Activation of Src by Protein Tyrosine Phosphatase 1B Is Required for
ErbB2 Transformation of Human Breast Epithelial Cells
Luis E. Arias-Romero,
3and Jonathan Chernoff
1Stephen P. Ethier,
1Fox Chase Cancer Center, Philadelphia, Pennsylvania;
School of Medicine, Detroit, Michigan; and
Indiana University School of Medicine, Indianapolis, Indiana
2Karmanos Cancer Institute, Department of Pathology, Wayne State University
3Department of Biochemistry and Molecular Biology,
Protein tyrosine phosphatase (PTP) 1B plays a major role in
inhibiting signaling from the insulin and leptin receptors.
Recently, PTP1B was found to have an unexpected positive
role in ErbB2 signaling in a mouse model of breast cancer, but
the mechanism underlying this effect has been unclear. Using
human breast epithelial cells grown in a three-dimensional
matrix, we found that PTP1B, but not the closely related
enzyme T-cell PTP, is required for ErbB2 transformation
in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B
expression, and increased expression of PTP1B activates Src
and induces a Src-dependent transformed phenotype. These
findings identify a molecular mechanism by which PTP1B
links an important oncogenic receptor tyrosine kinase to
signaling pathways that promote aberrant cell division and
survival in human breast epithelial cells. [Cancer Res
Protein tyrosine phosphatase (PTP) 1B is an abundant
cytoplasmic enzyme that plays a major role in down-regulating
insulin and leptin signaling (1). PTP1B binds to and dephosphor-
ylates the insulin receptor, thus terminating signals from this
receptor tyrosine kinase. Deletion of the Ptp1b gene in mice causes
hypersensitivity to insulin and is associated with marked increases
in tyrosine phosphorylation of the insulin receptor and its targets
(2, 3). PTP1B may also inhibit signaling from other receptor
tyrosine kinases such as the platelet-derived growth factor and
hepatocyte growth factor receptors (4–7). Consistent with its role
as an inhibitor of receptor tyrosine kinases, overexpression of
PTP1B in fibroblasts inhibits transformation by oncogenes that
increase tyrosine phosphorylation, including ErbB2, Src, Bcr-Abl,
and Crk, and also by Ras (8–11). In Src or Crk transformed cells,
overexpression of PTP1B is associated with loss of tyrosine
phosphorylation of key signaling proteins such as Crk and
The above results, coupled with effects of PTP1B on the insulin
receptor, gave rise to the idea that the main role of PTP1B is to act
as a brake to proliferative and metabolic signals. However, recent
data indicate that this idea is too limited. In fibroblasts, PTP1B is
required for the activation of the small GTPases Ras (12) and Rac
(13), enzymes that are generally associated with increased cell
proliferation and motility. In addition, PTP1B has also been shown
to activate Src by dephosphorylating the inhibitory Y527 site in the
COOH terminus of this kinase (13–16). Thus, at endogenous levels
of expression, PTP1B has certain progrowth properties in marked
contrast to its aforementioned ability to revert transformation by
various oncogenes when overexpressed. In vivo, Ptp1b-/-mice resist
transformation by the ErbB2 (HER-2/neu) oncogene, and trans-
genic overexpression of PTP1B in mammary cells is oncogenic
(17, 18). Moreover, in a significant fraction of human clinical
samples of ErbB2-positive breast cancer, amplification of chromo-
some 20q13 (containing the PTP1B gene) has been noted, with
increased expression of PTP1B (19, 20). These findings imply that
this enzyme plays a positive role in growth signaling in at least
some tissues and that PTP1B might therefore serve as a therapeutic
target in certain malignancies.
How does PTP1B contribute to oncogenesis in mammary cells?
PTP1B is known to up-regulate two growth-promoting pathways: it
activates Src and deactivates p62Dok, an inhibitor of the Ras/
mitogen-activated protein kinase pathway (21). Regarding these
substrates, it is at least theoretically plausible that the reported
in vivo effects of PTP1B loss on ErbB2-driven breast cancer can be
explained in terms of failure to activate Src. However, Kaminski
and colleagues have recently reported that Src activity is
dispensable for ErbB2-driven carcinogenesis (22). Another PTP1B
substrate, p62Dok, represents an alternate explanation for the
positive effects of PTP1B on transformation. When tyrosine
phosphorylated, this protein complexes with, and activates,
p120RasGAP, leading to decreased Ras and mitogen-activated protein
kinase activity (23, 24). Loss of PTP1B leads to hyperphosphor-
ylation of p62Dok, with consequent inactivation of Ras and its
downstream effectors. However, tissue samples from ErbB2/
Ptp1b-/-mice gave inconsistent results regarding the tyrosine
phosphorylation status of p62Dok(17, 18). For these reasons,
whether the observed effects of PTP1B on ErbB2-driven carcino-
genesis are due to the interactions of PTP1B with Src, p62Dok, or
unidentified substrates is not known.
In this article, we sought to clarify the molecular mechanism(s)
by which PTP1B contributes to ErbB2 signaling in breast epithelial
cells. We used a three-dimensional in vitro model to recapitulate
the architectural elements of breast acinar development while
retaining the ability to manipulate and analyze the pathways that
underlie the effects of PTP1B on ErbB2 signaling. Consistent with
its proposed role in oncogenic signaling, we found that activation
of ErbB2, but not ErbB1, increased PTP1B expression and that
small interfering RNA (siRNA)-induced reduction in PTP1B
expression or inhibition of PTP1B activity by small-molecule
inhibitors impeded the ability of activated ErbB2 to transform
these cells and to activate Src and its associated downstream
Note: Supplementary data for this article are available at Cancer Research Online
S. Saha, O. Villamar-Cruz, and S-C. Yip contributed equally to this work.
Requests for reprints: Jonathan Chernoff, Fox Chase Cancer Center, Philadelphia,
PA. Phone: 215-728-5319; Fax: 215-28-3616; E-mail: J_Chernoff@fccc.edu.
I2009 American Association for Cancer Research.
Cancer Res 2009; 69: (11). June 1, 2009
signaling targets. In addition, we found that the suppressive effects
of PTP1B loss could be bypassed by expression of mutationally
activated Src. Likewise, we found that overexpression of PTP1B in
breast epithelial cells distorted normal acinar morphology, causing
unchecked proliferation, and loss of polarity. These effects were
associated with Src activation and required the presence of protein
phosphatase activity and the Src-binding motif in the COOH
terminus of PTP1B. These results support a model in which PTP1B,
by activating Src, cooperates with ErbB2 in transforming mammary
Materials and Methods
Materials. Anti-ERK, anti-Akt, anti-Src, phospho-ERK, phospho-Akt,
phospho-Src, phospho-Y1248 ErbB2, phospho-Y1068 ErbB1, and phospho-
Y416 and Y527 Src antibodies were purchased from Cell Signaling
Technology. Monoclonal anti-hemagglutinin (12CA5) and anti-phosphotyr-
osine (PY20) antibodies were from BabCo and Transduction Laboratories,
respectively. Anti-Ki-67 was from Santa Cruz Biotechnology. Monoclonal
anti-T-cell PTP (TC-PTP) 3E2 (25) was a kind gift from Michel Tremblay.
Reconstituted basement membrane (Matrigel) was from BD Life Science.
PP2 was purchased from Calbiochem.
Cell lines and three-dimensional cell culture. 10A.ErbB2 cells (MCF-
10A cells expressing a chimeric form of ErbB2; ref. 26), 7.ErbB2 cells
(created by stable transduction of MCF7 cells with pMN.B2.F2.HA), and
10A.ErbB1 cells (created by stable transduction of MCF-10A cells with
pMN.B1.F2.HA; ref. 26) were maintained in DMEM/F-12 (Life Technolo-
gies) supplemented with 5% donor horse serum, 20 ng/mL EGF (Harlan
Bioproducts), 10 mg/mL insulin (Sigma), 1 ng/mL cholera toxin (Sigma),
100 mg/mL hydrocortisone (Sigma), 50 units/mL penicillin, and 50 mg/mL
streptomycin. For three-dimensional cultures, f5,000 cells were plated
atop reconstituted basement membrane in 8-well slide chambers as
described (26). To activate chimeric ErbB proteins, 1 mmol/L AP1510 was
added to the growth medium. BT-474, MDA-MB-231, and SUM190 cells
were grown in RPMI, 10% fetal bovine serum and Ham’s F12, 10% fetal
bovine serum, respectively.
Expression plasmids, transfection, and transduction. The retroviral
expression vector pMN.B1.F2.HA, encoding ErbB1, was obtained from
Muthuswamy and colleagues (26) and pWZL-PTP1B (5) was obtained from
Ben Neel. The PTP1BP309,310A(PTP-PA) and PTP1BD161A(PTP-DA) mutants
were subcloned into pWZL from pCMV6H (10). To generate viral stocks,
these vectors were transiently transfected into the Phoenix-Eco retroviral
packaging line,4and supernatants were collected 48 h later. For retroviral
transduction, 10A.ErbB2 cells were plated at 3 ? 105per 10 cm diameter
dish and then infected with high-titer retroviruses and screened by anti-
hemagglutinin or anti-PTP1B immunoblots.
RNA interference. The siRNA duplex targeted to PTP1B had the
sequence (sense) 5¶-CUUCCUAAGAACAAAAACCdTdT-3¶ and (antisense)
5¶-GGUUUUUGUUCUUAGGAAGdTdT-3¶. The siRNA duplexes targeted to
TC-PTP comprised a mixture of four different oligonucleotides (SMART
pool). Equal amounts of sense and antisense RNA oligonucleotides were
mixed and annealed according to the manufacturer’s instructions.
10A.ErbB2 cells were transfected with 40 AL of 20 Amol/L siRNA for PTP1B
or TC-PTP or the control siRNA duplex with Lipofectamine 2000 reagent for
12 h. All siRNAs were purchased from Dharmacon Research.
Immunoprecipitation, immunoblotting, and in-cell Westerns. Cells
were lysed in a buffer containing 50 mmol/L Tris-HCl (pH 8.0), 137 mmol/L
NaCl, 10% glycerol, 1% NP-40, 50 mmol/L NaF, 10 mmol/L h-glycerophos-
phate, 2 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl
fluoride, and 10 Ag/mL aprotinin. Lysates were clarified by centrifugation at
13,000 rpm for 10 min and protein concentrations were determined using a
bicinchoninic acid protein assay kit (Pierce Chemical). For immunopreci-
pitations, lysates were incubated with the appropriate antibodies for
3 h overnight at 4jC. Immune complexes were collected onto protein A-
Sepharose beads, washed extensively, resolved by SDS-PAGE, and trans-
ferred onto Immobilon-P membranes (Millipore). Immunoblots were
blocked with 5% bovine serum albumin or Carnation nonfat dry milk in
10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.05% Tween-20. After
incubation with appropriate primary and secondary antibodies, blots were
visualized using enhanced chemiluminescence reagents (Amersham
Biosciences). Quantification was carried out using NIH ImageJ software
version 1.40; data are expressed as relative units of phosphorylated protein
per total protein for each band. Anti-PTP1B and anti-hemagglutinin
antisera were used at 1:1,000 for immunoblotting. Anti-human PTP1B
monoclonal antibodies (FG6) were used at 1 Ag/mL for immunoprecipita-
tions and immunoblotting. All other antibodies were used at concentrations
as recommended by the supplier.
In-cell Westerns were done according to the manufacturer’s specifica-
tions.5In brief, cells were fixed in 4% methanol-free formaldehyde in PBS,
rinsed with PBS, blocked with 5% nonimmune rabbit or mouse serum in
PBS/0.1% Triton X-100 for 1 h, and incubated with primary antibodies for
2 h at 37jC. The cells were then rinsed three times in PBS for 5 min each
and then incubated in fluorochrome-conjugated secondary antibody diluted
1:500 (Alexa Fluor 680) or 1:800 (IRDye 800CW) in PBS/0.1% Triton X-100
for 1 h at room temperature in the dark. The cells were then rinsed three
times in PBS for 5 min each and scanned using a Li-Cor Odyssey device.
Immunofluorescence analysis. The acinar structures were fixed in 2%
paraformaldehyde at room temperature for 15 min and processed as
described by Muthuswamy and colleagues (26). Confocal analyses were
done with a Nikon TE2000 confocal microscopy system.
PTP1B is required for ErbB2-mediated transformation of
MCF-10A cells. To establish the functions of PTP1B in human
breast epithelial cells, we examined the effects of this phosphatase
in ErbB2 signaling in MCF-10A cells grown in three-dimensional
conditions. MCF-10A cells are immortalized, nontransformed cells
derived from a reduction mammoplasty, which form organized
acini when grown within three-dimensional matrices such as
reconstituted basement membrane (26, 27). In MCF-10A cells that
stably express an AP1510-activatable, chimeric form of ErbB2
(10A.ErbB2 cells), treatment with AP1510 caused characteristic
changes in acinar morphogenesis, proliferation, and luminal
apoptosis, resembling those seen in human ductal carcinoma of
the breast (ref. 26; Fig. 1A).
Next, we asked if endogenous PTP1B is required for the
phenotypic effects of ErbB2 on acinar development. We used
siRNA to reduce PTP1B expression in 10A.ErbB2 cells and assessed
the effects on acinar morphogenesis. Transfection of 10A.ErbB2
cells with siRNA directed against PTP1B resulted in f80% loss of
PTP1B expression, whereas a scrambled siRNA did not affect
PTP1B expression (Fig. 1C). Reduction of PTP1B did not have a
notable effect on basal rates of cell proliferation or apoptosis (data
not shown), nor did it affect acinar architecture; however, the
phenotypic effects of ErbB2 activation were blocked in cells treated
with siRNA against PTP1B (Fig. 1B). The siRNA against PTP1B did
not affect ErbB2 kinase activity (Fig. 1D), indicating that the
suppression of the multiacinar phenotype was mediated by events
downstream of this receptor tyrosine kinase.
TC-PTP is a close relative of PTP1B, and we therefore asked if
this phosphatase also affects ErbB2 signaling in 10A.ErbB2 cells.
Role of PTP1B in ErbB2 Transformation
Cancer Res 2009; 69: (11). June 1, 2009
We used siRNA to knock down TC-PTP expression and assessed its
effect on acinar development in three-dimensional cultures.
Despite effective reduction in TC-PTP expression by siRNA, acini
developed normally and were disrupted by activation of ErbB2
(Supplementary Fig. S1A). Thus, the effects of PTP1B on ErbB2
signaling are not shared with its closest relative, TC-PTP.
To rule out off-target effects and also to ensure that these effects
were due to loss of PTP1B activity rather than loss of the protein,
we carried out similar experiments using two different, highly
specific small-molecule inhibitors of PTP1B, compounds II and III
(28). Compounds II and III are cell-permeable analogues of the
most potent and selective PTP1B inhibitor (compound 40 in ref. 29)
Figure 1. PTP1B is required for ErbB2-mediated transformation of MCF-10A cells. A, MCF10A.ErbB2 cells were plated atop reconstituted basement membrane.
Beginning on day 3, 1 mmol/L AP1510 was added to the medium. The cells were fixed on day 12 and stained with Oregon green-phalloidin, Ki-67, or anti-cleaved
caspase-3. B, MCF10A.ErbB2 cells were transfected with control or PTP1B-specific siRNA and plated atop reconstituted basement membrane and processed as in A.
Percentage of unilamellar acini, Ki-67-positive, and anti-cleaved caspase-3-positive acini were scored based on assessment of 50 to 60 acini per well. C, anti-PTP1B
immunoblot. D, blot of activated ErbB2 and total ErbB2.
Cancer Res 2009; 69: (11). June 1, 2009
reported to date, which displays a Kivalue of 2.4 nmol/L for PTP1B
and exhibits several orders of magnitude of selectivity in favor of
PTP1B against a panel of PTPs (29). As with siRNA, chemical
inhibition of PTP1B suppressed the multiacinar effects of ErbB2
(Fig. 2). Together with the siRNA data, these experiments show that
PTP1B function is required by ErbB2 to induce a multiacinar
Molecular pathways affected by PTP1B in MCF-10A cells. We
next tested if PTP1B links ErbB2 to Src in MCF-10A cells. First, we
assessed Src activity in response to ErbB2. Three-dimensional
cultures of 10A.ErbB2 cells were treated with AP1510 and Src
activity assessed by immunoblot using phosphospecific antibodies
directed against Tyr416in Src. As expected, activation of ErbB2
induced activation of Src (Fig. 3). The expected reciprocal changes
were seen in Src Tyr527phosphorylation: AP1510-induced de-
creased Src Y527 phosphorylation (Supplementary Fig. S2A). In
contrast, in cells treated with siRNA against PTP1B, or a small-
molecule PTP1B inhibitor, Src activation by ErbB2 was markedly
suppressed as assessed by decreased Tyr416and increased Tyr527
phosphorylation (Fig. 3A; Supplementary Fig. S2A). Similar results
were seen regarding two other important signaling molecules that
are activated by Src, Akt and ERK (Fig. 3B and C). Notably,
inhibition of PTP1B did not indiscriminately affect other ErbB2-
induced signaling events, such as the tyrosine phosphorylation of
Shc, nor did it affect total tyrosine phosphorylation (Supplemen-
tary Fig. S2B and C). Also, the effects of PTP1B on ErbB2 signaling
were not confined to 10A.ErbB2 cells. As in 10A.ErbB2 cells,
addition of the specific PTP1B inhibitor compound II to the ErbB2-
expressing human breast cancer cell lines BT-474, MDA-MB-231, or
SUM190 resulted in marked loss of ERK activity (Supplementary
Activated Src bypasses requirement for PTP1B in ErbB2-
mediated transformation. Treatment of 10A.ErbB2 cells with the
Src inhibitor PP2 blocked the effects of ErbB2 activation on acinar
morphology (Fig. 4A). If Src activation downstream of ErbB2 and
PTP1B mediates the effects of ErbB2 on signal transduction, then a
constitutively active form of Src should bypass the need for either
ErbB2 or PTP1B in signaling, and loss of Src should block the
actions of these enzymes. We transduced 10A.ErbB2 cells with
SrcY527F, which lacks the inhibitory site of tyrosine phosphorylation
in the COOH terminus and is therefore constitutively active. These
cells display aberrant acini and elevated Akt and ERK activity even
in the absence of activated ErbB2 (Fig. 4B). In addition, these
Figure 2. Small-molecule inhibition of PTP1B blocks ErbB2-mediated
transformation of MCF-10A cells. MCF10A.ErbB2 cells plated atop reconstituted
basement membrane were treated with vehicle, 25 nmol/L compound II
(Comp II), or 250 nmol/L compound III (Comp III), plus 1 mmol/L AP1510, on day
3 as indicated, and fixed on day 12. Medium was replaced (with PTP1B inhibitors
and AP1510) every 3 d.
Figure 3. Molecular pathways affected by PTP1B in MCF-10A cells.
MCF10A.ErbB2 cells were transfected with control or PTP1B-specific siRNA and
plated atop reconstituted basement membrane. Cells were stimulated with
vehicle or AP1510 on day 3 and fixed on day 12. Where indicated, cells were
treated with specific small-molecule inhibitors of PTP1B or Src. The activity of
Src, Akt, and ERK was assessed by in-cell Western using phosphospecific
Role of PTP1B in ErbB2 Transformation
Cancer Res 2009; 69: (11). June 1, 2009
effects were also seen in Src-expressing cells treated with an
inhibitor of PTP1B (Fig. 4C).
Overexpression of PTP1B distorts acinar development via
activation of Src. Interestingly, we found that the expression
level of PTP1B increased markedly after treatment with AP1510
(Fig. 5A). Similar results were seen in MCF-7 cells expressing the
same AP1510-activatable form of ErbB2 (data not shown),
indicating that the effects on PTP1B are not confined to a single
type of breast epithelial cell line. These results are in line with
previous observations that ErbB2-transformed cells have elevated
PTP1B expression and suggest a causal relationship between these
events (30). In contrast, PTP1B expression was not altered in
10A.ErbB1 cells following activation of ErbB1, indicating that the
effect on PTP1B expression was specific to ErbB2 and not a general
response to growth stimuli (Fig. 5A). The ErbB2 effect on PTP1B
expression was reversible; withdrawal of AP1510 resulted in the
rapid decline in PTP1B expression (Supplementary Fig. S4).
If Src activation by PTP1B plays a role in ErbB2 signaling, then
activation or overexpression of PTP1B should affect acinar
development in a Src-dependent manner. To test this idea, we
examined the effects of PTP1B overexpression in 10A.ErbB2 cells.
These cells were infected with a retrovirus encoding no insert, wild-
type PTP1B, PTP1B-PA (a mutant that cannot bind Src homology
3-domain containing proteins, including Src; ref. 10), or PTP1B-DA
(a catalytically dead mutant). In all cases, f3-fold elevation in
PTP1B expression was observed, comparable with the degree of
PTP1B expression in cells following AP1510 treatment (Fig. 5B).
Overexpression of wild-type PTP1B, but not the PTP1B mutants,
was associated with a multilaminar phenotype (Fig. 5C). The
effects of wild-type PTP1B were blocked by the Src inhibitor PP2
(Fig. 5D), consistent with the idea that Src represents a key target
of PTP1B in breast epithelial cells.
In these studies, we used a three-dimensional breast epithelial
cell culture system to show that PTP1B plays a positive role in
ErbB2 signaling. Using this model, we were able to dissect the
Figure 4. Activated Src bypasses
requirement for PTP1B in Neu-mediated
transformation. A, MCF10A.ErbB2 cells
were treated with 10 mmol/L PP2 and
1 mmol/L AP1510. In-cell Western blots
were used to determine the activity of Src,
Akt, and ERK. B, MCF10A.ErbB2 cells
were transduced with a control retrovirus or
a retrovirus encoding activated c-Src.
Acinar morphology and activity of Src, Akt,
and ERK are shown. C, cells transduced
with activated c-Src were transfected
with scrambled siRNA or PTP1B-specific
siRNA. The activities of Src, Akt, and
ERK, as assessed by in-cell Westerns,
Cancer Res 2009; 69: (11). June 1, 2009
signaling events that underlie these important effects. We showed
that one of the primary targets for PTP1B activity in human breast
epithelial cells is the tyrosine kinase Src. This assertion rests on the
following observations: (a) the ErbB2-induced multiacinar pheno-
type, characterized by unrestrained proliferation, luminal cell
survival, and loss of polarity, is suppressed by decreased expression
or chemical inhibition of PTP1B but not by decreased expression of
the related phosphatase TC-PTP; (b) loss of PTP1B function blocks
the activation of Src and downstream signaling pathways by ErbB2;
(c) mutationally activated Src bypasses loss of PTP1B in ErbB2-
mediated transformation; and (d) activation of ErbB2 leads to
elevated expression of PTP1B and PTP1B overexpression distorts
mediated acinar morphogenesis; these effects require an intact Src
homology 3-binding motif and are blocked by a Src inhibitor. Taken
together, these findings support a model in which PTP1B
expression is increased by ErbB2 and that its activity is required
to activate Src by ErbB2 and that such activation is required for
transformation of breast epithelial cells.
These findings are compatible with recent in vivo experiments in
which loss of PTP1B was shown to reduce, and transgenic
overexpression increase, mammary oncogenesis in mice (17, 18).
However, our results contrast with earlier work from our laboratory
and others that showed that PTP1B overexpression suppresses
transformation by several oncogenes, including ErbB2 and Src
(8, 10, 11). How can these results be reconciled? One explanation
may lie in the choice of cells for analysis. In all of the prior work,
transformation was assayed in mouse or rat fibroblasts as opposed
to human mammary cells. It is well established that transformation
of human and murine cells has distinct signaling requirements
(31, 32). In addition, epithelial cells likewise use different signaling
networks than fibroblasts. For these reasons, it is likely that the
observed effects of PTP1B on ErbB2 signaling in human MCF-10A
cells are germane to breast epithelial cells and thus to breast
cancer but do not apply to all cell types.
We showed that PTP1B is required for Src activation by ErbB2 in
MCF-10A cells and that activated Src bypasses the need for PTP1B
in ErbB2-mediated transformation. These results suggest that Src is
a key target in mediating the positive role of PTP1B in ErbB2
oncogenesis. The role of Src in ErbB2 signaling has been
controversial. On the one hand, Src activation strongly correlates
with ErbB2 overexpression in human ductal carcinoma in situ (33),
and Src associates with activated ErbB2 in human breast cancer
cells in a Src homology 2 domain-dependent manner, resulting in
activation of Src (34). In addition, inhibition of Src in breast
epithelial cells suppresses ErbB2-mediated motility and soft-agar
growth (34–36). All of these studies suggest that Src plays an
important role in ErbB2 oncogenic signaling. On the other hand,
transgenic overexpression of the Csk gene, which encodes a protein
kinase that suppresses Src activation, does not affect ErbB2-
mediated breast cancer in mice (22). These latter findings point to
the possibility of a nonenzymatic role for Src in ErbB2 signaling.
Our findings are not inconsistent with this idea, because activation
of Src by PTP1B-catalyzed dephosphorylation of Y527, in addition
to its effects on kinase activity, also leads to conformational
changes in Src that could expose scaffolding motifs.
In clinical samples, it has been long noted that PTP1B expression
increases in several human tumor types, including breast and
ovarian cancers (20, 37). Furthermore, PTP1B expression is
elevated in breast epithelial cells transformed by ErbB2 (refs. 18,
30; Fig. 5A). It has been proposed that this increase in expression
represents the attempt of cells to counterincreased PTK activity. In
light of data that PTP1B is required for Ras and Rac activation
in vitro, and recent data that PTP1B is required for transformation
by Neu in mouse models of breast cancer, it is more likely that
elevated PTP1B contributes to transformation in certain human
Given that loss of PTP1B function attenuates carcinogenesis in
mouse models of breast cancer, it is plausible that PTP1B
inhibitors might prove useful in the therapy of certain cancers.
Figure 5. Overexpression of PTP1B alters acinar morphogenesis via activation
of Src. A, anti-phospho-ErbB1 or ErbB2, anti-PTP1B, and anti-actin immunoblots
from MCF10A.ErbB2 and MCF10A.ErbB1 cells, respectively, grown in the
absence or presence of AP1510. Numbers indicate fold expression relative to
unstimulated ErbB1 or ErbB2. B, a retrovirus bearing wild-type or mutant forms of
PTP1B was used to infect MCF10A.ErbB2 cells, which were then plated atop
reconstituted basement membrane. Cells were fixed on day 12. An anti-PTP1B
immunoblot is shown. Numbers indicate fold expression relative to endogenous
PTP1B. C, 4¶,6-diamidino-2-phenylindole and Oregon green-phalloidin staining
of acini overexpressing the indicated forms of PTP1B. D, effects of the Src
inhibitor PP2 on wild-type PTP1B-induced changes in acinar morphology.
Role of PTP1B in ErbB2 Transformation
Cancer Res 2009; 69: (11). June 1, 2009
Such a notion would have seemed heretical just a few years ago, as
copious previous data using overexpressed PTP1B in fibroblasts in
culture indicated that this protein acts as a suppressor of growth
factor signaling and that its loss might therefore be expected to
augment cell proliferation. Instead, the opposite seems to be true
in the case of the oncogene ErbB2 both in our three-dimensional
cell culture system and in ErbB2-driven transgenic mouse models
of breast cancer. Evidently, in these systems, the effects of PTP1B
on receptor tyrosine kinases are either confined to the insulin
receptor or are more than offset by positive effects on other
signaling elements such as Src. These findings highlight the
necessity of combining advanced cell-based and animal models
when probing complex signal transduction pathways.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Received 10/16/08; revised 3/23/09; accepted 3/24/09; published OnlineFirst 5/12/09.
Grant support: NIH grants R01 CA58836 (J. Chernoff), R01 CA69202 (Z-Y. Zhang),
R01 CA100724 (S.P. Ethier), and F32 DK079474 (S-C. Yip), Fox Chase Cancer Center
grant P30 CA006927, and an appropriation from the State of Pennsylvania.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Senthil Muthuswamy, Ben Neel, Michel Tremblay, and Ariad
Pharmaceuticals for the gifts of 10A.ErbB2 cells, plasmid pWZL-PTP1B, monoclonal
antibody 3E2, and AP1510, respectively, and Erica Golemis for comments on the
Cancer Res 2009; 69: (11). June 1, 2009
1. Bourdeau A, Dube N, Tremblay ML. Cytoplasmic
protein tyrosine phosphatases, regulation and function:
the roles of PTP1B and TC-PTP. Curr Opin Cell Biol
2. Elchebly M, Payette P, Michaliszyn E, et al. Increased
insulin sensitivity and obesity resistance in mice lacking
the protein tyrosine phosphatase-1B gene. Science 1999;
3. Klaman LD, Boss O, Peroni, OD, et al. Increased energy
expenditure, decreased adiposity, and tissue-specific
insulin sensitivity in protein-tyrosine phosphatase 1B-
deficient mice. Mol Cell Biol 2000;20:5479–89.
4. Liu F, Chernoff J. PTP1B associates with and is
tyrosine phosphorylated by epidermal growth factor
receptor in cultured cells. Biochem J 1997;327:139–45.
5. Haj FG, Markova B, Klaman LD, Bohmer FD, Neel BG.
Regulation of receptor tyrosine kinase signaling by
protein tyrosine phosphatase-1B. J Biol Chem 2003;278:
6. Lammers R, Bossenmaier B, Cool DE, et al. Differential
activities of protein tyrosine phosphatases in intact
cells. J Biol Chem 1993;268:22456–62.
7. Kakazu A, Sharma G, Bazan HE. Association of protein
tyrosine phosphatases (PTPs)-1B with c-Met receptor
and modulation of corneal epithelial wound healing.
Invest Ophthalmol Vis Sci 2008;49:2927–35.
8. Brown-Shimer S, Johnson KA, Hill DE, Bruskin AM.
Effect of protein tyrosine phosphatase 1B expression on
transformation by the human neu oncogene. Cancer Res
9. LaMontagne KRJ, Hannon G, Tonks NK. Protein
tyrosine phosphatase PTP1B suppresses p210 bcr-abl-
induced transformation of rat-1 fibroblasts and pro-
motes differentiation of K562 cells. Proc Natl Acad Sci
U S A 1998;95:14094–9.
10. Liu F, Chernoff J. Suppression of oncogene-mediated
transformation of rat 3Y1 fibroblasts by protein tyrosine
phosphatase 1B requires a functional SH3-ligand. Mol
Cell Biol 1998;18:250–9.
11. Woodford-Thomas TA, Rhodes JD, Dixon JE. Expres-
sion of a protein tyrosine phosphatase in normal and v-
src-transformed mouse 3T3 fibroblasts. J Cell Biol 1992;
12. Dube N, Cheng A, Tremblay ML. The role of protein
tyrosine phosphatase 1B in Ras signaling. Proc Natl
Acad Sci U S A 2004;101:1834–9.
13. Dadke S, Chernoff J. Protein-tyrosine phosphatase 1B
mediates the effects of insulin on the actin cytoskeleton
in immortalized fibroblasts. J Biol Chem 2003;278:
14. Bjorge JD, Pang A, Fujita DJ. Identification of protein-
tyrosine phosphatase 1B as the major tyrosine phos-
phatase activity capable of dephosphorylating and
activating c-Src in several human breast cancer cell
lines. J Biol Chem 2000;275:41439–46.
15. Anderie I, Schulz I, Schmid A. Direct interaction
16. Liang F, Lee SY, Liang J, Lawrence DS, Zhang ZY. The
role of protein-tyrosine phosphatase 1B in integrin
signaling. J Biol Chem 2005;280:24857–63.
17. Julien SG, Dube N, Read M, et al. Protein tyrosine
phosphatase 1B deficiency or inhibition delays ErbB2-
induced mammary tumorigenesis and protects from
lung metastasis. Nat Genet 2007;39:338–46.
18. Bentires-Alj M, Neel BG. Protein-tyrosine phospha-
tase 1B is required for HER2/Neu-induced breast cancer.
Cancer Res 2007;67:2420–4.
19. Tanner MM, Tirkkonen M, Kallioniemi A, et al.
Independent amplification and frequent co-amplifica-
tion of three nonsyntenic regions on the long arm of
chromosome 20 in human breast cancer. Cancer Res
20. Wiener JR, Kerns BJ, Harvey EL, et al. Overexpression
of the protein tyrosine phosphatase PTP1B in human
breast cancer: association with p185c-erbB-2 protein
expression. J Natl Cancer Inst 1994;86:372–8.
21. Tonks NK, Muthuswamy SK. A brake becomes an
accelerator: PTP1B-a new therapeutic target for breast
cancer. Cancer Cell 2007;11:214–6.
22. Kaminski R, Zagozdzon R, Fu Y, et al. Role of SRC
kinases in Neu-induced tumorigenesis: challenging the
paradigm using Csk homologous kinase transgenic
mice. Cancer Res 2006;66:5757–62.
23. Yamanashi Y, Baltimore D. Identification of the Abl-
and rasGAP-associated 62 kDa protein as a docking
protein, Dok. Cell 1997;88:205–11.
24. Kashige N, Carpino N, Kobayashi R. Tyrosine
phosphorylation of p62dok by p210bcr-abl inhibits
RasGAP activity. Proc Natl Acad Sci U S A 2000;97:
25. Ibarra-Sanchez MJ, Wagner J, Ong MT, Lampron C,
Tremblay ML. Murine embryonic fibroblasts lacking TC-
PTP display delayed G1 phase through defective NF-B
activation. Oncogene 2001;:4728–39.
26. Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge
JS. ErbB2, but not ErbB1, reinitiates proliferation and
induces repopulation in epithelial acini. Nat Cell Biol
27. Soule HD, Maloney TM, Wolman SR, et al. Isolation
and characterization of a spontaneously immortalized
human breast epithelial cell line, MCF-10. Cancer Res
28. Zhang ZY. Functional studies of protein tyrosine
phosphatases with chemical approaches. Biochim Bio-
phys Acta 2005;1754:100–7.
29. Shen K, Keng YF, Wu L, Guo XL, Lawrence DS,
Zhang ZY. Acquisition of a specific and potent
PTP1B inhibitor from a novel combinatorial library
and screening procedure. J Biol Chem 2001;276:
30. Zhai YF, Beittenmiller H, Wang B, et al. Increased
expression of specific protein tyrosine phosphatases in
human breast epithelial cells neoplastically trans-
formed by the neu oncogene. Cancer Res 1993;53:
31. Lim KH, Baines AT, Fiordalisi JJ, et al. Activation of
RalA is critical for Ras-induced tumorigenesis of human
cells. Cancer Cell 2005;7:533–45.
32. Hamad NM, Elconin JH, Karnoub AE, et al. Distinct
requirements for Ras oncogenesis in human versus
mouse cells. Genes Dev 2002;16:2045–57.
33. Wilson GR, Cramer A, Welman A, et al. Activated c-
SRC in ductal carcinoma in situ correlates with high
tumour grade, high proliferation and HER2 positivity. Br
J Cancer 2006;95:1410–4.
34. Muthuswamy SK, Siegel PM, Dankort DL, Webster
MA, Muller WJ. Mammary tumors expressing the neu
proto-oncogene possess elevated c-Src tyrosine kinase
activity. Mol Cell Biol 1994;14:735–43.
35. Sheffield LG. c-Src activation by ErbB2 leads to
attachment-independent growth of human breast epi-
thelial cells. Biochem Biophys Res Commun 1998;250:
36. Belsches-Jablonski AP, Biscardi JS, Peavy DR, Tice DA,
Romney DA, Parsons SJ. Src family kinases and HER2
interactions in human breast cancer cell growth and
survival. Oncogene 2001;20:1465–75.
37. Tanner MM, Grenman S, Koul A, et al. Frequent
amplification of chromosomal region 20q12–13 in
ovarian cancer. Clin Cancer Res 2000;6:1833–89.