Deregulated cell proliferation, resulting from disruption of cell cycle control, is characteristic of many cancers. In normal cells, cell cycle progression is mediated by a family of cyclin dependent kinases (Cdks) that are positively regulated by associated cyclins. The activities of these cyclin-Cdk complexes are regulated by two protein families: the inhibitors of Cdk4 (INK4) and the kinase inhibitor proteins (KIP). p27 is a KIP family member that can inhibit cyclin E-Cdk2 activity. It also plays a role in the assembly and nuclear import of cyclin D-Cdk4 in early G1. p27 has been shown to be deregulated in human cancers by accelerated proteolysis, sequestration in cyclin D-Cdk complexes, and mislocalization to the cytoplasm. The causes of these alterations are not fully understood, but result, at least in part, from changes in signal transduction pathways that alter p27 phosphorylation and function. Activation of both the Ras/Raf/ mitogen activated protein kinase (MAPK) and the phospho-inositol 3' kinase (PI3K) pathways have been shown to alter p27 function and to activate p27 degradation in different cell types. In this thesis, I have investigated the roles played by two kinases downstream of PI3K, protein kinase B (PKB) and p90 ribosomal S6 kinase (RSK1), in regulation of p27 function. I observed that PKB-mediated phosphorylation of p27 promotes p27-cyclin D1-Cdk4 assembly. p27 phosphorylation by RSK1 alters the interaction of p27 with cytoskeleton proteins to promote cell motility. I observed that PKB activation and the appearance of p27pT157 and p27pT198 in early G1 precede p27-cyclin D1-Cdk4 assembly. PI3K/PKB inhibition dissociates cellular p27-cyclin D1-Cdk4 and p27T157A, p27T198A and p27T157A/T198A bind cellular cyclin D1 and Cdk4 poorly. Cellular p27pT157 and p27pT198 co-precipitate with Cdk4 but not Cdk2. p27 phosphorylation by PKB increases the ability of p27 to assemble cyclin D1-Cdk4 in vitro, but yields inactive Cdk4. While Src does not affect p27's ability to assemble cyclin D1-Cdk4, Src treatment yields catalytically active p27-cyclin D1-Cdk4. Thus, while PKB dependent p27 phosphorylation promotes p27-cyclin D1-Cdk4 assembly, tyrosine phosphorylation of p27 is required for activation of p27-cyclin D1-Cdk4 complexes. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process. While RSK1 has been shown to phosphorylate p27, the key phosphorylation sites and the consequence of this phosphorylation event were not fully elucidated. I have shown that RSK1 activation in early G1 precedes p27 phosphorylation at T157 and T198 in synchronized cell populations. Overexpression of RSK1 causes resistance to G1 arrest by TGF-â. Moreover, cells overexpressing RSK1 show an increase in p27 phosphorylation at T198, increased p27 stability, and an increase in p27 binding to Cdk4. In addition, RSK1-transfectants have increased cytoplasmic p27, associated with increased cell motility and inhibition of RhoA. p27 phosphorylation by recombinant RSK1 increases p27 binding to RhoA, while p27T157A/T198A shows reduced association with RhoA in cells. Thus, phosphorylation of p27 at T198 by RSK1 promotes its binding to RhoA and loss of actin stress fiber stability. Oncogenic RSK1 activation may promote increased cancer cell migration and cancer metastasis. Taken together our results indicate that oncogenic activation of the PI3K pathway can contribute to loss of cyclin E-Cdk2 inhibitory action of p27 by at least two mechanisms. Activation of PKB and RSK1 signaling would promote cytoplasmic mislocalization of p27, p27-RhoA binding and inhibition of the RhoA pathway to augment cell motility. In addition, these phosphorylation events on p27 would increase the assembly of p27-cyclin D1-Cdk4 as a first step in a chain of events that would promote that nuclear import and activation of D-type cyclin Cdk complexes, shifting the equilibrium away from the Cdk2 inhibitory action of p27.

A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

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Les CDK/cyclines jouent un rôle majeur dans la progression du cycle cellulaire et dans le maintien de la prolifération des cellules cancéreuses, constituant ainsi des biomarqueurs clés et des cibles pharmacologiques attractives. Plus particulièrement, l’activité de CDK4/cycline D, kinase responsable de la progression de la phase G1 et de la transition G1/S, est dérégulée dans de nombreux cancers dont le mélanome. Cette hyperactivation est associée à des mutations, à l’amplification ou à la surexpression de CDK4, cycline D, p16INK4a ou encore pRb.Comme aucune approche sensible et directe n’existe pour évaluer l’activité de CDK4/cycline D dans des conditions physiologiques et pathologiques, le premier objectif de ma thèse a consisté à développer un biosenseur fluorescent permettant d’étudier cette kinase in vitro et in cellulo. Une fois caractérisé et validé in vitro, le biosenseur a été appliqué à la détection d’altérations de CDK4/cycline D dans des biopsies de peau humaine et de xénogreffes de mélanome dans des essais fluorescents d’activité kinase, ainsi que dans des cellules cancéreuses vivantes par microscopie de fluorescence et vidéo microscopie.Par ailleurs, peu d’inhibiteurs sont actuellement disponibles pour inhiber CDK4/cycline D et la plupart d’entre eux ciblent la poche de fixation de l’ATP. C’est pourquoi le second objectif de ma thèse a consisté à identifier des inhibiteurs non compétitifs de l’ATP, soit par élaboration rationnelle de peptides, soit par criblage de petites molécules. A cette fin, deux biosenseurs fluorescents ont été développés qui permettent d’identifier respectivement des composés ciblant l’interface entre CDK4 et cycline D ou des inhibiteurs allostériques capables de perturber la dynamique conformationnelle de CDK4. Des essais de criblage par fluorescence réalisés avec ces biosenseurs ont conduit à l’identification de touches qui ont été validées et caractérisées in vitro et dans des essais de prolifération cellulaire, et qui constituent des candidats prometteurs pour une chimiothérapie sélective du mélanome. / CDK/cyclins play a central role in coordinating cell cycle progression, and in sustaining proliferation of cancer cells, thereby constituting established cancer biomarkers and attractive pharmacological targets. In particular, CDK4/cyclin D, which is responsible for coordinating cell cycle progression through G1 into S phase, is a relevant target in several cancers including melanoma, associated with mutation of CDK4, cyclin D, p16INK4a and pRb.As there are no sensitive and direct approaches to probe CDK4/cyclin D activity in physiological and pathological conditions, the first goal of my thesis has consisted in engineering a fluorescent biosensor to probe this kinase in vitro and in cellulo. Once characterized and validated in vitro, the biosensor was applied to detect CDK4/cyclin D alterations in biopsies from human skin and melanoma xenografts in fluorescence-based activity assays, and in living cancer cells by fluorescence microscopy and timelapse imaging.Moreover, only few inhibitors are currently available to target CDK4/cyclin D and most of them bind the ATP pocket. As such, the second major goal of my thesis project has consisted in identifying non-ATP competitive inhibitors, either through rational design of peptides or by screening small molecule libraries. To this aim, two fluorescent biosensors were engineered which discriminate compounds that target the interface between CDK4 and cyclin D, or that perturb the conformational dynamics of CDK4, respectively, from ATP-pocket binding compounds. Fluorescence-based screening assays performed with these biosensors lead to identification of hits, which were validated and characterized in vitro and in cell proliferation assays, and which constitute promising candidates for selective chemotherapy in melanoma.