Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer
Transcript of Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer
Molecular Cell
Article
Identification of the Rac-GEF P-Rex1as an Essential Mediatorof ErbB Signaling in Breast CancerMaria Soledad Sosa,1,8 Cynthia Lopez-Haber,1,8 Chengfeng Yang,3 HongBin Wang,1 Mark A. Lemmon,2 John M. Busillo,4
Jiansong Luo,4 Jeffrey L. Benovic,4 Andres Klein-Szanto,5 Hiroshi Yagi,6 J. Silvio Gutkind,6 Ramon E. Parsons,7
and Marcelo G. Kazanietz1,*1Department of Pharmacology2Department of Biochemistry and Biophysics
University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA3Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA4Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA5Fox-Chase Cancer Center, Philadelphia, PA 19111, USA6Oral and Pharyngeal Cancer Branch, NIDCR, Bethesda, MD 20892, USA7Institute for Cancer Genetics, Columbia University Medical Center, New York, NY 10032, USA8These authors contributed equally to this work*Correspondence: [email protected]
DOI 10.1016/j.molcel.2010.11.029
SUMMARY
While the small GTPase Rac1 and its effectors arewell-established mediators of mitogenic and motilesignaling by tyrosine kinase receptors and havebeen implicated in breast tumorigenesis, little isknown regarding the exchange factors (Rac-GEFs)that mediate ErbB receptor responses. Here, weidentify the PIP3-Gbg-dependent Rac-GEF P-Rex1as an essential mediator of Rac1 activation, motility,cell growth, and tumorigenesis driven by ErbB recep-tors in breast cancer cells. Notably, activation ofP-Rex1 in breast cancer cells requires the conver-gence of inputs from ErbB receptors and a Gbg-and PI3Kg-dependent pathway. Moreover, we iden-tified the GPCR CXCR4 as a crucial mediator ofP-Rex1/Rac1 activation in response to ErbB ligands.P-Rex1 is highly overexpressed in human breastcancers and their derived cell lines, particularly thosewith high ErbB2 and ER expression. In addition to theprognostic and therapeutic implications, our findingsreveal an ErbB effector pathway that is crucial forbreast cancer progression.
INTRODUCTION
One of the hallmarks of breast cancer is the hyperactivation of
ErbB receptor signaling. The human ErbB family of tyrosine
kinase (TK) receptors consists of four members: ErbB1 (EGFR/
HER1), the orphan receptor ErbB2 (HER2), ErbB3 (HER3), and
ErbB4 (HER4). The combinatorial dimerization of ErbB receptors
and their distinct coupling to signaling adaptors and effectors
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create a complex network of signaling events that, when dysre-
gulated, leads to uncontrolled growth and transformation (Hynes
and Lane, 2005; Citri et al., 2003; Yarden and Sliwkowski, 2001).
Aberrant expression of both ErbB receptors and their growth
factor activators is a common feature in the progression of
many cancers. In breast cancer, overexpression of ErbB2 and
ligands such as TGF-a (ErbB1 ligand) or heregulins/neuregulins
(ErbB3/ErbB4 ligands) occurs with high frequency (Yarden and
Sliwkowski, 2001). Genetic abnormalities associated with breast
cancer also include gain-of-functionmutations of ErbB effectors,
such as PI3KCA gene mutations or PTEN deletions (Bose et al.,
2002; Bachman et al., 2004).
It is well established that members of the Rho family of small
GTP-binding proteins mediate ErbB responses. Rac GTPases
have been widely implicated in actin cytoskeleton reorganiza-
tion, migration, mitogenesis, transformation, and metastasis
(Jaffe and Hall, 2005). Rac inhibition impairs breast cancer cell
motility and proliferation in response to EGFR and ErbB3
ligands (Yang et al., 2006, 2008; Wang et al., 2006). The activity
of Rac is mainly regulated by guanine nucleotide exchange
factors (Rac-GEFs), which activate Rac by promoting the
exchange of GDP by GTP; guanine nucleotide dissociation
inhibitors (GDIs), which limit the access of Rac to GEFs; and
GTPase-activating proteins (GAPs), which lead to Rac inactiva-
tion by accelerating its intrinsic GTPase activity (Jaffe and Hall,
2005). TK receptors can signal through multiple mechanisms to
Rac-GEFs. Most notably, many Rac-GEFs depend on the PI3K
product PIP3 for their redistribution to membranes and activa-
tion (Rossman et al., 2005). Unlike Ras proteins, gain-of-func-
tion mutations in Rho GTPases are uncommon in cancer;
however, there is ample evidence for hyperactivation of the
Rac pathway in human cancer. For example, Rac-GAPs are
downregulated in human breast tumors (Yang et al., 2005),
and aberrant overexpression of Rac-GEFs contributes to
cancer progression and metastasis in various cancer types,
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 877
PR P-Rex1
V3 VAV3
A2 ARHGEF2
B3 BCAR3
A7 ARHGEF7
SW SWAP70
TR TRIO
S1 SOS1
V2 VAV2
S2 SOS2
E2 ELMOD2
D6 DEF6
V1 VAV1
D1 DOCK1
F2 FARP2
A6 ARHGEF6
A2 ALS2
T1 Tiam1
GE GEFT
T2 Tiam2
R1 RASGRF1
Ka KALRN
R2 RASGRF2
D2 P-Rex2/DEPDC2
E1 ELMOD1
M2 MCF2
PR V3 V2 S1 TR E2 A7 D1 B3SWS2 F2 A2 ALGE T1 D6 T2 R2 A6 V1 R1 M2 Ka D2 E1
0
50
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PR V3 B3 A2SWA7 S1 V2 E2 TR S2 D1 F2 D6 A6 V1 AL T1 T2 GER1 R2 Ka M2 E1 D2
0
50
100
B3 V3SWS1 V2 TR A7 S2 A2 F2 E2 D1 AL M2 D6 T1 T2 R2PR A6 R1 E1 KaGE V1 D2
0
50
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T-47D
MCF-7
MCF-10A
Ex
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Ex
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HCC1143
HCC202
MDA-MB-415
T-47D
UACC893
MDA-MB-361
BT-483
MCF-7
BT-474
0 50 100
Type ER ErbB2 Cell line
- -
- -
- +
- -
- -
- -
- -
- +
- -
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+ -
- +
+ +
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N
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P-Rex1 mRNA
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C GEF RNAi
Figure 1. P-Rex1 Is Upregulated in Breast
Cancer Cell Lines and Mediates the Rac1
Activation by HRG
(A) Array profiling of Rac-GEFs and accessory Rac
activators in breast cell lines. Expression was
determined by Q-PCR in 96-well plate arrays and
normalized to GAPDH. Data were expressed as
mean ± SD (n = 3).
(B) Q-PCR analysis of P-Rex1 expression, normal-
ized to 18S, is shown. Data, expressed as fold
change relative to MCF-10A cells, are presented
as mean ± SD (n = 3). Expression of P-Rex1 by
western blot and PREX1 gene copy number in
each cell line are shown. ND, not determined.
(C) P-Rex1 levels were obtained from Affymetrix
microarray data of 17 cell lines. The tumor type
from which each cell line was originated as well
as the ErbB2 and ER status are listed. N, normal;
B, basal type; L, luminal type.
(D) Rac-GEFs and other Rac modulators were
depleted from T-47D cells using validated siRNA.
After 16 hr, cells were serum starved for 48 hr
and stimulated with HRG (10 ng/ml, 5 min). Left
panel: Rac-GTP levels were determined with
a PBD pull-down assay. P-Rex1 mRNA levels
were determined by RT-PCR. Middle panel:
depletion of Rac-GEFs as determined by western
blot. Right panel: depletion of Rac-GEFs as deter-
mined by RT-PCR. Two additional experiments
gave nearly identical results. C, control RNAi.
Molecular Cell
P-Rex1 in Breast Cancer
including breast cancer (Minard et al., 2004; Fernandez-Zapico
et al., 2005). The Rac effector Pak1 is also hyperactive in human
breast tumors and promotes antiestrogen resistance (Holm
et al., 2006; Balasenthil et al., 2004; Felekkis et al., 2005). Dis-
secting the cellular mechanisms leading to dysregulation of
the Rac pathway in breast cancer is therefore highly relevant.
Nonetheless, the relevance of Rac-GEFs in breast cancer
progression remains elusive.
Here, we report the identification of phosphatidylinositol-3,4,
5-trisphosphate-dependent Rac exchange factor-1 (P-Rex1)
as an essential mediator of ErbB receptor-driven Rac responses
in breast cancer models. Signals from ErbB receptors and
GPCRs converge on P-Rex1 to mediate Rac1 activation.
Notably, there is a remarkable upregulation of P-Rex1 in human
breast tumors, thus underscoring the potential prognostic and
therapeutic implications of these findings.
878 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
RESULTS
P-Rex1 Is Upregulated in BreastCancer Cell Lines and MediatesRac Activation by HeregulinRac1 plays essential roles in breast
cancer cell motility, proliferation, and
tumorigenesis (Jaffe and Hall, 2005;
Yang et al., 2006, 2008). We reported
that EGF and the ErbB3 ligand heregulin
b1 (HRG) strongly activate Rac1 in
MCF-7 and T-47D breast cancer cells
(Yang et al., 2006). EGF and HRG also
activate Rac1 in other breast cancer cell lines as well as in
immortalized MCF-10A mammary cells (Figure S1A). Rac1 acti-
vation by HRG is inhibited by ectopic expression of the Rac-GAP
b2-chimaerin (Figure S1B). Activation of Rac1 by HRG in MCF-7
and T-47D cells is sustained and sensitive to the PI3K inhibitor
wortmannin (Yang et al., 2006). We aimed to identify the Rac-
GEF(s) implicated in this response. To this end, we designed
an array to determine the relative expression of 26 Rac-GEFs
and known GEF accessory proteins in breast cancer models
(‘‘Rac-GEF array’’). Surprisingly, Q-PCR analysis in MCF-7 and
T-47D cells using this array revealed very high levels of
P-Rex1, a PI3K- and Gbg-regulated Rac-specific exchange
factor. In striking contrast, nontransformed MCF-10A cells
have negligible P-Rex1 expression (Figure 1A). A distinct pat-
tern of expression for Rac-GEFs was observed in MDA-MB-
453, MDA-MB-468, and MDA-MB-231 breast cancer cells
A
B
C
D
Figure 2. P-Rex1 Is Overexpressed in Human Breast Cancer
(A) mRNA expression level of P-Rex1 in the five different tumor types. P-Rex1
average expression is the lowest in basal-like tumors (left) and the highest in
Luminal B subtype (right). p < 10�21 basal versus nonbasal.
(B) Analysis from 108 tumor samples is shown, using a Pearson correlation
test.
(C) The level of P-Rex1 expression is denoted by +. +, weak staining; ++,
medium staining; +++, strong staining. The information on metastasis was
available from 98 patients. *p = 0.008 versus normal; **p < 0.005 versus
normal; ***p = 0.004 versus normal; #p = 0.045 versus nonmetastatic primary
tumors. Differences in the IHC staining of human breast cancer specimens
were analyzed with the Fisher’s exact (one-sided) tests.
(D) Representative IHC stainings from normal mammary tissue and a P-Rex1-
positive breast tumor.
Molecular Cell
P-Rex1 in Breast Cancer
(Figure S1C). P-Rex1 was originally characterized in neutrophils
as a mediator of chemoattractant-induced responses via Rac2,
including motility and ROS production (Welch et al., 2002,
2005; Dong et al., 2005). To our knowledge, information on this
GEF in cancer models is limited, including in breast cancer.
A comparative analysis of P-Rex1 mRNA levels by Q-PCR
showed that BT-474, MCF-7, and T-47D cells, which derived
from luminal breast cancers, have 100- to 1000-fold higher
P-Rex1mRNA levels thanMCF-10A cells. MDA-MB-231, a basal
breast-cancer-derived cell line, showed essentially no P-Rex1
expression. P-Rex1 was slightly elevated in MDA-MB-453 and
MDA-MB-468 cells. P-Rex1 can be readily detected in MCF-7,
BT-474, and T-47D cells bywestern blot (Figure 1B). An indepen-
dent microarray gene profiling analysis of 17 breast cell lines
validated these findings and identified additional breast cancer
cell lines with high P-Rex1 expression. Interestingly, P-Rex1 is
preferentially expressed at high levels in cell lines of luminal
origin, whereas none of the cell lines derived from basal breast
cancers have significant P-Rex1 expression (Figure 1C). The
PREX1 locus is located in chromosome 20q13.13, a region
commonly amplified in human breast tumors and cell lines
(Hodgson et al., 2003; Mastracci et al., 2006; Kallioniemi et al.,
1994; Jonsson et al., 2007). Analysis of PREX1 copy number in
genomic DNA from breast cancer cell lines revealed amplifica-
tion particularly in MCF-7 cells and also in BT-474 cells, but
not in T-47D cells or in other breast cancer cell lines (Figure 1B).
To determine if P-Rex1 is implicated in Rac1 activation by
HRG, Rac-GEFs with expression >10% relative to P-Rex1
were depleted from T-47D cells using validated RNAi (SMART-
pool ON-TARGETplus, Dharmacon). Each RNAi duplex depleted
the corresponding target by >80%, as revealed by western blot
(for those proteins that can be readily detected in T-47D cells) or
RT-PCR. Remarkably, P-Rex1 RNAi depletion essentially abol-
ished HRG-induced activation of Rac1. For all other Rac-GEFs,
inhibition was <20% (Figure 1D). These results were validated
using four different P-Rex1 RNAi duplex sequences in T-47D
cells (Figure S1D) and MCF-7 cells (data not shown), thereby
minimizing the chance of ‘‘off-target’’ effects. The high P-Rex1
expression levels in breast cancer cell lines and the lack of
redundancy with other Rac-GEFs in the HRG response were
unanticipated.
P-Rex1 Is Overexpressed in Human Breast TumorsNext, we decided to examine the expression of P-Rex1 in human
breast tumors. PCR analysis of a breast cancer cDNA panel
(OriGene) showed P-Rex1 upregulation in a considerable num-
ber of tumors (Figure S2A). As P-Rex1 is highly expressed in
neutrophils (Welch et al., 2005), we needed to rule out that the
P-Rex1 signal originated from neutrophils potentially present in
the tumors. However, there was no correlation between the
expression of P-Rex1 and the neutrophil marker myeloperoxi-
dase. Indeed, in most cases samples with high P-Rex1 expres-
sion had very low or undetectable myeloperoxidase levels.
To further establish the clinical significance of these prelimi-
nary findings we carried out a separate analysis of P-Rex1
expression using the NKI microarray data set that established
the intrinsic gene signature of 295 samples from patients (Fan
et al., 2006). Interestingly, P-Rex1 mRNA levels were particularly
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elevated in luminal breast cancer specimens, while the basal
breast cancer group showed very low P-Rex1 levels. There
were no statistical differences between luminal A and luminal B
subtypes (Figure 2A). A positive correlation between estrogen
receptor (ER) and P-Rex1 expression was found (Figure 2B), in
agreement with gene expression analysis from Oncomine (Fig-
ure S2B). P-Rex1 expression was higher in ErbB2-positive
tumors (Figure 2B). The P-Rex1-related isozyme P-Rex2a was
recently reported to cooperate with PI3K signaling, and its
expression levels correlate with activating mutations in the
PI3KCA gene (Fine et al., 2009). However, using the same
cohort, we could not find any significant association between
increased P-Rex1 expression and PI3KCA mutations (data not
shown).
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 879
A
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Anti-ErbB3 AbControl IgG
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induction
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Figure 3. P-Rex1 Is an Essential Mediator of Rac Responses by ErbB Ligands in Breast Cancer Cells
(A) T-47D cells were transfected with validated P-Rex1 siRNA (P) or control duplexes (C). After 16 hr, cells were serum starved for 48 hr and then stimulated with
HRG (10 ng/ml, 5 min), EGF (100 ng/ml, 1 min), or TGF-a (10 ng/ml, 2.5 min). Densitometric values of Rac-GTP levels (normalized to total Rac) are presented as
mean ± SD (n = 5). *p < 0.001 versus control RNAi.
(B) Translocation of endogenous P-Rex1 by HRG in MCF-7 cells. Wortmannin (1 mM), a blocking anti-ErbB3 antibody, or control IgG (10 mg/ml) was added 1 hr
before HRG stimulation. Similar results were observed in more than ten individual cells in at least three different experiments.
(C) P-Rex1 RNAi inhibits ruffle formation in T-47D cells stimulated with HRG. The percentage of cells with ruffles was determined in at least 200 cells. Results were
expressed as mean ± SEM of three independent experiments. *p < 0.01 versus control RNAi (C).
Molecular Cell
P-Rex1 in Breast Cancer
880 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
Molecular Cell
P-Rex1 in Breast Cancer
To determine if P-Rex1 upregulation also occurs in other
cancer types, we used a commercial multicancer array (Ori-
Gene). Notably, P-Rex1 upregulation could be observed in other
tumor types, particularly thyroid, kidney, and prostate cancer.
Although at lower frequency, some cases of high P-Rex1 levels
were observed in other tumors such as esophageal, bladder,
colon, endometrial, and pancreatic cancer (Figure S2C).
Next, we screened paraffin-embedded tissue sections from
10 normal and 165 breast cancer patients by immunohisto-
chemistry (IHC). In agreement with the cDNA array, P-Rex1
was essentially undetectable in normal mammary samples.
On the other hand, P-Rex1 was detected in 58% of the tumor
specimens analyzed (Figure 2C). Importantly, P-Rex1 staining
was found specifically in the tumor cells, and no appreciable
P-Rex1 staining could be observed in the stroma or in normal
ducts (Figure 2D). While we observed a systematic increase
in P-Rex1 levels as a function of stage (Figure 2C), it is not
statistically significant, as determined by a sum-of-ranks anal-
ysis (p = 0.103). Nonetheless, P-Rex1 expression was statisti-
cally higher in primary tumors from patients that underwent
metastasis relative to those that did not (65% versus 46%,
p = 0.045). Analysis in lymph nodes from breast cancer patients
showed that 67% were P-Rex1 positive. Therefore, P-Rex1
upregulation occurs in primary breast tumors and their
metastases.
P-Rex1 Mediates ErbB Ligand-Driven Migrationand GrowthWe examined whether P-Rex1 could mediate Rac1 activation
downstream of EGFR. Like HRG, the EGFR ligands EGF and
TGF-a caused a marked elevation in Rac-GTP levels in T-47D
cells, which was reduced by silencing P-Rex1 expression using
RNAi (92%, 84%, and 88% inhibition for HRG, EGF, and TGF-a,
respectively) (Figure 3A). Similar inhibition (90%) was observed
in MCF-7 cells (data not shown). Whereas wortmannin impairs
Rac1 activation by ErbB ligands (Yang et al., 2006), activation
of the PI3K effector Akt1 remained unchanged both in P-Rex1-
depleted T-47D cells (Figure 3A) and MCF-7 cells (data not
shown), arguing for a PI3K-dependent but Akt-independent
mechanism for P-Rex1/Rac1 activation.
As membrane association is a prerequisite for the activation of
various Rac-GEFs, including P-Rex1 (Zhao et al., 2007; Ross-
man et al., 2005), we asked whether HRG could relocalize
P-Rex1 in breast cancer cells. Endogenous P-Rex1 distributed
diffusely throughout the cytoplasm in serum-starved MCF-7
cells (Figure 3B). HRG caused a pronounced peripheral translo-
cation of P-Rex1, particularly to membrane ruffles. Consistent
(D) Stable depletion of P-Rex1 in T-47D cells impairs Rac activation. Stably P-Rex
(#1–4). A representative Rac-GTP pull-down assay in response to HRG is shown
densitometry, is expressed as mean ± SD (n = 3).
(E) Impaired motility in P-Rex1-deficient T-47D cells, as determined with a Boyden
absence of HRG and expressed as mean ± SD of triplicate measurements. Two ad
virus (C) with HRG.
(F) Colony formation assays in P-Rex1-depleted T-47D cells. Experiments were
control cells growing in the absence of HRG. Data are expressed as mean ± SE
(G) MCF-10A cells were transfected with mammalian vectors encoding either P
starved for 48 hr and Rac-GTP levels were determined.
Molec
with our previous data that ErbB3, but not ErbB4, mediates
Rac1 activation by HRG (Yang et al., 2006), a blocking anti-
ErbB3 antibody prevented P-Rex1 translocation by HRG,
whereas control IgG (Figure 3B) or an anti-ErbB4 antibody
(data not shown) did not. P-Rex1 translocation by HRG was
inhibited by wortmannin, as expected from the PIP3 dependency
for P-Rex1 activation (Zhao et al., 2007; Barber et al., 2007).
Using GFP-fused P-Rex1 mutants, we found that deletion of
the DH-PH domain tandem abolished translocation by HRG,
which is consistent with the requirement of these domains for
P-Rex1 activation (Barber et al., 2007; Hill et al., 2005). The
DH-PH domain was sufficient to translocate to the cell periphery
in response to HRG (Figure S3A).
HRG causes a marked reorganization of the cytoskeleton and
induces a motile response in breast cancer cells in a PI3K-Rac1-
dependent manner (Adam et al., 1998; Yang et al., 2006). Inter-
estingly, ruffle formation in response to HRG was markedly
reduced in P-Rex1-depleted cells (Figure 3C). To determine
if P-Rex1 mediates motile responses, we generated stably
P-Rex1-depleted polyclonal T-47D andMCF-7 cell lines (Figures
3D and S3B) using four different P-Rex1 shRNA lentiviruses
(puromycin selected). Consistent with our transient knockdown
studies, stably P-Rex1-depleted cell lines were defective in
Rac1 activation (Figures 3D and S3B). Rac activation by HRG
can be rescued by an RNAi-insensitive P-Rex1 mutant (Fig-
ure S3C). Although no changes in the adhesive properties of
the cells were detected (data not shown), we observed a signifi-
cant impairment of migration in response to HRG, as determined
with a Boyden chamber (Figures 3E and S3D).
HRG has been implicated in breast cancer cell transformation
(Atlas et al., 2003). T-47D cells form colonies in soft agar, and
colony formation was significantly enhanced by HRG. In sharp
contrast, the ability of P-Rex1-depleted cells to grow in soft
agar in response to HRG was severely affected (Figure 3F),
thus arguing for a role of P-Rex1 in ErbB receptor-mediated
anchorage-independent growth. Importantly, P-Rex1 shRNA
depletion did not significantly affect the expression of other
Rac-GEFs (Figure S3E); therefore, the effects could be attributed
to specific P-Rex1 depletion.
Next, we examined the effect of overexpressing P-Rex1 in
MCF-10A cells, which express very low P-Rex1 levels. MCF-
10A cells were transfected with mammalian expression vectors
encoding P-Rex1, a DH-PH-deleted P-Rex1 mutant, or vector
alone. Overexpression of P-Rex1 elevated basal Rac-GTP levels
in MCF-10A cells (2.2-fold). On the other hand, expression
of DDH-PH-P-Rex1 did not change basal Rac-GTP levels
(Figure 3G).
1-depleted T-47D cells were generated using four different shRNA lentiviruses
. Fold induction in Rac-GTP levels normalized to total Rac, as determined by
chamber. Results are presented as fold increase relative to control cells in the
ditional experiments gave similar results. *p < 0.05 versus control shRNA lenti-
performed in quadruplicate. Results are presented as percentage relative to
M (n = 3). *p < 0.05 versus control RNAi (C) with HRG.
-Rex1 (WT), DDH-PH-P-Rex1, or empty vector. After 16 hr, cells were serum
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 881
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Rac-GTP
Total Rac
P-Rex1
Vinculin
MDA-MB-361BT-474 HCC1419
- C P-Rex1
- + - + - +
- C P-Rex1
- + - + - +
- C P-Rex1
- + - + - +
Total Rac
Rac GTP
- + - + ErbB2
ControlP-Rex1-
depleted
ErbB2
P-Rex1
Actin
- + - + ErbB2
ControlP-Rex1-
depleted
ErbB2
P-Rex1
Actin
Vinculin
EGFR
HER2
P-Rex1
C EGFR ErbB2 RNAi
EGFR
HER2
P-Rex1
C EGFR ErbB2 RNAi
0
1
2
3
4
5
6
7
8
9
RNAi - C P - C P - C P
Rac a
ctivatio
n
(fo
ld-in
cre
ase
)
BT- HCC MDA-
474 1419 MB-361
***
0
1
2
3
4
5
6
7
8
9
RNAi - C P - C P - C P
Rac a
ctivatio
n
(fo
ld-in
cre
ase
)
BT- HCC MDA-
474 1419 MB-361
***
Days
Tu
mo
r v
olu
me
(m
m3)
0 10
0
500
1000
1500
2000
Control shRNA
Parental
P-Rex1 shRNA #3
P-Rex1 shRNA #4
20 30 40
Parental
Contr
ol shRNA
P-R
ex1 s
hRNA
#4
P-R
ex1 s
hRNA
#3
Days
Tu
mo
r v
olu
me
(m
m3)
0 10
0
500
1000
1500
2000
Control shRNA
Parental
P-Rex1 shRNA #3
P-Rex1 shRNA #4
20 30 40
Parental
Contr
ol shRNA
P-R
ex1 s
hRNA
#4
P-R
ex1 s
hRNA
#3
0
5
10
15
Mig
ra
tio
n
(fo
ld-in
cre
as
e)
HRG - + - + - + - + - +
C #1 #2 #3 #4
P-Rex1 shRNA
* ***
0
5
10
15
Mig
ra
tio
n
(fo
ld-in
cre
as
e)
HRG - + - + - + - + - +
C #1 #2 #3 #4
P-Rex1 shRNA
* ***
5.4±0.1 1.4±0.2 1.5±0.3 1.9±0.5 1.7±0.3 Fold-
induction
5.4±0.1 1.4±0.2 1.5±0.3 1.9±0.5 1.7±0.3 Fold-
induction
C #1 #2 #3 #4
- + - + - + - + - + HRG
Rac-GTP
Total Rac
P-Rex1 shRNA
Vinculin
P-Rex1
Tumor growth after inoculation of BT-474
into the mammary fat pad of nude mice
Group n Incidence Tumor volume
(%) (mm3)
Control 15 7/15 (47%) 252 ± 113
P-Rex1 10 0/10 (0%) 0shRNA #3
G
Figure 4. P-Rex1 Is Required for ErbB2-Mediated Migration and Tumorigenesis
(A) T-47D cells (P-Rex1-depleted #3 and control from Figure 3E) were transfected with pcDNA3-ErbB2 or empty vector. After 24 hr, cells were serum starved for
48 hr and Rac-GTP levels were determined.
(B) BT-474, HCC1419, or MDA-MB-361 cells were transfected with validated P-Rex1 siRNA (P) or control duplexes (C). After 16 hr, cells were serum starved
for 48 hr and then stimulated with HRG (10 ng/ml, 5 min). Densitometric values of Rac-GTP levels (normalized to total Rac) are presented as mean ± SD (n = 3).
*p < 0.001 versus control RNAi.
Molecular Cell
P-Rex1 in Breast Cancer
882 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
Molecular Cell
P-Rex1 in Breast Cancer
P-Rex1 Is Required for ErbB2-Mediated Migration andTumorigenesisErbB2 overexpression is one of the most common genetic alter-
ations in breast cancer (Hynes and Lane, 2005), and Rac1 is
implicated in ErbB2-mediated mitogenesis and motility (Yang
et al., 2005, 2006; Lee et al., 2000; Ueda et al., 2004). As
P-Rex1 is prominently overexpressed in ErbB2-positive tumors,
and responses by HRG are mediated by ErbB3/ErbB2 dimers
(Citri et al., 2003), we speculated that P-Rex1 could be impli-
cated in ErbB2-driven activation of Rac1. As a first approach
to address this issue, we overexpressed ErbB2 both in control
and P-Rex1-depleted T-47D cells. As expected, ErbB2 overex-
pression led to elevated Rac-GTP levels in control T-47D cells.
However, this effect was not observed in P-Rex1-deficient cells
despite the similar levels of ErbB2 overexpression in both
cell lines (Figure 4A). Similar results were found in P-Rex-1-
depleted MCF-7/ErbB2 cells (data not shown). Next, we took
advantage of BT-474 cells, an ErbB2-overexpressing cell line
that expresses very high P-Rex1 levels (see Figures 1B and
1C). Transient RNAi depletion of P-Rex1 in these cells reduced
Rac1 activation by HRG by 88% (Figure 4B). Similar results
were observed in two other ErbB2-positive cell lines that express
very high P-Rex1 levels (HCC1419 andMDA-MB-361 cells, 90%
and 93% inhibition, respectively). We then generated BT-474 cell
lines in which P-Rex1 was stably silenced using shRNA lentivi-
ruses. In agreement with the transient depletion experiments,
these lines also had a defective Rac1 activation in response to
HRG (80%–90% inhibition) (Figure 4C). Moreover, migration
induced by HRG was impaired in P-Rex1-depleted BT-474 cells
(Figure 4D) or inP-Rex1-depletedMCF-7/ErbB2cells (FigureS4).
Neither overexpressing ErbB2 in T-47D cells (Figure 4A) nor
silencing ErbB2 or EGFR from BT-474 cells (Figure 4E) altered
P-Rex1 expression levels, suggesting that these receptors do
not modulate P-Rex1 expression.
As Rac1 is implicated in ErbB2 signaling (Yang et al., 2005,
2006; Lee et al., 2000), we next examined the relevance of
P-Rex1 overexpression in tumorigenesis. To this end, we as-
sessed the effect of P-Rex1 depletion on the growth of BT-474
xenografts in nude mice. Herceptin blocks the ability of BT-474
cells to form tumors in nude mice (Moulder et al., 2001), arguing
that the tumorigenic capacity of these cells is dependent
on ErbB2 signals. Athymic nude mice were injected s.c. with
BT-474 parental cells, control lentivirus-infected BT-474 cells,
or two P-Rex1-depleted BT-474 clones. While parental and
control BT-474 cells readily formed tumors in nude mice, the
tumorigenic ability of P-Rex1-depleted BT-474 cells was mark-
edly impaired (Figure 4F). We also compared the ability of control
(C) Stable depletion of P-Rex1 in BT-474 cells impairs Rac activation. Stably P-R
ruses (#1–4). A representative Rac-GTP pull-down assay in response to HRG is s
by densitometry, is expressed as mean ± SD (n = 3).
(D) Impaired motility in P-Rex1-deficient BT-474 cells, as determined with a Boyd
the absence of HRG, and expressed as mean ± SD of triplicate measurements. Tw
lentivirus (C) with HRG.
(E) Effect of EGFR or ErbB2 depletion on P-Rex1 levels, determined 72 hr after t
(F) Reduced tumorigenic potential of P-Rex1-depletedBT-474 cells in nudemice (
shRNA. Inset: representative tumors.
(G) Tumor growth in nude mice was monitored for 70 days after injection of contro
into the mammary fat pad. The tumor volume shown is that at the end of the exp
Molec
BT-474 cells and clone #3 to promote tumor formation in an
orthotopic model. As shown in Figure 4G, 47% of nude mice
developed mammary tumors upon inoculation in the mammary
fat pad of BT-474 cells transduced with control shRNA lentivirus,
whereas none of the mice developed tumors when injected
with P-Rex1-depleted BT-474 cells. Taken together, our results
suggest that P-Rex1mediates ErbB2-dependent Rac responses
both in cultured breast cancer cells as well as in vivo.
Gbg Subunits and PI3Kg Mediate the Activationof the P-Rex1/Rac1 Pathway by ErbB LigandsA unique feature of P-Rex1 is that it is dually regulated, activated
by the PI3K product PIP3 and Gbg subunits released upon acti-
vation of Gi-coupled receptors. Gbg alone causes partial activa-
tion of P-Rex1, and an additional PIP3 input is required for
membrane targeting and full activation of this Rac-GEF (Welch
et al., 2002; Barber et al., 2007; Mayeenuddin et al., 2006). While
ErbB and other TK receptors can relay signals via transactivation
of Gi-coupled receptors (Luttrell et al., 1995; Johnson et al.,
1986; Stanton et al., 1991; Hobson et al., 2001), a potential impli-
cation of GPCRs in the activation of P-Rex1/Rac1 signaling by
ErbB receptors has not been established. First, we used
pertussis toxin (PTX) to inhibit Gbg release from heterotrimeric
Gi proteins. Remarkably, Rac activation by HRG in PTX-treated
T-47D and MCF-7 cells was significantly reduced (Figure 5A).
PTX also inhibited the activation of Rac1 by EGF (data not
shown). Furthermore, PTX treatment reduced MCF-7 cell migra-
tion in response to HRG (see Figure 5C). These results implicate
Gbg subunits in the activation of Rac1 by ErbB receptors in
breast cancer cells expressing high P-Rex1 levels and argue
for a potential transactivation via Gi-coupled receptors for sig-
naling to P-Rex1/Rac1.
Gbg subunits released upon activation of Gi-coupled recep-
tors are known to directly activate PI3Kg, a type Ib PI3K
(Andrews et al., 2007). To determine a potential implication of
PI3Kg, we used the PI3Kg inhibitor 5-quinoxalin-6-ylmethy-
lene-thiazolidine-2,4-dione. This inhibitor dose-dependently
reduced HRG-induced Rac1 activation (Figure 5B). Moreover,
MCF-7 cell migration in response to HRG was greatly reduced
by the PI3Kg inhibitor and to a degree similar to PTX (Figure 5C).
As a second approach, we stably knocked down the catalytic
subunit of PI3Kg (p110g) from MCF-7 cells using two different
shRNA constructs. These two cell lines showed deficient activa-
tion of Rac1 by HRG (�60% inhibition) (Figure 5D) as well as
a reduced migratory response (Figure 5E). Thus, Rac1 activation
by HRG in P-Rex1-expressing breast cancer cells is mediated by
PI3Kg. PTX treatment was unable to reduce further Rac-GTP
ex1-depleted BT-474 cells were generated using four different shRNA lentivi-
hown. Fold induction in Rac-GTP levels normalized to total Rac, as determined
en chamber. Results are presented as fold increase relative to control cells in
o additional experiments gave similar results. *p < 0.001 versus control shRNA
ransfection of RNAi duplexes. Duplicate samples are shown.
tenmice/group). Results are expressed asmean ±SD. *p < 0.001 versus control
l (control shRNA lentivirus) or P-Rex1-deficient BT-474 cells (stable cell line #3)
eriment. Results were expressed as mean ± SD.
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 883
A
C
B
E
- - -
Rac-GTP
Total Rac
- + + - + + HRG
F
D
C #1 #2
HRG- + - + - +
Rac-GTP
Total Rac
PI3K
Vinculin
PI3K
shRNAC #1 #2
HRG- + - + - +
Rac-GTP
Total Rac
PI3K
Vinculin
PI3K
shRNA
+- ++++ +
Rac-GTP
Total Rac
Vinculin
0.0
1
0.0
3
0.1
0.3
1 PI3k inh ( M)
HRG
- -
+- ++++ +
Rac-GTP
Total Rac
Vinculin
0.0
1
0.0
3
0.1
0.3
1 PI3k inh ( M)
HRG
- -
0 0 0.01 0.03 0.1 0.3 1
0
20
40
60
80
100
Pi3k inh ( M)
HRG - + ++ + ++
% R
esp
on
se
Mig
ra
tio
n
(fo
ld
-in
crease)
HRG - + - + - +
C #1 #2
PI3K shRNA
* *
0
1
2
3
- + - + HRG
- - + + PTX
Rac-GTP
Total Rac
MCF-7
T-47D
Rac-GTP
Total Rac
0
1
2
3
4
5
Mig
ra
tio
n
(fo
ld
-in
crease)
HRG - + - + - +
PI3K
inh
-
**
PTX 7.2±0.5 3.2±0.8 3.6±0.1Fold-
induction
% R
esp
on
se
0
2 0
4 0
6 0
8 0
1 0 0
2 0
4 0
PTX - + - +
T-47D MCF-7
0
2 0
4 0
6 0
8 0
1 0 0
2 0
4 0
PTX - + - +
T-47D MCF-7
**
Figure 5. Involvement of Gbg Subunits and PI3Kg in Rac Activation by HRG
(A) Serum-starved T-47D and MCF-7 cells were treated with PTX (100 ng/ml, 24 hr) and then stimulated with HRG (10 ng/ml, 5 min). Top panel: representative
experiments. Bottom panel: densitometric analysis. Data are expressed as % of the HRG response in the absence of PTX and presented as mean ± SD (n = 3).
*p < 0.05 versus control.
(B) A PI3Kg inhibitor reduces Rac1 activation by HRG in MCF-7 cells. Densitometric analysis of the data (Rac-GTP normalized to total Rac1) is presented relative
to the effect in the absence of PI3Kg inhibitor.
(C) Inhibition of HRG-inducedMCF-7 cell motility by the PI3Kg inhibitor (1 mM) or PTX. Data from triplicate samples are presented as mean ± SD. *p < 0.05 versus
HRG (control). Results are presented as fold increase relative to control cells in the absence of stimuli and expressed as mean ± SD of triplicate measurements.
Two additional experiments gave similar results. *p < 0.05 versus control (C) with HRG.
(D) Stable depletion of PI3Kg fromMCF-7 cells impairs Rac1 activation. MCF-7 cells were transfected with two different plasmids encoding PI3Kg shRNA (#1 and
#2) or a shRNA plasmid control (C) and selected with puromycin. Rac-GTP levels in response to HRG (10 ng/ml, 5 min) are shown.
(E) Impaired cell motility in PI3Kg-depleted cells. Results are presented as fold increase relative to control cells in the absence of stimuli and expressed asmean ±
SD of triplicate measurements. Data from triplicates are presented as mean ± SD. Two additional experiments gave similar results. *p < 0.001 versus control (C)
with HRG.
(F) Serum-starved MCF-10A cells were treated with wortmannin (1 mM, 1 hr), the PI3Kg inhibitor (1 mM, 1 hr), or PTX (100 ng/ml, 24 hr), and Rac-GTP levels were
determined after stimulation with HRG.
Molecular Cell
P-Rex1 in Breast Cancer
884 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
Molecular Cell
P-Rex1 in Breast Cancer
levels in PI3Kg-depleted cells (Figure S5), arguing that Gbg and
PI3Kg are in the same pathway.
As nontransformed MCF-10A cells express very low P-Rex1
levels (see Figures 1B and 1C), we reasoned that Rac1 activation
by HRG in these cells should be insensitive to PTX or PI3Kg inhi-
bition. Remarkably, and in sharp contrast to MCF-7 cells, neither
PTX nor the PI3Kg inhibitor reduced Rac1 activation by HRG in
MCF-10A cells, even though wortmannin effectively impaired
this response (Figure 5F). Thus, in P-Rex1-deficient MCF-10A
cells, Rac1 activation by HRG is Gbg and PI3Kg independent.
These results argue for a differential utilization of Rac-GEFs in
P-Rex1-positive and P-Rex1-negative cells.
CXCR4 and EGFR Are Implicated in Rac1 Activationby HRG in Breast Cancer CellsNumerous studies have implicated Gi-coupled-receptors in
growth factor responses, including Rac activation and motility
(Johnson et al., 1986; Stanton et al., 1991; Luttrell et al., 1995;
Hobson et al., 2001). Most recently, studies in MDA-MB-435
cells (later reclassified to a melanoma cell line) showed that
ErbB2-induced migration and metastasis are mediated by
CXCR4, a Gi-coupled receptor for the chemokine SDF-1a/
CXCL12. Both SDF-1a and its receptor are highly expressed in
breast tumors and have been widely implicated in the progres-
sion of breast cancer. Moreover, there is a positive correlation
between CXCR4 and ErbB2 in human breast tumors (Muller
et al., 2001; Akekawatchai et al., 2005; Li et al., 2004). SDF-1a
caused a strong activation of Rac1 in MCF-7 cells, and conse-
quently, it induced a migratory response (Figure 6A). Rac1 acti-
vation by SDF-1a was dose-dependently reduced by the
PI3Kg inhibitor, and as expected, the CXCR4 inhibitor AMD-
3100 blocked the effect of the CXCR4 agonist (Figure 6A, left
panel). SDF-1a also stimulated MCF-7 cell migration, and this
effect was blocked by the PI3Kg inhibitor as well as by PTX (Fig-
ure 6A, right panel). Moreover, migration induced by SDF-1awas
abolished in the twoPI3Kg-depletedMCF-7 cell lines (Figure 6B).
Notably, when P-Rex-1 expression was silenced using RNAi,
both SDF-1a-induced Rac1 activation (Figure 6C, left panel)
andmigration (Figure 6C, right panel) were essentially abolished.
Thus, SDF-1a-induced activation of Rac1 in breast cancer cells
is mediated by P-Rex1.
In order to determine if CXCR4 is implicated in ErbB-driven
activation of Rac1, we silenced CXCR4 expression in MCF-7
cells using RNAi. Rac1 activation (Figure 6D, left panel) and
motility (Figure 6D, middle panel) by HRG were significantly
reduced (55% and 61% inhibition, respectively) in CXCR4-
depleted cells. Unlike the HRG effect, migration induced by
SDF-1awas essentially impaired (91% inhibition) in CXCR4-defi-
cient cells (Figure 6D, right panel). As Rac1 activation by HRG is
rapid (Yang et al., 2006), we reasoned that most likely it does not
involve the autocrine secretion of SDF-1a. In fact, blockade of
CXCR4 with the specific antagonist AMD-3100 was unable to
prevent HRG-induced Rac1 activation (Figure 6E, left panel) or
migration (Figure 6E, middle panel), indicating that these effects
are independent of ligand activation of CXCR4, whereas the
CXCR4 antagonist abolished SDF-1a-induced migration (Fig-
ure 6E, right panel). Moreover, AMD-3100 had no effect on
HRG-induced relocalization of endogenous P-Rex1 in MCF-7
Molec
cells (Figure 6F). In contrast, SDF-1a-induced translocation of
P-Rex-1 was effectively inhibited by AMD-3100. Both HRG-
and SDF-1a-induced translocation of P-Rex1 were impaired by
wortmannin or the PI3Kg inhibitor (Figure 6F), or by PI3Kg
RNAi depletion (Figure S6), whereas only the HRG effect was
blocked by an anti-ErbB3 antibody (Figure 6F). The lack of
involvement of SDF-1a was supported by the fact that AMD-
3100 was unable to inhibit BT-474 tumor growth in nude mice
(Figure 6G).
We then examined the activation status of CXCR4 in response
to HRG using phosphospecific antibodies against Ser324/325
and Ser330, residues that become phosphorylated in response
to SDF-1a and regulate CXCR4 signaling and trafficking
(Marchese and Benovic, 2001; Busillo et al., 2010). Notably,
a significant elevation in Ser324/325- and Ser330-CXCR4 phos-
phorylation was detected upon HRG treatment in T-47D cells
(Figure 7A), which was similar in magnitude to that induced by
SDF-1a (data not shown). Phosphorylation of these sites in
CXCR4 was also observed in response to EGF (data not shown).
It is known that CXCR4 becomes tyrosine phosphorylated upon
activation with SDF-1a (Vila-Coro et al., 1999). We found that in
MCF-7 cells, CXCR4 becomes tyrosine phosphorylated in
response to either SDF-1a or HRG, and only the effect of
SDF-1a was sensitive to AMD-3100 (Figure 7B). Since phos-
phorylated (activated) CXCR4 binds arrestins, we also examined
whether activation of ErbB3 receptors promotes binding of
arrestin to CXCR4 in MCF-7 cells using a bioluminescence
resonance energy transfer (BRET) approach (Busillo et al.,
2010). Remarkably, HRG promotes the association of arrestin2
to CXCR4 in a time-dependent manner and with a magnitude
similar to that observed with SDF-1a (Figure 7C).
In a previous study, we established that EGFR was required
for Rac activation by HRG in T-47D and MCF-7 cells (Yang
et al., 2006). Similar results were observed in BT-474 cells (Fig-
ure S7). To determine whether EGFR mediates CXCR4 phos-
phorylation, we used the EGFR inhibitor AG1478 and found
that it prevented phosphorylation of CXCR4 in Ser324/325 and
Ser330. AMD-3100 blocked CXCR4 serine phosphorylation
induced by SDF-1a but not by HRG (Figure 7D, left panel).
AG1478 also blocked CXCR4 tyrosine phosphorylation by
HRG to the same extent as an anti-ErbB3 blocking antibody
(Figure 7D, middle panel). Moreover, EGFR RNAi depletion
from BT-474 cells also impaired CXCR4 tyrosine phosphoryla-
tion (Figure 7D, right panel). These results implicate EGFR in
the transactivation of CXCR4 by stimulation of ErbB3 receptors.
Our results not only support the concept that CXCR4 becomes
activated in response to ErbB ligands independently of SDF-1a
but also strongly argue for the utilization of a CXCR4-dependent
pathway in the activation of P-Rex1/Rac1 by ErbB receptors.
DISCUSSION
P-Rex1 as a Mediator of Rac1 Activation by ErbBReceptors in Breast Cancer CellsOur studies provide evidence that the PI3K- andGbg-dependent
Rac-GEF P-Rex1 is an essential mediator of Rac1 activation and
migration in breast cancer cells by ErbB receptors. While many
GEFs for Rho GTPases were originally identified as oncogenes,
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 885
A
C
E
D
F G
B
Figure 6. CXCR4 Mediates Rac1 Activation by HRG
(A) Left panel: Rac-GTP levels in response to SDF-1a (10 nM, 5 min) in the presence of the PI3Kg inhibitor (0.1–1 mM) or AMD-3100 (10 mg/ml, 1 hr). Right panel:
Migration in response to SDF-1a (10 nM) was determined using a Boyden chamber in MCF-7 cells treated with the PI3Kg inhibitor (1 mM). Data from triplicates
(fold increase relative to control cells in the absence of stimuli) are presented as mean ± SEM of three independent experiments. *p < 0.001 versus controls with
SDF-1a.
Molecular Cell
P-Rex1 in Breast Cancer
886 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
Molecular Cell
P-Rex1 in Breast Cancer
they often act in cancer cells as transducers of upstream dysre-
gulated inputs. Persistent activation of Rac may arise as a con-
sequence of aberrant TK receptor hyperactivation or genetic
alterations leading to PI3K hyperactivation (PI3KCA mutations,
Pten deficiency, Ras mutations). Enhanced Rac activation due
to overexpression or hyperactivation of Rac-GEFs is also fre-
quent in cancer, as established for Vav1 in pancreatic cancer
and for Vav2 in head and neck squamous carcinoma (Fernan-
dez-Zapico et al., 2005; Patel et al., 2007).
The role of Rac-GEFs in human breast cancer progression
remains poorly understood. The identification of P-Rex1 as
a mediator of Rac1 activation in breast cancer cells was unantic-
ipated. P-Rex1 was originally identified in neutrophils (Dong
et al., 2005; Zhao et al., 2007; Welch et al., 2002, 2005), and
although some evidence suggested that P-Rex1 activation could
be dependent on TK activity (Zhao et al., 2007; Qin et al., 2009;
Yoshizawa et al., 2005), this GEF has been predominantly char-
acterized as a GPCR effector. Based on the limited information
on Rac-GEFs in breast cancer models, our original prediction
was that Tiam1, Trio, or Vav isoforms would have played a signif-
icant role in Rac1 activation by ErbB ligands. For example, Vav3
is upregulated in human breast tumors and mediates estrogen
mitogenic responses in breast cancer cells (Lee et al., 2008).
Interestingly, P-Rex1 RNAi does not affect Akt activation in
breast cancer cells. Very recently, it has been shown that the
P-Rex1 related isoform P-Rex2a, but not P-Rex1, inhibits Pten
phosphatase activity and consequently stimulates Akt phos-
phorylation and cell growth in breast cancer cells (Fine et al.,
2009). Thus, different P-Rex isoforms may be implicated in
breast cancer progression through remarkably distinct mecha-
nisms. Rac-GEF utilization may be exquisitely controlled by the
nature of the oncogenic input and the relative expression of
exchange factors in different cancer cell lines.
While the functional relationship between ErbB2 and Rac1
activation has not been fully investigated to date, the PI3K/
Rac/Pak1 axis plays an important role in actin cytoskeleton reor-
ganization in MCF-10A cells ectopically overexpressing ErbB2.
In this case, Vav2 seems to be critical for Rac activation, which
is consistent with the high expression of this GEF in MCF-10A
cells (see Figure 1A) (Ueda et al., 2004; Wang et al., 2006).
Remarkably, we found that P-Rex1 knockdown suppresses
Rac1 activation and motility in cell lines overexpressing ErbB2.
(B) Impaired migration of PI3Kg-depleted MCF-7 cells (see Figure 5) in response t
the absence of stimuli) are presented as mean ± SEM of three independent expe
(C) P-Rex1 mediates SDF-1a effects. Left panel: serum-starved P-Rex1-depleted
(10 nM, 5min), and Rac-GTP levels were then determined. Right panel: P-Rex1-de
control cells. Data from triplicates (fold increase relative to control cells in the abse
*p < 0.001 versus control with SDF-1a.
(D) Left panel: MCF-7 cells were transfected with either CXCR4 or control siRNA du
(10 ng/ml, 5 min), and Rac-GTP levels determined. Middle panel: CXCR4 deplet
motility in response to SDF-1a. Data from triplicates (fold increase relative to con
pendent experiments. *p < 0.001 versus control with HRG or SDF-1a.
(E) AMD-3100 does not inhibit Rac1 activation (left panel) or migration (middle pa
The concentration of AMD for the migration experiments was 10 mg/ml. Data from
sented as mean ± SEM of three independent experiments. *p < 0.01 versus vehi
(F) Translocation of endogenous P-Rex1 in MCF-7 cells after stimulation with eit
wortmannin (1 mM), the PI3Kg inhibitor (1 mM), AMD-3100 (10 mg/ml), or an anti-E
cells in three different experiments.
(G) The CXCR4 inhibitor AMD-3100 does not affect tumorigenicity of BT-474 cel
Molec
Moreover, silencing P-Rex1 from BT-474 cells profoundly
affects their ability to form tumors in nude mice, a finding of
particular relevance, taking into consideration that P-Rex1 is
preferentially expressed in ErbB2-positive tumors.
Overexpression of P-Rex1 in Human Breast CancerOur study revealed that P-Rex1 is highly expressed in a large
fraction of human breast tumors, preferentially the luminal
subtype, which are predominantly ER positive. Aberrant expres-
sion of P-Rex1 was detected in all stages of the disease,
although there is a tendency to increase with stage. The basal
subtype is characterized by a high abundance of triple-negative
(ER-, PR-, and ErbB2-negative) tumors and mostly does not
express P-Rex1. Although a significant correlation was also
foundwith ErbB2, it is not as strong aswith ER, possibly a conse-
quence of the relatively modest expression in the HER2+/ER�subtype. Notably, the rate of P-Rex1-positive tumors was higher
in patients that developed metastasis, and approximately two-
thirds of lymph node metastases were found to be P-Rex1 posi-
tive. A recent study showed that P-Rex1 is upregulated in the
metastatic prostate cancer cell line PC3 relative to nonmeta-
static LNCaP and CWR22RV1 cells, and in a small patient
sample analysis, higher expression was found in prostatemetas-
tasis relative to the corresponding primary prostate tumors (Qin
et al., 2009). It is conceivable that P-Rex1 overexpression con-
fers a motile advantage required for metastatic dissemination.
The human P-Rex1 gene (PREX1) is located in chromosome
20q.13. Amplification of this region occurs in 8%–29% of breast
tumors and correlates with poor clinical prognosis (Hodgson
et al., 2003; Mastracci et al., 2006; Kallioniemi et al., 1994; Jons-
son et al., 2007). As only a subset of breast cancer cell lines
present PREX1 gene amplification, alternative transcriptional,
translational, or posttranslational means may contribute to
P-Rex1 protein upregulation. Curiously, in silico analysis of the
P-Rex1 promoter revealed multiple ER-responsive elements,
and in addition, estradiol-mediated proliferation and Rac activa-
tion are markedly impaired in P-Rex1-deficient T-47D cells
(M.S.S. and M.G.K., unpublished data). Estrogen promotes
breast cancer proliferation via EGFR and CXCR4 transactivation
(Pattarozzi et al., 2008), thus suggesting that P-Rex1 may inte-
grate TK and GPCR inputs triggered by the estrogen response.
o SDF-1a (10 nM). Data from triplicates (fold increase relative to control cells in
riments. *p < 0.001 versus controls with SDF-1a.
MCF-7 cell line #3 or control cells (from Figure 3) were stimulated with SDF-1a
pleted cells have impairedmotility in response to SDF-1a (10 nM) compared to
nce of stimuli) are presented asmean ± SEMof three independent experiments.
plexes. After 16 hr, cells were serum starved for 48 hr and, stimulatedwith HRG
ion inhibits motility in response to HRG. Right panel: CXCR4 depletion inhibits
trol cells in the absence of stimuli) are presented as mean ± SEM of three inde-
nel) by HRG in MCF-7 cells, but it abolishes migration by SDF-1a (right panel).
triplicates (fold increase relative to vehicle in the absence of stimuli) are pre-
cle (Veh) + SDF-1a.
her HRG (10 ng/ml) or SDF-1a (10 nM) for 10 min, alone or in the presence of
rbB3 antibody (10 mg/ml). Similar results were observed in multiple individual
ls in nude mice (ten mice/group). Data are expressed as mean ± SD.
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 887
A B
C
D
E
HRG
AMD-3100
IgG
control Anti-CXCR4 Ab
SDF-1
++
++
++-- --
- - --
--- -
CXCR4
(Phospho-Tyr IP)
IP
CXCR4
(Total)
HRG
AMD-3100
IgG
control Anti-CXCR4 Ab
SDF-1
++
++
++-- --
- - --
--- -
CXCR4
(Phospho-Tyr IP)
IP
CXCR4
(Total)
AG1478
- + - + + + HRG
Anti-E
rbB3 A
b
ControlIg
G
-
CXCR4
(Phospho-Tyr IP)
CXCR4
(Total)
AG1478
- + - + + + HRG
Anti-E
rbB3 A
b
ControlIg
G
-
CXCR4
(Phospho-Tyr IP)
CXCR4
(Total)
Control EGFR RNAi
- ++ -
EGFR
CXCR4
(Total)
Vinculin
CXCR4
(Phospho-Tyr IP)
HRG
Control EGFR RNAi
- ++ -
EGFR
CXCR4
(Total)
Vinculin
CXCR4
(Phospho-Tyr IP)
HRG
Actin
Total
CXCR4
P-Ser324/5
-CXCR4
P-Ser330
-CXCR4
0 5 10 15
Time HRG (min)
Actin
Total
CXCR4
P-Ser324/5
-CXCR4
P-Ser330
-CXCR4
0 5 10 15
Time HRG (min)
P
HRG SDF-1
ErbB3CXCR4
Arrestin
No
BRET
BRET
P
HRG SDF-1
ErbB3CXCR4
Arrestin
No
BRET
BRET
- + - + - + - - HRG
- - - - - - + + SDF-1
AG
1478
AM
D-3
100
pSer324/5
-CXCR4
Total
CXCR4
pSer330
-CXCR4
AM
D-3
100
Vehic
le
Vehic
le
Figure 7. Activation of CXCR4 by HRG via EGFR
(A) Serum-starved T-47D cells were treated with HRG (10 ng/ml) for different times and subject to western blot with the indicated CXCR4 antibodies.
(B) BT-474 cells were treated with either HRG (10 ng/ml) or SDF-1a (10 nM) in the presence of absence of AMD-3100 (10 mg/ml). After the different treatments,
cells were subject to immunoprecipitation (IP) with an anti-CXCR4 antibody or IgG control. Immunoprecipitates were immunoblotted with either anti-phospho-
tyrosine or anti-CXCR4 antibodies.
(C) Left panel: MCF-7 cells were transiently transfected with RlucII-tagged CXCR4 and GFP10-arrestin2 as described in Experimental Procedures. Interactions
between CXCR4 and arrestin2 were measured by BRET2 following incubation with buffer (Control), SDF-1a (100 nM), or HRG (10 ng/ml). Both SDF-1a and HRG
stimulation resulted in rapid recruitment of arrestin2. Data are expressed as mean ± SD of triplicate samples. Two additional experiments gave similar results.
Right panel: schematic representation of the BRET assay.
Molecular Cell
P-Rex1 in Breast Cancer
888 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc.
Molecular Cell
P-Rex1 in Breast Cancer
P-Rex1 as a Mediator of CXCR4-Induced Rac1Activation in Breast Cancer Cells: Integration of TKand GPCR ResponsesA distinctive feature of P-Rex1 is that it is synergistically
regulated by PIP3 and membrane-bound Gbg proteins via the
DH-PH domain (Welch et al., 2002; Barber et al., 2007; Mayee-
nuddin et al., 2006). We found that in MCF-7 cells, P-Rex1 redis-
tributes to the plasma membrane in response to HRG via its
DH-PH domain. Our PTX results established the requirement
of Gbg subunits and a transactivation mechanism involving
Gi-coupled receptors in the ErbB receptor response. Curiously,
multiple studies have shown that TK responses are PTX sensi-
tive, including those mediated by ErbB receptors (Luttrell et al.,
1995; Hobson et al., 2001; Stanton et al., 1991; Johnson et al.,
1986). The insensitivity to PTX in MCF-10A cells argues that Gi
is not involved in mediating Rac1 activation by ErbB receptors
in normal cells and possibly in P-Rex1-negative breast cancer
cells.
The PIP3 component of P-Rex1 activation downstream of
ErbB receptors arises largely from a Gbg/PI3Kg pathway. Inputs
from Gbg subunits and PI3Kg may suffice for P-Rex1 activation
in response to agonist-directed GPCR activation, as inferred
from the full inhibitory effect of PI3Kg depletion on SDF-
1a-induced Rac activation and migration and as also described
in neutrophils (Zhao et al., 2007). It is conceivable that an addi-
tional input may be required for Rac activation by ErbB recep-
tors, possibly from type Ia PI3K. Type Ia PI3Ks are indeed
preferential effectors of ErbB2/ErbB3 dimers (Yarden and Sliw-
kowski, 2001; Citri et al., 2003; Hynes and Lane, 2005). Thus,
full activation of P-Rex1 by ErbB receptors may require the
convergence of inputs from type Ia and type Ib PI3Ks as well
as Gbg subunits, as depicted in the model presented in
Figure 7E.
A remarkable finding from our studies is the association of
CXCR4 with ErbB receptor-induced activation of P-Rex1/Rac
signaling. CXCR4 and its ligand SDF-1a have been widely impli-
cated in breast cancer cell proliferation, migration, and invasion.
CXCR4 and ErbB2 levels correlate in breast tumors (Li et al.,
2004; Akekawatchai et al., 2005; Muller et al., 2001). Interest-
ingly, in response to HRG, CXCR4 becomes phosphorylated
on serine residues that regulate signaling and trafficking of the
activated receptor, and in addition, it associates with arrestin,
a step normally required for proper receptor signaling, internal-
ization, and degradation upon activation (Busillo et al., 2010).
The inability of the CXCR4 antagonist AMD-3100 to affect
P-Rex1/Rac1 activation by HRG argues against the involvement
of an autocrine mechanism through SDF-1a. Notably, CXCR4
becomes tyrosine phosphorylated in response to HRG.
Although the implications of CXCR4 tyrosine phosphoryla-
tion are not completely understood, it has been shown that
CXCR4 activated by SDF-1a becomes tyrosine phosphorylated,
as we also show in Figure 7B, and inhibition of tyrosine phos-
(D) Left panel: Effect of AG1478 (1 mM) and AMD-3100 (10 mg/ml) on SDF-1a- andH
onCXCR4 tyrosine phosphorylation induced by HRG. As a control, the effect of HR
1 hr). Right panel: Effect of EGFR RNAi on HRG-induced CXCR4 tyrosine phosp
(E) Model: P-Rex1 mediates inputs from ErbB receptors in breast cancer cells th
Molec
phorylation prevents CXCR4 downstream signaling (Vila-Coro
et al., 1999). Notably, we found that EGFR is required for the
activation of CXCR4 by HRG. This fits with our previous model
showing that EGFR transactivation by HRG is a requirement
for Rac activation (Yang et al., 2006). One plausible scenario
is that CXCR4 is a direct substrate of EGFR. CXCR4 indeed
associates with other TK receptors, such as IGF-RI, in response
to IGF, leading to CXCR4 coupling to Gi (Akekawatchai et al.,
2005). We were unable to coimmunoprecipitate (coIP) EGFR
with CXCR4 (data not shown); however, CXCR4 may be phos-
phorylated by a TK downstream of EGFR, including soluble
TKs or JAKs (Andl et al., 2004). Disruption of this transactivation
mechanism may provide an alternative means of targeting the
Rac pathway.
Final RemarksIn summary, the data herein suggest that P-Rex1 is essential for
responses such as cell growth, migration, and tumorigenesis
driven by ErbB receptors. ErbB receptors relay signals to
P-Rex1/Rac1 through a Gi-PI3Kg-dependent pathway that
involves CXCR4 transactivation, strongly arguing for a role for
P-Rex1 as an integrator of TK and GPCR inputs in breast cancer
cells. Additionally, P-Rex1 upregulation is a molecular signature
of luminal breast tumors. In addition to the prognostic implica-
tions of these findings, our data also provide a strong basis for
considering the P-Rex1/Rac pathway an attractive target for
therapeutic intervention. Inhibitors of Rac-GEF/Rac interactions
with antitumorigenic activity have been developed (Nassar et al.,
2006; Vigil et al., 2010). This may be particularly relevant for
patients with ER-positive tumors that develop resistance to ther-
apies such as antiestrogens. Rac indeed mediates antiestrogen
resistance in MCF-7 cells, and importantly, a pharmacological
inhibitor of Rac abrogates antiestrogen resistance (Felekkis
et al., 2005), thus offering alternative therapeutic means for
treatment.
EXPERIMENTAL PROCEDURES
Reagents
HRG, TGF-a, and SDF-1a were purchased from R&D (Minneapolis). EGF was
purchased from BD Biosciences (San Jose, CA). Wortmannin was from LC
Laboratories (Woburn, MA). Pertussis toxin (PTX) and AMD-3100 were
obtained from Sigma. Wortmannin, AG1478, 5-quinoxalin-6-ylmethylene-thia-
zolidine-2,4-dione, and cholera toxin were from EMD/Calbiochem (Gibbs-
town, NJ).
Cell Culture
MCF-10A cells were obtained from the Karmanos Cancer Institute (Detroit)
and grown in DMEM/F12 supplemented with 10 mM HEPES, 10 mg/ml insulin,
20 ng/ml EGF, 100 ng/ml cholera toxin, 30 mM sodium bicarbonate, 0.5 mg/ml
hydrocortisone, and 5% fetal horse serum. All human breast carcinoma cell
lines were obtained from ATCC and grown in DMEM supplemented with
10% FBS (for MCF-7, T-47D, and BT-474 cells, the medium was supple-
mented with 0.2 U/ml bovine insulin).
RG-induced serine phosphorylation in CXCR4.Middle panel: Effect of AG1478
G is blocked by a blocking anti-ErbB3 antibody but not a control IgG (10 mg/ml,
horylation.
rough the transactivation of CXCR4-Gi-PI3Kg pathway.
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 889
Molecular Cell
P-Rex1 in Breast Cancer
Western Blot and IPs
Western blots were carried out essentially as previously described (Yang et al.,
2008). Antibodies and IP assays are described in the Supplemental
Information.
Pull-Down Assays, Phalloidin Staining, and Migration Assays
These studies were carried out essentially as described (Yang et al., 2006).
Rac-GEF Array
The Rac-GEF array was generated by SuperArray and contains validated
primers against Rac-GEFs and Rac-GEF modulators and a housekeeping
gene (GAPDH). Total RNA was extracted from cells using TRIzol (Invitrogen).
cDNA was generated using the RT2 First Strand Kit (SuperArray Bioscience
Corporation). An ABI Prism 7300 thermocycler was used for Q-PCR
determinations.
Tissue Arrays
P-Rex1 TaqMan primers were purchased from Applied Biosystems, mixed
with TaqMan Universal Master Mix (Applied Biosystems), and added to the
96-well Human Breast Cancer TissueScan Real-Time Panel II plate (OriGene;
Rockville, MD). Q-PCR was performed in an ABI Prism 7300 thermocycler.
Additionally, a 384-well TissueScan Cancer Survey panel (OriGene) was
used to determine P-Rex1 expression in 16 different tumor types, and
Q-PCR was performed using an ABI Prism 7900H thermocycler. Information
about the tumor samples can be found on the OriGene homepage (http://
www.origene.com).
RNAi and Generation of Cell Lines
siRNA delivery into breast cancer cells for transient knockdown was described
previously (Yang et al., 2006). shRNA sequences and details about the gener-
ation of cell lines and rescue experiments are described in Supplemental
Information.
Localization Studies
MCF-7 cells in coverslides were serum starved for 48 hr, stimulated with HRG
(10 ng/ml, 10 min), and fixed with 4% PFA. The anti-P-Rex1 antibody (1:250)
was then added for 1 hr, followed by a Cy2-conjugated goat anti-rabbit
secondary antibody (1:1500; Jackson Laboratory). Slides were mounted using
Vectashield and visualized with a Nikon TE2000-U fluorescence microscope.
MCF-7 cells were also transfected with pCEFL-EGFP-P-Rex1, pCEFL-
EGFP-DH-PH, pCEFL-EGFP-DEP-DEP, pCEFL-EGFP-PDZ-PDZ, pCEFL-
EGFP-DEP-DEP-PDZ-PDZ, or empty vector, and localization in response to
HRG was analyzed by real-time microscopy.
Growth in Soft Agar
3 3 103 cells were suspended in 0.7% granulated agar (BD Biosciences)
diluted in complete medium (23) and poured onto a 0.5% layer of agar. Fresh
mediumwas added every 3 days, and 29 days later colonies were stained with
MTT and counted.
Tumorigenesis Studies
For xenograft experiments, BT-474 cells (2 3 107 cells/mouse in 300 ml Matri-
gel) were injected into the flanks of 8-week-old female ovariectomized athymic
(nude) mice (Foxnnu, Harlan Laboratories, ten mice/group). A 17b-estradiol
pellet (1.7 mg, Innovative Research of America) was implanted s.c. 7 days
before injection. In some experiments, mice were injected with the CXCR4
antagonist AMD-3100 (0.625 and 1.25 mg/kg, s.c. once daily), as previously
reported (Rubin et al., 2003). For orthotopic growth experiments, 6-week-old
female SCIDmicewere injected into the fifthmammary glandwith BT-474 cells
(43 106 cells/mouse). Tumor formationwasmonitored by palpation and tumor
volume determined with a caliper. All animal experiments were carried out in
compliance with the institutions’ guidelines.
IHC
Detailed procedures for IHC can be found in Supplemental Information.
890 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier
BRET Assays
Detailed procedures for BRET can be found in Supplemental Information.
Statistical Analysis
Differences in the IHC staining of human breast cancer specimens were
analyzed with the Fisher’s exact (one-sided) tests. Microarray Pearson corre-
lations were performed on log2 ratios from Agilent 44K two-color gene expres-
sion microarrays on 108 human breast tumors for indicated genes after whole-
genome normalization. Data were analyzed using either a Student’s t test or
one-way analysis of variance (ANOVA).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
Supplemental References, and seven figures and can be found with this article
online at doi:10.1016/j.molcel.2010.11.029.
ACKNOWLEDGMENTS
This work is supported by grants R01CA74197, R01CA129133, and
R01CA139120 (NIH) and KG090522 (Susan Komen Foundation for the Cure)
to M.G.K. and by grant R01CA129626 to J.L.B. H.Y. and J.S.G. are supported
by the Intramural Program, NIDCR, NIH. R.E.P. is supported by grant
R01CA155117. We thank Andy Cucchiara (UPenn) for help with statistical
analyses and Celine Lefebvre (Columbia University) for support with microar-
ray data analysis.
Received: January 7, 2010
Revised: August 21, 2010
Accepted: October 18, 2010
Published: December 21, 2010
REFERENCES
Adam, L., Vadlamudi, R., Kondapaka, S.B., Chernoff, J., Mendelsohn, J., and
Kumar, R. (1998). Heregulin regulates cytoskeletal reorganization and cell
migration through the p21-activated kinase-1 via phosphatidylinositol-3
kinase. J. Biol. Chem. 273, 28238–28246.
Akekawatchai, C., Holland, J.D., Kochetkova, M., Wallace, J.C., and McColl,
S.R. (2005). Transactivation of CXCR4 by the insulin-like growth factor-1
receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells.
J. Biol. Chem. 280, 39701–39708.
Andl, C.D., Mizushima, T., Oyama, K., Bowser, M., Nakagawa, H., and Rustgi,
A.K. (2004). EGFR-induced cell migration is mediated predominantly by the
JAK-STAT pathway in primary esophageal keratinocytes. Am. J. Physiol.
Gastrointest. Liver Physiol. 287, G1227–G1237.
Andrews, S., Stephens, L.R., and Hawkins, P.T. (2007). PI3K class IB pathway.
Sci. STKE 2007, cm2.
Atlas, E., Cardillo, M., Mehmi, I., Zahedkargaran, H., Tang, C., and Lupu, R.
(2003). Heregulin is sufficient for the promotion of tumorigenicity and metas-
tasis of breast cancer cells in vivo. Mol. Cancer Res. 1, 165–175.
Bachman, K.E., Argani, P., Samuels, Y., Silliman, N., Ptak, J., Szabo, S.,
Konishi, H., Karakas, B., Blair, B.G., Lin, C., et al. (2004). The PIK3CA gene
is mutated with high frequency in human breast cancers. Cancer Biol. Ther.
3, 772–775.
Balasenthil, S., Sahin, A.A., Barnes, C.J., Wang, R.A., Pestell, R.G.,
Vadlamudi, R.K., and Kumar, R. (2004). p21-activated kinase-1 signaling
mediates cyclin D1 expression in mammary epithelial and cancer cells.
J. Biol. Chem. 279, 1422–1428.
Barber, M.A., Donald, S., Thelen, S., Anderson, K.E., Thelen, M., and Welch,
H.C. (2007). Membrane translocation of P-Rex1 is mediated by G protein
betagamma subunits and phosphoinositide 3-kinase. J. Biol. Chem. 282,
29967–29976.
Inc.
Molecular Cell
P-Rex1 in Breast Cancer
Bose, S., Crane, A., Hibshoosh, H., Mansukhani, M., Sandweis, L., and
Parsons, R. (2002). Reduced expression of PTEN correlates with breast cancer
progression. Hum. Pathol. 33, 405–409.
Busillo, J.M., Armando, S., Sengupta, R., Meucci, O., Bouvier, M., and
Benovic, J.L. (2010). Site-specific phosphorylation of CXCR4 is dynamically
regulated by multiple kinases and results in differential modulation of CXCR4
signaling. J. Biol. Chem. 285, 7805–7817.
Citri, A., Skaria, K.B., and Yarden, Y. (2003). The deaf and the dumb: the
biology of ErbB-2 and ErbB-3. Exp. Cell Res. 284, 54–65.
Dong, X., Mo, Z., Bokoch, G., Guo, C., Li, Z., and Wu, D. (2005). P-Rex1 is
a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils.
Curr. Biol. 15, 1874–1879.
Fan, C., Oh, D.S., Wessels, L., Weigelt, B., Nuyten, D.S., Nobel, A.B., van’t
Veer, L.J., and Perou, C.M. (2006). Concordance among gene-expression-
based predictors for breast cancer. N. Engl. J. Med. 355, 560–569.
Felekkis, K.N., Narsimhan, R.P., Near, R., Castro, A.F., Zheng, Y., Quilliam,
L.A., and Lerner, A. (2005). AND-34 activates phosphatidylinositol 3-kinase
and induces anti-estrogen resistance in a SH2 and GDP exchange factor-
like domain-dependent manner. Mol. Cancer Res. 3, 32–41.
Fernandez-Zapico, M.E., Gonzalez-Paz, N.C., Weiss, E., Savoy, D.N., Molina,
J.R., Fonseca, R., Smyrk, T.C., Chari, S.T., Urrutia, R., and Billadeau, D.D.
(2005). Ectopic expression of VAV1 reveals an unexpected role in pancreatic
cancer tumorigenesis. Cancer Cell 7, 39–49.
Fine, B., Hodakoski, C., Koujak, S., Su, T., Saal, L.H., Maurer, M., Hopkins, B.,
Keniry, M., Sulis, M.L., Mense, S., et al. (2009). Activation of the PI3K pathway
in cancer through inhibition of PTEN by exchange factor P-REX2a. Science
325, 1261–1265.
Hill, K., Krugmann, S., Andrews, S.R., Coadwell, W.J., Finan, P., Welch, H.C.,
Hawkins, P.T., and Stephens, L.R. (2005). Regulation of P-Rex1 by phospha-
tidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits. J. Biol. Chem.
280, 4166–4173.
Hobson, J.P., Rosenfeldt, H.M., Barak, L.S., Olivera, A., Poulton, S., Caron,
M.G., Milstien, S., and Spiegel, S. (2001). Role of the sphingosine-1-phosphate
receptor EDG-1 in PDGF-induced cell motility. Science 291, 1800–1803.
Hodgson, J.G., Chin, K., Collins, C., and Gray, J.W. (2003). Genome amplifica-
tion of chromosome 20 in breast cancer. Breast Cancer Res. Treat. 78,
337–345.
Holm, C., Rayala, S., Jirstrom, K., Stal, O., Kumar, R., and Landberg, G. (2006).
Association between Pak1 expression and subcellular localization and tamox-
ifen resistance in breast cancer patients. J. Natl. Cancer Inst. 98, 671–680.
Hynes, N.E., and Lane, H.A. (2005). ERBB receptors and cancer: the
complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354.
Jaffe, A.B., and Hall, A. (2005). Rho GTPases: biochemistry and biology. Annu.
Rev. Cell Dev. Biol. 21, 247–269.
Johnson, R.M., Connelly, P.A., Sisk, R.B., Pobiner, B.F., Hewlett, E.L., and
Garrison, J.C. (1986). Pertussis toxin or phorbol 12-myristate 13-acetate can
distinguish between epidermal growth factor- and angiotensin-stimulated
signals in hepatocytes. Proc. Natl. Acad. Sci. USA 83, 2032–2036.
Jonsson, G., Staaf, J., Olsson, E., Heidenblad, M., Vallon-Christersson, J.,
Osoegawa, K., de Jong, P., Oredsson, S., Ringner, M., Hoglund, M., and
Borg, A. (2007). High-resolution genomic profiles of breast cancer cell lines as-
sessed by tiling BAC array comparative genomic hybridization. Genes
Chromosomes Cancer 46, 543–558.
Kallioniemi, A., Kallioniemi, O.P., Piper, J., Tanner, M., Stokke, T., Chen, L.,
Smith, H.S., Pinkel, D., Gray, J.W., and Waldman, F.M. (1994). Detection
and mapping of amplified DNA sequences in breast cancer by comparative
genomic hybridization. Proc. Natl. Acad. Sci. USA 91, 2156–2160.
Lee, R.J., Albanese, C., Fu, M., D’Amico, M., Lin, B., Watanabe, G., Haines,
G.K., 3rd, Siegel, P.M., Hung, M.C., Yarden, Y., et al. (2000). Cyclin D1 is
required for transformation by activated Neu and is induced through an
E2F-dependent signaling pathway. Mol. Cell. Biol. 20, 672–683.
Molec
Lee, K., Liu, Y., Mo, J.Q., Zhang, J., Dong, Z., and Lu, S. (2008). Vav3 onco-
gene activates estrogen receptor and its overexpression may be involved in
human breast cancer. BMC Cancer 8, 158.
Li, Y.M., Pan, Y., Wei, Y., Cheng, X., Zhou, B.P., Tan, M., Zhou, X., Xia, W.,
Hortobagyi, G.N., Yu, D., and Hung, M.C. (2004). Upregulation of CXCR4 is
essential for HER2-mediated tumor metastasis. Cancer Cell 6, 459–469.
Luttrell, L.M., van Biesen, T., Hawes, B.E., Koch, W.J., Touhara, K., and
Lefkowitz, R.J. (1995). G beta gamma subunits mediate mitogen-activated
protein kinase activation by the tyrosine kinase insulin-like growth factor 1
receptor. J. Biol. Chem. 270, 16495–16498.
Marchese, A., andBenovic, J.L. (2001). Agonist-promoted ubiquitination of the
Gprotein-coupled receptor CXCR4mediates lysosomal sorting. J. Biol. Chem.
276, 45509–45512.
Mastracci, T.L., Shadeo, A., Colby, S.M., Tuck, A.B., O’Malley, F.P., Bull, S.B.,
Lam, W.L., and Andrulis, I.L. (2006). Genomic alterations in lobular neoplasia:
a microarray comparative genomic hybridization signature for early neoplastic
proliferationin the breast. Genes Chromosomes Cancer 45, 1007–1017.
Mayeenuddin, L.H., McIntire, W.E., and Garrison, J.C. (2006). Differential
sensitivity of P-Rex1 to isoforms of G protein betagamma dimers. J. Biol.
Chem. 281, 1913–1920.
Minard, M.E., Kim, L.S., Price, J.E., and Gallick, G.E. (2004). The role of the
guanine nucleotide exchange factor Tiam1 in cellular migration, invasion,
adhesion and tumor progression. Breast Cancer Res. Treat. 84, 21–32.
Moulder, S.L., Yakes, F.M., Muthuswamy, S.K., Bianco, R., Simpson, J.F., and
Arteaga, C.L. (2001). Epidermal growth factor receptor (HER1) tyrosine kinase
inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast
cancer cells in vitro and in vivo. Cancer Res. 61, 8887–8895.
Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M.E.,
McClanahan, T., Murphy, E., Yuan, W., Wagner, S.N., et al. (2001).
Involvement of chemokine receptors in breast cancer metastasis. Nature
410, 50–56.
Nassar, N., Cancelas, J., Zheng, J., Williams, D.A., and Zheng, Y. (2006).
Structure-function based design of small molecule inhibitors targeting Rho
family GTPases. Curr. Top. Med. Chem. 6, 1109–1116.
Patel, V., Rosenfeldt, H.M., Lyons, R., Servitja, J.M., Bustelo, X.R., Siroff, M.,
and Gutkind, J.S. (2007). Persistent activation of Rac1 in squamous carci-
nomas of the head and neck: evidence for an EGFR/Vav2 signaling axis
involved in cell invasion. Carcinogenesis 28, 1145–1152.
Pattarozzi, A., Gatti, M., Barbieri, F., Wurth, R., Porcile, C., Lunardi, G., Ratto,
A., Favoni, R., Bajetto, A., Ferrari, A., and Florio, T. (2008). 17beta-estradiol
promotes breast cancer cell proliferation-inducing stromal cell-derived
factor-1-mediated epidermal growth factor receptor transactivation: reversal
by gefitinib pretreatment. Mol. Pharmacol. 73, 191–202.
Qin, J., Xie, Y., Wang, B., Hoshino, M., Wolff, D.W., Zhao, J., Scofield, M.A.,
Dowd, F.J., Lin, M.F., and Tu, Y. (2009). Upregulation of PIP3-dependent
Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis. Oncogene
28, 1853–1863.
Rossman, K.L., Der, C.J., and Sondek, J. (2005). GEF means go: turning on
RHO GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol.
Cell Biol. 6, 167–180.
Rubin, J.B., Kung, A.L., Klein, R.S., Chan, J.A., Sun, Y., Schmidt, K., Kieran,
M.W., Luster, A.D., and Segal, R.A. (2003). A small-molecule antagonist of
CXCR4 inhibits intracranial growth of primary brain tumors. Proc. Natl. Acad.
Sci. USA 100, 13513–13518.
Stanton, R.C., Seifter, J.L., Boxer, D.C., Zimmerman, E., and Cantley, L.C.
(1991). Rapid release of bound glucose-6-phosphate dehydrogenase by
growth factors. Correlation with increased enzymatic activity. J. Biol. Chem.
266, 12442–12448.
Ueda, Y., Wang, S., Dumont, N., Yi, J.Y., Koh, Y., and Arteaga, C.L. (2004).
Overexpression of HER2 (erbB2) in human breast epithelial cells unmasks
transforming growth factor beta-induced cell motility. J. Biol. Chem. 279,
24505–24513.
ular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier Inc. 891
Molecular Cell
P-Rex1 in Breast Cancer
Vigil, D., Cherfils, J., Rossman, K.L., and Der, C.J. (2010). Ras superfamily
GEFs and GAPs: validated and tractable targets for cancer therapy?
Nat. Rev. Cancer 10, 842–857.
Vila-Coro, A.J., Rodrıguez-Frade, J.M., Martın De Ana, A., Moreno-Ortız, M.C.,
Martınez-A, C., and Mellado, M. (1999). The chemokine SDF-1alpha triggers
CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB
J. 13, 1699–1710.
Wang, S.E., Shin, I., Wu, F.Y., Friedman, D.B., and Arteaga, C.L. (2006). HER2/
Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by
transforming growth factor beta. Cancer Res. 66, 9591–9600.
Welch, H.C., Coadwell, W.J., Ellson, C.D., Ferguson, G.J., Andrews, S.R.,
Erdjument-Bromage, H., Tempst, P., Hawkins, P.T., and Stephens, L.R.
(2002). P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-
nucleotide exchange factor for Rac. Cell 108, 809–821.
Welch, H.C., Condliffe, A.M., Milne, L.J., Ferguson, G.J., Hill, K., Webb, L.M.,
Okkenhaug, K., Coadwell, W.J., Andrews, S.R., Thelen, M., et al. (2005).
P-Rex1 regulates neutrophil function. Curr. Biol. 15, 1867–1873.
Yang, C., Liu, Y., Leskow, F.C., Weaver, V.M., and Kazanietz, M.G. (2005).
Rac-GAP-dependent inhibition of breast cancer cell proliferation by beta2-
chimerin. J. Biol. Chem. 280, 24363–24370.
Yang, C., Liu, Y., Lemmon, M.A., and Kazanietz, M.G. (2006). Essential role for
Rac in heregulin beta1 mitogenic signaling: a mechanism that involves
892 Molecular Cell 40, 877–892, December 22, 2010 ª2010 Elsevier
epidermal growth factor receptor and is independent of ErbB4. Mol. Cell.
Biol. 26, 831–842.
Yang, C., Klein, E.A., Assoian, R.K., and Kazanietz, M.G. (2008). Heregulin
beta1 promotes breast cancer cell proliferation through Rac/ERK-dependent
induction of cyclin D1 and p21Cip1. Biochem. J. 410, 167–175.
Yarden, Y., and Sliwkowski, M.X. (2001). Untangling the ErbB signalling
network. Nat. Rev. Mol. Cell Biol. 2, 127–137.
Yoshizawa, M., Kawauchi, T., Sone, M., Nishimura, Y.V., Terao, M., Chihama,
K., Nabeshima, Y., and Hoshino, M. (2005). Involvement of a Rac
activator,P-Rex1, in neurotrophin-derived signaling and neuronal migration.
J. Neurosci. 25, 4406–4419.
Zhao, T., Nalbant, P., Hoshino, M., Dong, X., Wu, D., and Bokoch, G.M. (2007).
Signaling requirements for translocation of P-Rex1, a key Rac2 exchange
factor involved in chemoattractant-stimulated human neutrophil function.
J. Leukoc. Biol. 81, 1127–1136.
Note Added in Proof
In agreement with our data, a recent advanced online publication by Montero
et al. also found that P-Rex1 is implicated in ErbB signal transduction in breast
cancer cells: Montero, J.C., Seoane, S., Ocana, A., and Pandiella, A. (2010).
P-Rex1 participates in Neuregulin-ErbB signal transduction and its expression
correlates with patient outcome in breast cancer. Oncogene, in press. Pub-
lished online November 1, 2010. 10.1038/onc.2010.489.
Inc.