L’ensemble des tissus et des organismes vivants sont constitués de cellules dans lesquelles un certain nombre de décisions phénotypiques sont prises : division, différentiation, apoptose ou encore transformation. La biologie cellulaire s’est principalement concentrée sur la compréhension des déterminants moléculaires internes de ces décisions, mais il est important de considérer aussi l’existence de déterminants externes provenant des interactions intercellulaires qui sont essentielles à l’émergence de systèmes multicellulaires coordonnés. La compétition entre les déterminants internes et les déterminants externes est un aspect fondamental de la sociologie des communautés cellulaire menant à de possibles situations hautement individualisées ou, au contraire, à un effet collectif dominant. Ce travail de thèse a eu pour but de mettre en place une méthode permettant de mesurer la contribution relative de ces deux types de déterminants en les mettant en opposition. Pour cela, la stabilité collective d’un épithélium in vitro a été mise à l’épreuve grâce à l’induction hétérogène de la transition épithelio-mesenchymateuse (EMT) par le biais de la photoactivation du facteur de transcription Snail1. Les résultats principaux montrent que la réponse transcriptionelle de cellules induites à l’EMT dépend de la présence, ou non, de cellules avoisinantes non-induites. De la même manière, les cellules non-induites répondent de façon transcriptionelle à la présence de cellules induites. Ces effets de control mutuels introduisent la notion que la géométrie de la distribution d’une cause moléculaire donnée peut influencer la conséquence de cette même cause. Notre travail ouvre de nouvelle possibilités pour l’étude de la sociologie de communautés cellulaires hétérogènes, et une meilleure compréhension de phénomènes importants tel la suppression phénotypique ou encore les premiers instants de la carcinogenèse. / Tissues and organisms are built from cells in which important phenotype decisions are made: division, differentiation, apoptosis, and transformation. Cell biology has strongly focused on deciphering the internal molecular determinants of these decisions, but external information originating from intercellular interactions are key elements to coordinate multicellular physiology. The extent to which internal determinants dominate over external determinants or vice versa, is an essential feature of the sociology of cell communities, with possibly strong individualistic situations, or dominant collective effect. The present work was designed to set-up a method for assessing the relative contribution of internal vs. external determinant, by opposing these two classes of inputs. This is achieved by challenging the collective stability of an in vitro epithelium using the heterogeneous induction of the epithelial-to-mesenchymal transition (EMT) via the photoactivation of Snail1. The key results show that the transcriptional response of EMT-induced cells depends on the presence of non-induced cells in the culture. Conversely non-induced cells respond to the presence of induced cells. These mutual control effects lead to the notion that the geometry underlying the distribution of a given molecular cause strongly influences its consequence. Our work opens new perspectives for studying the sociology of heterogeneous cell communities, and better understand important phenomena such as phenotype suppression and or the onset of carcinogenesis.

Lors du développement d’une tumeur au sein d’un tissu, les cellules cancéreuses se retrouvent entourées par les cellules saines. Les interactions entre ces deux types cellulaires, transformé et normal, jouent un rôle important dans le devenir de la tumeur, mais restent à ce jour mal comprises. L’objectif de cette thèse a été de mettre en place des systèmes in vitro qui permettent d’étudier les interactions entre une population de cellules normales et une population de cellules transformées.Nous avons tiré profit d’une lignée de cellules épithéliales sensibles à la lumière, élaborée par Olivier Destaing (IAB, Grenoble). Lorsqu’elles sont exposées à la lumière bleue, ces cellules suractivent la protéine Src, connue pour être surexprimée dans de nombreux cancers. Sinon, elles gardent un phénotype normal. L’utilisation de ces cellules, appelées « OptoSrc », combinée à un dispositif optique, permet de créer des tissus mosaïques dans lesquels le motif des cellules mutées est déterminé par le motif d’illumination bleue. Notre système présente plusieurs avantages : le contrôle dans le temps et dans l’espace du motif de cellules transformées, mais aussi l’activation graduelle et réversible de l’oncoprotéine.Nous avons montré qu’en illuminant dans le bleu un îlot circulaire de cellules au sein d’une monocouche OptoSrc, les cellules activées s’extrudent collectivement, donnant naissance à un agrégat tri-dimensionnel cohésif surplombant la monocouche. Nous pouvons contrôler la taille et le temps d’apparition de ce sphéroïde en ajustant respectivement l’aire éclairée et la fréquence d’illumination. De plus, ce phénomène d’extrusion collective est réversible lorsque le stimulus de lumière bleue s’arrête. Finalement, nous avons montré que la formation de cet agrégat s’accompagne d’une diminution des E-cadhérines à la membrane, et de l’apparition de la vimentine, pour les cellules éclairées. Nos résultats suggèrent qu'un groupe de cellules surexprimant la protéine Src, au sein d’une monocouche de cellules normales, subit une transition epithéliale- mesenchymateuse partielle. / During the development of a tumour in a tissue, the cancer cells are surrounded by healthy cells. The interactions between these two cell types, transformed and normal, play an important role in the tumour stability, but remain to this day poorly understood. The aim of this thesis was to establish in vitro assays to study the interactions between populations of normal and transformed cells.We benefited from a light-sensitive cell line, constructed by Olivier Destaing (IAB, Grenoble). When they are exposed to blue light, these cells overactivate the protein Src, which is known to be overexpressed in many cancers. Otherwise, they keep a normal phenotype. Using these cells, called “OptoSrc”, in combination with an optical setup, we are able to create mosaic tissues in which the pattern of mutated cells is determined by the blue illumination pattern. Our system has several advantages: a selective control in time and space of the group of transformed cells, and a gradual and reversible activation of the oncoprotein.We have shown that when we illuminate a circular islet of cells from a monolayer of OptoSrc cells, the activated cells were collectively extruded, resulting in a cohesive three-dimensional aggregate on top of the monolayer. We can control the size and appearance time of this spheroid by tuning, respectively, the area and frequency of illumination. Besides, this collective extrusion is reversible when the blue light stimulation is stopped. Finally, we have shown that the formation of this three-dimensional aggregate coincides with the loss of E-cadherin at the membrane, as well as the apparition of vimentin, for the illuminated OptoSrc cells. Our results suggest that a group of cells overexpressing the protein Src, in a monolayer of normal cells, undergoes a partial epithelial-to-mesenchymal transition.

The invasion of cancer cells is an important aspect of cancer progression. Single tumor cells exhibit at least two types of invasion in 3D environment, mesenchymal and amoeboid invasion. Tumor cells can switch between these two modes of movement depending on cellular status and surrounding environment. Amoeboid-mesenchymal transition (AMT) is less explored then mesenchymal-amoeboid transition (MAT). We performed a proteomic analysis of amoeboid-mesenchymal transition of human melanoma cell line A375M2. We have induced amoeboid-mesenchymal transition by treatment with a ROCK inhibitor Y27632 in 3D matrigel matrices and in 2D environment. Induction of the amoeboid-mesenchymal transition has changed a level of expression of 92 proteins and a level of phosphorylation of 15 proteins. Expression of only 17 proteins and phosphorylation of 8 proteins was identically changed in both of these environments. We found that PKCα regulates amoeboid migration and that treatment of cells with a PKCα inhibitor Gö6976 induces amoeboid-mesenchymal transition. Analysis of the proteomics data have further shown that induction of AMT by the ROCK inhibitor Y27632 leads to activation of antiapoptotic signals and activation of signaling pathways involved in regulation of actin cytoskeleton especially regulation of focal...

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance. Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors. Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors. Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires. / The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.

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Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.

No description available.

No description available.

<p>  </p> <p>Breast cancer is a disease that impacts nearly one out of three women at some point in their life. Although the scientific community’s understanding of breast cancer development is actively researched, there is still a low 5-year survival rate of 30% following distant metastasis compared to the near 100% survival rate for localized disease. Epithelial to mesenchymal transition (EMT) is a known contributor to metastasis. Cells that undergo EMT shed cell-to-cell junctions and become fibroblastic like cells with differential extracellular matrix organization and increased mesenchymal gene expression. This change allows for greater cell motility and invasive potential, critical for metastasis. Our recent studies with single cell RNA sequencing demonstrate distinct populations of epithelial and mesenchymal cells. Several components of fibroblast growth factor receptor (FGFR) signaling are regulated during EMT. Fibroblast growth factor binding protein 1 (FGFBP1) is a known developmental factor that was observed at low expression in mesenchymal cells, with an unknown role in breast cancer. This study utilizes immunoblotting, mRNA analyses, immunofluorescence staining and novel 3D culture platform to investigate the regulation of FGFBP1 during EMT. FGFBP1 was consistently downregulated in HER2 transformed human mammary epithelial cells (HME2) during transforming growth factor β (TGF-Beta) induced EMT. Since FGFBP1 is acts as a secretory chaperone protein, secretion rate analysis was conducted at time periods throughout EMT showing rapid downregulation of secretion. Characterization of FGFBP1 regulation during EMT could lead to greater understanding of EMT and possibly a more sensitive marker for EMT relative to the current known markers.</p>