Loss of p53 promotes RhoA–ROCK-dependent cell migration and invasion in 3D matrices

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA–ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis

Matrix-Bound PAI-1 Supports Cell Blebbing via RhoA/ROCK1 Signaling

The microenvironment of a tumor can influence both the morphology and the behavior of cancer cells which, in turn, can rapidly adapt to environmental changes. Increasing evidence points to the involvement of amoeboid cell migration and thus of cell blebbing in the metastatic process; however, the cues that promote amoeboid cell behavior in physiological and pathological conditions have not yet been clearly identified. Plasminogen Activator Inhibitor type-1 (PAI-1) is found in high amount in the microenvironment of aggressive tumors and is considered as an independent marker of bad prognosis. Here we show by immunoblotting, activity assay and immunofluorescence that, in SW620 human colorectal cancer cells, matrix-associated PAI-1 plays a role in the cell behavior needed for amoeboid migration by maintaining cell blebbing, localizing PDK1 and ROCK1 at the cell membrane and maintaining the RhoA/ROCK1/MLC-P pathway activation. The results obtained by modeling PAI-1 deposition around tumors indicate that matrix-bound PAI-1 is heterogeneously distributed at the tumor periphery and that, at certain spots, the elevated concentrations of matrix-bound PAI-1 needed for cancer cells to undergo the mesenchymal-amoeboid transition can be observed. Matrix-bound PAI-1, as a matricellular protein, could thus represent one of the physiopathological requirements to support metastatic formation

Analysis of cell migration and its regulation by Rho GTPases and p53 in a three-dimensional environment.

International audienceCell migration plays a key role both in physiological conditions, such as tissue repair or embryonic development, and in pathological processes, including tumor metastasis. Understanding the mechanisms that allow cancer cells to invade tissues during metastasis requires studying their ability to migrate. While spectacular, the movements observed in cells growing on two-dimensional supports are likely only to represent a deformation of the physiological migratory behavior. In contrast, the analysis of cell migration on a support, which resembles the three-dimensional (3D) extracellular matrix, provides a more pertinent model of physiological relevance. This chapter provides protocols to assay the ability of cells to migrate or to invade a 3D matrix and to analyze their phenotypes. The invasion assay allows the quantification of tumor cell invasiveness, and the 3D migration assay permits the visual observation of the movements and morphology of migrating cells. This chapter also describes a method to examine the localization of different markers during 3D migration. Because Rho GTPases are clearly involved in migration and invasion, a protocol is supplied to evaluate their activation during cell migration. These techniques are especially suitable to elucidate the type of motility in a 3D matrix, particularly to discriminate between two different modes of migration adopted by cancer cells: blebbing versus elongation. Indeed, the way a cell moves may have important consequences for its invasiveness, as, for example, cancer cells adopt a rounded blebbing movement when deficient in p53

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles

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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

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

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

IFT proteins spatially control the geometry of cleavage furrow ingression and lumen positioning

Cytokinesis relies on central spindle organization and provides a spatial landmark for lumen formation. Here, the authors show that intraflagellar transport proteins are required for the localization of the cytokinetic regulator Aurora B and subsequent cleavage furrow ingression and lumen positioning

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles.

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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