Mise en évidence des propriétés chimiotactiques de l’oxygène pour des cellules épithéliales : implication du récepteur EGFR dans l’aérotaxie

Cell migration is a crucial process during embryonic development, wound healing, immune system but also metastasis. Success of these processes relies on the capacities of cells to sense an asymmetric signal, interpret it and orient themselves to migrate in a directed manner. In vivo, migration is guided by several signals from the cellular microenvironment. Hypoxia, or decrease in the level of tissue oxygen, is an important feature of the cellular environment in the embryo and in solid tumors. Owing to the limitation of oxygen diffusion, hypoxia often generates oxygen gradients in vivo. We have developed an original method in which epithelial cells themselves generate oxygen gradient in vitro. And interestingly, these cells are able to migrate directionally to higher oxygen concentrations. This aerotaxis ability is independent of mitochondrial respiration and hypoxia response pathway. The reactive oxygen species (ROS) would mediate the migratory response to the gradient. The asymmetric production of ROS between the front and the back of the cells would be at the origin of the differential activation of the EGFR receptor and the persistence of cells towards higher oxygen concentrations. This chemoattractant capacity of oxygen, known in bacteria, but not described for eukaryotic cells, could play a major role in embryonic development and in metastatic disseminationLa migration cellulaire dirigée est un processus crucial lors du développement embryonnaire, de la cicatrisation, de la réponse immunitaire mais aussi lors de la formation de métastases. La réussite de ces processus nécessite que les cellules perçoivent un signal asymétrique, l'interprète et s'oriente pour migrer de façon dirigée. In vivo, la migration est dirigée par de nombreux signaux du microenvironnement cellulaire. L'hypoxie, ou diminution du niveau d'oxygène tissulaire, est une caractéristique importante de l'environnement cellulaire dans l'embryon et dans les tumeurs solides. Du fait de la limitation de la diffusion de l'oxygène, l'hypoxie génère in vivo des gradients d'oxygène. Nous avons développé une méthode originale dans laquelle des cellules épithéliales génèrent elles-mêmes gradient d'oxygène in vitro. Et de façon très intéressante, ces cellules sont capables de migrer de façon directionnelle vers des concentrations en oxygène plus élevées. Cette capacité d'aérotaxie est indépendante de la respiration mitochondriale et des acteurs de réponse à l'hypoxie. Les dérivés réactifs de l'oxygène (ROS) seraient les médiateurs de la réponse migratoire au gradient. La production asymétrique de ROS entre l'avant et l'arrière des cellules serait à l'origine de l'activation différentielle du récepteur EGFR et de la persistance des cellules vers des concentrations plus importantes en oxygène. Cette capacité chimio-attractante de l'oxygène, connue chez les bactéries, mais non décrite pour des cellules eucaryotes, pourrait jouer un rôle majeur lors du développement embryonnaire et dans la dissémination métastatiqu

Identification of chemoattractant capacities of oxygen for epithelial cells : involvement of EGF receptor in aerotaxis

La migration cellulaire dirigée est un processus crucial lors du développement embryonnaire, de la cicatrisation, de la réponse immunitaire mais aussi lors de la formation de métastases. La réussite de ces processus nécessite que les cellules perçoivent un signal asymétrique, l'interprète et s'oriente pour migrer de façon dirigée. In vivo, la migration est dirigée par de nombreux signaux du microenvironnement cellulaire. L'hypoxie, ou diminution du niveau d'oxygène tissulaire, est une caractéristique importante de l'environnement cellulaire dans l'embryon et dans les tumeurs solides. Du fait de la limitation de la diffusion de l'oxygène, l'hypoxie génère in vivo des gradients d'oxygène. Nous avons développé une méthode originale dans laquelle des cellules épithéliales génèrent elles-mêmes gradient d'oxygène in vitro. Et de façon très intéressante, ces cellules sont capables de migrer de façon directionnelle vers des concentrations en oxygène plus élevées. Cette capacité d'aérotaxie est indépendante de la respiration mitochondriale et des acteurs de réponse à l'hypoxie. Les dérivés réactifs de l'oxygène (ROS) seraient les médiateurs de la réponse migratoire au gradient. La production asymétrique de ROS entre l'avant et l'arrière des cellules serait à l'origine de l'activation différentielle du récepteur EGFR et de la persistance des cellules vers des concentrations plus importantes en oxygène. Cette capacité chimio-attractante de l'oxygène, connue chez les bactéries, mais non décrite pour des cellules eucaryotes, pourrait jouer un rôle majeur lors du développement embryonnaire et dans la dissémination métastatiqueCell migration is a crucial process during embryonic development, wound healing, immune system but also metastasis. Success of these processes relies on the capacities of cells to sense an asymmetric signal, interpret it and orient themselves to migrate in a directed manner. In vivo, migration is guided by several signals from the cellular microenvironment. Hypoxia, or decrease in the level of tissue oxygen, is an important feature of the cellular environment in the embryo and in solid tumors. Owing to the limitation of oxygen diffusion, hypoxia often generates oxygen gradients in vivo. We have developed an original method in which epithelial cells themselves generate oxygen gradient in vitro. And interestingly, these cells are able to migrate directionally to higher oxygen concentrations. This aerotaxis ability is independent of mitochondrial respiration and hypoxia response pathway. The reactive oxygen species (ROS) would mediate the migratory response to the gradient. The asymmetric production of ROS between the front and the back of the cells would be at the origin of the differential activation of the EGFR receptor and the persistence of cells towards higher oxygen concentrations. This chemoattractant capacity of oxygen, known in bacteria, but not described for eukaryotic cells, could play a major role in embryonic development and in metastatic disseminatio

Redox regulation of EGFR steers migration of hypoxic mammary cells towards oxygen

International audienceAerotaxis or chemotaxis to oxygen was described in bacteria 130 years ago. In eukaryotes, the main adaptation to hypoxia currently described relies on HIF transcription factors. To investigate whether aerotaxis is conserved in higher eukaryotes, an approach based on the self-generation of hypoxia after cell confinement was developed. We show that epithelial cells from various tissues migrate with an extreme directionality towards oxygen to escape hypoxia, independently of the HIF pathway. We provide evidence that, concomitant to the oxygen gradient, a gradient of reactive oxygen species (ROS) develops under confinement and that antioxidants dampen aerotaxis. Finally, we establish that in mammary cells, EGF receptor, the activity of which is potentiated by ROS and inhibited by hypoxia, represents the molecular target that guides hypoxic cells to oxygen. Our results reveals that aerotaxis is a property of higher eukaryotic cells and proceeds from the conversion of oxygen into ROS

Combination of a discovery LC-MS/MS analysis and a label-free quantification for the characterization of an epithelial-mesenchymal transition signature

International audienceDisease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGF beta or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGF beta and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed.Biological SignificanceTo our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGF beta and K-Ras(v12). This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers

Reconstitution of cell migration at a glance

International audienc

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

SummaryThe RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins “master orchestrators” of differentiation that dynamically orchestrate several layers of gene expression

The apoptosis inhibitor Bcl-xL controls breast cancer cell migration through mitochondria-dependent reactive oxygen species production

International audienceThe Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer

TIF1γ interferes with TGFβ1/SMAD4 signaling to promote poor outcome in operable breast cancer patients.

International audienceThe Transforming growth factor β (TGFβ) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFβ1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFβ/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFβ1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFβ1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. 223 cases were assessable for TIF1γ, 204 for TGFβ1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFβ1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFβ1 was not associated with any of the traditional prognostic factors. TGFβ1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95% CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95 % CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFβ1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFβ1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95% CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95 % CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95 % CI: 1.4 to 5.4; p=0.005). There is a crosstalk between the TIF1γ and the TGFβ1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients