Squalene epoxidase drives NAFLD-induced hepatocellular carcinoma and is a pharmaceutical target

Abstract

Nonalcoholic fatty liver disease (NAFLD)–induced hepatocellular carcinoma (HCC) is an emerging malignancy in the developed world; however, mechanisms that contribute to its formation are largely unknown, and targeted therapy is currently not available. Our RNA sequencing analysis of NAFLD-HCC samples revealed squalene epoxidase (SQLE) as the top outlier metabolic gene overexpressed in NAFLD-HCC patients. Hepatocyte-specific Sqle transgenic expression in mice accelerated the development of high-fat, high-cholesterol diet–induced HCC. SQLE exerts its oncogenic effect via its metabolites, cholesteryl ester and nicotinamide adenine dinucleotide phosphate (NADP⁺). Increased SQLE expression promotes the biosynthesis of cholesteryl ester, which induces NAFLD-HCC cell growth. SQLE increased the NADP⁺/NADPH (reduced form of NADP⁺) ratio, which triggered a cascade of events involving oxidative stress–induced DNA methyltransferase 3A (DNMT3A) expression, DNMT3A-mediated epigenetic silencing of PTEN, and activation of AKT-mTOR (mammalian target of rapamycin). In human NAFLD-HCC and HCC, SQLE is overexpressed and its expression is associated with poor patient outcomes. Terbinafine, a U.S. Food and Drug Administration–approved antifungal drug targeting SQLE, markedly inhibited SQLE-induced NAFLD-HCC cell growth in NAFLD-HCC and HCC cells and attenuated tumor development in xenograft models and in Sqle transgenic mice. Suppression of tumor growth by terbinafine is associated with decreased cholesteryl ester concentrations, restoration of PTEN expression, and inhibition of AKT-mTOR, consistent with blockade of SQLE function. Collectively, we established SQLE as an oncogene in NAFLD-HCC and propose that repurposing SQLE inhibitors may be a promising approach for the prevention and treatment of NAFLD-HCC.

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Liu et al., Sci. Transl. Med. 10, eaap9840 (2018) 18 April 2018
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CANCER
Squalene epoxidase drives NAFLD-induced
hepatocellular carcinoma and is a
pharmaceutical target
Dabin Liu,1 Chi Chun Wong,1 Li Fu,1,2 Huarong Chen,1 Liuyang Zhao,1 Chuangen Li,1 Yunfei Zhou,1
Yanquan Zhang,1 Weiqi Xu,1 Yidong Yang,3 Bin Wu,3 Gong Cheng,4 Paul Bo-San Lai,4
Nathalie Wong,5 Joseph J. Y. Sung,1 Jun Yu1*
Nonalcoholic fatty liver disease (NAFLD)–induced hepatocellular carcinoma (HCC) is an emerging malignancy in
the developed world; however, mechanisms that contribute to its formation are largely unknown, and targeted
therapy is currently not available. Our RNA sequencing analysis of NAFLD-HCC samples revealed squalene epoxidase
(SQLE) as the top outlier metabolic gene overexpressed in NAFLD-HCC patients. Hepatocyte-specific Sqle trans-
genic expression in mice accelerated the development of high-fat, high-cholesterol diet–induced HCC. SQLE ex-
erts its oncogenic effect via its metabolites, cholesteryl ester and nicotinamide adenine dinucleotide phosphate
(NADP+). Increased SQLE expression promotes the biosynthesis of cholesteryl ester, which induces NAFLD-HCC cell
growth. SQLE increased the NADP+/NADPH (reduced form of NADP+) ratio, which triggered a cascade of events
involving oxidative stress–induced DNA methyltransferase 3A (DNMT3A) expression, DNMT3A-mediated epigenetic
silencing of PTEN, and activation of AKT-mTOR (mammalian target of rapamycin). In human NAFLD-HCC and HCC,
SQLE is overexpressed and its expression is associated with poor patient outcomes. Terbinafine, a U.S. Food and
Drug Administration–approved antifungal drug targeting SQLE, markedly inhibited SQLE-induced NAFLD-HCC
cell growth in NAFLD-HCC and HCC cells and attenuated tumor development in xenograft models and in Sqle
transgenic mice. Suppression of tumor growth by terbinafine is associated with decreased cholesteryl ester con-
centrations, restoration of PTEN expression, and inhibition of AKT-mTOR, consistent with blockade of SQLE func-
tion. Collectively, we established SQLE as an oncogene in NAFLD-HCC and propose that repurposing SQLE inhibitors
may be a promising approach for the prevention and treatment of NAFLD-HCC.
INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) affects 30 to 40% of the
adult population (1, 2), and its incidence is very high in obese indi-
viduals (75 to 100%) (3, 4). About 2.4 to 12.8% of NAFLD patients
with cirrhosis progress to NAFLD-associated hepatocellular carcinoma
(HCC) (4, 5). Given its prevalence in the general population and the
rising obesity epidemic, NAFLD is an emerging risk factor for HCC
(6). NAFLD is associated with high hepatic triglyceride and cholesterol
content (7). Excess cholesterol is lipotoxic, and it induces nonalcoholic
steatohepatitis, a severe form of NAFLD that strongly predisposes
to HCC development (8). To probe the molecular mechanism of NAFLD-
HCC, we performed RNA sequencing (RNA-seq) analysis of 17 paired
human NAFLD-HCC and adjacent normal tissues, which revealed
squalene epoxidase (SQLE) as an outlier gene markedly up-regulated
in NAFLD-HCC (25.2-fold). SQLE encodes a monooxygenase and
is the second rate-limiting enzyme in cholesterol biosynthesis by
catalyzing the first oxygenation step in sterol biosynthesis (9). Be-
cause hepatic cholesterol accumulation underlies the development
of NAFLD, we hypothesized that SQLE might play a crucial role in
NAFLD-HCC. However, the pathological role of SQLE in NAFLD-
HCC remains unclear.
Here, we evaluated the oncogenic role of SQLE in normal liver and
NAFLD-HCC cell lines in vitro and developed hepatocyte-specific
Sqle transgenic (tg) mice to assess its effect on NAFLD-HCC devel-
opment in vivo. We also elucidated the mechanism of SQLE action
in promoting NAFLD-HCC and assessed the therapeutic efficacy of
an SQLE inhibitor, terbinafine, for the treatment of this disease.
RESULTS
SQLE is overexpressed in NAFLD-HCC tissues
We performed RNA-seq analysis of 17 paired NAFLD-HCC tumor
and adjacent normal tissues. Reactome analysis of differentially ex-
pressed genes identified metabolism as a core set of pathways altered
in NAFLD-HCC. Among up-regulated metabolic genes, SQLE was
a top outlier gene and it was overexpressed in 16 of 17 paired NAFLD-
HCC samples (Fig.1, A and B), with 25.2-fold up-regulation. The
increase in SQLE mRNA was validated in an independent cohort of
10 paired NAFLD-HCC samples (Fig.1C). SQLE protein was also
increased in NAFLD-HCC (Fig.1D). We next determined the ex-
pression of Sqle in two obesity-associated NAFLD-HCC mouse models.
Sqle was up-regulated in all HCC tumors (six of six) from N,N-
diethylnitrosamine (DEN) and high-fat, high-cholesterol (HFHC)
diet–treated C57BL/6 mice (Fig.1E). Similarly, Sqle was up-regulated
in 8 of 10 HCC tumors from DEN-treated db/db mice (Fig.1E). SQLE
is therefore commonly overexpressed in human NAFLD-HCC and
1Institute of Digestive Disease and Department of Medicine and Therapeutics, State
Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK
Shenzhen Research Institute, The Chinese University of Hong Kong, 999077, Hong Kong.
2Guangdong Key Laboratory for Genome Stability and Human Disease Prevention,
Department of Pharmacology and Cancer Research Centre, School of Medicine,
Shenzhen University, Shenzhen 518060, China. 3Department of Gastroenterology,
The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong
510630, China. 4Department of Surgery, The Chinese University of Hong Kong,
999077, Hong Kong. 5Department of Anatomical and Cellular Pathology, The C hinese
University of Hong Kong, 999077, Hong Kong.
*Corresponding author. Email: junyu@cuhk.edu.hk
Copyright © 2018
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
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experimental NAFLD-HCC mouse models. In addition, we observed
that the copy number amplification of SQLE was positively correlated
with its mRNA expression in human NAFLD-HCC (Fig.1F), indicat-
ing that copy number gain contributes to up-regulation of SQLE.
Moreover, in silico analysis indicated that transcription factors MEIS1,
SREBP2, and EVI1 might bind to the SQLE promoter (fig. S1A). In
the NAFLD-HCC RNA-seq cohort, mRNA expression of SQLE was
positively correlated with MEIS1 (R = 0.559, P = 0.03) and SREBP2
(R = 0.542, P = 0.03), but not EVI1 (fig. S1B), suggesting that SQLE
expression may also be regulated by transcription factors, especially
SREBP2 and MEIS1. SREBP2 is a known transcriptional regulator of
SQLE (10). Knockdown of SREBP2 in LO2 (normal hepatocyte) and
HKCI2 (NAFLD-HCC) (11, 12) cells inhibited SQLE protein expres-
sion (fig. S2A). Predicted binding motif of MEIS1 is TGACA (fig. S2B),
and it is located −669 base pairs from the transcription start site (fig.
S2C). The interaction of MEIS1 with SQLE promoter was confirmed
using chromatin immunoprecipitation (ChIP)–polymerase chain re-
action (PCR) and luciferase reporter assays (fig. S2, D and E). MEIS1
overexpression in LO2 and HKCI2 cells induced SQLE mRNA and
protein expression, whereas knockdown of MEIS1 in LO2 and HKCI2
cells suppressed SQLE (fig. S2, F and G). Gene amplification and
transcription factors are hence the main drivers of SQLE overex-
pression in NAFLD-HCC.
SQLE promotes cell growth, regulates cell cycle progression,
and inhibits apoptosis in NAFLD-HCC cell lines
To assess the oncogenic effect of SQLE in human HCC, we con-
structed LO2 and HKCI10 (NAFLD-HCC) (11, 12) cell lines stably
expressing SQLE (Fig.2A). SQLE expression promoted cell growth,
as evidenced by cell growth curve and colony formation assays
(Fig.2, A and B). Conversely, knockdown of SQLE in HKCI2 (also
NAFLD-HCC) cells by short hairpin RNA (shRNA) inhibited cell
viability compared with shControl (Fig.2, C and D). Apart from
NAFLD-HCC cell lines, SQLE also promoted the growth and colony
formation of Huh7 HCC cell line, whereas the silencing of SQLE had
an opposite effect in BEL-7404 and HepG2 HCC cells (fig. S3, A and
B). Moreover, LO2 and HKCI10 cells overexpressing SQLE showed
an increase in S-phase cell population, with a concomitant decrease
in cells in the G1 phase (Fig.2E). In contrast, SQLE knockdown in
HKCI2 cells induced G1 arrest (Fig.2F). In HCC cell lines, manipu-
lation of SQLE expression had the same effect on cell cycle (fig. S4,
A and B), indicating that SQLE accelerated G1-S progression. Con-
sistent with these observations, SQLE induced protein expression of
cyclin D1 and proliferating cell nuclear antigen (PCNA) (Fig.2G).
SQLE also regulates apoptosis, and its overexpression suppressed
apoptosis in LO2 and HKCI10 cells and reduced the protein expression
of cleaved forms of caspase-7 and caspase-9, whereas SQLE knock-
down had an inverse effect in HKCI2 and BEL-7404 cells (Fig.2, H
to J, and fig. S4C). These results suggest that SQLE promoted cell
growth by activating cell cycle progression and inhibiting apoptosis.
Hepatocyte-specific tg SQLE expression in mice accelerates
NAFLD-HCC formation
To determine the relevance of SQLE in NAFLD-HCC development
in vivo, we constructed Sqle tg mice. Crossing Sqle tg mice to
Albumin- Cre (Alb-Cre) mice results in hepatocyte-specific Sqle ex-
pression (Fig.3A). To evaluate the role of Sqle in NAFLD-HCC, we
injected WT and Sqle tg mice with a single dose of DEN at days 10
to 13. Starting at the age of 6 weeks, mice were fed with HFHC diet
(Fig.3B). At 25 weeks of age, mice were sacrificed and the liver was
analyzed. Significantly more Sqle tg mice developed tumors (9 of
10) as compared to WT mice (2 of 10) (P = 0.003), and histological
Fig. 1. SQLE is overexpressed in NAFLD-HCC. (A) RNA-seq analysis of 18 paired NAFLD-HCC and adjacent normal tissues (left). SQLE was the top outlier gene among
the up-regulated metabolic genes (right). FPKM, fragments per kilobase of transcript per million mapped reads. (B) SQLE mRNA expression in 17 individual paired NAFLD-HCC
and adjacent normal samples (one paired sample was not available for analys is). (C) Increased SQLE mRNA and (D) protein expression in human NAFLD-HCC were validated
in an independent cohort. Arrows show SQLE protein. (E) Sqle mRNA expression was up-regulated in dietary and genetic NAFLD-HCC animal models: DEN-injected and
high-fat diet–treated wild-type (WT) mice (left) and DEN-treated db/db mice (right). (F) Analysis of correlation between SQLE gene copy number and mRNA expression in
17 paired NAFLD-HCC. Data are means ± SEM. (C to E) Paired two-tailed Student’s t tests were used. (F) The Pearson correlation coefficient was used.
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Fig. 2. SQLE promotes NAFLD-HCC cell growth. (A) Overexpression of SQLE in LO2 (normal liver) and HKCI10 (NAFLD-HCC) cells was confirmed by Western blot analysis. Ectopic
SQLE expression promoted cell viability (A) and colony formation (B) (n = 3, performed in triplicate). (C) SQLE knockdown in HKCI2 (NAFLD-HCC) cells was confirmed by Western
blot analysis. SQLE knockdown suppressed cell viability (C) and colony formation (D) (n = 3, performed in triplicate). (E) Cells were stained with propidium iodide (PI) and analyzed
by flow cytometry. Flow cytometry analysis indicated that SQLE expressio n decreased the numbe r of cells in G1 phase but increased the number of cells in S phase (n = 3, performed
in triplicate). (F) Cells were stained with PI and analyzed by flow cytometry. Flow cytometry analysis indicated that knockdown of SQLE in HKCI2 induced G1 arrest (n = 3, performed
in triplicate). (G) Western blot analysis indicated that SQLE overexpression increased PCNA and cyclin D1 expression, whereas SQLE knockdown had the opposite effects.
(H) SQLE overexpression reduced apoptosis, as determined by annexin V–phycoerythrin and 7-aminoactinomycin D (7-AAD) staining and flow cytometry (n = 3, performed in
triplicate). (I) SQLE knockdown in HKCI2 cells induced apoptosis, as determined by annexin V–phycoerythrin and 7-AAD staining and flow cytometry (n = 3, performed in triplicate).
(J) Western blot analysis showed that SQLE overexpression reduced the protein expression of cleaved forms of caspase-7 and caspase-9, whereas SQLE knockdown had the
opposite effects. Data are means ± SEM. Difference in cell viability between two groups was determined by repeated-measures ANOVA. Mann-Whitney U test or Student’s t test
was performed to compare the variables in two groups (colony formation, cell cycle, and apoptosis). *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 3. Hepatocyte-specific transgenic SQLE expression in mice accelerates HFHC diet–associated NAFLD-HCC. (A) Scheme for the generation of mice with
hepatocyte- specific Sqle overexpression. Western blot confirmed overexpression of SQLE in the livers of Sqle tg mice. GAPDH, glyceraldehyde-3-phosphate de-
hydrogenase. (B) Experimental design for DEN-injected and HFHC diet–induced mouse models of NAFLD-HCC. At the age of 10 to 13 days, WT or Sqle tg mice were
injected with a single dose of DEN. Starting at 6 weeks of age, mice were fed with HFHC diet until week 25 (top). H&E staining of WT and Sqle tg mouse livers (middle).
HCC tumor incidence and multiplicities in WT and Sqle tg mice (bottom). The red arrows show the tumor. Results are means ± SEM (n = 9 to 10). (C) Hepatocyte-specific
Sqle expression increased liver weight (left) and liver/body weight ratio (middle), but not body weight (right), in DEN-injected HFHC diet–treated mice. (D) Serum AFP
(left), ALT (middle), and AST (right) concentrations of WT and Sqle tg mice treated with DEN and HFHC diet. Results are means ± SEM (n = 9 to 10). (E) Ki-67 staining of
livers from DEN-injected and HFHC diet–treated WT and Sqle tg mice. The red arrows show the Ki-67–positive cells. T, tumor; N, adjacent normal. Results are means ±
SEM (n = 9 to 10). Mann-Whitney U test was used. Scale bars, 50 m. ***P < 0.001.
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examination [hematoxylin and eosin (H&E) staining] confirmed
HCC forma tion in the livers of Sqle tg mice, together with hallmarks
of fatty liver disease such as hepatocyte ballooning and inflammatory
cell infiltration (Fig.3B). Sqle tg mice also showed increased liver
weight and liver/body weight ratio but not body weight (Fig.3C).
Consistent with development of HCC, -fetoprotein (AFP), a serum
bio marker for liver cancer, was increased in Sqle tg mice (Fig.3D).
Serum concentrations of alanine aminotransferase (ALT) and as-
partate aminotransferase (AST), markers for liver inflammation and
injury, were also significantly higher in Sqle tg mice (P < 0.01; Fig.3D).
We next performed Ki-67 staining to assess cell proliferation (Fig.3E).
Compared to WT mouse liver tissues, nontumorous liver tissues
from Sqle tg mice showed increased cell proliferation, and tumors
derived from Sqle tg mice had the highest Ki-67 scores (Fig.3E),
which is consistent with our in vitro observations. We also per-
formed TUNEL [terminal deoxynucleotidyl transferase (TdT)–
mediated de oxyuridine triphosphate nick end labeling] staining and
Western blots to evaluate liver apoptosis. Compared to WT mouse
liver tissues, nontumor liver tissues from Sqle tg mice had higher
TUNEL scores (fig. S5A) and increased amounts of cleaved forms of
caspase-3 and caspase-7 (fig. S5B). Cytokine and Chemokines PCR
array indicated that Sqle overexpression increased the mRNA ex-
pression of pro inflammatory mediators, including Ccl-12, Ccl-20,
Ccr-5, Cxcl-9, Cxcr-4, Il-2, Il-4, Il-18r1, Osm, and Spp1 (fig. S5C).
Collectively, these data demonstrate that SQLE overexpression ex-
acerbates HFHC diet–induced NAFLD and promotes NAFLD- HCC
formation in mice by inducing inflammation and cell proliferation.
SQLE promotes cell growth via intracellular cholesterol/
cholesteryl ester accumulation
Given that SQLE is the rate-limiting enzyme for cholesterol bio-
synthesis, we hypothesized that its overexpression may deregulate
cholesterol metabolism in NAFLD-HCC. We first determined the
concentrations of cholesterol and cholesteryl esters in WT and Sqle tg
mice. With a normal diet, Sqle tg mice showed markedly enhanced
accumulation of liver free cholesterol and cholesteryl ester com-
pared to WT littermates (Fig.4A). Moreover, an HFHC diet together
with Sqle tg exacerbated liver free cholesterol and cholesteryl ester
concentrations (Fig.4A). In vitro, both intracellular free cholesterol
and cholesteryl ester were increased in LO2-SQLE and HKCI10-
SQLE cells compared to their control cells, especially for cholesteryl
ester (more than sevenfold) (Fig.4B). Conversely, SQLE knockdown
in HKCI2 and BEL-7404 reduced free cholesterol and cholesteryl ester
concentrations (Fig.4B). To determine whether cholesterol con-
tributed to the growth-promoting effect of SQLE, we cultured LO2
and HKCI2 cells in the presence of exogenous cholesterol. Cholesterol
promoted cell viability (Fig.4C). Analysis of intracellular cholesterol
content revealed that cholesteryl ester, but not free cholesterol, was
increased after cholesterol treatment (Fig.4C), suggesting that cho-
lesteryl ester is responsible for tumor-promoting effect of SQLE in
both LO2 and HKCI2 cell lines. Consistent with our hypothesis,
avasimibe (an acyl–coenzyme A: cholesterol acyltransferase inhibitor,
which blocks cholesterol ester synthesis) abolished growth-promoting
effects of cholesterol in LO2 and HKCI2 cells (Fig.4D). Moreover,
exogenous cholesteryl ester directly induced cell growth in LO2 and
HKCI2 cells (Fig.4E), thereby confirming an oncogenic role of this
metabolite in HCC. Finally, the incubation of SQLE-overexpressing
HKCI2 and HKCI10 cells with avasimibe repressed cell growth to
baseline rates (Fig.4F). Reduction in cholesteryl ester concentrations
confirmed the on-target inhibition by avasimibe (Fig.4G). To confirm
the correlation of cholesteryl ester with NAFLD-HCC, we mea-
sured the cholesterol/cholesteryl ester concentrations in 10 paired
NAFLD-HCC tumors and adjacent normal tissues (fig. S6, A and B).
We found increased concentrations of cholesteryl ester (P < 0.05)
and free cholesterol (P < 0.05) in primary NAFLD-HCC compared
to adjacent normal tissues. We also observed a positive correlation
between free cholesterol and cholesteryl ester in adjacent normal
tissues (R = 0.7354, P = 0.015) but not in HCC (R = 0.5379, P = 0.100)
(fig. S6C). Our data therefore indicate that SQLE induces cholesteryl
ester biosynthesis, which contributes to the pathogenesis of NAFLD-HCC.
SQLE suppresses PTEN and activates the PTEN/PI3K/AKT/
mTOR pathway in NAFLD-HCC
Next, we investigated the downstream molecular mechanisms
underlying the oncogenic function of SQLE using cancer pathway
luciferase reporter assays. Among several critical cancer-related gene
reporters, SQLE significantly suppressed FOXO3 reporter [phos-
phatidylinositol 3-kinase (PI3K)/AKT pathway–responsive reporter]
activity (P < 0.01) (Fig.5A and fig. S7, A and B), suggesting that SQLE
activated PI3K/AKT signaling. To elucidate the effect of SQLE on
the PI3K/AKT pathway, we profiled gene expression of LO2-vector
and LO2-SQLE cells using PI3K/AKT pathway PCR array. We
found that SQLE enhanced the expression of JUN but decreased the
expression of PTEN, CHUK, APC, GSK3B, and CDKN1B (Fig.5B).
Among these outlier genes, PTEN functions as an upstream regulator
of the PI3K/AKT pathway, whereas other candidate genes are down-
stream effectors of this pathway. Hence, we hypothesized that SQLE
activates PI3K/AKT pathway through silencing of PTEN. Western
blot revealed that overexpression of SQLE in LO2 and HKCI10 cells
silenced PTEN and induced expression of phosphorylated AKT
(p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR)
(Fig.5C). Reciprocally, silencing of SQLE induced PTEN but inhibited
p-AKT and p-mTOR expression in HKCl2 cells (Fig.5C). Notably,
Sqle tg mouse livers demonstrated PTEN loss and increased p-mTOR
expression as compared to WT mice (Fig.5D). To further corroborate
our findings, we analyzed expression of 10 genes that are negatively
regulated by PTEN. SQLE overexpression promoted expression of
PTEN downstream target genes, including BCL2, SP2, NFKB, KRAS,
ESR, AKT1, FGF21, FANCD, BRAF, and CD44, whereas SQLE knock-
down decreased the expression of these genes (Fig.5E). Together, our
data suggested that SQLE induced PTEN silencing, resulting in the
activation of PI3K/AKT/mTOR. Given that PTEN/PI3K/AKT/mTOR
pathway is correlated with tumorigenesis in multiple cancers (13, 14),
we postulated that SQLE promotes NAFLD-HCC by regulating this
pathway.
Oncogenic effects of SQLE depend on
PTEN/PI3K/AKT/mTOR pathway
We then sought to determine whether the PTEN/PI3K/AKT/mTOR
cascade is involved in mediating the oncogenic effect of SQLE. In
LO2 and HKCI10 cell lines, we silenced PTEN by small interfering
RNA (siRNA). Knockdown of PTEN in LO2 and HKCI10 cells in-
duced intracellular cholesteryl ester accumulation, cell viability, and
mTOR phosphorylation (Fig.5F), thereby recapitulating the effect
of SQLE. SQLE overexpression in PTEN-silenced cell lines did not
further promote cell growth, intracellular cholesteryl ester accumula-
tion, or mTOR phosphorylation (Fig.5F), suggesting that PTEN loss
plays a crucial role downstream of SQLE.
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To confirm involvement of the PTEN/
PI3K/AKT/mTOR cascade in SQLE-
induced tumorigenesis, we treated SQLE-
overexpressing cells with two PI3K
inhibitors (GDC0941 and BYL719) and
one mTOR inhibitor (rapamycin) (Fig.5G
and fig. S7C) in vitro. Consistent with
our hypothesis, the cell viability assay
demonstrated that inhibition of either
PI3K or mTOR nearly abolished SQLE-
mediated cell growth in both cell lines.
mTOR inhibition profoundly inhibited
SQLE-induced cholesterol acyltransferase
1/2 (SOAT1/2) expression (fig. S7D) and
suppressed the induction of cholesteryl
ester by SQLE (Fig.5G). Collectively, our
results suggest that SQLE-dependent
tumor cell growth and cholesteryl ester
accumulation are consequences of the
activation of the PTEN/PI3K/AKT/mTOR
signaling pathway.
SQLE silences PTEN via induction of
ROS-DNMT3A axis
Given that SQLE functions primarily as
a metabolic enzyme, we hypothesized
that SQLE might silence PTEN via its
metabolic products. SQLE is the second
rate-limiting enzyme of intracellular
cholesterol biosynthesis, and it functions
as a monooxygenase that uses an oxygen
atom from O2 to oxidize squalene and
simultaneously reduces the other oxygen
atom using reducing equivalents from
NADPH (reduced form of NADP+) to
generate NADP+ (nicotinamide adenine
dinucleotide phosphate) (9, 15). HMGCR,
the first rate-limiting enzyme of the
cholesterol biosynthesis pathway, also
consumes reducing equivalents in the
form of NADPH (15, 16). Consistent with
this notion, overexpression of SQLE sig-
nificantly increased the NADP+ to NADPH
ratio in vivo (P < 0.01) and in vitro (P < 0.01;
Fig.6, A and B). Blockade of HMGCR
using simvastatin reversed SQLE- induced
increase in NADP+/NADPH ratio, cho-
lesterol biosynthesis, and cell viability, in-
dicating that HMGCR- and SQLE- mediated
cholesterol biosynthesis contributed to the
increased NADP+/NADPH ratio and cell
viability (fig. S8A). Because NADPH is
essential for redox homeostasis, the ex-
haustion of NADPH by SQLE resulted
in oxidative stress induction (Fig.6B). To
identify the specific metabolite(s) in-
volved in the silencing of PTEN, we treated
SQLE- overexpressing HCC and NAFLD-
HCC cells with cholesterol, inhibitors of
Fig. 4. SQLE promotes intracellular cholesterol/cholesteryl ester accumulation, which induces tumor cell
growth. (A) Hepatocyte-specific Sqle expression increased liver free cholesterol (left) and cholesteryl ester (right)
concentrations in normal diet or DEN-injected, HFHC diet–treated mice. (B) SQLE overexpression in LO2 and HKCI10
cells increased intracellular free cholesterol and cholesteryl ester (left). Knockdown of SQLE in HKCI2 and an HCC cell
line (BEL-7404) decreased intracellular free cholesterol and cholesteryl ester (right) (n = 4, performed in triplicate).
(C) Exogenous cholesterol promoted cell growth and increased intracellular cholesteryl ester concentration but had
no effect on free cholesterol (n = 3, performed in triplicate). (D) Avasimibe abolished the proliferative effect of cholesterol
(n = 3, performed in triplicate). (E) Cholesteryl ester directly promoted cell growth (n = 2, performed in triplicate).
(F) Avasimibe abolished SQLE-induced cell growth and (G) reversed cholesteryl ester accumulation (n = 4, performed
in triplicate). Data are means ± SEM. The significance of the differences in cell growth rates was determined by repeated-
measures ANOVA. The significance of the difference in cholesterol concentrations was determined by Mann-Whitney
U test. *P < 0.05, **P < 0.01, ***P < 0.001.
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cholesteryl ester biosynthesis (avasimibe), and reactive oxygen spe-
cies (ROS) inhibitor [glutathione (GSH)]. Exogenous cholesterol
increased intracellular cholesteryl ester, and avasimibe decreased in-
tracellular cholesteryl ester content (Fig.4, C and G), but they both
failed to inhibit PTEN expression (fig. S8, B and C). GSH markedly
inhibited intracellular ROS (fig. S8D) and reversed SQLE-induced
silencing of PTEN (Fig.6C), suggesting that induction of ROS medi-
ates the effect of SQLE on PTEN silencing. Treatment with GSH also
suppressed conversion of cholesterol to cholesteryl esters, abolishing
SQLE-induced accumulation of cholesteryl esters while increasing
the concentration of free cholesterol (Fig.6D). These data indicate
that SQLE mediates the silencing of PTEN through induction of oxi-
dative stress.
Previous studies (17, 18) indicated an important role of oxidative
stress in shaping the epigenetic landscape. Given that the expres-
sion of PTEN is modulated by several epigenetic regulators (19, 20),
we postulated that SQLE may silence PTEN via ROS-induced epi-
genetic alterations. To identify epigenetic enzymes altered by SQLE,
Fig. 5. Oncogenic function of SQLE depends on PTEN/PI3K/AKT/mTOR. (A) SQLE activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, as indicated by FOXO3
luciferase reporter assay (n = 3, performed in triplicate). FOXO3 is a target of AKT, and PI3K/AKT pathway activation inhibited FOXO3 expression. (B) PI3K/AKT pathway
PCR analysis of LO2 cells overexpressing SQLE. PTEN is one of the outlier down-regulated genes. (C and D) Representative Western blot analysis confirmed that SQLE in-
duced PTEN silencing and activated downstream oncogenic signaling [phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR)] in vitro (C) and in vivo (D).
(E) mRNA expression of PTEN/PI3K/AKT/mTOR downstream effectors in SQLE-overexpressing LO2 cells and SQLE-knockdown BEL-7404 cells (n = 3, performed in triplicate).
(F) SQLE downstream effects on cholesteryl ester accumulation and cell growth depend on PTEN/mTOR. Knockdown of PTEN followed by overexpression of SQLE in LO2
and HKCI10 cells prevented the effects of SQLE on cholesteryl ester accumulation (left), cell viability (middle), and PTEN/mTOR signaling (right). (G) mTOR inhibitor rapa-
mycin suppressed the effect of SQLE on cholesteryl ester accumulation (left) (n = 4, performed in triplicate) and cell growth (right) (n = 3, performed in triplicate). Data are
means ± SEM. Mann-Whitney U test was used to assess the significance of the differences in cholesterol concentrations, mRNA expression, and luciferase reporter assay.
The significance of the difference between cell growth rates was determined by repeated-measures ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 6. SQLE silences PTEN through ROS-mediated DNA hypermethylation. (A) SQLE increased NADP+/NADPH ratio in Sqle tg mice. (B) SQLE increased NADP+/NADPH
ratio and ROS in LO2 and HKCI10 cells (n = 3, performed in triplicate). (C) GSH, a ROS scavenger, reversed SQLE-induced PTEN loss, as indicated by Western blot analysis.
(D) GSH abolished SQLE-induced accumulation of cholesteryl ester in LO2 and HKCI10 cells (n = 4, performed in triplicate). (E to I) SQLE silenced PTEN via ROS-mediated
DNMT3A expression. (E) Heat map of the Epigenetic Chromatin Modification Enzymes PCR Array using LO2 and HKCI2 cell lines overexpressing SQLE. (F) Quantitative PCR
and Western blot confirmed that mRNA (top left) and protein (top right) expression of DNMT3A were positively regulated by SQLE. Nuclear DNMT3A activity was also
induced by SQLE (bottom) (n = 3, performed in triplicate). (G) GSH reversed SQLE-induced DNMT3A expression. (H) Infinium HumanMethylation450 (450K) BeadChip
analysis of CpG methylation in LO2-vector and LO2-SQLE cell lines revealed increased global promoter methylation in SQLE-overexpressing cells (left). Pathway analysis
of hyper- and hypomethylated genes (right). (I) SQLE-induced PTEN silencing was reversed by DNMT3A knockdown (n = 3, performed in triplicate). (J) Schematic diagram
showing the mechanism of action of SQLE in NAFLD-HCC. SQLE increases cholesterol biosynthesis and NADP+/NADPH-related ROS. Increased ROS induces DNMT3A ex-
pression and activation, which mediates transcriptional silencing of PTEN via promoter methylation. PTEN loss activates AKT/mTOR to promote NAFLD-HCC. AKT/mTOR
activation also induces cholesteryl ester accumulation, which contributes to tumor cell growth. Data are means ± SEM. Mann-Whitney U test was used for comparing
means between two groups. *P < 0.05, **P < 0.01.
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we performed Epigenetic Chromatin Modification Enzymes PCR
Array on LO2 and HKCI2 cell lines with empty vector or SQLE.
SQLE overexpression in LO2 and HKCI2 cells increased mRNA ex-
pression of DNA methyltransferase 3A (DNMT3A) and histone de-
acetylase 5 (HDAC5) but suppressed HDAC9 (Fig.6E and table S1).
DNMT3A is a de novo DNMT that initiates CpG methylation and
is associated with tumorigenesis (21, 22). Quantitative PCR (qPCR)
and Western blot confirmed the up-regulation of DNMT3A mRNA
and protein in LO2 and HKCI10 cells overexpressing SQLE, whereas
the silencing of SQLE in HKCl2 and BEL-7404 cells repressed
DNMT3A expression (Fig.6F). In parallel, nuclear DNMT3A
activity was induced by SQLE overexpression in LO2 and HKCI10
cells but was reduced by SQLE knockdown in HKCI2 and BEL-7404
cells (Fig.6F). To determine whether SQLE-associated oxidative
stress mediates induction of DNMT3A, we treated SQLE-expressing
cells with GSH. As shown in Fig.6G, GSH reversed DNMT3A in-
duction by SQLE.
Next, we documented the epigenetic alterations induced by SQLE
in LO2 cells using the Illumina Infinium HumanMethylation450K
BeadChip. SQLE induced global promoter methylation in LO2 cells
(P < 0.0001; Fig.6H and tables S2 and S3). KEGG (Kyoto Encyclopedia
of Genes and Genomes) pathway analysis revealed that hypermethylated
genes are associated with cancer, including PI3K/PTEN/AKT and
p53 signaling pathways, whereas hypomethylated genes are related to
cell cycle and fatty acid metabolism (Fig.6H). To evaluate whether
PTEN was silenced by ROS-induced DNMT3A activation, we treated
SQLE-overexpressing cell lines with DNMT3A-specific siRNA. We ob-
served that knockdown of DNMT3A restored PTEN expression, suggest-
ing that DNMT3A-mediated DNA methylation results in transcriptional
silencing of PTEN (Fig.6I). Our findings therefore unveiled an SQLE-
ROS-DNMT3A-PTEN axis that contributes to HCC (Fig.6J).
In line with our in vitro findings, we observed a positive correlation
between SQLE and DNMT3A expression in our NAFLD-HCC (R =
0.275, P = 0.037), HCC (R = 0.229, P = 0.039), TCGA liver cancer
(R = 0.28, P < 0.0001), and Stanford HCC (R = 0.444, P < 0.0001)
cohorts (Fig.7A), suggesting that SQLE also regulates DNMT3A ex-
pression in human HCC. SQLE protein up-regulation was correlated
with DNMT3A protein up-regulation, PTEN silencing, and mTOR
phosphorylation in human NAFLD-HCC and HCC tissues (Fig.7B),
which was also confirmed in Sqle tg mice (Fig.7C). Together, these
data show that enhanced SQLE expression in vivo activates a DNMT3A/
PTEN/mTOR axis that drives NAFLD-HCC tumorigenesis.
SQLE expression is associated with poor survival
of HCC patients
We analyzed mRNA expression of SQLE in three independent HCC
cohorts (our Guangzhou cohort, TCGA, and Stanford). SQLE was
highly up-regulated in primary HCC as compared with adjacent
normal tissues (n = 91, P < 0.0001) as determined by qPCR (Fig.7D),
and its overexpression was validated in TCGA (n = 50, P < 0.0001)
and Stanford cohorts (n = 65, P < 0.0001) (Fig.7E). We then assessed
the clinical relevance of SQLE in human HCC. Multivariate Cox
proportional hazards regression analysis revealed that high SQLE
expression was an independent prognostic factor associated with
poor disease-specific survival [P < 0.0001; hazard ratio, 4.31; 95%
confidence interval (CI), 1.87 to 8.72] (Fig.7F and table S4). We
validated the prognostic relevance of SQLE in TCGA cohort (n = 330).
Kaplan-Meier curve showed that increased expression of SQLE mRNA
was associated with poor survival in HCC patients (P = 0.02) and
was an independent prognostic factor (P = 0.02; hazard ratio, 1.553;
95% CI, 1.042 to 2.314) (Fig.7G and table S5). These data indicate
that SQLE expression is associated with poor prognosis in HCC.
Pharmacological inhibition of SQLE suppressed
NAFLD-HCC growth
Given the important oncogenic role of SQLE in NAFLD-HCC, we
evaluated whether a specific SQLE inhibitor, terbinafine (used widely
to treat fungal infections in humans), can be repositioned for pre-
vention or treatment of NAFLD-HCC. We treated HKCI2 and
HKCI10 cells with different doses of terbinafine (25 nM to 50 M;
fig. S9, A to C). At 25 to 50 M, terbinafine markedly suppressed the
viability of HKCI2, HKCI10, and HepG2 cell lines, as determined
by cell growth and colony formation assays (Fig.8A and fig. S9, A
and B). Western blot indicated that terbinafine suppressed SQLE
and PCNA but restored PTEN expression (Fig.8B and fig. S9C).
Because terbinafine had no effect on mRNA expression of SQLE
(fig. S9D), we examined whether terbinafine might affect SQLE
protein degradation through ubiquitination or autophagy. HKCI2 and
HKCI10 cells were treated with terbinafine plus proteasome in-
hibitor (MG132) or autophagy inhibitors (chloroquine or bafilomycin
A1). Chloroquine (fig. S9E) or bafilomycin A1 (fig. S9F) restored
SQLE protein expression, whereas MG132 had no such effect (fig.
S9G). In keeping with this, terbinafine induced autophagy activation,
as evidenced by reduced p62 protein expression (fig. S9, E and F).
Collectively, these data suggest that terbinafine induces SQLE pro-
tein degradation by inducing autophagy activity. Furthermore,
terbinafine reduced free cholesterol and cholesteryl ester (Fig.8C).
Terbinafine also suppressed the expression and activity of DNMT3A
(fig. S9H), in line with the results of SQLE knockdown by RNA in-
terference (Fig.6F).
We next evaluated the efficacy of terbinafine in vivo. Terbinafine
significantly suppressed the growth of subcutaneous HepG2 xeno-
grafts (77.8%, P < 0.01; Fig.8D). We also assessed the survival of
mice harboring HepG2 xenografts (tumor size 400 mm3 as cutoff)
and found that terbinafine significantly prolonged the overall sur-
vival (P < 0.01; Fig.8E). Terbinafine also suppressed the growth of
orthotopic HKCI2 xenografts (>85%, P < 0.01; Fig.8F), in terms of
both tumor size and tumor weight. In these xenograft models, tumor
free cholesterol and cholesteryl ester were suppressed (Fig.8G). We
further validated the efficacy of terbinafine in Sqle tg mice injected
with DEN and fed with an HFHC diet (fig. S9I). Terbinafine treat-
ment significantly reduced tumor incidence [four of nine mice in
terbinafine group versus eight of nine mice in phosphate-buffered
saline (PBS); P < 0.05] and tumor number (P < 0.01; Fig.8H). H&E
staining of livers from the vehicle and terbinafine-treated mice con-
firmed a reduction in HCC tumorigenesis and cell proliferation by
terbinafine (Fig.8I). In Sqle tg mice, terbinafine decreased liver/
body weight ratio and liver and serum cholesterol concentrations
(Fig.8J). In parallel, terbinafine also inhibited NADPH oxidation,
thereby reducing the NADP+/NADPH ratio (Fig.8J). Moreover,
terbinafine inhibited SQLE and DNMT3A protein expression but
restored PTEN expression in the livers of Sqle tg mice (Fig.8K). Col-
lectively, these data indicate that terbinafine, by inhibiting SQLE,
suppressed the accumulation of liver cholesterol/cholesteryl ester
and blocked the SQLE-ROS-DNMT3A-PTEN oncogenic axis, ulti-
mately resulting in inhibition of hepatocarcinogenesis. Moreover,
no significant change was found in serum ALT and AST after terbin-
afine treatment, suggesting that terbinafine did not cause any liver
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Fig. 7. SQLE expression is increased in human HCC and correlates with poor survival. (A) SQLE mRNA expression in HCC was positively correlated with DNMT3A mRNA
expression. The correlation analysis between DNMT3A and SQLE mRNA expression was performed for the NAFLD-HCC cohort (n = 16) and validated in our Guangzhou
(n = 69), The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) (n = 423), and Stanford (n = 72) cohorts. Pearson correlation coefficient was used. (B and
C) Representative Western blot confirmed that SQLE overexpression up-regulated DNMT3A expression and mTOR phosphorylation and also silenced PTEN expression in
(B) NAFLD-HCC and HCC and (C) Sqle-induced mouse HCC. (D) SQLE gene expression in HCC and adjacent normal tissue was determined in our Guangzhou HCC cohort
(n = 91 pairs) and (E) validated in TCGA-LIHC (n = 50 pairs) and Stanford cohorts (n = 65 pairs). Paired two-tailed Student’s t tests were used. Data are means ± SEM. (F and
G) High SQLE expression correlates with poor survival in HCC. Kaplan-Meier survival analysis and Cox regression analysis of our cohort (high, n = 43; low, n = 45) (G), and
TCGA-LIHC (high, n = 155; low, n = 175) cohort based on predictive survival analysis.
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Fig. 8. SQLE inhibitor terbinafine suppresses NAFLD-HCC growth in vitro and in vivo. (A) Terbinafine treatment suppressed cell growth (left) and colony formation
(right) in NAFLD-HCC (HKCI2, HKCI10) and HepG2 cell lines (n = 3, performed in triplicate). (B) Terbinafine suppressed SQLE expression and reversed the silencing of PTEN,
as determined by Western blot (n = 3, performed in triplicate). (C) Terbinafine reduced the amounts of free cholesterol and cholesteryl ester in HCC cell lines (n = 4, per-
formed in triplicate). (D) Terbinafine (80 mg/kg per day, oral) inhibited growth of subcutaneous HepG2 xenografts, as evidenced by reductions in tumor volume and weight.
(E) Terbinafine increased the survival of mice harboring HepG2 xenografts. Kaplan-Meier analysis and log-rank test were used. (F) Terbinafine (80 mg/kg per day, oral)
attenuated the growth of orthotopic HKCI2 xenografts. Both tumor volume and weight were reduced. (G) Terbinafine decreased the amounts of free cholesterol and
cholesteryl ester in HepG2 xenografts and HKCI2 orthotopic nude mouse model. (H) Terbinafine (80 mg/kg per day, oral) suppressed tumorigenesis in DEN-injected and
HFHC diet–treated Sqle tg mice (left), in terms of both tumor incidence and tumor number (right). (I) H&E and Ki-67 staining of vehicle and terbinafine-treated livers. The
red arrows show the positive cells. Scale bars, 100 m (H&E) and 50 m (Ki-67). (J) Terbinafine treatment decreased liver/body weight ratio (left), liver and serum cholesterol
concentrations (middle), and NADP+/NADPH ratio (right). (K) Representative Western blot analysis showed that terbinafine suppressed Sqle expression and reversed the
effect of SQLE on downstream factors DNMT3A and PTEN. Data are means ± SEM. The significance of the difference between cell growth rates and tumor growth rates in nude
mice was determined by repeated-measures ANOVA. Mann-Whitney U test was used for comparing means between two groups. *P < 0.05, **P < 0.01, ***P < 0.001.
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injury or toxicity (fig. S9J). Pharmacological inhibition of SQLE is
hence a promising approach that should be safe and effective for the
prevention and treatment of NAFLD-HCC.
DISCUSSION
Here, we established SQLE as an oncogenic factor amplified in NAFLD-
HCC. SQLE exerts its effect through the action of two key downstream
metabolites, cholesteryl ester and NADP+, resulting in epigenetic re-
programming and activating the PTEN/PI3K/AKT/mTOR signal-
ing cascade to drive carcinogenesis in NAFLD-HCC cell lines and in
hepatocyte-specific Sqle tg mice. Treatment with terbinafine conferred
a therapeutic benefit in NAFLD-HCC, including cell culture and ani-
mal models, corroborating SQLE as a therapeutic target in this subset
of HCC.
RNA-seq revealed frequent overexpression of SQLE in NAFLD-
HCC. SQLE is located on chromosome 8q24.13, a genomic region
that is frequently amplified in multiple cancers (23–25). We observed
that SQLE up-regulation is associated with its gene amplification in
NAFLD-HCC. Moreover, in silico and in vitro analyses demon-
strated that MEIS1 is a transcription factor that drives SQLE over-
expression in NAFLD-HCC. In keeping with our findings (26, 27),
SQLE gene amplification and overexpression were observed in mul-
tiple NAFLD-HCC and HCC patient cohorts, consistent with an on-
cogenic role of SQLE in hepatocarcinogenesis.
Our findings reinforced the concept that cholesterol, especially
cholesteryl esters, has oncogenic properties (28, 29). Although SQLE
is a rate-limiting enzyme for cholesterol biosynthesis, SQLE overex-
pression caused a more notable rise in cholesteryl esters. Cholesteryl
esters were able to induce NAFLD-HCC cell growth (30). Inhibition
of cholesterol esterification using avasimibe abolished the growth-
promoting effect of SQLE or free cholesterol. Consistent with our
data, intracellular cholesteryl esters are associated with increased
cell growth and tumor aggressiveness in breast, pancreatic, and pros-
tate cancers (29, 31, 32). Our data thus identify SQLE as a mediator
of increased biosynthesis of cholesteryl esters, which is essential for
NAFLD-HCC cell growth.
In addition to cholesterol and cholesteryl ester biosynthesis, we
found that SQLE consumes reducing equivalents in the form of
NADPH, inducing oxidative stress. SQLE-mediated oxidative stress
then triggers a sequence of events that profoundly alters the epigenetic
landscape. First, oxidative stress stimulates DNMT3A expression and
activity, silencing PTEN via promoter methylation. PTEN loss, in
turn, activates the PI3K/AKT/mTOR pathway. PI3K/AKT/mTOR
promotes the biosynthesis of cholesteryl esters, thereby forming a
positive feedback mechanism. Our data imply that the mechanism
of action of SQLE is a direct consequence of its catalysis metabolites,
which then activate downstream oncogenic pathways.
We validated the oncogenic potential of SQLE in the context of
NAFLD-HCC in a model of hepatocyte-specific Sqle tg mice. Sqle tg
expression fully recapitulated the signaling cascades induced by
ectopic SQLE expression in vitro. Moreover, Sqle tg together with
an HFHC diet exacerbated the accumulation of liver cholesterol/
cholesteryl ester and induction of oxidative stress, which, in turn,
further triggers the downstream oncogenic signaling cascades. As a
consequence, Sqle tg expression in mice accelerated HCC develop-
ment in an experimental model of NAFLD-HCC, concomitant with
increased cell growth and the inhibition of apoptosis. Our results pro-
vide evidence that Sqle functions as an oncogene in NAFLD-HCC.
HCC patients suffer from poor survival because of a lack of
effective treatment options. Identification of additional treatment
strategies for HCC is therefore urgent and important. Our work
here revealed that terbinafine (SQLE inhibitor) has promising effi-
cacy in inhibiting NAFLD-HCC. Terbinafine is a U.S. Food and Drug
Administration–approved oral drug commonly used for the treat-
ment of fungal infection. We found that terbinafine suppressed
NAFLD-HCC cell viability in vitro. Using orthotopic NAFLD-HCC
xenografts and Sqle tg mice, we showed that terbinafine was effica-
cious and safe in inhibiting NAFLD-HCC growth in vivo. Terbinafine
has an excellent safety profile and relatively few adverse drug-drug
interactions (33). Besides blocking cholesterol biosynthesis, targeting
of SQLE by terbinafine is known to cause accumulation of squalene
(34), which exhibits both antioxidant and anticancer properties in
numerous animal models (35–37). Nevertheless, clinical testing in
humans will be required to validate the beneficial effect of terbinafine
in human NAFLD-HCC. Additional work will also be needed to de-
fine the mechanism of terbinafine-induced autophagy and its role
in hepatocarcinogenesis. Work by us and others (38–40) indicated
an association of cholesterol with the inhibition of autophagy, suggest-
ing that terbinafine might activate autophagy by suppressing intra-
cellular cholesterol accumulation. The detailed mechanism and
implication of terbinafine-induced autophagy will need to be eluci-
dated in future studies.
The impact of our findings was strengthened by the observation
that SQLE was consistently and extensively overexpressed in HCC
compared with adjacent normal tissues in independent HCC patient
cohorts. SQLE expression is an independent prognostic factor asso-
ciated with poor survival in HCC in our cohort and TCGA data set.
SQLE may therefore be useful as a prognostic biomarker for differ-
entiating patients according to their survival outcomes in human HCC.
In conclusion, our discovery of SQLE, an oncogene in NAFLD-HCC ,
has unraveled links between cholesterol metabolism, epigenetic
dysfunction, and hepatocarcinogenesis. Inhibition of SQLE may be
a promising therapy for the prevention and treatment of NAFLD-
associated HCC.
MATERIALS AND METHODS
Study design
This study was designed to investigate the biological function of
SQLE in NAFLD-HCC and assess the therapeutic efficacy of targeting
SQLE in NAFLD-HCC. Clinical impact of SQLE was determined in
human NAFLD-HCC and HCC cohorts. All human sample collec-
tion and study protocol were approved by the Clinical Research Ethics
Committee of the Chinese University of Hong Kong (CUHK) or the
Sun Yat-sen University of Medical Sciences. Biological function of
SQLE was assessed in vitro using NAFLD-HCC cell lines. For in vivo
experiments, WT mice and hepatocyte-specific Sqle tg mice were
injected with a single dose of DEN and fed an HFHC diet to inves-
tigate the oncogenic function of SQLE in NAFLD-HCC development.
Therapeutic efficacy of terbinafine, an SQLE inhibitor, was deter-
mined in NAFLD-HCC cell lines in vitro and in nude mice and Sqle
tg mice in vivo. For in vitro studies, three or four independent ex-
periments were performed in triplicate. For tg mice models, at least
6 to 10 male mice were randomly allocated into different treatment
groups. For the nude mouse model, at least five male mice were
randomly allocated per group. In vitro experiments were not blinded.
In vivo experiments were blinded.
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Human samples
Human NAFLD-HCC tumor tissues and adjacent normal tissues
were collected from patients with biopsy-proven NAFLD-HCC in
Prince of Wales Hospital, CUHK. Human HCC tumors and adjacent
normal tissues were collected during operations before any thera-
peutic intervention at the Third Affiliated Hospital of Sun Yat-sen
University (Guangzhou, China). Written informed consent was ob-
tained from all subjects, and the study protocol was approved by the
Clinical Research Ethics Committee of the Sun Yat-sen University of
Medical Sciences.
Animal model and treatment
Sqle tg mice (pCAG-loxp-stop-loxp-Rosa26-Sqle) were generated
by BIOCYTOGEN Company. Sqle-IRES (internal ribosomal entry
site)–eGFP (enhanced green fluorescent protein) was cloned into
Rosa26 WT allele to generate a gene-targeting vector. Then, the
Rosa26-Sqle-IRES-eGFP vectors were transfected into embryonic
stem cells with C57BL/6 background. After selection and identi-
fication by PCR and Southern blot, positive clones were injected
into mouse blastocysts to generate chimeric mice. Chimeric mice
were mated with WT C57BL/6 mice to obtain the Rosa26-Sqle
mice. To drive the hepatocyte-specific expression of Sqle, Rosa26-
Sqle mice were crossed to B6.Cg-Tg (Alb-Cre) 21Mgn/JNju mice
(Nanjing University). Sqle tg/Alb-Cre mice were confirmed by PCR
genotyping.
An orthotopic human NAFLD-HCC mouse model was estab-
lished using HKCI2 cells (11, 12). HKCI2 cells (1 × 107 cells in 0.1 ml
of PBS) were injected subcutaneously into the left dorsal flank of
4-week-old male Balb/c nude mice. Subcutaneous tumors were har-
vested once they reached about 10 mm3 and cut into 1.0 mm3 pieces.
One piece of a tumor was implanted into the left liver lobe in a sep-
arate group of nude mice (4 weeks old). Four weeks after implanta-
tion, these mice were divided into vehicle group (PBS, oral) and
terbinafine group (80 mg/kg, oral). Eight weeks after implantation,
the mice were sacrificed and examined. All animal studies were per-
formed in accordance with the guidelines approved by the Animal
Experimentation Ethics Committee of CUHK.
ChIP assay
A total of 1 × 107 LO2 cells stably transfected with Meis1-DYK
(OHu19435) were cross-linked with 1% formaldehyde for 10 min at
room temperature and quenched with 125 mM glycine. After soni-
cation, protein-DNA complexes were immunoprecipitated (IP) by
2 g of anti-DYK–tag antibody (no. 14793, Cell Signaling Technology)
or anti-rabbit immunoglobulin G antibody (Abcam) overnight with
rotation at 4°C, followed by the addition of 20 l of Dynabeads mag-
netic beads (Millipore) and incubation for 2 hours. Next, we washed
the antibody/chromatin complex by resuspending the beads in four
immune complex wash buffers (Millipore) with different salt con-
centrations (low salt, high salt, LiCl, and tris-EDTA buffer) step by
step. Then, we added 8 l of 5 M NaCl buffer to each IP and input
sample and incubated at 65°C overnight to reverse the DNA protein
cross-links. After elution, all samples were treated with 1 l of ribo-
nuclease A at 37°C for 30 min and incubated with 4 l of 0.5 M
EDTA, 8 l of 1 M tris-HCl, and 1 l of proteinase K at 45°C for 1 to
2 hours. Finally, the IP and input DNA were purified using spin
columns (Millipore). For target gene validation, PCR primers tar-
geting a region of the putative binding site were designed to detect IP
and input DNA. The sequences of primers used are listed in table S6.
Plasmids
pRL-cyto-megalovirus (pCMV)–SQLE (RC202008) and pCMV-entry
control plasmids, SQLE shRNA (TL309122V), and control shRNA
(pGFP-C-shlenti) plasmids were all ordered from OriGene. SQLE
promoter reporter clone (HPRM22529-OG04) was ordered from
GeneCopoeia. Meis1 (OHu19435) and control plasmids (pcDNA3.1-D YK)
were ordered from GenScript.
RNA interference
PTEN siRNA (siPTEN), DNMT3A siRNA (siDNMT3A), and negative
control (siControl) were ordered form Santa Cruz Company. siPTEN,
siDNMT3A, or siControl (50 nmol) was transfected into cells using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
instructions.
RNA extraction, semiquantitative reverse transcription PCR,
and real-time PCR analyses
Total RNA was extracted from cells and tissues using TRIzol Re-
agent (Molecular Research Center Inc.). Complementary DNA
(cDNA) was synthesized from 1 g of total RNA using Transcriptor
Reverse Transcriptase (Roche). Real-time PCR was performed
using an SYBR Green master mixture (Roche) on LightCycler 480
Instrument. Each sample was tested in triplicate. Ct method was
applied to determine the fold change in gene expression. Ct method
was applied to determine the relative expression of corresponding
genes.
Western blot analysis
Total protein was separated by SDS–polyacrylamide gel electrophoresis
(SDS-PAGE). The proteins in SDS-PAGE were transferred onto ni-
trocellulose membranes (GE Healthcare). The membrane was incu-
bated with primary antibodies overnight at 4°C (table S7) and then
with secondary antibody at room temperature for 1 hour. Proteins
of interest were visualized using ECL Plus Western blotting Detec-
tion Reagents (GE Healthcare).
Colony formation assay
For the cell colony formation assay, stably transfected cells (1000 per
well) were plated in six-well plates. After culturing for 5 to 7 days,
cells were fixed with 70% ethanol and stained with 0.5% crystal violet
solution. Colonies with more than 50 cells per colony were counted.
All experiments were conducted three times in triplicate.
Apoptosis and cell cycle analyses
Apoptosis was assessed using an annexin-phycoerythrin/7-amino-
actinomycin D staining kit (BD Biosciences). For cell cycle analysis,
cells were serum-starved overnight and stimulated with complete
medium for 4 to 8 hours. Cells were fixed in 70% ethanol, stained
with propidium iodide, and analyzed by flow cytometry.
Luciferase reporter assay
To check the interaction between MEIS1 and SQLE, SQLE promoter
reporter clone (SQLE 1200-GLuc, GeneCopoeia), which contains the
promoter of human SQLE 1.2 kb upstream of the transcriptional
start site, was transfected into MEIS1-transfected LO2 and HKCI2
cells. To investigate the signaling pathways modulated by SQLE, a
series of signaling pathway luciferase reporters were examined in
SQLE-transfected LO2 and HKCI10 cells, including p53-luc, AP1-luc,
WNT-luc, and FOXO3-luc.
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The cell lines (LO2 and HKCI10) were stably transfected with
pCMV-SQLE or pCMV-vector (1 × 105 cells per well) in 24-well
plates and cotransfected with luciferase reporter plasmid (0.2 g per
well) and pCMV-vector (5 ng per well) using Lipofectamine 2000
(Life Technologies). Cells were harvested 48 hours after transfec-
tion, and luciferase activity was analyzed by the Dual Luciferase Re-
porter Assay System (Promega).
Cholesterol/cholesteryl ester concentrations
Cells (106) or tissues (2 mg) were harvested, and cholesterol/
cholesteryl ester concentrations were detected by Cholesterol/
Cholesteryl Ester Quantification kit (ab65359, Abcam) according
to the manufacturer’s instructions. All experiments were conducted
three times in triplicate. Results were shown as means ± SEM.
NADP+/NADPH ratio
Cells (2 × 106) or tissues (50 mg) were harvested, and NADPH/
NADP+ ratio was quantified by the NADP/NADPH Assay Kit
(ab65349, Abcam) according to the manufacturer’s instructions. All
experiments were conducted three times in triplicate. Results were
shown as means ± SEM.
Reactive oxygen species
Intracellular ROS were quantified by the DCFDA Cellular ROS De-
tection Assay Kit (microplate) (ab113851, Abcam) according to the
manufacturer’s instructions. All experiments were conducted three
times in triplicate. Results were shown as means ± SEM.
Serum cholesterol, ALT, and AST
The serum cholesterol, ALT, and AST concentrations were detected
by the Catalyst One Chemistry Analyzer according to the manufac-
turer’s instructions (IDEXX). Thirty microliters of serum from WT
or Sqle tg mice was diluted to 90 l by physiological saline buffer.
The diluted samples and specific slides (cholesterol, ALT, and AST)
were then loaded into the analyzer for automatic analysis.
Serum AFP
Mouse serum AFP was detected by the mouse AFP/AFP ELISA Kit
according to the manufacturer’s instructions (MAFO00, R&D Sys-
tems). Serum (10 l) from WT or Sqle tg mice was diluted to 200 l
with Calibrator Diluent RD5-26 buffer (diluted 1:4). We then added
50 l of diluted samples, standard, and control to each microplate
well for further analysis.
Ki-67 staining
Paraffin slides from DEN-injected, HFHC-fed WT and Sqle tg mice
were used. Ki-67 signal was assessed by an anti–Ki-67 antibody
(ab833, Abcam). The proliferation index was determined by count-
ing the numbers of positive staining cells as percentages of the total
number of liver cells. At least 1000 cells were counted each time.
TUNEL staining
Paraffin slides from DEN-injected, HFHC-fed WT and Sqle tg mice were
used. TUNEL signal was assessed using the DeadEnd Colorimetric
TUNEL System (Promega). Briefly, we removed paraffin by xylene and
fixed slides in 4% paraformaldehyde in PBS. We then added 100 l
of TdT reaction mix to the tissue sections on the slides and incubated
the slides for 60 min at 37°C in a humidified chamber. After incuba-
tion with 100 l of streptavidin horseradish peroxidase (diluted 1:500
in PBS), 100 l of 3,3′-diaminobenzidine was added to the slides for
detection.
Statistical analysis
All statistical tests were performed using SPSS or GraphPad Soft-
ware. Data are presented as means ± SEM. The Pearson correlation
coefficient was used to evaluate the correlation between SQLE gene
amplification and expression in the clinical samples. Multiple group
comparisons were analyzed by one-way ANOVA. Overall survival
in relation to expression was evaluated by the Kaplan-Meier sur-
vival curve and the log-rank test. Mann-Whitney U test or Student’s
t test was performed to compare the variables in two groups. The differ-
ence in cell viability and tumor growth rate between the two groups of
nude mice was determined by repeated-measures ANOVA. P values
of <0.05 were taken as being statistically significant.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/10/437/eaap9840/DC1
Fig. S1. SQLE expression in NAFLD-HCC is controlled by transcription factors.
Fig. S2. Transcription factors SREBP2 and MEIS1 bind to SQLE promoter region and activate
SQLE gene expression.
Fig. S3. SQLE promotes HCC cell growth.
Fig. S4. SQLE in HCC cell lines promoted cell cycle progression at G1-S phase and inhibited
apoptosis induction.
Fig. S5. Sqle tg expression in mice induced apoptosis and the expression of proinflammatory
cytokines and chemokines.
Fig. S6. Cholesteryl ester and cholesterol concentrations are increased in NAFLD-HCC.
Fig. S7. Oncogenic function of SQLE is dependent on the PTEN/PI3K/AKT/mTOR pathway.
Fig. S8. SQLE silences PTEN expression through ROS-mediated DNA hypermethylation.
Fig. S9. SQLE inhibitor terbinafine suppressed NAFLD-HCC growth in vitro and in vivo.
Table S1. Epigenetic Chromatin Modification Enzymes PCR Array data.
Table S2. Hypermethylated genes in LO2-SQLE cells.
Table S3. Hypomethylated genes in LO2-SQLE cells.
Table S4. Clinicopathological features of SQLE mRNA expression in our HCC cohort.
Table S5. Clinicopathological features of SQLE mRNA expression in TCGA HCC cohort.
Table S6. Primers used in this study.
Table S7. Antibodies used in this study.
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Funding: This study was supported by the Research Grants Council (RGC)–General Research
Fund Hong Kong (14106415, 14101917, 14111216, and 14114615), Shenzhen Virtual University
Park Support Scheme to CUHK Shenzhen Research Institute, grant from Faculty of Medicine
CUHK, and direct grant from CUHK. Author contributions: D.L. was involved in the study
design, conducted the experiments, and drafted the paper; H.C., L.Z., C.L., and Y. Zhou
performed animal experiments; Y. Zhang and W.X. performed the experiments; Y.Y., B.W.,
G.C., and P.B.-S.L. collected human samples; N.W. performed human NAFLD-HCC genome
sequencing and provided NAFLD-HCC cell lines; L.F. and J.J.Y.S. designed and commented on
the study; C.C.W. drafted the paper and commented on the study; J.Y. designed and
supervised the study and critically revised the paper. Competing interests: The authors
declare that they have no competing interests.
Submitted 18 September 2017
Accepted 14 March 2018
Published 18 April 2018
10.1126/scitranslmed.aap9840
Citation: D. Liu, C. C. Wong, L. Fu, H. Chen, L. Zhao, C. Li, Y. Zhou, Y. Zhang, W. Xu, Y. Yang, B. Wu,
G. Cheng, P. B.-S. Lai, N. Wong, J. J. Y. Sung, J. Yu, Squalene epoxidase drives NAFLD-induced
hepatocellular carcinoma and is a pharmaceutical target. Sci. Transl. Med. 10, eaap9840 (2018).
by guest on April 24, 2018http://stm.sciencemag.org/Downloaded from

pharmaceutical target
Squalene epoxidase drives NAFLD-induced hepatocellular carcinoma and is a
Yidong Yang, Bin Wu, Gong Cheng, Paul Bo-San Lai, Nathalie Wong, Joseph J. Y. Sung and Jun Yu
Dabin Liu, Chi Chun Wong, Li Fu, Huarong Chen, Liuyang Zhao, Chuangen Li, Yunfei Zhou, Yanquan Zhang, Weiqi Xu,
DOI: 10.1126/scitranslmed.aap9840
, eaap9840.10Sci Transl Med
drug, as a potential intervention.
promotes the development of hepatocellular carcinoma and identified terbinafine, a clinically approved antifungal
cancer cells and mouse models of disease, the authors examined the pathway by which squalene epoxidase
squalene epoxidase, a metabolic enzyme involved in cholesterol biosynthesis. Using a combination of human
. discovered a role foret alhepatocellular carcinoma arising in the setting of nonalcoholic fatty liver disease, Liu
associated with an increase in the incidence of hepatocellular carcinoma. By studying the mechanism of
The rates of nonalcoholic fatty liver disease are increasing with the rising prevalence of obesity and are
Antifungal drug may help fight cancer
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