Metformin sensitizes sorafenib to inhibit postoperative recurrence and metastasis of hepatocellular carcinoma in orthotopic mouse models
You et al. Journal of Hematology & Oncology
Metformin sensitizes sorafenib to inhibit postoperative recurrence and metastasis of hepatocellular carcinoma in orthotopic mouse models
Abin You 1 4
Manqing Cao 0 3
Zhigui Guo 1 4
Bingfeng Zuo 2
Junrong Gao 5
Hongyuan Zhou 1 4
Huikai Li 1 4
Yunlong Cui 1 4
Feng Fang 1 4
Wei Zhang 1 4
Tianqiang Song 1 4
Qiang Li 1 4
Xiaolin Zhu 1 4
Haifang Yin 2
Huichuan Sun 0 3
Ti Zhang 1 4
0 Liver Cancer Institute and Zhongshan Hospital, Fudan University , 180 Fenglin Road, Shanghai 200032 , China
1 Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy , 24 Bin Shui Road, Hexi District, Tianjin 300060 , People's Republic of China
2 Research Center of Basic Medical Science, Tianjin Medical University , Qixiangtai Road, Heping District, Tianjin 300070 , China
3 Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education , 180 Fenglin Road, Shanghai 200032 , China
4 Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy , 24 Bin Shui Road, Hexi District, Tianjin 300060 , People's Republic of China
5 Academy of Medical Image, Tianjin Medical University , Tianjin 300070 , People's Republic of China
Background: Sorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma (HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy. Methods: The protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The relationship between HIF-2α and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry. Results: Our study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression. (Continued on next page)
(Continued from previous page)
Conclusions: Metformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis
after liver resection by regulating the expression of HIF-2α and TIP30.
Hepatocellular carcinoma (HCC) is the fifth most
frequently diagnosed cancer in men and the seventh in
women worldwide [
]. Surgical resection has been regarded
as the main treatment for HCC [
postoperative recurrence and metastasis are still the main obstacles
for long-term survival of HCC [
Sorafenib is the first-line oral multi-kinase inhibitor
acting on advanced liver cancer. Some evidence suggest that
sorafenib can block Raf⁄MEK⁄ERK signaling pathway to
inhibit tumor cell proliferation and it can also target the
tyrosine kinase receptor vascular endothelial growth factor
receptor-2 (VEGFR-2) or platelet-derived growth factor
receptor (PDGFR) to produce inhibition of angiogenesis
]. However, sorafenib has been proved to have limited
survival benefits with very low response rates because of
drug resistance [
]. Hypoxic environment in solid tumor
is one of the vital factors for the treatment of resistance
]. Many evidence indicate that hypoxia-inducible
factors (HIFs) are essential for tumor cells to adapt to low
oxygen environments [
]. Recent research findings
revealed that sorafenib could reduce the expression of
HIF-1α, promoting the hypoxic response switch from
HIF-1α- to HIF-2α-dependent pathways, resulting in
upregulation of HIF-2α [
]. The high expression of
HIF-2α induced by sorafenib is the main cause for
HCC cells resistant to therapy in hypoxia [
It has been reported that, under certain circumstances,
anti-angiogenic drugs could heighten invasiveness and
increase lymphatic or distant metastasis [
previous results demonstrated that relatively low dosages of
sorafenib promoted HCC invasion and metastasis by
downregulating tumor suppressor gene HTATIP2, also
known as TIP30, which is a 30-kDa human cellular
protein that was purified as a HIV-1 Tat-interacting protein
]. Until now, the relationship between HIF-2α and
TIP30 has yet not been reported.
Metformin is a widely used drug for the treatment of type
2 diabetes [
]. Recently, some articles pointed out that
metformin may be associated with reduced cancer incidence in
diabetic patients [
], including prostate, breast, pancreas,
and liver cancer [
]. In addition, it was also reported
that metformin could target NLK (Nemo-like kinase) to
inhibit non-small cell lung cancer (NSCLC) cell proliferation
and stemness [
]. Here, we showed that metformin could
increase the sensitivity of HCC cells to sorafenib and inhibit
HCC recurrence and metastasis after surgical resection.
Metformin-reversed tumor cells resistance to sorafenib in hypoxia
MHCC97H cells were incubated under hypoxia (CoCl2)
for 24 h, and then incubated with various concentrations
of sorafenib for another 48 h. Both the normoxic and
hypoxic cells were suppressed in a dose-dependent manner,
but the hypoxic cells were more resistant to sorafenib
treatment than the normoxic cells (Fig. 1a). Hypoxic cells
possessed a higher level of HIF-2α proteins expression
compared to the cells under normoxia which indicated
that sorafenib could promote HIF-2α expression while
metformin had little effect on the expression of HIF-2α
(Fig. 1b). In addition, the combination of metformin and
sorafenib inhibited HIF-2α expression to a large degree,
indicating that metformin could regain hypoxic cells to be
sensitive to sorafenib treatment (Fig. 1c).
TIP30 was regulated by HIF-2α at protein level under
hypoxic conditions in HCC cell lines
Our previous work demonstrated that sorafenib could
promote the invasive and metastatic potential of HCC
by downregulating the tumor suppressor gene HTATIP2
]. MHCC97H cell line with stable knocking-down or
overexpression of HIF-2α were established by lentivirus
infection. Several articles have suggested that TIP30
knockdown could result in delayed EGFR endocytic
degradation and prolonged phosphorylation of AKT
(Ser473) and ERK1/2 (Thr202/Tyr204), indicating that
downregulation of TIP30 enhances EGFR signaling
]. Here, we found that knocking-down of
HIF2α could not only upregulate TIP30 expression in
MHCC97H and Hep3B cell lines (Fig. 2a, Additional
file 1: Figure S1) but also decrease the expression
levels of EGFR and its associated downstream
molecules, such as phosphorylated extracellular
signalregulated kinases (ERK) and phosphorylated AKT, as
determined by Western blot analysis (Additional file 2:
Figure S2). However, knocking-down of TIP30 could
not cause obvious changes of HIF-2α expression
(Fig. 2b). Remarkably, knocking-down of HIF-2α
promoted TIP30 expression (Fig. 2c) and overexpression
of HIF-2α impaired TIP30 expression (Fig. 2d).
Chromatin immunoprecipitation (CHIP) assays were
performed to determine whether HIF-2α can bind to the
TIP30 promoter, MHCC97H cells were cultured for
6 h at 400 μM CoCl2 and fixed with formaldehyde, and
then cell nuclei were isolated and sonicated. The
sonicated chromatin lysates were immunoprecipitated with
anti-IgG or anti-HIF-2α antibodies. Extracted DNA
was used to amplify a 214-bp fragment of TIP30
promoter. The amplified 214-bp product was only observed
in DNA extracted from hypoxia exposed samples
immunoprecipitated with HIF-2α antibody. No product was
amplified when nonspecific rabbit anti-IgG was used
(Fig. 2e). All these results showed that HIF-2α could bind
to the TIP30 promoter under hypoxic conditions.
Combined metformin and sorafenib therapy suppressed
EMT process and promoted apoptosis in hypoxia
To investigate whether metformin could regain tumor
cells sensitive to sorafenib, we implemented Cell Counting
Kit-8 (CCK8) cell viability assay in MHCC97H cells. The
cells were treated with metformin or sorafenib at diverse
concentrations for 48 h, and the impact at different time
points was evaluated. Metformin or sorafenib
monotherapy significantly inhibited the proliferation of MHCC97H
cells in a dose- and time-dependent manner, and
combination of metformin and sorafenib could further suppress
this effect (Fig. 3a, b). We next explored the effects of
metformin and sorafenib on tumor cell apoptosis. Flow
cytometry analysis showed that metformin or sorafenib
alone had no significant effects on tumor cell apoptosis.
However, combination of metformin and sorafenib
obviously induced apoptosis of MHCC97H cells (Fig. 3c). Our
previous work suggested that relatively low dosages of
sorafenib promoted HCC invasion and metastasis by
downregulating tumor suppressor gene TIP30 [
Recent evidence revealed that decreased TIP30 expression
could lead to epithelial-mesenchymal transition (EMT),
as well as enhance motility and invasion of HCC cells
]. It has been noted that the EMT may play a major
role in tumor invasion and metastasis [
]. These evidence
are all in consistent with our results. We concluded that
the combination of metformin and sorafenib
synergistically impeded EMT and decreased metastasis of HCC
(Fig. 3d) which indicated that metformin could enhance
the sensitivity of tumor cells to sorafenib.
Metformin synergized with sorafenib to suppress postoperative intrahepatic recurrence and lung metastasis in orthotopic HCC implantation models
We next examined whether the growth of hepatocellular
carcinoma, implanted in their native environment in the
liver parenchyma, could also be affected by metformin
and sorafenib treatment. Orthotopic tumor xenografts
were generated as described in the methods. Fourteen
days after the orthotopic implantation, a second operation
was carried out to remove the lobe where the tumor was
implanted. Then, mice were randomly divided into four
groups. On the third day after tumor resection, the mice
were orally treated either with 0.9 % sodium chloride
(control), 30 mg/kg sorafenib (sorafenib), 200 mg/kg
metformin (metformin), or 30 mg/kg sorafenib in
combination with 200 mg/kg metformin (sorafenib + metformin)
once daily. As shown in Fig. 4a, b, the relapse of
vehicletreated tumors grew remarkably fast 37 days after
commencement of treatment. In contrast, the recurrent
tumors treated with sorafenib or metformin were
significantly (both P < 0.05) smaller than vehicle-treated
recurrent tumors. The combination of sorafenib and
metformin further suppressed recurrent tumor growth,
which were much smaller than the vehicle-treated
recurrent tumors (P < 0.01), and significantly smaller than the
recurrent tumors treated with sorafenib (P < 0.05) or
metformin (P < 0.05) monotherapies. Paraffin blocks of 10 %
buffered formalin-fixed samples of the lung were
prepared, and serial sections were cut at 5 μm and stained
with H&E to determine the presence of lung metastases.
Metastatic nodules were not significantly reduced in
sorafenib-treated mice. However, the number of
metastases was remarkably decreased when treated with
metformin in combination with sorafenib (Fig. 4c, d).
Combined therapy inhibited tumor proliferation, angiogenesis and EMT but promoted apoptosis in vivo
There were fewer Ki67 expression in the metformin or
sorafenib treatment groups compared with the control
groups, and the combination therapy had even fewer
Ki67 positive cells. Metformin or sorafenib alone
reduced the CD31 expression, while metformin synergized
with sorafenib could further decrease tumor
angiogenesis. A small number of TUNEL-positive cells were
detected in the control group, whereas plenty of apoptotic
tumor cells were detected in metformin or sorafenib
monotherapy, the combination therapy resulted in even
more TUNEL-positive cells (Fig. 5a). EMT-associated
proteins from tumor tissue were detected using Western
blot and combined therapy inhibited the process of
EMT by upregulating E-cadherin and downregulating
N-cadherin protein expression (Fig. 5b). In addition,
combined treatment groups inhibited HIF-2α expression
but upregulated TIP30 expression at protein level which
were consistent with the results in vitro (Fig. 5c).
Surgical operation is the primary treatment for clinical
HCC patients. However, recurrence and metastasis are
the major factors leading to poor prognosis [
Sorafenib is the first-line treatment for advanced hepatocellular
carcinoma. However, resistance of tumor cells to sorafenib
is one of the main reasons for drug utilization . It seems
that finding an effective way to inhibit the resistance may
improve the efficiency of sorafenib.
Hypoxia is a prevalent phenomenon in solid tumor in
which HIF-2α plays an important role. Previous results
indicated that HIF-2α was connected with tumorigenesis,
invasion, and metastasis [
]. Here, we revealed that
metformin could sensitize hypoxic HCC cells to sorafenib
and synergistically suppress the expression of HIF-2α. It
has been reported that decreased TIP30 was associated
with EMT , and our previous results pointed out that
sorafenib promoted EMT process by inhibiting TIP30
]. In the present study, we found that overexpression of
HIF-2α led to downregulation of TIP30 and subsequently
promoted the process of EMT, and knocking-down of
HIF-2α had the opposite effects. In vitro CHIP assays
showed a 214-bp band was amplified immunoprecipitated
with anti-HIF-2α antibodies under hypoxic conditions.
More broadly, our data support a role for TIP30 as a
unique HIF-2α target gene involved in the regulation of
cancer recurrence and metastasis.
The previous article reported that exosome-mediated
transfer of miR-122 via microRNA (miRNA)-modified
adipose tissue-derived mesenchymal stem cells (AMSCs)
can enhance the chemosensitivity of HCC cells [
results showed that combination of metformin and
sorafenib inhibited HIF-2α expression to a large degree,
indicating that metformin could regain hypoxic HCC cells to be
sensitive to sorafenib treatment in vitro. Subsequently, an
orthotopic xenograft mouse model was established to
explore the influence of metformin combined with sorafenib
on recurrence and metastasis after surgical resection of
HCC. The subcutaneous tumor was removed and cut into
about 2 mm3 slice for in situ tumor implantation, and we
tried to ensure the uniformity of the tumor as far as
possible before implantation. But unavoidably, there may be
some disadvantages because the variations may exist in
cancer cells. Zhang et al. indicated that arsenic trioxide
(As2O3) induced HCC cancer stem cells (CSCs)
differentiation, inhibited recurrence, and prolonged survival after
hepatectomy by targeting GLI1 expression [
]. Here, we
found that metformin in combination with sorafenib
could significantly inhibit the recurrence and metastasis of
primary liver cancer in mice after surgical resection.
Lower dosage of sorafenib have been found to promote
invasion and metastasis of HCC cells [
], and we showed
that combined therapy could obviously reduce this effect.
In general, the above features of metformin and the
encouraging results presented herein warrant future
investigation of the use of metformin for combating HCC,
especially in combination with sorafenib.
Our data indicate that a low dose of sorafenib increases
the expression of HIF-2α which downregulated the
expression of TIP30 and then promotes HCC invasion and
metastasis. Metformin may potentially act as an enhancer
of sorafenib to inhibit HCC recurrence and metastasis
after surgical resection.
Cell culture and drugs
The highly metastatic human hepatocellular carcinoma cell
line MHCC97H obtained from at the Liver Cancer Institute
of Fudan University was maintained in Dulbecco’s modified
eagle’s medium (DMEM, Gibco, UK), supplemented with
10 % fetal bovine serum, 100 units/ml penicillin, and
100 mg/ml streptomycin and cultured in a 37 °C incubator
with 5 % CO2 in the air. Sorafenib (Bayer Healthcare,
Leverkusen, Germany) was dissolved in dimethyl sulfoxide
at a final concentration of 20 mM, and metformin
(BristolMyers Squibb, China) was dissolved in PBS at a final
concentration of 1 M for in vitro assay. Small hairpin RNA
(ShRNA) construct against HIF2α (Cat. No.
HSH004903LVRH1GP) and control (Cat. No. CSHCTR001-LVRH1GP),
lentiviral plasmid overexpress HIF-2α (Cat. No.
EX-M0910Lv105-5), overexpress control (Cat. No. EX-eGFP-Lv-105),
and Lenti-Pac™HIV Expression Packaging Kit (Cat. No.
HPK-LvTR-20) were all purchased from GeneCopoeia
Western blot analysis
Cells were lysed with the lysate containing 25 mM
TrisHCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 %
Nadeoxycholate, 0.1 % sodium dodecyl sulfate (SDS), and
1 % Triton X-100, as well as protease and phosphatase
inhibitors. Protein concentration was measured with
Bradford reagent (Sangon Biotech, Cat. No. B724DB0009).
At least 20 μg of sample was used for detecting the
protein expression. The antibodies used were as follows:
antiHIF-2 alpha/EPAS1 rabbit polyclonal antibody (1:1000,
Novus, Cat. No. NB100-122), anti-TIP30 rabbit
monoclonal antibody (1:1000, abcam, Cat. No. ab177961),
anti-ECadherin rabbit polyclonal antibody (1:1000, abcam, Cat.
No. ab15148), and anti-N-Cadherin rabbit polyclonal
antibody (1:1000, abcam, Cat. No. ab18203).
Animals and in vivo experiment
Male BALB/c nude mice weighed 18–20 g and aged
4–6 weeks were purchased from the Animal Research
Center, Beijing, China. To construct a nude
subcutaneous tumor model, 1 × 107 MHCC97H cells were
resuspended in 0.2 ml PBS and injected into the left
flank of the mouse. When the tumor volume reached
about 1 cm in diameter, the subcutaneous tumor was
removed and cut into about 2 mm3 slice, and the pieces
from one tumor of one mouse were reimplanted into all
the subsequent mice. The recipient mice were
anesthetized with pentobarbital sodium salt (1 %, 25 mg/kg), and
a small piece of tumor was implanted into the left lobe of
the liver. Fourteen days after the orthotopic implantation,
a second operation was carried out to remove the lobe
where the tumor was implanted. Then, the mice were
randomly divided into four groups. On the third day,
after tumor resection, the mice were orally treated
either with 0.9 % sodium chloride (control), 30 mg/kg
sorafenib (sorafenib) [
], 200 mg/kg metformin
], or 30 mg/kg sorafenib in combination
with 200 mg/kg metformin (sorafenib + metformin) once
daily. All the drugs were dissolved in 0.9 % sodium chloride.
The mice were treated for 37 days and killed 48 h after the
last treatment. The tumor volume (V) was measured with
vernier caliper and calculated with V = 1/2(length × width2).
The lung tissues were fixed with 10 % formaldehyde, and
the HCC tumor tissues were cut into two parts for
subsequent fixation with 10 % formaldehyde or frozen resection.
The tumor tissue samples and lung tissues were fixed
by 10 % formalin, embedded with paraffin, and cut into
5-mm-thick sections. In order to observe the metastatic
node in the lung, the lung tissues were stained with
hematoxylin and eosin. For immunohistochemistry, the
tumor tissue sections were deparaffinized, rehydrated,
subsequently subjected to antigen retrieval with 121 °C
for 5 min, and incubated with 3 % H2O2 for 10 min to
inactivate endogenous peroxidase. The specimens were
blocked with 10 % goat serum for 1 h, and then
incubated with primary antibody of Ki67 or CD31 overnight
in 4 °C. The next day, the slice was incubated with
secondary antibody for 1 h at room temperature. Then,
the slice was colored by DAB Substrate-Chromogen
System. TUNEL was detected using the TUNEL
Apoptosis Detection Kit (KeyGENBioTECH, China, Cat. No.
Cell viability and apoptosis assay
MHCC97H cells were plated in 96-well plates at 4000
cells/well (n = 6) containing 100 μl of DMEM +10 %
FBS treated with 400 μM CoCl2 and cultured for
24 h, then incubated with sorafenib or metformin for
another 48 h. DMEM (100 μl) and CCK8 (10 μl) were
added to each well and incubated for 2 h. Then, the
absorbance was detected with a microplate reader at
a test wavelength of 450 nm.
Annexin V/PI was applied to investigate the impact of
sorafenib or metformin on cell apoptosis. After
treatments, the cells were added with 5 μl Annexin V and
10 μl PI staining supplied by the Annexin V-FITC
Apoptosis Detection Kit (SANGON, Cat. No. BS6336), then the
results were measured by flow cytometry using a FACS
flow cytometer (Becton Dickinson verse, San Jose, CA).
Chromatin immunoprecipitation assay
MHCC97H cells were fixed at 37 °C for 10 min with 1 %
formaldehyde. The cells were then collected and lysed
on ice for 30 min in cell lysis buffer (50 mM EDTA, 1 %
SDS, 50 mM Tris-HCl) containing protease inhibitors
and 1 mM PMSF. Nuclear chromatin was broken into
small fragments by sonicating the nuclear lysate on ice
using a Misonix Sonicator 3000 equipped with a
microtip. One percent of the volume of samples (input) was
saved for the subsequent PCR analysis. The samples were
precleared with protein A Sepharose (GE Healthcare, Cat.
No. 10043746). Equal aliquots of precleared chromatin
samples were incubated overnight at 4 °C with either
specific rabbit HIF-2α or nonspecific rabbit antiserum IgG.
Immune complexes were collected by incubation with
20 ml protein A Sepharose at 4 °C for 4 h. The complexes
were washed once with buffer I (1 % Triton X-100, 2 mM
EDTA, 150 mM NaCl, 20 mM Tris-HCl, 0.1 % SDS),
buffer II (1 % Triton X-100,2 mM EDTA, 50 mM NaCl,
20 mM Tris-HCl, 0.1 % SDS), buffer III (0.23 mM LiCl,
1 % NP40, 1 % deoxycholate, 1 mM EDTA, 10 mM
Tris-HCl), and TE buffer (10 mM Tris-HCl (pH =
8.0),1 mM EDTA), respectively. Immune complexes
were then eluted from the beads by incubation twice
under agitation at 37 °C with 100 μl of elution buffer
(0.1 M NaHCO3, 1 % SDS). The eluted material and
input was cross-linked at 65 °C for 6 h.
Immunoprecipitated DNA was purified by PCR purification kits.
Primers used for PCR correspond to the TIP30
promoter region, primers: 5′ primer: 5′-CAAACTTAGG
AAGGG TCGCG-3′; 3′ primer: 5′-ATCAGAGCATC
All data were expressed as mean ± SD. The Student t test
was used for the comparison of measurable variants of the
two groups. P < 0.05 was defined as statistically significant.
All statistical analyses were done using statistical software
(SPSS 13.0 for Windows; SPSS, Inc., Chicago, IL).
Additional file 1: TIP30 was regulated by HIF-2α at protein level in
Hep3B. (DOCX 66.9 kb)
Additional file 2: Knocking-down of HIF-2α decreased the expression
levels of EGFR and its associated downstream molecules by upregulating
TIP30 expression. (DOCX 55.5 kb)
CCK8: Cell Counting Kit-8; CHIP: chromatin immunoprecipitation;
EMT: epithelial-mesenchymal transition; HCC: hepatocellular carcinoma;
HIF-2α: hypoxia-inducible factors-2α; PDGFR: platelet-derived growth factor
receptor; TIP30: 30-kDa HIV Tat-interacting protein (also called CC3 or
HTATIP2); VEGFR-2: vascular endothelial growth factor receptor-2.
The authors declare that they have no competing interests.
ABY, MQC, ZGG, BFZ, JRG, HYZ, HKL, YLC, FF, WZ, TQS, QL, and XLZ carried
out all the experiments, prepared figures and drafted the manuscript. ABY,
HFY, AND HCS participated in data analysis and interpretation of results. TZ
designed the study, participated in data analysis and interpretation of results.
All authors read and approved the manuscript.
This work was supported by the National Natural Science Foundation of China
(No. 81372635, No. 81101871, No. 81572434, and No. 81201644) and the Major
Program of Natural Science Foundation of Tianjin (No. 11JCZDJC18800).
1. Torre LA , Bray F , Siegel RL , Ferlay J , Lortet-Tieulent J , Jemal A . Global cancer statistics, 2012 . CA Cancer J Clin. 2015 ; 65 ( 2 ): 87 - 108 .
2. Qi X , Wang D , Su C , Li H , Guo X . Hepatic resection versus transarterial chemoembolization for the initial treatment of hepatocellular carcinoma: a systematic review and meta-analysis . Oncotarget . 2015 ; 6 ( 21 ): 18715 - 33 .
3. Lai EC , Fan ST , Lo CM , Chu KM , Liu CL , Wong J . Hepatic resection for hepatocellular carcinoma . An audit of 343 patients. Ann Surg . 1995 ; 221 ( 3 ): 291 - 8 .
4. Chen WT , Chau GY , Lui WY , Tsay SH , King KL , Loong CC , et al. Recurrent hepatocellular carcinoma after hepatic resection: prognostic factors and long-term outcome . Eur J Surg Oncol . 2004 ; 30 ( 4 ): 414 - 20 .
5. Shah SA , Cleary SP , Wei AC , Yang I , Taylor BR , Hemming AW , et al. Recurrence after liver resection for hepatocellular carcinoma: risk factors, treatment, and outcomes . Surgery . 2007 ; 141 ( 3 ): 330 - 9 .
6. Cheng AL , Kang YK , Chen Z , Tsao CJ , Qin S , Kim JS , et al. Efficacy and safety <?show [?A3B2 twb= .27w?]?><?show [? A3B2 tlsb=- .09pt?] ?>of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial . Lancet Oncol . 2009 ; 10 ( 1 ): 25 - 34 .
7. Wilhelm SM , Carter C , Tang L , Wilkie D , McNabola A , Rong H , et al. BAY 43 - 9006 exhibits broad spectrum oral antitumor activity and targets the RAF/ MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis . Cancer Res . 2004 ; 64 ( 19 ): 7099 - 109 .
8 . Chang YS , Adnane J , Trail PA , Levy J , Henderson A , Xue D , et al. Sorafenib (BAY 43 -9006) inhibits tumor growth and vascularization and induces tumor <?show [?A3B2 twb= .27w?]?><?show [? A3B2 tlsb=- . 15pt?] ?>apoptosis and hypoxia in RCC xenograft models . Cancer Chemother Pharmacol . 2007 ; 59 ( 5 ): 561 - 74 .
9. Gadaleta-Caldarola G , Divella R , Mazzocca A , Infusino S , Ferraro E , Filippelli G , et al. Sorafenib: the gold standard therapy in advanced hepatocellular carcinoma and beyond . Future Oncol . 2015 ; 11 ( 16 ): 2263 - 6 .
10. Bottsford-Miller JN , Coleman RL , Sood AK . Resistance and escape from antiangiogenesis therapy: clinical implications and future strategies . J Clin Oncol . 2012 ; 30 ( 32 ): 4026 - 34 .
11. Gordan JD , Simon MC . Hypoxia-inducible factors: central regulators of the tumor phenotype . Curr Opin Genet Dev . 2007 ; 17 ( 1 ): 71 - 7 .
12. Menrad H , Werno C , Schmid T , Copanaki E , Deller T , Dehne N , et al. Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids . Hepatology . 2010 ; 51 ( 6 ): 2183 - 92 .
13. Zhao D , Zhai B , He C , Tan G , Jiang X , Pan S , et al. Upregulation of HIF2alpha induced by sorafenib contributes to the resistance by activating the TGF-alpha/EGFR pathway in hepatocellular carcinoma cells . Cell Signal . 2014 ; 26 ( 5 ): 1030 - 9 .
14. Paez-Ribes M , Allen E , Hudock J , Takeda T , Okuyama H , Vinals F , et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis . Cancer Cell . 2009 ; 15 ( 3 ): 220 - 31 .
15. Zhang W , Sun HC , Wang WQ , Zhang QB , Zhuang PY , Xiong YQ , et al. Sorafenib down-regulates expression of HTATIP2 to promote invasiveness and metastasis of orthotopic hepatocellular carcinoma tumors in mice . Gastroenterology . 2012 ; 143 ( 6 ): 1641 - 9 .
16. Kahn BB , Alquier T , Carling D , Hardie DG . AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism . Cell Metab . 2005 ; 1 ( 1 ): 15 - 25 .
17. Evans JM , Donnelly LA , Emslie-Smith AM , Alessi DR , Morris AD . Metformin and reduced risk of cancer in diabetic patients . BMJ . 2005 ; 330 ( 7503 ): 1304 - 5 .
18. Libby G , Donnelly LA , Donnan PT , Alessi DR , Morris AD , Evans JMM . New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes . Diabetes Care . 2009 ; 32 ( 9 ): 1620 - 5 .
19. Kisfalvi K , Eibl G , Sinnett-Smith J , Rozengurt E. Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth . Cancer Res . 2009 ; 69 ( 16 ): 6539 - 45 .
20. Liu B , Fan Z , Edgerton SM , Deng XS , Alimova IN , Lind SE , et al. Metformin induces unique biological and molecular responses in triple negative breast cancer cells . Cell Cycle . 2009 ; 8 ( 13 ): 2031 - 40 .
21. Kisfalvi K , Moro A , Sinnett-Smith J , Eibl G , Rozengurt E . Metformin inhibits the growth of human pancreatic cancer xenografts . Pancreas . 2013 ; 42 ( 5 ): 781 - 5 .
22. Donadon V , Balbi M , Mas MD , Casarin P , Zanette G . Metformin and reduced risk of hepatocellular carcinoma in diabetic patients with chronic liver disease . Liver Int . 2010 ; 30 ( 5 ): 750 - 8 .
23. Dong S , Zeng L , Liu Z , Li RL , Zou YY , Li Z , et al. NLK functions to maintain proliferation and stemness of NSCLC and is a target of metformin . J Hematol Oncol . 2015 ; 8 ( 1 ): 120 .
24. Li A , Zhang C , Gao S , Chen F , Yang C , Luo R , et al. TIP30 loss enhances cytoplasmic and nuclear EGFR signaling and promotes lung adenocarcinogenesis in mice . Oncogene . 2013 ; 32 ( 18 ): 2273 - 81 .
25. Zhang C , Mori M , Gao S , Li A , Hoshino I , Aupperlee MD , et al. Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors . Cancer Res . 2010 ; 70 ( 24 ): 10224 - 33 .
26. Zhu M , Yin F , Fan X , Jing W , Chen R , Liu L , et al. Decreased TIP30 promotes Snail-mediated epithelial-mesenchymal transition and tumor-initiating properties in hepatocellular carcinoma . Oncogene . 2015 ; 34 ( 11 ): 1420 - 31 .
27. Yang J , Weinberg RA . Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis . Dev Cell . 2008 ; 14 ( 6 ): 818 - 29 .
28. Patel SA , Simon MC . Biology of hypoxia-inducible factor-2alpha in development and disease . Cell Death Differ . 2008 ; 15 ( 4 ): 628 - 34 .
29. Gordan JD , Bertout JA , Hu CJ , Diehl JA , Simon MC . HIF-2alpha promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity . Cancer Cell . 2007 ; 11 ( 4 ): 335 - 47 .
30. Lou G , Song X , Yang F , Wu S , Wang J , Chen Z , et al. Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma . J Hematol Oncol . 2015 ; 8 ( 1 ): 122 .
31. Zhang KZ , Zhang QB , Zhang QB , Sun HC , Ao JY , Chai ZT , et al. Arsenic trioxide induces differentiation of CD133+ hepatocellular carcinoma cells and prolongs posthepatectomy survival by targeting GLI1 expression in a mouse model . J Hematol Oncol . 2014 ; 7 : 28 .
32. Buzzai M , Jones RG , Amaravadi RK , Lum JJ , DeBerardinis RJ , Zhao F , et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth . Cancer Res . 2007 ; 67 ( 14 ): 6745 - 52 .