-
Article
p53 deficiency-induced Smad1 upregulationsuppresses
tumorigenesis and causeschemoresistance in colorectal cancersXinsen
Ruan1,, Qiao Zuo2,, Hao Jia1,, Jenny Chau3, Jinlin Lin1, Junping
Ao4, Xuechun Xia1, Huijuan Liu1,Samy L. Habib5, Chuangang Fu2,*,
and Baojie Li1,*1 Bio-X Institutes, Key Laboratory for the Genetics
of Developmental and Neuropsychiatric Disorders, Ministry of
Education, Shanghai Jiao Tong University,
Shanghai 200240, China2 Department of Colorectal Surgery,
Changhai Hospital, Second Military Medical University, Shanghai
200433, China3 Institute of Molecular and Cell Biology, Proteos, 61
Biopolis Drive, Singapore 138673, Singapore4 State Key Laboratory
of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji
Hospital, Shanghai Jiao Tong University, School of Medicine,
Shanghai 200032, China5 Department of Cellular and Structural
Biology, University of Texas Health Science Center at San Antonio,
San Antonio, TX 78229, USA These authors contributed equally to
this work.
* Correspondence to: Baojie Li, E-mail: [email protected];
Chuangang Fu, E-mail: [email protected]
The DNA damage response helps to maintain genome integrity,
suppress tumorigenesis, and mediate the effects of radiotherapy
and
chemotherapy. Our previous studies have shown that Smad1 is
upregulated and activated by Atm in DNA damage response, which
can
further bind to p53 and promote p53 stabilization. Here we
report another aspect of the interplay between p53 and Smad1.
Comparison
of rectal tumor against paired paraneoplastic specimens and
analysis of >500 colorectal tumors revealed that Smad1was
upregulated
in tumor samples, which was attributable to p53 defects. Using
MEFs as a model, we found that knockdown of the elevated Smad1
in
p532/2 MEFs promoted cell proliferation, E1A/Ras-induced cell
transformation, and tumorigenesis. Mechanistic studies suggest
that
elevated Smad1 and momentary activation inhibit cell
proliferation by upregulating p57Kip2 and enhancing AtmChk2
activation.
Surprisingly, elevated Smad1 appears to have a negative effect
on chemotherapy, as colorectal tumors, primary cancer cells,
and
cell lines with Smad1 knockdown all showed an increase in
chemosensitivity, which could be attributable to elevated
p57Kip2.
These findings underscore the significance of Smad1p53
interaction in tumor suppression and reveal an unexpected role
for
Smad1 in chemoresistance of colorectal cancers.
Keywords: Smad1, p53, p57Kip2, chemoresistance, colorectal
cancer
Introduction
Tumor suppressor p53 is regarded as the guardian of the
genome. Activation of p53 by genotoxic stress or oncogene
activa-
tion turns on its target genes such as p21, Puma, and Bax to
induce
cell cycle arrest, apoptosis, and/or senescence, thus
maintaining
genome integrity and suppressing tumorigenesis caused by
accu-
mulation of mutations in oncogenes and tumor suppressor
genes
(Jackson and Bartek, 2009; Lord and Ashworth, 2012;
Reinhardt
and Schumacher, 2012). As such, p53 gene is mutated in .50%
of the primary tumors, which express mutant p53 molecules
that
either lose the normal function or display dominant-negative
effects (Vogelstein et al., 2000; Goh et al., 2011; Muller
and
Vousden, 2013). The lack of p53 function automatically leads
to
escape of cell senescence and immortalization and promotes
cell
transformation. In addition, p53 deficiency has been shown
to
affect cell differentiation in several cell types, including
neuron
and osteoblast (Ma et al., 2012; Liu et al., 2013), although
the
mechanisms by which p53 regulates cell differentiation
remain
under-explored.
Due to its critical roles in cell proliferation,
differentiation, and
death, the p53 expression needs to be tightly regulated. A
Mdm2p53 loop provides such a mechanism for fine-tuning p53
expression (Brooks and Gu, 2006). Moreover, the levels of
p53
are also influenced by the energy level, nutrition, and
other
growth conditions of the cell, in addition to the severity of
DNA
damage. For example, it has been shown that growth factors-
activated mTORS6K1 pathway has an influence on p53 induction
in response to DNA damage (Lai et al., 2010). In addition,
p53
deficiency leads to positive feedback regulation of the
expression
of genes that have redundant function as p53, e.g. p16INK4a
Received June 10, 2014. Revised November 4, 2014. Accepted
November 21, 2014.# The Author (2015). Published by Oxford
University Press on behalf of Journal of
Molecular Cell Biology, IBCB, SIBS, CAS. All rights
reserved.
doi:10.1093/jmcb/mjv015 Journal of Molecular Cell Biology
(2015), 7(2), 105118 | 105Published online March 10, 2015
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and p73 (Kravchenko et al., 2008; Leong et al., 2009; Lunghi et
al.,
2009; Hong et al., 2014). It is understandable that an
important
protein such as p53 needs to be tightly regulated and when
mutated, compensatory mechanisms need to kick in.
Our previous studies showed that Samd1 is upregulated and
further activated in response to DNA damage. DNA lesions
espe-
cially double-stranded DNA breaks activate the Atmp53 and
AtmChk2 pathways to induce cell cycle arrest and apoptosis
(Jackson and Bartek, 2009). DNA damage-induced Smad1 activa-
tion requires Atm and Atm-mediated phosphorylation of Smad1
on Ser239, which also promotes Smad1p53 interaction and
inhi-
bits Mdm2-mediated p53 ubiquitination, leading to p53
upregula-
tion (Chau et al., 2012). This provides a mechanistic
explanation
how BMPSmad1 signaling suppresses tumor development
(Howe et al., 2001; Hardwick et al., 2008; Thawani et al.,
2010;
Walsh et al., 2010; Tomlinson et al., 2011; Gao et al.,
2012;
Lubbe et al., 2012). In this study, we uncovered a novel aspect
of
the functional interaction between p53 and Smad1. We found
that Smad1 is upregulated in human colorectal tumor
specimens
compared with normal tissues or paired paraneoplastic
samples,
which is attributable to p53 deficiency. This elevation helps
to
curb cell proliferation, cell transformation, and tumor
formation,
by increasing the expression of cyclin-dependent kinase
inhibitor
p57Kip2, a Smad1 target gene (Jia et al., 2014), and
potentiating
AtmChk2 activation. In addition, elevated Smad1 appears to
render chemoresistance in tumor models and human rectal
cancer cells, which could be explained by increased p57Kip2
ex-
pression. These findings suggest that Smad1 elevation serves
as
a compensatory mechanism for p53 deficiency by potentiating
the activation of p53 parallel pathways, and that Smad1 plays
crit-
ical roles not only in tumor suppression but also in
chemosensitiv-
ity. In addition, parallel studies show that the functions of
Smad1 in
tumor suppression and chemosensitivity are not shared by
Smad5.
Results
Rectal tumor samples show Smad1 upregulation compared with
paraneoplastic tissues
Our previous studies have shown that Atm-mediated Smad1
Ser239phosphorylation promotes Smad1upregulation and activa-
tion and leads to p53 stabilization. To test the clinical
relevance of
this finding, we collected 22 rectal tumors (5 stage I samples,
6
stage II samples, 8 stage III samples, and 3 stage IV samples;
for
patient information, see Supplementary Table S1, sample 9
being discarded due to protein degradation) and
paraneoplastic
specimens and analyzed Smad1 expression. We found that both
protein (Figure 1A and B) and mRNA (Figure 1C and D) levels
of
Smad1 were significantly upregulated in the tumor samples
com-
pared with matched paraneoplastic samples. However, the
protein levels of Smad4, an important tumor suppressor
(Derynck et al., 2001), and Smad5 were not significantly
altered
(Supplementary Figure S1). Surprisingly, we found that
Smad1/
5/8 activation was reduced in most of the tumor samples even
in
the presence of elevated Smad1expression (Figure1A). This is
con-
sistent with previous findings that Smad1/5/8 activation is
inhib-
ited in colorectal tumor samples due to mutations in bone
morphogenetic protein (BMP) receptors (Sancho et al., 2004;
Kodach et al., 2008), and suggests that BMPSmad1 signaling
plays complex roles in tumor development and progression.
Smad1 upregulation is common in human colorectal cancer
samples
To validate the finding of Smad1 upregulation in tumors, we
col-
lected 542 human colorectal tumor specimens (stages I to IV)
and
53 normal tissues, which were obtained from Chinese Han
popula-
tion (for patient information, see Supplementary Table S2).
These
tissues were used to generate tissue arrays, which were
immuno-
histochemically stained for Smad1, and the levels of Smad1
were
scored (Supplementary Figure S2A). We found that Smad1 was
expressed at low levels in normal mucosa samples, yet tumor
patient samples expressed increased levels of Smad1 (from
17%
to 64%) (Table 1). At later stages, Smad1 levels appeared to
go
down. In the tumor samples with increased Smad1 expression,
immunohistochemical staining showed that Smad1was detectable
in both the cytoplasm and the nucleus, just like in normal
tissues
(Supplementary Figure S2B). These results indicate that
colorectal
tumors tend to upregulate Smad1 expression. The function of
Smad1 upregulation is the focus of present study.
Smad1 upregulation is attributable to p53 defects
Previous studies have shown that primary p532/2 osteoblasts
and neural stem cells (NSCs) displayed elevated Smad1
expres-
sion, which have an impact on osteogenic and neural
differenti-
ation, respectively (Ma et al., 2012; Liu et al., 2013). To
determine whether Smad1 upregulation is related to p53 in
tumor samples, we analyzed the protein levels of p53 in 22
pairs
of samples by western blot and found that p53 was either
down-
regulated or expressed in truncated forms in .70% tumor
samples (Figure 1A). Sequencing the p53 cDNA obtained from
22
samples confirmed the existence of various p53 mutations
(Supplementary Table S1). Five of the tumor samples carry
R175H mutations, and two carry G245R mutation. Moreover, 16
of them carry P72R, a common polymorphism of p53 gene that
is
associated with tumorigenesis (Aaltonen et al., 2001;
Olivier
et al., 2010), two of which also carry R248W and R282W
mutations.
Some of the tumors that do not carry mutations showed a
decrease
in p53 protein level. These results suggest that p53 defects
are
common in colorectal cancers.
Comparison of p53 down-regulation/mutations with Smad1
upregulation revealed that there exists a significant
correlation
between p53 defects and Smad1 upregulation (Figure 2A), sug-
gesting that p53 suppresses the expression of Smad1 in human
colorectal tumors. We then isolated primary rectal cancer
cells
from three patient samples, which showed a great reduction
in
p53 and an elevation in Smad1 compared with its paired
paraneo-
plastic sample (Figure 2B and Supplementary Figure S3A). The
sample shown in Figure 2B carries a R175H mutation in p53
gene. To test whether the increase in Smad1 is a result of p53
de-
ficiency, we ectopically expressed p53 using a retroviral vector
in
the tumor cells and found that p53, but not p53-R273H
mutant,
reduced the protein level of Smad1 (Figure 2C and D, and
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Supplementary Figure S3B). These results, taken together,
suggest
that p53 defects contribute to Smad1 elevation.
Knockdown of Smad1 promotes proliferation of p532/2 MEFs
p53 mutation is a frequent and early event in some tumor
types
(Rivlin et al.,2011; Vinall et al.,2012). Our previous studies
showed
that p53 is often mutated during MEF immortalization, a
process
that is required for cell transformation (Zhang et al., 2013a).
We
used primary MEFs to test the function of Smad1 elevation in
rela-
tion to p53deficiency. We found that primary p532/2MEFs
showed
elevated levels of Smad1 but not Samd5 (Figure 3A). It has
been
shown that p53 deficiency induced Smad1 upregulation in
Table 1 Expression of Smad1 in normal mucosa and stage IIV
colorectal cancer.
Characteristic Smad1 immunostaining distribution (%) Total
patients Positive rate Mean rank* P-value**
2 1 11 111
Normal mucosa 0 81.1 17.0 1.9 53 1 222.43
Stage I 0 77.8 22.2 0 45 1 229.83 0.002a
Stage II 0 31.1 64.0 4.9 286 1 383.36 ,0.001b
Stage III 0 45.6 50.6 3.8 158 1 336.49 0.003c
Stage IV 0 60.4 39.6 0 53 1 284.12 0.043d
n 595.*KruskalWallis test was used to determine significances
among the six groups (P, 0.001).
**MannWhitney U-test was used to determine significances between
each two groups.aCompared with the Normal mucosa group.bCompared
with the Stage I cancer group.cCompared with the Stage II cancer
group.dCompared with the Stage III cancer group.
Figure1Smad1 is upregulated in clinical rectal tumor samples.
(A) Total proteins of tumor (T) and paraneoplastic (P) samples
from22 rectal cancer
patients were extracted, and the levels of Smad1, p53, and
p-Smad1/5/8 were determined by western blot. (B) Quantitation data
for A. n 22.* P, 0.05 vs. paraneoplastic tissues. (C) Real-time PCR
was used to determine the mRNA levels of Smad1, showing
upregulation in human rectal
tumors. (D) Quantitation data for C. n 22. *P, 0.05 vs.
paraneoplastic tissues.
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osteoblasts and NSCs during differentiation via E2F1 (Ma et
al.,
2012). To confirm that p53 represses Smad1 expression under
normal growth conditions via the same mechanisms, we knocked
down E2F1 with siRNA in p532/2 MEFs, which also led to a de-
crease in Smad1 (Figure 3A). Importantly, Smad1 elevation is
ac-
companied by an increase in Smad1/5/8 activation in p532/2
MEFs. The findings that primary MEFs, osteoblasts, and NSCs
show co-elevation of Smad1expression and Smad1/5/8activation
in response to p53deficiency suggest that p53defects initially
lead
to elevated Smad1 expression and activation, which is later
deac-
tivated due to alteration in upstream regulators of BMPSmad1
signaling during tumorigenesis.
To test the function of Smad1 upregulation in p532/2 cells,
we
knocked down Smad1 in p532/2 MEFs using short hairpin RNA
(shRNA) expressed by a retroviral vector, with WT MEFs as a
control (Figure 3B). Four shRNA hairpins were tested and two
of
them (37928 and 30824) produced similar degrees of Smad1
knockdown as siRNA (Figure 3B and Supplementary Figure S4),
and thus were used further in this study. We found that
p532/2
MEFs with Smad1 knockdown showed enhanced proliferation
(Figure 3C), evidenced by an increase in the number of cells
after
the same number of cells were plated. Similar results were
obtained from Smad1-knockdown WT MEFs (Figure 3C). In
addition, analysis of the cell cycle profiles revealed that
Smad1
knockdown led to an increase in S phase especially in p532/2
cells (Figure 3D). These findings suggest that Smad1 may
regulate
cell proliferation in p53-independent manners.
Previous studies have demonstrated functional redundancy
between Smad1 and Smad5 and the doses of Smad1 and Smad5
alleles are critical in embryonic development (Arnold et al.,
2006;
Eivers et al., 2008). To test a possible dose effect on cell
prolifer-
ation, we knocked down Smad5 and found that this had no
signifi-
cant effect on cell proliferation of either p532/2 or WT
MEFs,
whereas knockdown of both Smad1 and Smad5 showed similar
results as Smad1 knockdown (Figure 3C), suggesting that
Smad1
and Smad5 play different roles in MEF proliferation. There
is
reported evidence that Smad1 and Smad5have distinct or even
op-
posite functions in vivo (Dick et al., 1999; Liu et al.,
2003;
McReynolds et al., 2007). Alignment of mouse Smad1 and Smad5
protein sequences reveals that the identity between these two
pro-
teins is 88%. The divergent region is the linker located
between
the MH1 and MH2 domains (Supplementary Figure S5), which
contains residues that are phosphorylated by various
kinases,
e.g. mitogen-activated protein kinases (MAPKs), glycogen
syn-
thase kinase (GSK), and Atm (Fuentealba et al., 2007;
Sapkota
et al., 2007; Eivers et al., 2008; Chau et al., 2012). A few
potential
Figure 2 Smad1 upregulation is correlated with and attributable
to p53 defects in clinical rectal tumor samples. (A) Pearsons
correlation analysis
shows that there exists a significant correlation between
Smad1upregulation and p53defects in rectal tumor samples. (B)
Primary tumor cells were
isolated from samples of rectal cancer patients, and Smad1 and
p53 levels were analyzed by western blot. (C) p53 or p53-R273H
mutant was
expressed in the primary tumor cells by retroviruses, with
pMSCV-vector as control, and the protein levels of p53 and Smad1
were determined
by western blot. (D) Quantification of p53 and Smad1 (normalized
to b-Actin). n 3. *P, 0.05 vs. retroviral vector-infected
cells.
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phosphorylation sites are not shared by Smad1 and Smad5
(Figure 3E), e.g. Ser181, Ser191, and Thr235. Yet, how the
linker
region including the phosphorylation sites determines
distinct
functions of Smad1 and Smad5 warrants further investigation.
Knockdown of Smad1 promotes transformation of p532/2 cells
We next tested whether Smad1 elevation in p532/2 MEFs has
any effect on cell transformation. Using a standard cell
transform-
ation procedure, we expressed E1A and RasV12 in p532/2
primary MEFs and cell transformation rates were scored. WT
MEFs were used as a control in these experiments. It was
found
that Smad1 knockdown enhanced transformation of p532/2
MEFs (Figure4A and B). Our previous studies have shown that
over-
expression of Smad1 inhibits cell transformation in p532/2 or
WT
MEFs (Chau et al., 2012). These results suggest that Smad1
eleva-
tion has an inhibitory effect on cell transformation in p532/2
cells.
Similar to the cell proliferation results (Figure3B), Smad5
knock-
down showed little effect on cell transformation rates
whereas
Figure 3 Knockdown of Smad1, but not Smad5, further promotes
proliferation of p532/2 MEFs. (A) Primary p532/2 MEFs showed an
increase in
Smad1 protein level, which could be brought down by E2F1
knockdown. Primary p53+/+ and p532/2 MEFs were transfected with
control or E2F1siRNA, and the levels of Smad1, Smad5, and
p-Smad1/5/8were analyzed by western blot after 2 days
post-transfection. (B) Western blot analysis
of Smad1and Smad5after the cells were infected with control,
Smad1 (shRNA37928), or Smad5 shRNA-expressing retrovirus. (C)
Viable cells were
determined by trypan blue staining of p53+/+ and p532/2 MEFs
with Smad1, Smad5, or Smad1/5 knockdown. n 6. *P, 0.05 vs.
vector-infected cells of the same genotype. (D) Smad1 knockdown led
to an increase in S phase and a decrease in G1 phase in p532/2
MEFs. n 6.*P, 0.05 vs. vector-infected cells. (E) Comparison of the
potential phosphorylation sites in the linker regions of Smad1 and
Smad5, with different
Ser or Thr residues framed.
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knockdown of both Smad1 and Smad5 gave rise to similar
results
as Smad1knockdown (Figure4A and B). These results
demonstrate
an inhibitory role for Smad1 in cell proliferation and
transform-
ation, a function not shared by Smad5.
Knockdown of Smad1 promotes tumor formation of p532/2 cells
We then tested whether Smad1 elevation in p532/2 MEFs has
any effect on tumorigenesis. The transformed MEFs described
in Figure 4A and B were transplanted into nude mice. Smad1
Figure 4 Knockdown of Smad1 enhanced Ras/E1A-induced
transformation and tumorigenesis of p532/2 MEFs. (A) Primary p53+/+
and p532/2
MEFs with Smad1, Smad5, or Smad1/5 knockdown were infected with
Ras/E1AV12-expressing retroviruses, and the colony forming units
were
stained with giemsa. (B) Statistics analysis of the numbers of
colony forming units. n 6. *P, 0.05 vs. control shRNA-infected
cells of thesame genotype. (C) p532/2 MEFs with Smad1, Smad5, or
Smad1/5 knockdown were immortalized with E1A/RasV12 and then were
transplanted
into nude mice. Tumor volumes were measured twice on Days 12 and
14 after the subcutaneous injection. (D) Same experiments were
performed
as in C except that p53+/+ MEFs were used and that tumor volumes
were measured on Days 15 and 17 after the subcutaneous injection. n
6.*P, 0.05 vs. control shRNA-infected cells of the same
genotype.
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knockdown in p532/2 MEFs accelerated tumorigenesis (Figure
4C),
which was similarly observed in WT MEFs (Figure 4D).
Histological
analysis of the tumors revealed that knockdown of Smad1
increased
the cellularity of tumor (Supplementary Figure S6). These
findings
suggest that Smad1 elevation in p532/2 cells helps to repress
cell
transformation and tumor formation via suppressing cell
prolifer-
ation, and thus is likely to act as a compensatory mechanism
for
p53deficiency, indicating that Smad1must repress cell
proliferation
via p53-independent mechanisms.
Although Smad5knockdown did not significantly alter cell
prolif-
eration and transformation, it inhibited tumorigenesis of
p532/2
cells but not WT cells. Knockdown of both Smad1 and Smad5
gave rise to results in between Smad1 knockdown and Smad5
knockdown (Figure 4C and D), suggesting that Smad5 plays a
role opposite to Smad1 in p532/2 tumor growth.
Smad1 knockdown sensitizes tumors to the chemotherapeutic
effects of Dox
We then asked whether elevated levels of Smad1 in tumor
cells have any effect on chemotherapeutic efficacy. We took
ad-
vantage of the tumor models developed in nude mice and
treated them with Dox for a week and monitored the size of
tumors. We started Dox treatment when the tumor sizes
reached
200 mm3. It was found that tumors derived from p532/2 cells
with Smad1knockdown showed increased sensitivity to Dox
treat-
ment (Figure 5A). Moreover, Smad1 knockdown also increased
the sensitivity to Dox treatment in tumors derived from WT
cells
(Figure 5B).
We found that Smad1 could be activated by Dox in transformed
MEFs, cancer cell lines, and tumors, which may regulate cell
prolif-
eration and/or death [(Chau et al., 2012) and data not shown].
We
stained the cancer samples for cell proliferation marker Ki67
and
found that Smad1 knockdown tumors showed a greater reduction
in the number of S phase cells than control tumors in response
to
Dox treatment (Supplementary Figure S7). Similarly,
transformed
MEFs with Smad1 knockdown also showed a modest decrease in
cell proliferation, judged by BrdU incorporation, and a
significant
decrease in cell survival rate in response to Dox treatment
(Figure 5C and D). These cells with Smad1 knockdown also
showed a decrease in cell survival rate in response to two
genotoxic
drugs 5-iodotubercidin and oxaliplatin (Figure 5E and F)
(Zhang
et al., 2013b). These results suggest that Smad1 knockdown
might improve chemosensitivity by inhibiting cell
proliferation
and survival.
We found that tumors derived from p532/2 cells with Smad5
knockdown showed decreased sensitivity to Dox treatment, and
knockdown of both Smad1 and Smad5 showed sensitivity similar
to control cells, in between Smad1 knockdown and Smad5
knock-
down (Figure 5A), suggesting that Smad5might also play a role
op-
posite to Smad1 in chemosensitivity.
Smad1 knockdown sensitizes colorectal cells and cell lines
to
genotoxic stress-induced cell death
To confirm the role for Smad1 in chemosensitivity, we tested
three colorectal tumor cell lines HCT116, Caco-2, and HT29.
Smad1 knockdown with siRNA in these cell lines resulted in a
de-
crease in BrdU incorporation, although to modest extents, and
sen-
sitized these cells to Dox-induced cell death, to a greater
extent
than in MEFs (Figure 6AC). Primary human rectal tumor cells
with Smad1 knockdown also showed a significant decrease in
cell proliferation and survival in response to Dox treatment
(Figure 6D and E). Smad1 knockdown also increased the
sensitivity
of primary cancer cells to anti-cancer drug oxaliplatin (Figure
6F).
Further analysis showed that Dox-induced cell death mainly
oc-
curred by apoptosis, with a small percentage of cells
undergoing
necrosis in cells with Smad1 knockdown (Supplementary Figure
S8A). It has been reported that Dox might kill cancer cells via
gen-
erating reactive oxygen species (ROS). We found that
pre-treating
the cells with 5 mM N-Acetylcysteine (NAC), a ROS scavenger,
only modestly inhibited Dox-induced cell death
(Supplementary
Figure S8B), suggesting that ROS may play a minor role in
Dox-induced cell death under this setting. Nevertheless, the
results derived from in vivo tumor model, colorectal cancer
lines,
and primary rectal cancer cells all indicate that reducing
Smad1
levels can increase chemosensitivity by inhibiting cell
proliferation
and survival, with the inhibition on survival more
pronounced.
Smad1 elevation/activation increases p57Kip2 expression and
AtmChk2 activation
We then wanted to understand howelevated Smad1expression/
activation inhibits cell proliferation and transformation in
p53-deficient cells? Our recent study indicates that CDK
inhibitor
p57Kip2 is a Smad1 target gene in DDR and it renders
chemoresis-
tance by suppressing cell death (Jia et al., 2014). In
consistent with
the increase in Smad1 expression and activation, we found that
the
levels of p57Kip2 were increased at basal level and in response
to
Dox in p532/2 MEFs, which was diminished by Smad1 knockdown
(Figure 7A). We have previously shown that p57Kip2 was
upregu-
lated in 22 rectal tumors compared with the matched
paraneoplas-
tic specimens (Jia et al., 2014), which were the same samples
used
in this study (Figure 1A and Supplementary Figure S1). We
com-
pared Smad1 upregulation and p57Kip2 upregulation in these
tumor samples and found that there exists a significant
asso-
ciation between these two events (Supplementary Figure S9A).
Knockdown of Smad1 in human rectal tumor cells also led to a
decrease in p57Kip2 (Supplementary Figure S9B), suggesting
that p57Kip2 elevation in colorectal tumors is mediated by
Smad1. Moreover, ectopic expression of Smad1 in WT MEFs led
to an increase in p57Kip2, which was not further increased
by
Dox treatment (Figure 7B). In addition, Dox-induced AtmChk2
ac-
tivation was enhanced in p532/2 MEFs, which could be impeded
by Smad1 knockdown (Figure 7A), and elevated expression of
Smad1 was able to potentiate AtmChk2 activation in response
to Dox (Figure 7B). These results suggest that the BMPSmad1
pathway, in addition to the Atmp53pathway, also plays a
positive
role in AtmChk2 activation in DDR. The increase in p57Kip2
ex-
pression and AtmChk2 activation may explain how elevated
Smad1 suppresses cell proliferation and transformation,
whereas
increased expression of p57Kip2 may mediate the effects of
Smad1 on chemoresistance.
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Figure5Smad1knockdown sensitizes tumors and transformed MEFs to
chemotherapy. (A) p532/2MEFs with Smad1, Smad5, or
Smad1/5knock-
down were immortalized with E1A/RasV12and then were transplanted
into nude mice. After14days, the tumor volumes were measured while
nude
mice were treated with 5 mg/kg Dox every other day for five
times. n 6. *P, 0.05 when Smad1 shRNA-expressing cells were
compared withcontrol shRNA-infected cells. (B) Same experiments
were carried out as in A except that p53+/+ MEFs were used and the
tumor volumes weremeasured after 17 days. n 6. *P, 0.05 when Smad1
shRNA-expressing cells were compared with control shRNA-infected
cells. (C) Smad1knockdown in transformed p532/2 or p53+/+ MEFs
resulted in decreased proliferation in response to Dox. p53+/+
cells were treated with0.1 mM Dox for 24 h, while p532/2 MEFs were
treated with 0.5 mM Dox for 24 h. Cell proliferation rates were
determined with BrdU assay, and
the percentage of BrdU-positive cells of each group was shown. n
6. *P, 0.05 vs. control shRNA-infected cells of the same genotype
underDox treatment. (D) Smad1 knockdown in transformed p532/2 or
p53+/+ MEFs resulted in a decrease in cell survival in response to
Dox treatment.Cell viability was determined with WST-1 assay. n 6.
*P, 0.05 vs. control shRNA-infected cells of the same genotype
under Dox treatment. (E)Smad1 knockdown in transformed p532/2 MEFs
resulted in a decrease in cell survival in response to oxaliplatin.
n 6. *P, 0.05 vs. untreatedcells. (F) Smad1 knockdown in
transformed p532/2 MEFs resulted in a decrease in cell survival in
response to 5-iodotubercidin (ITU) treatment.
n 6. *P, 0.05 vs. untreated cells.
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Figure 6 Smad1 knockdown leads to enhanced chemosensitivity in
colorectal tumor cell lines and primary human rectal cancer cells.
(A) Western
blot result shows that Smad1 levels were reduced by siRNA in
HCT116, Caco-2, and HT29 cells. (B) Smad1 knockdown led to a modest
decrease in
BrdU incorporation in response to Dox treatment in HCT116,
Caco-2, and HT29 cells compared with control siRNA-transfected
cells. These cells
were treated with 0.1 mM Dox for 24 h. Cell proliferation rates
were determined with BrdU assay, and the percentage of
BrdU-positive cells of
each group was shown. n 3. *P, 0.05 vs. control
siRNA-transfected cells under Dox treatment. (C) Colorectal cell
lines with Smad1 knockdownwere treated with Dox for48 h, and cell
viability was determined with WST-1 assay. n 3. *P, 0.05 vs.
control siRNA-transfected cells under Doxtreatment. (D) Primary
rectal cancer tumor cells with Smad1 knockdown were treated with
0.1 mM Dox for 24 h. Cell proliferation rates were deter-
mined with BrdU assay, and the percentage of BrdU-positive cells
was shown. Inset: western blot results showing knockdown of Smad1.
n 3.*P, 0.05 vs. control siRNA-transfected cells under Dox
treatment. (E) Primary rectal tumor cells with Smad1 knockdown were
treated with Dox for
48 h and cell viability was determined with WST-1 assay. n 3.
*P, 0.05 vs. control siRNA-transfected cells under Dox treatment.
(F) Smad1knockdown in human rectal cancer cells resulted in a
decrease in cell survival in response to oxaliplatin. n 3. *P, 0.05
vs. controlsiRNA-transfected cells under oxaliplatin treatment.
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Figure 7 Elevated Smad1 expression/activation promotes p57Kip2
expression and potentiates AtmChk2 activation. (A) Primary p532/2
MEFs
showed an increase in p57Kip2 expression and AtmChk2 activation,
which was diminished by Smad1 knockdown. Primary p53+/+ and
p532/2
MEFs were transfected with control or Smad1 siRNA for2days, and
were then challenged with Dox. The protein levels of Atm, Chk2,
p57Kip2, Actin,
Smad1, p-Atm, and p-Chk2were analyzed by western blot. Right
panels show quantitation data.n 3. *P, 0.05 vs. p53+/+ cells under
the sametreatment. **P, 0.05 vs. control siRNA-transfected cells of
the same genotype under the same treatment (color matched). (B)
Ectopic expression
of Smad1 in WT MEFs increased p57Kip2 expression and AtmChk2
activation. Primary WT MEFs were infected with Smad1-expressing
retrovirus
or empty retrovirus for2days, and were then challenged with Dox.
The protein levels of Atm, Chk2, p57Kip2, Actin, Smad1, p-Atm, and
p-Chk2were
analyzed by western blot. Right panels show quantitation data.n
3. *P, 0.05 vs. cells infected with empty vector under the same
treatment. (C)A diagram showing that elevated Smad1
expression/activation inhibits cell proliferation/transformation
via increasing p57Kip2 expression and
AtmChk2 activation, and causes chemoresistance via increasing
p57Kip2 expression.
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Discussion
The TGFb superfamily plays critical roles in development and
tissue homeostasis. The TGFb subfamily and its downstream
Smad2/3 have long been known to have anti-proliferative
activ-
ities. Smad2/3 can interact with p53 to induce the
expression
of target genes such as p21Cip1 to inhibit cell
proliferation
(Cordenonsi et al., 2003; Kortlever et al., 2006; Shirai et
al.,
2011; Samarakoon et al., 2013; Overstreet et al., 2014). The
BMP
subfamily also has tumor suppressive activities, especially in
colo-
rectal tissues (Ming Kwan et al., 2004; Pangas et al., 2008;
Neumann et al., 2011), although BMPs have been reported to
have pro-proliferative activity in certain cell types, which
can
be antagonized by TGFb (Goumans et al., 2003). Our previous
studies have shown that the BMPSmad1 pathway is activated
by Atm in response to DNA damage. Smad1 then interacts with
p53 and stabilizes p53. On top of that, this study reveals
that
when p53 is mutated or expressed at low levels, Smad1 is
upregu-
lated, which helps to suppress cell proliferation and
oncogenesis,
by increasing p57Kip2 expression and enhancing AtmChk2
activation (Figure 7C). Thus, Smad1 elevation may act as a
com-
pensation for p53 mutation/loss. These findings further
highlight
the significance of Smad1p53 interplay and suggest that
smad1
suppresses tumorigenesis with both p53-dependent and p53-
independent mechanisms.
While our previous study identified Smad1 as a DDR effector
molecule that interacts with and stabilizes p53, this study
sug-
gests that Smad1 can influence AtmChk2 activation as well,
especially in p53-deficient cells (Figure 7C). Smad1
knockdown
compromises AtmChk2 activation, while elevated expression of
Smad1 potentiates AtmChk2 activation. Atm activation mainly
occurs at the double-stranded DNA breaks, yet the activation
mechanism is currently unknown (Lord and Ashworth, 2012). We
found that Smad1 does not interact with Atm and is not
localized
to the DNA damage-induced foci (Chau et al., 2012). It is
possible
that BMPSmad1 may affect the expression of proteins that
help Atm foci recruitment and/or activation. Interestingly,
recent
studies revealed that the TGFbSmad2/3pathway is also
involved
in DDR (Wang et al., 2013; Barcellos-Hoff and Cucinotta,
2014). TGFb receptor activation is required for Atm
activation,
and Smad2 and Smad7 are localized on DNA damage-induced
nuclear foci (Kirshner et al., 2006; Zhang et al., 2006; Park et
al.,
2015). Yet, how BMPSmad1 signaling regulates Atm activation
and whether Smad7 mediates this effect of Smad1 await
further
investigation.
It has been reported that BMPSmad1 signaling is inhibited in
some human tumor types (Kodach et al., 2008; Chau et al.,
2012). However, our present study shows that Smad1 protein
level is upregulated in colorectal cancer samples. Based
upon
the findings that p53 deficiency leads to elevated Smad1
expres-
sion in primary MEFs, NSCs, and osteoblasts, which is
accompanied
by elevated Smad1/5/8activation, we conceive that during
tumori-
genesis, p53 defects lead to a compensatory Smad1
upregulation
and momentarily activation, which helps to suppress tumor
forma-
tion; late deactivation of BMP receptor due to further
mutations
facilitates tumor progression. Thus, our results and others
reveal
a dynamic change of Smad1 expression and activation in
colorectal
cancer, which appears to play critical roles in tumor
suppression.
This study also provides evidence to support the concept
that
there is a division of labor between Smad1 and Smad5 in
tumori-
genesis. It is generally believed that Smad1, 5, and 8 have
redun-
dant functions. Here we show that only Smad1 is upregulated
in
the absence of p53, and only Smad1 knockdown appears to
enhance cell transformation and tumor formation and
sensitize
tumor cells to Dox treatment. Moreover, only Smad1 is
activated
and upregulated in response to DNA damage (Chau et al.,
2012).
Functionally, while Smad5 knockdown did not significantly
affect
cell proliferation and cell transformation, it seems to play a
role op-
posite to Smad1 in tumor growth and chemosensitivity to Dox.
Mechanistically, the different functions of Smad1 and Smad5
could be caused by targeting different sets of genes or by
respond-
ing to different ligands, as revealed during embryonic
hematopoi-
esis (Liu et al., 2003; McReynolds et al., 2007).
While elevated Smad1 helps to inhibit cell proliferation and
tumorigenesis in p53-deficient cells, our findings suggest that
ele-
vated Smad1 also renders chemoresistance in tumors and tumor
cells. This may be explored to enhance chemosensitivity in
treat-
ment of colorectal cancer (Lee et al., 2011; Langenfeld et
al.,
2013). Our present and previous studies suggest that p57Kip2
may be an important mediator of the effects of elevated
Smad1
on chemoresistance (Figure 7C), based on the following
observa-
tions. Firstly, p57Kip2 is co-elevated with Smad1 in
colorectal
tumor samples. Secondly, p57Kip2 is a target gene of Smad1
and
is upregulated in response to chemotherapeutic drugs.
Thirdly,
knockdown of Smad1 or p57Kip2 enhances chemosensitivity in
p53-proficient or p53-deficient tumors.
In summary, analysis of Smad1 expression in .500 patient
tumor samples uncovered that Smad1 is often upregulated in
colo-
rectal cancers, which was attributable to p53defects.
Smad1eleva-
tion appears to act as a feedback compensatory mechanism in
p532/2 cells to help curb cell proliferation, cell
transformation,
and tumor formation, via increasing p57Kip2 expression and
AtmChk2 activation. Moreover, elevated Smad1 also causes
che-
moresistance, which may involve p57Kip2. Thus, Smad1might be
a
target to improve chemotherapeutic efficacy. Moreover, our
study
also reveals that Smad5 plays roles distinct from Smad1 in
tumor
growth and chemosensitivity.
Materials and methods
Patients and tissue samples
A total of 595 patients, who received the operation at the
Department of Colorectal Surgery, Changhai Hospital, Second
Military Medical University, Shanghai, China, between
December
1999 and December 2009, were collected in this study
(Supplementary Materials and methods and Table S2). Informed
consent had been obtained from all patients and the project
had
been approved by the local Ethics Committee.
Mice
p532/2 and Balb/c nude mice were bred and used, following
the
guidelines of mouse breeding and cage density expectations
for
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animal colonies at Bio-X Institutes, Shanghai Jiao Tong
University.
Mouse embryonic fibroblasts were isolated from E13.5 embryos
following a standard protocol.
Isolation of primary rectal tumor cells and cell cultures
A portion of rectal cancer tissue was cut into small pieces
and
enzymatically digested with 0.5% TrypsinEDTA (Gibco). The
dis-
persed tissue was filtered through a 100-mm cell strainer
(BD
Falcon), and the cells were washed with PBS and cultured.
The
primary tumor cells were cultured in DMEM/F12 (1:1), whereas
MEFs, HCT116, Caco-2, and HT-29 were cultured in DMEM, which
were supplemented with 10% FBS.
Smad1 knockdown
For transient Smad1 knockdown, siRNAs were used (Thermo
Scientific and Santa Cruz Biotechnology, Inc.). For stable
knock-
down of Smad1, four shRNA hairpins expressed by a retrovirus
vector (37928, 31427, 32418, and 30824, Dharmacon, Inc.)
were
tested. Two of them (37928 and 30824) could efficiently
knock
down Smad1 and were used further in this study. The
experiments
that involve stable Smad1 knockdown were carried out using
the
two shRNA hairpins with three repeats for each of them.
Immunohistochemistry and estimation of Smad1 expression
Expression of Smad1 innormal and tumortissues from
thepatients
was examined by immunohistochemistry using a monoclonal
anti-
body to Smad1 (ab108994, abcam, dilution 1:200) and
DAB-based
staining technique (Dako ChemMateTM EnvisionTM Kit). The
sections
were counterstained with Mayers hematoxylin (see
Supplementary
Materials and methods for scoring Smad1 protein levels).
BrdU labeling assay
Cell proliferation rate was determined with the BrdU assay
as
previously described (Jia et al., 2014).
Trypan blue exclusion assay
Cells treated with Dox were trypsinized from culture plates,
pooled with floating cells from medium, and centrifuged at
1000 g for 10 min at 48C. Trypan blue dissolved in
bufferedphosphate-buffered saline (pH 7.2) was added to the cell
cultures
to a final concentration of 0.04%. The number of live cells
was
counted using hemocytometer under a light microscope. The
results were expressed as percentage of live cells.
Cell viability assay
Cell viability was measured with the water-soluble
tetrazolium
salt (WST-1) assay (Roche Diagnostics), as previously
described
(Jia et al., 2014). IC50 was calculated from the cell survival
curves.
When IC50 could not be obtained from the survival curves,
especial-
ly of control cells, additional experiments using higher
concentra-
tions of Dox were carried out to determine the IC50 values.
Real-time PCR analysis
Total RNAwas extracted fromcells with TRIzol reagent
(Invitrogen)
following the manufacturers protocol. Complementary DNAs
were
synthesized with 0.5 mg of total RNA using iScript cDNA
Synthesis
Kit (Fermentas). The detection and quantification of target
mRNA
were performed with real-time PCR.
Western blot analysis
Western blot analysis was carried out as previously described
(Jia
et al., 2014). Anti-Smad1 (9743), Smad5 (9517), p-Smad1/5/8
(9511), p53 (2524), p-Atm (S1981) (4526S), p-Chk2 (Thr68)
(2661S),
Chk2 (2662), and E2F1 (3742) antibodies were purchased from
Cell
Signaling Technology. Anti-Actin (SC81178) antibody was
purchased
from Santa Cruz Biotechnology, Inc. Antibodies against Atm
(GTX70103) were from Genetex. The protein bands were
quantitated
using the software provided by FluorChem M system
(ProteinSimple)
and the average of three repeated experiments was shown.
Cell transformation and tumorigenesis
Cell transformation and tumorigenesis assays were carried
out
as previously described (Jia et al., 2014).
Statistical analysis
Associations between expression of Smad1 and clinicopa-
thological variables were analyzed by non-parametric
analysis,
using MannWhitney U-test for dichotomization variables and
KruskalWallis test for the others. All statistical analyses were
con-
ducted using SPSS17.0 statistical software. For other studies,
stat-
istical analysis was performed using an unpaired t-test.
Significant
association was defined when P, 0.05 compared with control.
Pearsons correlation analysis was used to determine the
correl-
ation of the expression levels of Smad1 and p53 using SPSS
17.0
software. R 0.6 is deemed significant association.
Supplementary material
Supplementary material is available at Journal of Molecular
Cell
Biology online.
Acknowledgements
We would like to thank Lina Gao (Shanghai Jiao Tong
University)
for technical assistance.
Funding
This work was supported by grants from the National Natural
Science Foundation of China (81130039, 31300684, and 81421
061), the National Key Basic Research Program of China
(2012CB966901 and 2014CB942900), and Program of Shanghai
Subject Chief Scientist (13XD1401900).
Conflict of interest: none declared.
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