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Mutant p53 Disrupts Role of ShcA Protein in Balancing Smad
Protein-dependent and -independent Signaling Activity of
Transforming Growth Factor-

(TGF-

)
*
□
S
Received for publication, May 27, 2011, and in revised form, October 26, 2011 Published, JBC Papers in Press, October 28, 2011, DOI 10.1074/jbc.M111.265397
Shu Lin
‡
, Lan Yu
‡
, Junhua Yang
‡
, Zhao Liu
‡
, Bijal Karia
‡§
, Alexander J. R. Bishop
द
, James Jackson
储
,
Guillermina Lozano
储
, John A. Copland**, Xiaoxin Mu
‡‡‡
, Beicheng Sun
‡‡
, and Lu-Zhe Sun
‡¶1
From the
‡
Department of Cellular and Structural Biology,
§
Greehey Children’s Cancer Research Institute, and
¶
Cancer Therapy and
Research Center, University of Texas Health Science Center, San Antonio, Texas 78229,
储
Department of Genetics, The University of
Texas M. D. Anderson Cancer Center, Houston, Texas 77030, **Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida
32224, and
‡‡
Key Laboratory of Living Donor Liver Transplantation, First Affiliated Hospital of Nanjing Medical University,
Nanjing, China 210009
Background: Biomarkers driving TGF-

from tumor-suppressing to tumor-promoting remain elusive.
Results: p53 mutation inhibits TGF-

-induced ShcA/ERK signaling and enhances Smad signaling. Elevated p-p52ShcA levels
shift the role of TGF-

from growth suppression to migration promotion.
Conclusion: Mutant p53 disrupts the role of ShcA in balancing Smad-dependent and -independent signaling activity of TGF-

.
Significance: Elevated p-p52ShcA levels are a promising biomarker for TGF-

as a tumor promoter.
Biomarkers are lacking for identifying the switch of trans-
forming growth factor-

(TGF-

) from tumor-suppressing to
tumor-promoting. Mutated p53 (mp53) has been suggested to
switch TGF-

to a tumor promoter. However, we found that
mp53 does not always promote the oncogenic role of TGF-

.
Here, we show that endogenous mp53 knockdown enhanced
cell migration and phosphorylation of ERK in DU145 prostate
cancer cells. Furthermore, ectopic expression of mp53 in p53-
null PC-3 prostate cancer cells enhanced Smad-dependent sig-
naling but inhibited TGF-

-induced cell migration by down-
regulating activated ERK. Reactivation of ERK by the expression
of its activator, MEK-1, restored TGF-

-induced cell migration.
Because TGF-

is known to activate the MAPK/ERK pathway
through direct phosphorylation of the adaptor protein ShcA and
MAPK/ERK signaling is pivotal to tumor progression, we inves-
tigated whether ShcA contributed to mp53-induced ERK inhi-
bition and the conversion of the role of TGF-

during carcino-
genesis. We found that mp53 expression led to a decrease of
phosphorylated p52ShcA/ERK levels and an increase of phos-
phorylated Smad levels in a panel of mp53-expressing cancer
cell lines and in mammary glands and tumors from mp53
knock-in mice. By manipulating ShcA levels to regulate ERK
and Smad signaling in human untransformed and cancer cell
lines, we showed that the role of TGF-

in regulating anchorage-
dependent and -independent growth and migration can be
shifted between growth suppression and migration promotion.
Thus, our results for the first time suggest that mp53 disrupts
the role of ShcA in balancing the Smad-dependent and -inde-
pendent signaling activity of TGF-

and that ShcA/ERK signal-
ing is a major pathway regulating the tumor-promoting activity
of TGF-

.
Transforming growth factor-

(TGF-

) is a tumor suppres-
sor during early tumor outgrowth. However, carcinogenesis-
mediated elevation of TGF-

production and signaling is often
tumor-promoting at later stages, leading to enhanced tumor
cell migration, invasion, and metastasis (1). Increased TGF-

is
often associated with the loss of the growth-inhibitory activity
of TGF-

and its conversion to promote malignant progression
of cancers (2), making TGF-

a potential therapeutic target.
Indeed, many preclinical studies have shown the efficacy of var-
ious types of TGF-

inhibitors in blocking tumor growth,
angiogenesis, and metastasis in animal models of human and
rodent cancer (3–5). However, biomarkers are lacking for sig-
nifying the complicated molecular alterations mediating the
switch of TGF-

from tumor suppression to tumor promotion
and for identifying appropriate cancer patients for therapy with
TGF-

inhibitors.
As a homodimeric polypeptide in humans and mice, TGF-

signals through cell surface receptors called TGF-

type I (RI)
2
and type II (RII) receptors to regulate multiple cellular func-
tions including cell proliferation, differentiation, migration,
and wound healing (1). RI and RII are transmembrane serine/
threonine kinase receptors that also contain tyrosine kinase
activity (6). Active TGF-

ligands bind first to RII, which then
*This work was supported, in whole or in part, by National Institutes of Health
Grants R01CA075253 (to L.-Z. S.), R01CA079683 (to L.-Z. S.), R01CA104505
(to J. A. C.), and P30CA054174-17, an NCI cancer center support grant (to
the Cancer Therapy and Research Center at the University of Texas Health
Science Center at San Antonio).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1 and 2.
1
To whom correspondence should be addressed: Dept. of Cellular and Struc-
tural Biology, University of Texas Health Science Center, 7703 Floyd Curl
Dr., Mail Code 7762, San Antonio, TX 78229-3900. Tel.: 210-567-5746; Fax:
210-567-3803; E-mail: sunl@uthscsa.edu.
2
The abbreviations used are: RI, TGF-

type I receptor; RII, TGF-

type II recep-
tor; mp53, mutant p53; SBE, Smad-binding element; R-Smad, receptor-
regulated class of Smads; DNRII, dominant-negative RII; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MMTV, murine
mammary tumor virus; p-ERK, phosphorylated ERK; *MEK-1, constitutively
active form of MEK-1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 51, pp. 44023–44034, December 23, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
DECEMBER 23, 2011•VOLUME 286 •NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 44023
recruits RI, leading to the phosphorylation and activation of RI.
Active RI directly phosphorylates the receptor-regulated class
of Smads (R-Smad), Smad2/3. Phosphorylated R-Smads in turn
associate with Smad4 and translocate into the nucleus to regu-
late the transcription of TGF-

-responsive target genes (1).
Besides Smad-mediated canonical signaling, TGF-

also sig-
nals through Smad-independent pathways including the Ras/
Raf/ERK pathway (7). The integration of TGF-

-mediated-
Smad-dependent and -independent signaling is believed to
contribute to the key events of TGF-

-induced tumor progres-
sion including ERK signaling-mediated cell migration (8, 9).
TGF-

activates Ras/ERK signaling through the direct phos-
phorylation of the adaptor protein ShcA (10). ShcA belongs to
the family of Shc adaptor proteins, which are substrates of
receptor tyrosine kinases (11). ShcA consists of three isoforms,
p46, p52, and p66. They are derived from two different tran-
scripts, called p66 and p52/p46 mRNAs (12). Compared with
p52ShcA, p46ShcA results from a different in-frame ATG tran-
script and is predominantly expressed in mitochondria with an
elusive role (13). Following the TGF-

engagement, tyrosine-
phosphorylated RI recruits and directly phosphorylates p52/
46ShcA proteins on tyrosine sites, leading to their association
with Grb2 adaptor protein and Sos GTP exchange factor (14).
The ShcA-Grb2-Sos complex activates Ras (15), thereby initi-
ating the sequential activation of c-Raf, MEK, and ERK1/2.
Among the three tyrosine phosphorylation sites at residues
239/240 (Tyr-239/240) and 317 (Tyr-317) in the CH1 domain
of p52/46ShcA, Tyr-317 plays the major role in Ras/ERK sig-
naling activation (11, 16). Clinical studies have shown that a
high amount of Tyr-317-phosphorylated p52/46ShcA alone
with a low amount of p66ShcA serves as an efficient predictor
for identifying aggressive breast tumors with a high risk of
recurrence (17, 18). High levels of TGF-

are also associated
with poor outcome of human cancer (1). However, it is unclear
whether ShcA-mediated activation of ERK signaling contrib-
utes to the switch of TGF-

function during carcinogenesis.
A recently published study has shown that the presence of
mutated p53 (mp53) in the DNA-binding domain in certain
cells together with additional Ras activation can switch TGF-

activity to that of a tumor promoter in the MDA-MB-231 breast
cancer cell line and H1299 non-small cell lung cancer cell line
(19). Somatic mutation-induced inactivation of p53 occurs in
about 50% of human cancers including breast and prostate can-
cer (20). p53 mutation is considered a biomarker of advanced
prostate cancer in which prostate cancer cells lose differenti-
ated phenotypes and transit from androgen-dependent to
androgen-independent growth (21). Rather than losing the
wild-type p53 (WTp53), the tumor with retention of mp53 has
been shown to be more aggressive and associated with poor
outcome in certain cancer types (22). For example, mp53 was
shown to enhance the cell migration and invasion in breast and
lung cancer cell lines (23). However, the role of mp53 in medi-
ating the key steps of cancer progression including cell migra-
tion and invasion is largely cell context-dependent and contro-
versial (24). For example, in human endometrial cancer cells,
the p53 R213Q mutation does not promote cell migration (25).
As shown in another study using the H1299 cell line, p53
R175H negatively regulates cell migration when TGF-

/Smad
signaling is repressed (26). At present, the cellular context for
mp53 to exert its oncogenic activity is underexplored. In addi-
tion, little is known about whether mp53 alone is sufficient to
activate the switch of TGF-

during tumor progression.
To gain a more thorough understanding of the effect of mp53
on tumor progression, particularly with respect to the role of
TGF-

signaling, we investigated whether mp53 contributes to
the conversion of the role of TGF-

in the regulation of cell
growth and migration. Here, we show that mp53 alone is not
sufficient to promote the oncogenic role of TGF-

. We further
demonstrate that mp53 disrupts the role of ShcA in altering the
signaling strength of TGF-

through ERK and Smad pathways
in certain human untransformed and cancer cell lines and mice
with mp53 knock-in. ShcA-mediated ERK signaling appears to
play a more dominant role in conferring the tumor-promoting
activity of TGF-

in the regulation of cell growth and migra-
tion. Our finding provides novel insight into the role of ShcA as
a promising biomarker in driving TGF-

signaling toward
tumor promotion.
EXPERIMENTAL PROCEDURES
Ethics Statement—All animal experiments were conducted
following appropriate guidelines. They were approved by the
Institutional Animal Care and Use Committee and monitored
by the Department of Laboratory Animal Resources at the Uni-
versity of Texas Health Science Center at San Antonio (proto-
col identification numbers 99142-34-11-A and 05054-34-01-A)
and the M. D. Anderson Institutional Animal Care and Use
Committee (protocol identification number 079906634).
Cell Culture—Human untransformed mammary epithelial
cell line MCF-10A was obtained from the Michigan Cancer
Foundation. These cells were grown in DMEM/F-12 supple-
mented with 5% horse serum, EGF, NaHCO
3
, hydrocortisone,
insulin, Fungizone, CaCl
2
, cholera toxin, and antibiotics.
Human prostate carcinoma cell lines PC-3, DU145, and 22Rv-1
and human breast cancer cell line BT20 was purchased from the
American Type Culture Collection (ATCC, Manassas, VA).
The human breast cancer MCF-7 control cell line and the dom-
inant-negative RII (DNRII)-transfected cell line were provided
by Dr. Michael G. Brattain (27). All these cells were cultured in
McCoy’s 5A medium with 10% fetal bovine serum (FBS) and
other supplements as described previously (28). The human
breast cancer cell line BT474 was obtained from ATCC and
cultured in DMEM (low glucose) with 10% FBS. Cells were
maintained at 37 °C in a 5% CO
2
humidified incubator.
Chemicals—The small RI kinase inhibitor HTS466284
reported previously to be an ATP-competitive inhibitor of RI
kinase (29) was synthesized by the Chemical Synthesis Core of
Vanderbilt University. U0126 is an MEK-1/2 inhibitor from
Calbiochem.
Plasmids and Transfection—p52/46ShcA plasmid was pur-
chased from Origene. p53 R175H-pCMV-Neo-Bam plasmid
was provided by Dr. Harikrishna Nakshatri. p53
R273H-pCDNA3 plasmid was provided by Dr. Zhi-Min Yuan.
Human WTp53 expression plasmid pRc/CMV hp53 was
obtained from Dr. Arnold Levine. Constitutively active MEK-1
plasmid was provided by Dr. Kun-Liang Guan. Stable transfec-
tion of p53 R175H into PC-3 cells was performed by using Lipo-
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

44024 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 51• DECEMBER 23, 2011
fectamine 2000 (Invitrogen). Forty-eight hours after the trans-
fection, G418 selection of neomycin-resistant cells was
conducted for 1 week. The sequence of p53 siRNA 1 is 5⬘-CCG
GAC GAU AUU GAA CAA UGG UUC A-3⬘, and the sequence
of p53 siRNA 2 is 5⬘-GCU-UCG AGA UGU UCC GAG AGC
UGA A-3⬘(Invitrogen). The sequence of ShcA siRNA is
5⬘-GAC UAA GGA UCA CCG CUU U-3⬘(Dharmacon, Lafay-
ette, CO). All transient transfections were performed by using
Lipofectamine 2000 according to the manufacturer’s protocol.
The pLKO.1-puro lentiviral RII shRNA and control shRNA
were purchased from Sigma. The sequence of RII shRNA is
5⬘-CCG GCC TGA CTT GTT GCT AGT CAT ACT CGA GTA
TGA CTA GCA ACA AGT CAG GTT TTG-3⬘. The process of
generating MCF-10A cells with stable knockdown of RII was
conducted according to the manufacturer’s protocol.
Immunoblotting Analysis—Immunoblotting analyses were
performed as described previously (30). Primary antibodies
were obtained from the following sources: p-Smad2, p-ERK
(Thr-202/Tyr-204), p-ShcA (Tyr-317), total-ERK, and MEK-
1/2 from Cell Signaling Technology (Danvers, MA); p-Smad3
from Epitomics (Burlingame, CA); total Smad2/3 from BD
Transduction Laboratories; p53 from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA); total ShcA from BD Biosciences; and RII
from Abcam (Cambridge, MA). Relative expression levels of the
indicated genes were quantified with ImageJ software (National
Institutes of Health).
Cell Migration Assay—Cell migration assays were performed
in 24-well Boyden chambers with 8-
m pore polycarbonate
membranes (BD Biosciences). Cells at the indicated number in
serum-free medium were seeded in the upper insert. Complete
medium with or without treatment was added in the lower
chamber. After 18 h, cells that had migrated through the mem-
brane were stained with the Hema 3 Stain 18 kit (Fisher Scien-
tific) according to the manufacturer’s protocol. Migrated cells
were counted under a microscope with 100⫻magnification.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bro-
mide (MTT) Assay—Cells were plated in a 96-well plate at 2,500
cells/well with or without TGF-

1 treatment. Two hours
before each time point, 50
l of MTT (2 mg/ml in PBS) was
added into each well, and cells were incubated at 37 °C for
another 2 h. DMSO (100
l) was added into each well after the
medium was removed. For dissolving the precipitate, the plate
was gently shaken on a shaker for 10 min. The absorbance was
measured at 595 nm with a microplate reader (BioTek Instru-
ment, Winooski, VT).
Soft Agar Colony Formation Assay—Cells in 1 ml of 0.4% low
melting agarose (Invitrogen) with culture medium were plated
at 6,000 cells/well on top of existing 0.8% agarose in 6-well
plates. The wells were covered with 1 ml of culture medium
containing various treatments and incubated at 37 °C in a 5%
CO
2
incubator for the indicated number of days. Visualized
colonies were counted after staining with p-iodonitrotetrazo-
lium violet (Sigma) overnight.
SBE-Luciferase Reporter Assay—The pSBE4-Luc plasmid
with Smad-responsive promoter and luciferase reporter gene
was used to measure the TGF-

-induced transcriptional activ-
ity (31). Cells at 100,000 cells/well were plated in 12-well plates.
After 24 h, the pSBE4-Luc plasmid (0.4
g) was transiently
co-transfected with a

-galactosidase expression plasmid (0.1
g) into the cells by using Lipofectamine 2000. TGF-

1 was
added to the transfected cells 5 h later. After an additional 20 h
of incubation, cells were lysed, and the activities of luciferase
and

-galactosidase of the cell lysates were assayed as described
previously (32). Luciferase activity was normalized to

-galac-
tosidase activity.
Animal Tissue Protein Extraction—C57BL/6 mice with a
heterozygous p53
R172H
mutation were described previously
(33). Heterozygous p53
R172P
mice were generated in a similar
way (34). They were crossed to C57BL/6 p
un/un
mice (The Jack-
son Laboratory, Bar Harbor, ME) (35). Heterozygous breeding
cohorts of p53
R172P/⫹
p
un/un
and p53
R172H/⫹
p
un/un
were inter-
crossed to produce the p53
R172P/R172P
p
un/un
,p53
R172H/R172H
p
un/un
, and p53
⫹/⫹
p
un/un
mice. MMTV-Wnt1 mice were bred
with p53
⫹/⫹
or p53
R172H/⫹
mice to generate MMTV-Wnt1
mice in p53
⫹/⫹
,p53
R172H/⫹
, and p53
R172H/R172H
backgrounds.
A fraction of tumors in the p53
R172H/⫹
background undergo
loss of heterozygosity (herein referred to as p53
R172H/O
) and
thus are functionally p53 mutants. Loss of heterozygosity anal-
ysis was performed as described previously (36). The tumor
sizes were measured regularly with a caliper in two dimensions.
Tumor volumes (V) were calculated with the equation V⫽(L⫻
W
2
)⫻0.5 where Lis length and Wis width. When tumors
reached a volume of ⬃500 mm
3
, mice were sacrificed, and the
tumors were collected.
The isolated mammary tissues from p53
R172P/R172P
p
un/un
,
p53
R172H/R172H
p
un/un
, and p53
⫹/⫹
p
un/un
mice or breast
tumors from MMTV-Wnt1-p53
⫹/⫹
,-p53
R172H/R172H
and
-p53
R172H/O
mice were snap frozen in liquid nitrogen. Proteins
for immunoblotting analysis were isolated from liquid nitrogen
by grinding tissues using T-Per extraction reagent (Thermo
Fisher Scientific Inc., Rockford, IL) according to the manufac-
turer’s protocol.
Statistical Analysis—Two-tailed Student’s ttests were used
to determine the significant difference between two mean val-
ues from the control and experimental data. All statistical anal-
ysis was performed with GraphPad Prism 3.03 software
(GraphPad Software, La Jolla, CA).
RESULTS
Mutant p53 Inhibits Cell Migration and Down-regulates ERK
Signaling in Prostate Cancer Cell Lines—To investigate
whether mp53 alone can promote tumor migration, we
knocked down mp53 in the human prostate cancer cell line
DU145 containing inactivating endogenous p53 P223L and
V274F mutations in its DNA-binding domain (37). We found
that instead of making cells less migratory mp53 knockdown
significantly enhanced cell migration accompanied by the acti-
vation of ERK via phosphorylation (p-ERK) (Fig. 1, Aand B).
Active ERK signaling is reported to be essential for tumor
metastasis progression including cell migration (9), suggesting
that the mp53 depletion-induced migration was likely caused
by the up-regulated ERK signaling. We further stably intro-
duced mutant p53 R175H with a mutation in its DNA-binding
domain into the p53-null human prostate cancer cell line PC-3
(PC-3/mp53). This resulted in the repression of cell migration
as well as the down-regulation of ERK signaling (Fig. 1, Cand
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

DECEMBER 23, 2011•VOLUME 286 •NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 44025
D). These data indicate that mp53 in certain prostate cell lines
attenuates cell migration, which is a key step of procancer
metastasis.
Mutant p53 Represses Oncogenic Role of TGF-

and Enhances
TGF-

/Smad Signaling in Prostate Cancer Cell Line—
The presence of mp53 has been shown to facilitate the tumor-
promoting activity of TGF-

in some breast cancer models (19).
Because mp53 was found not to enhance cell migration in our
studies with two prostate cancer cells, we explored whether
mp53 showed a different effect on the oncogenic role of TGF-

.
We observed that TGF-

significantly increased migration of
the control PC-3 cells, whereas it suppressed migration in the
PC-3/mp53 cells, suggesting that the activity of TGF-

is
affected by the presence of mp53 (Fig. 2A). Because PC-3 is a
tumorigenic cell line, we next performed a soft agar colony
formation assay to assess the effect of p53 R175H on the growth
of PC-3 cells in an anchorage-independent manner. We found
that the presence of p53 R175H repressed the anchorage-inde-
pendent growth ability of PC-3/mp53 cells compared with cells
without mp53. Interestingly, the colony formation was more
dramatically inhibited by TGF-

, and the treatment with
HTS466284, an RI kinase inhibitor (29), significantly stimu-
lated colony formation in PC-3/mp53 cells (Fig. 2, Band C). To
rule out the possibility that the p53 R175H-reduced cell migra-
tion in PC-3 cells was due to an altered cell growth rate, we
verified that the presence of p53 R175H in PC-3 cells showed
little effect on cell growth but that TGF-

treatment induced a
moderate growth inhibition in a dose-dependent manner (Fig.
2D). Thus, these results indicate that p53 R175H alone is not
sufficient to switch TGF-

to be more tumor-promoting in the
PC-3 cell line. Considering that TGF-

-inhibited anchorage-
dependent and -independent cell growth is mainly due to
Smad-dependent signaling (38, 39), we investigated whether
p53 R175H could alter the activation of TGF-

/Smad signaling
in PC-3/mp53 cells. We found that p53 R175H made PC-3 cells
more sensitive to TGF-

in a dose-dependent manner as evi-
denced by higher levels of TGF-

-induced phosphorylation of
Smad2/3 (Fig. 2E). Additionally, the transcriptional activity of
TGF-

was also increased in PC-3/mp53 cells when compared
with the control cells as detected with transfection of a Smad-
responsive promoter-luciferase reporter plasmid (SBE-Luc)
(Fig. 2F), further indicating that the presence of p53 R175H
enhanced the Smad-dependent signaling. These results
revealed that the addition of p53 R175H to PC-3 cells enhanced
TGF-

/Smad-signaling while inhibiting TGF-

-induced cell
migration.
Mutant p53 Represses Activation of ShcA/ERK Signaling—It
is suggested that Smad-dependent and -independent signaling
pathways work together to drive the key events of TGF-

-in-
duced cell migration and metastasis (8). However, our observa-
tions indicate that Smad-dependent signaling is not responsible
for the loss of TGF-

-induced migration in PC-3/mp53 cells as
reflected by the enhanced TGF-

/Smad signaling. Conse-
quently, we speculated that MAPK/ERK signaling might be
involved in the loss of TGF-

-induced cell migration of PC-3/
mp53 cells. Consistent with our observation above (Fig. 1C), we
found that the basal level of p-ERK was markedly reduced in the
PC-3/mp53 cells when compared with control cells (Fig. 3A).
Additionally, TGF-

was unable to further activate ERK in
PC-3/mp53 cells (Fig. 3A). To confirm that this observation was
specifically due to mp53 expression and not an aberrant
selection of a pool of antibiotic-resistant clones, we tran-
siently introduced p53 R175H into PC-3 cells and found that
p-ERK was greatly reduced when compared with PC-3 cells
transfected with a control plasmid or a WTp53 expression
plasmid (Fig. 3B). In parallel, p53 R175H knockdown in
PC-3/mp53 cells with p53 siRNA restored the p-ERK level
(Fig. 3C). We additionally used another p53 mutation in the
DNA-binding domain, p53 R273H, which also enhanced
TGF-

-stimulated phosphorylation of Smad2/3 and inhib-
ited the basal and TGF-

-stimulated p-ERK levels. Consis-
tently, p53 R273H also significantly attenuated TGF-

-in-
duced cell migration of PC-3 cells in comparison with the
control cells (Fig. 3Dand supplemental Fig. 1A). However,
exogenous human WTp53 expression in PC-3 cells showed
no effect on the level of TGF-

-induced cell migration, TGF-

-induced activation of ERK, or Smad signaling (Fig. 3Eand
supplemental Fig. 1B). Because tyrosine-phosphorylated
p52ShcA has been shown to positively mediate TGF-

-acti-
vated Ras/ERK signaling (10), we next examined whether the
mp53-inhibited phosphorylation of ERK was linked to the
down-regulation of p-p52ShcA. Indeed, the expression of
p53 R175H and p53 R273H, but not WTp53, in PC-3 cells
inhibited TGF-

-induced phosphorylation of p52ShcA
FIGURE 1. Mutant p53 inhibits cell migration and ERK signaling in pros-
tate cancer cell lines. A, immunoblotting analysis of the indicated gene
expression in DU145 cells, which were transfected with control or p53 siRNA
for 72 h. B, cell migration was assessed in DU145 cells 48 h after the transfec-
tion. Cells were plated at 40,000 cells/insert and incubated for 18 h as
described under “Experimental Procedures.” Data represent mean ⫾S.E.
from three inserts. *, p⬍0.05. C, immunoblotting analysis of the indicated
gene expression in PC-3 cells with stable introduction of p53 R175H expres-
sion plasmid (PC-3/mp53) or empty vector. D, cell migration was assessed in
PC-3/mp53 and control cells plated at 70,000 cells/insert and incubated for
18 h. Data represent mean ⫾S.E. from three inserts. *, p⬍0.05. T-ERK, total
ERK. Error bars represent S.E.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

44026 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 51• DECEMBER 23, 2011
compared with control cells (Fig. 3F). Thus, these data
revealed that mp53, but not WTp53, has the ability to down-
regulate ShcA-mediated ERK signaling.
Expression of MEK-1 or ShcA Restores Active ERK Level and
TGF-

-induced Cell Migration—To determine whether the
attenuated ERK signaling led to the loss of TGF-

-induced cell
migration in PC-3/mp53 cells, we examined whether TGF-

-
induced cell migration could be rescued by the reactivation of
ERK. As shown in Fig. 4A, ectopic expression of the ERK acti-
vator, a constitutively active form of MEK-1 (*MEK-1), was able
to restore the level of p-ERK in PC-3/mp53 cells. More impor-
tantly, compared with control plasmid-transfected cells, PC-3/
mp53 cells with *MEK-1 expression-rescued p-ERK became
responsive again to TGF-

in cell migration (Fig. 4B). These
results suggest the need for active ERK signaling to mediate the
migration-promoting activity of TGF-

in the presence of
mp53. To further confirm our results, we used DU145 cells to
test whether manipulating the active ERK by elevated ShcA
activation also affected TGF-

-induced cell migration in a dif-
ferent mp53-containing model system. We found that TGF-

slightly induced the phosphorylation of ShcA and did not
induce the phosphorylation of ERK (Fig. 4C). Neither was
TGF-

able to induce DU145 cell migration (Fig. 4D), reca-
pitulating the results with PC-3/mp53 (Fig. 2A). Conversely,
ectopic overexpression of p52/46ShcA in the presence of
TGF-

expression resulted in the clear activation of
p52ShcA and ERK as well as the stimulation of cell migra-
tion. These findings are highly consistent with the findings
from PC-3/mp53 cells with reactivated ERK (Fig. 4B). Our
observations thus far have demonstrated that TGF-

is able
to induce cell migration in the context of mp53 when
p-p52ShcA is elevated and that this correlates with activa-
tion of ERK. To verify that the increase of TGF-

-induced
cell migration after overexpression of p52/46ShcA was in
fact mediated by the activation of ERK, we treated the PC-3
cells with an ERK signaling inhibitor, U0126, and found that
TGF-

-induced cell migration was totally abolished (Fig.
4E). Thus, these findings indicate that TGF-

-induced cell
migration is dependent on its activation of ERK signaling in
prostate cancer cells in which mp53 tends to attenuate TGF-

-induced cancer malignancy.
FIGURE 2. Mutant p53 inhibits oncogenic role of TGF-

and enhances TGF-

/Smad signaling. A, cell migration was assayed with PC-3/mp53 and PC-3
control cells plated at 70,000 cells/insert with or without TGF-

1 (2 ng/ml) for 18 h. Data represent mean ⫾S.E. from three inserts. **, p⬍0.01; ***, p⬍0.0001.
B, soft agar colony formation ability was assessed in PC-3/mp53 and PC-3 control cells under the indicated TGF-

1 or HTS466284 (HTS) treatment for 12 days.
Data represent mean ⫾S.E. from three wells. *, p⬍0.05; **, p⬍0.01. C, representative pictures of soft agar colonies of PC-3/mp53 and PC-3 control cells were
taken after 12-day treatment with 2 ng/ml TGF-

1or0.1
MHTS466284. D, effect of TGF-

1 on cell proliferation was measured with the MTT assay at day 6 after
incubation with the indicated dose of TGF-

1. Data represent mean ⫾S.E. from five wells. A two-tailed ttest was performed to compare the mean of relative
cell number between PC-3/mp53 and PC-3 control cells for all treatments. *, p⬍0.05. E, immunoblotting analysis for the indicated gene expression was
performed in PC-3/mp53 and PC-3 control cells under TGF-

1 treatment for 40 min. F, SBE-luciferase assay was performed using PC-3/mp53 and PC-3 control
cells after TGF-

1 treatment as described under “Experimental Procedures.” Data represent mean ⫾S.E. from three independent transfections of relative
luciferase units normalized to

-galactosidase activity. *, p⬍0.05; **, p⬍0.01. T-Smad2/3, total Smad2/3. Error bars represent S.E.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

DECEMBER 23, 2011•VOLUME 286 •NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 44027
ShcA Alters Role of TGF-

in Cellular Migration and Anchor-
age-dependent and -independent Growth in Transformed Cells—
To this point, we have demonstrated that mp53-induced alter-
ation of Smad-dependent and -independent TGF-

signaling is
due to altered ShcA activation. Therefore, we next investigated
whether ShcA could serve as a biomarker in converting the role
of TGF-

from growth suppression to migration promotion in
cancer cells. To this end, we examined the effect of manipulat-
ing the expression level of ShcA in PC-3 cells on growth and
migration. Interestingly, we found that knockdown of ShcA iso-
forms by pan-ShcA siRNA decreased TGF-

-induced phos-
phorylation of ERK and increased TGF-

-induced phosphory-
lation of Smad2/3 (Fig. 5A). Conversely, the ectopic
overexpression of p52/46ShcA raised TGF-

-induced phos-
phorylated ERK levels and reduced TGF-

-induced phospho-
rylated Smad2/3 levels (Fig. 5B). Consistently, we found that
knockdown of p52/46ShcA enhanced, but ectopic overexpres-
sion of p52/46ShcA reduced, TGF-

-inhibited anchorage-de-
pendent (Fig. 5, Cand D) and -independent cell growth (Fig. 5,
Eand F). TGF-

-induced cell migration was significantly
diminished in ShcA-depleted cells but significantly increased in
p52/46ShcA-overexpressing cells (Fig. 5, Gand H). Because
anchorage-independent growth ability is associated with
tumorigenicity in vivo and cell migration is a key step of tumor
progression, our results suggest that ShcA attenuates the tumor
suppressor activity of TGF-

while enhancing its tumor pro-
moter activity.
ShcA Alters Role of TGF-

in Cellular Migration and Cell
Growth in Untransformed Cells—To further investigate the
effect of ShcA on TGF-

-mediated cellular growth and migra-
tion, we introduced pan-ShcA siRNA or p52/46ShcA cDNA
into untransformed MCF-10A cells, which lack complicated
alterations of tumor suppressor genes and oncogenes. By using
this spontaneously immortalized and non-tumorigenic human
mammary epithelial cell line, we were able to examine the tran-
sition of the role of TGF-

in the early stage of transformation.
We essentially observed the same phenotypic changes as in the
transformed PC-3 cells. Specifically, knockdown of ShcA iso-
forms down-regulated TGF-

-induced phosphorylation of
ERK and up-regulated TGF-

-induced phosphorylation of
Smad2/3 (Fig. 6A). In cells with knockdown of ShcA, we found
that TGF-

-inhibited cell growth was significantly enhanced
(Fig. 6C) and that TGF-

-induced cell migration was signifi-
cantly reduced (Fig. 6E) in comparison with the control
siRNA-transfected cells. In contrast, when compared with vehicle-
transfected cells, ectopic overexpression of p52/46ShcA aug-
mented TGF-

-induced phosphorylation of ERK (Fig. 6B) and
TGF-

-induced cell migration (Fig. 6F) but repressed TGF-

-in-
duced phosphorylation of Smad2/3 (Fig. 6B) and significantly
attenuated TGF-

-inhibited cell growth (Fig. 6D). Thus, our data
suggest that ShcA can alter the role of TGF-

in controlling cellu-
lar growth and migration by balancing its signaling between the
ERK and Smad pathways. We next asked whether TGF-

-induced
tyrosine phosphorylation of p52ShcA by RI requires RII. There-
fore, we knocked down RII in MCF-10A cells by using an RII
shRNA and found that the depletion of RII led to the attenuation of
TGF-

-induced phosphorylation of p52ShcA and ERK (Fig. 6G).
Additionally, we have previously shown that ectopic expression of
a DNRII blocked TGF-

signaling and reduced the level of active
ERK in the human MCF-7 breast cancer cell line, which contains a
high level of autocrine TGF-

activity (40). Interestingly, we found
that this DNRII-expressing MCF-7 cell line also has a lower level of
the tyrosine-phosphorylated p52ShcA than the control vector-
transfected cells (Fig. 6H). Thus, our observations suggest that RII
is required for the activation of p52ShcA by TGF-

.
Mutant p53 Positively Correlates with Activation of Smad-
dependent Signaling and Negatively Correlates with Active
ShcA/ERK Signaling in Human Cancer Cell Lines and Trans-
genic Mouse Models—To further generalize our findings, we
tested the correlation among the presence of mp53, activation
of ShcA/ERK signaling, and Smad-dependent signaling in a
FIGURE 3. ShcA-mediated ERK signaling is down-regulated in presence of
mutated p53. A, immunoblotting analysis for the indicated gene expression
was performed in PC-3/mp53 and PC-3 control cells after TGF-

1 treatment
for 40 min. B, immunoblotting analysis for p-ERK and p53 was performed in
PC-3 cells 72 h after transfection of p53 R175H expression plasmid, human
WTp53 expression plasmid, or the empty vector. C, immunoblotting analysis
for the indicated gene expression was performed in PC-3/mp53 cells 72 h
after transfection of p53 siRNA 1, p53 siRNA 2, or control siRNA. D, immuno-
blotting analysis for the indicated gene expression was assessed in PC-3 cells
72 h after transfection of a p53 R273H expression plasmid or empty vector
followed by TGF-

1 treatment for 40 min. E, immunoblotting analysis for the
indicated gene expression was assessed in PC-3 cells 72 h after transfection of
a human WTp53 expression plasmid or empty vector followed by TGF-

1
treatment for 40 min. F, immunoblotting analysis for the indicated gene
expression was conducted in PC-3/mp53 and PC-3 control cells and PC-3 cells
transfected with p53 R273H or WTp53 expression plasmid for 72 h followed
by TGF-

1 treatment for 40 min. T-ERK, total ERK; T-Smad2/3, total Smad2/3;
con; control.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

44028 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 51• DECEMBER 23, 2011
panel of human breast and prostate cancer cell lines, each
expressing varying p53 mutations, although all mutations were
within the DNA-binding domain. Human breast cancer cell
lines BT20 with p53 K132Q mutation, BT474 with p53 E285K
mutation (41), and DU145 were used in our study. We also
included human prostate cancer cell line 22Rv1 with p53
Q331R mutation in the dimerization domain (42). mp53 knock-
down in those cell lines increased the phosphorylation of
p52ShcA and ERK, whereas it decreased the phosphorylation of
Smad2 or Smad3 (Fig. 7A). In mammary gland tissues from
WTp53
⫹/⫹
,p53
R172H/R172H
mutant, or p53
R172P/R172P
mutant
(equivalent to human p53 R175H and R175P mutations,
respectively) female mice on a C57BL/6 p
un/un
genetic back-
ground, we found that the total ERK-normalized phosphoryla-
tion of ERK was down-regulated, whereas the total Smad2/3-
normalized phosphorylation of Smad3 was up-regulated in the
tissues bearing the mp53 when compared with WTp53-ex-
pressing tissues (Fig. 7B). We also normalized the p-ERK and
p-Smad3 expression levels with GAPDH protein levels and
obtained a similar outcome (supplemental Fig. 2A). The
p-ShcA expression levels were undetectable, and we speculated
that the normal murine mammary tissues might express low
levels of p-ShcA (data not shown). Furthermore, to evaluate the
correlation among mp53, ShcA/ERK signaling, and Smad sig-
naling in a more clinically relevant setting, we collected mam-
mary tumor tissues with WTp53
⫹/⫹
,p53
R172H/R172H
mutation,
or p53
R172H/O
(p53 R172H heterozygous with loss of heterozy-
gosity) on the background of the MMTV-Wnt1 transgenic
mice, a well established model of breast cancer development
and progression. The presence of mp53 showed negative cor-
relation with the active ShcA/ERK signaling and positive corre-
lation with the active Smad-dependent signaling (Fig. 7Cand
supplemental Fig. 2B). These observations further support the
conclusion that p53 mutation disrupts the role of ShcA in bal-
ancing the Smad-dependent and Smad-independent signaling
activity of TGF-

.
DISCUSSION
The cross-talk between p53 mutation and oncogenic Ras/
ERK signaling has been demonstrated to promote TGF-

-in-
duced cell migration and metastasis in certain breast cancer
models (19). However, it is still unclear whether mp53 alone can
act as a tumor promoter and cause TGF-

signaling to become
oncogenic. Several studies have revealed that the malignancy
gained from mp53 is cell context-dependent and controversial
(24). For example, mp53 can enhance the cell migration and
invasion via up-regulation of the epithelial-mesenchymal tran-
sition factor Slug in human non-small cell lung cancer and
breast cancer cell lines (23). In contrast, p53 R312Q mutation
does not positively regulate migration of human endometrial
cancer cells (25). p53 R175H mutation was shown to inhibit the
migration of the H1299 lung cancer cell line when its RII was
down-regulated and TGF-

/Smad signaling was repressed (26).
Here, we focused on the p53 DNA-binding domain mutations,
FIGURE 4. Expression of MEK-1 or ShcA restores active ERK level and TGF-

-induced cell migration. A, immunoblotting analysis for the indicated gene
expression was assessed in PC-3/mp53 cells 72 h after transfection of an *MEK-1expression plasmid or empty vector. B, cell migration was assayed in
PC-3/mp53 cells 48 h after *MEK-1 transfection. Cells were plated at 70,000 cells/insert with or without TGF-

1 (2 ng/ml) for 18 h. Data represent mean ⫾S.E.
from three inserts. *, p⬍0.05. C, 72 h post-transfection with an empty vector or p52/46ShcA expression plasmid, immunoblotting analysis for the indicated
gene expression was performed in DU145 cells with or without 2 ng/ml TGF-

1 treatment for 40 min. D, cell migration was assessed in DU145 cells 48 h after
transfection of p52/46ShcA or empty vector. Cells were plated at 40,000 cells/insert with or without TGF-

1 (2 ng/ml) for 18 h. Data represent mean ⫾S.E. from
three inserts. *, p⬍0.05. E, cell migration was assessed in PC-3 cells 48 h after transfection of p52/46ShcA or empty vector. Cells were plated at 100,000
cells/insert and treated with TGF-

1 (2 ng/ml) or U0126 (10
M) for 18 h. Data represent mean ⫾S.E. from three inserts. ***, p⬍0.0001. T-ERK, total ERK;
T-Smad2/3, total Smad2/3. Error bars represent S.E.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

DECEMBER 23, 2011•VOLUME 286 •NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 44029
which have been reported as the majority of p53 mutations in
human breast and prostate cancer cell lines (43). We found that
in DU145 and PC-3 human prostate cancer cells the endoge-
nous or ectopic expression of p53 with a point mutation in its
DNA-binding domain does not enhance the cell migration or
cause TGF-

to be more migration-promoting. Hence, our
results from these models indicate that mp53 alone does not
always function as a promoter of migration in already trans-
formed cells, and its function is likely context-dependent.
Indeed, p53 mutation has been shown to occur relatively late
during multistage oncogenic progression and often follows Ras
mutation-induced ERK signaling (44). It has been shown that
mp53 works together with oncogenic Ras to induce the expres-
sion of several protumorigenesis and prometastasis genes in
gene expression profiling studies (45). Our finding that mp53
repressed the oncogenic role of TGF-

in the model systems we
used suggests that additional signaling activation and genetic
alterations such as ERK signaling activation and attenuation of
TGF-

-induced growth inhibition appear necessary to collab-
oratively confer the tumor-promoting activity of mp53 and
TGF-

during cancer progression. Although the sequestration
of metastasis suppressor p63 by the formation of an mp53-
Smad-p63 ternary complex has been shown to enhance the
oncogenic activity of TGF-

(19), an additional mechanism
involving mp53 but not the formation of the ternary complex
has also been shown to drive TGF-

to become more tumor-
promoting (46). DU145 cells are negative for p63 expression
(47). Thus, the conversion of TGF-

is independent of the ter-
nary complex formation in DU145 cells with restored ShcA/
ERK signaling. The presence of mp53 repressed the basal level
of phosphorylated p52ShcA and also abolished the activation of
p52ShcA by TGF-

, suggesting that mp53 could be the
upstream regulator of p52ShcA. We speculate that the mecha-
nism by which mp53 down-regulates the phosphorylation of
p52ShcA is perhaps through an aberrant or dysregulated pro-
tein interaction. However, further investigation is needed to
elucidate the mechanism.
TGF-

receptors have been shown to possess dual tyrosine
and serine kinase activity and can directly tyrosine phosphoryl-
ate ShcA to activate Ras/MAPK signaling or serine phosphoryl-
ate ShcA with an unclear consequence (10). The same study
suggests that tyrosine phosphorylation of p66Shc, the inhibi-
tory isoform of ShcA, is more dependent on RI in untrans-
formed cells. On the other hand, it is unclear whether RI alone
is sufficient to induce tyrosine phosphorylation of p52ShcA. In
our study, the depletion of RII in the untransformed human
breast cells attenuated the TGF-

-induced activation of the
ShcA/ERK cascade as well as Smad signaling. DNRII has the
intact extracellular and transmembrane domains of the wild-
type RII and is capable of forming a functional complex with
TGF-

and RI. By introducing this DNRII into a human breast
cancer cell line that has autocrine TGF-

activity, we found that
the dysfunctional RII caused the inhibition of ShcA/ERK acti-
vation. Our observations suggest that RII is required for TGF-

to activate the p52ShcA/ERK pathway. It is worth mentioning
that other tyrosine kinase receptors such as epidermal growth
factor receptor and insulin receptor also have the ability to tyro-
sine phosphorylate p52/46ShcA and consequently activate Ras/
FIGURE 5. ShcA alters TGF-

-mediated Smad and ERK signaling and activ-
ity in human cancer cell. PC-3 cells were transfected with a pan-ShcA siRNA
or a p52/46ShcA expression plasmid as shown in Aand B, respectively. Sev-
enty-two hours post-transfection, immunoblotting analysis for the indicated
gene expression was performed using the transfected cells treated with or
without 2 ng/ml TGF-

1 for 40 min. Cand D, TGF-

1 (2 ng/ml)-induced cell
growth inhibition was measured with the MTT assay in cells 72 h after trans-
fection. Data are presented as the percentage of TGF-

-induced inhibition of
cell growth or colony formation (shown in Eand F) relative to untreated cells.
Data represent mean ⫾S.E. from four wells. Eand F, soft agar colony forma-
tion ability was assessed in cells 24 h after transfection. Cells were treated with
or without 2 ng/ml TGF-

1 for 6 days. Data represent mean ⫾S.E. from three
wells. Gand H, cell migration was assessed in cells 48 h after transfection. Cells
were plated at 70,000 cells/insert with or without TGF-

1 (2 ng/ml) for 18 h.
Data are presented as the -fold change of TGF-

-induced migration relative
to untreated cells. Data represent mean ⫾S.E. from three inserts. *, p⬍0.05; **,
p⬍0.01. T-ERK, total ERK; T-Smad2/3, total Smad2/3. Error bars represent S.E.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

44030 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 51• DECEMBER 23, 2011
ERK signaling (48). We speculate that RI and other tyrosine
receptors may compete for available ShcA. This competition
may further influence the signaling strength of TGF-

between
Smad and ERK signaling. Currently, how those pathways may
affect the Smad-dependent and -independent signaling of
TGF-

via p52/46ShcA is also under investigation.
The activation of TGF-

/Smad-dependent signaling
requires the serine/threonine kinase activity of RII and RI.
Tyrosine kinases, instead of serine/threonine kinases, have
been reported as the catalytic center of RI in certain structural
features (49). Conceivably, R-Smads and ShcA may compete for
RI kinase, and the balance between the tyrosine phosphoryla-
tion of ShcA and serine phosphorylation of R-Smads could
determine the signaling strength of TGF-

through the Smad-
independent and -dependent pathways, respectively.
It is well known that TGF-

-induced cell growth inhibition
in epithelial cells depends on Smad-dependent signaling (38,
39). Loss of Smad4 during cancer progression has been shown
to contribute to the resistance of TGF-

-inhibited cell growth,
which is considered one mechanism for the TGF-

to be
tumor-promoting (1). However, it is less clear what other
mechanistic biomarkers can indicate the change of TGF-

sig-
naling in favor of tumor progression during tumorigenesis. Our
study showed that knockdown of ShcA isoforms enhanced,
whereas overexpression of p52/46ShcA attenuated, TGF-

-in-
hibited anchorage-dependent and independent-growth of
tumor cells, implicating ShcA as a negative mediator of TGF-

-induced tumor suppression. For the Smad-dependent
TGF-

signaling, TGF-

-mediated cell cycle arrest involves a
Smad-dependent induction of cyclin-dependent kinase inhibi-
tors p15 and p21. On the other hand, the activation of MAPK/
ERK signaling is pivotal for the TGF-

-induced epithelial-mes-
enchymal transition and cell migration, both of which are
considered important steps of prometastatic progression (1).
Thus, it is conceivable that the mechanism by which p52/
46ShcA regulates TGF-

-induced cell migration or growth
inhibition is cell context-dependent. For DU145, PC-3, and
MCF-10A cells, we found that the cells are sensitive to TGF-

as evidenced by the increased p-Smad2/3 and that manipulat-
ing ShcA enhanced TGF-

-induced cell migration accompa-
nied by increased TGF-

/ShcA/ERK signaling and decreased
TGF-

/Smad signaling. Because TGF-

/Smad-dependent
stimulation of p21 expression has been observed in these cells
and p21 was shown to be up-regulated by the Smad3-FoxO
Forkhead transcription factor transcriptional complex (50), it is
likely that the altered cell growth we observed was due to the
manipulation of p52/46ShcA and altered Smad/FOXO/p21
cascade. On the other hand, in cancer cells with Smad4 muta-
tion or aberrant regulation of p21 and/or p15, the alteration of
Smad signaling due to a change in ShcA activity may not result
in TGF-

-inhibited cell growth.
It is believed that p66ShcA is not involved in the activation
of MAPK signaling (51). In fact, p66ShcA was shown to
FIGURE 6. ShcA alters TGF-

-mediated Smad and ERK signaling and activ-
ity in untransformed human cells. MCF-10A cells were transfected with a
pan-ShcA siRNA or a p52/46ShcA expression plasmid as shown in Aand B,
respectively. Seventy-two hours post-transfection, immunoblotting analysis
for the indicated gene expression was performed using the transfected cells
treated with or without 2 ng/ml TGF-

1 for 40 min. Cand D, TGF-

1 (2 ng/ml)-
induced cell growth inhibition was measured with the MTT assay in cells 72 h
after transfection. Data are presented as the percentage of TGF-

-induced
growth inhibition relative to the untreated cells. Data represent mean ⫾S.E.
from four wells. Eand F, cell migration was assayed in cells 48 h after transfec-
tion. Cells were plated at 70,000 cells/insert with or without TGF-

1 (2 ng/ml)
for 18 h. Data are presented as the fold change of TGF-

-induced migration
relative to untreated cells. Data represent mean ⫾S.E. from three inserts. *,
p⬍0.05; **, p⬍0.01. G, immunoblotting analysis of the indicated gene
expression was performed in MCF-10A cells with or without RII knockdown
after TGF-

1 treatment for 40 min. H, immunoblotting analysis for the indi-
cated gene expression was performed in MCF-7 cells with or without DNRII
expression. T-ERK, total ERK; T-Smad2/3, total Smad2/3; T-p52ShcA, total
p52ShcA; con, control. Error bars represent S.E.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

DECEMBER 23, 2011•VOLUME 286 •NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 44031
antagonize ERK activation via negative regulation of c-fos
promoter activity (52) and to be mainly involved in oxidative
stress signaling (53). Additionally, the role of p66ShcA in
tumor metastasis is controversial (54, 55). Therefore, the
reduction of the p-ERK level after ShcA knockdown with the
pan-ShcA siRNA was likely due to the depletion of
p52/46ShcA.
Consistent with a previous study demonstrating that
knockdown of ShcA or a dominant-negative form of ShcA
inhibited TGF-

-induced cell migration and invasion of
murine breast cancer cells bearing activated Neu/ErbB-2
receptor (56), our study showed that ShcA depletion-caused
down-regulation of ERK signaling resulted in the attenua-
tion of TGF-

-induced cell migration in human untrans-
formed and transformed cells. On the other hand, we found
that increased ShcA/ERK signaling augmented TGF-

-in-
duced cell migration. We further showed that in DU145 cells
the restored activation of p52/46ShcA/ERK signaling com-
pletely switched the role of TGF-

from suppression to pro-
motion of cell migration. ERK signaling-induced cell migra-
tion is a key sign of tumor progression (9); therefore, our
results provide a novel concept that the ShcA/ERK pathway
acts as a pivotal driver of the oncogenic role of TGF-

.Inan
unpublished study,
3
we found that knockdown of Smad4 in
MCF-10A cells moderately reduced TGF-

-induced cell
migration in comparison with control siRNA-transfected
cells, indicating that Smad-dependent signaling partially
contributes to TGF-

-induced cell migration. Here, we
showed that the ShcA-mediated increase of TGF-

-induced
migration was in the presence of down-regulated Smad sig-
naling. These observations suggest that ShcA-enhanced ERK
signaling appears to play a dominant role in TGF-

-induced
cell migration. Thus, the down-regulation of p-Smad2/3 is
not likely necessary for the ShcA/ERK signaling-mediated
increase of TGF-

-induced migration.
In our study, we found that mp53 positively correlates with
phosphorylation of R-Smads, whereas it negatively correlates
with phosphorylation of p52ShcA and ERK in a panel of
mp53-expressing breast and prostate cancer cell lines as well
as in mp53 mouse models. We demonstrated that ShcA
tipped the balance of the role of TGF-

in tumor progression
as evidenced by enhanced migration and attenuated prolif-
eration in the presence of TGF-

. The earlier clinical studies
reported that high levels of tyrosine-phosphorylated p52/
46Shc in primary tumors might actually identify patients
with malignant breast tumors (17, 18). Increased levels of
TGF-

are also positively related to poor outcome of human
cancer (1). Our finding that overexpression of ShcA
enhanced the tumor-promoting activity of TGF-

provides
insight in linking these two biomarkers to the disease out-
come. In the preclinical models, we and others have shown
that anti-TGF-

therapies are potent in treating cancer in
which TGF-

functions as a tumor promoter (3–5). How-
ever, the lack of knowledge about when TGF-

is switched
from a tumor suppressor to a tumor promoter is a major
3
S. Lin, L. Yu, J. Yang, and L.-Z. Sun, unpublished data.
FIGURE 7. Mutant p53 correlates with higher level of Smad-dependent
signaling and lower level of ShcA/ERK signaling in human cancer cell
lines and genetically altered mouse models. A, immunoblotting analysis
for the indicated gene expression was performed in BT-20, BT474, 22Rv1, and
DU145 cells 72 h after transfection of p53 siRNA 1 or control siRNA. B, immu-
noblotting analysis for the indicated gene expression was performed in the
tissue lysates from mammary gland of p53
R172H/R172H
p
un/un
(p53 R172H; n⫽2)
and p53
R172P/R172P
p
un/un
(p53 R172P; n⫽1) mice and the littermate p53
⫹/⫹
p
un/un
(WTp53; n⫽5). The scatter plot figures under the immunoblots are the
total ERK-normalized p-ERK levels and the total Smad2/3-normalized
p-Smad3 levels quantified with ImageJ software. C, immunoblotting analysis
for the indicated gene expression was performed in the tissue lysates from
mammary tumors of MMTV-Wnt1-WTp53
⫹/⫹
(WTp53; n⫽8), MMTV-Wnt1-
p53
R172H/R172H
(p53 R172H/H; n⫽4), and MMTV-Wnt1-p53
R172H/O
(p53
R172H/O; n⫽4) mice. The scatter plot figures under the immunoblots are the
total p52ShcA-normalized p-p52ShcA levels, total ERK-normalized p-ERK lev-
els, and the total Smad2/3-normalized p-Smad3 levels for each sample quan-
tified with ImageJ software. *, p⬍0.05. T-ERK, total ERK; T-Smad2/3, total
Smad2/3; T-p52ShcA, total p52ShcA.
Mutant p53 and ShcA Alter Oncogenic Role of TGF-

44032 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 •NUMBER 51• DECEMBER 23, 2011
obstacle for the utility of TGF-

antagonists for treating
metastatic carcinomas. Thus, our study indicates that the
TGF-

-dependent increase of tyrosine-phosphorylated p52/
46ShcA may be a promising biomarker for an effective anti-
TGF-

cancer therapy. Biomarkers identified by this type of
research may help cancer patients benefit from anti-TGF-

therapy in blocking tumor progression in the future.
Acknowledgments—We thank Dr. Andrew Hinck (University of Texas
Health Science Center at San Antonio, UTHSCSA) for the recombi-
nant TGF-

1, Dr. Michael G. Brattain (University of Nebraska,
Omaha) for the MCF-7 cell lines, Dr. Bert Vogelstein (Johns Hopkins
Oncology Center, Baltimore, Maryland) for the pSBE4-Luc plasmid,
Dr. Harikrishna Nakshatri (Indiana University, Indianapolis, IN) for
the p53 R175H plasmid, Dr. Zhi-Min Yuan (UTHSCSA) for the p53
R273H plasmid, Dr. Arnold Levine (Institute for Advanced Study,
Princeton, NJ) for the human WTp53 plasmid and Dr. Kun-Liang
Guan (University of California, San Diego) for the constitutively
active MEK1 plasmid.
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