P53 est mutée dans plus de la moitié des cancers humains et son inactivation est souvent associée à une résistance à la thérapie anti-cancer. Notre équipe a montré que dans le cas de tumeurs où p53 est inactive, la surexpression de la phosphatase Wip1 permettait la restauration de l’efficacité de la chimiothérapie, et une protection des tissus sains face aux effets secondaires du traitement. Afin d’améliorer cette stratégie, deux objectifs principaux ont été étudiés : trouver une protéine qui peut interagir avec la voie de signalisation de Wip1 et potentialiser son action dans cette stratégie thérapeutique, et trouver des molécules chimiques pouvant activer Wip1. Nous avons réalisé un criblage d’interférence à ARN de l’intégralité du kinome humain afin d’identifier plusieurs kinases, dont l’inhibition potentialise l’action anti-tumorale de Wip1. Cela a ainsi mis en évidence l’action de Wee1 et Hipk2, dont des inhibiteurs existent, sur la voie de signalisation de Wip1. Inhiber Wee1 avec une faible dose de MK-1775, un inhibiteur spécifique de cette kinase, a permis de réduire la concentration efficace de cisplatine, en entraînant une apoptose caspase-3-dépendante. De plus, combiner MK-1775 avec la surexpression de Wip1 n’a pas d’impact sur l’effet protecteur de cette phosphatase envers les tissus sains. Nous avons ensuite montré que le Vorinostat, un inhibiteur des histone-déacétylases, permettait une augmentation de la transcription de Wip1 dans des cellules de cancer du sein mutées pour p53. Ce travail de thèse a permis de mettre en évidence un moyen de potentialiser la stratégie de traitement des tumeurs p53-négatives basée sur la phosphatase Wip1. / P53 is mutated in more than half of human cancers and when inactivated is often associated with a resistance to anti-cancer therapy. Our team has hown that in the case of p53-negative tumors, overexpression of Wip1 phosphatase sensitizes tumor cells to chemotherapy, while protecting normal tissues from the side effects of the treatment. To improve this strategy, two main objectives were studied. Firstly, to find a protein which can interact with Wip1 pathway and potentiate its action in this therapeutic strategy. Secondly, to find a molecule which can activate Wip1. We realized a siRNA screening of the whole human kinome to identify several kinases, whose inhibition could potentiate anti-tumoral action of Wip1. We have shown that Wee1 and Hipk2, which both have available inhibitors, have an action on Wip1 pathway. Inhibiting Wee1 with a low dose of MK-1775, a specific inhibitor of this kinase, allowed us to decrease effective cisplatin concentration, inducing a caspase-3-dependent apoptosis. Moreover, the combination of MK-1775 with Wip1 overexpression does not impair the protective effect that this phosphatase provides towards normal tissues. We then showed that the Vorinostat, an HDAC inhibitor, induces an increase in Wip1 transcription in breast cancer cells with inactive p53. This work uncovered a way to potentiate the Wip1-based therapeutic strategy of p53-negative tumors.

Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....

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Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...

DNA damage response (DDR) represents a vital signaling network that protects genome integrity and prevents development of cancer. Therefore the study of DDR is of a crucial clinical importance and DDR proteins are promising therapeu- tic targets. Although the great advances have been made mapping out interac- tions between individual DDR proteins, better understanding of complex behav- ior of this network is still needed. One approach, which might help us in this task, is to describe the dynamics of key proteins under different conditions. The first objective of this study was to investigate whether the temporal dynamics of selected DDR proteins differ upon different genotoxic insults, particularly upon γ- irradiation and UV-C irradiation. We showed that under certain insult some DDR proteins exhibit a monotone continuous activation pulse, while the activation of others triggers a series of pulses. We observed a previously described pulsative dynamics of p53 after γ-irradiation in MCF7 cells. Interestingly, we detected a monotone increase of p53 in U2OS after γ-irradiation and similar dynamics upon UV-C irradiation. We suggest that p53 dynamics depends on the presence or ab- sence of effective negative feedback loops between the upstream p53-activating kinases and Wip1 phosphatase. In the second...