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

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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

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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.

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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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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

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0.0

3

0.1

0.3

1 PI3k inh ( M)

HRG

- -

+- ++++ +

Rac-GTP

Total Rac

Vinculin

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HRG

- -

0 0 0.01 0.03 0.1 0.3 1

0

20

40

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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

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2

3

4

5

Mig

ra

tio

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(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

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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

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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.