Autophagy promotes hepatocellular carcinoma cell invasion through activation of epithelial–mesenchymal transition

TL;DR: It is suggested that autophagy is critical for the invasion of HCC cells through the induction of EMT and that activation of TGF-β/Smad3-dependent signaling plays a key role in regulating autophophagy-induced EMT.

Abstract: Invasion of hepatocellular carcinoma (HCC) cells is a leading cause of intrahepatic dissemination and metastasis. Autophagy is considered to be an important mediator in the invasion of cancer cells. However, the precise contribution of autophagy to cancer cell invasion and underlying mechanisms remain unclear. Autophagy was induced in HepG2 and BEL7402 cells by starvation in Hank's balanced salt solution. Induction of autophagy inhibited the expression of epithelial markers and induced expression of mesenchymal markers as well as matrix metalloproteinase-9 stimulating cell invasion. Starvation-induced autophagy promoted the expression of epithelial-mesenchymal transition (EMT) markers and invasion in HepG2 and BEL7402 cells through a transforming growth factor-beta (TGF-β)/Smad3 signaling-dependent manner. The small interfering RNAs (siRNAs) for Atg3 or Atg7 and chloroquine inhibited autophagy of HepG2 and BEL7402 cells during starvation, resulting in suppression of EMT and diminished invasiveness of HCC cells. Administration of SIS3 also attenuated EMT and invasion of HepG2 and BEL7402 cells during starvation. Recombinant TGF-β1 was capable of rescuing EMT and invasion that was inhibited by siRNA for Atg3 and 7 in HepG2 and BEL7402 cells under starvation. These findings suggest that autophagy is critical for the invasion of HCC cells through the induction of EMT and that activation of TGF-β/Smad3-dependent signaling plays a key role in regulating autophagy-induced EMT. Inhibition of autophagy may represent a novel target for therapeutic interventions.

Summary

Introduction

• Hepatocellular carcinoma (HCC) is the most frequent primary cancer of the liver and the fifth most common solid tumor and the third leading cause of cancer-related deaths worldwide (1).
• The prognosis for patients with HCC has improved through the use of surgical resection, chemotherapy and interventional therapy.
• Invasion and metastasis are common characteristics of cancer cells.
• The exact contribution of autophagy into the promotion of cancer cell invasion during starvation has not been thoroughly investigated.
• During EMT, epithelial cells change phenotype from epithelial to mesenchymal, which is a process that includes expression of mesenchymal markers, actin cytoskeleton reorganization, loss of cell-to-cell junctions and pseudopod formation (13).

Cell culture and experimental design

• The human HCC cell line, HepG2, was purchased from ATCC (Manassas, VA).
• Autophagic activity was evaluated after 6 h of starvation performed in Hank’s balanced salt solution (HBSS, Hyclone).
• In addition, HCC cells transfected with small interfering RNA -Atgs (3 or 7) were treated with recombinant human TGF-β1 (20 ng/ml, R&D System, Minneapolis, MN) in HBSS for 6 h to evaluate the necessity of TGF-β1 in autophagy-induced EMT and invasion.
• Cells that were cultured in complete medium served as control.
• All siRNAs were synthesized and purified by GenePharma (Shanghai, China).

Cell apoptosis assay

• Apoptosis of HCC cell lines was quantitated by flow cytometry.
• Cells pretreated with chloroquine as well as transfected and non-transfected cells in Abbreviations: CK18, cytokeratin 18; EMT, epithelial–mesenchymal transition; HBSS, Hank’s balanced salt solution; HCC, hepatocellular carcinoma; MMP-9, matrix metalloproteinase 9; siRNA, small interfering RNA; TGF-β, transforming growth factor-beta.
• †These authors contributed equally to this work.
• D ow nloaded from https://academ ic.oup.com /carcin/article-abstract/34/6/1343/2463238 by guest on 09 M arch 2019 complete medium and HBSS for 6 h were harvested, washed and incubated with binding buffer containing propidium iodide (10 g/ml) and fluorescein isothiocyanate-labeled annexin V (Annexin V-APC) (Bender MedSystems, eBioscience, San Diego, CA) for 15 min at room temperature.
• Cells were incubated with the appropriate secondary antibody and visualized by peroxidase substrate in conjunction with fluorescein isothiocyanate or Cy3 .

Western blotting

• Western blotting was used to detect the expression of LC3, p62, Atg3, Atg7, E-cadherin, CK18, fibronectin, MMP-9, TGF-β1, Smad3 or phosphorylated Smad3 in transfected and non-transfected HCC cell lines with or without starvation.
• Protein expression levels were normalized against β-actin.
• Total RNA was reverse transcribed into first-strand cDNA using an iScript cDNA Synthesis kit (Bio-Rad, München, Germany).
• The following primer sequences were used—Atg3 sense: 5′-GGAAGAAGATG AAGATGAA-3′, antisense: 5′-CATAATCGTGGAGTCTGGTA-3′; Atg7 sense: 5′-CACA GATGGAGTAGCAGTT-3′, antisense: 5′-TCACAGGATTGGAGTAGGA-3′; E-cadherin (19) sense: 5′-ACACCCCCTGTTGGTGTCTTT-3′, antisense: 5′-TGTATGTGGCAATGCGTTCTC-3′; CK18 (20) sense: 5′-CAAAGCCTGAGTCCTGTCCT-3′, antisense: 5′-GAGATCCGGGAACCAGAG-3′; fibronectin (21) sense: 5′-GGAGCAAATGGCACCGAGATA-3′, antisense: 5′-GAGCTGCACATGTC.
• Cells that were seeded in the upper chamber with complete medium were served as control.

Statistical analysis

• After demonstration of homogeneity of variance with Bartlett test, one-way analysis of variance, followed by Student–Newman–Keuls test where appropriate, was used to evaluate the statistical significance.
• Values of P < 0.05 were considered statistically significant.

Autophagy promotes invasion of HCC cells

• Starvation or nutritional deficiency is a common factor that upregulates cell autophagy (4,5).
• HepG2 and BEL7402 cells were starved in HBSS, which contained neither nutrients nor serum for 6 h.
• These findings suggest that HCC cell invasion was dependent on autophagy under starvation.
• In addition, cell morphology changed from circular like to fibriform .
• These morphological alterations and changes in the expression of epithelial and mesenchymal markers as well as MMP-9 did not occur in autophagy-deficient cells with siRNA-Atgs (3 or 7) or chloroquine during starvation compared with control .

TGF-β/Smad3 signaling regulates autophagy-induced EMT

• TGF-β/Smad3 is one of the known signaling pathways that mediate EMT (16,17).
• The authors found that TGF-β1 and phosphorylated Smad3 D ow nloaded from https://academ ic.oup.com /carcin/article-abstract/34/6/1343/2463238 by guest on 09 M arch 2019 were not highly expressed by HepG2 and BEL7402 cells in control medium compared with starvation.
• Since inhibition of autophagy suppressed EMT and TGF-β/Smad3, the authors further investigated whether blockade of this signaling was capable of suppressing EMT of HCC cells with autophagy during starvation.
• Administration of the inhibitor of TGF-β/Smad3 signaling, SIS3 (2  μmol/l), inhibited the autophagy-induced phosphorylation of Smad3 in HepG2 and BEL7402 cells during starvation compared with control medium .
• As expected, SIS3 also decreased invasive number of HepG2 and BEL7402 cells during starvation when EMT was inhibited by inactivity of TGF-β/Smad3 signaling .

TGF-β1 rescues EMT and invasion in autophagy-deficient

• HCC cells Since autophagy-activated TGF-β/Smad3 signaling was associated with EMT in HCC cells under starvation, the authors further tested whether TGF-β1 was able to rescue EMT in autophagy-deficient HCC cells under starvation.
• (B) Representative western blots of Atg3 and Atg7.
• Cells cultured in complete medium without transfection were served as control.
• Data are representative of three independent experiments and shown as mean ± SEM, n = 4, *P < 0.05 versus control.
• These results suggest that autophagyinduced TGF-β1 signaling plays a crucial role in EMT and invasion of HCC cells under starvation .

Discussion

• Due to metastasis that results from invasion of HCC cells, it is difficult to cure many HCC patients by current therapeutic interventions (2,3).
• (B) Representative western blots of E-cadherin, CK18, fibronectin and MMP-9 in HepG2 and BEL7402 cells with or without SIS3.
• All data are representative of three independent experiments and shown as mean ± SEM, n = 4, *P < 0.05 versus control.
• All of these phenotypic changes arising from EMT facilitate cancer cell invasion.
• In summary, the findings reported here indicate that starvationinduced autophagy plays a crucial role in the invasion of HCC cells through activation of EMT, which involves the activation of TGF-β signaling.

Full text

© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Carcinogenesis vol.34 no.6 pp.1343–1351, 2013
doi:10.1093/carcin/bgt063
Advance Access publication February 21, 2013
Autophagy promotes hepatocellular carcinoma cell invasion through activation of
epithelial–mesenchymal transition
JunLi
1,†
, BinYang
2,†
, QiZhou
3,†
, YongzhongWu
4,†
,
DanShang
2,†
, YuGuo
2
, ZifangSong
2
,
QichangZheng
2
JunXiong
2,
*
1
Department of Breast Surgery, Cancer Hospital and Institute, Chongqing
400030, China,
2
Department of Hepatobiliary Surgery, Union Hospital,
Tongji Medical College, Huazhong University of Science and Technology,
1277 Jiefang Avenue, Wuhan 430022, China,
3
Department of Gynecologic
oncology and
4
Department of Radiotherapy, Cancer Hospital and Institute,
Chongqing 400030, China
*To whom correspondence should be addressed. Tel:+86 27 13995536848;
Fax: +86 27 85351676; Email: jxiongmd@gmail.com
Invasion of hepatocellular carcinoma (HCC) cells is a leading
cause of intrahepatic dissemination and metastasis. Autophagy is
considered to be an important mediator in the invasion of cancer
cells. However, the precise contribution of autophagy to cancer cell
invasion and underlying mechanisms remain unclear. Autophagy
was induced in HepG2 and BEL7402 cells by starvation in Hank’s
balanced salt solution. Induction of autophagy inhibited the
expression of epithelial markers and induced expression of mes-
enchymal markers as well as matrix metalloproteinase-9 stimu-
lating cell invasion. Starvation-induced autophagy promoted the
expression of epithelial–mesenchymal transition (EMT) markers
and invasion in HepG2 and BEL7402 cells through a transforming
growth factor-beta (TGF-β)/Smad3 signaling-dependent manner.
The small interfering RNAs (siRNAs) for Atg3 or Atg7 and chlo-
roquine inhibited autophagy of HepG2 and BEL7402 cells dur-
ing starvation, resulting in suppression of EMT and diminished
invasiveness of HCC cells. Administration of SIS3 also attenuated
EMT and invasion of HepG2 and BEL7402 cells during starva-
tion. Recombinant TGF-β1 was capable of rescuing EMT and
invasion that was inhibited by siRNA for Atg3 and 7 in HepG2
and BEL7402 cells under starvation. These ndings suggest that
autophagy is critical for the invasion of HCC cells through the
induction of EMT and that activation of TGF-β/Smad3-dependent
signaling plays a key role in regulating autophagy-induced EMT.
Inhibition of autophagy may represent a novel target for thera-
peutic interventions.
Introduction
Hepatocellular carcinoma (HCC) is the most frequent primary cancer
of the liver and the fth most common solid tumor and the third lead-
ing cause of cancer-related deaths worldwide (1). The prognosis for
patients with HCC has improved through the use of surgical resection,
chemotherapy and interventional therapy. However, metastasis result-
ing from the invasion of HCC signicantly reduces the efcacy of
these therapeutic interventions for a considerable number of patients
(2,3).
Autophagy is an evolutionarily conserved physiological process
in cells (4) that generates intracellular nutrients, growth factors and
energy to support cell survival and cellular activities during stress-
ors, such as starvation, hypoxia and growth factor withdrawal (4–6).
Invasion and metastasis are common characteristics of cancer cells.
In situ, cancer cells in solid tumors are exposed to starvation and/or
hypoxia, which is due to decient angiogenesis during tumor growth
(7,8). It has been observed that autophagy accelerates invasion of
cancer cells during starvation or hypoxia (9,10). However, the exact
contribution of autophagy into the promotion of cancer cell invasion
during starvation has not been thoroughly investigated.
Epithelial–mesenchymal transition (EMT) occurs during the pro-
gression of epithelial carcinogenesis (11,12). During EMT, epithe-
lial cells change phenotype from epithelial to mesenchymal, which
is a process that includes expression of mesenchymal markers, actin
cytoskeleton reorganization, loss of cell-to-cell junctions and pseudo-
pod formation (13). EMT increases the migratory capacity of epithe-
lial cells (13). In addition, EMT has been shown to play a key role in
the induction of cancer cell invasion and metastasis (14,15).
Transforming growth factor-beta (TGF-β)/Smad3 signaling is
a key regulator of EMT in many epithelial cell types. Activation of
TGF-β/Smad3 signaling in epithelial cells triggers alterations in mor-
phological and functional phenotypes from epithelial to mesenchy-
mal (16,17). Activation of TGF-β/Smad3 signaling is involved in the
metastasis of HCC (18). However, the role of TGF-β/Smad3 signaling
in the regulation of EMT and invasion of HCC has not been evaluated
during autophagy induced by starvation. Therefore, the regulation of
EMT and cell invasion by autophagy was investigated in two HCC
cell lines during starvation.
Materials and methods
Cell culture and experimentaldesign
The human HCC cell line, HepG2, was purchased from ATCC (Manassas, VA).
The HCC cell line, BEL7402, was obtained from the Institute of Cell Biology,
Chinese Academy of Sciences (Shanghai, China). Cell lines were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone,
Boston, MA) and 100 μg/ml each of penicillin and streptomycin (Gibco,
Invitrogen, Carlsbad, CA) in 5% CO
2
at 37
°
C. Autophagic activity was evalu-
ated after 6 h of starvation performed in Hank’s balanced salt solution (HBSS,
Hyclone). For the LC3 turnover assay, cell lines were cultured in complete
medium and HBSS both with chloroquine (5μM, Sigma–Aldrich, St Louis, MO)
for 6 h. HCC cell lines were also pretreated with chloroquine (5μM) for 2 h to
detect the effect of autophagy on EMT and invasion of HCC cells. The TGF-β/
Smad3 signaling inhibitor, SIS3 (2μmol/l, Sigma–Aldrich), was used to pretreat
each cell line in complete medium and HBSS for 30 min in order to assess the role
of TGF-β/Smad3 signaling in EMT and invasion of HCC cells. In addition, HCC
cells transfected with small interfering RNA (siRNA)-Atgs (3 or 7)were treated
with recombinant human TGF-β1 (20 ng/ml, R&D System, Minneapolis, MN)
in HBSS for 6 h to evaluate the necessity of TGF-β1 in autophagy-induced EMT
and invasion. Cells that were cultured in complete medium served as control.
siRNA synthesis and transfection
The cDNA sequence of the Atg3 and Atg7 gene was obtained from Genebank
(NM_022488 and NM_006395) and the respective targeting sequences of
three different siRNAs were designed using RNAi algorithm available online
(http://www.ambion.com/techlib/misc/siRNA_nder.html). All siRNAs were
synthesized and puried by GenePharma (Shanghai, China). Synthesized
siRNAs were transfected into HepG2 and BEL7402 cells by TransLipid
Transfection Reagent (Beijing, China) according to the manufacturer’s
instruction. The siRNA (sense: 5′-GGGAAAGGCACUGGAAGUG-3′,
antisense: 5′-CACUUCCAGUGCCUUUCCC-3′) with the greatest silencing
effect of Atg3 and the siRNA (sense: 5′-ACUAAAAGGGGCAAACUGCAG-3′,
antisense: 5′-GCAGUUUGCCCCUUUUAGUAG-3′) with the greatest
silencing effect of Atg7 were identied by real-time PCR and were
transfected into HepG2 and BEL7402 cells for further studies. The
siRNA (sense: 5′-UCAGACAUGCAACGUCAGCU-3′, antisense:
5′-AGCCUUACGGGAAUCGAAUA-3′) was served as the siRNA-vector
control. After 48 h of transfection, cells were used for experiments. Cell
viability was evaluated by trypan blue.
Cell apoptosisassay
Apoptosis of HCC cell lines was quantitated by ow cytometry. Cells pre-
treated with chloroquine as well as transfected and non-transfected cells in
Abbreviations: CK18, cytokeratin 18; EMT, epithelial–mesenchymal transi-
tion; HBSS, Hank’s balanced salt solution; HCC, hepatocellular carcinoma;
MMP-9, matrix metalloproteinase 9; siRNA, small interfering RNA; TGF-β,
transforming growth factor-beta.
†
These authors contributed equally to this work.
1343
Downloaded from https://academic.oup.com/carcin/article-abstract/34/6/1343/2463238 by guest on 09 March 2019

J.Li etal.
complete medium and HBSS for 6 h were harvested, washed and incubated
with binding buffer containing propidium iodide (10 g/ml) and uorescein
isothiocyanate-labeled annexin V (Annexin V-APC) (Bender MedSystems,
eBioscience, San Diego, CA) for 15 min at room temperature. Samples were
analyzed by ow cytometry (BD LSR II, BD Biosciences, San Jose, CA).
Immunouorescence
Immunouorescence was performed on cells pretreated with chloroquine and
on transfected and non-transfected cells in complete medium and HBSS for 6
h by using an avidin–biotin peroxidase complex method. Immunouorescence
for cells that were treated with SIS3 (2μmol/l) or recombinant human TGF-β1
(20 ng/ml) was utilized to evaluate the role of TGF-β/Smad3 signaling on EMT
and matrix metalloproteinase 9 (MMP-9) expression levels during starvation.
Briey, cells were xed in 4% paraformaldehyde and permeabilized using
Triton X-100. Cells were then treated with 3% hydrogen peroxide to inacti-
vate endogenous peroxidase. Non-specic binding was blocked in phosphate-
buffered saline containing 10% species-appropriate normal serum for 1 h at
room temperature. Primary LC3 antibody (1:200, Cell Signaling Technology,
Beverly, MA), E-cadherin antibody (1:100, Abcam, Cambridge, MA), cytoker-
atin 18 (CK18) antibody (1:100, Boster, Wuhan, China), bronectin antibody
(1:100, Santa Cruz Biotechnology, Santa Cruz, CA) and MMP-9 antibody
(1:200, Abcam) were applied overnight at 4°C in a humidied chamber. Cells
were incubated with the appropriate secondary antibody and visualized by
peroxidase substrate in conjunction with uorescein isothiocyanate or Cy3
(Boster). The number of LC3 puncta per high power eld was counted by an
independent pathologist in a blinded fashion.
Western blotting
Western blotting was used to detect the expression of LC3, p62, Atg3, Atg7,
E-cadherin, CK18, bronectin, MMP-9, TGF-β1, Smad3 or phosphoryl-
ated Smad3 in transfected and non-transfected HCC cell lines with or with-
out starvation. Cells were lysed in radioimmunoprecipitation assay (RIPA)
buffer supplemented with a protease inhibitor cocktail (Roche, Branford, CT)
and phosphatase inhibitor cocktail (Cell Signaling Technology). Total pro-
tein (30μg) from each sample was electrophoresed on 12% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis gels. After being transferred to
nitrocellulose membranes (Pierce, Thermo Fisher Scientic, Waltham, MA),
protein samples were incubated with the corresponding primary antibodies.
Blots were incubated with the appropriate horseradish peroxidase-conjugated
secondary antibodies and the membranes were developed with SuperSignal™
chemiluminescence reagent (Pierce, Thermo Fisher Scientic) according
to the manufacturer’s protocol. Protein expression levels were normalized
against β-actin. Optical density of the bands was quantied using NIH Image J
(Supplementary Figures III–VI, available at Carcinogenesis Online).
Quantitative reverse transcription–polymerase chain reaction
After 2 h of pretreatment with chloroquine and 48 h of transfection with siRNA-
control vector or siRNA-Atgs (3 or 7), cells were cultured in complete medium
and HBSS for 6 h. RNA was then isolated by TRIzol™ Reagent (Invitrogen)
according to the manufacturer’s protocol. Total RNA was reverse transcribed
into rst-strand cDNA using an iScript cDNA Synthesis kit (Bio-Rad, München,
Germany). RNA expression was analyzed by reverse transcription–polymerase
chain reaction using iQ SYBR Green Supermix in an iCycler Real-Time PCR
Detection System (Bio-Rad). The following primer sequences were used—Atg3
sense: 5′-GGAAGAAGATG AAGATGAA-3′, antisense: 5′-CATAATCGTGG-
AGTCTGGTA-3′; Atg7 sense: 5′-CACA GATGGAGTAGCAGTT-3′,
antisense: 5′-TCACAGGATTGGAGTAGGA-3′; E-cadherin (19) sense:
5′-ACACCCCCTGTTGGTGTCTTT-3′, antisense: 5′-TGTATGTGGC-
AATGCGTTCTC-3′; CK18 (20) sense: 5′-CAAAGCCTGAGTCCTGTCCT-3′,
antisense: 5′-GAGATCCGGGAACCAGAG-3′; bronectin (21) sense:
5′-GGAGCAAATGGCACCGAGATA-3′, antisense: 5′-GAGCTGCACATGTC
TTGGGAAC-3′; MMP-9 (22) sense: 5′-GCAAGCTGGACTCGGTCTTT-3′,
antisense: 5′-TGGCGCCCAGAGAAGAAG-3′. Expression was normalized to
that of β-actin.
Invasionassay
Cell invasion was analyzed using Matrigel-coated invasion chambers con-
taining 8 μm pore lters (BD Biosciences). Briey, after transfection with
siRNA-control vector or siRNA-Atgs or pretreatment with chloroquine, each
cell line was seeded at a density of 1 × 10
5
cells/well in the upper Matrigel-
coated chamber of 24-well plates. HBSS was placed in the upper chamber.
SIS3 (2μmol/l) or recombinant human TGF-β1 (20 ng/ml) added to the upper
chamber was utilized to detect the inuence of TGF-β/Smad3 signaling on
invasion. Complete medium was placed in the lower chamber as a chemotactic
agent. Cells that were seeded in the upper chamber with complete medium
were served as control. Invasion assay systems were incubated at 37°C with
5% CO
2
for 6 h. Cells that invaded the Matrigel to the bottom of lter were
stained with 2 μg/ml 4′,6-diamidino-2-phenylindole in phosphate-buffered
saline and counted under a uorescent microscope.
Statistical analysis
All data were presented as mean ± SEM. After demonstration of homogene-
ity of variance with Bartlett test, one-way analysis of variance, followed by
Student–Newman–Keuls test where appropriate, was used to evaluate the sta-
tistical signicance. Values of P<0.05 were considered statistically signi-
cant. Experiments were performed in triplicate.
Results
Autophagy promotes invasion of HCCcells
Starvation or nutritional deciency is a common factor that upregu-
lates cell autophagy (4,5). HepG2 and BEL7402 cells were starved
in HBSS, which contained neither nutrients nor serum for 6 h. The
LC3 puncta formation assay revealed that LC3 puncta signicantly
increased (93 ± 5, P < 0.05; 83 ± 4, P < 0.05) in cells under starva-
tion. Increased conversion of LC3-I to LC3-II revealed by LC3 turno-
ver assay and decreased expression of P62 conrmed that starvation
induced autophagy in HCC cells (Supplementary Figure I, available
at Carcinogenesis Online).
The impact of starvation on HCC cell invasion was evaluated. As
depicted in Figure1A, HepG2 and BEL7402 cells, which were cul-
tured with HBSS in the upper chambers, exhibited greater invasion
rates than those cultured with complete medium (60 875 ± 1051 ver-
sus 15 813 ±  for HepG2 cells, P < .; 60 500 ±  versus
15 500 ±  for BEL7402 cells, P < .). Since starvation-induced
autophagy in HCC cells, the effect of autophagy on cell invasion was
further evaluated. The autophagic activity in HepG2 and BEL7402
cells was inhibited by transfection of siRNA-Atg3 and siRNA-Atg7.
Silencing of Atg3 or Atg7 has been reported to effectively inhibit
autophagy in other cell types (23,24). Silencing of Atg3 and/or
Atg7 by siRNA was conrmed at both the RNA and protein level
(Figure1 and B). Lack of LC3-I to LC3-II conversion and decreased
P62 expression levels conrmed inhibition of autophagy in HCC
cells transfected with siRNA-Atg3 or siRNA-Atg7 during starvation
(Figure1C). Inhibition of autophagy by siRNA-Atg3 or siRNA-Atg7
signicantly reduced the invasiveness of HCC during 6 h of incu-
bation in HBSS compared with control complete medium. In addi-
tion, the pharmacological inhibitor of autophagy, chloroquine, also
attenuated the invasion activity of HepG2 and BEL7402 cells under
starvation (Figure2A). However, neither inhibition of autophagy by
siRNAs nor by chloroquine exhibited obvious alterations in the apop-
tosis rate compared with control (Figure2B). These ndings suggest
that HCC cell invasion was dependent on autophagy under starvation.
Autophagy promotes invasion of HCC cells by inducingEMT
EMT is reported to contribute to metastatic and invasive mechanisms of
cancer cell metastasis (14,15). To explore the mechanism through which
autophagy conferred invasion ability on HCC cells, the expression of
epithelial and mesenchymal markers as well as MMP-9was evaluated in
HepG2 and BEL7402 cells with or without autophagy inhibition during
starvation by western blotting and immunouorescence. The epithelial
markers, E-cadherin and CK18, were signicantly expressed, whereas
the mesenchymal marker, bronectin, and invasion associated protein,
MMP-9, were not highly expressed by HepG2 and BEL7402 cells in
complete medium. During starvation, the expression of E-cadherin and
CK18 was downregulated, whereas that of bronectin and MMP-9 was
upregulated in non-infected cells and cells with control siRNA-vector
(Figures 3 and 4). In addition, cell morphology changed from circular
like to briform (Figure4). However, these morphological alterations
and changes in the expression of epithelial and mesenchymal markers
as well as MMP-9 did not occur in autophagy-decient cells with
siRNA-Atgs (3 or 7)or chloroquine during starvation compared with
control (Figures 3 and 4).
TGF-β/Smad3 signaling regulates autophagy-inducedEMT
TGF-β/Smad3 is one of the known signaling pathways that mediate
EMT (16,17). We found that TGF-β1 and phosphorylated Smad3
1344
Downloaded from https://academic.oup.com/carcin/article-abstract/34/6/1343/2463238 by guest on 09 March 2019

Autophagy-dependent EMT
were not highly expressed by HepG2 and BEL7402 cells in control
medium compared with starvation. However, TGF-β1 and phos-
phorylated Smad3 were expressed in non-transfected cells and cells
transfected with control siRNA-vector during starvation. Inhibition of
autophagy by siRNA-Atgs (3 or 7)or by chloroquine in HCC cells
suppressed both phosphorylation of Smad3 and expression of TGF-
β1 in HBSS during starvation indicating that expression of TGF-β1
and activation of TGF-β/Smad3 signaling occurred in an autophagy-
dependent manner during starvation (Figure5A).
Since inhibition of autophagy suppressed EMT and TGF-β/Smad3,
we further investigated whether blockade of this signaling was capa-
ble of suppressing EMT of HCC cells with autophagy during star-
vation. Administration of the inhibitor of TGF-β/Smad3 signaling,
SIS3 (2 μmol/l), inhibited the autophagy-induced phosphorylation
of Smad3 in HepG2 and BEL7402 cells during starvation compared
with control medium (Figure5B). Furthermore, in contrast to control
medium, inhibition of TGF-β/Smad3 signaling by SIS3 suppressed
autophagy-dependent morphological and marker expression changes
in HCC cells under starvation (Figure5 and C). As expected, SIS3
also decreased invasive number of HepG2 and BEL7402 cells during
starvation when EMT was inhibited by inactivity of TGF-β/Smad3
signaling (Figure5D).
TGF-β1 rescues EMT and invasion in autophagy-decient
HCCcells
Since autophagy-activated TGF-β/Smad3 signaling was associated
with EMT in HCC cells under starvation, we further tested whether
TGF-β1 was able to rescue EMT in autophagy-decient HCC cells
under starvation. As depicted in Figure6 and B, HepG2 and BEL7402
cells transfected with siRNA-Atgs (3 or 7)exhibited changes in the
expression of EMT markers and MMP-9 under starvation compared
with control medium. In contrast to control medium, administration
of the exogenous TGF-β1 (20 ng/ml) downregulated expression of
epithelial markers and upregulated expression of bronectin and
MMP-9 in autophagy-decient HCC cells under starvation. Since
autophagy-dependent EMT was required for invasion of HCC cells
during starvation, we found that exogenous TGF-β1 was also capable
of promoting invasion of HepG2 and BEL7402 cells with siRNA-Atgs
Fig.1. siRNA-Atgs (3 or 7)inhibit autophagy of HCC during starvation. siRNA-Atg3 and siRNA-Atg7 inhibited Atg3 and Atg7 expression and LC3 conversion
as well as abrogated a decrease in P62 expression in HepG2 and BEL7402 cells under starvation. (A) Expression of mRNA for Atg3 and Atg7 was determined
by quantitative reverse transcription–polymerase chain reaction. mRNA levels were normalized to β-actin. (B) Representative western blots of Atg3 and Atg7.
(C) Representative western blots of P62 and LC3 conversion assay. Cells cultured in complete medium without transfection were served as control. Data are
representative of three independent experiments and shown as mean ± SEM, n=4, *P<0.05 versus control.
1345
Downloaded from https://academic.oup.com/carcin/article-abstract/34/6/1343/2463238 by guest on 09 March 2019

J.Li etal.
(3 or 7) under starvation. These results suggest that autophagy-
induced TGF-β1 signaling plays a crucial role in EMT and invasion
of HCC cells under starvation (Figure6C).
Discussion
Due to metastasis that results from invasion of HCC cells, it
is difcult to cure many HCC patients by current therapeutic
interventions (2,3). Several publications have described a critical
role for autophagy in the invasion of central neuronal system
tumor cells (10). However, relatively little is known regarding the
precise contribution of autophagy to the pathogenesis of HCC cell
invasion. Here, we provide the rst evidence that autophagy plays
a central role in the invasion of HCC cells by inducing EMT under
starvation. Autophagy promotes invasion of HepG2 and BEL7402
cells via inducing EMT of HCC cells under starvation. Furthermore,
autophagy induces expression of TGF-β1 and activates TGF-β/
Smad3 signaling during starvation. Inhibition of autophagy by
siRNA-Atgs (3 or 7) or pharmacological inhibition results in
inactivation of TGF-β/Smad3 signaling and the prevention of
EMT and invasion of HCC cells under starvation. On the other
hand, inhibition of TGF-β/Smad3 signaling pathway by SIS3
suppresses both EMT and invasion of HCC cells in the presence of
autophagy during starvation. Finally, administration of exogenous
recombinant TGF-β1 was sufcient to rescue the EMT and invasion
of autophagy-decient HCC cells under starvation. These ndings
suggest that invasion of HCC cells promoted by autophagy under
starvation is dependent on TGF-β/Smad3 signaling andEMT.
Autophagy occurs in variety of malignant tumor cells, including
HCC cells (25), glioma cells (10) and glioblastoma stem cells (26).
Under these pathological conditions, autophagy can be triggered
by various stimuli, such as starvation, hypoxia and growth factor
Fig.2. Inhibition of autophagy prevented invasion of HCC cells. Absence of autophagy in cells transfected with siRNA-Atg3 or siRNA-Atg7 or pretreatment of
chloroquine had no notable effect on cellular activity and apoptosis rate after 6 h of starvation in complete medium. However, there was a signicant reduction
of the invasiveness of HCC cells during starvation. (A) Invasive number of HepG2 and BEL7402 cells with or without siRNA-Atgs (3 or 7)and chloroquine.
(B) Representative ow cytometry of HepG2 and BEL7402 cells with or without siRNA-Atgs (3 or 7)and chloroquine. Cells cultured in complete medium were
served as control. Data are representative of three independent experiments and shown as mean ± SEM, n=4, *P<0.05 versus control.
1346
Downloaded from https://academic.oup.com/carcin/article-abstract/34/6/1343/2463238 by guest on 09 March 2019

Autophagy-dependent EMT
withdrawal (4–6). As the blood supply enriched solid tumor, starvation
and/or hypoxia that results from decient angiogenesis or usage of
chemotherapeutics appears to be responsible for autophagy in HCC
(7,8). Autophagy is a genetically regulated and nely orchestrated
process of selective cell survival and cell apoptosis. Both starvation and
hypoxia have been shown to be crucial initiators of this process (4). In
this study, HCC cell autophagy was successfully achieved by starvation
in HBSS (Supplementary Figure I, available at Carcinogenesis Online).
In contrast, silencing of Atg3 or Atg7 or pretreatment with chloroquine
increases resistance of HCC cells to starvation-induced autophagy.
Autophagy has been implicated in the pathogenesis of cancer cell
invasion. In glioblastoma stem cells, autophagy accelerates their
Fig.3. Autophagy-induced EMT in HCC cells during starvation. After transfection of siRNA-Atg3, siRNA-Atg7 or pretreatment of chloroquine, HepG2 and
BEL7402 cells were starved in HBSS for 6 h. Inhibition of autophagy by siRNA-Atgs or chloroquine prohibited EMT of hepatocarcinoma cells under starvation.
(A) Representative western blots of epithelial markers, E-cadherin and CK18, as well as MMP-9 and mesenchymal marker, bronectin, in these cells. (B)
Expression of mRNA for E-cadherin and CK18 as well as MMP-9 and bronectin was determined by quantitative reverse transcription–polymerase chain
reaction. mRNA levels were normalized to β-actin. Cells cultured in complete medium were served as control. Data are representative of three independent
experiments and shown as mean ± SEM, n=4, *P<0.05 versus control.
1347
Downloaded from https://academic.oup.com/carcin/article-abstract/34/6/1343/2463238 by guest on 09 March 2019

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Autophagy promotes hepatocellular carcinoma cell invasion through activation of epithelial–mesenchymal transition jun li1,†, bin yang2,†, qi zhou3,†, yongzhong wu4,†, dan shang2,†, yu guo2, zifang song2, qichang zheng2jun xiong2,*" ?

In this paper, the authors investigated the role of autophagy in hepatocellular carcinoma ( HCC ) invasion. 

Q2. What was used to detect the expression of LC3 in HCC cells?

Western blotting was used to detect the expression of LC3, p62, Atg3, Atg7, E-cadherin, CK18, fibronectin, MMP-9, TGF-β1, Smad3 or phosphorylated Smad3 in transfected and non-transfected HCC cell lines with or without starvation. 

Q3. What are the epithelial markers that are downregulated in HCC cells?

the epithelial markers, E-cadherin and CK18, were downregulated, whereas fibronectin and MMP-9 were upregulated in HepG2 and BEL7402 cells during starvation-induced invasion. 

Q4. What is the role of siRNA-Atgs in preventing EMT?

Inhibition of autophagy by siRNA-Atgs (3 or 7)  or pharmacological inhibition results in inactivation of TGF-β/Smad3 signaling and the prevention of EMT and invasion of HCC cells under starvation. 

Q5. What is the role of E-cadherin in the formation of intercellular junctions?

Its extracellular domain mediates interactions with E-cadherin molecules on adjacent cells to form intercellular junctions (29,30). 

Q6. What is the role of autophagy in HCC?

As the blood supply enriched solid tumor, starvation and/or hypoxia that results from deficient angiogenesis or usage of chemotherapeutics appears to be responsible for autophagy in HCC (7,8). 

Q7. What was used to pretreat each cell line in complete medium and HBSS for 30?

The TGF-β/ Smad3 signaling inhibitor, SIS3 (2 μmol/l, Sigma–Aldrich), was used to pretreat each cell line in complete medium and HBSS for 30 min in order to assess the role of TGF-β/Smad3 signaling in EMT and invasion of HCC cells. 

Q8. What is the role of E-cadherin in the formation of the adherens?

E-cadherin is the epithelial molecular marker, as it is responsible for establishment of the adherens junction, which forms a continuous adhesive belt below the apical surface (29). 

Q9. What is the role of fibronectin in the invasion of HCC cells?

loss of E-cadherin, expression of fibronectin and MMP-9 as well as pseudopod formation stimulated starvation-induced EMT may be prerequisites for invasion of HCC cells. 

Q10. What is the effect of inhibition of autophagy on EMT?

Since inhibition of autophagy suppressed EMT and TGF-β/Smad3, the authors further investigated whether blockade of this signaling was capable of suppressing EMT of HCC cells with autophagy during starvation. 

Q11. What is the role of TGF-1 in preventing invasion of HCC cells?

administration of exogenous recombinant TGF-β1 was sufficient to rescue the EMT and invasion of autophagy-deficient HCC cells under starvation. 

Q12. What is the role of TGF-1 in the invasion of HCC cells?

Since autophagy-dependent EMT was required for invasion of HCC cells during starvation, the authors found that exogenous TGF-β1 was also capable of promoting invasion of HepG2 and BEL7402 cells with siRNA-AtgsFig. 

Q13. Why is it difficult to cure HCC patients?

Due to metastasis that results from invasion of HCC cells, it is difficult to cure many HCC patients by current therapeutic interventions (2,3). 

Q14. What is the role of TGF- in the invasion of HCC cells?

In summary, the findings reported here indicate that starvationinduced autophagy plays a crucial role in the invasion of HCC cells through activation of EMT, which involves the activation of TGF-β signaling. 

Q15. What is the role of autophagy in the progression of HCC?

In support of their findings, indicating that autophagy plays a key role in the progression of HCC, other studies have reported that autophagy protects HCC through a beclin-1-dependent mechanism (36). 

Q16. What is the effect of SIS3 on autophagy?

in contrast to control medium, inhibition of TGF-β/Smad3 signaling by SIS3 suppressed autophagy-dependent morphological and marker expression changes in HCC cells under starvation (Figure 5 and C). 

Q17. How was the expression of mRNA for E-cadherin and CK18?

(B) Expression of mRNA for E-cadherin and CK18 as well as MMP-9 and fibronectin was determined by quantitative reverse transcription–polymerase chain reaction. 

Q18. What is the effect of SIS3 on the invasive number of HepG2 and?

As expected, SIS3also decreased invasive number of HepG2 and BEL7402 cells during starvation when EMT was inhibited by inactivity of TGF-β/Smad3 signaling (Figure 5D). 

Q19. What was the protocol used to develop the membranes?

Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies and the membranes were developed with SuperSignal™ chemiluminescence reagent (Pierce, Thermo Fisher Scientific) according to the manufacturer’s protocol. 

Q20. What is the role of TGF-1 in EMT?

These results suggest that autophagyinduced TGF-β1 signaling plays a crucial role in EMT and invasion of HCC cells under starvation (Figure 6C). 

Q21. What are the main factors that can trigger autophagy?

Under these pathological conditions, autophagy can be triggered by various stimuli, such as starvation, hypoxia and growth factorFig.