The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...

Les cellules détectent et intègrent une multitude de signaux d'instruction provenant de leur microenvironnement via un ensemble de récepteurs transmembranaires. Ces informations sont ensuite collectées au niveau des nœuds de signalisation intracellulaires pour être ensuite dispersées en cascades de signalisation afin de déterminer la destinée cellulaire. La manière dont un nœud de signalisation peut interpréter plusieurs stimuli et transmettre de manière spatio-temporelle les informations appropriées restent incomprises. Le proto-oncogène c-Src est une tyrosine kinase pléiotrope, un nœud signalisation essentiel au pilotage de nombreux processus cellulaires, tels que la migration, l'invasion, la dégradation et la division cellulaire. Nous avons développé une approche synthétique pour explorer la relation entre la structure de la SRC et la multiplicité des processus cellulaires qu’elle régule. Notre approche a abouti au découplage des différents modules composant la protéine SRC afin de comprendre l’impact de chacun d’eux sur son activité dans l’espace et dans le temps. Notre approche pour contrôler plusieurs états de la conformation SRC était la conception d’un OptoSrc capable à la fois de former des oligomères et d’être recruté à la membrane plasmique. Pour ce faire, nous avons modifié la structure de la SRC afin qu'elle soit potentiellement active dans le noir et nous l'avons fusionnée avec le CRY2 sensible à la lumière. La stimulation lumineuse induit l'hétérodimérisation CRY2 avec un CIBN ancré à la membrane plasmique et son homo-oligomérisation et déclenche une relocalisation de l’OptoSrc à la membrane plasmique ou son oligomérisation. Ce double système a permis de générer deux types de mobilitéz différentes au sein des adhérences focales à deux destins différents, la formation de lamellipodes dans un cas et la formation d’invadosomes dans l’autre. / Cells sense and integrate a multitude of instructional signals from their microenvironment through a diverse set of transmembrane receptors. This information is then collected at intracellular signaling nodes to later disperse down signaling cascades to drive cell fate. How one signaling node can interpret multiple stimuli and spatio-temporally transmit the appropriate information remains poorly understood. The proto-oncogene c-Src is a pleiotropic tyrosine kinase, is one such node essential for driving many cellular processes, such as migration, invasion, degradation, and cell division. We developed a synthetic approach to explore the relationship between SRC structure and the multiplicity of cellular processes it regulates. Our approach resulted in the decoupling of the different modules composing SRC protein to understand how each of them impacts its activity in space and time. Our approach to control multiple state of SRC conformation was the design of an OptoSrc both capable of forming oligomers and to be recruited at the plasma membrane. To do so, we modified SRC structure to be potentially active in the dark and fused it with light sensitive CRY2. Light stimulation induces CRY2 hetero-dimerization with a CIBN anchored at the plasma membrane and its homo-oligomerisation triggering relocalization of OptoSrc at the plasma and/or its oligomerization. This system generated two different type of mobility of OptoSrc inside focal adhesion inducing two different adhesion fates, the formation of invadosome in one case and the formation of lamellipodia on the other.

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Metastasis is the leading cause of death among cancer patients. The metastatic cascade, during which cancer cells from the primary tumor reach a distant organ and form multiple secondary tumors, consists of a series of events starting with cancer cells invasion through the surrounding tissue of the primary tumor. Invading cells may perform proteolytic degradation of the surrounding extracellular matrix (ECM) and directed migration in order to disseminate through the tissue. Both of the mentioned processes are profoundly affected by several parameters originating from the tumor microenvironment (extrinsic) and tumor cells themselves (intrinsic). However, due to the complexity of the invasion process and heterogeneity of the tumor tissue, the exact effect of many of these parameters are yet to be elucidated. ECM proteolysis is widely performed by cancer cells to facilitate the invasion process through the dense and highly cross-linked tumor tissue. It has been shown in vivo that the proteolytic activity of the cancer cells correlates with the cross-linking level of their surrounding ECM. Therefore, the first part of this thesis seeks to understand how ECM cross-linking regulates cancer cells proteolytic activity. This chapter first quantitatively characterizes the correlation between ECM cross-linking and the dynamics of cancer cells proteolytic activity and then identifies ß1-integrin subunit as a master regulator of this process. Once cancer cells degrade their immediate ECM, they directionally migrate through it. Bundles of aligned collagen fibers and gradients of soluble growth factors are two well-known cues of directed migration that are abundantly present in tumor tissues stimulating contact guidance and chemotaxis, respectively. While such cues direct the cells towards a specific direction, they are also known to stimulate cell cycle progression. Moreover, due to the complexity of the tumor tissue, cells may be exposed to both cues simultaneously, and this co-stimulation may happen in the same or different directions. Hence, in the next two chapters of this thesis, the effect of cell cycle progression and contact guidance-chemotaxis dual-cue environments on directional migration of invading cells are assessed. First, we show that cell cycle progression affects contact guidance and not random motility of the cells. Next, we show how exposure of cancer cells to contact guidance-chemotaxis dual-cue environments can improve distinctive aspects of cancer invasion depending on the spatial conformation of the two cues. In this dissertation, we strive to achieve the defined milestones by developing novel mathematical and experimental models of cancer invasion as well as utilizing fluorescent time-lapse microscopy and automated image and signal processing techniques. The results of this study improve our knowledge about the role of the studied extrinsic and intrinsic cues in cancer invasion. / Bioengineering / Accompanied by fourteen .avi files.

O câncer de próstata é o segundo câncer mais freqüentemente diagnosticado em homens. Durante o crescimento tumoral, as células neoplásicas interagem com a matriz extracelular (MEC). Analisamos o efeito de C16, peptídeo derivado da clivagem da MEC, sobre a migração, invasão e regulação dos invadopódios em células de câncer de próstata (DU145). Ensaios de migração e invasão demonstraram que C16 promoveu um aumento da atividade migratória e invasiva de células DU145 de maneira dose dependente. Demonstramos que o peptídeo estimula a fosforilação de Src. Ensaios de degradação em substrato fluorescente mostraram que C16 promoveu a formação de invadopódios de células DU145. O immunoblot nos revelou que este peptídeo também estimula a expressão de Tks4, Tks5, cortactina e MT1-MMP. C16 estimulou a produção de espécies reativas de oxigênio, importantes para o fenótipo invasivo das células tumorais. Nossos resultados sugerem que o peptídeo C16 regula migração, invasão, invadopódios e suas moléculas-chave e a geração de espécies reativas de oxigênio em células DU145. / Prostate cancer is the second most frequently diagnosed cancer in males. During tumor growth, neoplastic cells interact with the extracellular matrix (ECM) Our Laboratory has demonstrated that peptides derived from ECM cleavage are involved in migration, invasion and invadopodia formation in different tumor cell lines. Invadopodia activity depends on expression of the proteins Tks4, Tks5, cortactin, MT1-MMP, as well as reactive oxygen species (ROS) generation. Migration and invasion assays in chemotaxis chambers demonstrated that C16 increased migration and invasion activities of DU145 cells in a dose-dependent manner. We observed that the peptide stimulated phosphorylation of Src. Fluorescent substrate degradation assay showed that C16 increased invadopodia activity of DU145 cells. Immunoblot revealed that this peptide stimulated Tks4, Tks5, cortactin and MT1-MMP expression. Furthermore, C16 increased ROS production. Our results strongly suggested that C16 regulates migration, invasion, invadopodia key-molecules, and ROS generation in DU145 cells.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

Les oncogènes Bcr-Abl (p190bcr-abl et p210bcr-abl) sont issus d'une translocation chromosomique t(9,22) qui fusionne en phase les gènes bcr et c-abl. p210bcr-abl est généralement responsable de la Leucémie Myéloïde Chronique (LMC) alors que p190bcr-abl induit un sous type de Leucémie Aigue Lymphoblastique (LAL). La seule différence structurale entre ces deux protéines est la présence d'un domaine DH/PH au sein de p210bcr-abl activateur spécifique de RhoA. L'expression de Bcr-Abl dans la lignée Ba/F3 est associée au déclenchement d'une migration spontanée, dépourvue de directionnalité, sous la dépendance de la GTPase Rac1.L'activation de RhoA, spécifique des cellules Ba/F3p210, est associée à un phénotype migratoire amœboïde dans une matrice de Matrigel™ en 3D où les cellules Ba/F3p190 dépourvues de RhoA activé, présentent une mobilité de type roulement. Dans ce travail, nous avons mis en évidence que l'activation spécifique de ROCK1 par RhoA détermine deux voies parallèles et mutuellement indispensables pour le mouvement amœboïde : 1) la voie de la Chaine Légère de Myosine (CLM) 2) celle des protéines de la famille ADF (Actin Depolymerizing Factor), et plus particulièrement l'isoforme ADF/destrine. Nous démontrons également l'existence d'invadopodes spécifiquement dans les cellules Ba/F3p190, dont la formation est sous la dépendance de l'absence d'activation de RhoA corrélée à une augmentation de l'activation de Cdc42. Enfin nous démontrons que la voie de signalisation RhoA/ROCK est spécifiquement activée dans les progéniteurs hématopoïétiques CD34+ issus de patients atteints de LMC et ce, indépendamment de l'activité tyrosine kinase de Bcr-Abl. / Bcr-Abl chimeric oncogenes (p190bcr-abl and p210bcr-abl) result from the t(9,22) chromosomal translocation that fuse the bcr and the c-abl genes. p210bcr-abl and p190bcr-abl are associated with Chronic Myelogenous Leukemia (CML) and a subset of Acute Lymphoblastic Leukemia (ALL) respectively. The only difference between these two chimeras is the presence of a specific RhoA-GEF domain in the p210bcr-abl oncogene. Bcr-Abl expression in Ba/F3 lymphoblasts induces spontaneous migration of these cells without apparent directionality. Motility triggering of Bcr-Abl-expressing Ba/F3 depends on the RhoGTPase Rac1.RhoA activity is associated with a typical amoeboid movement of Ba/F3p210 cells embedded in Matrigel™ 3D matrix, whereas the Ba/F3p190 cells, devoid of RhoA activity, display a rolling-type motility. In this work we showed that activation of the RhoA effector ROCK1 triggers two parallel pathways which are both necessary for amoeboid movement: 1) the Myosin Light chain (MLC) pathway 2) ADF family proteins (Actin Depolymerizing Factor) pathway, specifically the ADF/destrin isoform. Besides, we showed that Ba/F3p190 cells could assemble invadopodia-like structures. The formation of these structures is driven by the reduction of RhoA activity associated with the absence of the DH/PH domain in p190bcr-abl and correlates with an increase in Cdc42 activity. We finally demonstrated that the RhoA/ROCK pathway is constitutively activated in CD34+ cells isolated from CML patients while not in their normal counterparts. We also demonstrated that this activation is independent of the tyrosine Kinase activity of Bcr-Abl.

Cette thèse traite du rôle de la protéine FAK (Focal Adhésion Kinase) dans les mécanismes d'invasion cellulaire. / This thesis is about the role of the protein FAK (Focal Adhesion Kinase) in the cellular mechanisms of invasion.