O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

O câncer cervical é uma neoplasia muito prevalente na população feminina e está associado à infecção pelo papilomavírus humano (HPV). As oncoproteínas E6 e E7 de HPV de alto risco são as principais responsáveis pelas alterações celulares que levam ao desenvolvimento deste tipo tumoral. A dipeptidil peptidase IV (DPPIV/CD26) é uma enzima que exerce importantes funções relacionadas à progressão tumoral. Diversos estudos demonstram alterações na expressão e atividade desta proteína em diferentes tipos de câncer. Tendo em vista a relação entre a DPPIV/CD26 e o câncer, e que ainda não existem estudos relacionando esta proteína ao câncer cervical, neste estudo inicialmente investigamos a expressão e atividade da DPPIV/CD26 em linhagens celulares de carcinoma cervical humano (SiHa, HeLa e C33A) e em queratinócitos imortalizados (HaCaT). Nossos resultados demonstram uma baixa expressão da DPPIV/CD26 nas linhagens celulares estudadas, sendo praticamente indetectável na linhagem HeLa. Foi verificada a atividade enzimática dipeptidilpeptidásica tanto ligada à membrana quanto solúvel em todas as linhagens. Na presença do inibidor de DPPIV/CD26 (fosfato de sitagliptina) observamos que a linhagem SiHa apresentou um aumento na migração celular, e assim sugerimos que ao menos em parte a migração nesta linhagem é regulada pela atividade enzimática da DPPIV/CD26. A fim de investigar a relação da expressão da DPPIV/CD26 com as oncoproteínas E6 e E7 do HPV, avaliamos sua expressão em queratinócitos normais e transduzidos com estas oncoproteínas. Verificamos que queratinócitos expressando E6 de HPV de alto risco apresentam uma redução na expressão da DPPIV/CD26, e esta regulação parece ser dependente da degradação da p53. Considerando que as linhagens celulares estudadas apresentam baixa expressão e atividade da DPPIV/CD26, para melhor compreender a importância da expressão desta proteína, nós induzimos a superexpressão da DPPIV/CD26 em linhagem de câncer cervical (HeLa) para posterior avaliação dos efeitos em diferentes mecanismos tumorais. Os resultados demonstram uma redução no crescimento de células expressando DPPIV/CD26, sendo este efeito independente da atividade enzimática. Além disso, foi demonstrado que a indução da expressão de DPPIV/CD26 não afeta os mecanismos de migração e adesão celular na linhagem HeLa. Sendo assim, acreditamos que o esclarecimento do papel da DPPIV/CD26 no contexto do câncer cervical possibilita que novas abordagens diagnósticas e terapêuticas sejam implementadas no futuro. / Cervical cancer is a very prevalent neoplasm in female population and is associated with human papillomavirus (HPV) infection. The high risk HPV E6 and E7 oncoproteins are responsible for cellular alterations that lead to the development of this tumor type. The dipeptidyl peptidase IV (DPPIV/CD26) is an enzyme that exerts important functions related to tumor progression. Several studies have shown changes in the expression and activity of this protein in different types of cancer. Considering the relationship between DPPIV/CD26 and cancer, and that there are still no studies relating this protein to cervical cancer, in the present study we first investigated the DPPIV/CD26 expression and activity in human cervical carcinoma cell lines (SiHa, HeLa and C33A) and in immortalized keratinocytes (HaCaT). Our results demonstrate a low DPPIV/CD26 expression in the studied cell lines, being almost undetectable in HeLa cell line. The dipeptidylpeptidasic enzymatic activity was verified both membrane bound and in the soluble form in all cell lines. In the presence of the DPPIV/CD26 inhibitor (sitagliptin phosphate) we observed that SiHa cell line showed an increase in cell migration, thus we suggest that at least in part cell migration in this cell line is regulated by DPPIV/CD26 enzymatic activity. In order to investigate the relationship between DPPIV/CD26 expression and HPV E6 and E7 oncoproteins, we evaluated the expression of this protein in normal keratinocytes or transduced with these oncoproteins. We have found that keratinocytes expressing high-risk HPV E6 present a reduction in DPPIV/CD26 expression, and this regulation appears to be dependent on p53 degradation. Considering that the cell lines studied have low DPPIV/CD26 expression and activity, in order to better understand the importance of the expression of this protein, we induced the DPPIV/CD26 overexpression in a cervical cancer cell line (HeLa) for further evaluation of the effects on different tumor mechanisms. The results demonstrate a reduction in cell growth of DPPIV/CD26 expressing cells, being this effect independent of the enzymatic activity. In addition, it has been demonstrated that the induction of DPPIV/CD26 expression does not affect the cell migration and adhesion mechanisms in the HeLa cell line. Thus, we believe that the elucidation of the DPPIV/CD26 role in the context of cervical cancer enables new diagnostic and therapeutic approaches to be implemented in the future.

Les kinases cycline-dépendantes (CDK) sont des protéines régulatrices essentielles du cycle cellulaire. Elles contrôlent la prolifération cellulaire et sont souvent déréglées dans les cancers. De nombreux inhibiteurs de CDKs ont été élaborés et sont actuellement le sujet d'essais cliniques. Bien que Cdk1 soit un régulateur essentiel de cycle cellulaire, Cdk2 n’est pas nécessaire pour la progression du cycle cellulaire, mais favorise la tumorigenèse. Par conséquent, Cdk2 est une cible thérapeutique prometteuse. L’utilisation des inhibiteurs de kinases pour modifier la prolifération cellulaire s’apparente à appliquer une sélection Darwinienne. Cette sélection peut être modélisée mathématiquement. Cette approche a montré que des avantages sélectifs, mêmes marginaux, peuvent être d'une importance majeure dans la compétition inter-cellulaire et la progression du cancer. Selon ce principe, nous avons fait l’hypothèse que le fait que la Cdk2 ait un rôle mineur dans la progression du cycle cellulaire lui confèrerait le statut de cible pertinente pour une thérapie du cancer. Selon cette hypothèse, son inhibition serait bien tolérée, permettant de réduire le niveau d’activité CDK et ainsi agir contre la prolifération déréglée des cellules. Nous avons supposé qu’au lieu d’éliminer entièrement les cellules les plus prolifératives, qui seraient les plus sensibles au traitement, il serait potentiellement intéressant de les exploiter pour concurrencer l’émergence des cellules résistantes, moins prolifératives. L'utilisation d'un traitement continu à faible dose avec les inhibiteurs Cdk2 pourrait permettre de maintenir cet équilibre. L'objectif de la thèse était d'étudier si Cdk2 confère un avantage prolifératif aux cellules cancéreuses, si les cellules peuvent développer une résistance aux inhibiteurs de CDKs, et si oui, déterminer quels étaient les mécanismes de résistance qui permettent de réduire le « fitness » des cellules prolifératives. Pour répondre à ces questions, nous avons généré des lignées cellulaires ayant des degrés variés de résistance à un inhibiteur spécifique de Cdk2 (inhibant également Cdk1 à des concentrations élevées). Nous avons caractérisé leur capacité à proliférer en comparaison avec des cellules parentales et des cellules isogéniques n’exprimant plus Cdk2 en raison d’un « knock-out » du gène. Bien que dans ces premières cellules le gène Cdk2 est retrouvé non muté et que l'expression de la protéine Cdk2 reste inaltérée, l'activité kinase de Cdk2 est diminuée. Les cellules résistantes à l’inhibiteur prolifèrent efficacement in vitro. Cependant, lors des expériences de compétition avec les cellules parentales, sensibles aux inhibiteurs, elles sont perdantes. Ceci montre que le développement d’une résistance à un inhibiteur de kinase entraîne un désavantage sélectif. Malgré une prolifération normale en l’absence de compétiteurs, ce désavantage est mis en évidence dans une population mixte, validant ainsi l’hypothèse de départ. Nous avons constaté que les Cdk2 KO et les cellules résistantes à l’inhibiteur (R50) ont un métabolisme altéré. Ces cellules sont sensibles à l'épuisement des nutriments et du glucose ainsi qu’à l'hypoxie, malgré un taux de consommation d'oxygène normal, ce qui indique une augmentation de la glycolyse aérobique. Les cellules R50 surexpriment la protéine Cdk6, ce qui peut contribuer à la résistance à l'inhibition Cdk2. De plus elles sont sensibles à l’inhibition des Cdk4/6, cibles référencées dans le traitement de certaines classes de cancer du sein. Enfin, les cellules Cdk2 KO présentent un point de contrôle de la phase S perturbé. Ces résultats suggèrent que des inhibiteurs pharmacologiques ciblant Cdk2 pourraient être synergique avec d’autres traitements, par exemple l’inhibition concomitante de la réplication de l'ADN, de la glycolyse, ou de Cdk6. Cela pourrait ainsi diminuer la prolifération des cellules cancéreuses et empêcher l’émergence d'une résistance thérapeutique. / Cyclin-dependent kinases (Cdk) are essential regulators of the cell cycle that support cell proliferation and are often deregulated in cancer. While Cdk1 is an essential regulator of the cell cycle, Cdk2 is not required for cell cycle progression but promotes tumorigenesis. Therefore, Cdk2 is a promising drug target. Many Cdk inhibitors have been developed and are currently undergoing clinical trials. Darwinian selection can be modelled mathematically, and such studies have shown that even marginal selective advantages can be of great importance in outcomes of cell-cell competition and cancer progression. We hypothesised that the non-essential role of Cdk2 for cell cycle progression may mean that it is a good target for cancer therapy as continual inhibition should be tolerated and should counteract deregulated cell proliferation in cancer. However, as with all chemotherapeutic agents, the development of clinical resistance is likely. We further hypothesized that applying a low-dose treatment with Cdk2 inhibitors should minimize chances of developing resistance, by maintaining competition between robustly proliferating cells that are sensitive to treatment, and resistant cells.The aim of the thesis was to investigate whether Cdk2 confers a proliferative advantage to cancer cells, whether cells can develop resistance to Cdk inhibitors, and if so, whether the mechanisms allowing resistance reduce cellular proliferative fitness.To answer these questions, we have created cell lines with varying degrees of resistance to a selective Cdk2 inhibitor (that at high doses, also inhibits Cdk1) and have characterised their proliferation capacity in comparison with parental cells and isogenic Cdk2 knockout cells. Although in these cells the Cdk2 gene is not mutated and the expression of Cdk2 protein remained unaltered, the kinase activity of Cdk2 is decreased. Similarly, Cdk2 gene knockout (Cdk2 KO) cells have reduced sensitivity to Cdk2 inhibition. Inhibitor-resistant cells proliferate efficiently but are outcompeted by parental, inhibitor-sensitive cells in competition experiments, confirming that inhibitor resistance entails a selective disadvantage. We found that the proliferation of both Cdk2 knockout and inhibitor-resistant (R50) cells is sensitive to nutrient and glucose depletion as well as hypoxia, despite a normal oxygen consumption rate, indicating increased aerobic glycolysis. R50 cells have highly upregulated Cdk6, which may contribute to resistance to Cdk2 inhibition. Moreover, they are sensitised to Cdk4/6 inhibition, which is currently authorised as a treatment for some classes of breast cancer. Finally, Cdk2 knockout cells have an impaired S-phase checkpoint. These results suggest that pharmacological inhibitors targeting Cdk2 might be synthetically lethal with other treatments, eg inhibition of DNA replication, of glycolysis, or of Cdk6. This might diminish cancer cell proliferation and prevent emergence of therapeutic resistance.

Les taux de guérison du cancer du sein sont en progression constante, toutefois l’apparition de métastases osseuses contribue à la morbidité et à la mortalité des femmes à un stade avancé et les traitements actuels ne sont que palliatifs. Afin de prévenir les métastases osseuses, il est essentiel de mieux comprendre les mécanismes moléculaires qui contrôlent les événements cellulaires précédant l'apparition de lésions squelettiques. Dans ce but, une analyse transcriptomique comparative a été réalisée entre une lignée de cellules de cancer du sein humaines MDA-MB-231 qui dissémine dans plusieurs organes sans site préférentiel, et une sous-population ostéotropique, les cellules B02, qui métastasent uniquement dans les os. Par comparaison à la lignée MDA-MB-231, les cellules B02 surexpriment Roundabout 4 (ROBO4), codant un récepteur cellulaire membranaire, appartenant à la famille des récepteurs de guidage axonal Roundabout. Physiologiquement, ROBO4 est impliqué dans l'angiogenèse, l'intégrité des vaisseaux sanguins et la migration des cellules souches hématopoïétiques dans la niche ostéoblastique. Afin de déterminer si ROBO4 peut, également, jouer un rôle de médiateur dans la localisation des cellules tumorales au sein de la moelle osseuse, nous avons invalidé ROBO4 dans les cellules B02 en utilisant une stratégie CRISPR / Cas9. In vivo, l'injection orthotopique de cellules B02 KO ROBO4 a entraîné la formation de tumeurs plus petites par rapport à celles formées par les cellules B02. De même, dans les os, on observe une forte diminution de la croissance tumorale et de l’étendue de l’ostéolyse. Des protocoles à court-terme de formation de micro-métastases ont également montré un rôle de ROBO4 dans les étapes précoces de la colonisation osseuse, probablement en agissant sur l'ancrage et/ou la survie des cellules tumorales. De plus, la colonisation d’une niche ostéoblastique humanisée implantée en sous-cutanée chez des souris NOD / SCID, par les cellules B02 KO ROBO4 a également été nettement réduite en comparaison aux cellules B02. Par conséquent, ROBO4 intervient dans la dissémination osseuse précoce de cellules cancéreuses du sein et dans la formation tumorale. In vitro, l’interaction des cellules B02 KO ROBO4 avec les cellules résidentes de la moelle osseuse est réduite par rapport à celle observée avec les cellules parentales B02. Les co-cultures de cellules B02 ou B02 KO ROBO4 en suspension avec les cellules de la moelle osseuse, conduisent à la formation de mammosphères hétérotypiques. Cependant, leurs tailles sont considérablement réduites suite à l'inhibition de ROBO4. Des résultats similaires sont observés lors de la culture homotypique de cellules B02 ou B02 KO ROBO4. On observe également une importante réduction de l’agrégation cellulaire lorsque ROBO4 est inhibée, suggérant un rôle de ROBO4 dans l’interaction cellulaire. De plus, les cellules B02 KO ROBO4 sont plus petites et plus isolées que les cellules B02 parentales, comme le montre l'analyse du cytosquelette d'actine. Ces résultats nous permettent d’émettre l’hypothèse que ROBO4 est une molécule d’adhérence jouant un rôle important dans la cohésion des cellules tumorales et dans l’interaction des cellules cancéreuses avec les cellules du microenvironnement osseux. De façon intéressante, l’inhibition de ROBO4, par utilisation d’un anticorps anti-ROBO4, réduit également la formation de sphéroïdes et la colonisation osseuse des cellules B02. L’ensemble de ces résultats suggèrent que ROBO4 pourrait être une nouvelle cible thérapeutique dans le traitement de tumeur primitive du sein et des métastases osseuses / The cure rates of breast cancer are steadily increasing; however, the occurrence of bone metastases contributes to the morbidity and mortality of women at an advanced stage and current treatments are only palliative. In order to prevent bone metastasis, it is vital to increase our understanding of the molecular mechanisms that control cellular events preceding the development of overt skeletal lesions. A comparative transcriptional analysis was performed between human MDA-MB-231 breast cancer cells that spread to several organs in animals, and a sub-population of the MDA-MB-231 cell line (B02) that metastasis only to bones. Compared to MDA-MB-231, B02 cells overexpress Roundabout 4 (ROBO4), encoding a cell surface receptor belonging to the Roundabout family of axonal guidance receptors. ROBO4 is involved in angiogenesis, blood vessel integrity and homing of hematopoietic stem cells in the osteoblastic niche. To determine whether ROBO4 could also mediate the homing of tumour cells in the bone marrow, we invalidate ROBO4 in B02 cells using a CRISPR/Cas9 strategy (KO ROBO4 B02). In vivo, orthotopic injection of KO ROBO4 B02 cells leads to smaller primary tumours compared to the ones formed by B02 control cell line. Moreover, in bone, ROBO4 invalidation induces a strong decrease of tumour growth, osteolysis occurrence and micrometastasis formation. Short-term protocols show the involvement of ROBO4 in the early-step of bone colonisation, probably by acting in tumour cell anchorage and/or survival. Furthermore, compared to B02 cells, the homing of KO ROBO4 B02 cells in a humanized osteoblastic niche implanted subcutaneously in NOD/SCID mice is also markedly reduced. Therefore, ROBO4 is involved in the early bone marrow dissemination of breast cancer cells and in tumor formation. According to this result, in vitro, the interaction of KO ROBO4 B02 cells with bone marrow resident cells is decreased, compared to what is observe with parental B02 cells. After the co-culture of B02 or KO ROBO4 cells in suspension with bone marrow resident cells, they both form heterotypic mammospheres. However, their size is substantially reduced in the absence of ROBO4. Similar findings were observed in homotypic culture of B02 or KO ROBO4 cells. An important decrease of tumor cell aggregation is also observed when ROBO4 is inhibited, suggesting a role of ROBO4 in cell interaction. Moreover, KO ROBO4 B02 cells are smaller and more isolated than parental B02 cells, as shown in actin cytoskeletal analysis. Altogether, these results provide strong evidence that ROBO4 is an adherent molecule which plays a crucial role in tumour cell cohesion and in the interaction of cancer cells with bone cells component. Interestingly, ROBO4 inhibition, using an anti-ROBO4 antibody, also reduces spheroid formation and bone colonization of B02 cells. Taken together, these results suggest ROBO4 as a new potential therapeutic target to treat primary breast tumours and bone metastasis

Le cancer du sein touche une femme sur neuf dans le monde et constitue un problème majeur de santé publique. L’identification de nouveaux biomarqueurs pour un traitement personnalisé pour les tumeurs du sein de plus mauvais pronostic, dites « triple-négatives », est extrêmement urgent. ATIP3, le produit majeur du gène candidat suppresseur de tumeurs MTUS1, a été identifié par l’équipe comme étant un biomarqueur des tumeurs du sein les plus agressives. De plus, ATIP3 inhibe la prolifération et la migration in vitro, ainsi que la progression tumorale et la formation de métastases in vivo et constitue une cible thérapeutique. ATIP3 est une protéine associée aux microtubules (MT) en interphase et au fuseau mitotique durant la mitose. Mon projet de thèse a pour objectif principal d’identifier les partenaires d’interaction d’ATIP3 impliqués dans ses mécanismes d’action antitumoraux. Dans une première partie, j’ai montré qu’ATIP3 interagit avec EB1, une protéine majeure de la dynamique du MT. L’interaction ATIP3-EB1 diminue l’accumulation d’EB1 à l’extrémité croissante du MT. Un nouveau mécanisme a été proposé dans lequel l’interaction ATIP3-EB1 réduit indirectement la vitesse d’échange d’EB1 à son site de liaison au bout plus du MT, ayant pour conséquence une diminution de la dynamique du MT. Dans une deuxième partie, j’ai montré qu’une déplétion d’ATIP3 induit une réduction de la taille du fuseau mitotique. Une analyse protéomique a permis d’identifier la kinésine Kif2A comme partenaire d’interaction d’ATIP3. ATIP3 forme un complexe avec Kif2A et Dda3 qui est dépendant d’une phosphorylation par Aurora kinase A. ATIP3 maintient la taille du fuseau en diminuant le recrutement de Kif2A et Dda3 au pôle de façon dépendante d’AurKA. ATIP3 régule donc négativement ses partenaires d’interaction. Enfin, dans une troisième partie, la relevance clinique du couple ATIP3-EB1 a été évaluée et j’ai montré que l’expression combinée des deux biomarqueurs ATIP3 et EB1 était associée à l’agressivité de la tumeur et à une survie diminuée. Ainsi, l’ensemble de mes travaux a permis de mettre en évidence de nouvelles cibles thérapeutiques afin de mettre en place des traitements personnalisés / Breast cancer is a leading cause of death by malignancy in women worldwide. The identification of new molecular markers for personalized treatment of poor prognosis breast tumors, such as those of the triple negative subtype, is urgently needed. Our team is leader in the study of ATIP3 protein, encoded by candidate tumor suppressor gene MTUS1. ATIP3 is down-regulated in 85% of triple negative breast tumors, and low levels of ATIP3 are associated with poor survival of the patients. We have shown that ATIP3 reduces proliferation and migration in vitro, and tumor growth and metastasis formation in vivo. ATIP3 localizes along the microtubule (MT) in interphase and on the mitotic spindle and spindle poles during mitosis. My PhD project aimed at identifying ATIP3 partners involved in its anti-tumoral effects. In the first part, I will present data showing that ATIP3 interacts with EB1, a major regulator of MT dynamics. ATIP3-EB1 interaction prevents EB1 accumulation at MT growing ends. I proposed a novel mechanism by which ATIP3-EB1 indirectly reduces EB1 turnover at its binding site at MT plus end, which consequently reduces MT dynamics. In the second part of my thesis, I showed that ATIP3 silencing induces reduced spindle length. In parallel, I identified the MT-depolymerizing kinesin Kif2A as an ATIP3 partner by proteomic analysis. ATIP3 forms a complex with Kif2A and Dda3 in an AurKA-dependent manner. I showed that ATIP3 maintains mitotic spindle size by inhibiting Kif2A and Dda3 recruitment at the spindle pole. My study also revealed a recriprocal regulation between ATIP3 and AurKA. Thus, ATIP3 negatively regulates its binding partners. Finally, in a third part, clinical relevance of ATIP3-EB1 in breast cancer has been evaluated and I showed that combinatorial expression of ATIP3 and EB1 is associated with tumor agressiveness and reduced patient survival. Altogether, this work highlighted new therapeutic targets to propose personalized treatments.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.

L’infarctus du myocarde (IDM), responsable du remodelage ventriculaire, peut conduire, s’il est délétère, à l’insuffisance cardiaque (IC), principale cause de mortalité à travers le monde. Les récentes découvertes ont montré l’implication des ARNs non codants, microARNs (miARNs) et les longs ARNs non codants (lncARNs), dans les processus physiologiques et pathologiques et notamment dans les maladies cardiovasculaires. L’objectif de ce travail a été d’étudier le potentiel des miARNs et des lncARNs en tant que biomarqueurs pronostiques et diagnostiques ainsi qu’en tant que cibles thérapeutiques dans l’IDM et le remodelage ventriculaire. Dans un premier temps, nous avons évalué le pouvoir diagnostique des miARNs sur une cohorte de patients présentant des douleurs thoraciques. Le miR-208b et le miR-499 ont montré une bonne capacité diagnostique de l’IDM, ne dépassant toutefois pas celle des troponines. Nous avons ensuite observé que le miR-150 présente une plus faible concentration dans le sang de patients avec un remodelage ventriculaire post-IDM par rapport aux patients sans remodelage, le positionnant comme un biomarqueur intéressant dans le pronostic de l’IC. Enfin, nous avons montré une régulation importante de plusieurs lncARNs dans le cœur de souris, 24 heures après IDM et 2 lncARNs, MIRT1 et MIRT2, ont été mis en avant pour leur association avec le remodelage. En conclusion, nos études ont montré l’utilité des ARNs non codants pour améliorer l’identification des patients à risque de développer une IC après IDM et ont permis également de mettre en évidence de nouvelles cibles thérapeutiques qui pourraient prévenir le remodelage ventriculaire post-IDM / Myocardial infarction (MI responsible for left ventricular remodeling which can be deleterious and the development of heart failure (HF). HF is one of the leading causes of mortality worldwide and despite many improvements, it remains a major challenge in clinical practice. Recent discoveries in genomics have showed the involvement of non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in physiological and pathological processes and notably linked to cardiovascular diseases. The goal of this work was to study the potential of miRNAs and lncRNAs as prognostic and diagnostic biomarkers and as therapeutic targets in MI and left ventricular remodeling leading to HF. First, we evaluated the diagnostic value of miRNAs in patients with chest pain. MiRNA-208b and miR-499 have shown a good diagnostic capacity for MI. However, these miARNs failed to improve the diagnosis of MI by troponins. MiRNA-208b could also predict patient mortality after MI but this capacity was modest. Then, we observed that miR-150 was present at a low level in the blood of patients with left ventricular remodeling post-MI compared to patients without remodeling. Therefore, miRNA-150 is an interesting prognostic biomarker. Finally, we have shown a significant regulation of several lncRNAs in mouse heart, 24 hours after MI, and 2 lncRNAs, MIRT1 and MIRT2, have been demonstrated for their association with left ventricular remodeling. In conclusion, our studies have shown the utility of non-coding RNAs to improve the identification of patients at risk of developing HF after MI and also allowed to identify potential therapeutic targets to prevent left ventricular remodeling

As enzimas desempenham papel fisiológico fundamental nos organismos, seja em condições normais e patológicas, o que as tornam atrativos alvos para intervenções terapêuticas. Pequenas moléculas capazes de inibir enzimas alvos são utilizadas para o tratamento de diversas doenças inflamatórias, câncer, AIDS, entre outras. Contudo, a identificação de pequenas moléculas inibidores ainda é uma etapa limitante no desenvolvimento de fármacos. Nesse contexto, o aprimoramento e novos ensaios robustos e confiáveis para acelerar a descoberta e a caracterização de novos inibidores é de extrema relevância. Nesta tese apresentamos o desenvolvimento de apropriados métodos para a quantificação direta da atividade das enzimas alvos Nucleosídeo Difosfato Quinase B (NDKb), humana (NME2) e de Leishmania major (LmNDKb), livres em solução ou imobilizadas em colunas capilares (ICER). A separação dos produtos e substratos da reação foi realizada por cromatografia de par iônico, que possibilitou a quantificação direta da atividade enzimática da NDKb livre em solução, método 1D-LC-UV. Já para a quantificação da atividade das enzimas imobilizadas foi elaborado um método on-line ICER-LC-UV, no qual a reação enzimática do ICER foi transferida à coluna analítica, permitindo a separação e quantificação da atividade de fosfotransferência. Utilizando esses métodos foi possível determinar o pH ótimo e os parâmetros cinéticos das enzimas livres e imobilizadas. Inibidores de NDKb de diferentes potências e mecanismos de ação foram utilizados para modulação do sistema ICER-LC-UV como ensaio de triagem de inibidores. O (-)-Epicatequina galato (ECG) foi utilizado como prova de conceito, para validar a aplicação do método on-line ICER-LC-UV na identificação e caracterização de inibidores para as enzimas alvo NME2 e LmNDKb. Em suma, a quantificação direta associada ao uso de enzimas imobilizadas representa um grande avanço aos métodos consolidados para o monitoramento da atividade enzimática e triagem de inibidores das enzimas alvo. / Enzymes play a key physiological role in organisms, either under normal and pathological conditions, which make them attractive targets for therapeutic interventions. Small molecules capable to inhibit specific target enzymes are used for the treatment of several inflammatory diseases, cancer, AIDS, among others. However, to identify small inhibitory molecules is still a limiting step in drugs discovery. In this context, the improvement and development of robust and reliable novel assays to accelerate the discovery and characterization of new inhibitors have become extremely relevant. This study describes an appropriate direct method to quantify the enzymatic activity of Nucleoside Diphosphate Kinase B (NDKb) from human (NME2) and Leishmania major (LmNDKb). It can be applied to free enzyme in solution or immobilized enzyme into silica capillary (ICER). The separation of both the substrates and products was achieved using ion-pair chromatography, it can be applied to direct quantification of free NDKb activity, method 1D-LC-UV. In order to quantify the activity of the immobilized enzymes, an on-line ICER-LC-UV method was developed. Initially, the enzymatic catalysis occurred into the ICER. Sequentially, the reaction mixture was transferred to an analytical column, where analytes were separated. From this method, it was possible to determine the optimum pH and kinetic parameters for the immobilized enzymes. Well stablished NDKb inhibitors with different strenght and mechanisms of action were used to modulate the on-line ICER-LC-UV method as an inhibitor screening assay. (-)-Epicatechin gallate was used as proof of concept in order to validate the application of the on-line ICER-LC-UV method to identify and characterize the target\'s enzymes NME2 and LmNDKb inhibitors. In summary, the direct quantification method coupled to the immobilized enzymes increases the likelihood of identifying the enzymatic activity and screening of inhibitors of the target enzymes when compared methods well described in the literature.

Les maladies rénales ont un impact socio-économique majeur sur la santé publique nécessitant le développement de nouvelles stratégies thérapeutiques. Le Récepteur à Domaine Discoïdine de type 1 (DDR1) est un récepteur non-intégrine des collagènes, à activité tyrosine-kinase. Son expression anormale est un facteur clé de la pathologie rénale qui promeut le développement de l’inflammation et de la fibrose.Ces travaux de thèse nous ont permis de démontrer que l'inhibition de DDR1 freinait la progression des maladies rénales dans trois modèles, dont l'un d'évolution aiguë, l'ischémie-reperfusion (I/R). Après I/R, les cellules épithéliales tubulaires proximales (CETP) exprimaient anormalement DDR1 et l'inhibition de ce récepteur empêchait l'acquisition d'un phénotype pro-inflammatoire par ce type cellulaire. Nous avons démontré in vitro que le stress du réticulum endoplasmique (RE), secondaire à l'hypoxie, était responsable de l'induction de DDR1, via l'activation du facteur de transcription CHOP. De plus, le profil d'expression de DDR1 dans des biopsies de patients transplantés était similaire à celui obtenu dans l'I/R expérimentale.Enfin, les résultats préliminaires obtenus dans un nouveau modèle de souris triples transgéniques ont montré l'installation d'une inflammation et d'une fibrose rénales secondaires à la surexpression génétiquement définie de DDR1 durant 4 semaines dans les cellules épithéliales tubulaires.En conclusion, nos résultats suggèrent que la surexpression de DDR1 joue un rôle délétère dans les néphropathies chroniques et aiguës, ce qui renforce l’intérêt du développement d’inhibiteurs spécifiques de DDR1 capables de bloquer la fonction de ce récepteur. / Renal diseases lead to severe long-term complications of kidney function and only few preventive and therapeutic options exist. Discoidin Domain Receptor 1 (DDR1) is a non-integrin collagen receptor expressed in several cell types within the kidney. Its abnormal expression has a deleterious role in experimental chronic kidney diseases (CKD) by promoting renal inflammation and fibrosis.The inhibition of DDR1 stopped the progression of renal disease in two models of experimental CKD and protected renal function and structure in a model of acute kidney disease, ischemia-reperfusion (I/R). DDR1 expression was strongly induced in proximal epithelial tubular cells (PETCs) after I/R. Moreover, isolated PETCs from DDR1 heterozygous mice after I/R did not acquire the pro-inflammatory phenotype displayed by PETCs from WT mice. Endoplasmic reticulum (ER) stress was responsible for DDR1 pathological expression in hypoxic PETCs after I/R through the activation of CHOP transcription factor. Interestingly, biopsies of transplant patients with prolonged ischemia during transplantation had a very similar expression profile of DDR1 in proximal tubules as in experimental I/R.Finally, DDR1 overexpression in epithelial tubular cells for four weeks, in a new conditional transgenic mouse model, led to the development of renal inflammation and fibrosis.To conclude, our results suggest that the genetically-induced or the pathological overexpression of DDR1 promotes renal inflammation and fibrosis. Thus, targeting DDR1 can be a promising strategy in the treatment of renal diseases.

Le paludisme reste une des principales causes de mortalité infantile dans le monde tropical. L'émergence continue des résistances du parasite aux anti-paludiques constitue un sérieux problème de santé publique. La recherche de nouvelles cibles thérapeutiques, basée sur une connaissance plus approfondie des mécanismes moléculaires de la vie du parasite, est une nécessité permanente dans un paradigme de « reine rouge » qui s'applique parfaitement à la capacité d'adaptation du parasite. La découverte récente d'une métacaspase de Plasmodium falciparum (PfMCA1) et la mise en évidence de son rôle potentiel dans l'apoptose du parasite, fait qu'elle est une cible thérapeutique contre le paludisme. Dans le but de mieux approfondir les connaissances sur cette protéine cible, nous avons voulu, dans un premier temps, déterminer la structure tridimensionnelle de PfMCA1, afin de confirmer les différentes structures prédites in silico, et chercher de nouvelles molécules candidates par le docking moléculaire. Cependant cet objectif n'a pas pu être atteint, à cause d'un phénomène d'autoclivage de la protéine suite à son expression, ce qui fait que nous n'avons pas réussi à récupérer la protéine. Dans un second temps, nous avons étudié la métacaspase de Plasmodium vivax (PvMCA1) en comparaison avec PfMCA1, et nous avons montré que les résidus histidine et cystéine dans la dyade catalytique sont bien conservés. Nous avons identifié un deuxième site potentiel dans le domaine catalytique de PvMCA1. A partir d'échantillons collectés en Mauritanie, au Soudan et à Oman, nous avons montré que les résidus histidine et cystéine, ainsi, que les résidus du second site du domaine catalytique de PvMCA1 sont très variables. Les mutations de ces résidus doivent faire l'objet d'étude approfondie de leurs effets sur la fonction de la protéine PvMCA1. Ce polymorphisme trouvé dans les résidus catalytiques de PvMCA1, doit-être évalué comme marqueurs moléculaires de résistance / Malaria remains one of the main causes of infant mortality in the tropical world.The continuous emergence of parasite resistant to drug treatment is a serious threat to public health. Exploring new therapeutics targets based on depth knowledge on molecular mechanism of the parasite’s life is utmost needed in a paradigm of « red queen», which applies perfectly on the ability of the parasitic adaptation. The recent discovery of metacaspase of Plasmodium falciparum (PfMCA1) and the demonstration of its potential role in apoptosis, make it a therapeutic target against malaria. In order to increase knowledge about this protein, we planned, to determine the three-dimensional structure of PfMCA1, to confirm the different structures predicted in silico, and to look for new drug using molecular docking. However, this goal was not reached, since autoprocessing occurred during expression, and we failed to obtain the full-length protein. Then we studied the metacaspase of Plasmodium vivax (PvMCA1) in comparison with PfMCA1 and, we shown that histidine and cysteine residues in the dyad catalytic are well conserved. We have identified a second potential site in the catalytic domain of PvMCA1. We shown that residues in both putative sites are highly polymorphic in samples from Mauritania, Sudan and Oman. Mutations on these residues need to be deeply studied for their effects on the PvMCA1 function. This polymorphism found in catalytic residues of PvMCA1should be evaluated as new molecular marker of resistance