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Published OnlineFirst June 1, 2012; DOI: 10.1158/0008-5472.CAN-11-2502
Vav3-Rac1 Signaling Regulates Prostate Cancer Metastasis with
Elevated Vav3 Expression Correlating with Prostate Cancer
Progression and Posttreatment Recurrence
Kai-Ti Lin, Jianli Gong, Chien-Feng Li, et al.
Cancer Res 2012;72:3000-3009. Published OnlineFirst June 1, 2012.
Updated version
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Published OnlineFirst June 1, 2012; DOI: 10.1158/0008-5472.CAN-11-2502
Cancer
Research
Molecular and Cellular Pathobiology
Vav3-Rac1 Signaling Regulates Prostate Cancer Metastasis
with Elevated Vav3 Expression Correlating with Prostate
Cancer Progression and Posttreatment Recurrence
Kai-Ti Lin1, Jianli Gong2, Chien-Feng Li4, Te-Hsuan Jang1, Wen-Ling Chen1,
Huei-Jane Chen1, and Lu-Hai Wang1,3
Abstract
Prostate cancer remains the second leading cause of cancer death in men in the Western world. Yet current
therapies do not significantly improve the long-term survival of patients with distant metastasis. In this study,
we investigated the role of the guanine nucleotide exchange factor Vav3 in prostate cancer progression and
metastasis and found that Vav3 expression correlated positively with prostate cancer cell migration and
invasion. Stimulation of the receptor tyrosine kinase EphA2 by ephrinA1 resulted in recruitment and tyrosine
phosphorylation of Vav3, leading to Rac1 activation as well as increased migration and invasion in vitro.
Reduction of Vav3 resulted in fewer para-aortic lymph nodes and bone metastasis in vivo. Clinically, expression
of Vav3 and EphA2 was elevated in late-stage and metastatic prostate cancers. Among patients with stage IIB
or earlier prostate cancer, higher Vav3 expression correlated with lower cumulative biochemical failure-free
survival, suggesting that Vav3 may represent a prognostic marker for posttreatment recurrence of prostate
cancer. Together, our findings provide evidence that the Vav3-mediated signaling pathway may serve as a
therapeutic target for prostate cancer metastasis. Cancer Res; 72(12); 3000–9. 2012 AACR.
Introduction
The Vav3 oncogene, the third member of the Vav family of
Rho GTPase nucleotide exchange factors (GEF; refs. 1–3), is
involved in various cellular signaling processes, acting through
its classical Dbl domain to activate the Rho family GTPases,
including RhoA, Rac1, and Cdc42 (3, 4). Various receptor
tyrosine kinases (RTK) activate Vav proteins (3, 5), resulting
in the opening up of the Dbl domain for its substrate (6).
Deficiency of Vav3 leads to increased bone mass density (7),
sympathetic hyperactivity, cardiovascular dysfunction (8), and
impaired wound healing (9).
The Eph receptors are the largest family of RTKs and have
significant roles in the regulation of cell attachment, cell shape,
and motility during development and pathologic conditions,
especially cancer metastases (10–12). One Eph receptor,
EphA2, is overexpressed in human prostate (13, 14) and other
Authors' Affiliations: 1Institute of Molecular and Genomic Medicine,
National Health Research Institutes, Miaoli County, Taiwan; 2Department
of Pharmacology, College of Physicians and Surgeons, Columbia University; 3Department of Microbiology, Mount Sinai School of Medicine, New
York; and 4Department of Pathology, Chi-Mei Foundation Medical Center,
Tainan, Taiwan
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Lu-Hai Wang, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan.
Phone: 886-37-246166 ex. 35300; Fax: 886-37-585-242; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-11-2502
2012 American Association for Cancer Research.
3000
cancers (15). The EphA2 receptor is required for the regulation
of cell motility, cell invasion, and in vivo metastasis in prostate
cancer (16) and it directly activates Vav2/3 in the regulation of
ephrinA1-induced angiogenesis (17). It is, therefore, a good
candidate for an upstream mediator of Vav3 activation and
subsequent signaling in prostate cancer progression.
Previous studies have extensively investigated the biochemical functions of Vav3; however, its role in tumorigenesis
remains unclear. Vav3 expression is increased in androgen
refractile prostate cancer cell lines and in prostate tumors
(18–20). Overexpression of Vav3 promoted prostate cancer cell
growth and enhanced the transcriptional activity of the androgen receptor (refs. 21, 22). Dong and colleagues further
described the involvement of Vav3 in the regulation of secretary phospholipase A2-IIa expression in prostate cancer (23). In
the study of Liu and colleagues, mice with targeted Vav3
overexpression in the prostate epithelium developed nonbacterial chronic prostatitis in the prostate gland, which was
associated with increased incidence of prostate cancer (24).
The involvement of Vav3 in the regulation of cytoskeletal
reorganization and increased Vav3 expression in prostate
cancer (18, 19) suggests that Vav3 has a role in prostate cancer
metastasis. Herein, the present study shows Vav3 involvement
in regulation of migration/invasion in prostate cancer cells,
mainly through the RTK-activated Vav3-Rac1 signaling axis.
In vivo analyses show that Vav3 knockdown in PC3 cells results
in significantly lower incidence of lymph node and bone
metastasis. Clinical analyses further suggest the prognostic
potential of Vav3 in early detection of posttreatment recurrence in prostate cancer patients. Overall, the findings of this
Cancer Res; 72(12) June 15, 2012
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Published OnlineFirst June 1, 2012; DOI: 10.1158/0008-5472.CAN-11-2502
EphA2-Vav3-Rac1 Signaling in Prostate Cancer Metastasis
study suggest the importance of the Vav3-Rac1 signaling
pathway in prostate tumor progression and metastasis.
Materials and Methods
Vectors, antibodies, and reagents
Plasmids including pHEF Vav3, Vav3-(6-10), dominant-negative (dn) RhoA, dnCdc42, dnRac1 or constitutively active (ca)
Rac1, and GST-PAK CRIB have been described previously
(3, 25). The PCDNA-EphA2 plasmid was amplified from PC3
cells and cloned into PCDNA3. Antibody information is provided in the Supplementary Material.
RNA interference and short hairpin RNA construction
Three Vav3-specific siRNAs were obtained from Invitrogen.
The target sequences are provided in Supplementary Table S2.
The sequences of the Vav3-specific short hairpin RNAs
(shRNA) are 50 -GGAAGGGTTCAGAACCTTA-30 and 50 -GAAGATCTCTATGACTGTG-30 . These sense and antisense
plus hairpin sequences were cloned into pSuper Vector
(Oligoengine).
Cell culture, transfections, and stable cell line generation
The LNCaP and PC3 cell lines were obtained from BCRC
(Hsinchu, Taiwan); C4-2 was a gift from Dr. Simon Hall of
Mount Sinai School of Medicine (New York, NY). The LNCaP
and C4-2 cells were maintained in RPMI medium (Invitrogen)
with 10% FBS (Biological Industries). The PC3 cells were
maintained in Dulbecco's Modified Eagle's Medium (DMEM;
Invitrogen) with 10% FBS. All cells were maintained at 37 C in
5% CO2. Cells were transfected using Lipofectamine 2000
(Invitrogen). The PC3 cell lines stably expressing pSupershControl and pSuper-shVav3 were established by selection
with Puromycin (Sigma-Aldrich). The stable clones used were
combined from 3 single clones selected from control or Vav3
shRNA–expressing cells.
with antibody against Vav3, together with protein G-sepharose
beads for 2 hours at 4 C. After 3 washes, the bound proteins
were eluted and analyzed using Western blotting. Protein
levels were quantified using densitometry and normalized to
Vav3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
or actin levels.
Assay of Rac1 activation
The glutathione-sepharose beads conjugated with GST-PAK
CRIB were purified and used as described (3). Cell lysates were
prepared in radioimmunoprecipitation assay (RIPA) buffer
(see Supplementary Material). The GST-PAK CRIB–conjugated
beads were used to pull-down GTP-Rac1 from the cell lysates
for 45 minutes at 4 C. The beads were washed 3 times. Bound
proteins were eluted and analyzed by Western blotting with
anti-Rac1. The levels of GTP-Rac1 were quantified using densitometry and normalized to total Rac1 levels.
Cell migration and cell invasion assay
Cell migration and invasion were assayed in 8.0-mm
Falcon Cell Culture Inserts with or without Matrigel (BD
Biosciences; ref. 25). For cells receiving ephrinA1 stimulation, 0.5 105 to 2 105 cells were starved overnight,
suspended in DMEM (300 mL), and plated in the 0.3-cm2
upper Transwell chamber. The bottom well was filled with
500 mL DMEM with 2 mg/mL preclustered ephrin-A1-Fc or
control Fc. For cells receiving serum stimulation, the bottom
well was filled with 500 mL DMEM with 10% FBS. After
incubation, nonmigrating cells were removed and membranes were stained with crystal violet. Photomicrographs
of 3 regions were captured from duplicated inserts, and the
numbers of cells were counted and normalized to the
control group. All experiments were repeated 3 times.
RNA purification and real-time reverse transcriptase
PCR
Illustra RNAspin Mini Kits (GE Healthcare Life Sciences)
were used to extract RNA, following protocols supplied by the
manufacturer. First-strand cDNA was generated using the
ReverTra Ace (TOYOBO). Real-time reverse transcriptase PCR
(RT)-PCR was carried out on a 1:10 dilution of cDNA, using
KAPA SYBR FAST qPCR Kits (KAPA Biosystems) and a CFX96
real-time PCR detection system (Bio-Rad). The mRNA levels
were then normalized to actin mRNA. All the primer sequences
used in this study are provided in Supplementary Table S2. All
experiments were repeated 2 times.
Animals and tumor cell injection
Male athymic BALB/c nude mice were purchased from
National Laboratory Animal Center, and Bio LASCO Taiwan
Co. Mice were injected with 1 106 PC3 cells stably expressing
Vav3 or control shRNA in the prostate as described (see
Supplementary Material; ref. 26). Two runs of implantation
were carried out independently and 6 to 8 mice were used per
group each time. Results from the experiments were combined
as the final data. Mice were sacrificed 28 to 35 days after tumor
cell injection. Tissue specimens were fixed, paraffin-embedded, serially sectioned, and stained with hematoxylin and eosin
(H&E). In the second run of mouse injections, bone marrow
cells from mouse thigh bones were collected and cultured in
DMEM with 10% FBS to detect bone metastatic cells from PC3
tumor cells in the prostate.
Immunoprecipitation and Western blot analysis
To activate EphA2, serum-starved cells were stimulated with
2 mg/mL preclustered ephrin-A1-Fc (R&D Systems) for 10
minutes. The ephrinA1-Fc or control Fc (Jackson ImmunoResearch) chimeras were preclustered by incubating with antihuman IgG (Jackson ImmunoResearch), in the ratio 1:2, at 4 C
for 1 hour before stimulation. Cells were lysed in lysis buffer
(see Supplementary Material). The cell lysates were incubated
Immunohistochemistry
Paraffin-embedded tissue sections of human prostate cancer specimens were obtained from Chi-Mei Medical Center
(Tainan, Taiwan; using an Institutional Review Board–
approved protocol) or from commercial prostate cancer tissue
arrays (US Biomax, Inc. and SuperBioChips). The slides were
stained with anti-Vav3 or anti-EphA2 using an automatic slide
stainer BenchMark XT (Ventana Medical Systems).
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Lin et al.
tion/invasion (Supplementary Fig. S1A), which correlated with
Rac1 activation (Supplementary Fig. S1B) in NIH3T3 cells. In
prostate cancer cells, individual transfection of low Vav3expressing LNCaP cells (Fig. 1B) with Vav3 or Vav3-(6-10) (Fig.
2B) resulted in significantly increased migration/invasion (Fig.
2C and D). The ability to induce both migration and invasion
was much higher for Vav3-(6-10) than for full-length Vav3,
indicating that the stimulus for Vav3 activation might be
important in Vav3-, but not Vav3-(6-10), mediated migration/invasion.
Subsequent testing of the small Rho GTPases revealed that
the dominant-negative form of Rac1 (dnRac1) significantly
inhibited Vav3-(6-10)-induced migration/invasion, whereas
dnCdc42 and dnRhoA did not (Figs. 2E and F). Using siRNAs
specifically targeting these Rho GTPases excluded the nonselective effects caused by the dominant-negative form (Supplementary Fig. S2A). Consistently, only cells with reduced Rac1
expression displayed significant inhibition of Vav3-(6-10)induced migration/invasion (Fig. S2B and S2C). Consistent
with increased migration/invasion, LNCaP cells expressing
Vav3, and especially Vav3-(6-10), displayed increased GTPRac1, the active form of Rac1 (Fig. 2G). Overall, these results
indicate that Vav3 promotes prostate cancer migration/invasion, mainly through activation of Rac1.
Patients and follow-up
Fifty radical prostatectomy specimens, no higher than stage
IIB, were retrospectively identified from Chi-Mei Medical
Center between 1998 and 2005. The detailed information on
these specimens is provided in the Supplementary Material.
Results
Upregulation of Vav3 and correlation with increased
migration/invasion in selected androgen-independent
prostate cancer cells
To evaluate Vav3 expression and its possible relation to
migration/invasion, the present study tested several prostate
cancer cell lines. Real time RT-PCR and Western blot analysis
both revealed higher Vav3 mRNA and protein expression in the
androgen-independent lines, PC3 and C4-2 (27), than in the
androgen-dependent line, LNCaP (Fig. 1A and B). These data
are consistent with previous reports of Vav3 overexpression in
prostate cancer (18, 19). Similarly, upregulation of EphA2
occurred in the 2 androgen-independent lines, especially in
PC3 (Fig. 1A and B).
Subsequent analyses evaluated the possible association of
migration/invasion with Vav3 expression, observing that both
migration and invasion occurred to significantly greater
extents in the high Vav3-expressing cells (C4-2 and PC3) than
in the low Vav3-expressing LNCaP cells (Fig. 1C and D). These
results suggest that Vav3 upregulation might lead to enhanced
migration and invasion abilities.
The ephrinA1 ligand stimulation of EphA2 receptor
results in recruitment of Vav3, tyrosine
phosphorylation of Vav3, Rac1 activation, and
increased migration/invasion
Activation of Vav3 occurs by way of Y173 phosphorylation by
several RTKs, including insulin, insulin-like growth factor (IGF)
1, EGF receptors, Ros (3), and EphA2 (17). To identify which
RTK(s) functions as the upstream activator for Vav3 in prostate
cancer cells, the present study tested several RTKs. The EGF
Role of Vav3 in promotion of migration/invasion by way
of Rac1 activation in LNCaP cells
To explore the role of Vav3 in migration/invasion, we first
showed that overexpression of Vav3 or Vav3-(6-10), the constitutively activated mutant (Fig. 2A; ref. 3), increased migra-
0
LNCaP C4-2
2
1
*
0
PC3
PC3
C
C
aP
LN
-2
C4
3
PC
Vav3
1 ± 0.1 2.5 ± 0.3 5.3 ± 0.4
EphA2
1 ± 0.1 7.7 ± 0.7 35.6 ± 2.2
GAPDH
Fold increase over control
B
LNCaP C4-2
D
***
***
0
-2
P
P
4
-C
L
a
NC
3
PC
2
***
1
**
0
PC3
Migration
8
L
Cancer Res; 72(12) June 15, 2012
*
12
4
AR
***
LNCaP C4-2
16
a
NC
3002
EphA2
20
15
10
5
0
Fold increase
1
*
Fold increase over control
**
2
EphrinA1
3
Fold increase
3
Fold increase
Vav3
Fold increase
A
LNCaP C4-2
PC3
Invasion
***
16
12
8
***
4
0
-2
P
Ca
C4
P-
LN
3
PC
Figure 1. Overexpression of Vav3
and correlation with migration/
invasion in androgen-independent
prostate cancer cells. A, real-time
RT-PCR analysis of mRNA levels in
LNCaP, C4-2, and PC-3 cells. Data
are presented as normalized mean
SD (n ¼ 3). , P < 0.05; , P < 0.01;
, P < 0.001. B, Western blot
analysis of Vav3 and EphA2 protein
levels in LNCaP, C4-2, and PC3
cells. Histograms represent
normalized mean SE (n ¼ 3). Cell
migration (C) and invasion (D)
assays were carried out. Cells were
incubated for 24 hours for
migration and 48 hours for invasion
assays. DMEM with 10% FBS
served as a chemoattractant.
Numbers represent normalized
mean SD (n ¼ 6). , P < 0.001.
a
NC
L
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Published OnlineFirst June 1, 2012; DOI: 10.1158/0008-5472.CAN-11-2502
EphA2-Vav3-Rac1 Signaling in Prostate Cancer Metastasis
1
0
PCDNA
G
**
***
6
6
***
4
**
2
0
PCDNA
6-10
LNCaP cells
NA
D
PC
***
***
Vav3
0)
-1
v3
Va
-(6
v3
Va
GTP-Rac1
4
2
0
dc
ho
2.0 ± 0.2
2.9 ± 0.2
C
R
1 ± 0.2
dn
+
10
10
42
1
ac
N
A
Total Rac1
6-
C
dn
6-10
Invasion
42
dc
ho
Vav3
Invasion
Fold increase over control
**
2
+
10
6-
6-
10
+
+
dn
dn
R
R
6-
ac
10
1
A
0
10
3
dn
1
***
R
2
D
Migration
A
**
***
3
6-
SH3
Fold increase over control
***
***
EF
SH2
F
Migration
pH
SH3
+
Actin
Fold increase over control
ZF
10
v3
Va
10.0 ± 1.1 6.8 ± 0.7
4
PH
6-
-1
-(6
v3
Va
Vav3
E
DH
10
0)
Vav3-(6-10)
1 ± 0.6
SH3
D
P
SH2
Fold increase over control
A
SH3
C
LNCaP cells
N
CD
ZF
PC
B
PH
dn
Vav3-(6-10)
DH
6-
AD
+
CH
Vav3
6-
A
Figure 2. The Vav3 oncoprotein mediates enhancement of cell migration and invasion by activating Rac1. A, schematic representation of Vav3 and Vav3-(6-10).
B, expression of Vav3 or Vav3-(6-10) in LNCaP cells 24 hours after transfection. Histograms represent normalized mean SE (n ¼ 3). C and D, the
LNCaP cells were transfected with Vav3 or Vav3-(6-10). Cells were incubated for 24 hours for migration (C) and 48 hours for invasion (D) assays.
DMEM with 10% FBS served as a chemoattractant. Numbers represent normalized mean SD (n ¼ 6). , P < 0.01; , P < 0.001. The LNCaP cells
cotransfected with Vav3-(6-10) plus dnRac1, dnRhoA, or dnCdc42 were incubated for 24 hours for migration (E) and 48 hours for invasion (F) assays.
Numbers represent normalized mean SD (n ¼ 6). , P < 0.01; , P < 0.001. G, Western blot analysis of GST-PAK immunoprecipitates (active GTP-Rac1) or
lysates from LNCaP cells expressing Vav3 or Vav3-(6-10). Histograms represent normalized mean SE (n ¼ 3).
receptor (EGFR) or Met (hepatocyte growth factor receptor)
activation, but not the insulin receptor (IR), is able to stimulate
Vav3 activity in LNCaP or PC3 cells to varying extents (Supplementary Fig. S3B–S3D). Activation of Vav3 by these receptors might contribute to Vav3-mediated cell migration/
invasion.
Other than those RTKs, the expression of EphA2 correlated
with Vav3 in prostate cancer cells (Fig. 1A and B). It stimulated
endogenous Vav3 tyrosine phosphorylation in the presence of
the preclustered EphA2 ligand, ephrinA1-Fc, in PC3 cells (Fig.
3A), and so was the overexpressed Vav3 in both PC3 and LNCaP
cells (Supplementary Fig. S3A). Endogenous Vav3 was associated with EphA2 upon ephrinA1-Fc stimulation (Fig. 3B). This
confirmed the ability of activated EphA2 to bind Vav2/3 (28).
Transfection of LNCaP cells with EphA2 or EphA2 plus Vav3
further tested whether EphA2 activation regulates Vav3-Rac1
www.aacrjournals.org
signaling. By ephrinA1 stimulation, Rac1 was activated in
EphA2-expressing cells; active Rac1 was markedly increased
in cells expressing EphA2 together with Vav3 (Fig. 3C). Consistently, upon ephrinA1-Fc stimulation, migration/invasion
was significantly greater in LNCaP cells overexpressing Vav3
than in unstimulated cells (Fig. 3D).
Reducing EphA2 expression in PC3 cells tested the requirement for EphA2 in Vav3 activation (Fig. 3E) with results
showing markedly reduced ephrinA1-induced Vav3 tyrosine
phosphorylation (Fig. 3F). The slightly decreased Vav3 upon
siEphA2 has been reproducibly observed. The reason for this is
unclear. However, the decrease of phosphorylated Vav3 is
much greater than that of Vav3. These data indicate that
EphA2 serves as a key upstream activator for Vav3-Rac1
signaling, leading to enhanced migration/invasion in prostate
cancer cells.
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B
hr
in
A
EphA2
Vav3
Vav3
1 ± 0.4 47.6 ± 9.6
__________
PC3
D
c
c
-F
-F
A1
A1
hr
Fold increase over control
in
in
ep
Fc
hr
ep
GTP-Rac1
Total Rac1
1 ± 0.1 2 ± 0.3 1.6 ± 0.3 2.7 ± 0.3
Vav3
________
________
EphA2+
EphA2+
PCDNA
Vav3
E
***
**
c
c
hr
ep
Fc
EphA2
Fc
hr
in
in
A1
A1
-F
-F
2
hA
Ep
si
si
C
on
tro
l
F
ep
Fc
IP: Vav3
pTyr
1 ± 0.5 8.8 ± 2.3
___________
PC3
C
ep
IP: Vav3
Fc
Fc
ep
hr
in
A
1Fc
1Fc
A
actin
1 ± 0.3 0.2 ± 0.04
__________
PC3
Vav3
1 ± 0.4 8.4 ± 1.9 0.5 ± 0.3 0.4 ± 0.1
__________
__________
siControl
Knockdown of Vav3 in PC3 cells results in decreased
migration/invasion and attenuated Rac1 activity
Knockdown experiments on high Vav3-expressing PC3
cells (Fig. 1A and B) evaluated the effects of reducing Vav3
expression on migration/invasion. The PC3 cells with
reduced Vav3 protein expression (Fig. 4A) showed more
than 50% reduction in serum-stimulated migration/invasion (Fig. 4B) and concordantly reduced GTP-Rac1 levels
(Fig. 4C). Expression of several matrix metalloproteinases
(MMP) decreased in PC3 cells upon Vav3 knockdown
(Supplementary Fig. S4A), indicating the potential role of
Vav3 in the regulation of MMP expression. Knockdown of
Vav3 resulted in attenuated ephrinA1-induced Rac1 activity
(Supplementary Fig. S4B) and decreased migration/invasion
(Supplementary Fig. S4C and S4D). Results indicated that
the activation of Rac1 by EphA2 signaling was less significant than serum-induced activation, suggesting that other
RTKs in serum may also mediate Vav3-Rac1 signaling in
3004
Cancer Res; 72(12) June 15, 2012
IP: Vav3
pTyr
siEphA2
Figure 3. Stimulation of EphA2 by
ephrin A1 results in recruitment
and phosphorylation of Vav3,
leading to Rac1 activation and
increased migration/invasion.
Following stimulation by
ephrinA1-Fc or Fc in PC3 cells for
10 minutes, endogenous Vav3
tyrosine phosphorylation (A) and
recruitment to EphA2 (B) was
evaluated by immunoprecipitation
(IP) with anti-Vav3 and
immunoblotting with anti-pTyr,
anti-EphA2, or anti-Vav3.
Histograms represent normalized
mean SE (n ¼ 3). C, Western blot
analysis of GST-PAK
immunoprecipitates (active GTPRac1) or lysates from LNCaP cells
overexpressing Rac1, EphA2, and
Vav3 in the presence of ephrinA1Fc or Fc for 10 minutes.
Histograms represent normalized
mean SE (n ¼ 3). D, the LNCaP
cells with Vav3 transfection were
starved overnight and incubated
for 24 hours for migration or 48
hours for invasion assays.
EphrinA1-Fc served as a
chemoattractant. Numbers
represent normalized mean SD
(n ¼ 6). , P < 0.01; , P < 0.001.
E, Western blot analysis showed
EphA2 expression in PC3 cells with
control or siEphA2 transfection.
Histograms represent normalized
mean SE (n ¼ 3). F, the PC3
cells were transiently transfected
with EphA2 or control siRNA,
and then Vav3 phosphorylation
was examined using
immunoprecipitation with
anti-Vav3 and immunoblotting
with anti-pTyr and anti-Vav3.
Histograms represent normalized
mean SE (n ¼ 3).
PC3 cells, such as EGFR or Met (Supplementary Fig. S3B
and S3D).
The C4-2 cells which expressed endogenous Vav3 plus AR
(Fig. 1A and B) showed attenuated EphA2-mediated Rac1
activity (Supplementary Fig. S4F) and decreased migration/
invasion (Supplementary Fig. S4G and S4H) with Vav3 knockdown (Supplementary Fig. S4E). Overall, results indicate that
Vav3 plays an important role in migration/invasion in prostate
cancer cells.
Prostate orthotopic implantation of PC3 cells with
reduced Vav3 expression slowed primary tumor growth
and reduced incidence of para-aortic lymph nodes and
bone metastases
To further investigate the role of Vav3 during prostate
cancer metastasis in vivo, approaches of xenografted orthotopic implantation in nude mice were established. Nude mice
were orthotopically injected with control or Vav3 knockdown
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EphA2-Vav3-Rac1 Signaling in Prostate Cancer Metastasis
Vav3
Actin
1 ± 0.2 0.4 ± 0.2
si
R
N
A
ca
R
ac
1
**
Va
v3
*
2
1.5
tro
l
si
R
N
A
C
C
on
Fold increase over control
si
R
N
A
B
Va
v3
C
on
tro
l
si
R
N
A
A
1
GTP-Rac1
0.5
0
Total Rac1
Control siVav3 Control siVav3
Migration
Invasion
1 ± 0.1 0.4 ± 0.01 3.3 ± 0.4
_____________
wt-Rac1
Figure 4. Reduced Vav3 expression in PC3 cells decreases Rac1 activation and cell migration/invasion. A, Western blot analysis showed Vav3 expression in
PC3 cells with control or Vav3 siRNA transfection. Histograms represent normalized mean SE (n ¼ 3). B, the PC3 cells transfected with control or Vav3 siRNA
were incubated for 16 hours for migration and 24 hours for invasion assays. DMEM with 10% FBS served as a chemoattractant. Numbers represent
normalized mean SD (n ¼ 6). , P < 0.05; , P < 0.01. C, Western blot analysis of GST-PAK immunoprecipitates (active GTP-Rac1) or lysates in PC3
cells with reduced Vav3 expression. The small Rho GTPase, caRac1, was used as a positive control. Histograms represent normalized mean SE (n ¼ 3). wt,
wild-type.
PC3 cells into the prostate. Mice were sacrificed 28 to 35 days
after injection. Table 1 summarizes the results of tumor growth
and metastasis. In the shVav3 group, the tumor volumes
reduced to 50% of the volumes in the shControl group. However, the in vitro cell proliferation rates in shControl and
shVav3 cells were similar (Supplementary Fig. S5C). As
expected, Vav3 expression was lower in the shVav3 group than
in the shControl group (Fig. 5A and D). Figure 5B shows the
histological morphology of the primary tumors.
Previous research has shown well-characterized PC3 cells
as the metastatic model for prostate orthotopic implantation (26), observing the development of local invasion, such
as seminal vesicle and lymph node metastases, 40 days after
PC3 cell injection. In the present study, metastases in paraaortic lymph nodes developed in all mice in the shControl
group; they developed in only 35.7% of mice in the shVav3
group (Table 1). Para-aortic lymph node metastases were
enlarged greater than 7-fold in the PC3-shControl group
(Table 1 and Supplementary Fig. S4B) and were located
inside the lymph node cavity (Fig. 5C, left). The PC3-shVav3
group displayed either no lymph node metastasis (Fig. 5C,
middle) or lymph node metastases smaller in size than in the
shControl group (Fig. 5C, right). A few PC3-shControl mice
produced metastases to distant organs, including the mes-
entery, spleen, and liver (Supplementary Fig. S5D–S5F); the
shVav3 group had no overt distant organ metastases. Following culture of cells derived from bone marrow, PC3
cells were detectable in half of shControl mice and undetectable in the shVav3 group. Genetic fingerprinting of the
bone metastatic PC3 cells by short tandem repeat assay
confirmed their lineage from the parental PC3 cells (Supplementary Table S1). Two bone metastatic cell lines showed
loss of an allele in the TH01 locus, indicating genome
instability. In summary, results indicate that reduction of
Vav3 expression in PC3 cells decreased tumor growth, as
well as lymph node and distant metastasis.
Further analyses verified Vav3 levels in the primary and
metastatic tumors of mice injected with PC3-shControl or
PC3-shVav3 cells. All mice in the PC3-shControl group developed para-aortic lymph node metastases (8 of 8); 6 of 15 mice
did so in the PC3-shVav3 group. Real time RT-PCR results
indicated a 3-fold reduction in Vav3 expression in primary
tumors in the shVav3 group compared with the control group
(Fig. 5D). Two of the metastasized lymph nodes in the shVav3
group displayed increased Vav3 expression compared
with their respective primary tumors; the others displayed
low Vav3 similar to their primary tumors. These data confirm
knockdown of Vav3 expression in the PC3-shVav3 tumors
Table 1. Tumorigenicity and incidence of prostate cancer metastases by PC3 cell implantation in the
prostate in nude mice
Xenograft
Average mouse weight, g
Average tumor size, mm3
Average para-aortic lymph nodes size, mm3
Para-aortic lymph nodes metastases (%)
Microscopic bone metastases (%)
www.aacrjournals.org
PC3-shControl
PC3-shVav3
P
22.31 1.56
88.95 55.79
5.32 3.03
100 (12/12)
50 (3/6)
21.49 2.06
46.95 31.25
0.73 0.48
35.7 (5/14)
0 (0/8)
0.27
0.03
<0.001
<0.001
0.02
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Lin et al.
C
A
v3
Va
sh
sh
C
on
tro
l
PC3 tumor
Vav3
T
Actin
0.2mm
0.2mm
shControl
shVav3
D
0.1mm
shControl
shVav3
Tumor
2
Lymph node
1.5
1
------------ ---------------------------------- --------- ------------------------------------------------ ----------------------------------------------------- --------------------------------------------------------------------------------------------- -------------------------- --------------------------------------------- --------------------------------------------------------------------------- ---------------------
0.5
Cell
lines
shCon
6
5
4
3
2
1
8
7
6
5
4
3
2
1
0
shCon
0.1mm
T
2.5
shVav3
T
Fold increase over shCon cells
B
0.2mm
shVav3
shVav3
Primary tumor :1.17 ± 0.43
Primary tumor :0.39 ± 0.14
Lymph node
Lymph node
:1.06 ± 0.38
:0.54 ± 0.35
Figure 5. Prostate orthotopic implantation of PC3 cells with reduced Vav3 expression decreases primary tumor growth and metastasis to para-aortic lymph
nodes. A, Western blot analysis showing Vav3 expression in PC3 primary tumor with control and Vav3 shRNA. B, histology of PC3 tumors (T; shControl or
shVav3) growing in the prostate. Images show H&E staining of the orthotopic tumors (200). C, para-aortic lymph nodes of nude mice orthotopically
implanted with PC3-shControl (left) or PC3-shVav3 cells (middle and right). Images show H&E staining of para-aortic lymph node sections (100). D, real-time
RT-PCR analysis of Vav3 expression in PC3 stable cell lines, primary tumors, and their corresponding metastasized para-aortic lymph nodes.
Normalized Vav3 levels were compared with levels in PC3-shControl cells. Numbers represent mean SD (n ¼ 3). The average Vav3 levels from
shControl (shCon) or shVav3 groups are listed below the graph (mean SD).
and suggest that upregulation of Vav3 might represent an
important mechanism in prostate tumor progression and
metastasis.
Expression of Vav3 and EphA2 correlates with prostate
cancer progression
In previous studies, both Vav3 and EphA2 were upregulated in prostate cancer specimens (13, 18). However, these
studies did not describe the relationship of this upregulation to different tumor stages. The present study evaluated
Vav3 and EphA2 expression in prostate cancer specimens
with defined stages and grades using immunohistochemistry, with grouping using the tumor–node–metastasis (TNM)
staging system. The evaluations included 75 primary prostate tumors, 15 metastatic tumors, and 16 adjacent nontumors. The staining intensities of both proteins correlated
well with the prostate cancer stage (Fig. 6A). As shown
in Fig. 6B, a high percentage of stage IV (85.7%) and
metastatic (80%) samples displayed both Vav3 and EphA2
expression. These data suggest the possible involvement of
Vav3 and EphA2 in the development of advanced prostate
cancer.
3006
Cancer Res; 72(12) June 15, 2012
The potential use of Vav3 as a prognostic marker of earlystage prostate cancer
Analysis of Vav3 expression in 50 primary radical prostatectomy specimens, of stage no higher than IIB, evaluated the
prognostic potential of Vav3 expression in early prostate
cancer. The overall 7-year biochemical failure-free survival
rate was significantly lower in patients with high Vav3 expression than in those with low Vav3 expression (60% vs. 90%; Fig.
6C). In patients with prostate cancer, the return of increased
levels of prostate-specific antigen (PSA) indicates treatment
failure, providing clinical evidence of tumor recurrence (29).
The Vav3 oncoprotein might, therefore, provide a suitable
clinical prognostic marker for the early prediction of prostate
cancer recurrence following radical prostatectomy.
Discussion
The present study indicates the significance of RTK-mediated activation of Vav3, especially the EphA2-Vav3-Rac1 signaling axis, in prostate cancer metastasis. Upon activation of
EphA2 or other RTKs, Vav3 is recruited to the receptor(s) and is
tyrosine-phosphorylated. The activated Vav3 then promotes
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Published OnlineFirst June 1, 2012; DOI: 10.1158/0008-5472.CAN-11-2502
EphA2-Vav3-Rac1 Signaling in Prostate Cancer Metastasis
A
Vav3
0.1mm
0.1mm
0.1mm
0.1mm
0.1mm
EphA2
0.1mm
0.1mm
0.1mm
N
II
0.1mm
III
M
C
1.0
Low Vav3 expression
0.8
High Vav3 expression
0.6
0.4
0.2
P = 0.0192
0.0
0
Rac1 activation among others, such as increased MMPs, to
regulate migration/invasion of prostate cancer cells in the
metastatic processes (Supplementary Fig. S6).
The Vav3 oncoprotein is a signaling transducer downstream
of RTKs in various signaling pathways. The study findings
indicated that EphA2 is a key upstream RTK activator of Vav3
in prostate cancer cells (Fig. 3A–D). In a similar manner to
Vav3, EphA2 expression markedly increased in androgenindependent prostate cancer cells (Fig. 1B) and in
advanced-stage human prostate cancers (Fig. 6A and B). In
a breast cancer model, EphA2-deficient mice displayed
decreased tumor volume, microvasculature density, and lung
metastasis (30). The EphA2 receptor might, therefore, play a
significant role in prostate cancer metastasis by activating
Vav3-Rac1 signaling. Other RTKs might also mediate prostate
cancer progression through Vav3 signaling, such as EGF and
Met receptors (Supplementary Fig. S3B and S3D).
www.aacrjournals.org
0.1mm
IV
B
Biochemical failure-free survival
Figure 6. Correlation of EphA2 and
Vav3 expression with late-stage
prostate cancer and patients with
higher Vav3 expression show higher
probability of prostate cancer
posttreatment recurrence. A and B,
immunohistochemical (IHC) analysis
of Vav3 and EphA2 expression in
different TNM stages of human
prostate cancer (B). Representative
microphotographs of
immunohistochemical staining
(200; A). The staining intensities
were evaluated by 2 independent
investigators. N, adjacent nontumor; M, distant metastasis. C,
retrospective analysis of Kaplan–
Meier biochemical failure-free
survival curves from 50 patients with
cancer stages no higher than IIB.
Patients were grouped into low
Vav3-expressing versus high Vav3expressing groups for follow-up
analysis.
20
40
60
80
100
120 (mo)
The EphA2 receptor is an important oncoprotein in prostate
cancer progression (16). However, the mechanism by which
EphA2 promotes prostate cancer progression remains unclear.
Previous research has suggested that EphA2 overexpression
promotes PC3 cell motility in a ligand-independent manner
(31), whereas ephrinA1-induced kinase activation leads to
inhibition of serum-induced AKT phosphorylation (31) and
integrin function (32), resulting in decreased migration and
adhesion ability. In the study of Pratt and Kinch, however,
ephrinA1-induced activation of EphA2 stimulated mitogenactivated protein (MAP)/extracellular signal-regulated kinase
(ERK) kinase signaling (33). Cancer cells with the EphA2
kinase–deficient mutant have shown reduced metastasis in
prostate carcinoma (16) and breast cancer (10), indicating the
importance of EphA2 kinase–dependent signaling in metastasis. The results of the present study indicate that Vav3 is
activated in an EphA2 kinase–dependent manner (Fig. 3A
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Lin et al.
and B), resulting in increased cell migration/invasion in LNCaP
(Fig. 3D), C4-2 (Supplementary Fig. S4G and S4H), and PC3
(Supplementary Fig. S4C and S4D) cells under serum-free
conditions. Although the exact mechanism by which EphA2
promotes cancer progression in vivo remains unclear, these
data suggest the significant involvement of EphA2 kinase–
dependent signaling in prostate cancer metastasis.
The Rac1 protein stimulates the formation of lamellipodia
and membrane ruffles to control cell motility (34). Accumulating evidence indicates the important role of Rac1-mediated
signaling in malignant transformation (35, 36). Elevated
expression of Rac1 in prostate cancer (37) and its suppression
of the cyclin-dependent kinase inhibitor p21 (CIP1) in prostate
cancer cells (38) suggest the involvement of Rac1 in prostate
cancer progression. Several Rac-specific GEFs have been linked
to tumor progression, such as Tiam1 (39) and DOCK3 (40). In
addition, Gao and colleagues have developed a small inhibitor,
which specifically interferes with interaction between Rac1
and its GEFs (41). Results from the present study indicate the
significance of another Rac-specific GEFs, Vav3, in prostate
cancer cell migration/invasion (Figs. 2–4) through the Vav3Rac1 signaling axis. Apart from effects on cell motility, preliminary data suggest that the increased invasive ability might
derive from the regulation of production of MMPs (Supplementary Fig. S4A); and in previous studies, Rac1 has been
shown to induce expressions of MMPs (42, 43). Overall, these
data provide evidence to suggest that a mechanism in prostate
cancer metastasis is Rac1-mediated.
In the study of Liu and colleagues, which developed
prostate-specific Vav3 transgenic mice, some of the adult
mice developed tumors (24) with no distant metastases.
Cancer progression involves multiple mutation events; thus,
it is not surprising that Vav3 is not the only driving force in
the metastatic process. In the present study, Vav3 knockdown in PC3 cells, which have intrinsic metastatic potential,
led to significantly lower incidence of para-aortic lymph
node and bone metastasis (Table 1), the prevalent sites of
prostate cancer metastasis.
In the orthotopically implanted mice, the primary tumors in
the PC3-shVav3 group were 2 times smaller than those in the
shControl group. However, the growth rates of PC3-shControl
and PC3-shVav3 cells were similar (Supplementary Fig. S5C).
The cause of the differences in tumor volume between these 2
groups is unclear. One possibility is angiogenesis. Hunter and
colleagues have shown EphA2-Vav2/3-Rac1 signaling involvement in angiogenesis (17), suggesting a role for Vav3 in
angiogenesis. The PC3-shVav3 tumor might be deficient in
angiogenesis processes because of reduced Vav3 expression.
Another possibility is that the in vivo tumor microenvironment
favors the growth of tumor cells in the presence of Vav3
signaling; for example, by way of Vav3-mediated cell–matrix
interaction. The observations of the present study of increased
Vav3 expression in some of the lymph node metastases from
the shControl and shVav3 groups (Fig. 5D) support the role of
Vav3 in promoting prostate cancer metastasis.
Biochemical failure-free survival is a valuable clinical parameter for the risk of development of local recurrence, distant
metastasis, and prostate cancer–specific mortality in prostate
cancer (29). In the present study, approximately 40% of
patients with high Vav3 expression developed PSA recurrence
within 80 months of radical prostatectomy (Fig. 6C), whereas
the low Vav3 group had almost no recurrence of elevated PSA
levels. These patients' cancer stages were no higher than stage
IIB, indicating the potential use of Vav3 prognostic marker of
early disease outcome following initial treatment. Given the
important clinical implications of this observation, further
analysis including a greater number samples is warranted.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank the Pathology Core Laboratory of the National Health
Research Institutes for H&E and immunohistochemical staining and Dr. Simon
Hall (Mount Sinai School of Medicine) for the gift of C4-2.
Grant Support
This work was supported by grants from the National Health Research
Institutes, Taiwan (99A1-MGPP11-014) and the Department of Health, Taiwan
(DOH100-TD-111-004).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 28, 2011; revised February 20, 2012; accepted March 5, 2012;
published OnlineFirst June 1, 2012.
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