Le système ubiquitine-protéasome joue un rôle central dans le contrôle de la progression du cycle cellulaire par la dégradation régulée de nombreuses protéines. Dans ce système, Fbw7 (aussi appelée Fbxw7, hCdc4, hAgo, Sel-10), est l'une des protéines F-box qui sert d'adaptateur de substrats pour l'une des plus importantes familles d'ubiquitine ligases : les complexes SCF (Skp1/Cullin/ F-box). Fbw7 assure la dégradation de plusieurs régulateurs positifs du cycle cellulaire : la cycline E, cMyc, c-Jun, Notch, Aurora A, mTOR, MCL1. En conséquence, l'altération des fonctions de Fbw7 conduit à des défauts de prolifération cellulaire, de différenciation et à de l'instabilité génomique. La mutation de Fbw7 dans les cancers entraîne une dérégulation de l'expression périodique cycline E qui n'est alors plus restreinte à la transition G1/S du cycle cellulaire. Nos résultats montrent qu'une isoforme, Fbw7, est exprimée dans les œufs de xénope matures arrêtés en métaphase II mais n'est pas fonctionnelle, expliquant la présence de grande quantité de cycline E dans les œufs à cette phase mitotique. Nous montrons que Fbw7 est maintenue inactive sous forme poly-ubiquitylée suite à sa phosphorylation par une PKC jusqu'à la fin des cycles embryonnaires rapides, au moment où la cycline E est brutalement dégradée. Nous montrons que la régulation négative de Fbw7 par PKC est conservée au cours des cycles cellulaires somatiques des cellules humaines, et contribue à l'expression périodique de la cycline E. Ces résultats mettent en évidence un nouveau mécanisme critique pour la régulation de Fbw7 au cours du cycle cellulaire et suggèrent que les fonctions de Fbw7 peuvent être altérées par une dérégulation de PKC, un phénomène observé dans de nombreux types de tumeurs humaines. / The ubiquitin-proteasome system plays a central role in the control of cell cycle progression through the regulated degradation of numerous critical proteins. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors. Fbw7 (also known as Fbxw7, hCdc4, hAgo, Sel-10) is one such F-box protein. It controls the stability and thus the levels of several positive regulators of the cell cycle, including cyclin E, cMyc, c-Jun, Notch, Aurora A, mTOR, Mcl1. As a consequence of its biological roles, alterations of the functions of Fbw7 lead to defects in cellular proliferation, differentiation and genetic instability. As seen in cancers, mutation of Fbw7 leads to deregulation of cyclin E expression, which is no more restricted to the G1-S phase boundary of the cell cycle. Here we report that Fbw7, although expressed in mature Xenopus eggs arrested in metaphase II, is not functional, explaining why cyclin E can be stockpiled in this mitotic-like phase. We found that, in these eggs as well as in early Xenopus embryos, Fbw7 is maintained under a PKC-dependent poly-ubiquitylated state until the end of the early rapid cleavage cycles where cyclin E is abruptly degraded. Importantly, we show that this PKC-dependent negative regulation of Fbw7 is conserved during human somatic cell cycles, resulting into the periodic expression of cyclin E. These findings reveal a novel mechanism critical for the temporal regulation of Fbw7 and suggest that the key functions of Fbw7 can be altered by PKC dysregulation, a mechanism known to occur in many types of human tumours.

Medulloblastomas are the most frequent malignant childhood brain tumors, arising in the posterior fossa of children. The overall 5-year survival is 70%, although children often suffer severe long-term side effects from standard medical care. To improve progression-free survival and quality of life for these children, finding new therapeutic targets in medulloblastoma is imperative. Medulloblastoma is divided in to four molecular subgroups (WNT, SHH, Group 3 and Group 4) based on key developmental pathways essential for the initiation and maintenance of tumor development. The MYC family of proto-oncogenes regulates cell proliferation and differentiation in normal brain. Aberrant expression of MYC proteins occurs commonly in medulloblastoma. Our studies on Group 3 medulloblastoma identify the transcription factor SOX9 as a novel target for the E3 ubiquitin ligase FBW7, and show that increased stability of SOX9 confers an increased metastatic potential in medulloblastoma. Moreover, SOX9-positive cells drive distant recurrences in medulloblastoma when combining two regulatable TetON/OFF systems. MYCN depletion leads to increased SOX9 expression in Group 3 medulloblastoma cells, and the recurring tumor cells are more migratory in vitro and in vivo. Segueing to treatment of medulloblastoma, we show that BET bromodomain inhibition specifically targets MYC-amplified medulloblastoma cells by downregulating MYC and MYC-transcriptional targets, and that combining BET bromodomain- and cyclin-dependent kinase- inhibition improves survival in mice compared to single therapy. Combination treatment results in decreased MYC levels and increased apoptosis, and RNA-seq confirms upregulation of apoptotic markers along with downregulated MYC target genes in medulloblastoma cells. This thesis addresses novel findings in transcription factor biology, recurrence and treatment in Group 3 medulloblastoma, the most malignant subgroup of the disease.

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