L’incidence du mélanome a augmenté de façon considérable lors des trente dernières années avec un doublement tous les dix ans. Le mélanome ne représente que 5% des cancers cutanés mais entraîne 80% de décès, ce qui constitue un problème majeur de santé publique. En effet, cette tumeur est extrêmement agressive et possède un fort potentiel métastatique. Dès l’apparition de métastases, le pronostic vital devient fortement défavorable. Malgré des avancées thérapeutiques majeures, de nombreux patients sont encore réfractaires à ces nouveaux traitements. La compréhension des mécanismes impliqués dans le développement de cette tumeur reste donc un enjeu de premier ordre. Le séquençage d'exomes a conduit à l'identification d'une mutation dans le gène RAC1 (la mutation P29S) constituant une des mutations somatiques les plus fréquentes dans le mélanome (après les mutations BRAFV600, NRASQ61 et NF1). RAC1 est une petite GTPase qui fonctionne dans plusieurs processus cellulaires. Dans des conditions physiologiques, l'activité de RAC1 est principalement contrôlée par des protéines activatrices de l'activité GTPase (GAPs) et des facteurs d'échange Nucléotidique (GEF). GAPs et GEFs contrôlent le niveau de RAC1-GTP et régulent donc son activité. L'activité de RAC1 est aussi dépendante de son niveau d'expression protéique qui est contrôlé par des E3 ubiquitine ligases, parmi lesquelles HACE1. HACE1 est considérée comme un suppresseur de tumeur. De façon inattendue, les données obtenues montrent clairement que HACE1 favorise les propriétés migratoires et tumorigéniques des cellules de mélanome. / Melanoma incidence has considerably increased over the last thirty years, with a doubling every ten years. Melanoma accounts for only 5% of cutaneous cancers but causes more than 80% of deaths, which is a major public health problem. Indeed, this tumor is extremely aggressive and has a high metastatic potential. After the onset of metastases, the prognosis becomes highly unfavorable. Despite major therapeutic advances, many patients are still refractory to these new treatments. Understanding the mechanisms involved in the development of this tumor and the identification of new therapies remain a major issue. The sequencing of exomes led to the identification of a mutation in the RAC1 gene (P29S) constituting one of the most frequent somatic mutations in melanoma (after the BRAFV600, NRASQ61 and NF1 mutations). RAC1 is a small GTPase that is involved in several key cellular processes. Under physiological conditions, the activity of RAC1 is mainly controlled by GTPase activating proteins (GAPs) and Nucleotide Exchange (GEF) exchange factors. GAPs and GEFs control the level of RAC1- GTP and thus regulate its activity. The activity of RAC1 is also dependent on its protein level of expression which is controlled by E3 ubiquitin ligases, including HACE1. HACE1 is considered a tumor suppressor. Unexpectedly, our data clearly show that HACE1 promotes migratory and tumorigenic properties of melanoma cells. Indeed, inhibition of HACE1 alters migration of melanoma cells in vitro, as well as in vivo pulmonary colonization in mice. Transcriptomic analysis of 4 melanoma cell lines demonstrated that HACE1 suppression inhibits ITGAV and ITGB1 expression.

La progression tumorale en cancer métastatique implique la perte de fonctions oncosuppressives, comme c'est le cas dans le mélanome. Une migration cellulaire aberrante est caractéristique de la progression du mélanome. SYK (Spleen tyrosine kinase) est une tyrosine kinase cytoplasmique impliquée dans la suppression tumorale du cancer du sein et du mélanome. Dans la peau, SYK est exprimée dans les mélanocytes mais est fréquemment réprimée épigénétiquement dans le mélanome. Nous avions pu montré que cette perte était associée à un échappement de la sénescence. Qu'elle puisse réguler la migration des cellules tumorales et la formation des métastases reste peu connu. Dans mes travaux j'ai utilisé des approches gain et perte de fonction pour analyser l'effet de SYK sur les mélanomes humains et murins. Respectivement, la réexpression et l'extinction de SYK diminue et augmente la migration, l'invasion et les métastases des cellules de mélanome. L'extinction de SYK induit notamment un phénotype et une signature mésenchymateuse. Notre étude dévoile ce rôle pour SYK dans la répression d'une adhérence dépendante des intégrines, points de tractions et plateforme de signalisation de la migration, et souligne l'importance la perte de SYK dans la formation de métastases. Pour clarifier le rôle de SYK dans la progression du mélanome, j'ai généré un modèle murin de KO conditionnel de SYK spécifique des mélanocytes concomitants à une perte de Pten et de l'activation de BrafV600E. Des résultats préliminaires suggèrent que la perte de SYK n'accélère pas la formation de mélanome dans ce contexte mutationnel mais mène à une invasion plus profonde des cellules tumorales dans le derme. / The progression of tumors to metastatic disease involves the loss of metastatic suppressor functions, as it is the case in melanoma. Thus, aberrant cell migration is a key feature of melanoma progression, and is required for metastasis. SYK (Spleen tyrosine kinase) is a cytoplasmic tyrosine kinase that has been implicated in tumor suppression of breast cancer and melanoma. In skin cells, SYK is found expressed in melanocytes but SYK is frequently downregulated in melanoma by epigenetic silencing. We showed previously that its loss has been associated with senescence escape. Whether it also regulates tumor cell migration and subsequent metastasis remains poorly understood. In this work we used gain- and loss-of-function approaches to analyze SYK’s effects on metastatic abilities of human and murine melanoma cells. Respectively, the reexpression or knockdown of SYK results in decreased or increased migration, invasion and metastasis of melanoma cells. Notably, SYK knockdown cells displayed a mesenchymal-like phenotype with upregulation of mesenchymal markers. Our study unveils a novel role for SYK in suppressing integrin-mediated adhesion, both a points of traction and a signaling platform during cell migration, and outlines the importance of SYK inactivation in acquisition of a metastatic phenotype. To clarify the role of SYK in melanoma formation and progression, we have generated a conditional Syk KO mouse model in melanoma based on melanocyte-specific Pten loss and BrafV600E activating mutation. Preliminary results suggest that Syk loss does not accelerate Pten/Braf-driven melanoma formation but leads to deep invasion of Braf/Pten tumor cells into the dermis.

Les glioblastomes (GB) sont des tumeurs cérébrales invasives, résistantes et qui récidivent systématiquement malgré un traitement associant chirurgie, radio- et chimiothérapie. Ces tumeurs de très mauvais pronostic, se caractérisent par une survie médiane de 15 mois. L'agressivité des GB est notamment due à la présence d'une sous-population de cellules souches (GSC). Les GSC sont caractérisées par leurs capacités d'auto-renouvellement, d'expression de différents marqueurs souches, de multipotence et de tumorigenèse. Elles sont fortement impliquées dans la résistance et la récidive tumorale et leur ciblage pourrait améliorer le traitement des GB. Au vu de la littérature et de résultats obtenus au laboratoire, l'intégrine ß8 est apparue comme une nouvelle cible potentielle de ces GSC. Cette intégrine est décrite comme possédant un rôle majeur dans la survie et l'auto-renouvellement des cellules progénitrices neurales saines et sa surexpression est associée à une diminution de la survie des patients. Nous avons alors émis l'hypothèse que l'intégrine ß8 pourrait être impliquée dans le maintien de l'état souche des GSC. J'ai démontré au cours de ma thèse que cette intégrine est surexprimée dans des primocultures de GSC issues de résections de GB ainsi que dans des coupes de tumeurs de patients. De plus, j'ai mis en évidence que ß8 est associée à l'état souche et à des fonctionnalités propres à ces cellules, notamment leur auto-renouvellement, leur viabilité, leur migration et leur radiorésistance. En inhibant sélectivement ß8 dans nos primocultures de GSC par si/shRNA, j'ai en effet observé in vitro une diminution de la formation de neurosphères et de la migration cellulaire ainsi qu'une augmentation de la différentiation et de la mort cellulaire, cette dernière étant potentialisée après irradiation. Enfin, in vivo, j'ai mis en évidence que l'inhibition de ß8 se traduit par une diminution de la tumorigenèse et une augmentation de la survie des souris. En conclusion, mes résultats de thèse permettent d'identifier l'intégrine ß8 comme une protéine membranaire nécessaire au maintien de l'état souche dans les GSC mais surtout comme une potentielle cible thérapeutique radiosensibilisante dans les GB. / Glioblastomas (GB) are invasive, resistant and recurrent brain tumors (median overall survival of 15 months) despite standard treatment including surgical resection, radio- and chemotherapy. This tumor aggressiveness could partly be explained by the presence into the tumor of Glioblastoma-Stem like Cells (GSC), characterized by their ability to self-renew, their higher expression of specific GSC markers, their multipotent aptitude and their high tumorigenic potential. They are strongly involved in tumor resistance and recurrence and their targeting could improve GB treatment. Regarding current literature but also transcriptomic results obtained in our lab, a specific ß8 integrin emerged as a potential selective target in GSC. We then hypothesized that ß8 integrin could be involved in stemness maintenance of GSC. I first demonstrated, during my doctoral thesis, that ß8 is overexpressed in primocultures of GSC isolated from patients resections and also in human GB samples. Moreover, I showed that this integrin could be associated with stemness and features unique to these cells, including self-renewal ability, viability, migration and radioresistance. Indeed, the selective inhibition of ß8 in GSC by si/shRNA resulted in vitro in a decrease of neurosphere formation and migration, associated with an increase of differentiation patterns and cell death, this one being potentiated after irradiation. Finally, in vivo, I showed that ß8 inhibition decreased tumorigenesis and increased mice survival. In conclusion, my doctoral results allow to identify ß8 integrin as a membrane protein essential for stemness maintenance of GSC but mostly as a new potential radiosensitizing therapeutic target for GB.

Le mélanome est un cancer en recrudescence depuis 10 ans. Il est devenu de ce fait une préoccupation majeure de santé publique. Au cours de la progression tumorale, l’expression des molécules d’adhérence (intégrines et cadhérines) ainsi que le récepteur à l’IGF de type I (IGF-IR) sont modulées. Notamment, il est observé un « switch des cadhérines » de la E vers la N, une surexpression des intégrines α2 et αv, et de l’IGF-IR. C’est dans cette optique que nous nous sommes intéressés aux dialogues croisés entre les molécules d’adhérence et l’IGF-IR. Dans un premier temps, nous avons mis en évidence un dialogue entre la N-cadhérine et l’intégrine αv lors de la migration des mélanomes. Dans un second temps, nous avons démontré que les dialogues entre l’IGF-IR, l’intégrine α2 et les cadhérines différent selon la nature de la cadhérine incriminée. Ces modifications de dialogues ont un impact important sur la propension migratoire des mélanomes. / Cutaneous malignant melanoma is an aggressive melanocyte malignancy that is characterized by early metastasis, bad prognosis, and poor survival. Altered cell-cell adhesion (cadherins), cell-extracellular matrix (integrins) interaction, and IGF type I receptor (IGF-IR) are playing an important role in melanoma progression. In particular, we observed a "cadherins switch" from E to N, and α2- αv-integrins and IGF-IR over-expression. In this optics, we were interested in the crosstalk between adhesion molecules and IGF-IR. First, we brought to light a crosstalk between N-cadherin and αv-integrin during melanoma migration. Secondly, we demonstrated that the dialogues between the IGF-IR, α2-integrin and cadherins are different according to the nature of the cadherin involved. Such modulation in the crosstalk may have an important impact on the migratory inclination of melanomas.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

β-adrenergic receptor (β-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVM). β1 integrin signaling plays a protective role in β-AR-stimulated apoptosis. Glycogen synthase kinase-3β (GSK-3β), a multifunctional serine/threonine kinase, negatively regulates cardiac hypertrophy. Here we show that β-AR stimulation (isoproterenol; 15 min) increases tyr216 phosphorylation and GSK-3β activity. Inclusion of LiCl, inhibitor of GSK-3β, in the reaction mix or expression of catalytically inactive GSK-3β (KM-GSK) inhibited β-AR-stimulated GSK-3β activity. Inhibition of tyrosine kinase using genistein or chelation of intracellular Ca2+ using BAPTA-AM inhibited β-AR-stimulated increases in tyr216 phosphorylation and GSK-3β activity. Inhibition of GSK-3β using pharmacological inhibitors or infection with KM-GSK decreased β-AR-stimulated cytosolic cytochrome C release and apoptosis. Expression of β1 integrins increased ser9 phosphorylation and inhibited β-AR-stimulated increase in GSK-3β activity. Wortmannin, inhibitor of PI3-kinase, reversed the effects of β1 integrins on GSK-3β activity and apoptosis. Purified active matrix metalloproteinase-2 (MMP-2), shown to interfere with β1 integrin signaling, increased GSK-3β activity, while inhibition of MMP-2 inhibited β-AR-stimulated increases in GSK-3β activity. β-AR stimulation induced nuclear accumulation of GSK-3β. β-AR stimulation (3 h) increased the expression of transcription factor Gadd153 (growth arrest- and DNA damage-inducible gene 153). These data suggest that β-AR stimulation increases GSK-3β activity. Activation of GSK-3β plays a pro-apoptotic role in β-AR-stimulated apoptosis via the involvement of mitochondrial death pathway. β1 integrins inactivate GSK-3β and play an anti-apoptotic role via the involvement of PI3-kinase pathway. The apoptotic effects of GSK-3β may be mediated, at least in part, via its nuclear localization and induction of pro-apoptotic genes, such as Gadd153.

Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.

Engulfment of dead cells by epithelial cells is crucial for the health of an organism. Defective engulfment can lead to serious conditions such as retinitis pigmentosa and asthma. In the Drosophila melanogaster ovary, protein starvation induces apoptotic cell death of germline cells, which are then engulfed by adjacent epithelial follicle cells. The follicle cells synchronously enlarge approximately four-to-five fold as they engulf the dying germline, suggesting that significant changes are required. However, the molecular changes needed to drive enlargement and engulfment by epithelial cells are still poorly understood. In this dissertation, I determined the role of integrins in engulfment, the interactions between the core engulfment machinery and the corpse processing pathway, and the roles of GTPases in engulfment by epithelial cells. First, I found that the integrin heterodimer, αPS3/βPS, becomes apically enriched and is required in the epithelial follicle cells for engulfment. αPS3/βPS is trafficked in a polarized fashion using much of the same machinery as migrating cells, suggesting similarities between engulfing and migrating cells. The canonical corpse processing pathway has been well-characterized, however, little is known about how the core engulfment machinery interacts with the corpse processing pathway. I found that the phagocytic receptor Draper is present on the phagocytic cup and early phagosomes, whereas integrins are maintained on the cell surface. Engulfment mutants fell into three distinct categories based on their specific effects on internalization and phagosome maturation, suggesting that components of the core engulfment machinery are required for distinct steps of corpse processing. Last, I investigated the roles of the Rho family GTPases during engulfment. Strikingly, Rac2 becomes induced during engulfment whereas Rac1 does not change its expression pattern. Both Rac1 and Rac2 are required for engulfment, however, Rac1 has defects in both enlargement and vesicle uptake whereas Rac2 only has defects in enlargement. Furthermore, I found that Rac1 and Rac2 may have differential effects on the Jun kinase signaling pathway, which suggests complexity in the regulatory network controlling engulfment in epithelial cells. Together, this work has provided a greater understanding of the molecular changes required within epithelial cells for proper engulfment. / 2016-12-01T00:00:00Z

2-ω-carboxyethylpyrrole (CEP) is a product of docosahexaenoic acid (DHA) oxidation, which forms covalent adducts with different proteins. CEP-modified proteins can interact with macrophage receptor, integrin αDβ2. This study aims to compare αDβ2 binding to its physiological ligand, fibrinogen, and CEP-modified fibrinogen, which is formed during inflammation. We hypothesize that modification of fibrinogen changes its ligand-binding properties to integrin αDβ2 which can affect macrophage migration and retention. Recombinant αD I-domain and αDβ2-transfected HEK293 cells were used for the experiments. Using biolayer interferometry, we found that the affinity of αD I-domain binding to fibrinogen-CEP was higher than fibrinogen and inhibited by the anti-CEP antibody. In agreement, αDβ2-transfected cells demonstrated stronger adhesion to fibrinogen-CEP and this adhesion was significantly inhibited by polyglutamic acid that mimics CEP-mediated binding. These findings suggest that αDβ2's interaction with DHA-modified extracellular matrix (ECM) proteins significantly increases macrophage adhesion and may serve for macrophage retention during chronic inflammation.

Invasion and metastasis of cancer is the leading cause of increased mortality. In addition, haematological malignancies (leukaemia and lymphoma) are a significant cause of morbidity and mortality in both children and adults. Therefore, new treatments which will inhibit cancer progression are required. Integrin adhesion receptors, particularly the RGD-binding integrin subfamily comprising αvβ3, αvβ5, αvβ6, αvβ8, αllbβ3, α5β1, α8β1 and αvβ1 are related to progress and spread of cancer and poor prognosis. Because of the importance of integrin biology in the regulation of cancer dissemination, the integrin receptors are being utilised as targets to regulate cancer progression. The goal of this study was to develop a dual αvβ3/ αIIbβ3 expressing model for testing integrin antagonists. Expression of αv, αIIb, and β3 integrin subunits was characterised using immunofluorescence and flow cytometry in a panel of cell lines. After characterising the expression of αv, αIIb and β3 integrin subunits in inducible and natural expression models (K562 and MCF-7 cells respectively), functional tests for cellular adhesion, detachment and migration were determined. Phorbol 12-myristate 13-acetate (PMA)-treated K562 cells showed increased adhesion on fibrinogen compared to untreated cells. Adhesion of cancer cells (K562 ± PMA and MCF-7) to fibrinogen was inhibited and detachment was induced by the known β3 antagonists, cRGDfV and GR104453. Migration of cancer cells (K562 without PMA and MCF-7) was inhibited by combination of the known β3 antagonists. A panel of 12 novel small molecules developed in the ICT was investigated for cytotoxicity and activity in the validated function assays. ICT9055 was the most potent antagonist in inhibition of cell adhesion, migration, and inducing cell detachment. The data presented in this thesis had selected models and assays for evaluating small molecule integrin antagonists and identified ICT9055 as a promising molecule to develop for further preclinical evaluation. / The Libyan Embassy; Omer Al Mukhtar University, Faculty of Medical Technology, Derna, Libya.