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All content in this area was uploaded by Ha Yong Song on Sep 21, 2016
Content may be subject to copyright.
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Content may be subject to copyright.
Oncotarget1
www.impactjournals.com/oncotarget
www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2016
SIRT2 deletion enhances KRAS-induced tumorigenesis in vivo
by regulating K147 acetylation status
Ha Yong Song1, Marco Biancucci2, Hong-Jun Kang1, Carol O’Callaghan1, Seong-
Hoon Park3, Daniel R. Principe3, Haiyan Jiang3, Yufan Yan1, Karla Fullner Satchell2,
Kirtee Raparia4, David Gius3 and Athanassios Vassilopoulos1
1 Department of Radiation Oncology, Laboratory for Molecular Cancer Biology, Robert H. Lurie Comprehensive Cancer Center,
Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
2 Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
3
Department of Radiation Oncology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern
University, Chicago, IL, USA
4 Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA
Correspondence to: Athanassios Vassilopoulos, email: athanasios.vasilopoulos@northwestern.edu
Correspondence to: David Gius, email: david.gius@northwestern.edu
Keywords: KRAS, SIRT2, acetylation, lung cancer, pancreas transformation
Received: August 23, 2016 Accepted: September 02, 2016 Published: September 13, 2016
ABSTRACT
The observation that cellular transformation depends on breaching a crucial
KRAS activity threshold, along with the nding that only a small percentage of cells
harboring KRAS mutations are transformed, support the idea that additional, not
fully uncovered, regulatory mechanisms may contribute to KRAS activation. Here we
report that Kras
G12D
mice lacking Sirt2 show an aggressive tumorigenic phenotype
as compared to Kras
G12D
mice. This phenotype includes increased proliferation, KRAS
acetylation, and activation of RAS downstream signaling markers. Mechanistically,
KRAS K147 is identied as a novel SIRT2-specic deacetylation target by mass
spectrometry, whereas its acetylation status directly regulates KRAS activity,
ultimately exerting an impact on cellular behavior as revealed by cell proliferation,
colony formation, and tumor growth. Given the signicance of KRAS activity as a
driver in tumorigenesis, identication of K147 acetylation as a novel post-translational
modication directed by SIRT2 in vivo may provide a better understanding of the
mechanistic link regarding the crosstalk between non-genetic and genetic factors in
KRAS driven tumors.
INTRODUCTION
Sirtuin genes are the human and murine homologs
of the S. cerevisiae Sir2 gene that have been shown to
regulate both replicative and overall lifespan [1]. While the
precise role of sirtuins in mammalian lifespan regulation
is yet to be fully dened, it was recently demonstrated that
male transgenic mice overexpressing SIRT6 live longer
than their wild-type littermates [2]. Despite the observed
discrepancies and the scarcity of detailed mechanisms
regarding the role of sirtuins in longevity, it is well
established that they do appear to direct critical acetylome
signaling networks responding to caloric restriction (CR)
[3], and following stress, several mice lacking one of the
sirtuin genes develop illnesses that mimic those observed
in humans that are strongly connected to increasing age
[4]. Consistent with this, mice lacking Sirt2 [5] develop
multiple epithelial malignancies, including pancreatic
ductal adenocarcinoma (PDAC) and lung adenocarcinoma
(LACA). Based on these ndings, it has been suggested
that sirtuins are energy/nutrient stress sensor proteins
that alter the activity of downstream signaling networks
and targets via post-translational modications (PTMs)
involving lysine deacetylation in response to specic types
of cellular stress.
It is well established that KRAS mutations are
observed in 95% of patients with PDAC [6, 7] and roughly
30% of LACA cases [7]. However, similar to most human
malignancies, it is also clear that additional aberrant
genetic and/or biochemical events are ultimately required
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for carcinogenesis. Interestingly, healthy people have cells
expressing oncogenic KRAS in dierent organs, including
the pancreas, colon, and lungs, at rates far exceeding the
rates of cancer development [8, 9]. In addition, whereas
the number of cells that ultimately are transformed is only
a small fraction of those expressing mutant Kras (mtKras)
in mouse models [10], there is emerging evidence
suggesting that KRAS activity is increased in cells
derived from mtKras-driven PDAC as compared to non-
transformed pancreas expressing mtKras [11]. Therefore,
identication of mechanisms which may contribute to
breaching a crucial enzymatic KRAS activity threshold to
initiate carcinogenesis, even in the presence of activating
KRAS mutations, may ll the critical gap in knowledge
related to KRAS-driven tumorigenesis.
Based on our previous nding that Sirt2-/- mice
develop spontaneous tumors, and a recent study showing
that KRAS is acetylated in cancer cells [12], we aimed
to determine the role of SIRT2 in KRAS-induced
tumorigenesis in vivo. Here we report that Sirt2
-/-
-Kras
G12D
mice show enhanced pancreas transformation as well as
lung tumorigenesis as compared to Kras
G12D
mice. These
phenotypes are associated with increased proliferation
and KRAS acetylation as well as downstream RAS-
activated signaling markers, suggesting carcinogenesis is
more aggressive when Sirt2 is deleted. These results can
be explained by the nding that the acetylation status of
K147 directed by SIRT2 regulates KRAS’s GTP-bound
“active state,” as shown by using mutants that mimic
either acetylation (K147Q) or deacetylation (K147R). The
eect of K147 acetylation on KRAS activity impacts both
the transformative and oncogenic properties of KRAS.
These results, together with the fact that K147 acetylation
can be detected in tissues in vivo, highlight the role of this
reversible PTM in regulating KRAS activity, and, more
importantly, identify for the rst time K147 acetylation as
an oncogenic modication directed by SIRT2.
RESULTS
Deletion of Sirt2 induces KRAS-mediated
pancreas transformation
To determine the role of SIRT2 in KRAS-induced
pancreas transformation, the pancreatic epithelium-
specic KrasG12D mice, which were generated using the
well-established LSL-KrasG12D knock-in mouse model
[13] and the Ptf1Cre driver line [14] to direct recombination
in pancreas, were crossed with Sirt2-/- mice [5] to
generate Sirt2+/+;LSL-KrasG12D;Ptf1Cre and Sirt2-/-;LSL-
KrasG12D;Ptf1Cre mice (referred to here as KrasG12D-Ptf1
and Sirt2-/--KrasG12D-Ptf1 mice, respectively). At 4 months
of age, the Sirt2-/--KrasG12D-Ptf1 mice exhibited a complete
loss of normal glandular architecture, with no discernible
normal tissue (Figure 1A right, see absence of normal
tissue (n)). This abnormal architecture was accompanied
by severe brosis (Figure S1A, right panel) as observed by
trichrome staining, especially around areas with detectable
pancreatic lesions. Histopathologic analyses revealed that
all mice developed pancreatic intraepithelial neoplasia
(PanIN) of dierent grades (Figure 1B). However, Sirt2
-/-
-Kras
G12D
-Ptf1 mice showed an increase in induction and
PanIN progression compared to KrasG12D-Ptf1 mice as
revealed by the amount and grade of these lesions both at
4 and 6 months of age (Figure 1C). This is consistent with
a tumor suppressor role of SIRT2 in the pancreas, which
is also in line with lower SIRT2 mRNA levels detected
in pancreatic adenocarcinoma compared to normal tissue
(Figure S1B).
To explore whether the Kras-induced pancreas
transformation found in Sirt2-/- mice is due to a cell
autonomous mechanism, we generated Sirt2 conditional
knockout (Sirt2
/
) mice (Figure S1C-E) that were crossed
with the LSL-KrasG12D;Ptf1Cre mouse model to study the
eect of pancreatic epithelium-specic Sirt2 deletion
in the KrasG12D knock-in mouse model. Histopathologic
analyses showed similar patterns of induction and PanIN
progression in both the Sirt2
/
-Kras
G12D
-Ptf1 and Sirt2
-/-
-
Kras
G12D
-Ptf1 mice (Figure 1D, 1E), suggesting that SIRT2
acts autonomously in pancreatic cells. Of note, some Sirt2
-
/--KrasG12D-Ptf1 mice (two out of ten) developed areas of
PDAC at 8 months of age (Figure 1F), an acceleration
of the timeframe of PDAC development in KrasG12D-
Ptf1 mice (≥1 year). In addition, these mice contained
areas of tumor cells that appeared disorganized and were
surrounded by dense brosis, as evidenced by a robust
desmoplastic response (Figure 1F).
Oncogenic KRAS activates a plethora of signaling
pathways, including the canonical Raf/MEK/ERK
pathway. Lesions from both KrasG12D-Ptf1 and Sirt2-/-
-KrasG12D-Ptf1 mice that were conrmed to be of pancreatic
origin after positive staining with CK19, were also stained
for phosphorylated ERK (pERK) as an indirect marker of
KRAS activity in these mice (Figure 1G). Sirt2
-/-
-Kras
G12D
-
Ptf1 pancreas showed higher pERK levels compared to
KrasG12D-Ptf1 mice, indicating that deletion of Sirt2 is
associated with increased downstream KRAS signaling.
Finally, proliferation analysis using BrdU labeling showed
active proliferation in Sirt2-/--KrasG12D-Ptf1 pancreas as
compared to KrasG12D-Ptf1 pancreas (Figure 1G, 1H).
All these data together further indicate that loss of Sirt2
induces KRAS-mediated pancreas transformation.
Deletion of Sirt2 increases KRAS-induced lung
adenocarcinoma
To determine the role of SIRT2 in KRAS-
dependent lung adenocarcinoma, the well-established
LSL-KrasG12D knock-in mouse model was crossed with
Sirt2-/- mice to generate Sirt2+/+;LSL-KrasG12D and Sirt2-
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Figure 1: Mice lacking Sirt2 and expressing mtKras exhibit enhanced development of PanIN and progression to PDAC.
A. Pancreata from KrasG12D-Ptf1 and Sirt2-/--KrasG12D-Ptf1 mice were harvested at 4 months of age (n = 5-8), and samples were xed and
analyzed by H&E staining for pancreas morphology. A representative image from each genotype is shown. Normal acinar tissue (n) is found
in KrasG12D-Ptf1 mice but not in Sirt2-/--KrasG12D-Ptf1 mice. Scale bar 200 μM (10x). B. Histology of the pancreas and dierent grades of
lesions found in the Sirt2-/--KrasG12D-Ptf1 mice. Representative images are shown: (a) normal tissue with both acinar cells and islets (i), (b)
PanIN1, (c) PanIN2; and (d) PanIN3. Scale bar 200 μM (10x). C. Quantication of lesions found at 4 months (left panel) and 6 months
(right panel) of age is presented. Data represent mean ± SEM. D. Pancreata from Sirt2-/--KrasG12D-Ptf1 (n = 5-8) and Sirt2/-KrasG12D-
Ptf1 (n = 4) mice were harvested at 2 months of age, and samples were xed and analyzed by H&E staining for pancreas morphology. A
representative image from lesions found in each genotype is shown. Scale bar 200 μM (10x). E. Quantication of normal tissue and lesions
found at 2 months of age in KrasG12D-Ptf1, Sirt2-/--KrasG12D-Ptf1, and Sirt2-/--KrasG12D-Ptf1 mice is presented. Data represent mean ± SEM.
F. Pancreata from the Sirt2-/--KrasG12D-Ptf1 mice that displayed mouse PDAC (mPDAC) were H&E stained, and a representative image
of PDAC is shown. Magnication (20x vs 10x left) of the mPDAC area is shown in the right panel. Scale bar 200 μM. G. Pancreata from
KrasG12D-Ptf1 and Sirt2-/- -KrasG12D-Ptf1 mice were isolated, and sections were subsequently IHC stained with antibodies against pERK (left,
5x, scale bar 200 μM), CK19 (middle, 20x, scale bar 100 μM) and BrdU (right, 20x, scale bar 100 μM). H. Quantication of BrdU-positive
cells found in pancreas of KrasG12D-Ptf1 and Sirt2-/--KrasG12D-Ptf1 mice. Data represent mean ± SD (n = 3), *p < 0.05.
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/-;LSL-KrasG12D mice (referred to here as KrasG12D and
Sirt2
-/-
-Kras
G12D
mice, respectively) followed by intranasal
administration of adenoviral Cre (adenoCRE) to induce
mtKras expression in the lungs [15]. Examination of the
lungs in a cohort of untreated Sirt2-/--KrasG12D mice as
well as KrasG12D and Sirt2-/--KrasG12D mice 4 months after
exposure to adenoCRE revealed that Sirt2-null mice had a
signicantly greater number of tumor lesions in the lungs
(Figure 2A, 2B), as well as larger tumor area (Figure 2C),
than KrasG12D mice.
Histopathologic examination of lungs at an earlier
time point after adenoCRE administration showed
similar results. In particular, 2 months after adenoCRE
administration, the Sirt2-/--KrasG12D mice lungs exhibited
increased numbers of tumor cell nests per 10x eld
(Figure S1F) and increased tumor size (Figure S1G).
Histopathological analyses showed that Sirt2-/--KrasG12D
mice clearly developed lung tumors (Figure S1H, two right
panels), as compared to mostly scattered areas of atypical
adenomatous hyperplasia (AAH) developed in the KrasG12D
mice (Figure S1H, two left panels). The tumors were
adenocarcinomas, as determined by thyroid transcription
factor-1 staining (data not shown). Furthermore, Sirt2-/--
KrasG12D serial lung sections showed nests of tumor cells
exhibiting increased in vivo pERK (Figure S1I, middle
panels) and Ki-67 staining (Figure S1I, right panels),
implying increased KRAS activity and tumorigenicity in
the lungs when Sirt2 is deleted. Collectively, these results
suggest that Sirt2 deletion enhances the progression of
oncogenic KRAS-induced lung adenocarcinomas.
Increased acetylation of KRAS due to Sirt2 loss is
associated with increased KRAS activity
To start unraveling the mechanistic details regarding
either the direct or indirect eect of SIRT2 on KRAS, we
checked whether there is a direct interaction between
the two proteins. Of note, we detected interaction of
the two proteins in both pancreas and lung (Figure 2D)
tissues found to exhibit enhanced KRAS-mediated
transformation and/or tumorigenicity when Sirt2 was
lost, as presented earlier. The presence of both proteins
in the same complex was further conrmed in reciprocal
co-immunoprecipitation experiments after overexpressing
both KRAS and SIRT2 in 293T cells (data not shown),
as well as after checking endogenous proteins in Sirt2+/+
primary MEFs (Figure S2A). Furthermore, KRAS was
detected to specically interact with SIRT2 but not other
members of the sirtuin family (Figure S2B, C) arguing
for a direct regulatory function of SIRT2 on KRAS.
More importantly, tissues depleted of Sirt2 exhibited a
signicant increase in KRAS acetylated levels which was
further associated with increased KRAS activity (Figure
2E-2G). In particular, GTP-bound “active” KRAS,
detected through a specic protein interaction with the
Raf1 Ras-binding domain (Raf1-RBD), was enriched in
the Sirt2-/- tissues. Under the same experimental conditions,
enhanced KRAS activity was associated with increased
pERK levels (Figure 2E-2G), further establishing the
positive eect of KRAS acetylation on its activity in vivo.
Taken together, these results favor the scenario of KRAS
being a SIRT2 deacetylation target in vivo. The generality
of the regulatory role of SIRT2 in KRAS signaling was
also conrmed in Sirt2-/- MEFs (Figure S2D). In addition,
in HCT116 cells, which express a wild-type and a mutant
Kras allele, infection with lenti-shSIRT2 increased both
KRAS activity, as evidenced by increased pERK levels
(Figure S2E), and colony formation, as revealed by
enhanced growth in soft agar (Figure S2F, G).
To establish KRAS as a legitimate SIRT2
deacetylation target, a series of cell culture experiments
were performed. To assess whether SIRT2 deacetylates
KRAS, 293T cells were co-transfected either with HA-
KRAS or HA-KRASG12V, with wild-type Flag-SIRT2, as
well as with p300/CBP based on our results showing
that these two are the main histone acetyl transferases
(HATs) to acetylate KRAS (Figure S2H). Following
immunoprecipitation with an anti-acetyl-lysine (Ac-K)
antibody, both wild-type and KRAS
G12V
were deacetylated
by SIRT2 (Figure S2I, lane 3 vs lane 2 and lane 6 vs lane
5, respectively). Increased KRAS activity upon treatment
with both nicotinamide, an inhibitor of all members of
the sirtuin family, and AGK2, a specic SIRT2 inhibitor
(Figure S2J), highlights the unique role of SIRT2
among other sirtuins as a regulator of KRAS activity.
Finally, KRAS activity was decreased in co-transfection
experiments using wild-type SIRT2 (SIRT2wt), but not a
SIRT2 deacetylation null mutant gene (SIRT2dn) (Figure
S2K), suggesting that SIRT2 regulates KRAS activity
through its deacetylation activity. Interestingly, the same
eect on KRASG12V activity was observed (data not
shown), implying that both wild-type and mutant KRAS
can be regulated through SIRT2-mediated deacetylation.
Together these results demonstrate that KRAS contains a
reversible acetyl-lysine and aberrant regulation of KRAS
when SIRT2 is lost may result in induced KRAS activity.
Acetylation status of K147 directs KRAS activity
and transformative properties
To identify specic SIRT2 target lysines, lysates
from 293T cells expressing either Flag-KRASG12V alone
(sample 1) or Flag-KRASG12V and SIRT2 (sample 2) were
run on a gel and bands corresponding to the molecular
weight of Flag-KRAS were excised and analyzed by
mass spectrometry (Figure S3A-C). In the absence of
SIRT2 (Figure S3A, sample 1), acetylated lysines 104
and 147 (K104, K147 in red circles) were detected with
the peptides identied under this condition (highlighted
in yellow). When SIRT2 was overexpressed (Figure S3A,
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Figure 2: Loss of Sirt2 enhances KRASG12D-induced lung adenocarcinoma and KRAS acetylation is associated with
increased activity in vivo. A.-C. The lungs from KrasG12D and Sirt2-/--KrasG12D mice (n = 5 for each genotype), four months after
intranasal administration of adenoCRE (Ad-Cre), were harvested, xed, sectioned, and H&E stained. A. Representative images from lungs
(2.5x) of control Sirt2-/--KrasG12D mice (untreated with adenoCRE), and lung tumors developed in KrasG12D and Sirt2-/--KrasG12D mice are
shown. Scale bar 50 μM. B. Tumor burden at 4 months in lungs from KrasG12D and Sirt2-/- -KrasG12D mice is presented. Data represent mean
± SEM, **p < 0.01. C. Higher magnication of lung histology in both KrasG12D (left, 10x) and Sirt2-/--KrasG12D (right, 10x) mice is shown.
Scale bar 200 μM. D. Endogenous KRAS was immunoprecipitated from lysates of either lung (left) or pancreas (right) tissues. Interaction
was conrmed by western blotting using anti-SIRT2 and anti-KRAS antibodies. Endogenous levels of both KRAS and SIRT2 are shown
as input. E. The lungs from KrasG12D and Sirt2-/--KrasG12D mice, 2 months after intranasal administration of adenoCRE, were harvested
and analyzed for KRAS acetylation and KRAS activity. KRAS acetylation was detected by immunoprecipitation with a pan anti-Ac-K
antibody, and KRAS activity was detected by immunoprecipitation with Raf1-RBD and by blotting for pERK. ERK, KRAS, and SIRT2
inputs are shown as controls, and actin and tubulin were used as loading controls. F. The pancreata from KrasG12D-Ptf1 and Sirt2-/--KrasG12D-
Ptf1 mice were harvested and analyzed for KRAS activity and KRAS acetylation as described in panel (E). G. Quantication of KRAS
activity, KRAS acetylation levels, and phosphorylation levels of ERK from panel (F). Data represent mean ± SEM.
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Figure 3: K147 acetylation status directed by SIRT2 regulates KRAS dependent proliferation. A. Kraslox MEFs were
infected with lenti-KRAS or lenti-KRASK147Q, and subsequently treated with 4-hydroxytamoxifen (4HT) to delete endogenous Kras. Next,
Sirt2 was knocked down by siRNA and cell proliferation was monitored. Data represent mean ± SD of three independent experiments,
****p < 0.0001. B. KRAS activity in lysates from cells used in (A) was detected by immunoprecipitation of GTP-bound KRAS using
Raf1-RBD agarose beads followed by immunoblotting using an anti-RAS antibody. KRAS, SIRT2 and GAPDH levels were checked by
immunoblotting as shown. C., D. Kraslox MEFs were infected with lenti-KRAS or lenti-KRASK147R and same experiments as described in
(A, B) were performed. For cell proliferation, data represent mean ± SD of three independent experiments, ****p < 0.0001. E. Kraslox
MEFs were infected with lenti-KRAS, lenti-KRASK147R, lenti-KRASK147Q (left), as well as lenti-KRASG12V, lenti-KRASG12V-K147R, and lenti-
KRASG12V-K147Q (right) and subsequently treated with 4-hydroxytamoxifen (4HT) to delete endogenous Kras. Active KRAS was detected
by immunoprecipitation of GTP-bound KRAS using Raf1-RBD agarose beads followed by immunoblotting using an anti-Flag antibody.
Levels of exogenously expressed KRAS proteins are shown in whole cell lysates by western blotting using an anti-Flag antibody. Actin and
tubulin are used as loading controls. F., G. The same cells as in (E) were used to check proliferation rate by measuring the number of cells
for 6 consecutive days (F) and by determining the colony formation ability after 21 days (G). For cell proliferation, data represent mean ±
SD of three independent experiments, ****p < 0.0001 KRASG12V/K147R and KRASG12V/K147Q vs KRASG12V MEFs, ****p < 0.0001 KRASK147Q vs
KRAS MEFs. H. Quantication of data in panel (G) is shown. Data represent the mean ± SEM of three independent experiments, *p < 0.05
KRASK147Q vs KRAS MEFs, ****p < 0.0001 KRASG12V/K147R vs KRASG12V MEFs.
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sample 2), K147 acetylation was undetectable in contrast
to K104 acetylation, implying that K147 is a SIRT2-
specic reversibly acetylated lysine target.
It has been previously shown that substitution of
a lysine with a glutamine mimics the acetylated state,
while substitution with an arginine mimics deacetylation
[16]. For this, we generated K147Q and K147R KRAS
mutants to determine the eect of K147 acetylation on
KRAS activity. The KRAS mutants were expressed
in Kraslox MEFs (Hras-/-;Nras-/-;Kraslox/lox;RERTnert/ert)
where the conditional Kraslox alleles are fully excised
upon expression of the Cre recombinase, inducible in
the presence of 4-hydroxytamoxifen (4HT), resulting
in Rasless MEFs [17]. In particular, Kraslox MEFs were
infected with lenti-KRAS, lenti-KRAS
K147R
, lenti-KRAS
K147Q
,
lenti-KRASG12V, lenti-KRASG12V-K147R and lenti-KRASG12V-
K147Q and subsequently treated with 4HT, resulting in cells
expressing only the lentiviral constructs of KRAS and
KRAS
G12V
. Cell proliferation rate was signicantly higher
in MEFs expressing KRAS after knocking down Sirt2
consistent with increased KRAS activity detected in these
cells, whereas decreased Sirt2 expression didn’t aect
proliferation in MEFs expressing either KRASK147R or
KRAS
K147Q
(Figure 3A-3D). Next we examined the impact
of K147 acetylation status in both wild-type KRAS and
mutant KRAS
G12V
. MEFs expressing KRAS
K147Q
exhibited
increased amounts of “active” GTP bound KRAS, as
measured by the Ras pulldown assay, compared to the
KRAS and KRASK147R infected cells (Figure 3E, left),
suggesting that acetylation enhances wild-type KRAS
activity. In cells expressing KRASG12V, expression of
KRASG12V-K147Q produced an increase in the amount of
active protein compared to KRASG12V
, whereas less
active protein was observed with expression of KRAS
G12V-
K147R. (Figure 3E, right). The eect of K147 acetylation
status on KRAS activity was conrmed in 293T cells
after transient overexpression of the KRAS and KRASG12V
acetylation mutants (data not shown). MEFs expressing
KRASK147Q exhibited increased proliferation as determined
by measuring the number of cells and colonies (Figure
3F-3H), compared to cells expressing either KRAS or
KRASK147R. In KRASG12V expressing MEFs, KRASG12V-K147Q
increased the proliferation rate, whereas KRASG12V-K147R
exerted the opposite eect (Figure 3F-3H).
To determine the impact of K147 acetylation
status on KRAS transformative properties, NIH3T3
cells expressing KRASG12V acetylation mutants after
knocking down endogenous Kras were constructed. Of
note, NIH3T3 cells expressing KRASG12V-K147Q exhibited
increased proliferation (Figure S4A-C), consistent with
previous results in MEFs, excluding the possibility that the
observed dierences in proliferation could be attributed
either to the dierent integration sites after viral infection
or to a cell type-specic phenomenon. More importantly,
these cells formed more colonies when grown both under
conuence and in soft agar (Figure 4A-4C), indicating
enhanced transforming activity of KRAS upon K147
acetylation. To further establish that K147 acetylation is
an oncogenic PTM, both NIH3T3 cells and Rasless MEFs
expressing the dierent KRASG12V acetylation mutants
were used for subcutaneous injections into nude mice, and
tumor growth was monitored. In accordance with the cell
culture results, KRASG12V-K147Q-expressing cells exhibited
a signicant increase in tumor growth rate relative to
cells expressing KRAS
G12V
, as evidenced by the increased
volume of tumors developed in these mice (Figure 4D-4H
and Figure S4D, E).
K147 acetylation alters the nucleotide exchange
kinetics of KRAS
K147 is located within the functionally important
G5 box, a critical peptide sequence for the formation
of the nucleotide binding site in RAS proteins [18].
Mutation of the adjacent A146 has been shown to activate
the transforming potential of KRAS by signicantly
increasing the nucleotide exchange rate [19]. To shed light
on the biochemistry related to the enhanced KRAS activity
detected when K147 is mutated to glutamine (resembling
the acetylated status), we employed a method using
uorescently labeled GDP and GTP analogues to measure
the nucleotide exchange kinetics of KRAS mutants
(Figure 5A). The increase in uorescence intensity when
N-methylanthraniloyl (mant)-labeled GDP and GTP bind
to puried KRAS, KRASK147Q, KRASG12V and KRASG12V/
K147Q allows monitoring of nucleotide exchange in real
time. Both KRASK147Q and KRASG12V/K147Q exhibited an
increased rate of nucleotide exchange compared to KRAS
and KRASG12V
, respectively (Figure 5B, 5C). Given the
higher intracellular concentration of GTP compared to
GDP, this may explain the increased GTP-bound “active”
KRAS detected when K147 is acetylated.
These results were conrmed when a similar
approach was followed using extracts from cells
expressing Flag-tagged KRAS or KRAS
K147Q
. In this case,
extracts were treated with 2 µM GDP, resulting in loading
of both KRAS and KRASK147Q with GDP, and exchange
reactions in the presence of EDTA - catalyzed o- were
carried out after adding increasing amounts of GTP.
GTP-bound KRAS and KRASK147Q were then assessed
by immunoprecipitation using Raf1-RBD agarose beads
(Figure 5D). In accordance with our previous results,
KRASK147Q showed enhanced GDP exchange for GTP
compared to KRAS (Figure 5E), suggesting that K147
acetylation may alter the nucleotide exchange kinetics
favoring the active KRAS state.
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K147 acetylation is a PTM that can be detected
in tissues
We previously observed increased KRAS
acetylation levels in tissues when Sirt2 is lost (Figure
2E-2G). However, acetylation was detected in those
experiments using a general pan Ac-K antibody. We now
tested whether K147 is specically acetylated in Sirt2-/-
tissues using a custom-made polyclonal antibody against
acetylated K147 (Ac-K147) of KRAS. The specicity
of the antibody against KRAS Ac-K147 was checked
Figure 4: K147 acetylation status regulates KRAS transformative properties. A., B. Transforming activity of NIH3T3 cells
expressing KRASG12V, KRASG12V-K147R and KRASG12V-K147Q after knocking down endogenous Kras was checked by testing colonies formed
when cells were grown under conuency (A) and in soft agar (B). C. Quantication of data in panel (B) is shown. Data represent the mean
± SD of three independent experiments, **p < 0.01 KRASG12V/K147R vs KRASG12V cells, ***p < 0.001 KRASG12V/K147Q vs KRASG12V cells. D.
NIH3T3 cells expressing KRASG12V, KRASG12V-K147R and KRASG12V-K147Q after knocking down endogenous Kras were injected subcutaneously
into nude mice, and tumor growth was monitored by measuring tumor volume (n = 8 tumor injections were performed for each cell line).
Data represent mean ± SEM, ****p < 0.0001 KRASG12V/K147Q vs KRASG12V tumors. E. Kraslox MEFs infected with lenti-KRASG12V, lenti-
KRASG12V-K147R and lenti-KRASG12V-K147Q followed by treatment with 4HT were injected subcutaneously into nude mice, and tumor growth
was monitored by measuring tumor volume (n = 8 tumor injections were performed for each cell line). Data represent mean ± SEM, ***p
< 0.001 KRASG12V/K147Q vs KRASG12V tumors. F. Number of tumors formed in nude mice injected with NIH3T3 cells expressing KRASG12V,
KRASG12V-K147R and KRASG12V-K147Q after knocking down endogenous Kras (upper) and Kraslox MEFs infected with lenti-KRASG12V, lenti-
KRASG12V-K147R, and lenti-KRASG12V-K147Q followed by treatment with 4HT (lower). G. Characteristic images of mice bearing the subcutaneous
tumors after injecting the dierent cells. H. Subcutaneous tumors were removed after sacricing the mice, and characteristic images of the
tumors are shown.
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Figure 5: The eect of K147 acetylation on the nucleotide exchange rate along with its detection in vivo support the
physiological signicance of this post-translational modication. A. Schematic representation of experimental results shown in
(B). Puried GDP-loaded KRAS is incubated in the presence of EDTA with uorescently labeled mant-GDP and mant-GTP. The nucleotide
exchange rate is monitored in real-time by measuring the increase in uorescence intensity after binding of mant-labeled nucleotides
to KRAS. B. Change in uorescence over time using the method described in (A) is shown for KRAS and KRASK147Q (left) as well as
KRASG12V and KRASG12V/K147Q (right). C. First-order rate constants for nucleotide exchange were determined for KRAS and KRASK147Q
(upper) as well as KRASG12V and KRASG12V/K147Q (lower). Data represent mean ± SEM. All readings were performed in triplicate. D.
Schematic representation of experimental results shown in E. Extracts from 293T cells expressing Flag-KRAS and Flag-KRASK147Q were
treated with 2 µM GDP, and exchange reactions in the presence of EDTA were carried out after adding excess amounts of GTP. GTP-bound
KRAS and KRASK147Q were nally assessed after immunoprecipitation using Raf1-RBD agarose beads. Immunoprecipitates following the
procedure described in (D) were run on a gel, transferred to a PVDF membrane, and immunoblotted using an anti-Flag antibody. Shorter
(upper) and longer (lower) exposures of the same membrane are shown. F. The pancreata from KrasG12D-Ptf1 and Sirt2-/--KrasG12D-Ptf1 mice
were harvested and analyzed for K147 acetylation by immunoprecipitation using an anti-Ac-K147 antibody followed by western blotting
with a KRAS antibody. G Pancreas tissue sections from KrasG12D-Ptf1 and Sirt2-/—KrasG12D-Ptf1 mice (n = 3) were H&E stained or stained
by IHC using an anti Ac-K147 antibody. Representative images are shown (5x). Scale bar 200 μM.
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in vitro via dot blot (Figure S5A) as well in cell culture
experiments (Figure S5B-E) where K147 acetylation
was detected both after overexpressing KRAS and after
checking endogenous KRAS. Therefore, after completing
the validation process, immunoprecipitation experiments
using the anti Ac-K147 antibody in pancreas from both
KrasG12D-Ptf1 and Sirt2
-/-
-Kras
G12D
-Ptf1 mice revealed that
K147 acetylation is a PTM that can be detected in vivo
(Figure 5F), highlighting the physiological signicance of
this modication. Then, after checking whether this newly
developed antibody is suitable for immunohistochemistry
(Figure S5F), pancreas from mice with the genotypes
described above were stained. Consistently, we detected
increased K147 acetylation in pancreas where Sirt2 is lost
(Figure 5G). Interestingly, besides pancreatic lesions that
were stained positively for K147 acetylation, the majority
of normal acinar cells in the Sirt2
-/—
KRAS
G12D
-Ptf1 mice
that were not yet transformed were stained strongly with
the anti Ac-K147 antibody, and this was further associated
with phosphorylation of ERK (Figure S5G), suggesting
that K147 acetylation may be an early event in KRAS-
induced tumorigenesis in the context of Sirt2 deletion.
Under these conditions, enhanced downstream KRAS
signaling could contribute to enhanced induction of acinar
to ductal metaplasia (ADM), as a result of the direct eect
of K147 acetylation on KRAS activity.
DISCUSSION
Based on the well-established observation that
increasing age is the most signicant risk factor for
tumor development, intense research eorts have focused
on the role played by sirtuins as the mechanistic link
between aging and tumorigenesis. Regarding SIRT2, we
have previously shown that Sirt2-decient mice develop
tumors in several tissues, providing the rst strong genetic
evidence that SIRT2 may function as a tumor suppressor
through its role in regulating the anaphase-promoting
complex/cyclosome (APC/C) [5]. Although additional
studies further support the tumor-suppressive role of
SIRT2 [20], it is worth mentioning that SIRT2 has been
shown to exert dual functions where it seems to have
oncogenic properties as well [21, 22]. This highlights the
complexity of sirtuin biology implying that additional
aberrant genetic and/or biochemical events are required
for and need to be identied to untangle the role of SIRT2
in tumorigenesis, especially within a tissue-specic and
genetic context.
Here, we demonstrate that SIRT2 functions as a
tumor suppressor in the context of KRAS-dependent
tumorigenesis. Mechanistically, we propose that K147
acetylation may increase KRAS activity and exerts a
signicant impact on cellular behavior as evidenced
by increased proliferation, colony formation ability,
anchorage independent growth and, nally, tumor growth
rate in cells expressing a K147Q KRAS mutant resembling
the acetylated state. These results, together with the
conrmed detection of K147 acetylation in tissues using
our anti-Ac-K147 KRAS antibody, reveal for the rst time
the role of K147 acetylation in vivo as a novel KRAS PTM
directed by SIRT2. Sirtuins are deacetylation enzymes
and as such they have been found to regulate a plethora
of substrates with SIRT2 not being an exemption. Thus,
it is expected that additional pathways may contribute
either synergistically or independently to KRAS-induced
tumorigenesis. In this study we provide evidence to further
establish the tumor-permissive phenotype observed in
mice lacking Sirt2, and we show that K147 KRAS
acetylation upon Sirt2 loss plays a signicant role in
driving cellular transformation and aberrant growth, at
least, in tissues where increased Ras activity has been
proven to be a driver for tumorigenesis.
KRAS is the most frequently mutated oncogene in
human cancer [23], which justies the intensive eorts
made to elucidate regulatory mechanisms, signaling
transduction, feedback loops, isoform dierences, and
heterogeneity regarding the mutational landscape, as well
as to exploit vulnerabilities in RAS-related tumors [24].
Here we show that K147 acetylation is a PTM directly
aecting KRAS activity rather than protein localization, as
commonly happens with other PTMs such as farnesylation
[25]. This nding, together with recently published studies
showing that both K104 acetylation [12] and K147 mono-
ubiquitination [26] regulate KRAS activity, even though
it is still unknown whether these PTMs play any role in
KRAS tumors in vivo, indicates that we are just starting
to understand and evaluate the contribution of PTMs to
ne-tuning KRAS activity. In this regard, the well-known
tumor suppressor p53 showcases how complex this
regulatory network of PTMs might be [27]. With regard
to KRAS, K147Q, which resembles K147 acetylation,
increases the SOS-independent nucleotide exchange rate,
whereas K147 mono-ubiquitination doesn’t aect intrinsic
nucleotide dissociation. Instead it impedes GAP-mediated
GTP hydrolysis, resulting in Ras activation [28]. The
dierence in the biochemistry implies that acetylation
and mono-ubiquitination exert their impact on KRAS
through dierent mechanisms. Moreover, if the eect
of acetylation was through regulating ubiquitination,
it would be expected that K147 acetylation (or K147
mutation) would prevent mono-ubiquitination, resulting in
decreased KRAS activity. However this is not supported
by our experimental results, which further establishes the
independent function of these PTMs. Regarding K104
acetylation, biochemical characterization revealed that
K104 acetylation renders KRAS more resistant to SOS-
dependent nucleotide exchange [12, 29], even though
the eect on KRAS activity hasn’t been tested directly.
Consistent with previous reports, we could detect K104
acetylation in cells overexpressing KRASG12V. However,
our mass spectrometry analysis revealed K147 acetylation
as a reversible lysine acetylation which is specically
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deacetylated by SIRT2. Based on our biochemical, cell
behavior and, most importantly, in vivo analyses, we
show that K147 acetylation may exert a positive eect
on KRAS activity (in contrast to the decreased KRAS
activity proposed upon K104 acetylation), which results
in enhanced KRAS-induced tumorigenesis in mouse
models. The opposing eect of the acetylation of these
two sites, at least at the biochemical level, indicates the
distinct nature of the PTMs, which might be reected
at the molecular and cellular levels with respect to
enzymes involved in acetylation/deacetylation as well as
physiological conditions under which these PTMs regulate
KRAS activity.
Consistent with previous studies showing that K147
is within a conserved sequence critical for the formation
of the nucleotide binding site [18], we found a signicant
increase in the nucleotide exchange rate when K147
was mutated to glutamine, which resembles acetylated
K147. Given the higher intracellular concentration of
GTP, it can be suggested that acetylation of wild-type
KRAS may result in a more frequent auto-activation
by spontaneously exchanging GDP for GTP, leading to
increased aberrant RAS signaling. This could increase
mutant KRAS activity as well, based on the observation
that most KRAS mutants retain some intrinsic GTPase
activity, despite their insensitivity to GAP-mediated GTP
hydrolysis, implying that increased nucleotide exchange
could positively regulate KRAS activity. The idea that an
increase in nucleotide exchange can act synergistically
with decreased GTPase activity is supported by data
showing that strongly activating mutations in terms of
transforming potential represent a combined eect of
reduced GTPase activity and increased exchange [19].
Taking this into account and in the context of a recently
proposed classication of dierent KRAS mutations
based on biochemical characterization [30], we could
predict that some KRAS mutations, such as G12C, G12D,
and G12V, would be aected more by K147 acetylation,
based on the undetectable eect on nucleotide exchange
and the modest eect on GTP hydrolysis compared to
other mutations. Moreover, the nding that KRAS K147
seems to be a specic deacetylation target of SIRT2 raises
some novel, yet undiscovered, possibilities regarding
the crosstalk between non-genetic and genetic factors in
KRAS-induced tumors. Taking into consideration that
sirtuins respond to both environmental stress and nutrient
availability, whereas their activity is altered with aging,
it would be trivial to determine in the future whether the
SIRT2-directed acetylation status of K147 can, at least
in some part, mediate the eects of non-genetic factors
known to aect KRAS-induced tumorigenesis [31], [32],
[33] providing an additional layer of regulation.
Taking into account the regulatory role of SIRT2,
we present here evidence to further establish the tumor
suppressor role of SIRT2 in a KRAS-specic context by
proposing that the acetylation status of K147 may direct
activity and transformative properties. Both in vitro assays
and in vivo mouse models, as well as data showing that
K147 acetylation can be detected in vivo, underscores
the signicance of unraveling the molecular and cellular
events related to PTMs which may directly ne-tune
KRAS activity. Given the so far unsuccessful attempts to
therapeutically target KRAS, stemming from the diculty
of predicting which of the many downstream eector
pathways is engaged under specic conditions, the deeper
understanding of mechanisms regulating RAS activity
itself could ll critical knowledge gaps in the RAS biology
eld. Furthermore, identication of K147 acetylation as
a novel post-translational modication directed by SIRT2
may provide a better understanding of the mechanistic link
regarding the crosstalk between non-genetic and genetic
factors in KRAS driven tumors. Thus it is reasonable to
suggest that conditions known to aect sirtuin activity
may further regulate KRAS activity and signal output
through K147 acetylation even in the presence of mutant
KRAS. In this regard and towards possible therapeutic
implications, our in vivo experimental results suggest that
decreasing KRAS acetylation through increased SIRT2
activity could be an approach to limiting tumorigenic
potential of KRAS-driven tumors.
MATERIALS AND METHODS
Mice
A description of all mice used in this study, as well
as details regarding the generation of the Sirt2
/
mice, can
be found in the Supplementary Materials and Methods
section. Mice were housed, fed and treated in accordance
with the guidelines approved by the Northwestern
University IACUC.
Histology/immunohistochemistry
Lung and pancreas tissues were harvested and xed
with 10% formalin. Tissues were paran embedded, and
4 μm sections were cut and stained with hematoxylin
and eosin (H&E) in the Mouse Histology & Phenotyping
Laboratory (MHPL) at Northwestern University. Details
regarding the protocol for immuhostochemistry can be
found in the Supplementary Materials and Methods.
Cell culture
NIH/3T3 (ATCC® CRL 1658™), 293T (ATCC®
CRL-3216™) and HCT116 (ATCC® CCL-247™) cells
were maintained in DMEM medium supplemented with
10% fetal bovine serum (FBS) with antibiotics at 37°C
in a humidied atmosphere containing 5% CO
2
and 95%
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O2. Cell lines were authenticated using CellCheck by
IDEXX Bioresearch, and tested for mycoplasma using
PlasmoTest™ - Mycoplasma Detection Kit (InvivoGen,
Inc). Hras
-/-
;Nras
-/-
;Kras
lox/lox
;RERT
ert/ert
MEFs were kindly
provided by Mariano Barbacid (Spanish National Cancer
Research Centre) and were maintained as described
previously [17]. Immortalized Sirt2+/+ and Sirt2-/- MEFs
were made as described previously [5]. Cell lines were
cultured for less than 20 passages and frozen down from
the rst 3 passages for further experiments.
In vivo BrdU incorporation
For in vivo BrdU labeling, mice were injected
intraperitoneally with 50 mg/kg of BrdU in PBS, pH 7.6,
and were sacriced 1.5 h later. Pancreata were isolated
and xed in 10% formalin, embedded in paran, and
processed by routine procedures. BrdU incorporation was
detected by immunohistochemistry using BrdU antibody
(Sigma) according to the manufacturer’s instructions.
RAS activity
GTP-bound “active” KRAS was determined by
using the Thermo Scientic Active Ras Pull-Down
and Detection Kit (Thermo Scientic) according to
the manufacturer’s instructions. A GST-fusion protein
of the RAS-binding domain (RBD) of Raf1 along with
glutathione agarose resin is used to specically pull down
active RAS.
Intranasal infection
After anesthetization of KrasG12D and Sirt2-/-;KrasG12D
mice using isouorane, replication-decient adenovirus
expressing Cre recombinase (adenoCRE; Gene Transfer
Vector Core, University of Iowa) was administered
intranasally. A detailed description of the protocol can be
found in the Supplementary materials and methods
Immunoprecipitation
Cells or tissue samples were lysed using
immunoprecipitation (IP) buer (25mM Tris-HCl pH
7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 5%
glycerol). After protein quantication using the Bradford
assay, cell extracts (500 µg - 1 mg total protein) were
incubated overnight with appropriate antibodies followed
by incubation with protein A or G agarose beads for
4 h at 4 oC. After washing ve times with IP buer,
immunocomplexes were resolved using SDS-PAGE and
analyzed by western blotting. For immunoprecipitation
the antibodies mentioned below were used: Ras (Thermo
Scientic), KRAS (Proteintech), Ac-K (Immunechem),
HA (Sigma), and Ac-KRAS-K147 (Eurogentec).
Cell proliferation
For measuring cell proliferation, the MTT-
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] proliferation assay, cell number counting and
colony formation ability were determined using the assays
described in Supplementary Materials and Methods.
Cellular transformation
Transformation ability of NIH3T3 cells was
assessed by checking colonies formed by conuent cells
as well as by observing anchorage-independent growth in
soft agar. A detailed description of the assays used can be
found in the Supplementary Materials and Methods.
Tumor growth analysis
NIH3T3 cells (1 × 106) or MEFs (2 × 106) were
subcutaneously injected in each ank of 4-6 week-old
male nude athymic mice (nu/nu) (Jackson Laboratory) in
200 µL PBS. Tumor sizes were determined every 1-2 days
(NIH3T3) or 3-4 days (MEFs) by measuring the length (l)
and the width (w) of each tumor with an electronic caliper.
Tumor volume (V) was calculated using the formula V =
lw2/2. Mice were housed, fed, and treated in accordance
with the guidelines approved by the Northwestern
University IACUC.
Nucleotide exchange assay
Each puried KRAS protein was exchanged in 20
mM Tris pH 7.5, 150 mM NaCl, 5 mM b-mercaptoethanol
using PD10 desalting columns (GE Healtcare). Protein
samples were, next, incubated with 5 mM GDP (50-
fold molar excess) in the presence of 20 mM EDTA.
For the nucleotide exchange assay, 2 µM GDP-loaded
KRAS and 4 µM mant-GTP or mant-GDP were mixed in
the assay buer (20 mM Tris pH 7.5, 150 mM NaCl, 5
mM β-mercaptoethanol, 10 mM MgCl2, 20 mM EDTA)
and dispensed into a 384-well plate. Fluorescence was
measured every 4 s for 15 min at excitation/emission
set to 360 nm/440 nm in a Spectramax M3 plate reader
(MolecularDevice). Data were exported and analyzed
using Graphpad Prism (GraphPad Software, Inc., La Jolla,
CA). All readings were performed in triplicate.
Statistical analysis
Statistical signicances were determined by
comparing means of dierent groups using unpaired t test
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or two-way ANOVA followed by post-tests. Graph Pad
Prism 6 (Graphpad Software Inc. La Jolla, CA) was used
for statistical analysis.
ACKNOWLEDGMENTS
We would like to thank the members of the
Vassilopoulos lab for critically reading the manuscript
and Melissa Stauer for editing the manuscript. We
thank Harold L. Moses (Vanderbilt University), Mark R.
Phillips (New York University), Lewis C. Cantley (Weil
Cornell Medical College) and Mariano Barbacid (Spanish
National Cancer Research Centre) for providing mice,
plasmids, and cells used in this study. Also we would
like to thank Kristie Linsey Rose (MSRC Proteomics
Laboratory, Vanderbilt University) for assistance with
the mass spectrometry analysis as well as Paul J. Grippo
(Gastroenterology and Hepatology, Department of
Medicine, UIC) and members of his lab for helping us
with preparation and examination of pancreas histological
sections.
CONFLICTS OF INTEREST
The authors report no conicts of interest pertaining
to this study.
FINANCIAL SUPPORT
D. Gius was supported by NCI-1R01CA152601-01,
1R01CA152799-01A1, 1R01CA168292-01A1 and the
Hirshberg Foundation. A. Vassilopoulos was supported
by R01CA182506-01A1, a Lefkofsky Family Foundation
Innovation Research Award and the Lynn Sage
Foundation.
Editorial note
This paper has been accepted based in part on peer-
review conducted by another journal and the authors’
response and revisions as well as expedited peer-review
in Oncotarget.
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