14320061 ( Dewi Mirotun N)

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

by guest on June 7, 2015http://jmcb.oxfordjournals.org/

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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.

114 | Ruan et al.


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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.

ReferencesAaltonen, L.M., Chen, R.W., Roth, S., et al. (2001). Role of TP53 P72R polymorph-

ism in human papillomavirus associated premalignant laryngeal neoplasm.

J. Med. Genet. 38, 327.

Arnold, S.J., Maretto, S., Islam, A., et al. (2006). Dose-dependent Smad1,

Smad5 and Smad8 signaling in the early mouse embryo. Dev. Biol. 296,

104118.

116 | Ruan et al.


Dow

nloaded from

Barcellos-Hoff, M.H., and Cucinotta, F.A. (2014). New tricks for an old fox: impact

of TGFb on the DNA damage response and genomic stability. Sci. Signal. 7,

re5.

Brooks, C.L., and Gu, W. (2006). p53ubiquitination: Mdm2and beyond. Mol. Cell

21, 307315.

Chau, J.F., Jia, D., Wang, Z., et al. (2012). A crucial role for bone morphogenetic

protein-Smad1 signalling in the DNA damage response. Nat. Commun.3, 836.

Cordenonsi, M., Dupont, S., Maretto, S., et al. (2003). Links between tumor sup-

pressors: p53 is required for TGF-b gene responses by cooperating with

Smads. Cell 113, 301314.

Derynck, R., Akhurst, R.J., and Balmain, A. (2001). TGF-b signaling in tumor sup-

pression and cancer progression. Nat. Genet. 29, 117129.

Dick, A., Meier, A., and Hammerschmidt, M. (1999). Smad1 and Smad5 have dis-

tinct roles during dorsoventral patterning of the zebrafish embryo. Dev. Dyn.

216, 285298.

Eivers, E., Fuentealba, L.C., and De Robertis, E.M. (2008). Integrating positional

information at the level of Smad1/5/8. Curr. Opin. Genet. Dev. 18, 304310.

Fuentealba, L.C., Eivers, E., Ikeda, A., et al. (2007). Integrating patterning

signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell

131, 980993.

Gao, H., Chakraborty, G., Lee-Lim, A.P., et al. (2012). The BMP inhibitor Coco

reactivates breast cancer cells at lung metastatic sites. Cell 150, 764779.

Goh, A.M., Coffill, C.R., and Lane, D.P. (2011). The role of mutant p53 in human

cancer. J. Pathol. 223, 116126.

Goumans, M.J., Valdimarsdottir, G., Itoh, S., et al. (2003). Activin receptor-like

kinase (ALK)1 is an antagonistic mediator of lateral TGFb/ALK5 signaling.

Mol. Cell 12, 817828.

Hardwick, J.C., Kodach, L.L., Offerhaus, G.J., et al. (2008). Bone morphogenetic

protein signalling in colorectal cancer. Nat. Rev. Cancer 8, 806812.

Hong, B., Prabhu, V.V., Zhang, S., et al. (2014). Prodigiosin rescues deficient p53

signaling and antitumor effects via upregulating p73 and disrupting its inter-

action with mutant p53. Cancer Res. 74, 11531165.

Howe, J.R., Bair, J.L., Sayed, M.G., et al. (2001). Germline mutations of the gene

encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat.

Genet. 28, 184187.

Jackson, S.P., and Bartek, J. (2009). The DNA-damage response in human biology

and disease. Nature 461, 10711078.

Jia, H., Cong, Q., Chua, J.F., et al. (2014). p57Kip2 is an unrecognized DNA

damage response effector molecule that functions in tumor suppression

and chemoresistance. Oncogene, doi: 10.1038/onc.2014.287.

Kirshner, J., Jobling, M.F., Pajares, M.J., et al. (2006). Inhibition of transforming

growth factor-b1 signaling attenuates ataxia telangiectasia mutated activity

in response to genotoxic stress. Cancer Res. 66, 1086110869.

Kodach, L.L., Wiercinska, E., de Miranda, N.F., et al. (2008). The bone morpho-

genetic protein pathway is inactivated in the majority of sporadic colorectal

cancers. Gastroenterology 134, 13321341.

Kortlever, R.M., Higgins, P.J., and Bernards, R. (2006). Plasminogen activator

inhibitor-1 is a critical downstream target of p53 in the induction of replicative

senescence. Nat. Cell Biol. 8, 877884.

Kravchenko, J.E., Ilyinskaya, G.V., Komarov, P.G., et al. (2008). Small-molecule

RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent

salvage pathway. Proc. Natl Acad. Sci. USA 105, 63026307.

Lai, K.P., Leong, W.F., Chau, J.F., et al. (2010). S6K1 is a multifaceted regulator of

Mdm2 that connects nutrient status and DNA damage response. EMBO J. 29,

29943006.

Langenfeld, E., Hong, C.C., Lanke, G., et al. (2013). Bone morphogenetic protein

type I receptor antagonists decrease growth and induce cell death of lung

cancer cell lines. PLoS One 8, e61256.

Lee, Y.C., Cheng, C.J., Bilen, M.A., et al. (2011). BMP4 promotes prostate tumor

growth in bone through osteogenesis. Cancer Res. 71, 51945203.

Leong, W.F., Chau, J.F., and Li, B. (2009). p53 Deficiency leads to compensatory

up-regulation of p16INK4a. Mol. Cancer Res. 7, 354360.

Liu, B., Sun, Y., Jiang, F., et al. (2003). Disruption of Smad5 gene leads to

enhanced proliferation of high-proliferative potential precursors during em-

bryonic hematopoiesis. Blood 101, 124133.

Liu, H., Jia, D., Li, A., et al. (2013). p53 regulates neural stem cell proliferation and

differentiation via BMP-Smad1 signaling and Id1. Stem Cells Dev. 22,

913927.

Lord, C.J., and Ashworth, A. (2012). The DNA damage response and cancer

therapy. Nature 481, 287294.

Lubbe, S.J., Pittman, A.M., Olver, B., et al. (2012). The 14q22.2 colorectal cancer

variant rs4444235 shows cis-acting regulation of BMP4. Oncogene 31,

37773784.

Lunghi, P., Costanzo, A., Mazzera, L., et al. (2009). The p53 family protein p73

provides new insights into cancer chemosensitivity and targeting. Clin.

Cancer Res. 15, 64956502.

Ma, G., Li, L., Hu, Y., et al. (2012). Atypical Atm-p53 genetic interaction

in osteogenesis is mediated by Smad1 signaling. J. Mol. Cell Biol. 4,

118120.

McReynolds, L.J., Gupta, S., Figueroa, M.E., et al. (2007). Smad1 and

Smad5 differentially regulate embryonic hematopoiesis. Blood 110,

38813890.

Ming Kwan, K., Li, A.G., Wang, X.J., et al. (2004). Essential roles of BMPR-IA sig-

naling in differentiation and growth of hair follicles and in skin tumorigenesis.

Genesis 39, 1025.

Muller, P.A., and Vousden, K.H. (2013). p53 mutations in cancer. Nat. Cell Biol.

15, 28.

Neumann, J.C., Chandler, G.L., Damoulis, V.A., et al. (2011). Mutation in the type

IB bone morphogenetic protein receptor Alk6b impairs germ-cell differenti-

ation and causes germ-cell tumors in zebrafish. Proc. Natl Acad. Sci. USA

108, 1315313158.

Olivier, M., Hollstein, M., and Hainaut, P. (2010). TP53 mutations in human

cancers: origins, consequences, and clinical use. Cold Spring Harb.

Perspect. Biol. 2, a001008.

Overstreet, J.M., Samarakoon, R., Meldrum, K.K., et al. (2014). Redox control of

p53 in the transcriptional regulation of TGF-b1 target genes through SMAD

cooperativity. Cell. Signal. 26, 14271436.

Pangas, S.A., Li, X., Umans, L., et al. (2008). Conditional deletion of Smad1 and

Smad5 in somatic cells of male and female gonads leads to metastatic tumor

development in mice. Mol. Cell. Biol. 28, 248257.

Park, S., Kang, J.M., Kim, S.J., et al. (2015). Smad7 enhances ATM

activity by facilitating the interaction between ATM and Mre11-Rad50-Nbs1

complex in DNA double-strand break repair. Cell. Mol. Life Sci. 72,

583596.

Reinhardt, H.C., and Schumacher, B. (2012). The p53 network: cellular and

systemic DNA damage responses in aging and cancer. Trends Genet. 28,

128136.

Rivlin, N., Brosh, R., Oren, M., et al. (2011). Mutations in the p53 tumor suppres-

sor gene: important milestones at the various steps of tumorigenesis. Genes

Cancer 2, 466474.

Samarakoon, R., Dobberfuhl, A.D., Cooley, C., et al. (2013). Induction of renal

fibrotic genes by TGF-b1 requires EGFR activation, p53 and reactive oxygen

species. Cell. Signal. 25, 21982209.

Sancho, E., Batlle, E., and Clevers, H. (2004). Signaling pathways in intestinal

development and cancer. Annu. Rev. Cell Dev. Biol. 20, 695723.

Sapkota, G., Alarcon, C., Spagnoli, F.M., et al. (2007). Balancing BMP

signaling through integrated inputs into the Smad1 linker. Mol. Cell 25,

441454.

Shirai, Y.T., Ehata, S., Yashiro, M., et al. (2011). Bone morphogenetic protein-2

and -4 play tumor suppressive roles in human diffuse-type gastric carcinoma.

Am. J. Pathol. 179, 29202930.

Thawani, J.P., Wang, A.C., Than, K.D., et al. (2010). Bone morphogenetic proteins

and cancer: review of the literature. Neurosurgery 66, 233246.

Tomlinson, I.P., Carvajal-Carmona, L.G., Dobbins, S.E., et al. (2011). Multiple

common susceptibility variants near BMP pathway loci GREM1, BMP4, and

BMP2 explain part of the missing heritability of colorectal cancer. PLoS

Genet. 7, e1002105.

Vinall, R.L., Chen, J.Q., Hubbard, N.E., et al. (2012). Initiation of prostate cancer in

mice by Tp53R270H: evidence for an alternative molecular progression. Dis.

Model. Mech. 5, 914920.



Dow

nloaded from

Vogelstein, B., Lane, D., and Levine, A.J. (2000). Surfing the p53 network. Nature

408, 307310.

Walsh,D.W.,Godson,C.,Brazil,D.P.,etal. (2010).ExtracellularBMP-antagonist regu-

lation in development and disease: tied up in knots. Trends Cell Biol.20,244256.

Wang, M., Saha, J., Hada, M., et al. (2013). Novel Smad proteins localize to

IR-induced double-strand breaks: interplay between TGFband ATM pathways.

Nucleic Acids Res. 41, 933942.

Zhang, S., Ekman, M., Thakur, N., et al. (2006). TGFb1-induced activation of ATM

and p53 mediates apoptosis in a Smad7-dependent manner. Cell Cycle 5,

27872795.

Zhang, M., Li, L., Wang, Z., et al. (2013a). A role for c-Abl in cell senescence and

spontaneous immortalization. Age (Dordr) 35, 12511262.

Zhang, X., Jia, D., Liu, H., et al. (2013b). Identification of 5-Iodotubercidin as a

genotoxic drug with anti-cancer potential. PLoS One 8, e62527.

118 | Ruan et al.


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