PDGF and Y27632 impair cancer cell invasion.

<p>The invasiveness of SW620 colon cancer cells following treatment or not (control) with PDGF and/or Y27632 was quantified by using Matrigel invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of at least three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, ns non-significant.</p

Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity

<div><p>Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.</p> </div

Cofilin phosphorylation increases in the most invasive colon cancer cell lines.

<p>Cells were lysed and the amount of phosphorylated Cofilin and of total Cofilin present in the lysates was analysed by immunoblotting. (a) Representative immunoblot. (b) Quantification of Cofilin phosphorylation. Histograms represent the ratios of the phospho-Cofilin signal over the total Cofilin signal. Values are the mean +/− SD (error bars) of three independent experiments.</p

PDGF and Y27632 treatments promote elongation of colon cancer cells.

<p>(a) Still DIC time-lapse images of cells treated or not (control) with PDGF and Y27632 (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s001" target="_blank">videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s005" target="_blank">S5</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s006" target="_blank">S6</a>). Frames show the change in morphology (from round to a more elongated shape) of the cells at the beginning of the movies. Scale bars, 10 µm. (b) DIC time-lapse images of SW620 cells before and after treatment with PDGF + Y27632 (from video S7). Frames show the change in cell morphology at the indicated times. Scale bars, 10 µm. (b) Quantification of cell elongation. A cell was considered elongated when its longest dimension was twice the shortest one and when it showed at least one protrusion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344-SanzMoreno1" target="_blank">[23]</a>. Histograms represent the percentage of elongated SW620 cells following treatment or not (control) with PDGF and/or Y27632 for 24 hours. Values are the mean +/− SD (error bars) of three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. (d) Cdc42 activity was determined following treatment or not (control) with PDGF for 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of four independent experiments and the statistical significance was calculated using the unpaired t-test. *p<0.05. (e) Rac1 activity was determined following treatment or not (control) with PDGF for 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of four independent experiments and the statistical significance was calculated using the unpaired t-test. *p<0.05. (f) SW620 cells were lysed and the amount of phosphorylated Myosin Light Chain 2 (MLC2) and of total MLC2 present in the lysates was determined by immunoblotting at different time-points (0 to 30 minutes) following treatment with 10 µM Y27632. Shown is a representative immunoblot.</p

The combined treatment with PDGF and Y27632 induces the re-establishment of E-cadherin junctions.

<p>(a) Representative E-cadherin staining in colon cancer cells treated or not (control) with PDGF and Y27632 for 24 hours. Scale bars, 10 µm. (b) Quantification of E-cadherin junctions. We scored as positive every cell showing at least one E-cadherin junction with one or more neighbouring cells. Histograms represent the percentage of SW620 colon cancer cells with E-cadherin junctions following treatment or not with PDGF and/or Y27632 for 24 hours. Values are the mean +/− SD (error bars) of three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001.</p

Kaplan–Meier curves of metastasis-free survival in the cohort of 36 patients with rectal cancer stratified according to delta133p53 expression (low/high).

<p>Kaplan–Meier curves of metastasis-free survival in the cohort of 36 patients with rectal cancer stratified according to delta133p53 expression (low/high).</p

Delta133p53ß physically interacts with RhoB.

<p>(a) Immunoblot analysis showing the specific co-immunoprecipitation of MYC-tagged delta133p53ß and endogenous RhoB or RhoC, to a lower extent, but not RhoA. (b) Immunoblot analysis showing the co-immunoprecipitation of endogenous RhoB and delta133p53ß with anti-RhoB antibodies. Arrow shows co-immunoprecipitation of delta133p53ß isoform. (c) <i>In-vitro</i> binding assay showing the direct interaction between recombinant RhoB-GST fusion protein and delta133p53ß, but not α or γ. (d) Cellular fractionation showing RhoB cytoplasmic re-localization in SW620 cells transfected with shRNAS against delta133p53 compared with shControl (mock-transfected cells). (e) Immunofluorescence analysis of RhoB localization in control SW620 cells (shControl) or after transfection with shdelta133p53. Arrows indicate examples of RhoB cytoplasmic localization. Scale bar: 10μm. (f) Confocal images showing the co-localization of RhoB and delta133p53ß in the nucleus of SW480 cells that overexpress delta133p53ß. Arrows indicate examples co-localization of RhoB and delta133p53ß in the nucleus. Scale bar: 10μm.</p

Patients’ characteristics.

<p>Patients’ characteristics.</p

Delta133p53ß protects cancer cells from camptothecin-induced apoptosis.

<p>(a) Apoptosis assay showing the sensitivity to 5μM camptothecin of mock-transfected (control) SW620 cells and after transfection with siRNAs against RhoB. Results are expressed as the apoptosis ratio of untreated versus camptothecin-treated cells and represent the mean ± SEM of four independent experiments; *, p<0.05. (b) Immunoblot showing RhoB downregulation in siRhoB-transfected SW620 cells compared with mock-transfected cells (control). (c) Apoptosis assay to test the sensitivity of SW480 and SW620 cells to 5μM camptothecin. Results are expressed as the percentage of apoptotic cells relative to all Hoechst-positive cells and represent the mean ± SEM of three independent experiments; *, p<0.05. (d) Apoptosis assay showing the sensitivity to 5μM camptothecin of mock-transfected (control) and SW480 cells that stably express delta133p53α, ß or γ. Results are expressed as the percentage of apoptotic cells relative to all Hoechst-positive cells and represent the mean ± SEM of three independent experiments; *, p<0.05. (e) Apoptosis assay showing the sensitivity to 5μM camptothecin of mock-transfected and SW480 cells that stably express delta133p53ß. Results are expressed as the percentage of apoptotic cells relative to all Hoechst-positive cells and represent the mean ± SEM of four independent experiments; *, p<0.05.</p

Immobilized, active PAI-1 maintains the RhoA/ROCK pathway activation.

<p>(<b>A</b>) Percentage of blebbing SW620 cells seeded on PAI-1 14-1b or collagen in the presence of the ROCK inhibitor Y27632 (Y27) (10 µM) at 19 h time-point after seeding. Data are the mean ± s.e.m. of three independent experiments; **: <i>P</i><0.01. (<b>B</b>) Detection of cleaved Caspase 3 by immunoblotting in SW620 cells seeded on non-coated plates (control), PAI-1 14-1b or collagen 19 h and 24 h after seeding. SW620 cells treated with 1 µM staurosporin (St.) for 24 h were used as positive control. β-Tubulin served as loading control. (<b>C</b>) RhoA activity and proportion of blebbing cells in SW620 cells seeded on PAI-1 14-1b or collagen at different time-points after seeding. Values were normalized to the effects at 3 h for each condition. Data are the mean ± s.e.m. of four independent experiments. (<b>D</b>) Quantification following immunoblotting of active RhoA relative to total RhoA in SW620 cells seeded on PAI-1 14-1b or collagen for 19 h. Data are the mean ± s.e.m. of three independent experiments; ***: <i>P</i><0.001. (<b>E</b>) Quantification following immunoblotting of phosphorylated MLC (MLC-P) relative to β-Tubulin (loading control) expression in SW620 cells seeded on PAI-1 14-1b or collagen for 19 h. Data are the mean ± s.e.m. of three independent experiments; *: <i>P</i><0.05. (<b>F</b>) Percentage of ROCK1 colocalization with PDK1 relative to total ROCK1 expression in blebbing SW620 cells seeded on PAI-1 14-1b or collagen for 30 min. Data are the mean ± s.e.m. of the analysis of ten blebbing cells in each microenvironment; **: <i>P</i><0.01. (<b>G</b>) Expression of PDK1 and ROCK1 in blebbing SW620 cells seeded on PAI-1 14-1b or collagen for 30 min. The colocalization of PDK1 and ROCK1 is shown and their signal distributions are compared. Bar, 5 µm.</p