Le cancer du sein est une pathologie hétérogène au plan clinique et au moins 5 sous-types moléculaires ont pu être définis sur la base de différences d’expression ARNm. Ces sous-types présentent des différences de profils d’anomalies génomiques et de méthylation des cytosines. Ces différences génétiques et épigénétiques s’expliqueraient par des types cellulaires d’origines distincts au sein de l’épithélium mammaire, toutefois, ceci n’a pas été confirmé clairement à ce jour. Alternativement, il a été proposé que l’activation de voies oncogéniques différentes pouvait avoir un impact significatif sur les modifications génétiques ou épigénétiques. Dans ce travail nous avons voulu vérifier cette hypothèse en l’appliquant à un modèle de cellules épithéliales mammaires normales primaires humaines, que nous avons isolé des à partir de glandes mammaires. Ces cellules ont été transformées en deux étapes par transduction avec (i) un shARN ciblant TP53, (ii) un oncogène. Nous avons sélectionné 3 oncogènes qui activent des voies de signalisations distinctes CCNE1, HRAS-v12 et WNT1. Nous avons établi un modèle de transformation tumorale en trois étapes, cellules normales, immortalisées et transformées, permettant de suivre les modifications moléculaires associées à chaque étape et de vérifier si l’activation de voies oncogéniques distinctes produisait des profils d’anomalies différents. Les différents modèles ont été analysés par CGH-array, RRBS, transcriptome et miRNA à des temps de culture définis.Nos résultats montrent que l’activation de la voie RAS aboutit à des profils d’anomalies génétiques et de méthylation des CpG radicalement différents de ceux obtenus après surexpression des gènes CCNE1 et WNT1. Ces différences apparaissent très rapidement après transduction des oncogènes alors que les profils des cellules CCNE1 et WNT1 divergent plus tardivement. Enfin, l’inactivation de p53 n’induit pas par elle-même une instabilité élevée, mais produit un contexte de plasticité favorable aux modifications génétiques et épigénétique.Par ailleurs, nous avons noté des différences phénotypiques entre les HMEC RAS (mésenchymateuses) et les HMEC CCNE1 et les HMEC WNT1 (épithéliales). Dans ce travail, je montre que les HMEC shp53 immortalisées présentent une plasticité phénotypique, une partie des cellules entrant en EMT spontanément, l’autre restant épithéliales. J’ai montré que la transduction RAS sélectionnait les cellules ayant effectué une EMT, alors que la transduction de CCNE1 ou WNT1 sélectionnait les cellules épithéliales. J’ai cherché à identifier les déterminants de ces changements phénotypiques et mes résultats suggèrent qu’ils résultent d’une balance entre une signalisation TGFB1/BMP1, qui favorise l’EMT, et BMP4/WNT7 qui favorise la MET. / Breast cancer is a heterogeneous pathology. Based on the differences of mRNA expression, at least five molecular subtypes have been defined. These subtypes show differences in profiles of genomic abnormalities and CpG methylation. These molecular subtypes are thought to originate from different cell lineages in the mammary gland. However, this has not yet been clearly demonstrated. Alternatively, it has been proposed that the activation of different oncogenic pathways could have a significant impact on genetic or epigenetic changes.We wanted to verify this hypothesis by applying it to a normal primary human mammary epithelial cells (HMEC) model, which we isolated from normal mammary explants. These cells were transformed in two step process by sequential transduction of (i) a shRNA targeting TP53, (ii) an oncogene. We selected 3 oncogenes that activate distinct signaling pathways CCNE1, HRAS-v12 and WNT1. Our tumor transformation model was established in three-step, normal, immortalized and transformed cells, allowing us to monitor the molecular changes associated with each step and to verify whether the activation of distinct oncogenic pathways produced different profiles of genetic and epigenetic modifications. The different models were analyzed at defined culture times by CGH-array, RRBS, transcriptome and miRNA. Our results show that genetic abnormalities and CpG methylation profiles are different between cells where the RAS pathway was activated and cells overexpressing WNT1 or CCNE1. These differences appear rapidly after oncogene transduction, whereas the profiles of the CCNE1 and WNT1 cells diverged later. Finally, inactivation of p53 by itself does not induce high instability, but produces a context of plasticity favorable to genetic and epigenetic changes.In addition, we noted phenotypic differences between HMEC RAS (mesenchymal) and HMEC CCNE1 and HMEC WNT1 (epithelial). In this work, I show that the immortalized HMEC shp53 exhibit a phenotypic plasticity, where some cells enter a spontaneous EMT and the others remain epithelial. I showed that RAS transduction selected cells that are undergoing an EMT, whereas transduction with CCNE1 or WNT1 selected the epithelial cells. I have sought to identify the determinants of these phenotypic changes and my results suggest that a balance exists between TGFβ1 / BMP1 signaling, which promotes EMT, and BMP4 / WNT7a which promotes TEM.

La migration cellulaire est un phénomène essentiel au développement, à l'immunité et à la cicatrisation. Pourtant, l'activation des programmes de migration en dehors des situations physiologiques peut avoir des effets néfastes. Par exemple, la migration cellulaire permet aux cellules d'une tumeur primaire d'envahir de nouveaux territoires et d'installer des tumeurs secondaires ou métastases. Lorsqu'une migration cellulaire est initiée à partir d'un tissu épithélial, ces cellules doivent acquérir des caractéristiques mésenchymateuses. Pour cela, elles diminuent leur adhérences cellule-cellule, perdent leur polarité apico-basale, réorganisent leur cytosquelette, changent d'adhérence à la matrice et modifient la composition et l'organisation de la matrice. C'est ce qu'on appelle la transition épithélio-mésenchymateuse (TEM). La famille des Métalloprotéinases Matricielle (MMP) est connue pour participer au remodelage de la matrice. Les MMPs sont au nombre de 25 et sont sécrétées ou membranaires. L'une de ces MMP membranaires est MMP14 ou MT1-MMP. Elle participe à la migration physiologique et pathologique via la dégradation de composants de la matrice. Elle dégrade également des protéines non matricielles sécrétées ou membranaires. De plus, MMP14 agit indépendamment de son activité catalytique en régulant par exemple l'activation de petites GTPases, de voies de signalisation et en contrôlant l'expression de gênes. Cependant, beaucoup d'études sur MMP14 ont été faites in vitro et ex vivo et il n'est pas clair si toutes les fonctions de MMP14 sont retrouvées in vivo. Plus spécifiquement les fonctions possibles de MMP14 dans la TEM et la migration in vivo sont encore mal définies. Nous proposons d'utiliser les crêtes neurales (CN) de l'embryon de poulet comme modèle pour étudier MMP14 au cours de la TEM et de la migration in vivo. Les CN sont des cellules embryonnaires retrouvées dans la partie dorsale du tube neural. Les CN réalisent une TEM pour quitter le tube neural avant de parcourir de longues distances et donner de nombreux types cellulaires. Les CN se séparent en deux populations, les CN céphaliques retrouvées dans la tête et les CN troncales dans le reste de l'embryon. Ces deux populations de CN réalisent des TEM différentes, avec une TEM rapide et massive pour les CN céphaliques et plus lente et en continue pour les CN troncales. Même si ces TEM sont différentes, elles présentent une diminution des jonctions cellulaires, une perte de la polarité apico-basale, un changement d'adhérence à la matrice et une réorganisation de la matrice. Une particularité des CN troncales est la localisation du noyau en position basale de l'épithélium juste avant la sortie du tube neural. Plusieurs substrats de MMP14 sont retrouvés dans la TEM et la migration des CN et une étude a montré par PCR la présence de l'ARNm de MMP14 dans les CN céphaliques de poulet. L'objectif de la thèse est d'explorer la fonction de MMP14 au cours de la TEM et de la migration des CN. Nous avons montré que MMP14 est exprimée dans les deux populations de CN au cours de la TEM et de la migration. / Cell migration is an essential event during embryonic development, immunity and wound healing. Furthermore, the activation of migration program in non-physiologic conditions can have side effects. For example, cell migration promotes invasion of primary tumor cells in new territories and the formation of secondary tumors or metastasis. When an epithelial tissue initiates migration, epithelial cells need to gain mesenchymal attributes. To this end, they decrease their cell-cell adhesions, loss their apico-basal polarity, reorder their cytoskeleton, change their matrix adhesions and modify the matrix composition and organization. This event is named epithelial-mesenchymal transition (EMT). The family of Matrix Metalloproteinase (MMP) is known to reshape the matrix. MMP family is composed of 25 members which are secreted or linked to the membrane. One of the membrane-bound MMP is MMP14 or MT1-MMP. MMP14 is known to promote physiological and pathologic cell migration by inducing degradation of numerous matrix components. MMP14 cleaves also non-matrix proteins which are secreted or membrane-bound. Moreover, MMP14 can act independently of its catalytic activity for example in the regulation of small GTPases, signaling pathway and in gene expression control. However, the vast majority of MMP14 related studies were conducted in vitro or ex vivo and it is not clear whether some of its functions occur in vivo. More specifically, MMP14's putative functions in EMT and migration are still ill-defined. We propose to use the Neural Crest (NC) of chick embryo as model to study MMP14 during in vivo EMT and migration. NC is an embryonic cell population located in the dorsal part of the neural tube. NC cells realize an EMT to leave the neural tube before performing a long-distance migration and producing a myriad of cell types as neurons, bones and cartilages of the face and pigment cells. NC cells are divided in two populations, the cephalic NC in embryo's head and the trunk NC in the posterior part. The cephalic NC perform a fast and massive EMT while the trunk NC's EMT is slower and continuous. Although the EMT are different, they conserve common characteristics with a decrease of cell junctions, a loss of the apico-basal polarity, a change of matrix adherence and a rearrangement of the matrix. One particularity of trunk NC is the epithelium basal position of the nucleus just prior their exit from the neural tube. Many MMP14's substrates are found during NC EMT and migration and a study suggested by PCR that chick cephalic NC express MMP14 mRNA. The goal of this thesis is to explore the function of MMP14 during chick NC EMT and migration. Our results show that MMP14 is expressed by the two populations of NC during EMT and migration. Moreover, MMP14 cell localization changes from apical to basal during EMT. Loss of function experiments show that MMP14 is needed for NC EMT. Our rescues with various MMP14 versions indicate that: 1/ the cytoplasmic domain is not essential, 2/ the extracellular domain is needed and 3/ the catalytic activity is not required for EMT. MMP14 is involved in the control of cell junctions by a switch between cadherin-6B and cadherin-7 but not in the remodeling of the matrix during NC EMT. We have also showed that MMP14 is necessary for the change of cell polarity during EMT. Furthermore, we have showed that MMP14 is needed for the formation of matrix adherence. In conclusion, our study shows that MMP14 is involved in NC EMT and migration and that NC are a good model to investigate MMP14 function in vivo.

Cells can release lipid bilayer vesicles of endosomal and plasma membrane origin, which are known as exosomes or extracellular vesicles (EVs). EVs contain diverse shuttling lipids, RNA and transmembrane proteins, and play an important role in communicating between neighboring or distant cells. Breast cancer is the most commonly diagnosed malignancy, with over 2 million new cases in 2018, and is the leading cause of cancer mortality in women all over the world. Some observational studies have suggested that breast cancer is more likely to develop among women who have type 2 diabetes; the association is clear in postmenopausal women. Moreover, women with type 2 diabetes diagnosed before, at the same time, or after breast cancer diagnosis, have decreased overall survival compared to women without diabetes. The most recent medical studies provide more clues as to why breast cancer is more common and has poorer prognosis in type 2 diabetes patients, by pointing out the role of insulin-resistant adipocytes in the etiopathology. Here, we demonstrate how insulin-resistant adipocytes engage crosstalk with breast cancer cells through EVs in the microenvironment and drive the tumor cells to be more metastatic and aggressive. These progression mechanisms and the effects of insulin-resistant adipocytes on breast cancer cells require Bromodomain and ExtraTerminal (BET) proteins – an important epigenetic pathway. Targeting this pathway may help reduce morbidity and mortality of women with breast cancer and type 2 diabetes.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21642号 / 医博第4448号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 小川 誠司, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24136号 / 医博第4876号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 波多野 悦朗, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Digital image processing is widely used in the field of time-lapse microscopy and biological research to provide statistical data of cellular dynamics. The data can provide more comprehensive understanding of the molecular phenomenon. Further, digital image processing enables rapid and consistent quantification of qualitative observations. The image processing model examined here provides a study to identify structures called retraction fibers (RFs) that are formed during epithelial-mesenchymal transition (EMT) [1], an important developmental process which also occurs during cancer metastasis. Quantifying RF formation is an important task for biologists studying cellular regulation of EMT. This thesis work uses digital image processing and computer vision algorithms to detect and track each RF in image sequences of cells undergoing EMT that are captured using time-lapse microscopy. The algorithms isolate the RFs with reasonable precision. Statistical information is generated about these automatically detected RFs, such as the number formed during a particular time window, lifetime of each, and their geometric dimension. This information can in turn be used by biologists to quantitatively measure the extent of EMT under different test conditions. Biologists feel that the information thus obtained may help clarify the molecular interactions of cell migration and will aid in developing methods of preventing cancer metastasis. Experimental results show that this methodology has significant potential in helping biologists determine RF behavior during EMT.

Development is punctuated by morphogenetic rearrangements of epithelial tissues, including complete detachment of individual motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling; yet, neither the precise series of actin rearrangements at cell-cell junctions that accompany EMT, nor the machinery that controls actin rearrangement during EMT, have been identified. This work represents a detailed study of junctional actin reorganization in cells undergoing EMT, identifies actin regulatory proteins that control this actin reorganization, and defines the specific function of one regulatory protein, zyxin, in EMT. Using immunofluorescence and live cell imaging of HGF induced scattering of MDCK cells, dynamic actin rearrangement events occurring during EMT are characterized. Junctional actin characteristic of cell-cell adherent cells is rearranged into contractile medial actin networks linked to the junctional membrane in the initial steps of cell scattering. This actin rearrangement is accompanied by dynamic redistribution of specific actin regulatory proteins, namely α-actinin and zyxin-VASP complexes. α-Actinin mediates higher order structure of junctional actin. Zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction membranes. Zyxin regulation of actin-membrane linkage controls whether cell migration during EMT occurs independently in solitary cells or is coordinated through tissues. The functional analysis employed here uses novel, quantitative methods that define specific cellular EMT ‘phenotypes’ to reveal the precise role of zyxin in EMT. Constitutive active zyxin mutants exhibit persistent actin-membrane linkages and a scattering phenotype in which cells migrate without loss of cell-cell adhesion. Zyxin is proposed to regulate EMT progression by regulating disruption or maintenance of actin membrane linkages at cell-cell junctions. Zyxin alters the ability of cells to fully detach and migrate independently during EMT and may be an important regulator of morphogenetic plasticity.

Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and migrating as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-Met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. This study addresses this shortcoming in a number of ways.A high-throughput in vitro drug screen was employed to identify proteins necessary in this HGF-induced signaling. Cells were tested for reactivity to HGF stimulation in a Boyden chamber assay. This tactic yielded several small molecules that block HGF-induced scattering, including a calcium channel blocker. Patch clamping was used to determine the precise effect of HGF stimulation on Ca2+ signaling in MDCK II cells. Cell-attached patch clamping was employed to detect Ca2+ signaling patterns, and channel blockers were used in various combinations to deduce the identity of Ca2+ channels involved in EMT. The results of these experiments show that HGF stimulation results in sudden and transient increases in calcium channel influxes. These increases occur at predictable intervals and rely on proper tubulin polymerization to appear, as determined through the use of a tubulin polymerization inhibitor. Though multiple channels occur in the membranes of MDCK II cells, noticeably TRPV4 and TrpC6, it is TrpC6 that is specifically required for HGF-induced scattering. These HGF-induced calcium influxes through TrpC6 channels drive a transient increase in NFAT-dependent gene transcription which is required for HGF-induced EMT. This was determined through the use of luciferase-based NFAT reporter assays and confirmed through confocal immunofluorescence. Using a small-molecule inhibitor of WNK kinase, it was determined that loss of WNK kinase function is sufficient to prevent HGF-induced EMT. Furthermore, patch-clamp analysis demonstrated that WNK kinase significantly increases channel opening at the surface of MDCK cells, indicating a possible mechanism of action for c-Met inhibition, but leaving doubt as to whether WNK kinase is in fact normally involved in c-Met signaling, or whether it is simply permissive.

No description available.