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

PAI-1 and functional blockade of SNAI1 in breast cancer cell migration

12 pages, 5 figures.-- PMID: 19055748 [PubMed].-- et al.[Introduction]: Snail, a family of transcriptional repressors implicated in cell movement, has been correlated with tumour invasion. The Plasminogen Activation (PA) system, including urokinase plasminogen activator (uPA), its receptor and its inhibitor, plasminogen activator inhibitor type 1(PAI-1), also plays a key role in cancer invasion and metastasis, either through proteolytic degradation or by non-proteolytic modulation of cell adhesion and migration. Thus, Snail and the PA system are both over-expressed in cancer and influence this process. In this study we aimed to determine if the activity of SNAI1 (a member of the Snail family) is correlated with expression of the PA system components and how this correlation can influence tumoural cell migration.[Methods]: We compared the invasive breast cancer cell-line MDA-MB-231 expressing SNAI1 (MDA-mock) with its derived clone expressing a dominant-negative form of SNAI1 (SNAI1-DN). Expression of PA system mRNAs was analysed by cDNA microarrays and real-time quantitative RT-PCR. Wound healing assays were used to determine cell migration. PAI-1 distribution was assessed by immunostaining.[Results]: We demonstrated by both cDNA microarrays and realtime quantitative RT-PCR that the functional blockade of SNAI1 induces a significant decrease of PAI-1 and uPA transcripts. After performing an in vitro wound-healing assay, we observed that SNAI1-DN cells migrate more slowly than MDA-mock cells and in a more collective manner. The blockade of SNAI1 activity resulted in the redistribution of PAI-1 in SNAI1-DN cells decorating large lamellipodia, which are commonly found structures in these cells.[Conclusions]: In the absence of functional SNAI1, the expression of PAI-1 transcripts is decreased, although the protein is redistributed at the leading edge of migrating cells in a manner comparable with that seen in normal epithelial cells.This work was supported by the CNRS ACI Program "Complexité du vivant" (grant # 050009DR11) and by the Evry Genopole grant "Aide à l'acquisition d'équipement semi-lourd" 2007 and 2008.Peer reviewe

Dynamique du microenvironnement cellulaire

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Detection and counting of "in vivo" cells to predict cell migratory potential

International audienceIn this paper, we present a work which is performed by biologists and computer scientists both. The aim of this work is to evaluate the migratory potential of cancerous cells. Cancer is characterised by primary tumour. When some cells move they create new tumours, which are called metastases. It is very important to understand this migration process in order to be able to arrest it and increase the chances of a cure. Today, biologists analyse images from different cell cultures and manually count one by one the cells present therein. It is a hard and fastidious work, so here we present some algorithms to automatically perform these tasks of detection and counting. The images that we have are very low contrasted, with a gradient of illumination, and the cells are numerous and tightly aggregated. In this paper different algorithms are evocated and results compared for about 150 images comprising more than 65,000 cells

The PAI-1 swing: Microenvironment and Cancer cell migration

International audienceCancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process we need to study each of the levels involved, what allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymoamaeboid transition. This matricellular protein should be considered as a potential launcher of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair

International audienceIn vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment

Synergy between PAI-1 and Snail in tumoral migration

International audienceDuring epithelial-mesenchymal transition (EMT), cells undergo structural changes resulting in loss of polarity, contact with neighbouring cells and acquisition of mobility. Members of the Snail family act as transcriptional repressors; their important role in EMT has been demonstrated both during embryogenesis and oncogenesis. In several tumours including breast cancers, Snail has been correlated with tumour invasivity, partly on its ability to directly repress transcription of genes whose products are involved in cell-adhesion. The Plasminogen Activation system (PAs), including urokinase (uPA), its receptor (uPAR), and its inhibitor (PAI-1), also plays a pivotal role in cancer invasion and metastasis, either through proteolytic degradation or by non proteolytic modulation of cell adhesion and migration. Thus, Snail and PAs both influence those processes. Moreover, Snail and many of the components of the PA system are over-expressed in cancer. In this study we aimed to determine if PAI-1's expression is correlated with Snail activity and how this correlation can influence tumoral cell migration. Therefore we compared the invasive breast cancer MDA-MB-231 Snail positive cell-line with a dominant negative form of Snail (Snail negative cells). Expression of PAI-1 messengers was performed by cDNA microarrays and real time quantitative RT-PCR. A wound healing assay was used to determine migration and PAI-1's distribution was assessed by immunostaining. We showed a significant decrease in PAI-1 messengers in Snail negative cells compared to Snail positive cells. We also observed that Snail (-) cells migrate more slowly than Snail (+) cells and in a more organised and collective pathway. We also examined changes in PAI-1's localization. We observed a homogeneous immunolabelling of PAI-1 in Snail (+) but not in Snail (-) cells where, on the contrary, PAI-1 expression appeared restricted to the outermost edge of the wound. We conclude that, in the absence of functional Snail, expression of PAI-1 transcripts is decreased, but PAI-1 protein is relocated at the leading edge of migration. The decrease migration in Snail (-) cells, was observed together with a more collective behaviour opposed to the individual cell translocation observed during metastasis. Taken together, these findings show a synergy between PAI-1 and Snail, two factors implicated independently in tumoral migration and shed a new light on our understanding of Snail's multiple actions during the metastatic process

Matrix-bound PAI-1, an extracellular modulator of cell functions: a novel member of the matricellular protein family

International audiencePAI-1 is a SERPIN found either soluble or bound to vitronectin in the matrix. In both cases, PAI-1 is a fast acting inhibitor for uPA. By that mean PAI-1 modulates all the processes influenced by uPA, such as development, wound healing, cancer cell migration, proteolysis, and in general, cell matrix interactions. In cell adhesion, PAI-1 plays directly a contradictory role: 1. interfering in Vitronectin/Integrin linking, i.e. in de adhesion, 2. as a member of the tripartite complex uPAR/uPA/PAI-1, promotes the linking of the cell membrane to ECM, i.e. intermediate adhesion. PAI-1 has also been evoked in another key cell function, migration and we previously showed that cancer cells of high invasivity were parallely increasing their migration when surrounded by a PAI-1 coat (1). As such, PAI-1 already appeared as an extracellular modulator of cell function. Here we present several additional experimental arguments for considering PAI-1 as a novel member of the matricellular protein family (2). 1. In an in vitro epithelial cell wound-healing model, where the main process is cell migration, a) we have shown that antibodies directed against PAI-1 were able to decrease the rate of closure; b) PAI-1 was expressed as an ECM-bound protein within 6 hours after wounding. 2. In epithelial colon cancer cells, a) matrix PAI-1 was able to reorganize the actin cytoskeleton (producing actin rings) and lead to the modification of the Rho/Rock pathway; b) matrix PAI-1 induces cell blebbing and amaeboid migration, a strong modification of cell behaviour. 3. In invasive mammary cancer cells, a) the expression of PAI-1 mRNAs was correlated with the presence of the transcription factor SNAIL, a major pro-migratory factor involved in embryogenesis, and cancer invasion; b) matrix PAI-1 induced an increase in steady state PAI-1 mRNA levels in cells where the expression of SNAIL has been switched off. These data strongly suggest matrix PAI-1 as an extracellular modulator of cell function, referring to the main family of matricellular protein

Matrix-bound PAI-1, an extracellular modulator of cell functions: a novel member of the matricellular protein family

International audiencePAI-1 is a SERPIN found either soluble or bound to vitronectin in the matrix. In both cases, PAI-1 is a fast acting inhibitor for uPA. By that mean PAI-1 modulates all the processes influenced by uPA, such as development, wound healing, cancer cell migration, proteolysis, and in general, cell matrix interactions. In cell adhesion, PAI-1 plays directly a contradictory role: 1. interfering in Vitronectin/Integrin linking, i.e. in de adhesion, 2. as a member of the tripartite complex uPAR/uPA/PAI-1, promotes the linking of the cell membrane to ECM, i.e. intermediate adhesion. PAI-1 has also been evoked in another key cell function, migration and we previously showed that cancer cells of high invasivity were parallely increasing their migration when surrounded by a PAI-1 coat (1). As such, PAI-1 already appeared as an extracellular modulator of cell function. Here we present several additional experimental arguments for considering PAI-1 as a novel member of the matricellular protein family (2). 1. In an in vitro epithelial cell wound-healing model, where the main process is cell migration, a) we have shown that antibodies directed against PAI-1 were able to decrease the rate of closure; b) PAI-1 was expressed as an ECM-bound protein within 6 hours after wounding. 2. In epithelial colon cancer cells, a) matrix PAI-1 was able to reorganize the actin cytoskeleton (producing actin rings) and lead to the modification of the Rho/Rock pathway; b) matrix PAI-1 induces cell blebbing and amaeboid migration, a strong modification of cell behaviour. 3. In invasive mammary cancer cells, a) the expression of PAI-1 mRNAs was correlated with the presence of the transcription factor SNAIL, a major pro-migratory factor involved in embryogenesis, and cancer invasion; b) matrix PAI-1 induced an increase in steady state PAI-1 mRNA levels in cells where the expression of SNAIL has been switched off. These data strongly suggest matrix PAI-1 as an extracellular modulator of cell function, referring to the main family of matricellular protein