoryla-
tion by the core MTS1/2-LATS1/2 kinase cascade [13].
Mounting evidence suggests that aberrant YAP expression

or activity is involved in cancer initiation and progression [12,
14]. It has been reported that 5–10 % of human HCC cases
show YAP gene amplification as part of a chromosome 11q22
amplicon [15] and that approximately 60 % of human HCC
cases are associated with increased YAP activity [16, 17].
Additionally, it has been shown that YAP is critical for liver
tumorigenesis [18–21]. YAP may also promote resistance to
targeted therapy. Lin et al. reported, for example, that YAP
promotes resistance to RAF and MEK inhibitors in cancer cell
lines harboring BRAF, KRAS or NRAS activating mutations
[22]. In HCC, YAP has additionally been reported to be in-
volved in sorafenib resistance [23–25], although the mecha-
nism underlying this resistance still needs to be clarified.
In this study, we explored the role of YAP in sorafenib
resistance of HCC. We found that YAP contributes to sorafe-
nib resistance by upregulating the expression of survivin.
Targeting YAP may be a strategy to improve the antitumor
effect of sorafenib in HCC.
2 Materials and methods
2.1 Cell culture and reagents
Huh-7, HepG2 and LO2 cells were obtained from the
American Type Culture Collection (ATCC). Sorafenib resis-
tant cell lines (Huh-7R and HepG2R) were obtained from
Shanghai Aolu Biotechnology Co. Ltd. The cell lines were
maintained in Dulbecco’s Modified Eagle Medium (DMEM;
GIBCO BRL) supplemented with 10 % (v/v) FBS, 100 U/ml
penicillin and 100 U/ml streptomycin. Cultures were main-
tained at 37oC in a humidified atmosphere with 5 % CO2.
Sorafenib and Verteporfin were purchased from Selleck
Chemicals. Antibodies directed against PARP, YAP, p-
YAP(S127), survivin, Bcl-xl and Histone H3 were purchased
from Cell Signaling Technology Inc., whereas an antibody
directed against GAPDH was purchased from Santa Cruz
Biotechnology Inc. and an antibody directed against Flag
from Sigma.
2.2 Cell viability assay
Cells were seeded in 96-well plates (4,000 cells/well) and
incubated overnight for attachment. Next, they were treated
with the indicated agents in 10 % FBS-supplemented medium
for 72 hours. This medium was replaced with cell counting
kit-8 (CCK-8; Merck) at 37oC for 2 hours after which absor-
bance was measured at 450 nm.
2.3 Immunofluorescence assay
For immunofluorescence analysis, cells were seeded in cham-
ber slides and subsequently fixed in methanol for 10 min at
Sun et al.
room temperature and permeabilized with 5 % bovine serum
albumin in PBST. Next, the cells were exposed to a primary
anti-YAP antibody 1:200 diluted in PBST containing 5 %
bovine serum albumin overnight at 4oC. After washing three
times with PBS for 10 min, a secondary antibody (Alexa Fluor
488-goat anti-rabbit) 1:200 diluted in PBST was added and
incubated for 1 h at room temperature. Next, the cells were
washed in PBS and mounted using 4,6-diamidino-2-
phenylindole (DAPI) to counterstain DNA. Images were col-
lected using a confocal microscope (Olympus FV-1000).
2.4 Colony formation assay
For colony formation assessment, cells were seeded into 6-
well plates (500 cells per well) and next treated with sorafenib
and verteporfin, alone or in combination. The medium was
replaced with fresh medium containing the respective reagents
every three days. After treatment for 10 days, the medium was
removed and the cell colonies were fixed with 4 % parafor-
maldehyde for 20 minutes and stained with crystal violet
(0.1 % in 20 % methanol) for 30 minutes. Next, they were
washed slowly with running water and air dried. To record
the results, pictures were taken using a digital camera, and the
numbers of cell clones with more than 50 cells were counted
under a microscope.
2.5 Plasmids and transfection
Plasmids encoding human YAP and survivin were cloned into
a pcDNA3.1 vector equipped with a Flag-tag. For transient
expression, plasmids were transfected with lipofetamine 2000
(Invitrogen) for 24 hours after which the cells were processed
with the indicated reagents as described above.
2.6 RNA interference
Target siRNA was produced by GenePharma (Suzhou,
China) and transfected using Lipofectamine RNAiMAX
Transfection Reagent (Invitrogen) according to the manufac-
turer’s protocol. A non-targeting siRNA was used as negative
control. The target sequence used was: survivin (5’-
AAGGAGAUCAACAUUUUCA-3′). For stable YAP
knockdown the following Addgene plasmids were used:
pLKO1-shYAP#1(27,368) and pLKO1-shYAP#2 (27,369).
2.7 Quantitative RT-PCR
Total RNA was extracted using TRIZOL Reagent (Invitrogen)
and subsequently reverse transcribed into cDNA using M-
MLVReverse Transcriptase (Promega). Real-time PCR was
carried out using FastStart Universal SYBR Green Master
(Roche) after which cDNA amplification was measured using
a StepOne RT-PCR System (Applied Biosystems). The
YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin
primers used were: CTGF-forward: 5′AGGAGTGGGTGTGT
GACGA3′ CTGF-reverse: 5′CCAGGCAGTTGGCT
CTAATC3′; CYR61-forward: 5′CCTTGTGGACAGCC
AGTGTA3′ CYR61-reverse: 5′ACTTGGGCCGGTAT
TTCTTC3′; Survivin-forward: 5′GAGGCTGGCTTCAT
CCACTG3′ Survivin-reverse: 5′ATGCTCCTCTATCG
GGTTGTC3′;GAPDH-forward: 5′CTCCTGCACCACCA
ACTGCT3′ GAPDH-reverse: 5′GGGCCATCCACAGT
CTTCTG3′.
2.8 Apoptosis assay
Apoptotic rates were detected by flow cytometry using an
Annexin V-fluorescein isothiocyanate (FITC) apoptosis de-
tection kit (BD Biosciences). Briefly, cells were collected after
different treatments after which the assay was performed ac-
cording to the manufacturer’s instructions. Samples were an-
alyzed immediately using a Cytomics FC500 flow cytometer
(Beckman Coulter).
2.9 Caspase activity assay
Caspase activity was determined using a Caspase-Glo® 3/7 lu-
minescent assay (Promega), according to the manufacturer’s
instructions. After allowing the reagent and 96-well plates con-
taining cells to equilibrate to room temperature, 100 µl Caspase-
Glo® 3/7 Reagent was added to each well of a white-walled 96-
well plate containing 100 µl blank, negative control cells or
treated cells in culture medium. Next, the contents of the
wells was gently mixed using a plate shaker at 300-500 rpm
for 30 seconds and incubated at room temperature for 2 hours.
Luminescence of each sample was measured using a plate-
reading luminometer as directed by the luminometer manufac-
turer. The assays were performed in triplicate, and mean values
were obtained based on the results of 3 independent assays.
2.10 Xenograft tumor assay
For the establishment of a xenograft tumor model, nude mice
(BALB/c nu/nu, 5-week-old females) were injected subcuta-
neously in the dorsal flank with 5 × 10 6 HepG2 cells
suspended in 0.1 ml serum-free medium. When the tumors
reached 100 to 200 mm3, the mice were randomly divided
into four groups receiving (i) vehicle, (ii) sorafenib (50 mg/kg)
orally once daily, (iii) verteporfin (100 mg/kg) intraperitone-
ally every other day or (iv) the combination of sorafenib and
verteporfin, respectively. Tumor volumes were measured ev-
ery 4 days using calipers and their volumes calculated using
the following formula: tumor volume = π/6 (L×W2). Mice
were sacrificed on day 32, after which the tumors were dis-
sected and analyzed. The animal experiments were approved
by the Ethics Committee of the First Affiliated Hospital of
Zhengzhou University.
2.11 Immunohistochemistry (IHC)
Xenograft tumors were fixed in 4 % paraformaldehyde (PFA),
embedded in paraffin, sectioned and stained with hematoxylin
and eosin (H&E). Immunohistochemical staining of the tis-
sues was performed using anti-survivin (CST, 1:100 dilution)
and anti-Ki-67 (Abcam, 1:100 dilution) primary antibodies
and an ABC Elite immunoperoxidase detection kit according
to the manufacturer’s instructions.
2.12 Statistical analysis
The data were analyzed using SPSS 16.0 software. Student’s t
test was used to compare the mean of different groups and
p < 0.05 was considered statistically significant.
3 Results
3.1 Sorafenib induces apoptosis and activates YAP in
HCC cells
Sorafenib has the potential to inhibit tumor growth, progres-
sion, metastasis and angiogenesis [26]. We found that sorafe-
nib induced apoptosis in Huh-7 and HepG2 cells and that
cleaved PARP levels were strongly increased in a dose- and
time- dependent manner (Fig. 1a-d). Previous work indicated
that YAP may play an important role in tumorigenesis by
regulating cell proliferation and apoptosis [10, 27, 28]. Here,
we first examined YAP protein expression in a human hepatic
cell line (LO2) and several commonly used liver cancer cell
lines, and found that YAP was highly expressed in the liver
cancer cell lines (Supplemental Fig. 1). Next, YAP expression
was evaluated after sorafenib treatment in Huh-7 and HepG2
cells. Using Western blotting, no detectable differences in
total YAP protein levels were found, whereas a substantial
decrease in YAP Ser127 phosphorylation after sorafenib treat-
ment was noted (Fig. 1a-d). YAP phosphorylation at Ser127
mediated by the Hippo pathway has been reported to mainly
lead to its cytoplasmic sequestration and degradation [29].
Therefore, we also assessed the localization of YAP by sepa-
rating proteins from the cytoplasm and nucleus and found that
sorafenib increased the nuclear accumulation of YAP (Fig. 1e-
f). To further confirm activation of YAP in the sorafenib treat-
ed cells, we measured YAP target gene expression. We found
that sorafenib significantly increased the transcription of
CTGF and CYR61, two known YAP/TEAD target genes
(Fig. 1g-h).
In addition, we examined the effect of sorafenib on apopto-
sis in sorafenib resistant HCC cells (Huh-7R and HepG2R).
Compared with the primary cells, the cleavage of PARP in-
duced by sorafenib was weak in Huh-7R and HepG2R cells
(Supplemental Fig. 2a-b). After sorafenib treatment, no

Fig. 1 Sorafenib promotes YAP nuclear accumulation and activates YAP
in HCC cells. a, b Huh-7 and HepG2 cells were exposed to the indicated
doses of sorafenib for 24 h. Next, the cells were collected for Western
blotting. c, d Huh-7 and HepG2 cells were treated with 5 µM sorafenib
for the indicated times. Next, the cells were collected for Western
significant difference in YAP protein and YAP Ser127 phos-
phorylation levels were observed (Supplemental Fig. 2a-b).
Compared with Huh-7 and HepG2 cells, more YAP nuclear
localization in Huh-7R and HepG2R cells was noted, but so-
rafenib did not increase the nuclear accumulation of YAP
(Supplemental Fig. 2c-d). Correspondingly, we found that so-
rafenib did not significantly increase the transcription of CTGF
and CYR61 in the sorafenib resistant cells (Supplemental
Fig. 2e-f). These results suggest that YAP had a high basal
activity in the sorafenib resistant cells, which were resistant to
sorafenib-induced apoptosis.
Taken together, these data indicate that sorafenib pro-
motes YAP nuclear accumulation and activation in HCC
cells, and that activated YAP may be involved in sorafenib
resistance.
3.2 YAP attenuates the sensitivity of HCC cells to
sorafenib
Previous work indicates that YAP can regulate apoptosis [30,
31]. To investigate the biological significance of YAP in
sorafenib-induced apoptosis, we knocked down YAP expres-
sion in Huh-7 and HepG2 cells using two independent short
hairpin RNAs (shRNAs) (Supplemental Fig. 3). We found
that sorafenib had no significant effect on YAP protein and
mRNA expression (Fig. 2a, Supplemental Fig. 4a-b), but that
YAP knockdown did promote sorafenib-induced apoptosis, as
indicated by induction of both PARP cleavage and caspase
activity in HCC cells (Fig. 2a-c). LO2 is a normal hepatocyte
Sun et al.
blotting. e, f Cytoplasmic and nuclear proteins were separated, after
which YAP localization was detected by Western blotting. g, h mRNA
expressions of CTGF and CYR61 analyzed by qRT-PCR. The results are
presented as mean ± SEM (n = 3) for each treatment. *p < 0.05,
**p < 0.01, ***p < 0.001
cell line, expressing a low level of endogenous YAP. We
found that exogenous YAP overexpression inhibited
sorafenib-induced apoptosis in LO2 cells (Fig. 2d-e). We also
assessed sorafenib-induced apoptosis by performing flow cy-
tometry using Annexin V-FITC/PI double staining. Apoptotic
cell rates were assessed 48 hours after sorafenib treatment. We
found that YAP knockdown increased about three-fold the
number of apoptotic Huh-7 and HepG2 cells (Fig. 2f-i), while
YAP overexpression reduced the number of apoptotic LO2
cells by 60 % (Fig. 2j-k). Since YAP could suppress
sorafenib-induced apoptosis, we hypothesized that YAP may
promote sorafenib resistance in HCC cells. Using a CCK-8
assay, we found that YAP knockdown enhanced the cytotox-
icity of sorafenib in Huh-7 and HepG2 cells (Supplemental
Fig. 5a-b). Conversely, we found that YAP overexpression
reduced the cytotoxicity of sorafenib in LO2 cells
(Supplemental Fig. 5c). Collectively, these data indicate that
YAP plays a crucial role in mediating the sensitivity of HCC
cells to sorafenib.
3.3 Survivin acts as a downstream mediator of YAP in
sorafenib‐induced apoptosis
It has been reported that YAP can transcriptionally upregulate
the expression of specific anti-apoptotic factors, including
BCL-xL [22, 32] and survivin [33, 34] in certain cell types.
We reasoned that YAP may enhance the expression of anti-
apoptotic factors to promote survival and sorafenib resistance
of HCC cells. Indeed, we found that YAP knockdown resulted
YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin
Fig. 2 YAP attenuates the sensitivity of HCC cells to sorafenib. a-c Huh-
7 and HepG2 cells transfected with shYAP were treated with sorafenib
for 24 h. Next, PARP and YAP expression was analyzed by Western
blotting and caspase activities were determined. d, e LO2 cells
transfected with flag-YAP plasmids were treated with sorafenib for
24 h. Next, PARP, YAP and Flag expression was analyzed by Western
in decreased expression of survivin, both at the protein and
mRNA level, specifically after sorafenib treatment in Huh-7
and HepG2 cells (Fig. 3a-d). Conversely, we found that exog-
enous YAP overexpression increased survivin expression
(Fig. 3e-f). The level of BCL-xL was, however, not markedly
blotting and caspase activities were determined. f- k Huh-7 and HepG2
cells with YAP knockdown and LO2 cells with YAP overexpression
were treated with sorafenib and subsequently subjected to flow cytome-
try. Columns, indicating the total percentage of K2 and K4 cells, represent
the average of three independent experiments. The results are presented as
mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001
affected, indicating a potential link between YAP and survivin
in sorafenib treated HCC cells.
To substantiate the putative functional link between YAP
and survivin, HCC cells were treated with small interfering
RNA (siRNA) to knockdown survivin expression. The data

Fig. 3 YAP promotes the expression of survivin to inhibit sorafenib-
induced apoptosis. a-d Huh-7 and HepG2 cells transfected with shYAP
were treated with sorafenib for 24 h. Next, protein and mRNA expression
were analyzed using Western blotting and qRT-PCR. e, f LO2 cells
transfected with flag-YAP were treated with sorafenib for 24 h. Next,
protein and mRNA expression were analyzed using Western blotting
obtained clearly showed that survivin silencing significantly
increased sorafenib-induced PARP cleavage in Huh-7 and
HepG2 cells (Fig. 3g). Moreover, we found that survivin si-
lencing rescued the inhibitory effect of YAP overexpression
on apoptosis in LO2 cells (Fig. 3h), while survivin overex-
pression reduced sorafenib-induced apoptosis promoted by
YAP knockdown in HepG2 cells (Fig. 3i). Together, these
data support the notion that survivin serves as a downstream
mediator of YAP in sorafenib-induced apoptosis.
3.4 Verteporfin enhances sorafenib cytotoxicity and
reverses sorafenib resistance
Verteporfin, a photosensitizer clinically used in photodynamic
therapy, has been reported to abrogate the interaction between
YAP and TEAD, thereby inhibiting YAP transcriptional ac-
tivity [35]. Here, we hypothesized that verteporfin may en-
hance sorafenib cytotoxicity in HCC cells by inhibiting
YAP. We found that in Huh-7 and HepG2 cells the combina-
tion of verteporfin and sorafenib significantly decreased
survivin expression and enhanced PARP cleavage (Fig. 4a-
Sun et al.
and qRT-PCR. g Huh-7 and HepG2 cells transfected with sisurvivin were
treated with sorafenib for 24 h and analyzed using Western blotting.
h LO2 cells transfected with different combinations of flag-YAP and si-
survivin were treated with sorafenib and analyzed using Western blotting.
i HepG2 cells transfected with different combinations of shYAP and flag-
survivin were treated with sorafenib and analyzed using Western blotting
b). Subsequent cell viability analyses revealed that verteporfin
significantly increased the cytotoxicity of sorafenib in Huh-7
and HepG2 cells (Fig. 4c-d). Furthermore, we found that the
combination of verteporfin and sorafenib resulted in a marked
reduction in colony formation of HCC cells compared with
verteporfin or sorafenib alone (Fig. 4e-f). Verteporfin also
significantly enhanced sorafenib-induced apoptosis in Huh-7
and HepG2 cells (Fig. 4g).
Next, we repeated the above experiments in sorafenib re-
sistant cells. Surprisingly, we found that verteporfin could
reverse sorafenib resistance in Huh-7R and HepG2R cells.
In Huh-7R and HepG2R cells, the combination of verteporfin
and sorafenib reduced survivin expression and enhanced
PARP cleavage, while sorefinib alone had a weak effect
(Supplemental Fig. 6a-b). Verteporfin also significantly in-
creased the cytotoxicity of sorafenib in Huh-7R and
HepG2R cells (Supplemental Fig. 6c-d). Furthermore, we
found that the combination of verteporfin and sorafenib result-
ed in decreased colony formation and increased apoptosis,
while sorefinib alone had no significant effect (Supplemental
Fig. 6e-g).
YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin
Fig. 4 YAP inhibitor verteporfin promotes sorafenib cytotoxicity in HCC
cells. a, b Huh-7 and HepG2 cells were treated with sorafenib in
combination with or without verteporfin for 24 hours. Next, the cells
were analyzed using Western blotting. c, d Huh-7 and HepG2 cells
were treated with different combinations of sorafenib and verteporfin
for 72 h. Next, cell viabilities were measured using a CCK-8 assay. e,
f Huh-7 and HepG2 cells were treated with different combinations of
Together, these findings indicate that verteporfin can in-
crease the sensitivity of HCC cells to sorafenib and reverse
sorafenib resistance.
3.5 Verteporfin enhances the in vivo antitumor
activity of sorafenib
To verify whether the synergistic effect of sorafenib and
verteporfin may have clinical implications, we evaluated the
effect of verteporfin on the antitumor activity of sorafenib
in vivo. To this end, BALB/c (nu/nu) mice were injected sub-
cutaneously with HepG2 cells and divided randomly into 4
groups. Tumor-bearing mice were treated with vehicle, soraf-
enib (50 mg/kg) orally once daily, verteporfin (100 mg/kg)
intraperitoneally every other day or the combination of soraf-
enib and verteporfin. All animals tolerated the treatments well
without observable signs of toxicity, and had stable body
weights throughout the course of the study. We found that
the combination of sorafenib and verteporfin significantly de-
layed tumor growth compared with sorafenib or verteporfin
alone (Fig. 5a-b). Accordingly, immunohistochemical stain-
ing revealed that the expression levels of survivin and Ki-67
were markedly decreased by the combination treatment of
sorafenib and verteporfin (Fig. 5c-e). Thus, sorafenib in com-
bination with verteporfin exhibits an increased antitumor
sorafenib and verteporfin. Next, cells viabilities were analyzed using a
colony formation assay. Columns represent the average of three indepen-
dent experiments. g Huh-7 and HepG2 cells were treated with different
combinations of sorafenib and verteporfin for 48 h. Next, the cells were
analyzed using flow cytometry. Columns represent the average of three
independent experiments. The results are presented as mean ± SEM (n =
3). *p < 0.05, **p < 0.01, ***p < 0.001
activity in vivo and these effects are, at least in part, due to
the inhibition of YAP and survivin (Fig. 6).
4 Discussion
Liver cancer is one of the leading causes of cancer-related
death worldwide and, currently, it has the fastest rising inci-
dence rate of all cancers. Although sorafenib represents the
standard first-line treatment for advanced HCC, sorafenib re-
sistance is a major concern due to a shortage of alternative
systemic treatment options [36]. In this study, we show that
activated YAP in HCC cells correlates with the therapeutic
effect of sorafenib. Survivin was found to be a critical medi-
ator of YAP in sorafenib resistance. Combination with the
YAP inhibitor verteporfin significantly improved the antitu-
mor activity of sorafenib both in vitro and in vivo. These
findings suggest that inhibiting YAP may be a strategy to
improve the efficacy of sorafenib in HCC.
Research over the past decade has revealed a critical role of
the Hippo/YAP pathway in both normal organ development
and cancer. As such, YAP is emerging as an attractive thera-
peutic target for cancer. Previous studies have shown that
YAP contributes to the development of cancer resistance
[37, 38]. However, the direct relationship between YAP and
sorafenib resistance in HCC is currently poorly understood.
 Sun et al.

Fig. 5 Verteporfin enhances the

anti-cancer effect of sorafenib in
HepG2 xenograft models.
a HepG2 cells were subcutane-
ously implanted in nude mice, af-
ter which the mice were treated as
described in Materials and
methods. Tumor growth curves in
each group were determined.
b Tumor weights were measured
after collection. c‐e H&E and im-
munohistochemical staining for
Ki-67 and survivin in xenograft
tumor samples from each group.
Scale bar = 20 µm. The results are
presented as mean ± SEM (n = 6)
for each group. *p < 0.05,
**p < 0.01, ***p < 0.001

Here, we show that YAP promotes sorafenib resistance and
that YAP inhibition enhances the antitumor activity of soraf-
enib. Notably, we identified survivin as a crucial mediator of
YAP in inducing sorafenib resistance in HCC cells. Survivin
is a member of the inhibitor of apoptosis protein family that is
widely overexpressed in human cancers and well known for
its capacity to drive evasion from apoptosis. Thus, our find-
ings not only confirm a regulatory relationship between YAP
and survivin, but also provide evidence that survivin serves as
an executor of YAP in promoting sorafenib resistance.
Targeting YAP has become an exciting yet challenging
goal for cancer therapy. Verteporfin is a small molecule inhib-
itor of the YAP-TEAD interaction, and several studies have
used verteporfin as an effective inhibitor to suppress YAP-
induced tumorigenesis [39–41]. Recently, drugs such as met-
formin and statins have been shown to be able to effectively
YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin
Fig. 6 Diagram depicting YAP-
mediated sorafenib resistance in
HCC. Sorafenib suppresses tumor
growth by inhibiting the RAS/
RAF/MEK/ERK pathway.
Meanwhile, sorafenib activates
YAP, which contributes to soraf-
enib resistance by increasing the
expression of survivin
target upstream pathways and indirectly inhibit YAP activity
[42, 43], indicating that inhibition of YAP may become clin-
ically feasible.
Collectively, our study shows that YAP promotes sorafenib
resistance in HCC cells through downstream regulation of
survivin. Combination of YAP inhibitors with sorafenib may
be a promising therapeutic strategy for advanced HCC.
Abbreviations HCC, Hepatocellular carcinoma; IHC,
Immunohistochemistry; QRT-PCR, quantitative real-time polymerase
chain reaction; siRNA, Small interfering RNA; YAP, Yes-associated
protein
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s13402-021-00595-z.
Author contributions TS designed the study and prepared the manu-
script; WM and HP performed most of the experiments; TS and LJ ana-
lyzed the data and competed the figures; QW revised the manuscript. All
authors read and approved the final manuscript.
Funding This project was supported by the National Natural Science
Foundation of China (No. 81874188) and the Science and Technology
Project of Henan province of China (No. 202102310119).
Data availability The data and materials supporting the conclusion of this
manuscript are included in the article.
Declarations
Ethics approval and consent to participate All experiments were per-
formed in accordance with the standards of the ethics committee of the
First Affiliated Hospital of Zhengzhou University.
Consent for publication Not applicable.
Conflict of interest The authors declare that they have no competing
interests.
References
1. F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A.
Jemal, Global cancer statistics 2018: GLOBOCAN estimates of
incidence and mortality worldwide for 36 cancers in 185 countries.
CA Cancer J. Clin. 68, 394–424 (2018)
2. R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019. CA
Cancer J. Clin. 69, 7–34 (2019)
3. J. Zucman-Rossi, A. Villanueva, J.C. Nault, J.M. Llovet, Genetic
landscape and biomarkers of hepatocellular carcinoma.
Gastroenterology 149, 1226-1239.e1224 (2015)
4. W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A.
Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015. CA Cancer
J. Clin. 66, 115–132 (2016)
5. J.M. Llovet, S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.F.
Blanc, A.C. de Oliveira, A. Santoro, J.L. Raoul, A. Forner, M.
Schwartz, C. Porta, S. Zeuzem, L. Bolondi, T.F. Greten, P.R.
Galle, J.F. Seitz, I. Borbath, D. Haussinger, T. Giannaris, M.
Shan, M. Moscovici, D. Voliotis, J. Bruix, S.I.S. Group,
Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med.
359, 378–390 (2008)
6. M.S. Copur, Sorafenib in advanced hepatocellular carcinoma. N.
Engl. J. Med. 359, 2498; author reply 2498–2499 (2008)
7. J.F. Dufour, The evasive promise of antiangiogenic therapy. J.
Hepatol. 51, 970–972 (2009)
8. M.A. Worns, P.R. Galle, HCC therapies–lessons learned. Nat. Rev.
Gastroenterol. Hepatol. 11, 447–452 (2014)
9. C. Berasain, Hepatocellular carcinoma and sorafenib: too many
resistance mechanisms? Gut 62, 1674–1675 (2013)

10. K.F. Harvey, X. Zhang, D.M. Thomas, The Hippo pathway and
human cancer. Nat. Rev. Cancer 13, 246–257 (2013)
11. J. Huang, S. Wu, J. Barrera, K. Matthews, D. Pan, The Hippo
signaling pathway coordinately regulates cell proliferation and ap-
optosis by inactivating Yorkie, the Drosophila Homolog of YAP.
Cell 122, 421–434 (2005)
12. F.X. Yu, B. Zhao, K.L. Guan, Hippo pathway in organ size control,
tissue homeostasis, and cancer. Cell 163, 811–828 (2015)
13. B. Zhao, K. Tumaneng, K.L. Guan, The Hippo pathway in organ
size control, tissue regeneration and stem cell self-renewal. Nat.
Cell Biol. 13, 877–883 (2011)
14. F. Zanconato, M. Cordenonsi, S. Piccolo, YAP/TAZ at the roots of
cancer. Cancer Cell 29, 783–803 (2016)
15. M. Overholtzer, J. Zhang, G.A. Smolen, B. Muir, W. Li, D.C.
Sgroi, C.X. Deng, J.S. Brugge, D.A. Haber, Transforming proper-
ties of YAP, a candidate oncogene on the chromosome 11q22
amplicon. Proc. Natl. Acad. Sci. U. S. A. 103, 12405–12410 (2006)
16. T. Zhang, J. Zhang, X. You, Q. Liu, Y. Du, Y. Gao, C. Shan, G.
Kong, Y. Wang, X. Yang, L. Ye, X. Zhang, Hepatitis B virus X
protein modulates oncogene Yes-associated protein by CREB to
promote growth of hepatoma cells. Hepatology 56, 2051–2059
(2012)
17. J. Tao, D.F. Calvisi, S. Ranganathan, A. Cigliano, L. Zhou, S.
Singh, L. Jiang, B. Fan, L. Terracciano, S. Armeanu-Ebinger, S.
Ribback, F. Dombrowski, M. Evert, X. Chen, S.P.S. Monga,
Activation of beta-catenin and Yap1 in human hepatoblastoma
and induction of hepatocarcinogenesis in mice. Gastroenterology
147, 690–701 (2014)
18. W. Kim, S.K. Khan, J. Gvozdenovic-Jeremic, Y. Kim, J. Dahlman,
H. Kim, O. Park, T. Ishitani, E.H. Jho, B. Gao, Y. Yang, Hippo
signaling interactions with Wnt/beta-catenin and Notch signaling
repress liver tumorigenesis. J. Clin. Invest. 127, 137–152 (2017)
19. S.M.E. Weiler, F. Pinna, T. Wolf, T. Lutz, A. Geldiyev, C. Sticht,
M. Knaub, S. Thomann, M. Bissinger, S. Wan, S. Rossler, D.
Becker, N. Gretz, H. Lang, F. Bergmann, V. Ustiyan, T.V. Kalin,
S. Singer, J.S. Lee, J.U. Marquardt, P. Schirmacher, V.V.
Kalinichenko, K. Breuhahn, Induction of chromosome instability
by activation of yes-associated protein and forkhead box M1 in
liver cancer. Gastroenterology 152, 2037-2051.e2022 (2017)
20. W.C. Yuan, B. Pepe-Mooney, G.G. Galli, M.T. Dill, H.T. Huang,
M. Hao, Y. Wang, H. Liang, R.A. Calogero, F.D. Camargo,
NUAK2 is a critical YAP target in liver cancer. Nat. Commun. 9,
4834 (2018)
21. S. Zhang, D. Zhou, Role of the transcriptional coactivators YAP/
TAZ in liver cancer. Curr. Opin. Cell Biol. 61, 64–71 (2019)
22. L. Lin, A.J. Sabnis, E. Chan, V. Olivas, L. Cade, E. Pazarentzos, S.
Asthana, D. Neel, J.J. Yan, X. Lu, L. Pham, M.M. Wang, N.
Karachaliou, M.G. Cao, J.L. Manzano, J.L. Ramirez, J.M. Torres,
F. Buttitta, C.M. Rudin, E.A. Collisson, A. Algazi, E. Robinson, I.
Osman, E. Munoz-Couselo, J. Cortes, D.T. Frederick, Z.A. Cooper,
M. McMahon, A. Marchetti, R. Rosell, K.T. Flaherty, J.A. Wargo,
T.G. Bivona, The Hippo effector YAP promotes resistance to RAF-
and MEK-targeted cancer therapies. Nat. Genet. 47, 250–256
(2015)
23. T.Y. Zhou, L.H. Zhuang, Y. Hu, Y.L. Zhou, W.K. Lin, D.D. Wang,
Z.Q. Wan, L.L. Chang, Y. Chen, M.D. Ying, Z.B. Chen, S. Ye, J.S.
Lou, Q.J. He, H. Zhu, B. Yang, Inactivation of hypoxia-induced
YAP by statins overcomes hypoxic resistance tosorafenib in hepa-
tocellular carcinoma cells. Sci. Rep. 6, 30483 (2016)
24. H. Xia, X. Dai, H. Yu, S. Zhou, Z. Fan, G. Wei, Q. Tang, Q. Gong,
F. Bi, EGFR-PI3K-PDK1 pathway regulates YAP signaling in he-
patocellular carcinoma: the mechanism and its implications in
targeted therapy. Cell Death Dis. 9, 269 (2018)
25. A.M. Gomes, T.S. Pinto, C.J. da Costa Fernandes, R.A. da Silva,
W.F. Zambuzzi, Wortmannin targeting phosphatidylinositol 3-
Sun et al.
kinase suppresses angiogenic factors in shear-stressed endothelial
cells. J. Cell. Physiol. 235, 5256–5269 (2020)
26. F. Pitoia, F. Jerkovich, Selective use of sorafenib in the treatment of
thyroid cancer. Drug Des. Devel. Ther. 10, 1119–1131 (2016)
27. R. Johnson, G. Halder, The two faces of Hippo: targeting the Hippo
pathway for regenerative medicine and cancer treatment. Nat. Rev.
Drug Discov. 13, 63–79 (2014)
28. D. Pan, The hippo signaling pathway in development and cancer.
Dev. Cell 19, 491–505 (2010)
29. Z. Meng, T. Moroishi, K.L. Guan, Mechanisms of Hippo pathway
regulation. Genes Dev. 30, 1–17 (2016)
30. D.D. Shao, W. Xue, E.B. Krall, A. Bhutkar, F. Piccioni, X. Wang,
A.C. Schinzel, S. Sood, J. Rosenbluh, J.W. Kim, Y. Zwang, T.M.
Roberts, D.E. Root, T. Jacks, W.C. Hahn, KRAS and YAP1 con-
verge to regulate EMT and tumor survival. Cell 158, 171–184
(2014)
31. A. Kapoor, W. Yao, H. Ying, S. Hua, A. Liewen, Q. Wang, Y.
Zhong, C.J. Wu, A. Sadanandam, B. Hu, Q. Chang, G.C. Chu, R.
Al-Khalil, S. Jiang, H. Xia, E. Fletcher-Sananikone, C. Lim, G.I.
Horwitz, A. Viale, P. Pettazzoni, N. Sanchez, H. Wang, A.
Protopopov, J. Zhang, T. Heffernan, R.L. Johnson, L. Chin, Y.A.
Wang, G. Draetta, R.A. DePinho, Yap1 activation enables bypass
of oncogenic Kras addiction in pancreatic cancer. Cell 158, 185–
197 (2014)
32. J. Rosenbluh, D. Nijhawan, A.G. Cox, X. Li, J.T. Neal, E.J.
Schafer, T.I. Zack, X. Wang, A. Tsherniak, A.C. Schinzel, D.D.
Shao, S.E. Schumacher, B.A. Weir, F. Vazquez, G.S. Cowley, D.E.
Root, J.P. Mesirov, R. Beroukhim, C.J. Kuo, W. Goessling, W.C.
Hahn, beta-Catenin-driven cancers require a YAP1 transcriptional
complex for survival and tumorigenesis. Cell 151, 1457–1473
(2012)
33. W. Zhang, Y. Gao, F. Li, X. Tong, Y. Ren, X. Han, S. Yao, F.
Long, Z. Yang, H. Fan, L. Zhang, H. Ji, YAP promotes malignant
progression of Lkb1-deficient lung adenocarcinoma through down-
stream regulation of survivin. Cancer Res. 75, 4450–4457 (2015)
34. K. Ma, Q. Xu, S. Wang, W. Zhang, M. Liu, S. Liang, H. Zhu, N.
Xu, Nuclear accumulation of Yes-Associated Protein (YAP) main-
tains the survival of doxorubicin-induced senescent cells by pro-
moting survivin expression. Cancer Lett. 375, 84–91 (2016)
35. Y. Liu-Chittenden, B. Huang, J.S. Shim, Q. Chen, S.J. Lee, R.A.
Anders, J.O. Liu, D. Pan, Genetic and pharmacological disruption
of the TEAD-YAP complex suppresses the oncogenic activity of
YAP. Genes Dev. 26, 1300–1305 (2012)
36. B. Zhai, X.Y. Sun, Mechanisms of resistance to sorafenib and the
corresponding strategies in hepatocellular carcinoma. World J.
Hepatol. 5, 345–352 (2013)
37. C.A. Hall, R. Wang, J. Miao, E. Oliva, X. Shen, T. Wheeler, S.G.
Hilsenbeck, S. Orsulic, S. Goode, Hippo pathway effector Yap is an
ovarian cancer oncogene. Cancer Res. 70, 8517–8525 (2010)
38. J.M. Huang, I. Nagatomo, E. Suzuki, T. Mizuno, T. Kumagai, A.
Berezov, H. Zhang, B. Karlan, M.I. Greene, Q. Wang, YAP mod-
ifies cancer cell sensitivity to EGFR and survivin inhibitors and is
negatively regulated by the non-receptor type protein tyrosine phos-
phatase 14. Oncogene 32, 2220–2229 (2013)
39. A. Perra, M.A. Kowalik, E. Ghiso, G.M. Ledda-Columbano, L. Di
Tommaso, M.M. Angioni, C. Raschioni, E. Testore, M. Roncalli, S.
Giordano, A. Columbano, YAP activation is an early event and a
potential therapeutic target in liver cancer development. J. Hepatol.
61, 1088–1096 (2014)
40. M.E. Garcia-Rendueles, J.C. Ricarte-Filho, B.R. Untch, I. Landa,
J.A. Knauf, F. Voza, V.E. Smith, I. Ganly, B.S. Taylor, Y. Persaud,
G. Oler, Y. Fang, S.C. Jhanwar, A. Viale, A. Heguy, K.H.
Huberman, F. Giancotti, R. Ghossein, J.A. Fagin, NF2 loss pro-
motes oncogenic RAS-induced thyroid cancers via YAP-
dependent transactivation of RAS proteins and sensitizes them to
MEK inhibition. Cancer Discov. 5, 1178–1193 (2015)
YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin

41. E. Ciamporcero, H. Shen, S. Ramakrishnan, S. Yu Ku, S. Chintala,
L. Shen, R. Adelaiye, K.M. Miles, C. Ullio, S. Pizzimenti, M. Daga,
G. Azabdaftari, K. Attwood, C. Johnson, J. Zhang, G. Barrera, R.
Pili, YAP activation protects urothelial cell carcinoma from
treatment-induced DNA damage. Oncogene 35, 1541–1553 (2016)
42. T. Moroishi, C.G. Hansen, K.L. Guan, The emerging roles of YAP
and TAZ in cancer. Nat. Rev. Cancer 15, 73–79 (2015)
43. N. Gronich, G. Rennert, Beyond aspirin-cancer prevention with
statins, metformin and bisphosphonates. Nat. Rev. Clin. Oncol.
10, 625–642 (2013)
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