Christelle Anguille

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.

TNFα induces sequential activation of Cdc42- and p38/p53-dependent pathways that antagonistically regulate filopodia formation

Cell migration is an essential function in various physiological processes, including tissue repair and tumour invasion. Repair of tissue damage requires the recruitment of fibroblasts to sites of tissue injury, which is mediated in part by the cytokine tumour necrosis factor α (TNFα). As dynamic rearrangements of actin cytoskeleton control cell locomotion, this implicates that TNFα is a potent coordinator of cellular actin changes. We have investigated the role of TNFα in regulating the cortical actin-containing structures essential for cell locomotion called filopodia. Kinetic analysis of TNFα-treated mouse embryonic fibroblasts (MEFs) revealed a dual effect on filopodia formation: a rapid and transient induction mediated by Cdc42 GTPase that is then counteracted by a subsequent sustained inhibition requiring activation of the mitogen-activated protein kinase p38 but not Cdc42 activity. This inhibition also involves the tumour suppressor p53, given that it is activated in response to TNFα following the same time course as the decrease of filopodia formation. This functional activation of p53, measured by transcription induction of its target p21WAF1(p21), is also associated with p38 kinase-dependent phosphorylation of p53 at serine 18. Furthermore, TNFα did not inhibit filopodia formation in MEFs treated with the transcription inhibitor actinomycin D, in p53-deficient MEFs, or MEFs expressing p53 mutants H273 or H175, which supports a role for the transcriptional activity of p53 in mediating TNFα-dependent filopodia inhibition. Our data delineate a novel inhibitory pathway in which TNFα prevents filopodia formation and cell migration through the activation of the mitogen-activated protein kinase (MAPK) p38, which in turn activates p53. This shows that TNFα on its own initiates antagonistic signals that modulate events linked to cell migration.

Cooperative anti-invasive effect of Cdc42/Rac1 activation and ROCK inhibition in SW620 colorectal cancer cells with elevated blebbing activity.

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.

Analysis of Cell Migration and Its Regulation by Rho GTPases and p53 in a Three-Dimensional Environment

Cell 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.

Postprandial triglyceride‐rich lipoproteins promote invasion of human coronary artery smooth muscle cells in a fatty‐acid manner through PI3k‐Rac1‐JNK signaling

SCOPE The aim was to investigate the effect of postprandial triglyceride-rich lipoproteins (TRLs) with different fatty acid compositions on human coronary artery smooth muscle cell (hCASMC) invasion and to identify the molecular pathways involved. METHODS AND RESULTS TRLs were isolated from the plasma of healthy volunteers after the ingestion of single meals enriched in MUFAs, saturated fatty acids (SFAs), or PUFAs. hCASMC invasion was analyzed using transwell chambers with Matrigel. TRLs-SFAs provoked the highest invasion, followed by TRLs-MUFAs and TRLs-PUFAs. Inhibition studies with Orlistat showed that invasion was dependent on the fatty acid composition of the TRLs. Fatty acids incorporated into the cell membranes strongly associated with cell invasion. Pull-down assays showed that TRLs-SFAs were able to increase Rac1 activity via inhibition of RhoA-dependent signaling. Chemical inhibition and siRNA studies showed that Rac1, PI3k, JNK, and MMP2 regulates TRL-SFA-induced hCASMC invasion. CONCLUSION We demonstrate for the first time that TRLs induce hCASMCs invasion in a fatty acid dependent manner. This effect in TRLs-SFAs is mediated by the PI3k-Rac1-JNK, RhoA, and Rac1-MMP2 pathways. The ingestion of MUFA, compared to other dietary fatty acids such as SFA, could be considered as a nutritional strategy to reduce the atherosclerotic plaque formation.

Co-crystallization and preliminary X-ray diffraction studies of Lathyrus ochrus isolectin I with di- and trisaccharides, and a biantennary octosaccharide.

Isolectin I (LOL I) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of four different oligosaccharides from an N-acetyllactosaminic type biantennary glycan of human lactotransferrin. The crystals containing putative complexes of LOL I with two different disaccharides are isostructural with the saccharide-free LOL I form (space group P2(1)2(1)2, a = 135.8 A, b = 63.1 A and c = 54.5 A). The LOL I-trisaccharide complex crystallizes in the same space group with small but significant changes in the cell dimensions: a = 136.9 A,b = 63.4 A and c = 54.6 A. Both crystal forms diffract strongly up to at least 1.8 A resolution. One functional entity, an alpha 2 beta 2 tetramer in the asymmetric unit (Mr = 52,000) give a Vm of 2.2 A3/dalton, or a solvent content of approximately 44%. The putative LOL I-octosaccharide complex crystallizes in the monoclinic space group C2 with cell dimensions a = 78.3 A, b = 75.4 A, c = 103.9 A and beta = 92 degrees. The crystals diffract to a resolution of 2.3 A and are suitable for crystallographic investigations. An alpha 2 beta 2 tetramer complexed to two octosaccharides (Mr = 55,000) in the asymmetric unit leads to a Vm value of 2.8 A3/dalton (57% solvent).

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

Cytokinesis mediates the physical separation of dividing cells and, in 3D epithelia, provides a spatial landmark for lumen formation. Here, we unravel an unexpected role in cytokinesis for proteins of the intraflagellar transport (IFT) machinery, initially characterized for their ciliary role and their link to polycystic kidney disease. Using 2D and 3D cultures of renal cells, we show that IFT proteins are required to correctly shape the central spindle, to control symmetric cleavage furrow ingression and to ensure central lumen positioning. Mechanistically, IFT88 directly interacts with the kinesin MKLP2 and is essential for the correct relocalization of the Aurora B/MKLP2 complex to the central spindle. IFT88 is thus required for proper centralspindlin distribution and central spindle microtubule organization. Overall, this work unravels a novel non-ciliary mechanism for IFT proteins at the central spindle, which could contribute to kidney cyst formation by affecting 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.

Correction: The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner

[This corrects the article DOI: 10.1371/journal.pone.0172125.].