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, 123 (Pt 3), 401-12

Dynamic Regulation of ROCK in Tumor Cells Controls CXCR4-driven Adhesion Events

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Dynamic Regulation of ROCK in Tumor Cells Controls CXCR4-driven Adhesion Events

Amanda P Struckhoff et al. J Cell Sci.

Abstract

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.

Figures

Fig. 1.
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).
Fig. 2
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 μM, 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-paxillin-containing 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.
Fig. 3.
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 μM 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 μm.
Fig. 4.
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 μM) or CXCL12 (10 nM) + Y-27632 (10 μM) 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 LiveCell™ 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.
Fig. 5.
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 μg total lysates per lane). Immunoblot shown is a representative blot from three independent experiments.
Fig. 6.
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 μN/cm2, for a total of 20 minutes at 37°C. (A) Cells were pretreated with 10 nM CXCL12 or 10 μM 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 μm. (C) MCF7-CXCR4 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.
Fig. 7.
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 TNFα (see Materials and Methods) in a Bioptechs FCS2 flow chamber, at a rate of 20 μN/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 μm. (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.
Fig. 8.
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.

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