Microenvironnement cellulaire, PAI-1 et migration cancéreuse

Cancer 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 one of the levels involved, which 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 mesenchymo-amaeboid 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. To cite this article: M. Malo et al., C. R. Biologies 329 (2006).     

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.     

SPARC and tumor growth: Where the seed meets the soil?

Matricellular proteins mediate interactions between cells and their extracellular environment. This functional protein family includes several structurally unrelated members, such as SPARC, thrombospondin 1, tenascin C, and osteopontin, as well as some homologs of these proteins, such as thrombospondin 2 and tensascin X. SPARC, a prototypic matricellular protein, and its homolog hevin, have deadhesive effects on cultured cells and have been characterized as antiproliferative factors in some cellular contexts. Both proteins are produced at high levels in many types of cancers, especially by cells associated with tumor stroma and vasculature. In this Prospect article we summarize evidence for SPARC and hevin in the regulation of tumor cell growth, differentiation, and metastasis, and we propose that matricellular proteins such as these perform critical functions in desmoplastic responses of tumors that culminate in their dissemination and eventual colonization of other sites.     

Inhibition of PAI-1 expression in breast cancer carcinoma cells by siRNA at nanomolar range

Plasminogen activator inhibitor type I (PAI-1) plays a central role in metastatic behavior by increasing cells' migratory capacities as shown in several tumoral cell lines. Moreover, in vivo high expression of this factor helps tumoral growth, both by its role in extracellular matrix remodeling and by favoring angiogenesis. High levels of PAI-1 are correlated with bad prognosis in several cancers, particularly in breast cancer. The effect of PAI-1 upon angiogenesis is also involved in atherosclerosis, in which high levels of PAI-1 expression are observed. Breast carcinoma MDA MB 231 cells are known for both having important metastatic capacities and expressing high levels of PAI-1. We have demonstrated in these cells that the transfection of PAI-1 specific small interfering RNAs (siRNA) specifically inhibited the expression of this factor by 91%. We evaluated siRNA activity by determining PAI-1 mRNA level, as well as intracellular and extracellular PAI-1 protein by using RT Q-PCR, Western blot and ELISA analyses, respectively. Data confirmed inhibition at mRNA levels (primary aim of interference), intracellular protein, and secreted PAI-1, the latter being operative successfully in the cell microenvironment. The lipidic vector Delivery Liposomes System (DLS) used was adapted to siRNA delivery as observed by particle size distribution analysis, confocal microscopy and transfection into MDA MB 231, in the presence of serum. SiRNA activity was clearly detected at concentrations as low as 10 nM. Moreover, the low cytotoxicity of this vector makes it a good candidate for future in vivo siRNA delivery.     

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.     

Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01.

Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.