Oncogene (2006) 25, 2285–2296
& 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00
ORIGINAL ARTICLE
RhoC GTPase is required for PC-3 prostate cancer cell invasion but not
motility
H Yao1, EJ Dashner1, CM van Golen2 and KL van Golen1
1
Division of Hematology/Oncology, The Department of Internal Medicine, The University of Michigan Comprehensive Cancer
Center, Ann Arbor, MI, USA and 2The Department of Neurology, The University of Michigan Medical School, Ann Arbor, MI, USA
It is projected that in 2005, approximately 220 900 men
will be newly diagnosed with carcinoma of the prostate
(CaP). Men who are diagnosed with locally advanced or
metastatic disease undergo androgen ablation therapy and
most will relapse and progress within 18 months.
Metastasis to bone is the major clinical concern during
CaP progression, as it is associated with intractable pain,
bone fracture and paralysis resulting from spinal cord
compression. Therefore, an understanding of the key
mechanisms involved in CaP cell bone metastasis is vital
to development of novel treatments. The Rho GTPases are
molecular switches involved in cell survival, motility and
invasion. Increased expression of RhoC GTPase is linked
to enhanced metastatic potential in multiple cancers;
however, the role of RhoC GTPase in CaP metastasis has
not been addressed. In the current study, we demonstrate
that RhoC GTPase is expressed and active in PC-3 CaP
cells. RhoC inhibition, either pharmacologically with C3
exotransferase or molecularly through expression of a
dominant-negative RhoC, promotes IGF-I stimulated
random motility but decreases in vitro invasion and
experimental metastases. Inhibition of RhoC activity
results in drastic morphologic changes and alterations in
the expression and distribution of focal adhesion-related
proteins. These data suggest that RhoC inhibition leads to
activation of other GTPases involved in nondirected
motility and that expression of active RhoC is required
for the invasive phenotype of PC-3 cells.
Oncogene (2006) 25, 2285–2296. doi:10.1038/sj.onc.1209260;
published online 28 November 2005
Keywords: RhoC GTPase; Rac1 GTPase; insulin-like
growth factor-1; collagen 1
Introduction
This year alone, an estimated 29 900 men will die of
prostate cancer (CaP) in the United States, and the vast
Correspondence: Assistant Professor KL van Golen, Department of
Internal Medicine, The University of Michigan Cancer Center, 200
Zina Pitcher Place, R-4048A Kresge II, Ann Arbor, MI 48109-0548.
E-mail: Kgolen@umich.edu
Received 8 August 2005; revised 13 October 2005; accepted 13 October
2005; published online 28 November 2005
www.nature.com/onc
majority of them will have extensive bone metastasis
(Alcover et al., 1994; Abate-Shen and Shen, 2000;
American Cancer Society, 2005). Metastatic bone
lesions are associated with intractable pain, bone
fracture and spinal compression that can lead to
paralysis (Cancer Management: A Multidisciplinary
Approach, 1999; Thalmann et al., 2000). Currently,
the mechanisms that underlie CaP metastasis are poorly
understood. Therefore, molecules involved in CaP cell
motility and invasion must be identified to design novel
therapeutic strategies for metastatic CaP. Recent studies
have focused on Ras homology (Rho) kinase (a.k.a.
ROCK), the downstream effector protein of the mono-
meric G-proteins RhoA and RhoC (Somlyo et al., 2000,
2003). The ROCK inhibitor Wf-536, used in conjunction
with the matrix metalloproteinase inhibitor Marimastat,
significantly inhibits experimental tumor growth, tumor
angiogenesis and increased survival of mice bearing PC-
3 xenotransplants (Somlyo et al., 2003). Other investi-
gators have demonstrated that treatment of PC-3 cells
with the ROCK inhibitor Y27632 retards in vitro PC-3
cell migration (Somlyo et al., 2000). These results
underscore the importance of the molecules that lie
upstream of ROCK, specifically, the Rho GTPases.
The Rho GTPases belong to the Ras superfamily of
small GTP binding proteins and act as molecular switches
involved in all aspects of cellular motility and invasion,
including polarity, cytoskeletal organization and signal
transduction from the extracellular environment (Kjoller
and Hall, 1999; Price and Collard, 2001; Ridley, 2001;
Takai et al., 2001). Like Ras, Rho cycles between an
inactive GDP-bound and active GTP-bound state that
can activate downstream effector proteins (Olson, 1996;
Geyer and Wittinghofer, 1997). To be fully effective in
producing an invasive phenotype, Rho proteins need to
complete a full GTPase cycle and continue to cycle,
transitioning between inactive and active states (Hall,
1990; Moorman et al., 1999; Sander et al., 1999; Danen
et al., 2000). John Collard and his group elegantly
demonstrated a reciprocal relationship of activation
between RhoA GTPase and Rac1 (Sander et al., 1999;
Zondag et al., 2000). Therefore, although one isoform of
Rho may predominate in a cancer cell, activation of other
GTPases must occur during motility, migration and
invasion, to effectively reorganize the cytoskeleton form-
ing stress fibers, lamellipodia and filopodia (Nobes and
Hall, 1995; Hall, 1998; Price and Collard, 2001).
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RhoC GTPase is a Rho protein implicated in the
progression and metastasis of several cancers, including
breast, ovarian, pancreas, gastric cancer, hepatocellular
carcinoma, bladder, colorectal and cutaneous melano-
ma (Moscow et al., 1994; del Peso et al., 1997; Suwa
et al., 1998; Fritz et al., 1999; van Golen et al., 1999,
2000; Imamura et al., 1999; Clark et al., 2000; Sahai and
Marshall 2002; Carr et al., 2003; Horiuchi et al., 2003;
Kamai et al., 2003; Marionnet et al., 2003; Shinto et al.,
2003; Wang et al., 2003; Kondo et al., 2004). Our
laboratory has demonstrated that ectopic expression of
wild-type RhoC in immortalized human mammary
epithelial cells leads to a metastatic phenotype (van
Golen et al., 2000a, b). We have also demonstrated that
RhoC can be used as a prognostic marker to identify
small breast tumors (o1 cm) with a high likelihood to
metastasize (Kleer et al., 2002). Recently our attention
has turned to the potential role of RhoC in CaP
metastasis, particularly metastasis to bone.
In several aggressive cancers, including CaP, activa-
tion of the Rho GTPases, including RhoC, occurs
through protein tyrosine kinase growth factor receptor
stimulation (van Golen, 2003). Motility of many cancer
cell types is stimulated by growth factors such as insulin-
like growth factor I (IGF-I) (Sahai and Marshall, 2002).
Increased levels of the type I IGF receptor (IGF-IR) are
found in the majority of primary and metastatic CaP
patient tumors (Hellawell et al., 2002). Functional IGF-
IR is expressed on PC-3 cells (Iwamura et al., 1993;
Kimura et al., 1996), and bone is a rich source of IGF-I,
which is thought to act as a chemoattractant in CaP
bone metastasis (Cooper and Pienta, 2000). CaP cells
further upregulate IGF-IR expression upon localization
to bone (Rubin et al., 2004). Although the link between
the IGF system and CaP progression is well established,
recent new drug developments have led to renewed
pharmaceutical interest in IGF signaling pathways as
therapeutic targets (Zhang and Yee, 2004; Garber, 2005;
Mitsiades and Mitsiades, 2005).
In the present study, we demonstrate that RhoC is
highly expressed and active in the human PC-3 cell line,
which is derived from a bone metastasis. Furthermore,
RhoC is responsible for IGF-I-mediated invasion of PC-
3 cells but not for IGF-I-stimulated motility. Inhibition
of RhoC activity leads to increased Rac1 and Cdc42
expression and activation. This in turn leads to an
increase in linear motility, but decreased directed
migration and invasion. Finally, inhibition of RhoC
activity results in a change in the expression and cellular
distribution of focal adhesion-associated proteins. These
data provide insight into the potential mechanisms of
CaP cell invasion, a key component in the metastatic
cascade.
Results
Rho GTPase expression in CaP cells
We previously demonstrated that RhoC GTPase is a
putative mammary oncogene responsible for the motile
and invasive capabilities of aggressive breast cancer cells
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Figure 1 Expression of RhoA and RhoC GTPases in LNCaP,
DU145 and PC-3 CaP cell lines. Panel a shows the semiquantitative
RT-PCR analysis of 5 mg total RNA using primers specific for
RhoA, RhoC and GAPDH (as a loading control). No noticeable
differences were seen in RhoA expression amongst the three cell
lines tested, whereas only the LNCaP and PC-3 cells expressed
RhoC mRNA. Panel b shows the Western blot and GTPase
activation for the RhoA and RhoC proteins. A GST-fusion protein
of the Rho binding domain of rhotekin was used to selectively pull
out active RhoA or RhoC. Actin was used as a loading control of
the immunoblot analysis. No differences in RhoA protein
expression or activation were observed for the three lines. Only
the PC-3 cell line expressed active GTP-bound RhoC protein.
Actin was used as a loading control.
(van Golen et al., 1999, 2000a, b, 2002). Increasing
evidence from several laboratories suggests that RhoC
expression is associated with particularly aggressive,
metastatic cancers; however, this has never been
reported for CaP (Moscow et al., 1994; del Peso et al.,
1997; Fritz et al., 1999; Imamura et al., 1999; Clark
et al., 2000; Sahai and Marshall, 2002; Carr et al., 2003;
Horiuchi et al., 2003; Kamai et al., 2003; Marionnet
et al., 2003; Shinto et al., 2003; Wang et al., 2003;
Kondo et al., 2004). We hypothesized that RhoC may be
involved in CaP metastasis. To answer this question, we
screened the LNCaP, DU145 and PC-3 cell lines for
RhoC expression. RhoA GTPase, a close relative of
RhoC, has been implicated in PC-3 cell invasion;
therefore, we also examined RhoA expression. RhoA
GTPase is highly expressed at the mRNA (Figure 1a,
left panel) and protein (Figure 1b, left panel) level in all
three CaP cell lines tested. However, RhoC message was
detected only in the LNCaP and PC-3 cell lines
(Figure 1a, right panel). Active, GTP-bound RhoA
was detected in all three prostate cancer cell lines. In
contrast, only the PC-3 cell line, which was originally
derived from a skeletal metastasis, expressed active
RhoC protein (Figure 1b, right panel). Although the
LNCaP cell line has detectable levels of RhoC message,
it does not express appreciable levels protein, suggesting
that RhoC expression and activation may be associated
with the PC-3 metastatic phenotype.
RhoC GTPase activity affects PC-3 cell motility
and invasion
To determine whether expression of RhoC facilitates
cellular motility stimulated by elements found in the
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H Yao et al
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bone environment, we compared cell motility using a
colloidal blue-fluorescent bead random motility assay
and measured the area of phagokinetic tracks produced
by the cells (Figure 2a). The conditions of this assay
were adjusted to test motility in response to insulin-like
growth factor-I (IGF-I) stimulation on a collagen type I
(col I)-coated surface. Both IGF-I and collagen I are
present in the bone microenvironment and are impli-
cated in facilitating CaP dissemination, adherence and
survival (Kiefer and Farach-Carson, 2001; Rubin et al.,
2004; Chung et al., 2005; Yin et al., 2005). Each of the
three CaP cell lines tested have functional IGF-IR
(Kimura et al., 1996). At 16 h after stimulation with
5 nM IGF-I, the PC-3 cells produced unique circular
phagokinetic tracks many times the diameter of their cell
bodies (Figure 2a). Interestingly, when PC-3 cells were
pretreated with C3 exotransferase, a potent inhibitor of
Rho GTPases, and then stimulated with IGF-I, PC-3
cells no longer displayed a circular motility but became
extremely linearly mobile (Figure 2a). Quantitation of
the phagokinetic tracks produced by PC-3 cells demon-
strated a significant increase in area in cells treated with
C3 exotransferase prior to IGF-I stimulation
(Figure 2a). This observation is contrary to what we
have observed in other cell systems such as breast (van
Golen et al., 2000a, b). LNCaP cells are nonmotile, and
DU145 cells demonstrate moderate linear motility that
is inhibited by C3 exotransferase treatment. Since C3
exotransferase has specific affinity for the Rho proteins
(RhoC>RhoA>>RhoB) and very little affinity for
Rac1 or Cdc42, these data suggest that RhoC activity
may negatively modulate PC-3 cell random linear
motility (Aktories and Hall, 1989; Aktories et al.,
1989; Aktories, 1997). In contrast, since RhoC is not
expressed in the DU145 cells, inhibition of RhoA by C3
exotransferase treatment retards DU145 random linear
motility.
Next, we determined whether C3 treatment also
affected migration in response to a chemoattractant Figure 2 Effect of Rho GTPase activation on PC-3 cell motility,
(also termed directed motility). In this assay a Transwell migration and invasion. Panel a shows a comparison of the motile
chamber was used to determine the migratory capabil- capabilities of the CaP cells using a blue-fluorescent bead random
motility assay after stimulation with IGF-I on a collagen I-coated
ities of PC-3 cells through a filter with 8 mm pores using surface. In this assay, a colloid of blue-fluorescent polymer beads
5 nM IGF-I as a chemoattractant (Figure 2b). We found are overlaid onto 96-well plates coated with collagen I. The cells are
that the PC-3 cells were highly migratory towards IGF- allowed to attach for 1 h in serum-free medium and then stimulated
I. However, in contrast to the random motility assay with 5 nM IGF-I. After growth factor stimulation, motile cells
phagocytize the colloid and the area of the resulting phagokinetic
described above, the ability of cells to migrate in tracks are measured 24 h later. To determine the role of Rho
response to IGF-I is significantly inhibited by C3 proteins in conferring a motile phenotype, cells were treated
exotransferase treatment. overnight with 10 mg C3 exotransferase, a potent inhibitor of Rho
Similar results were observed when the cells were proteins. Rho inhibition significantly increased the PC-3 cells linear
tested for haptotactic migration through an 8 mm filter random motility (*P ¼ 0.001). Panel b demonstrates the results of
directed migration, haptotaxis (with a gradient of 1 nM in the top
with a gradient of 1 nM IGF-I in the upper chamber to chamber to 5 nM IGF-I in the lower chamber) or invasion assay
5 nM IGF-I in the lower chamber (Figure 2b). Untreated through a Matrigel or collagen I-coated filter. In each assay the
PC-3 cells move readily toward the higher IGF-I cells were placed in the top of a Transwell filter and migrated
concentration; however, C3 treatment abrogates this towards a chemoattractant. In contrast to the random motility
assay, C3 treatment (gray bars) significantly decreased PC-3
affect. movement as compared with the untreated cells (black bars,
Finally, the cells were tested for their ability to invade *Po0.05).
through a Matrigel- or collagen I-coated filter in
response to 5 nM IGF-I as a chemoattractant. In these
systems, the PC-3 cells are highly invasive (Figure 2b). motility and invasion in PC-3 cells are particularly
C3 exotransferase treatment significantly inhibits CaP regulated by different GTPases. Specifically, RhoC
cell invasion. Taken together, these data suggest that activity is required for directional motility toward a
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 Requirement of RhoC GTPase for prostate cancer invasion
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chemoattractant (i.e. migration, haptotaxis and inva-
sion), but limits linear random motility.
Since C3 exotransferase also inhibits RhoA GTPase,
stable dominant-negative RhoC-expressing PC-3 (PC-3/
dnRhoC) cells were produced. Dominant-negative
GTPases act by entering into a nonproductive interac-
tion with Rho guanine exchange factors (GEFs), the
proteins responsible for catalyzing the exchange of GDP
for GTP on the GTPase. Given that RhoC and RhoA
are 91% homologous on the protein level, we speculated
that they may share a common activating GEF, and
therefore expression of a dominant-negative RhoC
would affect RhoA activity. No significant differences
in active or total RhoA levels were detected in the PC-3/
dnRhoC cells compared with the parental PC-3 or PC-3/
pcDNA3.1 vector control cells (Figure 3b). In contrast
to the observed RhoA activity, GTP-bound RhoC is
reduced by B65% in the RhoC/dnRhoC cells and
B60% in C3 exotransferase-treated PC-3 cells com-
pared with the parental and vector control and cell lines
(Figure 3a). However, there are no significant changes in
total RhoC levels. These data, taken from four separate
analyses, suggest that RhoC and RhoA, although highly
homologous, are activated by distinct RhoGEFs in the
PC-3 cells. Therefore, expression of a dominant-negative
RhoC specifically decreases activity of RhoC without
affecting RhoA activation. No differences in monolayer
cell growth were observed between the parental, vector
and dnRhoC-expressing PC-3 cells (Figure 3c).
To determine whether expression of dominant-nega-
tive RhoC recapitulates the effect of C3 exotransferase
treatment on PC-3 cells, we performed a random
motility assay as before. Parental PC-3, PC-3/
pcDNA3.1 vector control and PC-3/dnRhoC stable

Figure 3 Expression of a dominant-negative RhoC in PC-3 cells. RhoC containing a T19N mutation was introduced into PC-3 and
stable clones selected. The data are from a representative polyclonal population. Panel a shows the result of triplicate analysis of RhoC
protein expression (dark gray bars) and activation (light gray bars). RhoC activation was compared with a vector control transfectant
left either untreated or treated with C3 exotransferase. C3 treatment also leads to decreased RhoC activation comparable to the
dnRhoC expressing line. Panel b shows the result of triplicate analysis of a GTPase activation assay and Western blot analysis of
RhoA. This was performed to demonstrate that the dominant-negative version of RhoC did not interfere with RhoA activation. No
significant difference in RhoA expression (dark gray bars) or activation (light gray bars) was observed in the parental, vector control or
dominant RhoC expressing PC-3 cells. Panel c is an MTT assay comparing the monolayer growth rates of parental (’), vector control
(m) and dnRhoC () PC-3 cells.

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Figure 4 RhoC GTPase inhibits PC-3 linear random motility but
promotes directed migration and invasion. Panel a shows the
quantitation of the area of phagokinetic tracks produced in a
random motility assay comparing wild type, vector control and
dnRhoC expressing PC-3 cells. The motility assay was performed
using collagen I as a base substrate and 5 nM IGF-I to stimulate
motility for 16 h. Similar to what was observed when the PC-3 cells
were treated with the Rho inhibitor C3 exotransfersase, expression
of dnRhoC led to increased linear motility. Panel b shows a
comparison of parental (light gray bars), vector control (dark gray
bars) and dominant-negative RhoC (black bars) PC-3 cells in a
directed motility, Matrigel and collagen I invasion assays.
Compared with the control cell lines, the PC-3/dnRhoC cells
demonstrated a significant decrease in directed migration and
invasion (*Po0.05).
transfectants were plated onto blue fluorescent beads
overlaid onto a collagen I-coated surface, stimulated
with 5 nM IGF-I and analysed 24 h later. Similar to C3
exotransferase-treated PC-3 cells, PC-3/dnRhoC trans-
fectants displayed increased linear motility (Figure 4a).
Quantitation of the areas of phagokinetic tracks from
three separate assays demonstrates that the PC-3/
dnRhoC cells have a threefold increase in the area of
their phagokinetic tracks as compared with the control
PC-3 cells.
The effect of dnRhoC on migration and invasion was
evaluated as described previously. Migration and inva-
sion in PC-3/dnRhoC cells are approximately 2- and
2.5-fold less, respectively, than that of the control PC-3
cells (Figure 4b). This suggests that RhoC activation
promotes PC-3 cell-directed migration and invasion, but
limits linear random motility.
RhoC GTPase activity affects PC-3 cell morphology
One observable consequence of inhibiting RhoC
GTPase activity is a profound change in PC-3 cell
Requirement of RhoC GTPase for prostate cancer invasion
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morphology. Figure 5A shows phase contrast, laser
confocal and scanning electron micrographs of parental
PC-3 (panels a, d and g), PC-3/pcDNA3.1 (panels b, e
and h) and PC-3/dnRhoC (panels c, f and i) cells. Both
control cell lines are relatively flat with very broad
lamellipodia nearly encompassing the circumference of
the cell. In contrast, the PC-3/dnRhoC cells are
elongated with smaller lamellipodia located on the
leading edge of the cell.
Immunofluorescent staining for actin and the cyto-
skeletal protein vinculin demonstrates profound differ-
ences between PC-3 and PC-3/dnRhoC cells when
grown on collagen I and stimulated with IGF-I
(Figure 5A, panels j–o). In PC-3 cells the cytoskeleton
appears diffuse and appears branched leading to broad
deformation of the cells’ lamellipodia. Vinculin that
does not appear to be associated with actin bundle ends
is localized in the center of the parental PC-3 cell. In
contrast, colocalized actin and vinculin in PC-3/
dnRhoC cells are isolated to the tips of the protrusions
at the leading and trailing ends of the cell. Actin is
highly organized along the periphery of the cells in an
arrangement reminiscent of concave actin bundles and
the bulk of vinculin is localized throughout the main
body of PC-3/dnRhoC cells.
The change in PC-3 morphology is reminiscent of the
epithelial to mesenchymal transition (EMT) described
for other tumor cell types (Chang et al., 2003; Bockhorn
et al., 2004). Figure 5B shows the Western blot analysis
for molecular markers associated with EMT: ZO-1, E-
and N-cadherin. As typically described for other cell
types undergoing EMT, a switch in expression of E- to
N-cadherin and a decrease in ZO-1 are observed in the
PC-3/dnRhoC cells (Figure 5B), suggesting that RhoC
may act as a molecular switch for an EMT-like change
in PC-3 cells.
RhoC GTPase activation influences expression
and activation of Rac1 and Cdc42
A reciprocal relationship between RhoA and Rac1/
Cdc42 activity has been described for motile NIH3T3
cells (Sander et al., 1999; Zondag et al., 2000). To
determine whether other members of the Rho GTPase
family are affected by changes in RhoC activity, we
performed Western blot analysis and GTPase activity
assays for Rac1 and Cdc42. Both Rac1 and Cdc42 are
implicated in cellular motility, specifically through the
formation of lamellipodia and filopodia, respectively,
and are therefore prime candidates for mediating the
observed changes in the morphologic characteristics of
PC-3 cells (Ridley and Hall, 1992a, b; Ridley et al., 1992;
Nobes and Hall, 1995). The process of linear motility
requires coordinated changes in Rac1 and Cdc42
activity. Therefore, we determined the expression and
activation profile of these two GTPases over an
extended time course using conditions similar to the
random motility assay. Serum-starved PC-3 and PC-3/
dnRhoC cells were plated onto collagen I, stimulated
with IGF-I and analysed 0, 5, 15, 60, 120 and 180 min
after stimulation. Quantitative changes in Rac1 and
Cdc42 expression and activation are observed in PC-3/
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Figure 5 Inhibition of RhoC GTPase alters the morphology of PC-3 cells. Panel A shows phase (a–c), laser confocal (d–f) and
scanning electron (g–i) micrographs of PC-3 (a, d and g), PC-3/pcDNA3.1 (b, e and h) and PC-3/dnRhoC (c, f and i). Panels j–o show
immunofluorescent (IF) staining of vinculin (j and m) and rhodamine-phallodin for actin stress fibers (k and n) with merge (l and o).
Cells were grown on collagen I, stimulated with 5 nM IGF-I and imaged as described in the Materials and methods section. Panel B
shows the Western blot analysis of 30 mg total protein from PC-3, vector control and PC-3/dnRhoC cells for expression of EMT-
associated proteins ZO-1, E- and N-cadherin. Densitometry measurements are the averages of three independent experiments. The
value for the PC-3 parental cells was set at 1 and all other values were measured against it.

dnRhoC cells compared with parental PC-3 cells
(Figure 6A). In the PC-3 cell line, a low, persistent level
of Rac1 and Cdc42 activation is observed throughout
the time course (Figure 6A). In contrast to parental
cells, PC-3/dnRhoC cells display a marked increase in
expression and activation of both Rac1 and Cdc42 at
60 min after IGF-I stimulation, which persists up to
180 min (Figure 6A). Of particular note is a striking
increase in Cdc42 expression from 0 to 60 min after
stimulation.
Interestingly, levels of phosphorylated Rac1 and
Cdc42 are reduced in PC-3/dnRhoC- and C3-treated
PC-3 cells grown on collagen I and stimulated with IGF-
I for 60 min (Figure 6B). Rac1 and Cdc42 are
phosphorylated on serine 71 and in vitro assays suggest
that phosphorylation of Rac1 inhibits the exchange of
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GDP for GTP (Kwon et al., 2000). Thus, decreased
phosphorylation of Rac1 may contribute to an increase
in Rac1 and Cdc42 activity.
RhoC GTPase activity modulates focal adhesion protein
activation and localization
The phenotypic changes observed in PC-3/dnRhoC cells
are identical to those previously described for cells
transfected with a paxillin deletion mutant (West et al.,
2004). Specifically, CHO.K1 fibroblasts transfected with
paxillin lacking the LD4 domain, which binds paxillin
kinase linker (PKL), demonstrate increased linear
motility, decreased invasion and increased, persistent
Rac1 activation (West et al., 2004). With this in mind,
we determined the effect of RhoC activation on focal
adhesion proteins. Total and phosphorylated focal
 Requirement of RhoC GTPase for prostate cancer invasion
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Figure 6 Expression and activation of Rac1 and Cdc42 in PC-3 cells are increased when RhoC GTPase is not active. Panel A, to
determine the duration of Rac1 and Cdc42 expression and activation, serum-starved PC-3 and PC-3/dnRhoC cells were plated on
collagen I, stimulated with IGF-I and harvested at 0, 5, 15, 60, 120 and 180 min. Immunoblot analysis is shown in (a). An aliquot of
30 mg total protein was taken to determine total Rac1 or Cdc42 levels; the remainder of the protein lysates was used to determine levels
of active GTPase. Densitometry was performed on each of the samples and a ratio of GTP versus total protein was determined and is
shown for the duration of the time course (b). PC-3 cells (’) are represented by the dashed line and the dnRhoC cells (m) by the solid
line. A separate panel (c) shows the changes for Rac1 observed in the first 15 min of the experiment. The t ¼ 0 value of parental PC-3
cells was set to 1 and all other values were measured against it. The values shown are averages from at least three separate experiments.
Panel B shows the immunoblot analysis for total and serine 71 phosphorylated Rac1 and Cdc42. PC-3, PC-3/pcDNA3.1 vector and
PC-3 dnRhoC cells were grown on collagen I, stimulated with 5 nM IGF-I and protein harvested 60 min later. A 50 mg aliquot of each
protein was analysed by immunoblotting with monoclonal antibodies specific for Rac1, Cdc42 and phospho-Rac1/Cdc42. GAPDH
was included as a loading control.

adhesion kinase (FAK) and phosphorylated paxillin
levels were decreased in the PC-3/dnRhoC cells, whereas
total paxillin and PKL protein levels essentially
remained unchanged (Figure 7A). More interestingly,
immunoprecipitation experiments demonstrate that the
interaction of PKL with paxillin is decreased in PC-3/
dnRhoC cells (Figure 7B). In contrast, FAK and PKL
or FAK and paxillin coimmunopreciptated, indicating
that their interaction was not altered (Figure 7A).
Next, colocalization of paxillin with FAK (panel a),
PKL with FAK (panel b) and paxillin with PKL (panel
c) was determined (Figure 7C). All of the focal adhesion
proteins colocalize within the cell, but the distribution of
these proteins is altered in PC-3/dnRhoC cells compared
with the parental PC-3 line (Figure 7C). Fluorescent
localization demonstrated that FAK and PKL coloca-
lize in both PC-3 and PC-3/dnRhoC cells. Paxillin also
colocalized with both FAK and PKL in these cells.
However, in the PC-3/dnRhoC cells, PKL is found
independent of paxillin in the cytoplasm of the cells
(white arrows; Figure 7C vi). These data suggest that the
changes in cell motility, migration and invasion due to
inhibition of RhoC activity may be because of altera-
tions in the localization, activity and interaction of focal
adhesion proteins. Furthermore, taken with the altera-
tions seen in the distribution of the actin cytoskeleton,
these data may indicate turnover of focal adhesions to
focal complexes (Kaverina et al., 2002).
Discussion
The ability of CaP cells to invade and metastasize,
particularly to bone, has been an area of intense
research. Bone contains high levels of IGF ligands,
which are intimately involved in normal bone turnover
and are implicated in metastasis (Chung et al., 2005).
IGF-I not only acts as a mitogenic factor for cells, but
also stimulates cellular motility (reviewed in (Cooper
and Pienta, 2000; van Golen, 2003)). IGF-I potentially
regulates tumor cell motility through the activity of the
Rho GTPases (van Golen, 2003). In particular, IGF-I
stimulation of Rac1 in neuroblastoma cells and RhoA
and RhoC in breast cancer cells is well studied (Jackson
et al., 2001; Meyer et al., 2001; Tanno et al., 2001; Lopez
and Hanahan, 2002). Several laboratories, including our
own in the present study, have demonstrated that IGF-I
acts as a chemoattractant for CaP cells Cooper and
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Figure 7 Inhibition of RhoC leads to changes in expression and distribution of focal adhesion proteins. Panel A shows the Western
blot analysis for phosphorylated and total focal adhesion kinase (FAK), paxillin and paxillin kinase linker (PKL). Aliquots of 50 mg
total protein were analysed for phosphorylated and total protein levels. Relative ratios of phosphorylated to total protein levels are
shown for FAK and paxillin. Relative levels of total PKL are also shown. Densitometry measurements are the averages of three
independent experiments. The value for the PC-3 parental cells was set at 1 and all other values were measured against it. Panel B
interaction of focal adhesion proteins was determined by immunoprecipitation of 500 mg total protein that was pre-cleared with normal
IgG followed by immunoblotting. Normal IgG negative controls did not produce bands when immunoblotted with antibodies specific
for FAK, paxillin or PKL. Measurement of the relative levels of PKL detected in the paxillin immunopreciptiations is shown. The
value for the parental PC-3 cells was set at 1 and all other values were measured against it. Measurements are the average of three
independent experiments. Panel C shows the immunfluoresence for (a) paxillin (i and iv) and FAK (ii and v) with merge (iii and vi), (b)
PKL (i and iv) and FAK (ii and v) with merge (iii and vi), (c) paxillin (i and iv) and PKL (ii and v) with merge (iii and vi). The arrows
indicate that PKL is not associated with paxillin.

Pienta (2000). Furthermore, collagen I, an integral
component of mineralized bone matrix, affects CaP cell
migration and invasion through integrin activation
(Keely et al., 1998; Jaffe and Hall, 2002). Through
interplay of growth factor and integrin signaling, Rho
Oncogene
GTPases are temporally and spatially activated, allow-
ing tumor cells to successfully invade, survive and grow
at distant sites (Keely et al., 1998; Jaffe and Hall, 2002).
Thus, growth factor and integrin signaling provide two
potential stimuli for Rho activation (Cooper and Pienta,

2000). Therefore, we chose to investigate the role of
RhoC GTPase in IGF-I and/or collagen I-stimulated
motility, migration, invasion and morphological altera-
tions in PC-3 cells.
Clearly, the Rho GTPases play a central role in tumor
cell invasion and metastasis (Sahai and Marshall 2002).
To date, few studies have attempted to define the Rho
GTPase profile of metastatic CaP cells. A recent study
by Hodge et al. (2003) suggests that increased RhoA
expression and activity are responsible for NF-kB-
mediated PC-3 cell invasion. The invasive capabilities
of highly metastatic PC-3 variants, but not of low
metastatic variants, were increased by introducing a
constitutively active form of RhoA into the cells (Hodge
et al., 2003). Our data suggest that RhoA is a
component in the motile and invasive phenotype of
PC-3 cells; however, we were unable to find a difference
in RhoA expression in our PC-3/dnRhoC transfectants
that could account for the in vitro differences in invasion
detected, compared with parental PC-3 cells. Other
groups have demonstrated that RhoA and Cdc42
activation is involved in cytoskeletal reorganization of
CaP cells. Activation of the G-protein-coupled receptors
called protease-activated receptors (PARs) results in
activation of RhoA and other members of the Rho
GTPase family (Greenberg et al., 2003). Stimulation of
LNCaP cells with PAR-activating peptides, trypsin or
thrombin leads to increased stress fiber formation and
filopodia formation (Greenberg et al., 2003). Similar
cytoskeletal reorganization and activation of RhoA and
Cdc42 is observed when PC-3U cells (a subline of PC-3)
are stimulated with transforming growth factor-b
(Edlund et al., 2002). Although these studies demon-
strate a role for these GTPases in CaP cell cytoskeletal
reorganization, they have not fully made the connection
with motility, invasion or metastasis. Further, it is
becoming increasingly evident that the vast majority of
human tumors overexpress RhoA, but overexpression of
RhoC is limited to particularly aggressive and metastatic
forms of cancer (Moscow et al., 1994; del Peso et al.,
1997; Fritz et al., 1999; Imamura et al., 1999; Clark
et al., 2000; Sahai and Marshall, 2002; Carr et al., 2003;
Horiuchi et al., 2003; Kamai et al., 2003; Marionnet
et al., 2003; Shinto et al., 2003; Wang et al., 2003;
Kondo et al., 2004) The PC-3 cells, derived from a bone
metastasis, were the only CaP cell line to express active
RhoC protein.
Interestingly, we demonstrate that when PC-3 cells are
tested for random motility using collagen I as a
substrate and IGF-I as a stimulant; the phagokinetic
tracks produced suggest that the cells are anchored at
one end allowing them to only move in a circular
pattern. Yet, they are highly motile in directed migra-
tion, haptotaxis and invasion assays. In contrast, when
RhoC is inhibited, the cells become highly randomly
motile but do not invade. Changes in PC-3 cell
morphology, reminiscent of EMT described for other
tumor cell types, is observed when RhoC is inhibited
(Chang et al., 2003; Bockhorn et al., 2004). Accom-
panying the changes in morphology, we distinguish
changes in protein expression of the EMT markers ZO-
Requirement of RhoC GTPase for prostate cancer invasion
H Yao et al
2293
1, E- and N-cadherin. These data suggest that in PC-3
cells, RhoC may act as a determinant switch for EMT.
Collard’s group was the first to demonstrate that
RhoA GTPase and Rac1 are in a reciprocal relationship
of activation during cellular motility and invasion
(Sander et al., 1999; Zondag et al., 2000). In the current
study, we demonstrate that when RhoC is inhibited in
PC-3 cells there is a marked upregulation of Rac1 and
Cdc42 expression and activation. In addition, in PC-3/
dnRhoC cells, we observe changes in expression,
activation and distribution of focal adhesion proteins.
These changes likely represent a switch from focal
adhesions, which are associated with stable adhesion, to
focal complexes, present during active cell motility, that
are typically formed through Rac1 and Cdc42 activity
(Kaverina et al., 2002). The changes in morphology,
Rac1 GTPase activation, random motility and invasion
are similar to those observed in paxillin deletion mutants
(West et al., 2004). Deletion of the LD4 domain of
paxillin results in redistribution of PKL to the cyto-
plasm of CHO.K1 fibroblasts. As a result, cellular
morphology changes, cellular protrusiveness increases,
random motility increases and invasion decreases,
suggesting an increase in focal complex formation.
These cells also have increased Rac1 expression and
prolonged Rac1 activity. Therefore, disrupted interac-
tion between paxillin and PKL promotes prolonged Rac
activity and extended linear motility. In our study, we
observe a decreased interaction between paxillin and
PKL in the PC-3/dnRhoC cells. We have measured a
slight decrease in PKL expression in the PC-3/dnRhoC
cells, which could account for the decreased paxillin/
PKL interaction. However, we also observed a large
amount of PKL in the cytoplasm of the cells suggesting
that PKL is localized away from paxillin.
Paxillin is present in both focal complexes and focal
adhesions and is one of the earliest proteins recruited to
these structures by either integrin engagement or growth
factor stimulation (Turner, 2000; Zaidel-Bar et al.,
2003). Rac-1 promotes focal complex formation,
whereas Rho kinase inhibition induces focal adhesion
disassembly, also resulting in increased focal complex
formation (Zaidel-Bar et al., 2003). Our observations
suggest that high levels of active RhoC in cells may
induce paxillin/PKL interaction, thus reducing the
prolonged Rac activation associated with focal complex
formation, leading to a reduction in cellular protrusions
and associated random motility. Since focal complexes
are rather short-lived structures, active RhoC inhibition
of focal complex formation could in turn promote more
directed migration through stable focal adhesion for-
mation and/or extended interactions with ECM.
In summary, our data suggest an inverse relationship
of active RhoC with Rac1 and Cdc42 in PC-3 cells.
RhoC activity promotes IGF-I-stimulated migration
and invasion while limiting linear motility. In contrast,
when RhoC activation is inhibited, Rac1 and Cdc42
activity increase, promoting linear motility. Concur-
rently, there is a switch reminiscent of an epithelial to
mesenchymal morphology. Expression, activation and
localization of focal adhesion proteins are also affected.
Oncogene
 Requirement of RhoC GTPase for prostate cancer invasion
H Yao et al
2294
Thus, RhoC appears to act as a critical and dynamic
switch controlling PC-3 cell linear and directed move-
ment. Our laboratory is currently exploring the con-
vergence of growth factor and integrin downstream
signaling and its effect on Rho GTPase activation.
Materials and methods
Cell culture
All CaP cell lines were obtained from American Type Culture
Collection (Manassas, VA, USA). The LNCaP cells were each
maintained in 90% RPMI-1640 with 2 mM L-glutamine, 1.5 g/l
sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1 mM
sodium pyruvate and 10% fetal bovine serum (FBS, Gibco,
Carlsbad, CA, USA). The DU145 cells were propagated in
Eagles minimal essential medium with Earles BSS, 2 mM L-
glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM nonessential
amino acids, 1.0 mM sodium pyruvate and 10% FBS (Gibco).
The PC-3 cells were maintained in Ham’s F-12 medium with
1.5 g/l sodium pyruvate, 2 mM L-glutamine and 10% FBS
(Gibco). PC-3 transfectants were maintained in the same
medium as the parental PC-3 cells with the addition of 250 mg/
ml Neomycin (Gibco). All cell lines were maintained at 371C in
a 10% CO2 incubator. C3 exotransferase was introduced into
cells as previously described using a lipid transfer-mediated
method and treated for 16 h before analysis (van Golen et al.,
2000).
Plasmids and transfections
Dominant-negative RhoC (dnRhoC) in pcDNA3.1 was
obtained from Gutherie cDNA Resource center (Sayre, PA,
USA). Empty pcDNA3.1 was used as a vector control
transfectant (Invitrogen, Carlsbad, CA, USA). The dnRhoC
or empty vector was introduced into PC-3 cells using FuGene6
transfection reagent (Roche, Indianapolis, IN, USA) and
selecting cells in 250 mg/ml Neomycin. Expression of the
transgene was confirmed by RT-PCR.
Anchorage-independent growth assays
For anchorage-independent growth assays, a 2% stock of
sterile, low-melt agarose was diluted 1:1 with 2  MEM.
Further dilution to 0.6% agarose was made using 10% FBS-
containing growth medium and 1 ml added to each well of a
six-well plate as a base layer. The cell layer was prepared by
diluting agarose to 0.3, 0.6 or 0.9% with 103 cells in 2.5% FBS-
containing growth medium/1.5 ml/well. A 1 ml layer of
medium was maintained on top of the agar to provide
nutrients. Colonies X100 mm in diameter were counted after
2-week incubation at 371C in a 10% CO2 incubator.
RT-PCR
Semi-quantitative RT-PCR for RhoC and RhoA expression
was performed as previously described using primers specific
for RhoC, RhoA or as a control, GAPDH (van Golen et al.,
2000a, b). Briefly, total RNA was harvested from cells using
Trizol reagent (Life Technologies, Gaithersburg, MD, USA)
and cDNA was made using the AMV-reverse transcriptase kit
(Promega, Madison, WI, USA). RhoC and RhoA transcripts
were PCR amplified from cDNA aliquots using a 1:1000
dilution of Rho-specific primers mixed with GAPDH primers.
PCR products were separated on a 1.2% TAE agarose gel and
visualized with ethidium bromide. The relative intensities of
the Rho and GAPDH bands were measured using an Alpha
Imager 2200 (Alpha Innotech, Co., San Leandro, CA, USA).
Oncogene
MTT assay
Monolayer growth was measured using MTT (Sigma Chemical
Co.) as previously described (van Golen et al., 2000). Briefly,
cells were plated at densities ranging from 100 to 10 000 cells in
200 ml growth medium in six replicate wells of a 96-well plate
and incubated in a 371C incubator under 10% CO2. Cell
growth was measured at 0, 1, 2, 3 and 5 days after plating. For
the 0 daytime point plates were centrifuged at 1000 r.p.m. in
Sorvall refrigerated tissue culture centrifuge. A 40 ml aliquot of
5 mg/ml MTT was added to the cultures for 1 h at 371C. The
medium was aspirated and 100 ml DMSO (Sigma Chemical
Co.) was added to each well to solubilize the formazon dye.
Absorbance was read on a Dynatech microplate reader at
590 nm.
Western blot analysis
Protein was harvested from cells using RIPA buffer as
previously described (van Golen et al., 2000a, b). Lysates were
separated by SDS–PAGE on a 12% gel, transferred to
nitrocellulose, blocked and probed with a polyclonal antibody
for RhoC (Kleer et al., 2002) or monoclonal antibodies for
RhoA (Cytoskeleton, Denver, CO, USA), Rac1, Cdc42 and
phospho-Rac1/Cdc42 (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), E-cadherin (a gift from Dr Peggy Wheelock), N-
cadherin, PKL (BD Transduction Laboratories, San Diego,
CA, USA), FAK (Santa Cruz and Biolegend, San Diego, CA,
USA), phospho-FAK, paxillin and phospho-paxillin (Bio-
legend). After incubation with a goat anti-rabbit-HRP or goat
anti-mouse-HRP antibody (Santa Cruz), immunoblots were
developed with ECL, exposed to Hyperfilm (Amersham,
Piscataway, NJ, USA) and images recorded on an Alpha
Image 90 documentation system (Alpha Innotech, San
Leandro, CA, USA). Actin or GAPDH were used as loading
controls (Sigma, St Louis, MO, USA). Whenever possible
blots were first probed with the phospho-specific antibody,
stripped and reprobed with an antibody to total protein.
Densitometry was performed using Scion Image version 1.62.
Values for the PC-3 parental line were set at 1 and all other
values were measured against it.
GTPase activation assays
Rho activation assay reagents were supplied to us by John
Collard, PhD, at The Netherlands Cancer Institute (Evers
et al., 2000; Diekmann and Hall, 1995) and used to determine
activation of RhoC in PC cells. Cells, 5–10  106 cells/assay,
were grown in fresh serum-containing medium for 16 h and
lysed with ice-cold GST-FISH buffer (10% Glycerol, 50 mM
Tris pH 7.4, 100 mM NaCl, 1% NP-40 and 2 mM MgCl2).
Protein lysates, 900 ml of 1 mg/ml, were precleared with
glutathione sepharose for 1 h at room temperature. The
cleared lysates were incubated with GST-rhotekin-sepharose
conjugate at 41C for 30 min. The agarose conjugate was
collected by centrifugation, resuspended in Laemelli buffer,
separated by SDS–PAGE, transferred to nitrocellulose and
probed with a RhoC antibody developed by our laboratory
(Kleer et al., 2002). Densitometry was performed using an
Alpha Image 950 imaging system.
Motility, migration, haptotaxis and invasion assays
Random motility assays were also performed using blue
fluorescent bead motility HitKit (Cellomics, Pittsburg, PA,
USA), which is based on the colloidal gold random motility
assay (Albrecht-Buehler, 1977; van Golen et al., 2000a, b).
Cells, 200/well in SFM, were seeded onto a lawn of blue
fluorescent beads in a BSA-coated 96-well plate (BD Bio-
sciences). Cells were stimulated 1 h later with 10% FBS,

incubated 6 h at 371C and fixed. Cells were visualized by
fluorescent microscopy, digitally recorded and the areas of
phagokenetic tracks measured using Scion Image.
The directed migration assay was performed using an
uncoated Transwell filter with 8 mm pores (Costar, Corning,
NY, USA). The lower chamber of the filter was filled with
either serum-free medium or serum-free medium containing
5 nm IGF-I. The Matrigel and collagen I invasion assays were
performed as previously described using precoated Transwell
filters with 8 mm pores (Hendrix et al., 1987; van Golen et al.,
2000a, b). Cells were harvested and resuspended in serum-free
medium containing 0.1% BSA at a concentration of
3.75  105 cells/ml, and 0.5 ml was added to the top chambers.
The haptotaxis assay was performed similar to the migration
assay, except that 1 nM IGF-I was added to the top chamber
with the cells. The chambers were incubated for 24 h at 371C in
a 10% CO2 incubator. The cell suspension was aspirated from
the top chamber of all three assays. Excess Matrigel or
collagen was removed from the invasion assay filter using a
cotton swab. The filters were then cut away from the Transwell
assembly, fixed top side down with methanol to a glass
microscope slide, stained with H&E and the entire surface of
the filters counted. In the directed migration, haptotaxis and
invasion assays, the number of cells that moved in the serum-
free controls was considered as background and was sub-
tracted from the number of cells counted in the samples
containing chemoattractant.
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed as
previously described (van Golen et al., 2002). Briefly, cells
(12 000) grown on collagen I were fixed with buffered 2.5%
glutaraldehyde for 1 h, rinsed with phosphate-buffered saline
(PBS) and postfixed for an additional hour with buffered
osmium tetroxide. After dehydration in ascending strengths of
ethanol, the cells were critical-point dried, mounted onto
standard SEM stubs and gold-sputter coated. They were
viewed using an AMRAY 1000-B scanning electron micro-
scope housed at the University of Michigan Microscopy and
Imaging Laboratories (MIL).
Immunofluorescence
PC-3 and PC-3/dnRhoC cells were grown on collagen I-coated
coverslips (BD Biosciences, San Diego, CA, USA) until 70%
confluence, serum-starved for 16 h, stimulated with 5 nM IGF-I
for 60 min, rinsed three times with 1  PBS pH 7.4 and fixed
with 4% paraformaldehyde in PBS for 20 min at room
temperature. After fixation, cells were rinsed three times with
1  PBS and blocked with 5% normal goat serum (NGS,
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Abbreviations
CaP, prostate cancer; Rho, Ras homology; IGF-I, insulin-like
growth factor I; col I, collagen type I; FAK, focal adhesion
kinase; PKL, paxillin kinase linker; EMT, epithelial to
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Acknowledgements
This work was supported in part by the University of
Michigan Comprehensive Cancer Center support grant (5
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Specialized Program of Research Excellence (P50 CA69568,
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