Le mélanome est le cancer de la peau le plus rare, mais celui qui cause le plus de mortalité. L’étapecritique de sa progression est l’angiogenèse, processus détourné par la tumeur pour s’oxygéner et senourrir. Les cellules tumorales peuvent alors former des métastases dans les ganglions lymphatiques,notamment. D’autre part, la tumeur inhibe les réponses immunitaires de l’hôte à son encontre. Tousces mécanismes font intervenir le monoxyde d’azote (NO ). Le but de ce projet a été d’approfondir lerôle du NO dans l’angiogenèse et le recrutement leucocytaire, mécanismes basés sur lareconnaissance cellulaire de l’endothélium.Nous avons montré que le NO est nécessaire à l’angiogenèse, mais que des doses plus fortes sontanti-angiogéniques. Cet effet inhibiteur peut s’expliquer par la diminution des interactions cellules-cellules,et l’inhibition de l’expression de PECAM-1/CD31, principalement.D’autre part, nous avons établi que l’adhésion des leucocytes est inhibée par le NO ce qui a été reliéà la modulation de l’expression des molécules capables de fixer des chimiokines : lesglycosaminoglycannes et les récepteurs de chimiokines, ainsi qu’à la sous-expression des moléculesd’adhésion CD34, ICAM-2/CD102 et VCAM-1/CD106.En conclusion, le NO est capable de réguler des mécanismes cellulaires majeurs de la progressiontumorale, par modulation de l’expression des molécules de surface de l’endothélium. Tout au long dece travail, nous avons observé que les différences de réponse sont dépendantes des doses de NO etdu type cellulaire, ce qui démontre le rôle pivot du NO dans la progression du cancer. / Melanoma is the rarest skin cancer, but one that causes the most of deaths. The critical step of itsprogression is angiogenesis, a physiological tumor-activated process which allows the delivery ofoxygen and nutrients. Tumor cells may then metastasize to the lymph nodes, in particular. Moreover,the tumor inhibits the host immune responses toward itself. All these mechanisms are regulated bynitric oxide (NO ). The aim of the project was to deepen the role of NO in angiogenesis andleukocyte recruitment, two mechanisms based on endothelial cell recognition.We have shown here that NO is necessary for angiogenesis, but that high concentrations are antiangiogenic.This inhibitory effect of NO can be attributed to the decrease of cell-cell interactions andthe inhibition of the PECAM-1/CD31 expression, mainly.Besides, we have demonstrated that leukocyte adhesion on endothelium is inhibited by NO . This canbe explained by the modulation of the expression of molecules able to bind to chemokines:glycosaminoglycans and chemokine receptors, as well as by the down-regulation of the adhesionmolecules CD34 and ICAM-2/CD102 VCAM-1/CD106.In conclusion, NO is able to regulate the main cellular mechanisms of tumor progression bymodulating the expression of surface molecules on endothelial cells. Throughout this work, we haveobserved that these modulations depend on NO concentrations and on the cell type, demonstratingthe pivotal role of NO in cancer progression.

Le mélanome est le cancer de la peau le plus rare, mais celui qui cause le plus de mortalité. L'étapecritique de sa progression est l'angiogenèse, processus détourné par la tumeur pour s'oxygéner et senourrir. Les cellules tumorales peuvent alors former des métastases dans les ganglions lymphatiques,notamment. D'autre part, la tumeur inhibe les réponses immunitaires de l'hôte à son encontre. Tousces mécanismes font intervenir le monoxyde d'azote (NO ). Le but de ce projet a été d'approfondir lerôle du NO dans l'angiogenèse et le recrutement leucocytaire, mécanismes basés sur lareconnaissance cellulaire de l'endothélium.Nous avons montré que le NO est nécessaire à l'angiogenèse, mais que des doses plus fortes sontanti-angiogéniques. Cet effet inhibiteur peut s'expliquer par la diminution des interactions cellules-cellules,et l'inhibition de l'expression de PECAM-1/CD31, principalement.D'autre part, nous avons établi que l'adhésion des leucocytes est inhibée par le NO ce qui a été reliéà la modulation de l'expression des molécules capables de fixer des chimiokines : lesglycosaminoglycannes et les récepteurs de chimiokines, ainsi qu'à la sous-expression des moléculesd'adhésion CD34, ICAM-2/CD102 et VCAM-1/CD106.En conclusion, le NO est capable de réguler des mécanismes cellulaires majeurs de la progressiontumorale, par modulation de l'expression des molécules de surface de l'endothélium. Tout au long dece travail, nous avons observé que les différences de réponse sont dépendantes des doses de NO etdu type cellulaire, ce qui démontre le rôle pivot du NO dans la progression du cancer.

The control of phosphatidylinositol 3, 4, 5-trisphosphate [PtdIns(3,4,5)P3] level depends on the activities of both PI kinase and PtdIns(3,4,5)P3 phosphatases: 5-phosphatase like SHIP1 and SHIP2, and 3-phosphatase like PTEN. The ubiquitous SH2 domain containing inositol 5-phosphatase SHIP2 contains both a series of protein interacting domains and the ability to dephosphorylate PtdIns(3,4,5)P3. Previous reports obtained in SHIP2 deficient mice have shown that SHIP2 is involved in the control of insulin sensitivity and reducing weight gain on fatty diet. <p><p>Since SHIP2 is a lipid phosphatase as well as a docking protein, the initial aim that emerged in the lab was to measure the inositol lipid levels in SHIP2 +/+ and deficient cells and compare the levels of 3-phosphoinositides PtdIns(3,4,5)P3 and PtdIns(3,4)P2. At first, we developed mouse embryonic fibroblasts (MEF) as a cellular model. Amongst various stimuli tested, surprisingly, only serum showed an obvious difference in terms of PtdIns(3,4,5)P3 level. This lipid was significantly up regulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P3 levels in heterozygous fibroblast cells was intermediate between the +/+ and -/- cells. Serum stimulated PI3K activity appeared to be comparable between +/+ and -/- cells. Moreover, PKB, but not MAP kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The up regulation of PKB activity in serum stimulated cells was totally reversed in the presence of the PI3K inhibitor LY-294002, in both +/+ and -/- cells.<p><p>Reactive oxygen species (ROS) have emerged as physiological mediators of many cellular responses. H2O2 mimics some effects of insulin in a number of cell culture systems. It also inactivates tyrosine phosphatase activities including PTEN. In addition, in Swiss 3T3 fibroblasts, Gray et al reported that exposure of the cells to H2O2 resulted a huge increase in PtdIns(3,4)P2 level. The authors suspected that the effect was attributed to a inositol 5-phosphatase activity. We thus exposed our cells to H2O2 in order to address the question of the role of SHIP2 in response to oxidative stress.<p><p>We worked on the same SHIP2 MEF model, stimulated by H2O2: at 15 min, PtdIns(3,4,5)P3 was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P2 could be detected at 15 or 120 min incubation of the cells with H2O2 (0.6 mM). PKB activity was upregulated in SHIP2 -/- cells in response to H2O2 and therefore follows the regulation of PtdIns(3,4,5)P3. As for serum, the PI3K activity appeared to be comparable between +/+ and -/- cells. The levels of PTEN and type I 4-phosphatase [an enzyme that acts on PtdIns(3,4)P2] remained unchanged between the two types of cells. SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role in the control of PtdIns(3,4,5)P3 level in response to H2O2: the decrease in PtdIns(3,4,5)P3, observed in SHIP2 expressing cells, was no longer seen in cells infected with a catalytic mutant of this enzyme. <p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished