Rôle de la protéine ARF6 dans le processus invasif du cancer du sein.

The ability of cancer cells to traffic through the extracellular matrix relies on the action of the membrane-anchored matrix metalloprotease MT1-MMP. MT1-MMP is exocytosed to invadopodia, the actin-based membrane protrusions responsible for matrix degradation. The small GTP-binding protein ARF6 is known to coordinate post-endocytic recycling and actin cytoskeletal organization at the plasma membrane and was shown to be up-regulated in breast cancer cells. In my PhD work I showed that ARF6 and two of its effectors JIP3 and JIP4 are required for MT1-MMP endosomes intracellular positioning and exocytosis at invadopodia and consequently for tumor cells ability to remodel the matrix and invade through a three-dimensional matrix environment. ARF6, through the interaction with JIP3/4, negatively controls the activity of the minus-end-directed microtubule motor dynactin/dynein, thus negatively regulating the clearance and inward movement of MT1-MMP endosomes from the cell periphery. In human samples ARF6 is accumulated at the plasma membrane, together with MT1-MMP, in a subset of highly aggressive breast carcinomas, thus corroborating the ARF6-JIP3/JIP4-MT1-MMP axis in breast cancer invasion. In a second study I addressed the contribution of ARF6 activation on actin cytoskeleton remodeling in breast cancer cells. ARF6 links epidermal growth factor receptor signaling to Rac1 activation and targeting to the leading edge where it activates the SCAR/WAVE complex and regulates ventral actin polymerization during lamellipodia extension. Collectively my work identifies novel molecular mechanisms through which ARF6 contributes to the invasive program of breast tumor cellsLa migration des cellules tumorales à travers la matrice extracellulaire dépend de l'activité d'une métalloprotéase matricielle, MT1-MMP, ancrée à la membrane plasmique. MT1-MMP accumule aux invadopodes, des protrusions membranaires à base d'actine responsables de la dégradation de la matrice. La petite protéine G ARF6 est impliquée dans la régulation du trafic membranaire et dans le remodelage du cytosquelette d'actine. Dans mon travail de thèse, j'ai montré qu'ARF6 et deux de ses protéines effectrices JIP3 et JIP4, sont nécessaires à l'exocytose de MT1-MMP au niveau des invadopodes et, par conséquent, à la capacité des cellules tumorales à remodeler la matrice extracellulaire et migrer à travers un environnement matriciel tridimensionnel. ARF6, à travers son interaction avec JIP3/4, contrôle négativement l'activité du complexe dynactine/dynéine, un moteur moléculaire qui se déplace en direction du bout (-) des microtubules, et donc la clairance des endosomes MT1-MMP à partir de la périphérie cellulaire. En plus dans des échantillons humaines ARF6 est accumulée au niveau de la membrane plasmique, avec MT1-MMP, dans un sous-groupe de carcinomes mammaires agressifs, en confirmant donc l'implication d'un axe ARF6-JIP3/JIP4-MT1-MMP dans le processus invasif du cancer du sein. Dans une deuxième étude, j'ai montré que l'hyperactivation d'ARF6 induit un réarrangement important du cytosquelette d'actine à la surface ventrale des cellules tumorales mammaires et contribue à l'activation et au ciblage de Rac1 au front cellulaire. Mon travail a permis d'identifier de nouveaux mécanismes moléculaires par lesquels ARF6 contribue au programme invasif des cellules tumorales mammaires

ARF6 Promotes the Formation of Rac1 and WAVE-Dependent Ventral F-Actin Rosettes in Breast Cancer Cells in Response to Epidermal Growth Factor

<div><p>Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.</p></div

Hyperactivation of ARF6 induces formation of ventral self-expanding F-actin and cortactin-rich rosette-like structures in MDA-MB-231 breast adenocarcinoma cells.

<p><b>(A)</b> Immunofluorescence microscopy micrographs of MDA-MB-231 cells (left panel) or MDA-MB-231 cells stably expressing ARF6T157N (upper right panel) or ARF6T27N (lower right panel) stained for cortactin. Arrowheads, ventral cortactin-positive rosettes; arrows, cortactin-enriched lamellipodia; asterisks, endosomal cortactin-rich puncta. Scale bar, 5 μm. <b>(B)</b> Percentage of cells displaying cortactin-positive rosettes was scored in the three cell populations. Values are mean ± SEM from three (MDA-MB-231 and MDA-MB-231/ARF6T27N cells) and seven (MDA-MB-231/ARF6T157N cells) independent experiments, scoring 50 cells for each cell population in each experiment. Comparisons were made with a Student’s t-test. ***, P < 0.001, *, P < 0.05 as compared to parental MDA-MB-231 cells. <b>(C-D)</b> Still images of TIRFM time-lapse sequences of MDA-MB-231 cells expressing ARF6T157N plated on gelatin. Cells were transiently transfected to express DsRed-cortactin. Scale bars, 10 μm. Galleries correspond to the boxed regions of the still images. Time is in min. Scale bars, 5 μm. <b>(E)</b> Still image of a time-lapse sequence of MDA-MB-231 cell expressing ARF6T157N-GFP (green) and cortactin-DsRed (red) plated on gelatin and imaged by confocal spinning disk microscopy. Scale bar, 10 μm. <b>(F)</b> The gallery corresponds to the boxed region in E. Time is in seconds. Scale bar, 5 μm. <b>(G)</b> Kymograph of ARF6T157N-GFP (green) and cortactin-DsRed (red)-positive rosette. The line used for kymograph analysis is shown in the still image in E.</p

Formation of cortactin-positive ventral rosettes requires the Arp2/3 complex and SCAR/WAVE.

<p><b>(A-D)</b> MDA-MB-231 cells stably expressing ARF6T157N plated on cross-linked gelatin were fixed and stained for the indicated proteins. Images were acquired by wide-field microscopy. Scale bar, 10 μm. Insets are higher magnification of the boxed regions. Scale bar 5 μm. <b>(E)</b> Cells stably expressing ARF6T157N treated with the indicated siRNAs for 72 hours were plated on cross-linked gelatin, fixed and stained for cortactin. Scale bar, 10 μm. <b>(F)</b> The percentage of cells displaying cortactin-positive rosettes was scored. Values are mean ± SEM from at least four independent experiments, scoring about 200 cells for each cell population. Comparisons were made with a Student’s t-test. ns, non significant, ***, P < 0.001 (compared to siNT-treated cells).</p

EGF stimulation triggers the formation of ventral F-actin structures in MDA-MB-231 cells.

<p><b>(A)</b> MDA-MB-231 cells were plated on cross-linked gelatin, serum starved over-night and stimulated with EGF for 30’ sec up to 10 min as indicated. Then cells were fixed and stained for cortactin and images were acquired by wide-field microscopy. Arrows point to nascent cortactin-positive rosettes. Scale bar, 10 μm. <b>(B)</b> MDA-MB-231 cells were treated as in <b>A</b> and the percentage of cells displaying cortactin-positive rosettes was scored. Values are mean ± SEM from duplicate samples in two independent experiments scoring 100 cells for each cell population in each experiment. Comparisons were made with a Student’s t-test. ***, P < 0.001 (compared with serum-starved MDA-MB-231 cells). <b>(C)</b> Gallery from a time-lapse sequence of MDA-MB-231 cells expressing DsRed-cortactin after EGF-treatment. Arrows point to nascent cortactin-positive rosette. Time is in sec. Scale bar, 10 μm.</p

Cortactin-rich rosettes are ARF6-dependent and correlate with membrane protrusion formation.

<p><b>(A)</b> Still images of TIRFM time-lapse sequences of MDA-MB-231 cells treated with non-targeting or ARF6 siRNAs and transfected with GFP- or DsRed-cortactin, respectively. Arrowheads, ventral cortactin-positive rosettes; arrows, cortactin-enriched lamellipodia. Scale bars, 10 μm. <b>(B-C)</b> Frequency of cortactin-positive rosette <b>(B)</b> and membrane protrusion formation <b>(C)</b> in the indicated cell populations was calculated by scoring rosettes or protrusions occurring per cell and per hour. Values are mean ± SEM from two independent experiments. <i>n</i> represents the number of cells scored for each cell population. Comparisons were made with a Mann-Whitney (two-tailed) t-test. ***, P < 0.001, **, P < 0.01 (compared with siNT-treated cells). <b>(D)</b> Correlation between number of rosettes (x-axis) and protrusions (y-axis) scored during time-lapse sequences of control siNT-treated cells (n = 47). Correlation was calculated with a Spearman test. <b>(E)</b> Still images of TIRFM movies of MDA-MB-231 cells treated as in A showing extension of membrane protrusions. Kymograph views were obtained from the white lines in the still images between the first and twentieth frame with a 1 min interval between each frame. <b>(F)</b> Speed of membrane protrusion extension in the indicated cell populations was calculated by dividing the distance of protrusion (vertical axis in the kymographs) by the time (horizontal axis). Values are mean ± SEM from two independent experiments. <i>n</i> represents the number of protrusions scored for each cell population. Comparisons were made with a Mann-Whitney (two-tailed) t-test. ***, P < 0.001 (compared with siNT-treated cells). <b>(G-I)</b> Epifluorescence images of MDA-MB-231 cells <b>(G)</b> and ARF6T157N-expressing cells <b>(H-I)</b> stained for cortactin (red) and plated on FITC-labelled gelatin (cyan). Scale bar 10 μm. Magnification of the boxed regions of the merged images and gelatin channel are shown in the right panels. Scale bar 5 μm. <b>(J, K)</b> Percentage of degradative cells <b>(J)</b> and degradation index <b>(K)</b> calculated by normalizing the degradation area by the cell area in the different cell populations. Values are mean ± SD from two replicate values from one experiment. <i>n</i> represents the number of cells scored for each cell population. Comparisons were made with Student’s t-test. Ns, non significant, **, P < 0.01 (compared with MDA-MB-231 cells).</p

Rac1 localizes in ventral actin-rich rosettes and ARF6 is required for Rac1 activation and recruitment.

<p><b>(A)</b> MDA-MB-231 cells stably expressing ARF6T157N were plated on cross-linked gelatin, fixed and stained for F-actin and Rac1. Upper insets are higher magnifications of boxed regions. Lower insets show rosettes from another cell. Scale bars, 10 μm and 5 μm (insets). <b>(B)</b> ARF6 immunoblotting analysis of MDA-MB-231 cell lysates after indicated siRNA treatments. Anti-β-actin was used as loading control. <b>(C)</b> MDA-MB-231 cells were treated with the indicated siRNAs for 72 hours, plated on gelatin and then serum-starved for 12–16 hrs. After 15 min treatment with EGF, cells were fixed and stained for F-actin and Rac1. Insets are higher magnification of boxed regions. Scale bars, 10 μm. Arrowheads, peripheral F-actin-rich rosette. <b>(D)</b> Quantification of Rac1 fluorescence intensity profile was done along a 160-pixel line drawn perpendicularly to the cell edge as shown in insets. Intensity profiles along the line from at least 50 cells per condition from two independent experiments were averaged and normalized to the highest fluorescence intensity value in the siNT-treated cells. Comparison between the two mean peak values (corresponding to the cell edge) was made with a two-way ANOVA test. ***, P < 0.001 (compared to siNT-treated cells). <b>(E)</b> MDA-MB-231 cells were treated with the indicated siRNAs for 72 hrs, plated on gelatin, serum-starved and treated with EGF for 1 or 15 min. Levels of GTP-bound ARF6 (blue bars) and Rac1 (purple bars) in the different conditions were measured with G-LISA assay. Values are normalized mean ± SEM from replicate samples from two independent experiments. Comparisons were made with a Student’s t-test. **, P < 0.01, ***, P < 0.001 (compared with siNT-treated cells).</p

Cellular and Molecular Mechanisms of MT1-MMP-Dependent Cancer Cell Invasion

Metastasis is responsible for most of cancer-associated deaths. Accumulating evidence based on 3D migration models has revealed a diversity of invasive migratory schemes reflecting the plasticity of tumor cells to switch between proteolytic and nonproteolytic modes of invasion. Yet, initial stages of localized regional tumor dissemination require proteolytic remodeling of the extracellular matrix to overcome tissue barriers. Recent data indicate that surface-exposed membrane type 1?matrix metalloproteinase (MT1-MMP), belonging to a group of membrane-anchored MMPs, plays a central role in pericellular matrix degradation during basement membrane and interstitial tissue transmigration programs. In addition, a large body of work indicates that MT1-MMP is targeted to specialized actin-rich cell protrusions termed invadopodia, which are responsible for matrix degradation. This review describes the multistep assembly of actin-based invadopodia in molecular details. Mechanisms underlying MT1-MMP traffic to invadopodia through endocytosis/recycling cycles, which are key to the invasive program of carcinoma cells, are discussed.Fil: Castro Castro, Antonio. Instituto Pasteur; FranciaFil: Marchesin, Valentina. Imagine Institute of Genetic Diseases; FranciaFil: Monteiro, Pedro. University of London; Reino UnidoFil: Lodillinsky, Catalina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossé, Carine. Institute Curie; Francia. Centre National de la Recherche Scientifique; FranciaFil: Chavrier, Philippe. Institute Curie; Francia. Centre National de la Recherche Scientifique; Franci

Molecular Basis for Autosomal-Dominant Renal Fanconi Syndrome Caused by HNF4A

International audienceHNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). Here, we find that expression of the conserved fly ortholog dHNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type dHNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. Our data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors