Research Article
401
Dynamic regulation of ROCK in tumor cells controls
CXCR4-driven adhesion events
Amanda P. Struckhoff1, Jason R. Vitko1, Manish K. Rana1, Carter T. Davis1, Kamau E. Foderingham1,
Chi-Hsin Liu1, Lyndsay Vanhoy-Rhodes2, Steven Elliot2, Yun Zhu2, Matt Burow2 and Rebecca A. Worthylake1,*
1
Departments of Oral Biology and Pharmacology, LSU Health Sciences Center, New Orleans, LA, 70112, USA
Department of Medicine, Tulane University Medical Center, New Orleans, LA 70112, USA
2
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 2 November 2009
Journal of Cell Science 123, 401-412 Published by The Company of Biologists 2010
doi:10.1242/jcs.052167
Summary
CXCR4 is a chemokine receptor often found aberrantly expressed on metastatic tumor cells. To investigate CXCR4 signaling in tumor
cell adhesion, we stably overexpressed CXCR4 in MCF7 breast tumor cells. Cell attachment assays demonstrate that stimulation of
the receptor with its ligand, CXCL12, promotes adhesion of MCF7-CXCR4 cells to both extracellular matrix and endothelial ligands.
To more closely mimic the conditions experienced by a circulating tumor cell, we performed the attachment assays under shear stress
conditions. We found that CXCL12-induced tumor cell attachment is much more pronounced under flow. ROCK is a serine/threonine
kinase associated with adhesion and metastasis, which is regulated by CXCR4 signaling. Thus, we investigated the contribution of
ROCK activity during CXC12-induced adhesion events. Our results demonstrate a biphasic regulation of ROCK in response to adhesion.
During the initial attachment, inhibition of ROCK activity is required. Subsequently, re-activation of ROCK activity is required for
maturation of adhesion complexes and enhanced tumor cell migration. Interestingly, CXCL12 partially reduces the level of ROCK
activity generated by attachment, which supports a model in which stimulation with CXCL12 regulates tumor cell adhesion events by
providing an optimal level of ROCK activity for effective migration.
Key words: CXCL12, CXCR4, ROCK, Flow, Metastasis, Tumor recruitment
Introduction
Metastasis is the result of a complex set of events that culminate
in tumor cell colonization of distal tissues, resulting in deadly
consequences. During the metastatic process, tumor cells acquire
the ability to survive and proliferate in a new tissue environment
(Steeg, 2006). To access these new tissues, metastatic cells display
migratory capacities not present in their normal cell counterparts
(Friedl and Wolf, 2003; Wang et al., 2005). One of these migratory
behaviors is the ability to be trafficked into secondary organs.
Although the mechanisms that govern organ-selectivity of metastasis
are multi-faceted, one important mechanism is the recruitment of
tumor cells by chemokines (Ben-Baruch, 2008; Dittmar et al., 2008;
Gassmann and Haier, 2008). Chemokines are potent regulators of
cellular trafficking within the body, particularly in the case of
directing hematopoietic cell migration. The chemokine receptor
CXCR4 is a potent motomorphogen and is essential not only for
hematopoiesis, but also for regeneration of multiple solid tissues
(Kucia et al., 2005). The expression of CXCR4 is normally
restricted to hematopoietic cells and committed stem cells; however,
aberrant expression of this chemokine receptor has been shown to
be correlated with tumor metastasis (Balkwill, 2004; Zlotnik,
2006). Significantly, blocking CXCR4 signaling inhibits breast
tumor cell metastasis in an intravenous model system (Muller et
al., 2001). It is thought that metastatic tumor cells can hijack the
CXCR4 trafficking signals needed for normal tissue reorganizing
activities, thereby gaining entry to distal organs as potential sites
for colonization.
Rho GTPase signals are responsible for many cell migration
activities (Heasman and Ridley, 2008; Titus et al., 2005; Wennerberg
and Der, 2004). The Ser/Thr kinases, ROCKI and ROCKII (also
known as ROCK-1 and ROCK-2), are RhoA effectors that have
been associated with metastasis in animal models (Hakuma et al.,
2005; Itoh et al., 1999; Lin et al., 2007; Nakajima et al., 2003a;
Nakajima et al., 2003b; Narumiya et al., 2009; Ogawa et al., 2007;
Takamura et al., 2001; Wong et al., 2009; Xue et al., 2007; Ying
et al., 2006). Several lines of investigation implicate ROCKs in the
invasive behavior of metastatic tumor cells (Cardone et al., 2005;
Itoh et al., 1999; Joshi et al., 2008; Kamai et al., 2003; Kitzing et
al., 2007; Li et al., 2006; Wilkinson et al., 2005). Recent studies
have demonstrated that RhoA and ROCK promote invasion of
matrix in response to CXCL12 (Amine et al., 2009; Azab et al.,
2009; Molina-Ortiz et al., 2009). ROCKs also play a role in the
regulation of invasive pseudopodia and/or invadopodia in metastatic
tumor cells. ROCK activity is required for organization of the matrix
surrounding advancing tumor cells to promote invasion in three
dimensions (Provenzano et al., 2008) and ROCKs have been shown
to be required for an ‘amoeboid’ mode of motility that is used by
many metastatic cells to invade through three-dimensional matrix
(Rosel et al., 2008; Sahai and Marshall, 2003; Torka et al., 2006;
Wyckoff et al., 2006). Thus, one way that ROCKs are thought to
contribute to metastasis is by stimulating invasiveness; either by
the promoting invasive protrusions, or remodeling the matrix.
In addition to a role in tumor invasiveness, ROCKs are also
known to regulate cell migration in response to extracellular cues,
including those mediated by chemokines (Moyer et al., 2007;
Samaniego et al., 2007; Tan et al., 2006; Vicente-Manzanares et
al., 2002). Regulation of integrin adhesion is an important
mechanism by which chemokines regulate cellular recruitment.
CXCL12, the ligand for CXCR4, is known as a ‘capture’
chemokine because of its ability to induce arrest of circulating
hematopoietic cells by the vascular endothelium (Alon and Ley,
2008; Laudanna and Alon, 2006; Ley, 2003). Although tumor cell
Journal of Cell Science
402
Journal of Cell Science 123 (3)
recruitment is probably more complex than leukocyte recruitment
and can involve interactions with platelets and other hematopoietic
cells (Borsig, 2008; de Visser et al., 2006; Witz, 2008), tumor cell
recruitment shares several features of leukocyte recruitment
(Balkwill, 2004; Ben-Baruch, 2008; Dittmar et al., 2008; Miles
et al., 2008). Notably, tumor cell trafficking can be directed by
chemokines, and direct adhesive interactions between tumor cells
and the endothelial ligands including selectins, intercellular
adhesion molecule 1 (ICAM-1) and vascular cell adhesion
molecule (1VCAM-1) have been shown to contribute to tumor
cell arrest (Gassmann and Haier, 2008; Glinsky et al., 2003; Jones
et al., 2007; Kobayashi et al., 2007; Zetter, 1993).
Subsequent to arrest, chemokines are further thought to regulate
chemotaxis by spatially coordinating integrin adhesions, such that
adhesion occurs at the front and de-adhesion occurs at the back
end of the migrating cell (Imhof and Aurrand-Lions, 2004;
Kinashi, 2007; Worthylake and Burridge, 2001). Integrins cluster
together with many associated proteins in the cytoplasm to form
adhesion complexes that contain both structural and signaling
molecules that regulate adhesion behavior. The highly dynamic
nature of these adhesion complexes controls the formation of
nascent adhesions and their maturation to more stable integrin
adhesions during initial attachment and subsequent cell migration.
Thus, CXCR4 has the potential to regulate recruitment through
two distinct adhesion mechanisms: initial arrest of circulating
tumor cells, and subsequent reorganization of the adhesion
complexes to support chemotaxis.
ROCKs are important regulators of integrin adhesions, promoting
the development of large integrin adhesion complexes (Riento and
Ridley, 2003). Several chemokines, including CXCL12, have been
shown to regulate the activity of RhoA and ROCKs; however, their
specific effect on ROCK activity is context dependent. In some
systems, CXCL12 induces ROCK activity whereas in others the
chemokine inhibits ROCK activity (Amine et al., 2009; Arakawa
et al., 2003; Moyer et al., 2007; Tan et al., 2006; VicenteManzanares et al., 2002). These discrepancies can probably be
explained by the use of different cell types, experimental conditions,
and the time course examined. CXCL12 is known to induce rapid
changes in cell adhesion, cell shape and cell migratory capacities,
which correspond to underlying changes in signaling pathways
(Kucia et al., 2004). During a dynamic process such as cell
migration, it is very likely that the effect of CXCL12 on ROCK
activity varies over time and might be further influenced by the
particular cellular context. Therefore, CXCL12 regulation of ROCK
activity is a potential means of controlling tumor cell adhesive
events. However, studies to date have not specifically investigated
the role of ROCK activity in CXCL12-induced adhesive events
during the early phases of cellular attachment, which is thought to
be an important first step in tumor cell recruitment.
To investigate the role of ROCK in CXCR4-driven tumor cell
adhesion during recruitment, we overexpressed CXCR4 in MCF7
breast tumor cells and analyzed their behavior in response to
adhesion stimulated by CXCL12. To more closely mimic the
conditions of a circulating tumor cell, we specifically designed our
experiments to examine the transition from suspension to adhesion.
Our results show that CXCL12 promotes adhesion in two ways.
First, it increases initial cellular attachment to both extracellular
matrix and endothelial adhesion molecules. Second, it promotes the
maturation of integrin adhesion complexes following attachment.
Analysis of the role of ROCK activity in these processes reveals
an unexpected biphasic role for ROCK activity during adhesion.
Initially, inhibition of ROCK activity is required to allow cellular
attachment. Following attachment, re-activation of ROCK is
required for maturation of adhesion complexes and subsequent
migration. Interestingly, although CXCL12-induced adhesion
maturation and migration require ROCK activity, CXCL12 reduces
the level of ROCK re-activation. This suggests a model in which
CXCL12 promotes adhesion maturation and migration by providing
an optimal level of ROCK activity. Finally, we have examined tumor
cell attachment under physiological flow conditions, and find that
constitutively active ROCK blocks tumor cell attachment to an
endothelial monolayer, highlighting the importance of the transient
decrease in ROCK activity during tumor cell adhesion.
Results
CXCR4 stimulation increases MCF7 cell attachment
Chemokines, such as CXCL12, are known to increase leukocyte
arrest on the endothelium by stimulating integrin adhesion. In order
to identify the role of the CXCR4-CXCL12 axis in tumor cell
adhesion, we used cell attachment assays to evaluate whether
CXCR4 activation stimulated MCF7 breast cancer cell adhesion.
We expressed CXCR4 in MCF7 cells (MCF7-CXCR4) to a level
equivalent to metastatic MDA-MB 361 cells (supplementary
material Fig. S1). MCF7-CXCR4 cells labeled with CellTrackerTM
Green were put into suspension, incubated for 5 minutes with or
without 10 nM CXCL12, and then plated onto 96-well plates
coated with a panel of adhesion molecules for 20 minutes. We
limited the time of attachment to 20 minutes to restrict our analysis
to early adhesion events as a model for tumor cell attachment.
We found that CXCL12 increased the attachment of MCF7CXCR4 cells to collagen 1 (Fig. 1A). For comparison, the
attachment of MCF7-vector-control cells is also shown. We found
that overexpression of CXCR4 in MCF7 cells promotes adhesion
to collagen 1 even in the absence of CXCL12 stimulation. The
binding to collagen 1 was very efficient and was subsequently
used to normalize the attachment data and serve as a reference
point for comparison between different experiments. We next
analyzed binding to laminin and several endothelial cell ligands
expected to be important for tumor cell adhesion and found that
CXCL12 stimulated adhesion to E-selectin, ICAM-1 and VCAM1 (between 38% and 60% of plated cells attached depending on
the ligand; Fig. 1B). This is an important result because it
demonstrates that stimulation of CXCR4 can enhance adhesion
to endothelial ligands which might be important for tumor cell
migration during metastasis.
Because ROCK is known as a potent promoter of adhesion
complexes, we next wanted to determine whether basal and
CXCL12-induced early adhesion events were dependent on ROCK
activity. We pretreated MCF7-CXCR4 cells with Y-27632, an
inhibitor of ROCKI and II. (Note that we are not distinguishing
between the two ROCK isoforms because the reagents used do
not discriminate between them.) We found that inhibition of ROCK
with Y-27632 increased MCF7-CXCR4 cell attachment to collagen
1 (30%) and ICAM-1 (45%). In addition, we found that the ROCK
inhibitor increased cell attachment even over the levels observed
with CXCL12 stimulation (Fig. 1C). Inhibition of ROCK similarly
increased attachment to laminin (32%), VCAM (35%) and Eselectin (50%); supplementary material Fig. S2). Because ROCK
activity is associated with the formation of robust integrin
adhesions, the finding that ROCK activity was not only
dispensable, but actually promoted tumor cell attachment was
unexpected.
Journal of Cell Science
ROCK in CXCR4-driven tumor cell adhesion
403
Fig. 1. Effect of CXCL12 and ROCK signaling on tumor cell attachment.
The relative amount of cells that attached in 20 minutes was determined (see
Materials and Methods) and the data plotted (values are from triplicate wells in
three to seven separate experiments). (A)Attachment to collagen 1 was
measured in the presence or absence of 10 nM CXCL12, and was plotted as
arbitrary fluorescence units. Subsequently, the attachment data was normalized
to collagen 1 control, which was included in each experiment as a point of
reference. (B)Attachment to laminin and endothelial adhesion molecules.
(C)Attachment to collagen 1 or ICAM-1 when ROCK activity was inhibited
with Y-27632. CXCL12 promoted adhesion to all surfaces tested, but
inhibition of ROCK did not block, and instead enhanced attachment (*P<0.05
and **P<0.01, as determined by Student’s t-test).
CXCL12 enhances adhesion complexes in a ROCKdependent fashion
Existing evidence for integrin adhesions being promoted by ROCK
is largely based on studies that monitor the size of individual integrin
adhesion complexes as an experimental readout; whereas our initial
experiments were specifically designed to analyze attachment of
whole cells. Thus, we next examined the effect of CXCL12
stimulation on individual adhesion complexes within single cells.
Because we are interested in the initial phase of tumor cell
attachment, we used Total internal reflectance fluorescence (TIRF)
microscopy to examine the formation of adhesion complexes,
marked by GFP-paxillin during the first 20 minutes after cell plating.
In control cells, GFP-paxillin was clearly incorporated into
adhesion complexes, visible by TIRF microscopy. By 20 minutes,
cells were well spread and the adhesion complexes were distinct
and visible, primarily at the cell periphery with a few located on
the interior of the cell (Fig. 2A). We also observed that in addition
to effects on adhesion complexes, CXCL12 stimulation led to
increased cell protrusions and a less symmetric, more elongated
morphology (Fig. 2A). CXCL12 stimulation enhanced adhesion
complexes as measured by a 79% increase in the number of adhesion
complexes, and a 144% increase in the area occupied by adhesions
(Fig. 2B,C).
To determine the effect of ROCK on adhesion complexes, we
included the ROCK inhibitor, Y-27632, during cell attachment.
Inhibition of ROCK led to accumulation of numerous small
adhesion complexes that were restricted to a thin band along the
cell periphery (Fig. 2A). Although there was a striking effect on
Fig. 2 CXCL12 enhances adhesion complexes, which requires ROCK
activity. MCF7-CXCR4 cells were transfected with GFP-paxillin, then
suspended and allowed to adhere to collagen-1-coated coverslips on Bioptechs
plates maintained at 37°C on the microscope stage. GFP-paxillin-expressing
MCF7-CXCR4 cells were pretreated with 10 nM CXCL12 (10 nM, 5
minutes), Y-27632 (10mM, 10 minutes) or Y-27632 followed by CXCL12
prior to plating to collagen-1-coated dishes. TIRF images were collected
within 20 minutes of attachment to show the pattern of GFP-paxillincontaining adhesion complexes. (A)Control cells had a relatively round
morphology with adhesions primarily on the periphery; CXCL12 treated cells
were more asymmetric, with adhesions in the center of the cell, as well as the
periphery. The total area of adhesions (B) and the total number of adhesion per
cell (C) were quantified using ImageJ software (n9 cells from three
independent experiments). Treatment with CXCL12 or CXCL12 + Y-27632
significantly increased the total area (*P<0.5) of adhesions as compared with
control cells. Treatment with CXCL12 also significantly increased the number
of adhesions per cell (*P<0.5). It was not possible to resolve the number of
adhesions per cells in Y-27632- or CXCL12 + Y-27632-treated cells, therefore
these data are not shown in C.
the morphology of the adhesions that form with Y-27632, the total
area occupied by adhesions was increased by Y-27632 (Fig. 2B),
which might explain why cell attachment does not require ROCK
activity. The morphology of the adhesions induced by CXCL12
was blocked by Y-27632, indicating that ROCK activity was
necessary to form larger adhesions (Fig. 2A). At this early time
following adhesion (20 minutes), the cells treated with Y-27632
appeared larger with relatively regular edges; the asymmetry
induced by CXCL12 stimulation is lost when ROCK is inhibited.
404
Journal of Cell Science 123 (3)
Thus, these findings show that inhibition of ROCK during cell
attachment leads to small clusters of GFP-paxillin-containing
complexes near the periphery, analogous to the ROCK-independent
adhesions formed during membrane extension (Rottner et al.,
1999). These observations led us to propose that whereas ROCK
is dispensable for CXCL12-driven attachment and nascent adhesion
formation, re-activation of ROCK is required for adhesion complex
maturation.
ROCK inhibition prevents formation of mature adhesion
complexes
Journal of Cell Science
To test the idea that the small adhesion complexes that accumulate
upon ROCK inhibition are immature adhesion complexes, we used
two markers of early and mature adhesion complexes: tyrosinephosphorylated paxillin [Y(P)-Pax] and zyxin, respectively (ZaidelBar et al., 2007b). We found that adhesion complexes, marked by
vinculin, are differentially marked by Y(P)-Pax and zyxin following
treatment with Y-27632. In untreated, control cells, adhesion
complexes formed within the first 20 minutes of attachment are
positive for vinculin, Y(P)-Pax and zyxin (Fig. 3A). However, when
cell attachment occurs in the presence of Y-27632, the adhesions
are smaller (as visualized by vinculin staining) and positive for Y(P)Pax, but negative for zyxin (Fig. 3B). We obtained a similar result
when cells were stimulated by 10 nM CXCL12. The small adhesions
that formed in CXCL12 cells treated with the ROCK inhibitor were
positive for Y(P)-Pax, but negative for zyxin, indicating that the
enhancement of adhesions promoted by CXCL12 requires
maturation that is dependent upon ROCK activity. This result
highlights the distinct requirements for ROCK activity during
CXCL12-promoted adhesion: ROCK activity is dispensable for both
initial cellular attachment and formation of nascent adhesions, but
ROCK activity is required for adhesion maturation which is
probably important for cell migration.
ROCK activity is required for migration
To test whether ROCK activity was required for tumor cell
migration in response to CXCL12, we monitored migration by phase
contrast microscopy over a 4 hour time course (See Materials and
Methods for details). MCF7-CXCR4 cells were plated onto
collagen-1-coated MatTek plates in the presence or absence of
CXCL12 and images were collected every 2.5 minutes for
4 hours. Fig. 4A and B show the tracks of a representative
group of cells (full movies are included in supplementary
material Movies 1-4). Measurement of migration by net
displacement distance shows that the migration activity of
the CXCL12-treated cell population was increased relative
to that of the control cell population (P0.001). The CXCL12induced increase in migration activity was significantly
attenuated in the presence of Y-27632 (P0.014),
demonstrating that ROCK activity is required for efficient
cell migration. Interestingly, we found that Y-27632 did not
block membrane activity because there were numerous,
active membrane extensions (Fig. 4A and supplementary
material Movies 3 and 4), but the morphology was abnormal.
The most striking effect of CXCL12 stimulation was the
increase in the percentage of cells that moved long distances.
Fig. 4C shows the distribution of net cell displacement for
the populations of cells under each treatment condition.
Whereas 19% of the CXCL12-treated cell population
migrated >80 mm, none of the control cell population
migrated this far. The data also demonstrate that ROCK
activity is required for CXCL12-enhanced migration, because
none of the cells treated with both CXCL12 and Y-27632
migrated >80 mm.
Fig. 3. ROCK inhibition prevents formation of mature adhesion
complexes. Immunofluorescence images of MCF7-CXCR4 cells
plated for 20 minutes on collagen-1-coated coverslips (A). MCF7 cells
were detached from culture plates and incubated at 37°C for 5 minutes
with 10 nM CXCL12 (B), 10 minutes with 10mM Y-27632 (C) or 10
minutes with Y-27632 followed by 5 minutes with CXCL12 (D). Cells
were then plated onto collagen-1-coated coverslips in treatment media.
After adhering for 20 minutes, cells were fixed with paraformaldehyde
and incubated with vinculin, zyxin or FITC-conjugated
phosphotyrosine (Y118) paxillin. White boxes indicate regions of the
cells shown enlarged above the respective panel. Pretreatment with
Y-27632 resulted in small, peripheral adhesion complexes. These small
Y-27632-induced adhesion complexes were positive for Y(P)-Pax, but
negative for zyxin, indicating that ROCK activity is necessary for
adhesion maturation. Inhibition of ROCK by Y-27632 also prevented
the formation of mature adhesions in the presence of CXCL12. Scale
bars: 10 mm.
Journal of Cell Science
ROCK in CXCR4-driven tumor cell adhesion
405
Fig. 4. CXCL12 promotes migration, which requires ROCK activity. (A)MCF7-CXCR4 cells were plated onto collagen-1-coated MatTek plates in the
presence or absence of CXCL12 (10 nM), Y-27632 (10mM) or CXCL12 (10 nM) + Y-27632 (10mM) and DIC images were collected every 150 seconds for 4
hours. Cells were maintained during image capture at 37°C and 5% CO2 using a LiveCellTM environmental chamber. (B)Individual cell positions in sequential DIC
images were determined using Slidebook cell tracking software, and x-y coordinates [with starting points adjusted to (0,0)] were plotted. The cells that were
analyzed are indicated by letters in A. (C)Pie charts representing the fraction of cells undergoing different levels of net displacement were generated from the
populations of cells under each treatment condition. Data was pooled from four separate experiments, with n99 (control), n63 (CXCL12), n80 (Y-27632); and
n75 (CXCL12 + Y-27632). Statistical analysis comparing the net displacement of the entire population of cells exposed to each experimental treatment was
performed using Fisher’s exact test. Pairwise statistical analysis was performed for each treatment condition and the resulting P values are provided in the table on
the right.
Biphasic ROCK activity during tumor cell attachment
Because initial CXCL12-stimulated tumor cell attachment is
promoted by ROCK inhibition, but subsequent events, such as
maturation of adhesion complexes and enhanced cell migration,
are blocked by ROCK inhibition, we examined the kinetics of
ROCK activity during the transition from suspension to adhesion.
To measure ROCK activity, we used phosphorylation of two
downstream targets of ROCK signaling, myosin phosphatase target
subunit (MYPT) and cofilin, as readouts for ROCK activity during
early adhesion events. As shown in Fig. 5A, cell attachment
induced a brief but pronounced decrease in ROCK activity within
5 minutes of cell attachment. Following this brief decrease in
activity, ROCK signaling was re-activated to basal levels in
response to attachment.
It is known that CXCL12 and other chemokines can regulate
RhoA and ROCK activity, however, the kinetics of regulation are
dependent upon the specific experimental system used (Arakawa
et al., 2003; Moyer et al., 2007; Tan et al., 2006; VicenteManzanares et al., 2002). To determine how CXCL12 affected the
ROCK activity during tumor cell attachment, MCF7-CXCR4 cells
were pretreated with 10 nM CXCL12 5 minutes prior to plating
onto collagen-1-coated dishes, and the phosphorylation state of
MYPT and cofilin were measured as indicators of ROCK activity.
We found that CXCL12 decreased the overall levels of ROCK
activity, although the pattern of ROCK activity in response to
attachment was retained (Fig. 5A). Quantification of the blots is
provided in supplementary material Fig. S4A,B. We next measured
the levels of GTP-RhoA using an ELISA-based activity assay (Fig.
5B,C). As with ROCK activity, cellular attachment induced a
transient decrease in RhoA activity, followed by re-activation.
Pretreatment with 10 nM CXCL12 decreased the activity of RhoA
at early time points of tumor cell adhesion, mirroring the effect of
CXCL12 on ROCK activity. These findings are different from that
observed in adherent cell cultures, where CXCL12 has been shown
to enhance RhoA and ROCK activity (Amine et al., 2009; Azab et
al., 2009; Molina-Ortiz et al., 2009). For comparison, we performed
analogous assays in steady state adherent cells and found that
CXCL12 increased ROCK activity in this context, consistent with
other studies (supplementary material Fig. S5). Thus, the influence
of CXCL12 on ROCK activity is dependent upon the specific
406
Journal of Cell Science 123 (3)
Journal of Cell Science
cellular context, and in newly attaching cells, CXCL12 reduces the
activity of ROCK.
Because there is often a reciprocal relationship between ROCK
and Rac activity, we also measured the effect of CXCL12 on Rac
activation, following cell attachment. We found that 5 minutes after
attachment there was a brief, 25% increase in Rac activity that
returned to baseline by 10 minutes (supplementary material Fig.
S4C). However, 10 nM CXCL12 had no effect on Rac activity under
these conditions, suggesting that although initial adhesion is
correlated with a transient increase in Rac activity, the ability of
CXCL12 to promote tumor cell adhesive events does not occur
through modulation of Rac.
It is important to note that phosphorylation of MYPT and cofilin
represents the balance of phosphorylation by ROCK and
dephosphorylation by cognate phosphatases. The rapid kinetics of
changes in ROCK target phosphorylation are an indication of how
dynamically ROCK signaling pathways are regulated. Lower
MYPT phosphorylation levels suggest that CXCL12 tips the
balance more towards dephosphorylation; although preserving the
biphasic pattern of ROCK activity in response to adhesion. Even
though CXCL12 treatment resulted in less ROCK activity, the
reduced activity was sufficient for maturation of adhesions (Fig.
3). Cell migration is dependent upon a balance between integrin
adhesion and myosin contractility, such that the most efficient
migration occurs when the balance between adhesion and
contraction is optimal (Gupton and Waterman-Storer, 2006).
Because ROCK influences both integrin adhesion and myosin
contractility, the reduced ROCK activity in CXCL12-treated cells
might correspond to the optimal levels of ROCK activity needed
to enhance cell attachment, adhesion maturation and migration of
MCF7-CXCR4 cells.
CXCR4 stimulation increases MCF7-CXCR4 cell
attachment under flow conditions
Fig. 5. Biphasic ROCK activity during tumor cell attachment. (A)MCF-7
CXCR4 cells were suspended, incubated with or without 10 nM CXCL12, and
plated on collagen-1-coated 10 cm2 dishes for the indicated times at 37°C.
Dishes were washed to remove non-adherent cells and the remaining adherent
cells were lysed with ice-cold modified RIPA buffer with protease and
phosphatase inhibitors. Phosphospecific MYPT and cofilin antibodies were
used as indicators of ROCK activity. There was a brief but pronounced
decrease in ROCK kinase after which ROCK activity recovered to just above
initial levels. CXCL12 decreased the level of ROCK activity over all time
points measured, although the kinetic pattern of ROCK activity, including the
transient decrease in ROCK upon initial attachment, was retained. A
representative blot from three independent experiments is shown.
Quantification of the western blots is provided in supplementary material Fig.
S4. The phosphorylated (P)-ERK blot indicates that suppression of P-MYPT
and P-cofilin is not due to a global effect on signaling. Actin and ERK1/2
expression are shown as a loading control. (B)CXCR4 cells were treated as
described for A. RhoA activity in cellular lysates was determined by an
ELISA-based protocol. Data plotted are the average of duplicate samples from
three independent experiments, and the error bars represent the standard error
between the three experiments. Treatment with CXCL12 significantly
decreased (**P<0.01) RhoA activity prior to and during the initial replating
period (*P<0.05). (C)Immunoblot analysis indicates expression of RhoA in
cell lysates analyzed for RhoA activity was equivalent (20mg total lysates per
lane). Immunoblot shown is a representative blot from three independent
experiments.
Together our data show that CXCL12 induces attachment to a wide
range of adhesive ligands, including both extracellular matrix and
endothelial cell adhesion molecules. The goal of the study was to
determine if CXCR4-CXCL12 signaling could influence adhesive
activity of tumor cells transitioning from suspension to adhesion.
Thus, we measured the adhesion of the MCF7-CXCR4 cells under
physiological flow conditions following stimulation with 10 nM
CXCL12.
MCF7-CXCR4 cells were pumped through a laminar flow
chamber in the presence or absence of CXCL12, as described in
the Materials and Methods. We found that the flowing tumor cells
attached to the collagen 1 under shear stresses ranging from 5.0 to
40 mN/cm2. Based on published data, we selected 20 mN/cm2 for
subsequent analysis (Liang and Dong, 2008; Ngo et al., 2008). Our
data shows that CXCL12 leads to a marked, 2.8-fold (180%)
increase in the number of cells attached under flow (Fig. 6A). In
fact, the increase is significantly more than what was observed in
the static attachment assays (26%; Fig. 1). This is probably because
shear stress provides a more stringent environment for attachment,
and the requirement for CXCL12 signaling becomes more essential.
Inhibition of ROCK activity with Y-27632 did not block adhesion
induced by CXCL12, similar to what we observed with the static
adhesion assays in Fig. 1. Microscopic analysis of the morphology
of the newly adherent cells (Fig. 6B) showed that CXCL12
enhances cell spreading, whereas Y-27632 impairs spreading under
flow conditions. Thus, CXCL12 and Y-27632 both promote
attachment under flow, but only CXCL12 supports cell spreading.
Journal of Cell Science
ROCK in CXCR4-driven tumor cell adhesion
407
Fig. 6. Role of ROCK activity in attachment to collagen 1 under flow. MCF7-CXCR4 cells were processed as in Fig. 2 for the attachment assay. Cells were then
flowed over collagen-1-coated coverslips in a Bioptechs FCS2 flow chamber at a rate equivalent to 20mN/cm2, for a total of 20 minutes at 37°C. (A)Cells were
pretreated with 10 nM CXCL12 or 10mM Y-27632 as previously described. DIC images were then captured for ten different fields on the coverslip, and the
number of attached cells was counted manually. Graphs depict the number of adherent cells per coverslip, normalized to the control condition, and the average of
three to five independent experiments is plotted. Note that both CXCL12 and the ROCK inhibitor lead to significant increases in the number of attached cells.
(*sample pairs for which P<0.05 and **sample pairs for which P<0.01; Student’s t-test.) (B)Following the attachment assay, coverslips were fixed and stained for
F-actin to examine the morphology of the cells. The control cells remain relatively round, but the CXCL12 cells are well spread. By contrast, the cells treated with
Y-27632 (abbreviated as Y in some panels) are more spread than controls, but clearly different from those treated with CXCL12. Scale bar: 100mm. (C) MCF7CXCR4 cells were transfected with either GFP alone as a control, or GFP-CA ROCK. Following 20 minutes of attachment under flow conditions, fluorescent
images were captured for 10 different fields on the coverslip and the number of attached cells was counted manually. Graphical analysis was performed as in A,
and represent the average of three independent experiments. (D) Following the attachment assay, cells transfected with GFP (control) or GFP-CA ROCK were
fixed and stained for F-actin to examine the morphology of the cells. Both GFP and GFP-CA ROCK cells remain relatively round.
MCF7-CXCR4 tumor cell attachment to endothelium under
flow conditions
We have used collagen 1 matrix as an initial surface to measure
adhesion under flow conditions because we find it to form a robust
surface for MCF7-CXCR4 cell adhesion and it provides an important
condition for comparing our static attachment assay results. However,
to more closely mimic the conditions for tumor cell recruitment, we
performed analogous experiments in which the lower surface of our
laminar flow cell was composed of an endothelial cell monolayer. A
confluent monolayer of endothelial cells (HUVECs) was stimulated
with TNFa for 3 hours prior to the introduction of MCF7-CXCR4
cells (see Materials and Methods for details). In these experiments,
the MCF7-CXCR4 tumor cells were fluorescently labeled with
CellTrackerTM Green prior to the attachment assay, so they could be
easily discerned from the endothelial monolayer (Fig. 7B). Fig. 7A
shows that stimulation of suspended tumor cells with 10 nM CXCL12
increased the number of tumor cells attached to the activated
endothelium by 2.3-fold. Thus, tumor cells expressing CXCR4 have
a greatly enhanced chance of adhering to activated endothelium when
stimulated with CXCL12. Inhibition of ROCK with Y-27632 also
increased attachment to the endothelium by nearly twofold, and did
not block CXCL12-induced attachment (Fig. 7A), showing that
ROCK activity is not required for initial tumor cell attachment under
flow conditions.
Inhibition of ROCK activity is required for MCF7 cell
attachment
The observations that ROCK activity is dispensable for tumor cell
attachment, coupled with the decrease in ROCK activity during early
adhesion time points, led us to hypothesize that the transient decrease
in ROCK activity that occurs following tumor cell adhesion is
required for cell attachment. To test the hypothesis that attachment
under flow conditions requires the inhibition of ROCK, we
expressed a fluorescently tagged constitutively active mutant of
ROCK (GFP-CA ROCK) in MCF7-CXCR4 cells and measured the
attachment of the active-ROCK-transfected cells as compared with
GFP control transfectants under flow conditions. Fig. 6C shows
that GFP-CA ROCK expression reduced attachment to collagen 1
by 51%, as compared with GFP controls, and prevented an increase
in attachment in response to CXCL12. The morphology of the GFPCA ROCK cells that did attach to the substratum was relatively
round and unspread, even in the presence of CXCL12,
Journal of Cell Science
408
Journal of Cell Science 123 (3)
Fig. 7. Role of ROCK activity in attachment to endothelial monolayers under flow. MCF7-CXCR4 cells were processed as in Fig. 2 for the attachment assay.
Cells were then flowed over endothelial (HUVEC) monolayers that had been stimulated with TNFa (see Materials and Methods) in a Bioptechs FCS2 flow
chamber, at a rate of 20mN/cm2, for 20 minutes at 37°C. Tumor cells were distinguished from the endothelial monolayer by labeling with CellTracker Green, and
phase-contrast and fluorescent images of 10 separate fields per sample were collected. (A)Graphs depict the number of attached cells per experimental condition,
normalized to the control condition, and represent the average from three independent experiments. Note that CXCL12 significantly increases the attachment of
tumor cells by the endothelium. (*indicates sample pairs for which P<0.05 and **sample pairs for which P<0.01; Student’s t-test.) (B) Representative images of
control and CXCL12-treated MCF7-CXCR4 cells attached to the endothelial monolayer that were used for quantification. The fluorescent images of the tumor
cells are overlaid on the phase contrast image of the endothelial monolayer. Scale bar: 200mm. (C) Graphs depict the number of attached cells per experimental
condition, normalized to the control condition, and represent the average from three independent experiments. Note that expression of GFP-CA ROCK significantly
decreases attachment of tumor cells to the endothelium in both unstimulated and CXCL12 treatment conditions.
demonstrating that too much ROCK activity prevents both
attachment and spreading in response to CXCL12 (Fig. 6D).
We then examined attachment to endothelial cell monolayers and
found that tumor cells expressing GFP-CA ROCK reduced basal
attachment by 27%, as compared with GFP controls. Strikingly,
GFP-CA ROCK expression completely blocked attachment in
response to CXCL12 (Fig. 7C). These results demonstrate that
CXCL12 promotes the binding of flowing tumor cells to an
activated endothelial monolayer in a manner that requires inhibition
of ROCK. This is an important result because it demonstrates that
inhibition of ROCK is required for tumor cell attachment. In other
words, not only is ROCK activity unnecessary, but ROCK
inactivation must occur to allow initial cellular attachment. The
finding that active ROCK potently blocks attachment to endothelial
cells in response to CXCL12, coupled with the findings that
CXCL12 reduces RhoA and ROCK activity levels, support the
model that inhibition of ROCK is a previously unrecognized
signaling event during chemokine-promoted recruitment of CXCR4expressing tumor cells.
Discussion
To better understand the signaling mechanisms that promote
metastasis, we designed a system to examine the role of ROCK
signaling in CXCR4-driven breast tumor adhesive events.
Interestingly, we found that ROCK plays a biphasic role in the
regulation of adhesion during tumor cell attachment. Initially,
ROCK inhibition is required for cell attachment; but subsequently,
re-activation of ROCK is required for maturation of adhesion
complexes and migration (Model, Fig. 8). These two distinct
requirements for ROCK activity during adhesive events are
reflected in the kinetics of ROCK activity. Specifically, analysis
of ROCK activity following adhesion shows two distinct phases:
a rapid initial decrease in ROCK activity followed by subsequent
re-activation (Fig. 5). Interestingly, CXCL12 reduces the activity
levels of ROCK at both phases. This suggests that CXCL12
signaling acts in concert with attachment signals to fine-tune
ROCK activity for optimal tumor cell adhesive events, as too much
or too little activity blocks tumor cell behavior. To more accurately
model the environment of tumor cell recruitment, we performed
adhesion assays under conditions of shear stress and examined
adhesion to extracellular matrix and endothelial cell monolayers
(Figs 6, 7). Under conditions of shear stress, the ability of the
chemokine CXCL12 to stimulate tumor cell adhesion and
spreading was even more pronounced than in the static adhesion
assays (Figs 1, 6). Further analysis of cell attachment under flow
conditions showed that expression of active ROCK blocked
attachment, demonstrating that not only is ROCK activity
dispensable for cellular attachment, but inhibition of activity is
required for attachment (Figs 6, 7).
Although many factors are likely to contribute to the recruitment
of tumor cells to specific organs during metastasis, one mechanism
is that adhesion promoted by CXCL12 plays a fundamental role in
the process (Balkwill, 2004; Ben-Baruch, 2008; Dittmar et al., 2008;
Miles et al., 2008; Witz, 2008). CXCL12 is a known ‘capture’
chemokine for hematopoietic cell recruitment, and as such functions
as a gatekeeper for cellular trafficking into specific tissues. The
aberrant trafficking characteristics of malignant tumor cells are often
thought to be via appropriation of mechanisms normally used during
Journal of Cell Science
ROCK in CXCR4-driven tumor cell adhesion
Fig. 8. Model for biphasic role of ROCK during tumor attachment. Curves
illustrate typical ROCK activity profile over a timecourse of MCF7-CXCR4
cell attachment. In the first phase, the ROCK activity rapidly decreases,
corresponding to the initial step of tumor cell attachment. At this stage,
ROCK-independent nascent adhesions form on the cell periphery. In the
second phase, ROCK activity increases; this is necessary for maturation of the
adhesions, as well as appropriate regulation of membrane protrusions and cell
migration. We find that either too much or too little ROCK activity blocks
CXCL12-promoted tumor cell behavior at different phases. In the situation
where CXCL12-CXCR4 signaling is stimulated, overall ROCK activity levels
are lower. Thus, we propose that CXCL12 modulates levels of ROCK activity
regulated by attachment, to provide an optimal level of ROCK activity
necessary for tumor cell migration.
hematopoietic cell recruitment by the vascular endothelium (Kucia
et al., 2005; Miles et al., 2008); although tumor cell recruitment
involves other factors, including interactions with hematopoietic
cells in the bloodstream (Borsig, 2008; de Visser et al., 2006). Direct
adhesive interactions between tumor cells and the endothelial lining
of the vasculature have been shown to contribute to tumor cell arrest
(Gassmann and Haier, 2008; Gassmann et al., 2009; Glinskii et al.,
2005; Glinsky, 2006), and there are multiple examples of tumor
cells using endothelial adhesion molecules, including selectins,
ICAM-1 and VCAM-1, during metastasis (Kobayashi et al., 2007;
Zetter, 1993). Following initial cell attachment, the dynamics of
the adhesion complexes are important for subsequent cell migration,
and the proteins making up the adhesion complexes can be regulated
by chemokine signals to promote migration (Kinashi, 2007). To
investigate specific mechanisms by which CXCL12 signaling can
promote metastasis, we focused on the regulation of tumor cell
adhesive events by CXCL12.
We first investigated how ROCK signaling contributed to
CXCR4-promoted tumor cell attachment. To test the requirement
for ROCK activity, we measured cell attachment in the presence
of the ROCK inhibitor Y-27632. We found that ROCK activity was
not required for attachment in response to CXCL12. In
complementary experiments, we expressed a constitutively active
mutant of ROCK, which markedly reduced cell attachment to both
matrix and endothelial monolayers under flow conditions (Figs 6,7).
Previous studies have shown that RhoA activity is decreased upon
integrin engagement; however, these studies did not measure the
activity of specific RhoA effectors nor the effect on cell attachment.
Thus, our results extend the previous findings by demonstrating
409
that adhesion triggers a transient decrease in ROCK activity, and
identify ROCK inhibition as a previously unrecognized requirement
for attachment of tumor cells.
Although inhibition of ROCK was necessary for initial tumor
cell attachment, subsequent re-activation of ROCK was required
for downstream responses to CXCL12. Paxillin-containing adhesion
complexes were enhanced by CXCL12 through a mechanism that
required ROCK activity (Fig. 2). Adhesion complexes can contain
at least 150 different proteins, involving both structural and signaling
components (Zaidel-Bar et al., 2007a). Investigations into regulation
of integrin adhesion complexes have shown that several cytoskeletal
and signaling systems are coordinated to control adhesion dynamics.
Integrin adhesions are regulated by the structure of the matrix
outside the cell, as well as actin and microtubule dynamics, myosin
II contractility, and signaling modifiers within the cell (Broussard
et al., 2008; Gupton and Waterman-Storer, 2006; Webb et al., 2004)
(Choi et al., 2008; Even-Ram et al., 2007; Vicente-Manzanares et
al., 2007). The interplay between these systems leads to the
formation of nascent adhesions in the lamellipodium, followed by
maturation of the nascent adhesions into larger, dynamic complexes
which support forward movement of the lamella to advance cell
motility (Giannone et al., 2007; Gupton and Waterman-Storer, 2006;
Vicente-Manzanares et al., 2007). Our results show that ROCK
activity is required for the maturation of nascent adhesions in
response to CXCL12 and subsequent cell migration (Figs 3,4). The
requirement for ROCK activity during adhesion maturation and cell
migration is reflected in the activity assays that show ROCK is reactivated following the initial decrease in activity (Fig. 5). Together,
our data show that although ROCK activity is inhibited during cell
attachment, its activity must return for CXCL12 to regulate adhesion
complexes and cell migration.
Our results show that ROCK has a biphasic response to adhesion:
a transient decrease, followed by a return to activity levels greater
than or equal to baseline. This is similar to the pattern of RhoA
activity in response to integrin engagement (Arthur et al., 2000;
Ren et al., 1999) where a transient decrease in RhoA activity was
shown to be necessary for cell spreading, although the effects on
cell attachment were not assessed. Arthur et al. identified activation
of p190RhoGAP as a mediator of the decrease in RhoA activity
(Arthur et al., 2000). Interestingly, CXCL12 decreases the overall
levels of ROCK activity, while retaining the transient decrease
pattern induced by adhesion (Fig. 5B). One possibility is that
CXCR4 signaling activates tyrosine kinases, such as Src or Arg,
that activate p190RhoGAP, leading to inhibition of RhoA-GTP
loading and lower ROCK activity (Bradley et al., 2006; Chang et
al., 1995; Fincham et al., 1999). In this way, CXCR4 could act in
concert with integrin signaling to modulate ROCK activity to
regulate tumor cell adhesion events that support motility.
We found that CXCL12-induced migration requires ROCK
activity; however, CXCL12 reduces the level of ROCK activity
generated by adhesion. This seemingly paradoxical result suggests
that CXCL12 leads to an optimal level of ROCK activity to enhance
migration (Model, Fig. 8). In the context of current models for
leading edge behavior during migration (Choi et al., 2008; Giannone
et al., 2007; Gupton and Waterman-Storer, 2006), CXCL12 might
fine-tune ROCK activity to regulate cycles of lamellipodial and
adhesion dynamics, favoring advance of the lamella and persistent
migration. Further studies are designed to probe how CXCR4
regulates lamellipodial and adhesion dynamics, as a means to
understanding mechanisms by which modulation of ROCK activity
contributes to tumor cell recruitment.
410
Journal of Cell Science 123 (3)
Metastasis is clearly an important health concern and CXCR4
and ROCK have each been identified as contributing factors. ROCK
is a multifunctional kinase: it receives input from a wide range of
extracellular signals and is responsible for cellular responses
including regulation of the actomyosin cytoskeleton, cell growth
and survival, and gene transcription. The mechanisms by which
extracellular signals direct ROCK to act at the right place and at
the right time to achieve the appropriate biological outcome are
presently undefined. Likewise, the effects of CXCR4 are pleiotropic.
Although ROCK and CXCR4 might impinge on multiple steps of
tumor cell metastasis, our studies implicate tumor cell adhesion to
the endothelium as an effect of aberrant CXCL12 signaling, and
identify ROCK-dependent regulation of cell adhesion events as a
possible mechanism by which CXCR4 promotes tumor cell
recruitment and metastasis. Continued investigation into how
CXCR4 regulates ROCK signaling to promote tumor cell adhesion
and recruitment has the potential for identifying target molecules
for blocking CXCR4-driven metastasis.
Materials and Methods
Journal of Cell Science
Reagents
Recombinant ICAM-1, VCAM-1, CXCL12 and TNFa were purchased from R&D
Systems (Minneapolis, MN). Anti-phospho-myosin phosphatase (MYPT; Thr696)
was purchased from Millipore (Billerica, MA). Anti-phospho-ERK (1:2000) and antiphospho-cofilin [Ser3, -cofilin, -ERK, actin and -MYPT (all at 1:1000) were obtained
from Cell Signaling (Danvers, MA)]. Y-27632, anti-zyxin (1:100) and anti-vinculin
(1:400) were obtained from Sigma-Aldrich (St Louis, MO). FITC-labeled phosphopaxillin antibody (Tyr118; used at 1:50 dilution) was purchased from Biosource
(Camarillo, CA). CellTracker cell-permeable dye and Lipofectamine 2000 were both
purchased from Invitrogen (Carlsbad, CA). Collagen 1 and reduced-growth-factor
Matrigel were obtained from BD Biosciences (Bedford, MA). An N-terminal GFP
cloning vector was generated by inserting the GFP gene [from EGFP-C1 (Clontech,
Mountain View, CA)] into pcDNA3 (Invitrogen) then it was converted to a Destination
vector with a conversion kit (Invitrogen) following the manufacturer’s instructions
(kindly provided by Keith Burridge, UNC Chapel Hill, NC). The resulting nGFPDEST vector was then used to subclone the constitutively active mutant of ROCKII
(pCAG-myc-D3-ROCK2 used as a template) as a GFP-fusion protein.
Cell culture
MCF7 breast adenocarcinoma cell lines were routinely cultured in DMEM
supplemented with 10% FCS, GlutaMax, insulin, non-essential amino acids, and
antibiotics (BD Biosciences). The MCF7-CXCR4 and MCF7-vector control cell lines
were generated by lentiviral transduction of the human CXCR4 gene, or empty vector,
followed by selection with G418 to generate a stable clonal cell line. HUVEC from
pooled donors and EGM2 medium were purchased from Lonza (Basel, Switzerland).
Primary cell human umbilical vein endothelial cells (HUVEC) cultures were used
between passages 3 and 6 and monolayer cultures obtained by seeding at a density
of 2500 cells/cm2 such that the cells formed a confluent monolayer upon plating
(Worthylake et al., 2001).
Attachment assays
Ninety-six-well plates were coated with the indicated basement membranes (all at
10 mg/ml) for 2 hours prior to assay. ECM-coated assay plates were then blocked
with blocking buffer [serum free medium (SFM) plus 1% BSA] at 37°C for 30 minutes
prior to assay. MCF7-CXCR4 cells were labeled with CellTracker (cell-permeable,
fluorescent dye, 1:5000 dilution) for 30 minutes followed by starvation with blocking
buffer for 2 hours. Cells were then incubated with or without 10 mM Y-27632 for 10
minutes, and/or 10 nM CXCL12 (5 minutes). Trypsinized cells were seeded in 96well plates (60,000 cell/well) and allowed to adhere at 37°C for 20 minutes, they
were then washed with PBS, and fixed in 3.7% formaldehyde for 15 minutes.
Fluorescence was detected using a fluorescence plate reader (Molecular Devices,
Sunnyvale, CA; ex492nm/em517nm). Adhesion assays were performed three or more
times and each data point is the average of three wells per experiment. Background
binding to blocked-only wells was subtracted, and the relative level of adhesion
determined by normalizing to collagen 1 control binding, which was included in every
experiment as a standard.
Attachment assay under physiological shear stress
Flow experiments were performed with the FCS2 closed chamber from Bioptechs
(Butler, PA). Microaqueduct slides (Bioptechs) were coated with collagen 1 (10 mg/ml)
overnight at 37°C, 5% CO2. 40 mm coverslips were placed on the microaqueduct
slide separated by inside gaskets of 0.1. Shear stress () was calculated using the
equation (6Qm)/(wh2), where Q is the volumeric flow rate, m is the viscosity of the
fluid, w is the slide width, and h is the height of the flow chamber (Rops et al., 2007).
5⫻105 cells/ml were incubated with or without CXCL12 (10 nM, 5 minutes) or Y27632 (10 mM) in SFM, which remained in the solution during the flow experiments.
In some cases, cells were transfected with nGFP-DEST vector alone, or nGFP-CA
ROCK (transfection described above) and used in flow-based attachment assays after
48 hours. MCF7-CXCR4 cells were flowed through the chamber at a shear stress of
2 dynes/cm2 for 20 minutes at 37°C, after which images from ten regions of the
chamber were taken and the number of attached cells per field were counted manually.
Images were acquired with a 10⫻ (NA0.45) objective on an Olympus IX81
microscope, equipped with a Hamamatsu OrcaER camera, run by Slidebook software.
ROCK activity assay (Immunoblot)
To assess ROCK activity during adhesion, cells were serum-starved for 2 hours, then
detached using 0.05% trypsin, pooled and centrifuged at 800 g, for 5 minutes, and
then incubated alone or with 10 nM CXCL12 for 5 minutes at 37°C. Cells were then
plated in incubation medium onto collagen-1-coated (10 mg/ml) culture dishes and
allowed to adhere for 5-60 minutes at 37°C. The cells were then washed with PBS
to remove any unattached cells and the remaining adherent cells were lysed with icecold modified RIPA buffer with protease and phosphatase inhibitors (Sigma). Equal
amounts of protein were loaded on 10% polyacrylamide gels, transferred to PVDF
membranes, blocked for 1 hour with 5% milk-TBS, and incubated with
phosphospecific antibodies to MYPT or cofilin.
For analysis of adherent cells (supplementary material Fig. S5) MCF7-CXCR4
cells were plated 2 days prior to the experiment, then serum starved for 2 hours prior
to the addition of 10 nM CXCL12 for the indicated times. Following CXCL12
treatment, cells were then washed with PBS and lysed with ice-cold modified RIPA
buffer with protease and phosphatase inhibitors. Equal amounts of protein were loaded
onto polyacrylamide gels for immunoblot analysis. For analysis of suspension cells
(supplementary material Fig. S5) MCF7-CXCR4 cells were suspended as for the
attachment assays, incubated for 10 minutes, then 10 nM CXCL12 was added for
the indicated times. To demonstrate that the phosphorylation of MYPT and cofilin
were dependent upon ROCK activity, we performed the same assay in the presence
of Y-27632 (supplementary material Fig. S5). Following incubation, cells were pelleted
at 500 g for 5 minutes and lysed with ice-cold modified RIPA buffer with protease
and phosphatase inhibitors (Sigma). Equal amounts of protein were loaded onto
polyacrylamide gels for immunoblot analysis.
RhoA and Rac activity assays
To measure Rho and Rac GTP levels, cells were suspended and then replated as
described for the ROCK activity assay (see above) in the presence or absence of 10
nM CXCL12. Rho and Rac activity were measured by G-LISA assay (Cytoskeleton,
Boulder, CO), with strict adherence to the manufacturer’s guidelines. The G-LISA
reports levels of active RhoA and Rac normalized by protein input levels. Additionally,
equivalent amounts of cellular lysates were taken from RhoA activity lysates and
were analyzed by immunoblotting to ensure RhoA expression did not change during
the time frame of the experiment.
Immunocytochemistry
MCF7-CXCR4 cells were plated onto collagen-1-coated coverslips (10 mg/ml) and
allowed to adhere at 37°C for 20 minutes with 10 nM CXCL12, 10 mM Y-27632.
As a control, we also tested an alternative ROCK inhibitor, H1152 (supplementary
material Fig. S3). Following the adhesion period, cells were washed with ice-cold
PBS and then fixed with 4% paraformaldehyde in PBS. After washing three times
with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS, washed once
with PBS, and 4% normal goat serum was used as a blocking agent. Fixed cells were
then incubated with primary antibodies [anti-phospho-paxillin (1:50), anti-vinculin
(1:400) or anti-zyxin (1:100)] for 2 hours, followed by a 1-hour incubation with the
appropriate secondary antibody (goat anti-rabbit or anti-mouse Alexa Fluor 488 or
Alexa Fluor 594; Jackson ImmunoResearch, West Grove, PA). In some experiments,
cells were stained for F-actin with Alexa Fluor 594-phalloidin. Coverslips were then
mounted onto glass slides using Prolong AntiFade Gold (Invitrogen). Images were
obtained using an Olympus IX81 with a 60⫻ (NA1.4) oil immersion objective,
equipped with a Hamamatusu EM camera, and processed using SlideBook software
(Intelligent Imaging Innovations, Denver, CO). Images were further processed using
Adobe Photoshop, using only linear adjustments of the signal.
Total internal reflectance fluorescence (TIRF) microscopy
Cells were transfected overnight with GFP-paxillin using Lipofectamine 2000
transfection reagent (Invitrogen) at a ratio of 1:3 (mg/ml) DNA:Lipofectamine
according to manufacturer’s protocol. The following day, cells were starved in SFM
for 2 hours, detached from plates, and replated for 20 minutes on collagen-1-coated
Bioptechs Delta T dishes. During attachment and imaging, cells were maintained at
37°C using a Bioptechs Delta T temperature-controlled stage adaptor and heated lid.
Images were collected on an Olympus IX71 microscope with a 60⫻ TIRFM objective
(NA1.49). Samples were excited with an argon laser (488 nm) and fluorescent images
captured with a Hamamatsu OrcaER digital camera, using Slidebook Software.
Assessment of cell motility
MCF7-CXCR4 cells were detached, pretreated with CXCL12 (10 nM, 5 minutes),
Y-27632 (10 mM, 10 minutes) or Y-27632 (10 mM, 10 minutes) followed by CXCL12
ROCK in CXCR4-driven tumor cell adhesion
(10 nM, 5 minutes), and plated onto collagen-1-coated MatTek dishes. Phase-contrast
images of newly adherent cells were taken at 2.5-minute intervals beginning 20
minutes after initial plating, and lasting for 4 hours using an Olympus IX71 microscope
with a 20⫻ objective (NA0.87). Adhering cells were maintained at 37°C, 5% CO2
using a LiveCell Environmental Chamber (NEUE Group, Ontario, NY). Cell position
in sequential images was determined using Slidebook software and x-y coordinates
of individual cells were plotted with starting points adjusted to (0,0). Net displacement
was calculated from the distance from the starting (x,y) position (t20 minutes) to
final (x,y) position (t4 hour).
FACS analysis
Journal of Cell Science
MCF7, MCF7-CXCR4 and MDA-MB 361 cell cultures were dissociated, then divided
into samples that were fixed or fixed and permeabilized, all steps were performed
with BD Biosciences Dissociation, Fixation and Permeabilization solutions for Flow
Cytometry analysis following the manufacturer’s instructions. Each of these was then
incubated with anti-CXCR4 (10 mg/ml MAB171, R and D Systems), or no primary
antibody, followed by anti-mouse-FITC (Jackson ImmunoResearch). 1⫻106 cells/ml
were analyzed by flow cytometry using a BD CantoII flow cytometer, and the mean
fluorescence intensity value was used for comparison of total CXCR4 expression
(fixed and permeabilized samples) and surface CXCR4 expression (fixed only)
between the different cell types.
We appreciate all of the input from the members of the Cell Adhesion
and Migration Group at LSU Health Sciences Center. In addition, we
would like to thank Chris Turner for the GFP-paxillin construct,
Stephania Cormier and Dahui You for the flow cytometry analysis, and
Don Mercante for the statistical analyses. The project was supported
by grant number P20RR020160 from the National Center for Research
Resources, grant number P20RR018766 from the National Center for
Research Resources, grant number LEQSF(2006-09)-RD-A-16 from
the Louisiana Board of Regents, and Seed Grant Funding from the
Louisiana Cancer Research Consortium. The content is solely the
responsibility of the authors and does not necessarily represent the
official views of the granting agencies. Deposited in PMC for release
after 12 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/3/401/DC1
References
Alon, R. and Ley, K. (2008). Cells on the run: shear-regulated integrin activation in
leukocyte rolling and arrest on endothelial cells. Curr. Opin. Cell Biol. 20, 525-532.
Amine, A., Rivera, S., Opolon, P., Dekkal, M., Biard, D. S. F., Bouamar, H., Louache,
F., McKay, M. J., Bourhis, J., Deutsch, E. et al. (2009). Novel anti-metastatic action
of Cidofovir mediated by inhibition of E6/E7, CXCR4 and Rho/ROCK signaling in
HPV<suP>+</suP> tumor cells. PLoS ONE 4, e5018.
Arakawa, Y., Bito, H., Furuyashiki, T., Tsuji, T., Takemoto-Kimura, S., Kimura, K.,
Nozaki, K., Hashimoto, N. and Narumiya, S. (2003). Control of axon elongation via
an SDF-1{alpha}/Rho/mDia pathway in cultured cerebellar granule neurons. J. Cell Biol.
161, 381-391.
Arthur, W. T., Petch, L. A. and Burridge, K. (2000). Integrin engagement suppresses
RhoA activity via a c-Src-dependent mechanism. Curr. Biol. 10, 719-722.
Azab, A. K., Azab, F., Blotta, S., Pitsillides, C. M., Thompson, B., Runnels, J. M.,
Roccaro, A. M., Ngo, H. T., Melhem, M. R., Sacco, A. et al. (2009). RhoA and Rac1
GTPases play major and differential roles in stromal cell-derived factor-1-induced cell
adhesion and chemotaxis in multiple myeloma. Blood 114, 619-629.
Balkwill, F. (2004). The significance of cancer cell expression of the chemokine receptor
CXCR4. Semin. Cancer Biol. 14, 171-179.
Ben-Baruch, A. (2008). Organ selectivity in metastasis: regulation by chemokines and
their receptors. Clin. Exp. Metastasis 25, 345-356.
Borsig, L. (2008). The role of platelet activation in tumor metastasis. Expert Rev.
Anticancer. Ther. 8, 1247-1255.
Bradley, W. D., Hernandez, S. E., Settleman, J. and Koleske, A. J. (2006). integrin
signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP
and recruitment to the membrane. Mol. Biol. Cell 17, 4827-4836.
Broussard, J. A., Webb, D. J. and Kaverina, I. (2008). Asymmetric focal adhesion
disassembly in motile cells. Curr. Opin. Cell Biol. 20, 85-90.
Cardone, R. A., Bagorda, A., Bellizzi, A., Busco, G., Guerra, L., Paradiso, A., Casavola,
V., Zaccolo, M. and Reshkin, S. J. (2005). Protein kinase a gating of a pseudopodiallocated RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell
lines. Mol. Biol. Cell 16, 3117-3127.
Chang, J. H., Gill, S., Settleman, J. and Parsons, S. J. (1995). c-Src regulates the
simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP
following epidermal growth factor stimulation. J. Cell Biol. 130, 355-368.
Choi, C. K., Vicente-Manzanares, M., Zareno, J., Whitmore, L. A., Mogilner, A. and
Horwitz, A. R. (2008). Actin and alpha-actinin orchestrate the assembly and maturation
of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 10, 10391050.
411
de Visser, K. E., Eichten, A. and Coussens, L. M. (2006). Paradoxical roles of the immune
system during cancer development. Nat. Rev Cancer. 6, 24-37.
Dittmar, T., Heyder, C., Gloria-Maercker, E., Hatzmann, W. and Zanker, K. S. (2008).
Adhesion molecules and chemokines: the navigation system for circulating tumor
(stem) cells to metastasize in an organ-specific manner. Clin. Exp. Metastasis 25, 1132.
Even-Ram, S., Doyle, A. D., Conti, M. A., Matsumoto, K., Adelstein, R. S. and Yamada,
K. M. (2007). Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.
Nat. Cell Biol. 9, 299-309.
Fincham, V. J., Chudleigh, A. and Frame, M. C. (1999). Regulation of p190 Rho-GAP
by v-Src is linked to cytoskeletal disruption during transformation. J. Cell Sci. 112, 947956.
Friedl, P. and Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape
mechanisms. Nat. Rev. Cancer 3, 362-374.
Gassmann, P. and Haier, J. (2008). The tumor cell-host organ interface in the early onset
of metastatic organ colonisation. Clin. Exp. Metastasis 25, 171-181.
Gassmann, P., Hemping-Bovenkerk, A., Mees, S. and Haier, J. (2009). Metastatic tumor
cell arrest in the liver-lumen occlusion and specific adhesion are not exclusive. Int. J.
Colorectal Disease 24, 851-858.
Giannone, G., Dubin-Thaler, B. J., Rossier, O., Cai, Y., Chaga, O., Jiang, G.,
Beaver, W., Dobereiner, H. G., Freund, Y., Borisy, G. et al. (2007). Lamellipodial
actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561575.
Glinskii, O. V., Huxley, V. H., Glinsky, G. V., Pienta, K. J., Raz, A. and Glinsky, V. V.
(2005). Mechanical entrapment is insufficient and intercellular adhesion is essential for
metastatic cell arrest in distant organs. Neoplasia 7, 522-527.
Glinsky, V. V. (2006). Intravascular cell-to-cell adhesive interactions and bone metastasis.
Cancer Metastasis Rev. 25, 531-540.
Glinsky, V. V., Glinsky, G. V., Glinskii, O. V., Huxley, V. H., Turk, J. R., Mossine, V.
V., Deutscher, S. L., Pienta, K. J. and Quinn, T. P. (2003). Intravascular metastatic
cancer cell homotypic aggregation at the sites of primary attachment to the endothelium.
Cancer Res. 63, 3805-3811.
Gupton, S. L. and Waterman-Storer, C. M. (2006). Spatiotemporal feedback between
actomyosin and focal-adhesion systems optimizes rapid cell migration. Cell 125, 13611374.
Hakuma, N., Kinoshita, I., Shimizu, Y., Yamazaki, K., Yoshida, K., Nishimura, M.
and Dosaka-Akita, H. (2005). E1AF/PEA3 activates the Rho/Rho-associated kinase
pathway to increase the malignancy potential of non-small-cell lung cancer cells. Cancer
Res. 65, 10776-10782.
Heasman, S. J. and Ridley, A. J. (2008). Mammalian Rho GTPases: new insights into
their functions from in vivo studies. Nat. Rev. Mol. Cell. Biol. 9, p. 690.
Imhof, B. A. and Aurrand-Lions, M. (2004). Adhesion mechanisms regulating the
migration of monocytes. Nat. Rev. Immunol. 4, 432-444.
Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T. and Narumiya, S. (1999).
An essential part for Rho-associated kinase in the transcellular invasion of tumor cells.
Nat. Med. 5, p. 221.
Jones, J., Marian, D., Weich, E., Engl, T., Wedel, S., Relja, B., Jonas, D. and Blaheta,
R. A. (2007). CXCR4 chemokine receptor engagement modifies integrin dependent
adhesion of renal carcinoma cells. Exp. Cell Res. 313, 4051-4065.
Joshi, B., Strugnell, S. S., Goetz, J. G., Kojic, L. D., Cox, M. E., Griffith, O. L., Chan,
S. K., Jones, S. J., Leung, S. P., Masoudi, H. et al. (2008). Phosphorylated caveolin1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration
and invasion. Cancer Res. 68, 8210-8220.
Kamai, T., Tsujii, T., Arai, K., Takagi, K., Asami, H., Ito, Y. and Oshima, H. (2003).
Significant association of Rho/ROCK pathway with invasion and metastasis of bladder
cancer. Clin. Cancer Res. 9, 2632-2641.
Kinashi, T. (2007). Integrin regulation of lymphocyte trafficking: lessons from structural
and signaling studies. Adv. Immunol. 93, 185-227.
Kitzing, T. M., Sahadevan, A. S., Brandt, D. T., Knieling, H., Hannemann, S.,
Fackler, O. T., Grosshans, J. and Grosse, R. (2007). Positive feedback between
Dia1, LARG, and RhoA regulates cell morphology and invasion. Genes Dev. 21,
1478-1483.
Kobayashi, H., Boelte, K. C. and Lin, P. C. (2007). Endothelial cell adhesion molecules
and cancer progression. Curr. Med. Chem. 14, 377-386.
Kucia, M., Jankowski, K., Reca, R., Wysoczynski, M., Bandura, L., Allendorf, D. J.,
Zhang, J., Ratajczak, J. and Ratajczak, M. Z. (2004). CXCR4-SDF-1 signalling,
locomotion, chemotaxis and adhesion. J. Mol. Histol. 35, 233-245.
Kucia, M., Reca, R., Miekus, K., Wanzeck, J., Wojakowski, W., Janowska-Wieczorek,
A., Ratajczak, J. and Ratajczak, M. Z. (2005). Trafficking of normal stem cells and
metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1CXCR4 axis. Stem Cells 23, 879-894.
Laudanna, C. and Alon, R. (2006). Right on the spot. Chemokine triggering of integrinmediated arrest of rolling leukocytes. Thromb Haemost. 95, 5-11.
Ley, K. (2003). Arrest chemokines. Microcirculation 10, 289-295.
Li, B., Zhao, W. D., Tan, Z. M., Fang, W. G., Zhu, L. and Chen, Y. H. (2006). Involvement
of Rho/ROCK signalling in small cell lung cancer migration through human brain
microvascular endothelial cells. FEBS Lett. 580, 4252-4260.
Liang, S. and Dong, C. (2008). Integrin VLA-4 enhances sialyl-Lewisx/a-negative
melanoma adhesion to and extravasation through the endothelium under low flow
conditions. Am. J. Physiol. Cell Physiol. 295, C701-C707.
Lin, M. T., Lin, B. R., Chang, C. C., Chu, C. Y., Su, H. J., Chen, S. T., Jeng, Y. M.
and Kuo, M. L. (2007). IL-6 induces AGS gastric cancer cell invasion via activation
of the c-Src/RhoA/ROCK signaling pathway. Int. J. Cancer 120, 2600-2608.
Journal of Cell Science
412
Journal of Cell Science 123 (3)
Miles, F. L., Pruitt, F. L., van Golen, K. L. and Cooper, C. R. (2008). Stepping out of
the flow: capillary extravasation in cancer metastasis. Clin. Exp. Metastasis 25, 305324.
Molina-Ortiz, I., Bartolome, R. A., Hernandez-Varas, P., Colo, G. P. and Teixido, J.
(2009). Overexpression of E-cadherin on melanoma cells inhibits chemokine-promoted
invasion involving p190RhoGAP/p120ctn-dependent inactivation of RhoA. J. Biol.
Chem. 284, 15147-15157.
Moyer, R. A., Wendt, M. K., Johanesen, P. A., Turner, J. R. and Dwinell, M. B. (2007).
Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and
restitution in model intestinal epithelia. Lab. Invest. 87, p. 807.
Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan,
T., Murphy, E., Yuan, W., Wagner, S. N. et al. (2001). Involvement of chemokine
receptors in breast cancer metastasis. Nature 410, 50-56.
Nakajima, M., Hayashi, K., Egi, Y., Katayama, K., Amano, Y., Uehata, M., Ohtsuki,
M., Fujii, A., Oshita, K., Kataoka, H. et al. (2003a). Effect of Wf-536, a novel ROCK
inhibitor, against metastasis of B16 melanoma. Cancer Chemother. Pharmacol. 52, 319324.
Nakajima, M., Hayashi, K., Katayama, K., Amano, Y., Egi, Y., Uehata, M., Goto, N.
and Kondo, T. (2003b). Wf-536 prevents tumor metastasis by inhibiting both tumor
motility and angiogenic actions. Eur. J. Pharmacol. 459, 113-120.
Narumiya, S., Tanji, M. and Ishizaki, T. (2009). Rho signaling, ROCK and mDia1, in
transformation, metastasis and invasion. Cancer Metastasis Rev. 25, 65-76.
Ngo, H. T., Leleu, X., Lee, J., Jia, X., Melhem, M., Runnels, J., Moreau, A. S., Burwick,
N., Azab, A. K., Roccaro, A. et al. (2008). SDF-1/CXCR4 and VLA-4 interaction
regulates homing in Waldenstrom macroglobulinemia. Blood 112, 150-158.
Ogawa, T., Tashiro, H., Miyata, Y., Ushitora, Y., Fudaba, Y., Kobayashi, T., Arihiro,
K., Okajima, M. and Asahara, T. (2007). Rho-associated kinase inhibitor reduces tumor
recurrence after liver transplantation in a rat hepatoma model. Am. J. Transplant 7, 347355.
Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M. and Keely, P. J. (2008).
Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCKdependent matrix reorganization. Biophys. J. 95, 5374-5384.
Ren, X. D., Kiosses, W. B. and Schwartz, M. A. (1999). Regulation of the small GTPbinding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578-785.
Riento, K. and Ridley, A. J. (2003). Rocks: multifunctional kinases in cell behaviour. Nat.
Rev. Mol. Cell. Biol. 4, 446-456.
Rops, A. L., Jacobs, C. W., Linssen, P. C., Boezeman, J. B., Lensen, J. F., Wijnhoven,
T. J., van den Heuvel, L. P., van Kuppevelt, T. H., van der Vlag, J. and Berden, J.
H. (2007). Heparan sulfate on activated glomerular endothelial cells and exogenous
heparinoids influence the rolling and adhesion of leucocytes. Nephrol. Dial Transplant
22, 1070-1077.
Rosel, D., Brabek, J., Tolde, O., Mierke, C. T., Zitterbart, D. P., Raupach, C., Bicanova,
K., Kollmannsberger, P., Pankova, D., Vesely, P. et al. (2008). Up-regulation of
Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of
protrusive forces. Mol. Cancer Res. 6, 1410-1420.
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between Rac and Rho in the
control of substrate contact dynamics. Curr. Biol. 9, 640-648.
Sahai, E. and Marshall, C. J. (2003). Differing modes of tumour cell invasion have distinct
requirements for Rho/ROCK signalling and extracellular proteolysis. Nat. Cell Biol. 5,
711-719.
Samaniego, R., Sanchez-Martin, L., Estecha, A. and Sanchez-Mateos, P. (2007).
Rho/ROCK and myosin II control the polarized distribution of endocytic clathrin
structures at the uropod of moving T lymphocytes. J. Cell Sci. 120, 3534-3543.
Steeg, P. S. (2006). Tumor metastasis: mechanistic insights and clinical challenges. Nat.
Med. 12, 895-904.
Takamura, M., Sakamoto, M., Genda, T., Ichida, T., Asakura, H. and Hirohashi, S.
(2001). Inhibition of intrahepatic metastasis of human hepatocellular carcinoma by Rhoassociated protein kinase inhibitor Y-27632. Hepatology 33, 577-581.
Tan, W., Martin, D. and Gutkind, J. S. (2006). The G{alpha}13-Rho Signaling axis is
required for SDF-1-induced migration through CXCR4. J. Biol. Chem. 281, 39542-39549.
Titus, B., Schwartz, M. A. and Theodorescu, D. (2005). Rho proteins in cell migration
and metastasis. Crit. Rev. Eukaryot. Gene Expr. 15, 103-114.
Torka, R., Thuma, F., Herzog, V. and Kirfel, G. (2006). ROCK signaling mediates the
adoption of different modes of migration and invasion in human mammary epithelial
tumor cells. Exp. Cell Res. 312, 3857-3871.
Vicente-Manzanares, M., Cabrero, J. R., Rey, M., Perez-Martinez, M., Ursa, A., Itoh,
K. and Sanchez-Madrid, F. (2002). A role for the Rho-p160 Rho coiled-coil kinase
axis in the chemokine stromal cell-derived factor-1{alpha}-induced lymphocyte
actomyosin and microtubular organization and chemotaxis. J. Immunol. 168, 400-410.
Vicente-Manzanares, M., Zareno, J., Whitmore, L., Choi, C. K. and Horwitz, A. F.
(2007). Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and
IIB in migrating cells. J. Cell Biol. 176, 573-580.
Wang, W., Goswami, S., Sahai, E., Wyckoff, J. B., Segall, J. E. and Condeelis, J. S.
(2005). Tumor cells caught in the act of invading: their strategy for enhanced cell motility.
Trends Cell Biol. 15, 138-145.
Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J.
T. and Horwitz, A. F. (2004). FAK-Src signalling through paxillin, ERK and MLCK
regulates adhesion disassembly. Nat. Cell Biol. 6, p. 154.
Wennerberg, K. and Der, C. J. (2004). Rho-family GTPases: it’s not only Rac and Rho
(and I like it). J. Cell Sci. 117, 1301-1312.
Wilkinson, S., Paterson, H. F. and Marshall, C. J. (2005). Cdc42-MRCK and Rho-ROCK
signalling cooperate in myosin phosphorylation and cell invasion. Nat. Cell Biol. 7, p.
255.
Witz, I. P. (2008). Tumor-microenvironment interactions: dangerous liaisons. Adv. Cancer
Res. 100, 203-229.
Wong, C. C., Wong, C. M., Tung, E. K., Man, K. and Ng, I. O. (2009). Rho-kinase 2
is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion.
Hepatology 49, 1583-1594.
Worthylake, R. A. and Burridge, K. (2001). Leukocyte transendothelial migration:
orchestrating the underlying molecular machinery. Curr. Opin. Cell Biol. 13, 569-577.
Worthylake, R. A., Lemoine, S., Watson, J. M. and Burridge, K. (2001). RhoA is required
for monocyte tail retraction during transendothelial migration. J. Cell Biol. 154, 147160.
Wyckoff, J. B., Pinner, S. E., Gschmeissner, S., Condeelis, J. S. and Sahai, E. (2006).
ROCK- and myosin-dependent matrix deformation enables protease-independent tumorcell invasion in vivo. Curr. Biol. 16, 1515-1523.
Xue, F., Zhang, J. J., Qiu, F., Zhang, M., Chen, X. S., Li, Q. G., Han, L. Z., Xi, Z. F.
and Xia, Q. (2007). Rho signaling inhibitor, Y-27632, inhibits invasiveness of metastastic
hepatocellular carcinoma in a mouse model. Chin. Med. J. 120, 2304-2307.
Ying, H., Biroc, S. L., Li, W. W., Alicke, B., Xuan, J. A., Pagila, R., Ohashi, Y., Okada,
T., Kamata, Y. and Dinter, H. (2006). The Rho kinase inhibitor fasudil inhibits tumor
progression in human and rat tumor models. Mol. Cancer Ther. 5, 2158-2164.
Zaidel-Bar, R., Itzkovitz, S., Ma’ayan, A., Iyengar, R. and Geiger, B. (2007a). Functional
atlas of the integrin adhesome. Nat. Cell Biol. 9, 858-867.
Zaidel-Bar, R., Milo, R., Kam, Z. and Geiger, B. (2007b). A paxillin tyrosine
phosphorylation switch regulates the assembly and form of cell-matrix adhesions. J.
Cell Sci. 120, 137-148.
Zetter, B. R. (1993). Adhesion molecules in tumor metastasis. Semin. Cancer Biol. 4, 219229.
Zlotnik, A. (2006). Chemokines and cancer. Int. J. Cancer 119, 2026-2029.