Combined inhibition of AURKA and HSF1 suppresses proliferation
and promotes apoptosis in hepatocellular carcinoma by activating
endoplasmic reticulum stress
Zetian Shen1,2 & Li Yin1 & Han Zhou2 & Xiaoqin Ji2 & Changchen Jiang2 & Xixu Zhu2 & Xia He1
Received: 26 October 2020 /Accepted: 2 June 2021
# Springer Nature Switzerland AG 2021
Abstract
Purpose In this study we aimed to assess the anti-tumor effect of co-inhibition of Aurora kinase A (AURKA) and heat shock
transcription factor 1 (HSF1) on hepatocellular carcinoma (HCC), as well as to explore the mechanism involved.
Methods Expression of AURKA and HSF1 in primary HCC tissues and cell lines was detected by immunohistochemistry (IHC),
qRT-PCR and Western blotting. AURKA was knocked down in HepG2 and BEL-7402 HCC cells using lentivirus-mediated
RNA interference. Next, CCK-8, clone formation, transwell and flow cytometry assays were used to assess their viability,
migration, invasion and apoptosis, respectively. The expression of proteins related to cell cycle progression, apoptosis and
endoplasmic reticulum stress (ERS) was analyzed using Western blotting. In addition, in vivo tumor growth of HCC cells was
assessed using a nude mouse xenograft model, and the resulting tumors were evaluated using HE staining and IHC.
Results Both AURKA and HSF1 were highly expressed in HCC tissues and cells, while being negatively related to HCC
prognosis. Knockdown of AURKA significantly inhibited the colony forming and migrating capacities of HCC cells. In addition,
we found that treatment with an AURKA inhibitor (Danusertib) led to marked reductions in the proliferation and migration
capacities of the HCC cells, and promoted their apoptosis. Notably, combined inhibition of AURKA and HSF1 induced HCC cell
apoptosis, while increasing the expression of ERS-associated proteins, including p-eIF2α, ATF4 and CHOP. Finally, we found
that co-inhibition of AURKA and HSF1 elicited an excellent in vivo antitumor effect in a HCC mouse model with a relatively low
cytotoxicity.
Conclusions Combined inhibition of AURKA and HSF1 shows an excellent anti-tumor effect on HCC cells in vitro and in vivo,
which may be mediated by ERS. These findings suggest that both AURKA and HSF1 may serve as targets for HCC treatment.
Keywords Aurora kinase A (AURKA) . Heat shock transcription factor 1 (HSF1) . Hepatocellular carcinoma . Apoptosis .
Endoplasmic reticulum stress
1 Introduction
Liver cancer is one of the most common malignancies, rank￾ing sixth in incidence and fourth in mortality worldwide [1].
Hepatocellular carcinoma (HCC) is the main type of liver
cancer, accounting for about 75-85 % of all cases [1]. HCC
exhibits high fatality, high metastasis and high invasion rates
[2]. Although significant progress has been made in recent
years in its treatment, including surgical resection, transplan￾tation and the application of cytotoxic drugs, the prognosis of
HCC patients is still poor [3]. The pathogenesis of HCC is
known to involve multiple signaling pathways, but the molec￾ular mechanisms underlying its malignant progression have
remained unclear. As such, it is of great clinical significance
to identify more effective biomarkers for early diagnosis as
well as novel targets for tailor-made therapies.
Aurora kinase A (AURKA), a cell cycle regulating en￾zyme, starts to accumulate in the S phase of the cell cycle
and rises rapidly in the late G2 and M phases until the G1
phase of the following cycle [4]. During the process of chro￾mosome separation, AURKA participates in the formation of
* Xia He
[email protected]; http://www.jszlyy.com.cn/
1 The Affiliated Cancer Hospital of Nanjing Medical University,
Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, 42
Baiziting, Nanjing, Jiangsu 210009, China
2 Department of Radiation Oncology, Jinling Hospital, Medical
School of Nanjing University, 210002 Nanjing, Jiangsu, China
Cellular Oncology
microtubules and/or the stability of the spindle pole, promotes
maturation of the centrosome, and ensures appropriate mitotic
cell cycle progression [5]. It has been reported that the expres￾sion of AURKA is increased in various cancers, including
breast cancer, colorectal cancer, bladder cancer, head and neck
cancer and HCC, being related to clinical stage, local lymph
node metastasis and distant metastasis [6–11]. AURKA can
accelerate cell cycle progression and promote tumor metasta￾sis by activating several signaling pathways [12]. Meanwhile,
it can promote tumor progression by accelerating epithelial￾mesenchymal transition and regulating tumor angiogenesis
[13, 14]. Given the notion that AURKA may serve as a target
for the treatment of HCC, further studies on the role of
AURKA in HCC and its underlying mechanisms are
warranted.
As an important transcriptional regulator, heat shock tran￾scription factor 1 (HSF1) is essential for normal developmen￾tal processes, while its abnormal expression has been found to
be closely related to the occurrence and severity of malignant
tumors [15]. It has been found that the expression level and
transcriptional activity of HSF1 in HCC are higher than those
in normal liver tissues [16]. HSF1 regulates the expression of
genes related to the growth and development of cancer cells
by mediating the transcription of heat shock proteins [17]. In
the meantime, it can promote drug resistance of HCC cells by
regulating the level of autophagy [18]. Here, we aimed to
investigate the effect of co-inhibition of AURKA and HSF1
on HCC cells in vitro and in vivo and to explore its underlying
mechanism in order to, ultimately, develop a new therapy for
HCC.
2 Materials and methods
2.1 HCC specimens
Three pairs of HCC tissues and adjacent normal tissues were
obtained from the Department of Pathology, Jinling Hospital,
Medical School of Nanjing University. The patients did not
receive any chemotherapy, immunotherapy or radiotherapy
prior to specimen collection. This study was approved by
the Ethics Committee of Jinling Hospital, and written in￾formed consent was obtained from all the patients.
2.2 Cell culture
HepG2 cells and BEL-7402 cells were purchased from the
Cell Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China), and maintained in Roswell Park
Memorial Institute (RPMI)-1640 medium (Gibco, Scotland,
UK) containing 10 % fetal bovine serum (FBS, Sigma, St.
Louis, MO, USA), 100 U/ml penicillin, and 100 µg/ml strep￾tomycin. All cells were cultured at 37 °C with 5 % CO2.
2.3 Transfection assay
A small-interfering RNA (siRNA) targeting AURKA and a
negative control were purchased from RIBOBIO (Suzhou,
China). The target sequence of the AURKA siRNA was 5′-
ATGCCCTGTCTTACTGTCA-3 ′ , while the
siN05815122147 NControl_05815 (standard) from
RIBOBIO served as a siRNA control (Ctrl). HepG2 cells
and BEL-7402 cells were transfected with AURKA-siRNA
or Ctrl-siRNA using Lipofectamine® RNAiMAX Reagents
(Thermo Fisher Scientific) according to the manufacture’s
protocol. After 48 h of transfection, the cells were collected
and subjected to Western blotting.
2.4 Cell viability assay
Cell viability was measured using a Cell Counting Kit-8
(CCK-8) assay following the manufacturer’s instructions.
Cells were seeded into 96-well plates at a density of 5000
cells/well and treated with various concentrations of
Danusertib. After 48 h of culture, 20 µl CCK-8 solution was
added to each well for cell viability detection. Absorbance at
450 nm was measured using a microplate reader.
2.5 Clone formation assay
Cells were seeded into 6-well plates at a density of 500 cells/
well in RPMI-1640 serum-free medium. The growth medium
of each well was carefully replaced with fresh medium every 2
days for two consecutive weeks. Next, the cells were washed
with PBS and stained with crystal violet, after which the
clones in each well were photographed and counted.
2.6 Cell migration and invasion assays
Cells in a logarithmic growth phase were seeded on a
Transwell upper chamber or on a Matrigel-coated Transwell
upper chamber in serum-free medium. The lower chambers
contained DMEM medium with 10 % FBS. After 48 h, non￾migrated/invaded cells were gently removed from the upper
chamber using cotton swabs, after which the migrated/
invaded cells were stained using 0.1 % crystal violet and
counted under a microscope.
2.7 Cell cycle analysis
Cells were seeded in 6-well plates at a density of 4 × 105 cells/
well. After treatment with Danusertib, the cells were washed
with PBS and incubated with RNase A solution (100 µl) for
30 min at 37 °C. Next, 400 µl propidium iodide (PI) was
added to the cells and, after an additional 30 min incubation
at room temperature, the DNA content of the cells was
Shen et al.
measured using flow cytometry (Guava easyCyte HT;
Millipore, USA) to determine cell cycle progression.
2.8 Cell apoptosis assay
Cells in a logarithmic growth phase were washed with PBS
and resuspended at a density of 1 × 106 cells/ml. Next, cell
apoptosis was determined using an eBioscience™ Annexin
V Apoptosis Detection Kit (eBioscience, Thermo Fisher
Scientific, Inc., USA) according to the manufacturer’s proto￾col, in conjunction with flow cytometry.
2.9 qRT-PCR assay
Total RNA was isolated using TRIzol reagent (Invitrogen;
Thermo Fisher Scientific, Inc.) after which cDNA was syn￾thesized using a TransScript One-Step gDNA Removal and
cDNA Synthesis SuperMix (Transgen Biotech, China) fol￾lowing the manufacturer’s protocol. Subsequently, the
cDNAs were amplified using an UltraSYBR Mixture
(CWBIO, China). The following PCR conditions were used:
95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec,
60 °C for 40 sec. The primer sequences used were as follows:
AURKA: forward, 5’-CTGCATTTCAGGACCTGTTA
AGG-3’ and reverse, 5’-AACGCGCTGGGAAGAATTT-3’;
HSF1: forward, 5’-GACCAAGCTGTGGACCCTC-3’ and
reverse, 5’-CACTTTCCGGAAGCCATACAT-3’; GAPDH:
forward, 5’-TGTGGGCATCAATGGATTTGG-3’ and re￾verse, 5’-ACACCATGTATTCCGGGTCAAT‑3’.
2.10 Western blotting
Proteins from the indicated cells were extracted using RIPA
lysis buffer (Beyotime, China), after which protein concentra￾tions were determined using a BCA Protein Assay Kit (cat. no.
P0010; Beyotime, China). Next, the extracted proteins were
separated using SDS-PAGE and transferred onto PVDF mem￾branes (Millipore, Bedford, MA, USA). The resulting mem￾branes were incubated with the following primary antibodies
overnight at 4 °C: anti-AURKA (1:1000, Cell Signaling
Technology, Inc.), anti-HSF1 (1:1000, Cell Signaling
Technology, Inc.), anti-CDC2 (1:1000, Cell Signaling
Technology, Inc.), anti-cyclin B1 (1:1000, Cell Signaling
Technology, Inc.), anti-cleaved caspase-3 (1:1000, Cell
Signaling Technology, Inc.), anti-cleaved PARP (1:1000,
Cell Signaling Technology, Inc.), anti-ATF4 (1:1000, Cell
Signaling Technology, Inc.), anti-p-EIF2α (1:1000, Cell
Signaling Technology, Inc.), anti-EIF2α (1:1000, Cell
Signaling Technology, Inc.) and anti-CHOP (1:1000, Cell
Signaling Technology, Inc.). After being washed with TBST
three times, the membranes were incubated with horseradish
peroxidase (HRP)-conjugated secondary antibodies (1:1000,
A0208 Beyotime, China) for 1 h at room temperature.
GAPDH (Cell Signaling Technology, Inc.) served as a load￾ing control. Optical densities of the bands were measured
using an ECL detection system (Tanon, Shanghai, China),
after which the bands were scanned and analyzed using
Image J software (National Institutes of Health, Bethesda,
USA).
2.11 Mouse xenograft model
Female BALB/c nude mice (6-week-old) were obtained from
Hangzhou Ziyuan Experimental Animal Technology Co.
LTD and housed in well-ventilated rooms under specific
pathogen-free conditions. The animal experiments were ap￾proved by the Ethics Committee of Jinling Hospital (approval
no: 2020JLHGKJDWLS-140).
A HCC xenograft model was generated by subcutaneous
injection of HepG2 cells into the right flanks of the mice. After
12 days, the mice were randomly divided into 4 groups and
treated with KRIBB11, Danusertib or KRIBB11 combined
with Danusertib, respectively. The body weights and tumor
volumes of the mice were recorded every two days. One
month after inoculation, all the mice were sacrificed and the
xenografts were recovered and weighted. In addition, heart,
kidney and liver tissues were harvested. Xenograft tumor vol￾umes were calculated using the formula (V) = 0.5 × l × w2
, in
which w and l represent the short diameter and long diameter,
respectively. Finally, the tissues and xenografts were embed￾ded in paraffin and stained with hematoxylin-eosin (HE) and
for immunohistochemical (IHC) biomarkers.
2.12 HE staining
Paraffin-embedded sections were dewaxed, rehydrated and
stained with hematoxylin. After being rinsed with running
water, the sections were stained with eosin, dehydrated
through an ethanol gradient, cleared with xylene and sealed
with neutral gum. Next, the sections were evaluated by light
microscopy (Nikon, Japan).
2.13 Immunohistochemistry
Paraffin-embedded sections were dewaxed with dimethyl
benzene and rehydrated in dH2O. Next, the slides were im￾mersed in 0.01 M citrate buffer (pH 6.0), heated under high
pressure for 2 min and incubated with the following primary
antibodies overnight at 4 °C: anti-AURKA, anti-HSF1, anti￾cleaved caspase-3, anti-Ki-67 (Cell Signaling Technology,
Inc.) and anti-ATF4. After incubation with the primary anti￾bodies, the slides were subjected to incubation with a second￾ary antibody and DAB staining (Beijing Zhongshan Golden
Bridge Biotechnology Co, Ltd, Beijing, China). Finally, the
slides were counterstained with hematoxylin, dehydrated and
Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular…
sealed in neutral gum. The staining results were evaluated by
light microscopy (Nikon, Japan).
2.14 Statistical analysis
The data are presented as mean ± SD and were analyzed using
SPSS 20.0 software. Group comparisons were performed
using Student’s t-test or one-way ANOVA with Tukey post￾test. Correlation analyses were carried out using Pearson cor￾relation test. P < 0.05 was considered statistically significant.
3 Results
3.1 High AURKA expression is associated with a poor
HCC prognosis
To investigate the correlation between AURKA expression
and HCC prognosis, first 3 pairs of primary HCC tissues
and adjacent normal tissues were tested for AURKA expres￾sion using qRT-PCR, Western blotting and IHC, respectively.
We found that the expression level of AURKA in the HCC
tissues was significantly higher than that in the adjacent nor￾mal counterparts (Fig. 1A-C). Subsequent analysis of data in
the online Gepia database (gepia.cancer-pku.cn/detail.php?)
confirmed that AURKA was highly expressed in HCC tissues
compared with their adjacent normal samples (Fig. 1D). In
addition, we found that the overall survival of HCC patients
with a high AURKA expression was significantly shorter than
that of those with a low AURKA expression (Fig. 1E). These
data indicate that a high AURKA expression is related to a
poor prognosis of HCC patients.
3.2 AURKA promotes the proliferation and
migration/invasion of HCC cells
To test whether AURKA has a pro-oncogenic potential for
HCC, we generated AURKA-knockdown HepG2 and Bel-
7402 cells (Fig. 2A) and subsequently assessed their clone
forming and migrative/invasive capacities. We found that
AURKA knockdown significantly decreased the clone
forming ability of HepG2 and Bel-7402 cells, and markedly
inhibited the migration and invasion of these cells (Fig. 2B￾D). Together, the results suggest that AURKA may play an
important role in regulating HCC cell proliferation and migra￾tion/invasion.
3.3 AURKA inhibition down-regulates proliferation
and migration/invasion and promotes apoptosis of
HCC cells
Next, we investigated the effect of AURKA inhibition on the
biological behavior of HCC cells. For this purpose, HepG2
and Bel-7402 cells were treated with various concentrations of
the AURKA inhibitor Danusertib. We found that Danusertib
effectively inhibited the proliferation of HepG2 and Bel-7402
cells in a concentration-dependent manner (Fig. 3A). In addi￾tion, we found that the clone formation, migration and inva￾sion capacities were significantly reduced in the Danusertib
groups compared with the control groups (Fig. 3B-D), being
consistent with the data obtained above with AURKA￾knockdown HepG2 and BEL-7402 cells. Subsequently, flow
cytometry was used to determine the effects of Danusertib on
the apoptosis of HepG2 and Bel-7402 cells. We found that
HepG2 and Bel-7402 cells treated with Danusertib showed
increased apoptotic rates, while being arrested in the G2/M
phase of the cell cycle (Fig. 4A and C). Additionally, we
found that Danusertib treatment led to a significant decrease
in the expression of CDC2 and cyclin B1, and a marked in￾crease in the expression of cleaved PARP and cleaved
caspase-3 (Fig. 4B, D). Taken together, these data indicate
that inhibition of AURKA attenuates the proliferation and
induces the apoptosis of HCC cells.
3.4 High HSF1 expression is associated with a poor
HCC prognosis and positively correlates with AURKA
expression
Next, we found that HSF1 was highly expressed in HCC
tissues (Fig. 5A-D), and that the overall survival of HCC pa￾tients with a high HSF1 expression was significantly shorter
than that of those with a low HSF1 expression (Fig. 1E).
Subsequent correlation analysis revealed that HSF1 expres￾sion was significantly positively correlated with AURKA ex￾pression in HCC. These observations led us to speculate that
simultaneous inhibition of HSF1 and AURKA may suppress
the development of HCC.
3.5 Co-inhibition of HSF1 and AURKA induces
apoptosis in HCC cells by activating endoplasmic
reticulum stress
To explore the cancer-promoting mechanisms underlying
HSF1 and AURKA, HepG2 and Bel-7402 cells were treated
with the HSF1 inhibitor KRIBB11, Danusertib and KRIBB11
combined with Danusertib, respectively, after which Western
blotting was used to detect the expression of proteins related to
endoplasmic reticulum stress. We found that both ATF4 ex￾pression and EIF2α phosphorylation in HepG2 and Bel-7402
cells treated with KRIBB11 or Danusertib were increased, and
that combination treatment with KRIBB11 and Danusertib led
to marked increases in the expression of ATF4, CHOP and p￾EIF2α (Fig. 6A). These data indicate that co-inhibition of
AURKA and HSF1 activates endoplasmic reticulum stress
in HCC cells. We next generated ATF4-knockdown HepG2
and Bel-7402 cells and treated these cells with or without
Shen et al.
KRIBB11 + Danusertib. We found that ATF4 knockdown
was rescued and led to apoptosis after co-administration of
KRIBB11 and Danusertib (Fig. 6B-C). Likewise, we found
that the increased expression of ATF4 and apoptosis caused
by the co-administration can be rescued by TUDCA, an in￾hibitor of endoplasmic reticulum stress (Fig. 6D-E).
Collectively, these findings suggest that combined inhibition
of AURKA and HSF1 induces apoptosis in HCC cells, pre￾sumably by activating endoplasmic reticulum stress.
3.6 Co-inhibition of HSF1 and AURKA attenuates
in vivo tumorigenesis and tumor growth of HCC cells
Finally, we analyzed the in vivo pro-oncogenic activity of
AURKA and HSF1 in HCC xenografted nude mice co￾Fig. 1 High AURKA expression
is associated with a poor HCC
prognosis. (A)
Immunohistochemical staining
for AURKA in HCC and adjacent
normal tissues. (B) Western blot
and (C) qRT-PCR analysis of
AURKA expression in HCC and
adjacent normal tissues; GAPDH
was used as internal control. (D)
AURKA expression in HCC and
adjacent normal tissues deduced
from online datasets. (E) Kaplan￾Meier analysis of overall survival
of HCC patients with low or high
AURKA expression. *p < 0.05;
**p < 0.01
Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular…
Fig. 2 AURKA promotes the proliferation and migration of HCC cells.
(A) Western blot analysis of AURKA expression in HepG2 and Bel-7402
cells treated with siRNA targeting AURKA or a negative control;
GAPDH was used as internal control. (B) Representative colony forma￾tion images of HepG2 and Bel-7402 cells treated with siRNA targeting
AURKA or a negative control. (C-D) Transwell assays to detect the (C)
migration and (D) invasion of HepG2 and Bel-7402 cells treated with
siRNA targeting AURKA or a negative control. *p < 0.05; **p < 0.01
Shen et al.
Fig. 3 AURKA inhibitor Danusertib inhibits the proliferation and
migration of HCC cells. (A) CCK-8 assay to determine the proliferation
of HepG2 and Bel-7402 cells treated with different concentrations of
Danusertib (0, 0.05, 0.1, 0.25, 0.5, 1 and 2 µM) for 48 h. (B)
Representative colony formation images of HepG2 and Bel-7402 cells
treated with different concentrations of Danusertib (0, 0.5 and 1 µM) for
48 h. (C-D) Transwell assays to detect the migration (C) and invasion (D)
of HepG2 and Bel-7402 cells treated with different concentrations of
Danusertib (0, 0.5 and 1 µM) for 48 h. *p < 0.05; **p < 0.01;
***p < 0.001
Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular…
Shen et al.
administered with KRIBB11 and Danusertib. We found that
there were no significant differences in body weights of the
mice among the four groups (Fig. 7A). Additionally, we found
that the xenografted tumor volumes in the four groups in￾creased in a time-dependent manner, while the tumor growth
of the xenografts in the KRIBB11 + Danusertib group was
significantly slower than that in other three groups (Fig. 7B).
Next, we removed and weighted the xenografted tumors from
each mouse at the end of the experiment. Strikingly, we found
that the xenografts in the KRIBB11 + Danusertib group were
significantly smaller and lighter than those in the control (Ctrl)
group (Fig. 7C). Subsequent HE staining revealed that there
were no significant differences in pathological morphologies
of heart, kidney and liver tissues among the four groups, sug￾gestive of a low drug toxicity in the KRIBB11 + Danusertib
group (Fig. 7D). Besides, immunohistochemical analyses
showed that compared with the Ctrl group, the KRIBB11 +
Danusertib group exhibited a reduced expression of Ki-67, as
well as a markedly increased expression of cleaved caspase-3
and ATF4 (Fig. 7E). Together, these results indicate that co￾inhibition of AURKA and HSF1 attenuates the tumorigenesis
of HCC cells.
4 Discussion
AURKA plays a key role in regulating mitosis, and the activ￾ity of AURKA significantly increases during transition from
the G2 to the M phase of the cell cycle [4]. In addition, it has
been reported that AURKA is closely related to the occurrence
and development of various malignancies, such as endometri￾al cancer, colorectal adenocarcinoma and oral cancer [19–21].
Here, we found that AURKA is highly expressed in primary
HCC tissues and cells, and is significantly related to a poor
prognosis of HCC. In recent years, AURKA has been sug￾gested to serve as an effective target for anti-cancer therapy
due to its important role in cell cycle regulation and the avail￾ability of small molecule inhibitors [22]. Since the discovery
of the first AURKA inhibitor (ZM447439) as a potential drug
for targeted cancer therapy, more than 30 AURKA inhibitors
have been introduced [22]. Here, we evaluated the effect of the
AURKA inhibitor Danusertib on HCC cells, and found that
Danusertib treatment led to a significant dose-dependent inhi￾bition of their proliferation, migration and invasion. In addi￾tion, we found that Danusertib induced G2/M cell cycle arrest
by activating the Cdc2/cyclin B1 pathway and promoted ap￾optosis by increasing the cleavage of PARP and caspase-3.
Overall, we found that Danusertib exhibited marked anti￾tumor effects on HCC cells, suggestive of a potential of
AURKA as a target for HCC treatment.
HSF1 has been reported to be a potential therapeutic target
for liver cancer. It can promote liver cancer cell proliferation,
inhibit apoptosis and suppress anti-tumor immunity, and is
involved in regulating cell cycle progression, cell growth
and colony formation [23]. Here, we found that HSF1 is high￾ly expressed in HCC tissues, and that there is a significant
correlation between HSF1 and AURKA expression.
Additional in vivo studies on co-inhibition of AURKA and
HSF1 revealed that, compared to the control group, a HCC
nude mouse model treated with both Danusertib and
KRIBB11 displayed a marked inhibition in tumor growth with
a relatively low drug-associated cytotoxicity. These results
support a superior therapeutic efficacy of co-administration
of AURKA and HSF1 inhibitors.
In order to additionally investigate the mechanism under￾lying HCC cell apoptosis induced by co-inhibition of
AURKA and HSF1, we chose to analyze endoplasmic reticu￾lum stress (ERS) in HCC cells treated with both AURKA and
HSF1 inhibitors. The occurrence of HCC is closely associated
with chronic inflammation and liver cirrhosis related to liver
disease. These factors can induce ERS through oxidative
stress, inflammation and the occurrence of gene mutations
[24]. Early ERS can alleviate stress-induced cell damage by
activating the unfolded protein response (UPR). It has been
reported that HCC can adapt to various unfavorable environ￾mental conditions by activating the UPR, thereby contributing
to tumor cell survival [24]. Sustained ERS may, however,
trigger apoptosis. Lin et al. [25] found that HepG2 cells may
exhibit a stress response and an increase in apoptosis induced
by tunicamycin, thereby linking ERS to the induction of HCC
cell apoptosis. Jin et al. [26] noted that, while mice fed with
hexavalent chromium showed increases in the expression of
GRP78, ATF6 and CHOP in liver tissues, which subsequently
promoted apoptosis, there was a correlation between the
above effects and the dosage of hexavalent chromium. In ad￾dition, several reports have shown that the CHOP pathway of
ER stress plays an important role in kaempferol-induced apo￾ptosis of HCC cells [27]. Here, we found that co-inhibition of
AURKA and HSF1 in HCC cells led to increased expression
of ERS-related proteins, including ATF4, p-eIF2α and
CHOP. Phosphorylation of eIF2α could theoretically cause
selective translation of ATF4. Moreover, prolonged ERS
may lead to the induction of several pro-apoptotic genes me￾diated by ATF4-CHOP, further promoting apoptosis [28]. The
ƒFig. 4 AURKA inhibitor Danusertib induces HCC cell apoptosis. (A)
Flow cytometric cell cycle detection in HepG2 and Bel-7402 cells treated
with different concentrations of Danusertib (0, 0.5 and 1 µM) for 20 h. (B)
Western blot analysis of CDC2 and Cyclin B1 expression in HepG2 and
Bel-7402 cells treated with different concentrations of Danusertib (0, 0.5
and 1 µM) for 20 h; GAPDH was used as internal control. (C) Flow
cytometric detection of apoptosis in HepG2 and Bel-7402 cells treated
with different concentrations of Danusertib (0, 0.5 and 1 µM) for 24 h.
(D) Western blot analysis of cleaved PARP and cleaved caspase-3 ex￾pression in HepG2 and Bel-7402 cells treated with different concentra￾tions of Danusertib (0, 0.5 and 1 µM) for 24 h; GAPDH was used as
internal control. *p < 0.05; **p < 0.01; ***p < 0.001
Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular…
Fig. 5 High HSF1 expression is associated with a poor HCC prognosis
and positively correlates with AURKA expression. (A)
Immunohistochemical staining for HSF1 in HCC and adjacent normal
tissues. (B) Western blot and (C) qRT-PCR analyses to determine HSF1
expression in HCC and adjacent normal tissues; GAPDH was used as
internal control. (D) HSF1 expression in HCC and adjacent normal tis￾sues deduced from online datasets. (E) Kaplan-Meier analyses of the
overall survival of HCC patients with low or high HSF1 expression. (F)
Correlation between HSF1 and AURKA expression. *p < 0.05;
**p < 0.01
Shen et al.
Fig. 6 HSF1 inhibitor KRIBB11 combined with AURKA inhibitor
Danusertib induces HCC apoptosis by activating endoplasmic reticulum
stress. (A) Western blot analysis of endoplasmic reticulum stress-related
protein expression in inhibitor treated HepG2 and Bel-7402 cells; GAPDH
was used as internal control. (B) Western blot analysis of ATF4 expression in
HepG2 cells, ATF4 knockdown HepG2 cells, Bel-7402 cells and ATF4
knockdown Bel-7402 cells administered with or without KRIBB11 +
Danusertib; GAPDH was used as internal control. (C) Flow cytometry to
determine apoptosis of HepG2 and ATF4 knockdown HepG2 cells, Bel-
7402 cells and ATF4 knockdown Bel-7402 cells treated with or without
KRIBB11 + Danusertib. (D) Western blot analysis of ATF4 expression in
inhibitor treated HepG2 and Bel-7402 cells; GAPDH was used as internal
control. (E) Flow cytometric detection of apoptosis of inhibitor treated
HepG2 and Bel-7402 cells. *p < 0.05; **p < 0.01; ***p < 0.001
Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular…
Shen et al.
above observations led us to propose that co-inhibition of
AURKA and HSF1 may induce HCC cell apoptosis by acti￾vating endoplasmic reticulum stress, thereby inhibiting tumor
progression.
Abbreviations AURKA, aurora kinase A; HSF1, heat shock transcrip￾tion factor 1; HCC, hepatocellular carcinoma; IHC, immunohistochemis￾try; ERS, endoplasmic reticulum stress; FBS, fetal bovine serum; siRNA,
small-interfering RNA; CCK-8, Cell Counting Kit-8; HE, hematoxylin￾eosin; UPR, unfolded protein response
Authors’ contributions ZS and XH conceived and designed this study;
LY, HZ, XJ and CJ performed the experiments and data analyses; ZS and
XZ drafted the manuscript; XH provided critical comments, suggestions
and revised the manuscript. All authors read and approved the final ver￾sion of the manuscript.
Funding This work was supported by grants from the Nanjing Municipal
Science and Technology Committee of Jiangsu Province, China (grant
number: 201803050) and the Jiangsu Post-doctoral Research Funding
Program, China (grant number: 2020Z305).
Data availability The datasets used and/or analyzed during the current
study are available from the corresponding author upon reasonable
request.
Declarations
Conflict of interest The authors declare that they have no competing
interests.
Ethics approval and consent to participate This study was approved by
the Ethics Committee of Jinling Hospital, and written informed consent
was obtained from all participating patients. The animal experiments were
approved by the Ethics Committee of Jinling Hospital (approval no:
2020JLHGKJDWLS-140).
Consent for publication Not applicable.
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Fig. 6 HSF1 inhibitor KRIBB11 combined with AURKA inhibitor
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