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SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in hepato-cellular carcinoma
Zheng Yun Zhang, Doopyo Hong, Seung Hoon Nam, Jong Man Kim, Yong HanPaik, Jae Won Joh, Choon Hyuck David Kwon, Jae Berm Park, Gyu-SeongChoi, Kyu Yun Jang, Cheol Keun Park, Sung Joo Kim
PII: S0168-8278(14)00546-7DOI: http://dx.doi.org/10.1016/j.jhep.2014.08.007Reference: JHEPAT 5284
To appear in: Journal of Hepatology
Received Date: 28 January 2014Revised Date: 23 July 2014Accepted Date: 7 August 2014
Please cite this article as: Zhang, Z.Y., Hong, D., Nam, S.H., Kim, J.M., Paik, Y.H., Joh, J.W., Kwon, C.H.D., Park,J.B., Choi, G-S., Jang, K.Y., Park, C.K., Kim, S.J., SIRT1 regulates oncogenesis via a mutant p53-dependentpathway in hepatocellular carcinoma, Journal of Hepatology (2014), doi: http://dx.doi.org/10.1016/j.jhep.2014.08.007
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SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in
hepatocellular carcinoma
Zheng Yun Zhang MD, PhD1,2,*; Doopyo Hong PhD2,*; Seung Hoon Nam Ms2;
Jong Man Kim MD4; Yong Han Paik MD, PhD5; Jae Won Joh MD4; Choon
Hyuck David Kwon MD4; Jae Berm Park MD4; Gyu-Seong Choi MD4; Kyu Yun
Jang MD, PhD6; Cheol Keun Park MD, PhD7; Sung Joo Kim, MD, PhD2,3,4
1Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth
People's Hospital, Shanghai, China
2Transplantation Research Center, Samsung Medical Center, Samsung
Biomedical Research Institute, Sungkyunkwan University School of Medicine,
Seoul, Republic of Korea
3Sarcoma Research Center, Samsung Medical Center, Samsung Biomedical
Research Institute, Sungkyunkwan University School of Medicine, Seoul,
Republic of Korea
4Department of Surgery, Samsung Medical Center, Samsung Biomedical
Research Institute, Sungkyunkwan University School of Medicine, Seoul,
Republic of Korea
5Department of Medicine, Samsung Medical Center, Samsung Biomedical
Research Institute,, Sungkyunkwan University School of Medicine, Seoul,
Republic of Korea
6Department of Pathology, Chonbuk National University Medical School and
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Research Institute of Clinical Medicine, Jeonju, Republic of Korea
7Department of Pathology, Samsung Medical Center, Samsung Biomedical
Research Institute, Sungkyunkwan University School of Medicine, Seoul,
Republic of Korea
*Zheng Yun Zhang and Doopyo Hong contributed equally to this work.
Key words: SIRT1; AMPK; mTOR; metformin; p53
Corresponding author: Sung Joo Kim, MD, PhD
E-mail: [email protected]
Telephone: +82-2-2148-7308
Fax: +82-2-2148-7379
Address reprint requests to Sung Joo Kim, MD, PhD. Department of Surgery,
Samsung Medical Center, Sungkyunkwan University School of Medicine, 50
Ilwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea.
Co-corresponding author: Cheol Keun Park PhD MD
E-mail: [email protected]
Telephone: +82-2-3410-2766
Fax: +82-2-2148-7379
Address reprint requests to Sung Joo Kim, MD, PhD. Department of
Pathology,
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Samsung Medical Center, Sungkyunkwan University School of Medicine, 50
Ilwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea.
Electronic word count: 5364 words (abstract; 247 words)
Number of figures and tables: 4 figures and 3 tables
.
The authors have no conflicts of interest.
Financial support: this study was supported by a Sungkyunkwan University
grant, Research Fund (GT10), 2012 and Shanghai Pujiang
Program (14PJ1407300).
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Abstract
Background & Aims: SIRT1 is a class III histone deacetylase that plays
diverse roles in various cancers. However, the clinical significance of SIRT1 in
hepatocellular carcinoma (HCC) remains unknown.
Methods: We analyzed p53 mutations and the activation of SIRT1 in 252
hepatitis B virus-positive HCC cases. None of the patients had been subjected
to pre-operative treatment.
Results: We assessed 57 p53 mutations from 248 HCC tissues. Activated
SIRT1 (phosphorylated form of Ser47) predicted a longer RFS but not a
longer OS with mutant p53 (RFS: P = 0.007, OS: P=0.280). In multivariate
analysis, activated SIRT1 remained a significant predictor of longer RFS
(OR=0.307, CI: 0.143-0.660, P=0.002). Analysis of 248 paired specimens
revealed a significant correlation between activated SIRT1 (Ser47) and
activated AMPK (Thr172) in HCC tissues harboring mutant p53 (P=0.003, n =
57). The combination of these 2 parameters was a powerful predictor for a
good prognosis in such patients. In in vitro studies, SIRT1 inactivation
stimulated the growth of HCC cells bearing mutated p53 by suppressing
AMPK activity and subsequently enhancing mammalian target of rapamycin
(mTOR) activity, resulting in induction of p70S6K1 activation in HCC cells.
Metformin, an AMPK activator, more strongly suppressed cell growth in p53-
mutant cell lines with inactive SIRT1 than in p53-mutant cell lines with active
SIRT1.
Conclusions: SIRT1 exerted anti-carcinogenic effects via the AMPK-mTOR
pathway in HCC in the context of mutant p53. Metformin could be a
therapeutic drug for HCC in patients with mutated p53, inactivated SIRT1, and
AMPK expression.
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Introduction
Hepatocellular carcinoma (HCC) is the fifth most common malignant
tumor worldwide and the third most common cause of cancer-related deaths
[1]. Despite the establishment of liver resection as a first-line treatment for
HCC, patients with HCC still face a high incidence of postoperative recurrence
and unsatisfactory survival rates. Consequently, identification of the biological
molecules and pathways involved in the oncogenesis of HCC remains a major
focus of researchers in this field.
Silent mating-type information regulation 2 homolog 1 (SIRT1), an
NAD+-dependent histone deacetylase, mediates cell survival via the
deacetylation of key cell cycle molecules and apoptosis regulatory proteins [2,
3]. SIRT1 deacetylase is also involved in the cell stress response and can
directly deacetylate p53, a critical tumor suppressor protein associated with
the DNA damage repair system and cell cycle arrest in response to DNA
damage. SIRT1 has also been reported to promote telomere maintenance via
the regulation of telomerase and the sheltering complex (4) and plays an
important role in genome stability [4, 5, 6, 7]. Recently, SIRT1 has been
shown to act as an oncoprotein in many cancers, including B-cell lymphoma,
ovarian cancer, and breast cancer. In addition, SIRT1 has been reported to
function as a tumor promoter in HCC [8, 9, 10, 11, 12, 13, 14].
AMP-activated protein kinase (AMPK) functions as a type of biosensor
in living cells, protecting cells from stresses such as energy depletion and
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maintaining energy homeostasis via the down-regulation of catabolism [5].
Through these activities, AMPK is generally thought to be vital for proper
metabolism in living organs and has been reported to act as a tumor
suppressor via a pathway involving liver kinase B1 (LKB1). However, the role
of SIRT1 in AMPK signaling and the clinical relevance of SIRT1/AMPK
signaling in HCC remains unknown.
This study sought to identify the relevance of SIRT1, AMPK, and p53
in HCC using in vitro, in vivo and clinical studies. Our data show that SIRT1
inhibits the progression of HCC via AMPK in the context of mutant p53 and
may therefore serve as a prognostic factor for HCC patients with mutant p53.
Materials and methods
Detailed materials and methods are described in the Supplementary
Materials and Methods.
Results
SIRT1 and AMPK activity in primary HCC and paired adjacent nontumor
liver tissue
To identify differences in the activation of SIRT1 and AMPK in primary
HCC and paired adjacent nontumor tissues, we performed western blot for the
phosphorylation of Ser47 in SIRT1, which has been suggested to mediate the
activation of SIRT1 [15, 16], and for Thr172 in AMPK. More phosphorylated
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SIRT1 and less phosphorylated AMPK were observed in tumor tissues than in
nontumor tissues (from N2 to T; Supplementary Fig. 1). These results suggest
that SIRT1 activity may be increased, whereas AMPK activity is decreased,
during carcinogenesis.
SIRT1 expression and activity in HCC and nontumor tissues
To further establish the significance of SIRT1 expression and activity
in HCC, we measured SIRT1 expression and activity by western blotting in
primary HCC (T) and paired adjacent nontumor liver tissues (N2 tissues,
abbreviated as N below) in patient group 2 (n = 130; Supplementary Fig. 2A).
Approximately one-half of the examined cases (53.8%) had SIRT1 expression
in HCC tissue, and this level was significantly higher than that observed in
nontumor tissues (7.7%; P < 0.001). Similarly, a significantly higher proportion
of cases (38.5%) demonstrated activated SIRT1 in tumor tissues than in
nontumor tissues (6.2%; P < 0.001; Supplementary Fig. 2B). In particular, 10
of 130 nontumor tissues exhibited SIRT1 expression, among which 8 tissues
(80%) were positive for activated SIRT1. In contrast, 70 of 130 tumor tissues
exhibited SIRT1 expression, among which 50 tissues (71.4%) were positive
for activated SIRT1, suggesting that activated SIRT1 is correlated with SIRT1
expression in nontumor and HCC tissues (Supplementary Fig. 2C). In addition,
there were significantly higher expression levels of activated SIRT1 in HCC
than in nontumor tissues (Supplementary Fig. 2D).
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Next, large-scale analysis of SIRT1 expression and activity was
performed using western blots of an additional 122 resectable HCC and
paired adjacent nontumor liver tissues from patient group 3. The
clinicopathological parameters influencing overall and relapse-free survival
(OS and RFS, respectively) in these HCC patients (n =252) are summarized
in Supplementary Table 1. No prognostic significance of SIRT1 expression or
activation was found (SIRT1 expression: P = 0.153 for OS and P = 0.932 for
RFS; activated SIRT1: P = 0.717 for OS and P = 0.795 for RFS;
Supplementary Fig. 3). Multivariate analysis of the clinicopathological
parameters influencing OS and RFS are shown in Supplementary Table 2.
These data support the results of previous studies and suggest that SIRT1
has no significant effect on survival in HCC patients.
Identification of p53 mutations in HCC and nontumor tissues
To further understand the role of SIRT1 activation in HCC, we
examined p53 mutations in our HCC samples because SIRT1 has been
shown to mediate p53 signaling via deacetylation [3].
Detailed information describing these p53 mutations is provided in
Supplementary Table 3. Fifty-eight of 252 (23.0%) HCC tissues harbored p53
mutations, whereas only 4 of 252 (1.6%) nontumor tissues demonstrated
mutated p53 (Supplementary Table 4), indicating that p53 mutations are more
frequent in HCC than nontumor tissues. Among the 4 cases, 3 cases had p53
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mutation only in the nontumor tissues, but not in the HCC tissues. However
one case had p53 mutation in both of nontumor and HCC tissue, suspected to
have Li Fraumeni Syndrome with no information. All about 4 mutations, we
regarded that nontumor tissues were contaminated with tumor cells.
Therefore, we excluded these 4 cases from this study thereafter (total n = 248,
57 have p53 mutation in tumor tissues).
Identification of p53 loss of function
To identify mutations leading to the loss of p53 function, we analyzed
the identified p53 mutations using the p53.free.fr database, which is available
at http://p53.free.fr, and the MUT-TP53 program (Supplementary Table 3). To
define the effects of these mutations on the biological activity of p53, we
performed western blotting for p21 expression, the most important p53 target.
Among 57HCC tissues with p53 mutations, we failed to detect p21 expression
in all but 1 case (Supplementary Fig. 4). These data validate the notion that
tissues with p53 mutations have no p53 function or biological activity,
indicating that these 57 HCC tissues with p53 mutations were useful used for
further studies.
p53 mutations are associated with SIRT1 and predict patient survival
The clinicopathological characteristics of 248 HCC cases according to
p53 mutation status are summarized in Supplementary Table 5. The
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clinicopathological characteristics of patients with p53 mutations did not
significantly differ from those with wild-type p53. However, a significantly
higher proportion of tissues with mutant p53 demonstrated activated SIRT1
than did tissues with wild type p53 (63.2% vs. 31.4%, respectively, P < 0.001;
Table 1), suggesting that activated SIRT1 was correlated with p53 mutation
status. Next, we performed survival analysis by stratifying the cases according
to p53 mutational status, and the results showed that activated SIRT1 did not
predict OS or RFS in cases with wild-type p53 (P = 0.565 and P = 0.330,
respectively, n = 191; Fig. 1A,B). However, activation of SIRT1 predicted
longer RFS but not OS in cases with mutant p53 (RFS: P = 0.007, n = 57; OS:
P = 0.280, n = 57; Fig. 1C, D). In univariate and multivariate Cox regression
analysis, activated SIRT1 in HCC independently predicted a lower risk of
disease recurrence in p53 mutant cases (odds ratio [OR]: 0.307; 95%
confidence interval [CI]: 0.143 – 0.660; P = 0.002; Table 2). However,
activated SIRT1 did not serve as an independent risk factor for patients with
wild-type p53 (Supplementary Table 6). Moreover, activated SIRT1 was
negatively associated with satellite nodules in HCC in the context of mutant
p53 (OR: -0.291, P=0.028; Supplementary Table 7), and neither p53 mutation
nor activated SIRT1 was correlated with disease stage (Supplementary Table
8).
AMPK expression and activity in HCC and nontumor tissues
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AMPK has been shown to act as a tumor suppressor and be
associated with SIRT1 signaling [16]. Therefore, to analyze the correlation
between SIRT1 and AMPK in HCC formation, we examined AMPK expression
and activity in HCC tissues (n = 248). Western blotting analysis indicated that
100% of the cases had positive AMPK expression in both nontumor and HCC
tissues (Fig. 2A). Moreover, a significantly higher proportion of nontumor
tissues exhibited activated AMPK (97.0% versus 47.2% in nontumoral tissues
versus HCC tissues, respectively; P < 0.001; Fig. 2B). In addition, the relative
expression level of total AMPK in nontumor tissue was significantly higher
than that in HCC tissues (Fig. 2C).
Analysis of 248 paired specimens revealed a strong correlation between
activated SIRT1 and activated AMPK in HCC tissues harboring mutant p53 (n
= 57) but not in HCC tissues with wild-type p53 (n = 191; Table 3). Moreover,
HCC patients with mutant p53, activated SIRT1, and activated AMPK
demonstrated significantly better prognoses in terms of RFS than patients
with inactive SIRT1 and AMPK (Fig. 1E). In patients with wild-type p53,
disease outcomes were not noticeably different in pairwise comparisons (Fig.
1F). These data suggest that simple evaluation of SIRT1 and AMPK activation
may allow for more accurate predictions for the prognosis of HCC patients
with mutant p53.
The role of SIRT1 in the p53 pathway in HCC cells
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To confirm our clinical outcomes, we conducted in vitro and in vivo
studies using HepG2 HCC cells, which express wild-type p53, and PLC5 HCC
cells, which express mutant p53. We also used the p53-null HCC cell line
Hep3B to confirm our results following transfection with wild-type or mutant
p53. SIRT1 silencing by shRNA resulted in the acetylation of p53 in HepG2
(HepG2-shSIRT1) and PLC5 (PLC5-shSIRT1) cells. In HepG2 cells, p21 was
upregulated, demonstrating that p53 signaling was activated, whereas p21
was not induced in PLC5 cells despite the activation of p53. To further confirm
the above findings, Hep3B cells were transfected with a retrovirus expressing
wild-type or mutant (H168R) p53 (p53WT or p53MUT, respectively) and a
lentivirus containing shSIRT1. Consistent with our previous results, p53
signaling induced p21 expression in Hep3B-shSIRT1-p53WT cells but not in
Hep3B-shSIRT1-p53MUT cells despite p53 acetylation as mediated by SIRT1
knockdown (Supplementary Figs. 5A and 7A). Because knockdown of SIRT1
does not provide a full explanation for the role of SIRT1 in p53 signaling, we
over-expressed the SIRT1 gene in all of the cell lines. In contrast to SIRT1
knockdown in HepG2 cells, SIRT1 over-expression in HepG2 cells (HepG2-
hSIRT1) yielded different results: although SIRT1 was activated, p53
activation was unchanged, and p21 retained its activation compared with in
scramble transfected HepG2 cells. By contrast in PLC5 cells (PLC5-hSIRT1),
the acetylated form of p53 was present at lower levels when comparing with
scramble transfected cells (PLC5-scramble), and p21 was not detected in
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either cells. In Hep3B (Hep3B-hSIRT1) cells, the activation of p53 and p21
was below detectable levels.(Supplementary Fig. 6B). These results show
that wild type p53 has bypass signaling from the deacetylation activity of
SIRT1, repressed conditions generated by SIRT1 over-expression, in addition
this was confirmed by the increasing level of p21 expression in HepG2 cells.
However, the results of SIRT1 overexpression in mutant p53 cell lines agree
with results from knockdown cells. From these results, SIRT1 knockdown or
over-expression results in the alteration of p53 acetylation, suggesting that
SIRT1 acts as a deacetylase regardless of the p53 genotype.
The role of SIRT1 in the AMPK-mTOR pathway
AMPK is an important protein involved in sensing and mediating
metabolic stress and has been shown to play a crucial regulatory role in
normal and malignant cells. Moreover, activation of AMPK has been shown to
suppress mammalian target of rapamycin (mTOR) signaling and negatively
control malignant transformation and cell proliferation [17, 18, 19]. We found
that silencing SIRT1 results in a decrease in phosphorylated AMPK and an
increase in mTOR and p70S6K1 phosphorylation in PLC5 cells. In contrast,
silencing SIRT1 in HepG2 cells (expressing wild-type p53) results in
increased AMPK phosphorylation and leads to a reduction in the phospho-
mTOR and phospho-p70S6K1 levels. Consistent with these findings, the
results obtained for the Hep3B-shSIRT1-p53MUT cells were similar to those
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for PLC5 cells, whereas the results for the Hep3B-scramble-p53WT cells were
similar to those for HepG2 cells (Supplementary Figs. 5B and 7B). To identify
the cause of the reduction in mTOR phosphorylation in HepG2-shSIRT1 and
Hep3B-shSIRT1-p53WT cells, we analyzed the expression and activation of
components of the PTEN-PI3K-AKT pathway. SIRT1 knockdown led to up-
regulation of PTEN expression and down-regulation of AKT signaling, and this
effect was not observed in PLC5-shSIT1 and Hep3B-shSIRT1-p53MUT cells
(Supplementary Figs. 5C and 7C). To further confirm these results, we
observed these pathways in SIRT1 over-expressing cell lines. Similar to the
SIRT1 knockdown results, the activated AMPK level was induced by SIRT1
overexpression in HepG2-hSIRT1 cells. Moreover, despite the increased
phopho-AMPK levels, there was induction of activated mTOR and p70S6K1,
an mTOR target protein. In addition, SIRT1 over-expressing HepG2 cell lines
had increased AKT activation. In contrast, PLC5-hSIRT1 and Hep3B-hSIRT1
cells had inverted results compared with SIRT1 knockdown cells (PLC5-
shSIRT1, Hep3B-shSIRT1-p53MUT) (Supplementary Fig. 6A, B). Given the
above results, we asked whether there is an unknown factor that modulates
AMPK and SIRT1 and hypothesized that SESTRINs are the most likely
candidates in p53 wild type HCC cell lines.
SESTRIN1 and SESTRIN2 activate AMPK, suppress the activation of
mTORC1, and mediate many p53-dependent processes, including
metabolism and the generation of reactive oxygen species (ROS). Thus,
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these factors act as linkers between p53 and AMPK signaling [20]. Therefore,
we sought to determine whether the inhibition of sestrin1 and sestrin2
mediates crosstalk between SIRT1 and AMPK in HCC cells expressing wild-
type p53. As expected, knockdown of SESTRIN1/2 resulted in a decrease in
phospho-AMPK and phospho-SIRT1 signals. Moreover, treatment with
metformin, an AMPK activator, increased SIRT1 phosphorylation in
SESTRIN1/2-knockdown cells, suggesting that there may be crosstalk
between AMPK and SIRT1 signaling in these cells (Supplementary Fig. 5D).
Effects of SIRT1 on cell proliferation and colony formation in vitro
To address the effects of SIRT1 on cell proliferation, we measured cell
proliferation and colony formation following transfection with two shSIRT1
constructs in the HepG2, PLC5, and Hep3B cell lines and SIRT1 over-
expression constructs in the same cell lines. Significant inhibition of cell
proliferation was observed in SIRT1-knockdown HepG2 cells, whereas
enhanced proliferation was observed in SIRT1-knockdown PLC5 cells (P <
0.01; Fig. 3A, B). SIRT1 over-expressing HepG2 cells did not have
augmented proliferation compared with HepG2-scramble cells. However, for
PLC5 and Hep3B cells, the proliferation rates were significantly suppressed
(P < 0.01; Supplementary Fig. 6C).
SIRT1 knockdown reduced the number and size of HepG2 colonies
but increased the number and size of PLC5 colonies as determined by colony
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formation assays (P < 0.01; Fig. 3C, D). In HepG2-hSIRT1 cells, SIRT1 had
no effect in colony formation assays, but results from p53 mutant cells
revealed its inhibitory effects on colony formation ability (PLC5-hSIRT1, P <
0.01: Hep3B-hSIRT1, P < 0.01; Supplementary Fig. 6D). Similarly, Hep3B-
shSIRT1-p53MUT cells had a higher proliferation rate and better colony-
forming ability than Hep3B-scramble-p53MUT cells, whereas Hep3B-
shSIRT1-p53WT cells had a lower proliferation rate and worse colony-forming
ability compared with Hep3B-scramble-p53WT cells (P < 0.01). In a control
experiment, Hep3B-scramble-p53MUT cells demonstrated a higher
proliferation rate and better colony-forming ability than Hep3B-scramble-
p53WT cells (P < 0.01). Taken together, these results suggest that SIRT1
knockdown by shRNA promotes carcinogenesis in mutant p53 HCC cells
(Supplementary Fig. 7D, E).
Effects of SIRT1 on tumor growth in vivo
To test the effects of SIRT1 on tumor formation in mice, HepG2 and
PLC5 cells were transfected with a shSIRT1 lentivirus vector and then
subcutaneously injected into right flanks of NOG mice. HepG2-shSIRT1 cells
formed tumors that were significantly smaller than those of control cells (P <
0.01), whereas PLC5-shSIRT1 cells formed tumors that were significantly
larger than those of control cells (P < 0.01; Fig. 3E). Interestingly, tumors
derived from HepG2-shSIRT1 cells exhibited similar AMPK expression to the
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control, whereas tumors derived from PLC5-shSIRT1 cells exhibited lower
AMPK expression than the control (P < 0.01; Fig. 3F). These data confirm the
effects of SIRT1 on tumorigenesis in vivo.
Metformin suppresses the growth of HCC cells in vitro
To evaluate the effects of AMPK activation on cell proliferation in p53-
mutant HCC cells, HepG2-scramble, HepG2-shSIRT1, PLC5-scramble, and
PLC5-shSIRT1 cells were treated with different doses of metformin, an AMPK
activator, for 0, 24, and 48 h. Metformin was effective in inhibiting cell growth
when used at different concentrations and different times in HepG2-scramble
and PLC5-shSIRT1 cells. Interestingly, metformin treatment resulted in a
greater reduction in the proliferation of PLC5-shSIRT1 cells compared with
PLC5-scramble cells (Fig. 4A, B). To explore the mechanisms underlying the
effects of metformin, we analyzed the activation and expression of various
pathway components following treatment of cells with 100 mM metformin for
24 h. These results showed that metformin induced the phosphorylation of
acetyl CoA carboxylase (ACC), a specific AMPK target, in all cell lines and
induced an increase in AMPK phosphorylation in all cell lines except HepG2-
shSIRT1 cells. Notably, within 24 h, metformin also inhibited the
phosphorylation of mTOR in HepG2-scramble, PLC5-scramble, and PLC5-
shSIRT1 cells (Fig. 4C, D).
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Metformin suppressed the growth of HCC tumors in mice
As shown above, metformin demonstrated a strong influence on the
suppression of PLC5-shSIRT1 cells in vitro. Therefore, we hypothesized that
AMPK activation via metformin treatment may suppress the in vivo growth of
PLC5-shSIRT1 tumors in mice. Two weeks after the inoculation of PLC5-
scramble and PLC5-shSIRT1 cells in mice, metformin treatment (150
mg/kg/day) was initiated. As expected, there was a significant reduction in the
growth of tumors in mice after 6 weeks of treatment with metformin (P < 0.01)
compared with the untreated control group. Furthermore, metformin therapy
led to an even greater reduction in the growth of tumors generated from
PLC5-hSIRT1 cells than those generated from PLC5-scramble cells (Fig. 4E).
Additionally, tumor tissues derived from PLC5-shSIRT1 cells exhibited a
higher induction of phospho-AMPK and phospho-mTOR compared with those
derived from PLC5-scramble cells (P < 0.01; Fig. 4F). These data indicate that
metformin specifically inhibits the growth of tumors derived from cells
harboring mutant p53 and inactive SIRT1.
Discussion
The role of SIRT1 in tumorigenesis is controversial, and previous studies
have indicated that the function of SIRT1 may be tumor-type specific and
depend on the stage of oncogenesis. In this report, we demonstrated
significant roles for SIRT1, AMPK, and p53 in HCC using in vitro, in vivo, and
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clinical studies. Overall, our data demonstrate that the role of SIRT1 depends
on the p53 mutation status of HCC cells and tumor tissues, which supports
the need for biomarker evaluation in the prognosis and treatment evaluation
of HCC patients. SIRT1 is generally thought to disrupt cell cycle control and
promote tumor progression through p53 deacetylation and inactivation,
suggesting that p53 is a vital intermediate between SIRT1 and SIRT1’s
oncogenic activity [4, 5, 6, 7]. Recently, dynamic interactions have been
reported between AMPK and SIRT1 via the cellular NAD+ levels [21]. In
addition, SIRT1 deacetylates LKB1, inducing augmented AMPK
phosphorylation and activation [22, 23].
Based on our results and previous data, we hypothesize that SIRT1 is
required to induce the phosphorylation of AMPK and the inhibition of the
mTOR pathway [24, 25]. Indeed, SIRT1 inhibition by shRNA activated the
phosphorylation of mTOR (Ser2448) via decreased phospho-AMPK in the
mutant p53 cells, PLC5-shSIRT1.
The details of the mechanism involving p53, phospho-AMPK (Thr172) and
phospho-SIRT1 (Ser47) remains unclear. Our current findings provide some
understanding for the connection between the p53-SESTRIN1/2-AMPK
pathways [20]. Baik et al. argued that mTOR inhibits SIRT1 via the
phosphorylation of SIRT1 (Ser47) while promoting A431 cell survival, and
these results are different from those of previous studies [26, 27]. The
importance of individual SIRT1 targets may depend on the cell process and
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cell type studied. However, it has been shown that phospho-SIRT1 can adjust
for cell survival by directly interacting with indispensable factors (i.e., mTOR)
[28]. In this context, our results indicate that phospho-SIRT1 (Ser47) remains
responsive to stress-induced activation in mutant p53 HCC. Furthermore,
phospho-SIRT1 inhibits HCC growth via a certain tumor-suppressive pathway.
AMPK activation via SIRT1 (phospho-SIRT1) may play an important role
in preventing mTOR activation in p53 mutant HCC. Despite the complex
regulation of mTOR in HCC cells, metformin data show that enhanced AMPK
phosphorylation is sufficient to decrease p70S6K translational activity via
mTOR inactivation in p53 mutant HCC cells and mice. Therefore, AMPK
activation by metformin is a potential strategy for enhancing HCC targeting
(Fig. 4G). Metformin has been investigated as an anticancer drug for
decreasing the HCC risk of diabetes patients. We observed a tumor growth
suppressive effect in mice during metformin treatment. Our results suggest
further investigation of this drug as an approach for targeting HCC. In addition,
these may aid in the development of more selective therapeutic approaches
for HCC patients in the future.
Acknowledgments
We thank Prof. Ben C.B. Ko (Department of Anatomical and Cellular
Pathology, the Chinese University of Hong Kong) and Prof. Yick-Pang Ching
(Department of Anatomy, the University of Hong Kong) for advice during the
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preparation of this manuscript and critically reviewing the manuscript.
22
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Table 1 Correlation between activated SIRT1 and p53 mutation in HCC
(n=248)
Activated SIRT1 P
+ -
p53 mutation + 36 (63.2%) 21 (36.8%)
- 60 (31.4%) 131 (68.6%) <0.001
28
Table 2 Univariate and multivariate analysis of factors influencing relapse-free survival (RFS) in HCC with mutant p53 (n=57)
Independent factor
Univariate multivariate
Exp(B) 95% CI for Exp(B) P Exp(B) 95% CI for Exp(B) P
Gender 0.597 0.316-1.128 0.112
Age 1.624 0.863-3.053 0.132
AFP 1.654 0.874-3.130 0.122
Tumor number 0.554 0.295-1.041 0.066 0.267 0.130-0.549 <0.001
Tumor size 1.540 0.826-2.869 0.174
Edmondson grade 1.327 0.471-3.739 0.592
Fibrous capsule formation 0.597 0.289-1.230 0.162
Microvascular invasion 1.367 0.679-2.752 0.381
Portal vein invasion 1.077 0.331-3.500 0.902
Serosa invasion 1.029 0.248-4.276 0.969
Satellite nodule 3.199 1.658-6.174 0.001 3.479 1.606-7.534 0.002
AJCC staging 1.343 0.714-2.527 0.360
29
Activated SIRT1 0.432 0.229-0.814 0.009 0.307 0.143-0.660 0.002
AFP, alpha-fetoprotein; AJCC, American Joint Committee on Cancer; CI, confidence interval
30
Table 3 The correlation between activated SIRT1 and activated AMPK in HCC
with wild type and mutant p53 (n=248)
p53 WT Activated AMPK P
+ -
Activated SIRT1 + 26 (43.3%) 34 (56.7%)
- 71 (54.2%) 60 (45.8%) 0.212
p53 MUT Activated AMPK P
+ -
Activated SIRT1 + 18 (50.0%) 18 (50.0%)
- 2 (9.5%) 19 (90.5%) 0.003
WT, wild type; MUT, mutant
31
Figure Legends
Fig. 1 Stage-specific postoperative survival as calculated by the Kaplan-Meier
method. Activated SIRT1 was not a prognostic indicator for overall survival
(OS) or relapse-free survival (RFS) in cases with wild-type. A P-value less
than 0.05 was considered significant (A, B). (C) Activated SIRT1 was not a
prognostic indicator for overall survival (OS) but was a prognostic indicator for
relapse-free survival (RFS) in cases with mutant p53 (D). (E) In HCC patients
with mutant p53, activated SIRT1 and activated AMPK demonstrated a
significantly better prognosis in terms of RFS than in patients with inactive
SIRT1 and AMPK. (F) In patients with wild-type p53, neither activated SIRT1
nor activated AMPK predicted a better RFS in pair wise comparisons. A P-
value less than 0.05 was considered significant. *the number at risk on the
last day of contact
Fig. 2 AMPK expression and activation in nontumor and HCC tissues. (A) The
total and phosphorylated AMPK protein pattern in nontumor and HCC tissues
(B) Quantification of the percentage of AMPK and activated AMPK-positive
cases according to the results in (A). (C) Relative expression of total AMPK (t-
AMPK) and phosphorylated AMPK (p-AMPK) in HCC and nontumor tissues.
Fig. 3 Cell growth and proliferation assays and the effect of SIRT1 knockdown
on tumor growth in vivo. The proliferation and colony-forming ability of HepG2
cells (A, C) and PLC5 cells (B, D) were analyzed after SIRT1 knockdown (*, p
< 0.01). SIRT1 knockdown and control cells (both HepG2 and PLC5) were
subcutaneously injected in the right flanks of NOG mice (E). Tumor volumes
were measured after 6–8 weeks. (*, P < 0.01) Immunohistochemical analysis
of AMPK activity in tumors derived from SIRT1-knockdown and scramble-
transfected HepG2 and PLC5 cells (F). (*, P < 0.01)
Fig. 4 Restriction of cancer cell growth in vitro and reduced HCC xenograft
tumor growth in vivo by metformin. Cell proliferation was measured in (A)
HepG2 and (B) PLC5 cells. Phosphorylation of the indicated targets was
32
analyzed in (C) HepG2 and HepG2-shSIRT1 and (D) PLC5 and PLC5-
shSIRT1 cells. Western blot data are representative of at least 3 independent
experiments. *, P < 0.01. (E) PLC5 and PLC5-shSIRT1 cells were
subcutaneously injected into mice. Metformin treatment was started 2 weeks
later. Tumor volumes were determined every 2 weeks. (F)
Immunohistochemical analysis and quantification of AMPK and mTOR
phosphorylation in mouse tumors. (*P < 0.01) and (G) schematic summary of
the different roles of SIRT1 in HCC as suggested by our current data.
Metformin may be more effective at treating a specific class of HCC in which
p53 is mutated and SIRT1 and AMPK are inactive.
Figure 1
F
Figure
Figure 2
A
B
C
Figure 2
0
0.5
1
1.5
2
2.5
3
0h 24h 48h 72h
Ab
so
rb
an
ce O
D 4
50(m
m)
HepG2-scramble
HepG2-shSIRT1-1
HepG2-shSIRT1-2
* *
SIRT1
Actin
Figure 3
A
0
0.5
1
1.5
2
2.5
3
0h 24h 48h 72h
Ab
so
rban
ce O
D 4
50(m
m)
PLC5-scramble
PLC5-shSIRT1-1
PLC5-shSIRT1-2
*
*
SIRT1
Actin
Figure 3
B
0
1
2
3
4
5
6
7
HepG2-scramble HepG2-shSIRT1-1 HepG2-shSIRT1-2
co
lob
y n
um
ber(
>=
1m
m)
*
*
Figure 3
C
0
1
2
3
4
5
6
7
PLC5-scramble PLC5-shSIRT1-1 PLC5-shSIRT1-2
co
lob
y n
um
ber(
>=
1m
m)
*
*
Figure 3
D
0
0.5
1
1.5
2
2.5
3
HepG2 PLC5
Tu
mo
r vo
lum
e(c
m3)
scramble
shSIRT1* *
scramble scramble
shSIRT shSIRT
Figure 3
E
scramble scramble shSIRT1 shSIRT1
HepG2 PLC5
SIRT1
phosphor-AMPK
Figure 3
F
0
20
40
60
80
100
120
SIRT1 phosphor-AMPK
Po
sti
ve s
tain
ing
(%)
HepG2-scramble
HepG2-shSIRT1
PLC5-scramble
PLC5-shSIRT1
* * *
Figure 3
F
Figure 4
Figure 4
Metformin
(100 mmol/l, 24h) - +
HepG2
t-SIRT1
p-ACC
t-ACC
β-actin
t-AMPK
P-SIRT1
P-AMPK
- +
HepG2-shSIRT1
t-mTOR
p-mTOR
Figure 4
C
- +
PLC5
Metformin
(100 mmol/l, 24h)
t-SIRT1
p-ACC
t-ACC
β-actin
t-AMPK
P-SIRT1
P-AMPK
- +
PLC5-shSIRT1
t-mTOR
p-mTOR
Figure 4
D
0
0.5
1
1.5
2
2.5
3
0W 2W 4W 6W 8W
Tu
mo
r vo
lum
e(c
m3)
PLC5-scramble:control,n=4
PLC5-scramble:metformin 150mg/kg/Day,n=6
PLC5-shSIRT1:control,n=5
PLC5-shSIRT1:metformin 150mg/kg/Day,n=8
*
*
*
Metformin treatment
Figure 4
E
Figure 4
F
Figure 4
G