Enterocyte-Specific Inactivation of SIRT1 Reduces Tumor
Load in the APC+/minMouse Model
Vid Leko1, Gemma J. Park1, Uyen Lao1, Julian A. Simon1,2, Antonio Bedalov1,3*
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2Human Biology Division, Fred Hutchinson Cancer
Research Center, Seattle, Washington, United States of America, 3Departments of Medicine and Biochemistry, University of Washington, Seattle, Washington, United
States of America
SIRT1 is a mammalian NAD+-dependent histone deacetylase implicated in metabolism, development, aging and
tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1
on polyp formation in the intestines of APC+/minmice, a commonly used model for intestinal tumorigenesis, yielded
conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve
the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency
confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion.
We crossed APC+/minmice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in
the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm2vs. 23.3 mm2, p=0.01), average polyp
size (0.24 mm2vs. 0.51 mm2, p=0.005) and the number of polyps .0.5 mm in diameter (14 vs. 23, p=0.04), indicating that
SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased
numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor
cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC+/minmouse model of intestinal tumorigenesis.
Citation: Leko V, Park GJ, Lao U, Simon JA, Bedalov A (2013) Enterocyte-Specific Inactivation of SIRT1 Reduces Tumor Load in the APC+/minMouse Model. PLoS
ONE 8(6): e66283. doi:10.1371/journal.pone.0066283
Editor: Robert W. Sobol, University of Pittsburgh, United States of America
Received October 19, 2012; Accepted May 8, 2013; Published June 14, 2013
Copyright: ? 2013 Leko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (NIH), CA 129132, to AB. (http://www.nih.gov/). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
SIRT1 is a mammalian NAD+-dependent histone deacetylase
that plays important roles in ageing, metabolism, development,
neurodegeneration and tumorigenesis (reviewed in [1–6]). Con-
sidering the multitude of cellular pathways it affects, SIRT1
appears to play a rather complex role in the biology of cancer, and
evidence supports both tumor promoting and tumor suppressing
SIRT1 was first implicated in tumorigenesis by the finding that
it deacetylates and down-regulates the tumor suppressor p53
under conditions of genotoxic stress, decreasing its pro-apoptotic
activity and promoting survival of cells that have accumulated
DNA damage [9,10]. Acetylation of p53 turned out to be a critical
posttranslational modification, one that controls many functions of
the p53 protein [11,12]. SIRT1 was later found to deacetylate and
regulate several other proteins that share similar roles in cellular
stress responses (e.g. Ku70, p73, FoxO3a, FoxO4 and E2F1[13–
17]), while small-molecule inhibitors of SIRT1 were shown to
exhibit antitumor activity, suggesting that pharmacological inhi-
bition of SIRT1 could be therapeutically beneficial in a subset of
human cancers [18–23]. SIRT1 has also been proposed to
participate in tumorigenesis through epigenetic silencing of tumor
suppressor genes . Coupled with the observation that SIRT1
expression levels are increased in many human tumors (e.g. colon
cancer) and usually associated with poor prognosis in these
patients [25–31], these findings suggest that SIRT1 acts as a tumor
Paradoxically, a growing body of evidence suggests that SIRT1
may suppress tumor development. SIRT1 was found to be
important for maintaining genome stability, loss of which is a
hallmark of cancer , and to mediate DNA damage repair
[33,34]. Furthermore, global overexpression of SIRT1 led to
reduced incidence of some age-related tumors and protection from
metabolic-syndrome driven liver cancer . SIRT1+/2p53+/2
mice developed spontaneous tumors at higher rates than their
p53+/2controls , while p53+/2mice overexpressing SIRT1
demonstrated decreased incidence of thymic lymphoma and
increased survival following c-radiation . Despite the results
of these animal studies, mutations in SIRT1 gene have never been
documented in human tumors, indicating that SIRT1 may not
behave as a typical tumor suppressor. However, consistent with its
potential anti-oncogenic role, SIRT1 expression was found to be
decreased in a subset of human cancers [33,36].
The APC+/minmouse model mimics the early events of colon
cancer in humans  and is widely used to test the effects of
potential oncogenes and tumor suppressors on formation of
intestinal tumors. Heterozygous APC+/minmice inherit a nonsense
mutation in one copy of the tumor suppressor gene APC
(designated as APCmin) and lose the remaining wild-type allele
after birth, resulting in nuclear translocation and constitutive
activation of the crucial Wnt-signaling effector b-catenin and
PLOS ONE | www.plosone.org1 June 2013 | Volume 8 | Issue 6 | e66283
subsequent formation of numerous tumors (polyps) in the intestines
[38,39]. When a SIRT1 transgene was overexpressed in the
intestinal epithelium, APC+/minmice developed fewer intestinal
polyps, which was attributed to the increased deacetylation and
nuclear exclusion of b-catenin  and subsequently decreased
proliferation rates of the tumor cells. However, since the
expression of exogenous SIRT1 in the enterocytes surpassed
physiological levels by several fold and because gene overexpres-
sion can sometimes phenocopy gene loss-of-function through a
dominant interfering effect, it was important to examine the effect
of SIRT1 deficiency on polyp formation in the same model.
mice exhibiting whole-body SIRT1
deficiency developed a similar number of intestinal polyps as the
APC+/mincontrols, but demonstrated significantly decreased
average polyp size, indicating that SIRT1 could actually promote
tumor growth . However, as it was known that SIRT1-null
mice that survived weaning exhibit severe growth retardation and
decreased circulatory levels of free Insulin-like Growth Factor 1
(IGF-1), a known promoter of tumor growth [33,42–44], concerns
were raised that systemic adaptations to global SIRT1 deficiency
may have masked the true effect of intestinal SIRT1-deficiency
and protected these animals from tumor development.
In order to eliminate potentially confounding influences of
global SIRT1 deletion and resolve the controversy regarding the
exact role of SIRT1 in intestinal tumorigenesis, we crossed APC+/
minmice with mice harboring SIRT1-inactivation restricted to the
intestinal epithelium. Unlike their whole-body deficient counter-
parts, mice with intestine-specific SIRT1 deletion demonstrated
wild-type expression of SIRT1 in extra-intestinal tissues and
exhibited normal survival rates without detectable developmental
defects. We found that these mice exhibited significant reduction
in polyp sizes and developed significantly fewer polyps .0.5 mm
in diameter than their controls. Additionally, tumors from SIRT1-
deficient mice exhibited significantly increased apoptotic rates,
without affecting their proliferation, indicating that SIRT1
promotes survival of tumor cells. Taken together, our findings
indicate that SIRT1 plays a tumor-promoting role in the mouse
intestinal tumor model.
We generated mice in which endogenous SIRT1 has been
specifically inactivated in the intestinal epithelium using the Villin-
Cre system  and crossed them with APC+/minmice to generate
SIRT12/2) and APC+/minSirt1+/+(APC+/minSirtco/coVil-
Cre2/2, or SIRT1+/+) mice. APC and Cre genotypes were
confirmed by PCR prior to weaning, while the deletion of SIRT1
was confirmed at the time of sacrifice by a western blot analysis of
intestinal mucosa scrapings. A truncated version of the SIRT1
protein confirmed that exon 4 of SIRT1 gene, which encodes the
catalytic domain of the protein, was successfully deleted in the
enterocytes. Other tissues from APC+/min
exhibited a band of the wild-type size, confirming that the deletion
was intestine-specific (Figure 1A). Importantly, we noted that
APC+/minSIRT12/2mice were born at expected Mendelian
frequencies and did not exhibit increased postnatal mortality or
developmental defects and phenotypic features of mice with
systemic deletion of SIRT1.
At the age of 120 days, SIRT12/2and SIRT1+/+mice were
sacrificed and their entire intestines examined for polyps
(Fig. 1B,C). We found that the average size of the largest polyp
in SIRT12/2cohort was significantly smaller than in the control
group (1.360.6 mm vs. 2.260.9 mm, p,0.05) and that polyps
from SIRT12/2mice exhibited a marked decrease in average
(0.235 vs. 0.510 mm2, Figure 2A) and total surface areas (9.3 vs.
23.3 mm2, Figure 2B) throughout the small intestine (Figure 2C,
D). The majority of tumors were found to reside in the distal
segment of the small intestine, while only a few were detected in
the colon (Figure 2E). Of note, tumors in the colon were excluded
from the analysis because their peduncular appearance made the
assessment of the exact tumor size difficult (although SIRT12/2
mice had qualitatively smaller polyps in the colon as well). When
we compared tumor size distribution between SIRT12/2and
SIRT1+/+mice we found that only 2% of adenomas in SIRT12/2
mice were larger than 1.0 mm in diameter, whereas 12% of
tumors in the control group reached this size (p=0.0076). We
found that the number of polyps in the small intestine that
surpassed 0.5 mm was also significantly lower in SIRT12/2
animals (Figure 2F), while the total polyp number and the number
of very small tumors, measuring 0.4 mm or less in diameter, was
not significantly different between two genotype groups (Figure 2G,
H). These results indicate that SIRT1 deficiency reduces overall
polyp burden in the intestine of APC+/minmice primarily by
curtailing their size.
We next examined expression of proliferative and apoptotic
markers within size-matched tumors from SIRT12/2
SIRT1+/+animals. According to staining for the proliferation
marker Ki-67, we observed no differences in the proliferation rates
between the two genotype groups (Figure 1D). However, polyps
from SIRT12/2animals exhibited an increased number of cells
expressing the apoptotic marker cleaved caspase-3 (Figure 1E),
indicating that the observed reduction in tumor size is attributable
to increased apoptosis of SIRT12/2cells, which is consistent with
previously published findings in colon cancer cell lines .
In order to gain an insight into a mechanism by which SIRT1
promotes intestinal tumorigenesis, we analyzed the effect of SIRT1
inhibition on Wnt and p53 signaling pathways. We first analyzed
b-catenin expression in adenomas of APC+/minmice and observed
increased expression levels and nuclear accumulation of b-catenin
regardless of the intestinal SIRT1 deletion (Figure 3A). Next we
wanted to determine whether loss of SIRT1 affected Wnt signaling
in normal appearing intestinal crypts, where the remaining wild-
type copy of the APC gene keeps overall nuclear b-catenin levels
low and confined to the cell membrane in all the non-basal cells.
We compared the number of nuclear b-catenin -positive cells per
crypt in mice with and without intestinal SIRT1 deletion, and
found that mice with the deletion had significantly fewer cells per
crypt then their wild-type counterparts (Figure 3B,C), suggesting
the SIRT1serves as an activator of Wnt signaling. Furthermore, to
measure the effect of SIRT1 inhibition on b-catenin mediated
transactivation, we analyzed the transcriptional output of T-cell
factor/lymphoid enhancer factor (TCF/LEF)-driven luciferase
reporter gene using a TOP-GLOW assay  in SW480 colon
cancer cell line, which is known to exhibit a loss-of-function
mutation in the APC gene and constitutive activation of Wnt
pathway . SIRT1 inhibition with both EX527, a selective
small-molecule SIRT1 inhibitor , and cambinol, a non-
selective SIRT1 and SIRT2 inhibitor , led to reduction in
TCF-mediated transcription (Figure 3D). We also observed
decreased TCF/LEF transcriptional activity in SW480 cells upon
(Figure 3E), further indicating that SIRT1 activates Wnt signaling
and consecutively promotes intestinal tumorigenesis in the APC+/
Next we assessed the role of p53 acetylation in SIRT1-driven
intestinal tumorigenesis by employing Hct116 colon cancer cell
line with constitutive activation of b-catenin but without mutations
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org2June 2013 | Volume 8 | Issue 6 | e66283
Figure 1. Enterocyte-specific SIRT1 deletion increases the rate of apoptosis in the intestinal tumors of APC+/minmice. (A) A
representative western blot showing expression of the wild-type SIRT1 protein in the intestinal epithelium of APC+/minSIRT1+/+mice (first lane) and a
truncated version in the epithelium of APC+/minSIRT12/2mice (second lane). Liver cells of the APC+/minSIRT12/2mice (third lane), as well as other
tissues (not shown) express the wild-type protein. Pan-actin immunostaining served as a loading control. (B) Representative photographs of unfixed
small intestines (distal segments) showing similar polyp number for APC+/minSIRT1+/+(right) and APC+/minSIRT12/2(left) mice. Scale bar indicates
5 mm length. (C) Representative photomicrographs of typical polyps from the two groups of mice, stained with hematoxylin and eosin. Scale bar
indicates 100 mm length. (D) Representative photomicrographs and a bar graph showing Ki-67 immunohistochemical staining of polyp sections from
APC+/minSIRT1+/+(left) and APC+/minSIRT12/2(middle) mice. Scale bar indicates 100 mm length. Proliferation index for polyps from APC+/minSIRT1+/+
and APC+/minSIRT12/2mice (right), expressed as a fraction of Ki-67 positive cells within each polyp. Bars represent means 6 SEM, n=20 polyps per
group. No statistically significant difference was observed. (E) Representative photomicrographs and a bar graph showing activated (cleaved)
caspase-3 immunohistochemical staining of polyp sections from APC+/minSIRT1+/+(left) and APC+/minSIRT12/2(middle) mice. Scale bar indicates
100 mm length. Absolute numbers of apoptotic (caspase-3 positive) cells per high power field (400 x) for polyps from SIRT1+/+and SIRT12/2mice
(right). Bars represent means 6 SEM, n=25 polyps per group. ***p,0.001.
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org3 June 2013 | Volume 8 | Issue 6 | e66283
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org4June 2013 | Volume 8 | Issue 6 | e66283
in p53 gene . As previously shown in other cell culture models
, we saw that inhibition of SIRT1 in these colon cancer cells
lead to p53 hyperacetylation (Figure 3F), suggesting that down-
regulation of p53 could be an additional cellular mechanism by
which SIRT1 promotes tumor growth.
Taken together, our results show that enterocyte-specific
inactivation of SIRT1 reduces tumor load in the intestines of
APC+/minmice by decreasing both overall tumor size and the
number of larger tumors, and they suggest that SIRT1 acts as a
tumor promoter by suppressing apoptosis of tumor cells in this
mouse model, through mechanisms that include both activation of
Wnt signaling pathway and inhibition of p53 activity.
In order to bypass systemic effects related to global deletion of
SIRT1, especially those associated with reduced circulatory levels
of IGF-1, and focus instead on its intestine-specific role only, we
generated APC+/minmice harboring enterocyte-specific inactiva-
tion of SIRT1 and examined their susceptibility to forming
intestinal polyps. These APC+/minSIRT12/2mice, which were
born at expected Mendelian frequencies and demonstrated
unperturbed development and growth, exhibited a significant
decrease in the number of tumors .0.5 mm in diameter, a
dramatic reduction in average and total polyp surface areas, and
an increased fraction of apoptotic cells within polyps. We also
found that SIRT12/2mice had similar frequencies of very small
tumors as SIRT1+/+controls and similar total numbers of polyps,
indicating that SIRT1 could play a role in tumor progression and
growth, rather than influencing the formation of early neoplastic
lesions. This phenotype is congruent with one previously reported
for systemic SIRT1 knockout animals , which showed that
tumor size is significantly decreased upon SIRT1 inactivation,
whereas the total number of intestinal polyps remained the same.
This surprisingly robust finding shows that SIRT1 inactivation,
regardless of differences in genetic background (C57BL/6 with
some contribution of 129X1/SvJ vs. mixed C57BL/6/129X1/
SvJ/CD1) and age of sacrifice (4 months vs. 12 months) in these
two studies, leads to marked changes in tumor growth rate. Our
results suggest that the tumor attenuation previously observed in
APC+/minmice carrying a systemic deletion of SIRT1  was a
tissue-specific effect of SIRT1-deficiency. However, these findings
are seemingly at odds with the findings of Firestein et al. ,
which showed that intestine-specific overexpression of SIRT1
reduced the number of polyps in the intestines of APC+/minmice
(tumor size was not analyzed). Thus, SIRT1 overexpression and
SIRT1 loss-of-function resulted in similar phenotypes, a phenom-
enon frequently observed in gene overexpression studies (reviewed
in ) that may be explained by changes in the stoichiometry of
protein-protein interactions and disassembly of multi-protein
complexes leading, seemingly paradoxically, to inhibition of the
overexpressed protein. The facts that SIRT1 functions in several
multi-protein complexes and that its expression in the study by
Firestein et al.  surpassed physiological levels by several fold
make this explanation highly plausible. However, we noted that
intestine-specific SIRT1 overexpression in the study by Firestein
et al. did not create an exact phenocopy of the enterocyte-specific
deletion, as the total number of polyps was reduced 3–4 fold in the
former study, while our model demonstrated only 2 fold reduction
among tumors .0.5 mm in size, without affecting the total
number of tumors. Furthermore, unlike in the SIRT1-overexpres-
sion study, which did not analyze cell survival and apoptosis, we
found no differences in proliferation rates between SIRT12/2and
SIRT1+/+mice, but rather an increased rate of apoptosis in the
SIRT12/2group. This is consistent with the current paradigm in
which SIRT1 plays a role as a pro-survival and anti-apoptotic
mediator, as discussed in the next section. It is possible that supra-
physiological levels of SIRT1 in the overexpression study engaged
cellular mechanisms outside the classical SIRT1-mediated signal-
ing pathways that are normally not active when SIRT1 is
expressed at physiological levels. Based on cell culture studies,
Firestein et al. attributed the polyp number reduction to reduction
in transcriptional activity of b-catenin, which is a target of Wnt
signaling. In contrast, our results suggest that SIRT1 actually
promotes Wnt signaling, both in vivo and in vitro. Apart from the
use of different cell lines, we cannot account for the discrepancy
between the two in vitro results. However, as indicated by a
growing body of evidence, the role of SIRT1 in Wnt signaling
appears to be complex, as different studies had also associated
SIRT1 with activation of the Wnt pathway, as discussed in greater
detail below. As for the discrepancy in the in vivo results, we do not
believe that the observed differences in proliferation rates between
the two studies are attributable to the age of the animals or
differences in the intestinal sections scored, as in both studies polyp
analysis was carried out throughout the small intestine of the
animals sacrificed at the same age. However, there is a possibility
that the disparity in proliferation rates could be at least partially
due to subtle differences in genetic backgrounds, as the Firestein
study was carried out in C57BL/6 mice, while our study utilized
C57BL/6 animals with a contribution from the 129X1/SvJ
Several previously described activities of SIRT1, particularly
those related to modulation of p53 activity [9,10], c-myc
[51,52,53,54], canonical Wnt signaling  and epigenetic control
, may account for the reduction in tumor size we observed in
the intestines of APC+/minSIRT12/2mice. SIRT1 has been
shown to promote cell survival during genotoxic stress through
deacetylation and down-regulation of p53 and several other stress-
response proteins (reviewed in ). Moreover, SIRT1 was also
implicated in promoting intestinal tumorigenesis through its
interactions with the tumor suppressor HIC1 (Hypermethylated
in Cancer 1), a transcriptional repressor frequently inactivated in
human colon cancer though epigenetic means, namely CpG island
Figure 2. Enterocyte-specific SIRT1 deletion reduces tumor size and number of tumors . .0.5 mm in diameter. (A) Average surface area
of polyps in the small intestine. Total polyps surface area for each mouse was divided by total number of polyps to obtain average polyp surface area.
Each dot represents an individual animal; n=11 per group, p=0.00019. (B) Total surface area of polyps in the small intestine; samples are as in panel
A. Polyp surface was calculated as a square of tumor radius multiplied by p; surface areas of all the polyps in each mouse were then added up to
obtain total polyp surface. Each dot represents an individual animal; n=11 per group, p=0.01. (C) Average surface area of polyps according to the
location in the small intestine. Bars represent means 6 SEMs, n=11 per group. (D) Total surface area of polyps according to the location in the small
intestine. Bars represent means 6 SEMs. (E) Distribution of tumors according to specific intestinal location. No statistically significant differences were
observed. (F) Number of polyps .0.5 mm in diameter for the entire small intestine; the groups are the same as in panel A. Each dot represents an
individual animal; n=11 per group, p=0.044. (G) Total number of the polyps in the intestines of APC+/minSIRT12/2(gray) and APC+/minSIRT1+/+mice
(white). Each dot represents an individual animal; n=11 per group. No statistically significant difference was observed. (H) Number of tumors in the
whole small intestine according to diameter. Bars represent means 6 SEMs; n=11 per group. Significant difference in tumor number in APC+/min
SIRT12/2(gray) and APC+/minSIRT1+/+was observed only for 1.0–1.9 mm polyp size.
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org5 June 2013 | Volume 8 | Issue 6 | e66283
Figure 3. SIRT1 inactivation inhibits Wnt and promotes p53 a pathway. A) Representative photomicrographs showing b-catenin
immunohistochemical staining of intestinal sections from APC+/minSIRT1+/+and APC+/minSIRT12/2mice. Scale bar indicates 50 mm length. Polyps
from both groups demonstrate intense cytoplasmic and nuclear staining for b-catenin. B) Bar graph with frequencies of crypts with indicated number
of cells with nuclear b-catenin staining in normal appearing mucosa of APC+/minSIRT1+/+and APC+/minSIRT12/2mice. The average number of b-
catenin positive cells per crypt is reduced in APC+/minSIRT12/2mice compared to APC+/minSIRT1+/+animals 2.161.2 vs 3.261.3 (p=1.561025, at
least 100 crypts per genotype were scored). C) Representative photomicrographs of normal appearing mucosa from APC+/minSIRT1+/+and APC+/min
SIRT12/2mice demonstrating a reduced number of basal cells with nuclear b-catenin in APC+/minSIRT12/2animals. Arrows are directed toward
representative basal cells with nuclear b-catenin. Scale bar indicates 25 mm length. D) Inhibition of SIRT1 with EX527 (2 mM) and cambinol (50 mM)
reduces activity of the TCF/LEF driven firefly luciferase reporter (TOP FLASH) transiently transfected into SW480 cells. TOP FLASH luciferase reporter
contains minimal promoter along with three TCF binding sites, which have been mutated in FOP FLASH reporters. Bars represent means 6 SEM of
relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org6 June 2013 | Volume 8 | Issue 6 | e66283
hypermethylation of its promoter [56,57]. Heterozygosity for
HIC1 in mice with a mutation in APC was shown to induce
transcriptional de-repression of SIRT1 in enterocytes and to
promote polyp formation, possibly through down-regulation of
p53. Intriguingly, both HIC1 and SIRT1 have been shown to be
direct transcriptional targets of p53, suggesting the presence of a
HIC1-SIRT1-p53 autoregulatory loop. Our observation that
intestinal SIRT1-deficiency decreases polyp load and increases
apoptosis in the APC+/minmodel, and that pharmacological
inhibition of SIRT1 in APC-mutated colon cancer cell lines leads
to p53 hyeperacetylation, provide additional support for impor-
tance of such an autoregulatory loop in the intestinal tumorigen-
SIRT1 was previously proposed to directly deacetylate b-
catenin, which, depending on cell context, may promote or inhibit
b-catenin activity [40,58]. Besides participating in b-catenin
deacetylation, there are several indirect mechanisms by which
SIRT1 may promote Wnt signaling. SIRT1 downregulates the
activity of p53, which has been shown to downregulate Wnt
signaling by promoting b-catenin phosphorylation and degrada-
tion . HIC1, whose expression could be influenced by the
above described autoregulatory loop, has been shown to antag-
onize canonical Wnt signaling by associating with TCF4 and b-
catenin and sequestering them in an inactive complex within the
nucleus . Additionally, SIRT1 has been shown to up-regulate
all three mammalian Disheveled proteins and thereby promote
Wnt signaling . Finally, down-regulation of SIRT1 in breast
and colon cancer cell lines leads to re-expression of the Wnt
pathway inhibitors, SFRP1 and SFRP2 (Secreted Frizzled-like
Proteins 1 and 2), which are frequently inactivated in human
cancers through epigenetic means , as discussed in greater
detail below. Our findings that the lack of SIRT1 decreases the
number of basal crypt cells expressing the active form of b-catenin
and that SIRT1-inhibition reduces b-catenin mediated transcrip-
tional activity in vitro, suggest that SIRT1 may indeed promote
adenoma formation through increased Wnt signaling. However,
further work is required to determine which of the proposed
pathways is critical for promoting Wnt signaling in the course of
Clonal evolution, which dictates the progression from normal
epithelium to polyp and ultimately invasive cancer, requires
progressive accumulation of both new genetic lesions and
epigenetic alterations that cause transcriptional silencing of tumor
suppressor genes (reviewed in ). Besides serving as an
established model for intestinal tumorigenesis, APC+/minmice
were the first animal model used to demonstrate that genetic or
pharmacological interference with DNA methylation inhibits
tumor formation, thus establishing a role of epigenetics in tumor
formation and the feasibility of targeting epigenetic mechanisms
for cancer therapy [62,63]. Besides DNA methyl transferases and
class I and II histone deacetlyases (HDAC), SIRT1 (a class III
HDAC) has been shown to participate in gene silencing in colon
cancer cell lines , both synergistically with DNA methyl
transferases and independently, without appreciable alterations in
DNA methylation level. Among genes repressed in a SIRT1-
dependent manner was a set of genes frequently inactivated by
DNA methylation in human cancers, providing direct evidence
that SIRT1 controls expression of tumor suppressor genes. Besides
the Wnt inhibitors SFRP1 and SFRP2, genes up-regulated upon
inhibition of SIRT1 included E-cadherin, a cell adhesion
mediating gene, and MLH1, a mismatch repair gene whose
inactivation leads to a mutator phenotype. Together, these
findings suggest that abrogation of epigenetic repression that
accompanies polyp progression could be one of the mechanisms
for the reduced tumor burden that we observed in APC+/min
SIRT12/2animals, raising the exciting possibility that SIRT1
inhibitors, similarly to inhibitors of DNA methyltransferase and
class I/II HDACs, could be used for epigenetic therapy of cancer.
Materials and Methods
APC+/min SIRT12/2 (APC+/min Sirt1co/co Vil-cre+/2)
Homozygous B6;129-Sirt1tm1Ygu/J mice carrying conditional
deletion of Sirt1 exon 4 (Sirt1co/co, available from Jackson
Laboratories, stock No. 008041), which encodes the catalytically
active domain of the protein, were crossed with homozygous
B6.SJL-Tg(Vil-cre)997Gum/J mice (a gift from Dr. William
Grady (FHCRC), but also available from Jackson Laboratories,
stock No. 004586) to generate heterozygous Sirt1+/coVil-cre+/2
mice. Resulting heterozygotes were interbred to generate Sirt1co/
coVil-cre+/2mice, which were subsequently crossed with SIRT
1co/comice to generate Sirt1co/coVil-cre+/2mice and their
littermate controls (Sirt1co/coVil-cre2/2). Both groups were then
crossed with APC+/minmice (available from Jackson Laboratories,
stock No. 002020) to generate APC+/minSirtco/coVil-cre+/2
SIRT12/2) and APC+/min
(APC+/minSIRT1+/+) mice. APC and Vil-cre genotypes were
confirmed by PCR carried out on DNA isolated from clippings
prior to weaning (PCR primer sequences are available upon
All animal work was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol for this study was approved by the Fred Hutchinson
Cancer Research Center Institutional Animal Care and Use
Committee (file 1505).
Animal Pathology and Histopathology
The gastrointestinal tract of the sacrificed 16-week old animals
was promptly excised and cut, with mucosal side up, into 3
segments (colon, distal and proximal small intestine), flushed with
ice cold phosphate-buffered saline (PBS, Invitrogen), pinned open
and fixed in 10% formalin, neutral buffered (Sigma) for 45 minutes
in a dissection tray. Fixed intestine was then examined under the
dissection microscope by two researchers who, blinded from the
genotyping data, counted the polyps, determined their diameter
with a caliper and calculated their surface. For histopathology,
fixed intestine segments were placed onto a dry board and gently
transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. **p,0.01. E) SW480 cells with shRNA-
mediated downregulation of SIRT1 (top: western blot for SIRT1 and actin) exhibit reduced activity of the transiently transfected TCF/LEF driven firefly
luciferase reporter (TOP FLASH). Bars represent means 6 SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the
thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out
in quadruplicate. *p,0.05. F) Immunoblot for acetyl-p53, p53 and actin from cells treated with etoposide, SIRT1 inhibitor EX527 or the combination
of the two drugs. Inhibition of SIRT1 leads to p53 hyperacetylation in Hct116 colon cancer cell line. Hyperacetylation of p53 is observed in cells
treated with EX527 and a combination of EX527 and etoposide. Etoposide alone modestly induces p53 acetylation.
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e66283
rolled into Swiss rolls and placed in 10% NBF for 24 hours. The
rolls were then switched over into 70% ethanol and submitted for
paraffin-embedding and hematoxylin/eosin, Ki-67 (rat anti-mouse
Ki67, Dako, M7249, 1:50 dilution) and cleaved caspase-3 staining
(rabbit anti-mouse, Biocare Medical, CP 229B, 1:50 dilution).
Microscopic images were obtained using Nikon E800 microscope
and analyzed by two researchers independently. Fractions of Ki-
67 and caspase-3 positive cells were determined using Image J
program (Wayne Rasband, National Institutes of Health).
Tissue Culture Studies
Hct116 and SW480 cell lines were obtained from ATCC and
grown in Dulbecco’s Modified Eagle Medium (Life Technologies)
supplemented with 10% Fetal Calf Serum. TOP GLOW reporter
plasmid that contains the firefly luciferase gene under control of
minimal c-Fos promoter, along with three TCF binding sites,
mutated in a control FOP GLOW reporter , were obtained
from Dr. Hans Clevers. Herpes simplex virus thymidine kinase
promoter driven renilla luciferase reporter, pRL-TK (Promega)
was used for transfection normalization. Transient transfections
were carried out using Lipofectamine (Invitrogen) and firefly and
renilla luciferase activities measured using Dual Luciferase
Reporter Assay System (Promega). EX527 was obtained from
Sigma and cambinol was synthesized in our laboratory. shRNA
specific for SIRT1 (shRNA
SW480 cells as a lentiviral particle using pLKO.1 vector and
puromycin selectable marker.
After flushing and opening the large intestine, the mucosal
surface was gently scraped off using a glass slide, snap frozen in
liquid nitrogen and placed at 280uC. Upon thawing, each sample
was resuspended in half its volume of RIPA buffer (1% NP-40,
0.1% SDS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl 0.5%
Sodium Deoxycholate,1 mM EDTA), sonicated, incubated on ice
for 30 minutes and spun down to remove debris. A BCA Protein
assay (Pierce) was performed to determine the protein concentra-
tion; 25 mg of each sample was then mixed with the equal amount
of SDS gel loading buffer (26) containing 100 mM Tris-Cl
(pH 6.8), 4% (w/v) sodium dodecyl sulfate, 0.2% (w/v) bromo-
phenol bluem 20% (v/v) glycerol, 200 mM DTT (dithiothreitol)
and loaded onto 7.5% polyacrylamide gel. The protein was then
transferred to a nitrocellulose membrane and subsequently probed
with the rabbit anti-mouse SIRT1 antibody (Anti-SIRT1,
Millipore 07-131). In tissue culture studies the following antibodies
were used: rabbit polyclonal anti-SIRT1 (Millipore, 07-131),
mouse monoclonal anti-p53 (DO-1 Santa Cruz Biotechology),
rabbit polyclonal anti-acetyl p53 (Cell Signaling, Acetyl-p53
(Lys379), catalogue number 2570). Anti-actin staining was
performed to ensure equal loading between the samples (Pan
Actin, ACTN05, NeoMarkers).
Standard two-tailed Student’s t-test was used for comparisons
between two experimental groups, with a p value of ,0.05
considered as significant.
We are grateful to Dr. William Grady, FHCRC, and the members of his
laboratory for providing us with Vil-cre mice and assistance with polyp
scoring, Drs. Eric Foss, William Grady and Elizabeth Kwan, for carefully
reading the manuscript and providing us with helpful suggestions.
Conceived and designed the experiments: AB VL. Performed the
experiments: VL UL GJP. Analyzed the data: VL GJP AB JS. Contributed
reagents/materials/analysis tools: VL GJP JS. Wrote the paper: AB VL.
1. Guarente L (2007) Sirtuins in aging and disease. Cold Spring Harb Symp Quant
Biol 72: 483–488.
2. Imai S, Guarente L (2010) Ten years of NAD-dependent SIR2 family
deacetylases: implications for metabolic diseases. Trends Pharmacol Sci 31:
3. Herranz D, Serrano M (2010) SIRT1: recent lessons from mouse models. Nat
Rev Cancer 10: 819–823.
4. Brooks CL, Gu W (2009) How does SIRT1 affect metabolism, senescence and
cancer? Nat Rev Cancer 9: 123–128.
5. Bonda DJ, Lee HG, Camins A, Pallas M, Casadesus G, et al. (2011) The sirtuin
pathway in ageing and Alzheimer disease: mechanistic and therapeutic
considerations. Lancet Neurol 10: 275–279.
6. Mahajan SS, Leko V, Simon JA, Bedalov A (2011) Sirtuin modulators. Handb
Exp Pharmacol 206: 241–255.
7. Song NY, Surh YJ (2012) Janus-faced role of SIRT1 in tumorigenesis.
Ann N Y Acad Sci 1271: 10–19.
8. Herranz D, Maraver A, Canamero M, Gomez-Lopez G, Inglada-Perez L, et al.
(2012) SIRT1 promotes thyroid carcinogenesis driven by PTEN deficiency.
9. Luo J, Nikolaev AY, Imai S, Chen D, Su F, et al. (2001) Negative control of p53
by Sir2alpha promotes cell survival under stress. Cell 107: 137–148.
10. Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, et al. (2001)
hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107:
11. Li T, Kon N, Jiang L, Tan M, Ludwig T, et al. (2012) Tumor suppression in the
absence of p53-mediated cell-cycle arrest, apoptosis, and senescence. Cell 149:
12. Tang Y, Zhao W, Chen Y, Zhao Y, Gu W (2008) Acetylation is indispensable
for p53 activation. Cell 133: 612–626.
13. Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, et al. (2004) Calorie
restriction promotes mammalian cell survival by inducing the SIRT1
deacetylase. Science 305: 390–392.
14. Dai JM, Wang ZY, Sun DC, Lin RX, Wang SQ (2007) SIRT1 interacts with
p73 and suppresses p73-dependent transcriptional activity. J Cell Physiol 210:
15. Ford J, Jiang M, Milner J (2005) Cancer-specific functions of SIRT1 enable
human epithelial cancer cell growth and survival. Cancer Res 65: 10457–10463.
16. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, et al. (2004)
Mammalian SIRT1 represses forkhead transcription factors. Cell 116: 551–563.
17. Wang C, Chen L, Hou X, Li Z, Kabra N, et al. (2006) Interactions between
E2F1 and SirT1 regulate apoptotic response to DNA damage. Nat Cell Biol 8:
18. Heltweg B, Gatbonton T, Schuler AD, Posakony J, Li H, et al. (2006) Antitumor
activity of a small molecule inhibitor of human Sir2 enzymes. Cancer Res 66:
19. Ota H, Tokunaga E, Chang K, Hikasa M, Iijima K, et al. (2006) Sirt1 inhibitor,
Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK
signaling in human cancer cells. Oncogene 25: 176–185.
20. Kojima K, Ohhashi R, Fujita Y, Hamada N, Akao Y, et al. (2008) A role for
SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145
cells. Biochem Biophys Res Commun 373: 423–428.
21. Lara E, Mai A, Calvanese V, Altucci L, Lopez-Nieva P, et al. (2009) Salermide,
a Sirtuin inhibitor with a strong cancer-specific proapoptotic effect. Oncogene
22. Li L, Wang L, Li L, Wang Z, Ho Y, et al. (2012) Activation of p53 by SIRT1
inhibition enhances elimination of CML leukemia stem cells in combination with
imatinib. Cancer Cell 21: 266–281.
23. Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, et al. (2008) Discovery,
in vivo activity, and mechanism of action of a small-molecule p53 activator.
Cancer Cell 13: 454–463.
24. Pruitt K, Zinn RL, Ohm JE, McGarvey KM, Kang SH, et al. (2006) Inhibition
of SIRT1 reactivates silenced cancer genes without loss of promoter DNA
hypermethylation. PLoS Genet 2: e40.
25. Lee H, Kim KR, Noh SJ, Park HS, Kwon KS, et al. (2011) Expression of DBC1
and SIRT1 is associated with poor prognosis for breast carcinoma. Hum Pathol
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org8June 2013 | Volume 8 | Issue 6 | e66283
26. Nosho K, Shima K, Irahara N, Kure S, Firestein R, et al. (2009) SIRT1 histone
deacetylase expression is associated with microsatellite instability and CpG island
methylator phenotype in colorectal cancer. Mod Pathol 22: 922–932.
27. Bradbury CA, Khanim FL, Hayden R, Bunce CM, White DA, et al. (2005)
Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of
expression that changes selectively in response to deacetylase inhibitors.
Leukemia 19: 1751–1759.
28. Hida Y, Kubo Y, Murao K, Arase S (2007) Strong expression of a longevity-
related protein, SIRT1, in Bowen’s disease. Arch Dermatol Res 299: 103–106.
29. Huffman DM, Grizzle WE, Bamman MM, Kim JS, Eltoum IA, et al. (2007)
SIRT1 is significantly elevated in mouse and human prostate cancer. Cancer
Res 67: 6612–6618.
30. Jang KY, Hwang SH, Kwon KS, Kim KR, Choi HN, et al. (2008) SIRT1
expression is associated with poor prognosis of diffuse large B-cell lymphoma.
Am J Surg Pathol 32: 1523–1531.
31. Cha EJ, Noh SJ, Kwon KS, Kim CY, Park BH, et al. (2009) Expression of
DBC1 and SIRT1 is associated with poor prognosis of gastric carcinoma. Clin
Cancer Res 15: 4453–4459.
32. Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, et al. (2008)
SIRT1 redistribution on chromatin promotes genomic stability but alters gene
expression during aging. Cell 135: 907–918.
33. Wang RH, Sengupta K, Li C, Kim HS, Cao L, et al. (2008) Impaired DNA
damage response, genome instability, and tumorigenesis in SIRT1 mutant mice.
Cancer Cell 14: 312–323.
34. Yuan Z, Zhang X, Sengupta N, Lane WS, Seto E (2007) SIRT1 regulates the
function of the Nijmegen breakage syndrome protein. Mol Cell 27: 149–162.
35. Herranz D, Munoz-Martin M, Canamero M, Mulero F, Martinez-Pastor B, et
al. (2010) Sirt1 improves healthy ageing and protects from metabolic syndrome-
associated cancer. Nat Commun 1: 3.
36. Wang RH, Zheng Y, Kim HS, Xu X, Cao L, et al. (2008) Interplay among
BRCA1, SIRT1, and Survivin during BRCA1-associated tumorigenesis. Mol
Cell 32: 11–20.
37. Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, et al. (1992)
Multiple intestinal neoplasia caused by a mutation in the murine homolog of the
APC gene. Science 256: 668–670.
38. Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, et al. (1993)
Association of the APC gene product with beta-catenin. Science 262: 1731–
39. Munemitsu S, Albert I, Souza B, Rubinfeld B, Polakis P (1995) Regulation of
intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-
suppressor protein. Proc Natl Acad Sci U S A 92: 3046–3050.
40. Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, et al. (2008) The
SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.
PLoS One 3: e2020.
41. Boily G, He XH, Pearce B, Jardine K, McBurney MW (2009) SirT1-null mice
develop tumors at normal rates but are poorly protected by resveratrol.
Oncogene 28: 2882–2893.
42. McBurney MW, Yang X, Jardine K, Hixon M, Boekelheide K, et al. (2003) The
mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis.
Mol Cell Biol 23: 38–54.
43. Lemieux ME, Yang X, Jardine K, He X, Jacobsen KX, et al. (2005) The Sirt1
deacetylase modulates the insulin-like growth factor signaling pathway in
mammals. Mech Ageing Dev 126: 1097–1105.
44. Wu Y, Yakar S, Zhao L, Hennighausen L, LeRoith D (2002) Circulating insulin-
like growth factor-I levels regulate colon cancer growth and metastasis. Cancer
Res 62: 1030–1035.
45. el Marjou F, Janssen KP, Chang BH, Li M, Hindie V, et al. (2004) Tissue-
specific and inducible Cre-mediated recombination in the gut epithelium.
Genesis 39: 186–193.
46. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, et al. (1997)
Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC2/
2 colon carcinoma. Science 275: 1784–1787.
47. Ilyas M, Tomlinson IP, Rowan A, Pignatelli M, Bodmer WF (1997) Beta-catenin
mutations in cell lines established from human colorectal cancers. Proc Natl
Acad Sci U S A 94: 10330–10334.
48. Napper AD, Hixon J, McDonagh T, Keavey K, Pons JF, et al. (2005) Discovery
of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med
Chem 48: 8045–8054.
49. Liu Y, Bodmer WF (2006) Analysis of P53 mutations and their expression in 56
colorectal cancer cell lines. Proc Natl Acad Sci U S A 103: 976–981.
50. Prelich G (2012) Gene overexpression: uses, mechanisms, and interpretation.
Genetics 190: 841–854.
51. Menssen A, Hermeking H (2012) c-MYC and SIRT1 locked in a vicious cycle.
Oncotarget 3: 112–113.
52. Menssen A, Hydbring P, Kapelle K, Vervoorts J, Diebold J, et al. (2012) The c-
MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the
SIRT1 deacetylase form a positive feedback loop. Proc Natl Acad Sci U S A 109:
53. Yekkala K, Baudino TA (2007) Inhibition of intestinal polyposis with reduced
angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression. Mol
Cancer Res 5: 1296–1303.
54. Marshall GM, Liu PY, Gherardi S, Scarlett CJ, Bedalov A, et al. (2011) SIRT1
promotes N-Myc oncogenesis through a positive feedback loop involving the
effects of MKP3 and ERK on N-Myc protein stability. PLoS Genet 7: e1002135.
55. Holloway KR, Calhoun TN, Saxena M, Metoyer CF, Kandler EF, et al. (2010)
SIRT1 regulates Dishevelled proteins and promotes transient and constitutive
Wnt signaling. Proc Natl Acad Sci U S A 107: 9216–9221.
56. Chen WY, Wang DH, Yen RC, Luo J, Gu W, et al. (2005) Tumor suppressor
HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage
responses. Cell 123: 437–448.
57. Mohammad HP, Zhang W, Prevas HS, Leadem BR, Zhang M, et al. (2011)
Loss of a single Hic1 allele accelerates polyp formation in Apc(Delta716) mice.
Oncogene 30: 2659–2669.
58. Simic P, Zainabadi K, Bell E, Sykes DB, Saez B, et al. (2013) SIRT1 regulates
differentiation of mesenchymal stem cells by deacetylating beta-catenin. EMBO
Mol Med 5: 430–440.
59. Levina E, Oren M, Ben-Ze’ev A (2004) Downregulation of beta-catenin by p53
involves changes in the rate of beta-catenin phosphorylation and Axin dynamics.
Oncogene 23: 4444–4453.
60. Valenta T, Lukas J, Doubravska L, Fafilek B, Korinek V (2006) HIC1 attenuates
Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies.
EMBO J 25: 2326–2337.
61. Grady WM, Carethers JM (2008) Genomic and epigenetic instability in
colorectal cancer pathogenesis. Gastroenterology 135: 1079–1099.
62. Eads CA, Nickel AE, Laird PW (2002) Complete genetic suppression of polyp
formation and reduction of CpG-island hypermethylation in Apc(Min/+)
Dnmt1-hypomorphic Mice. Cancer Res 62: 1296–1299.
63. Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, et al. (1995)
Suppression of intestinal neoplasia by DNA hypomethylation. Cell 81: 197–205.
SIRT1 Inactivation Reduces Polyps in APCmin Mice
PLOS ONE | www.plosone.org9 June 2013 | Volume 8 | Issue 6 | e66283