<div>Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men. In this study, I identify two combinational therapeutics to treat PCa – the combination of enzalutamide and simvastatin, and the combination of GSK126 and metformin, both of which strongly suppress PCa cell growth in vitro and in vivo via inhibiting androgen receptor (AR), an important oncogenic driver for the PCa progression. Simvastatin leads to more AR degradation when combined with enzalutamide. For the combination of GSK126 and metformin, the interaction between enhance of zeste homolog2 (EZH2) and AR is interrupted by GSK126, re-sensitizing EZH2 to metformin. Meanwhile, GSK126 inhibits EZH2’s activity. </div><div><br></div><div>Polo-like kinase 1 (PLK1), a cell cycle regulator, is usually overexpressed in non-small-cell lung cancer (NSCLC). Here, we report that PLK1 overexpression promotes the development of Kras^G12D and Trp53^fl/fl (KP)-driven lung adenocarcinoma (LADC). KP mice harboring transgenic PLK1 (KPPI) display heavier tumor burden, poorer tumor differentiation, and lower survival than KP mice. Mechanistically, PLK1 overexpression enhances the activity of MAPK pathway, via upregulating RET expression in a kinase-dependent manner. Supporting our findings, PLK1 knockout in KP mice reduces RET gene expression, inhibits MAPK pathway activity, and strongly delays LADC development. Therefore, these data reveal that PLK1 functions as an oncogene in KP-driven LADC.</div><div> </div><div><br></div>

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18431号 / 生博第311号 / 新制||生||41(附属図書館) / 31289 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 藤田 尚志, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Dans les régions de fortes endémicités, 70-80% des carcinomes hépatocellulaires sont induits par le VHB. Bien qu’un vaccin prophylactique très efficace existe, il n’est d’aucune utilité pour les 250 millions de personnes chroniquement infectées. Les traitements actuels pour contrôler l'infection chronique par le VHB montrent des limites et le besoin de nouvelles thérapies se fait ressentir. La Polo-like kinase-1 (PLK1), qui joue un rôle essentiel dans la mitose et est surexprimée dans de nombreux cancers, représente une cible prometteuse. Outre son rôle lors de la division cellulaire, PLK1 est impliquée dans la régulation de l'expression des gènes en interphase. Il a été montré que la protéine X du VHB (HBx) active PLK1 dans des modèles de cellules murines. Cependant, il restait à déterminer si PLK1 jouait un rôle au niveau de la réplication du VHB dans des hépatocytes quiescents. Des études récentes ont mis en évidence un lien positif entre l'activation de PLK1 et la réplication du VHB. Le but de ce projet de thèse a été d'étudier le(s) mécanisme(s) par le(s)quel(s) PLK1 jouait un rôle positif sur la réplication virale, avec pour objectif futur d'explorer l’inhibition de PLK1 comme cible antivirale. L'interaction entre PLK1 et la réplication du VHB a d'abord été décrite à l'aide du modèle HepAD38. Dans ce contexte, l'ADN viral est intégré dans le génome hôte, sous le contrôle d'un système d'expression Tet-off. La transcription de l'ARN prégénomique (pgRNA), à la base de la réplication virale, est initiée par la suppression de tétracycline. Dans ce contexte, l'augmentation de l'expression de PLK1 est corrélée avec la régulation négative de deux protéines; SUZ12 et ZNF198, faisant partie de complexes de remodelage de la chromatine. L'inhibition de PLK1 bloque la réplication du VHB, en agissant au niveau de la transcription virale. D'autre part, dans les modèles de réplication du VHB qui miment au mieux une infection, comprenant les hépatocytes primaires humains (PHH) et les cellules non transformées/différenciées HepaRG (dHepaRG), où le VHB se réplique dans des cellules quiescentes, nous avons mis en évidence que: 1) L'inhibition pharmacologique de PLK1 bloque la réplication virale, semblablement en perturbant l’encapsidation du pgRNA via une interaction avec la protéine core du VHB (HBc). 2) Un knocking-down de PLK1 en utilisant des ARN interférents délivrés par nanoparticules lipidiques résulte en une forte baisse de la production de pgRNA et dans la sécrétion des antigènes HBeAg/HBsAg, sans impact sur la viabilité cellulaire. Ce projet a donc permis la preuve de concept que PLK1 pouvait être une cible thérapeutique afin de controler la réplication du VHB. De plus, grâce à la technologie de délivrance par nanoparticules lipidiques d’ARN interférents, nous avons pu cibler spécifiquement les hépatocytes, augmentant de ce fait la spécificité et l’efficacité de nos traitements. Un travail sur la compréhension précise des méchanismes cellulaires impliqués permettra de mieux cerner cette interaction hôte/virus afin de poursuivre le développement de stratégies antivirales innovantes portant sur l’inhibition de PLK1. De manière significative, l'inhibition de PLK1 est non toxique pour les cellules quiescentes par rapport à des cellules cancéreuses à fort taux réplicatif, ce qui fait de PLK1 une cible thérapeutique attrayante. Des inhibiteurs spécifiques sont déjà en essais cliniques pour certains cancers (e.g., Volasertib pour le traitement de la leucémie myéloïde aiguë) et pourraient servir de thérapie bimodale dans le cadre de patients infectés par le VHB, en inhibant la réplication virale, ainsi qu’en prévenant l'émergence de cellules néoplasiques. L'inhibition de la PLK1 est une approche antivirale innovante, qui, en combinaison avec les thérapies actuelles de type IFN-α ou analogues nucléotidiques offre de grandes promesses pour endiguer l'infection chronique par le VHB mais également prévenir les événements carcinogéniques / In highly HBV endemic regions, 70-80% of hepatocellular carcinoma cases are attributable to this virus. Despite the existence of an HBV vaccine, the World Health Organization estimates 240 million individuals are chronically infected with HBV worldwide. Current antiviral treatments to control chronic HBV infections, and consequently reduce the incidence of liver cancer, are ineffective. New and effective therapies are needed not only for fighting the virus but also to prevent HCC emergence or progression. The polo-like-kinase 1 (PLK1), which plays pivotal roles in mitosis and is over-expressed in many human cancers, represents a promising druggable target in oncology. Beside its role during cell division, PLK1 is also thought to be involved in gene expression regulation during interphase. It was shown that the X protein (HBx) could activate PLK1 in murine cell transformation models. Yet it remained to be determined whether PLK1 could also play a role for HBV replication in non-dividing hepatocytes. Our, and collaborators, recent studies have identified a positive link between PLK1 activation and HBV replication. The goal of this thesis project was to investigate the mechanism(s) by which PLK1 exerts a positive effect on HBV replication, with the future goal of exploring PLK1 as an antiviral target. The interplay between PLK1 and HBV replication was firstly described using the HepAD38 cellular model of HBV replication. In this context, the HBV DNA is stably integrated into the host genome, under control of a Tet-off expression system. Transcription of HBV pregenomic RNA (pgRNA), the template of viral replication, is initiated by tetracycline removal. It has been shown that in HBV-replicating HepAD38 cells, increased PLK1 expression correlates with down-regulation of two proteins that are components of chromatin modifying complexes; SUZ12 protein of the PRC2 complex, and ZNF198 of the LSD1-CoREST-HDAC1 complex. PLK1 inhibition was described to inhibit HBV replication by reducing viral transcription. How PLK1 regulates HBV transcription remains unknown. On the other hand, in HBV replication models that resemble physiologic HBV infection, comprised of Primary Human Hepatocytes (PHH) and non-transformed/differentiated HepaRG cells (dHepaRG), where HBV replicates in non-transformed and non-dividing cells, thus enabling the study of the inter-phasic role of PLK1, irrespective of its well-established cell division implication, we have demonstrated that: 1) A pharmacological inhibition of PLK1 suppressed HBV replication by a different mechanism, likely targeting the packaging of pgRNA by the HBV core antigen (HBc). 2) Knocking-down PLK1 using siRNA delivered by lipid nanoparticles (LNP siPLK1) results in a strong drop of HBV DNAs, RNAs and HBe/HBsAg secretion without affecting the cell viability. This thesis project brought the proof of concept that PLK1 could be a drug target in HBV infection. Furthermore, the use of LNP allowed us to improve the delivery of siPLK1 to hepatocytes. Significantly, PLK1 inhibition is not toxic to quiescent cells in comparison to fast growing cancer cells, rendering PLK1 an attractive therapy target. High level of viremia in chronic HBV patients is a risk factor for progression to liver cancer. PLK1 specific inhibitors are already in clinical trials for other types of cancer (e.g., acute myeloid leukaemia) and could serve as bimodal therapy in HBV infected patients, by inhibiting virus replication as well as preventing emergence and spreading of neoplastic cells. This project was part of a full-working group of experts and thus, has beneficiated of a strong support. The proximity of the oncology-specialized hospital, the Centre Léon Bérard provided us with fresh hepatic biopsy [etc...]

Background: Oesophagogastric cancers generally present late in life with advanced disease and carry a poor prognosis. Few patients receive curative treatment. Polo-like Kinase 1 (PLK1) is a mitotic kinase with regulatory functions at the G2/M cell cycle phase transition. In mammalian cells, PLK1 phosphorylates and activates FOXM1, a forkhead transcription factor at the G2/M cell cycle phase transition. FOXM1 then promotes transcription of multiple gene products, including PLK1 and CCNB1, which then act individually or in complexes to further phosphorylate FOXM1 generating a positive feedback loop driving the cell into M phase. Aims: We aimed to assess the expression of PLK1 and FOXM1 in oesophageal and gastric cancer patients. Secondly we aimed to investigate the expression and inter- relationship of PLK1 and FOXM1 in oesophageal cell lines during the cell cycle. Results: FOXM1 and PLK1 are expressed in oesophageal cell lines and demonstrate cross-regulatory interactions. Inhibition of PLK1 leads to the decreased expression of FOXM1 and it’s target gene in oesophageal cell lines. FOXM1 and PLK1 are also concomitantly overexpressed in a large proportion of oesophageal and gastric carcinoma’s at both the protein and mRNA level. Other FOXM1 target genes including, CCBN1, AURKB and CKS1 are co-expressed in a similar manner. In a homogenous cohort of patients who underwent surgery, the expression of PLK1 and AURKB was prognostic for overall survival. Conclusions: This study has demonstrated that FOXM1 and a number of target genes including PLK1 are coordinately expressed in a proportion of oesophageal and gastric carcinomas. This suggests that chemotherapeutic treatments that target this pathway may be of clinical utility.

L’ubiquitination est une modification post-traductionnelle impliquée dans de nombreux mécanismes cellulaires. L’E3-ubiquitine ligase CULLIN 3 (CUL3) est un régulateur essentiel de la progression mitotique, ubiquitinant d’importants régulateurs mitotiques et contrôlant leur localisation subcellulaire. Plus particulièrement, notre travail décrit le rôle de la nouvelle E31 ligase CUL3/KLHL22 dans la régulation de l’activité localisée de Polo-like kinase 1 (PLK1) et de ce fait dans l’établissement d’une progression mitotique précise. Néanmoins, les mécanismes moléculaires qui régissent la reconnaissance de son substrat par CUL3 demeurent inconnus. L’activité catalytique de PLK1 ne semble pas être nécessaire à son interaction avec KLHL22, mais aussi bien son domaine kinase que Polo-box (PBD) suffisent à co-purifier KLHL22. Des mutations au niveau du motif DFG, situé en amont du domaine kinase,et du tryptophane 414 au sein du PBD semblent influer sur la reconnaissance de KLHL22. Les résultats obtenus montrent les premières indications biochimiques du mode d’interaction du complexe CUL3/KLHL22/PLK1. / Ubiquitination is a post-translational modification involved in many cellular processes. The E3 ubiquitin-ligase based on CULLIN 3 protein (CUL3) is an essential regulator of mitotic division in human cells by ubiquitinating several important mitotic regulators and controlling their subcellular localization. In particular, our work described the role of novel CUL3/KLHL22 E3-ligase in regulation of localized activity of Polo-like kinase 1 (PLK1) and there by faithful mitotic progression. However, the molecular mechanisms of substrate recognition by CUL3 remain unknown. The catalytic activity of PLK1 may not be required for binding KLHL22 but both the kinase and the Polo-box domains are sufficient to co-purify KLHL22. Mutating the DFG motif within the kinase domain and the tryptophan 414 within the PBD influence the binding to KLHL22. These results provide first insights into molecular mechanisms of CUL3/KLHL22/PLK1complex.

Optineurine ("Optic neuropathy-inducing", Optn) est une protéine exprimée de façon ubiquitaire chez les vertébrés et impliquée dans de nombreux processus cellulaires tels que la régulation du trafic vésiculaire associée à l'appareil de Golgi, la réponse immunitaire innée ou l'autophagie des bactéries. Mon travail de thèse a permis de caractériser une nouvelle fonction d'Optn dans la régulation du cycle cellulaire. Plus précisément, j'ai pu montrer qu'Optn était un régulateur négatif de Polo-like kinase 1 (Plk1), une kinase qui joue un rôle clef dans chacune des étapes de la mitose : de la prophase à la cytokinèse. Les résultats présentés dans cette thèse montrent qu'Optn est phosphorylée par Plk1 sur la sérine 177 en début de mitose provoquant le détachement d'Optn de l'appareil de Golgi et son accumulation dans le noyau. Nous avons montré que la phosphorylation et la translocation nucléaire d'Optn étaient requises pour permettre la régulation négative de Plk1 par le complexe phosphatase MYPT1-PP1 au cours de la mitose. Les conséquences fonctionnelles de la déplétion d'Optn et donc de l'hyperactivité de Plk1 sont des défauts de cytokinèse et de ségrégation des chromosomes, aboutissant à l'apparition de cellules plurinucléées. En conclusion, nos résultats mettent en évidence un mécanisme de rétrocontrôle négatif par lequel Plk1 module la localisation d'Optn pendant la mitose pour réguler sa propre activité

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Hereditary non-polyposis colon cancer syndrome (HNPCC) is associated with an increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. HNPCC results from a germline mutation in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, PMS1, PMS2, MSH6, MSH3 and MLH3. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. The goal of this project was to study MMR deficiency in ovarian cancer at both the clinical and molecular level. The first aim was to examine the frequency of MMR loss in a large patient cohort and investigate the clinical consequences of MMR deficiency. The second aim was to describe the molecular characteristics of MMR deficiency in ovarian cancer cell lines and establish an in vitro cell line model of MMR deficiency in ovarian cancer. The third aim was to identify synthetic lethal strategies for the treatment of ovarian cancer to maximise cytotoxicity in a MMR-deficient background. In order to characterise the clinical consequences of MMR deficiency, a large patient cohort was studied with regard to MMR status. Three tissue microarrays consisting of 581 ovarian tumours were constructed, and expression of the four most frequently lost MMR proteins: MLH1, MSH2, PMS2 and MSH6 were detected by immunohistochemistry. Afterwards, MMR status and histology subtypes were analysed in combination with the associated clinical data. The overall incidence of MMR deficiency (loss of any MMR protein) was 15.7%, with PMS2 being the most frequently lost protein (9.7%). In addition, MMR deficiency tended to appear in a grouped fashion: MLH1 with PMS2; MSH2 with MSH6. Patients with non-serous subtypes of ovarian cancer, clear cell or mucinous especially, had higher incidence of MMR deficiency compared to patients with serous ovarian cancer. Overall MMR deficient patients were more likely to be diagnosed at early stages compared with MMR proficient patients, and this is probably due to the association between MMR deficiency and non-serous histology. However, platinum-based treatment for patients with MMR deficiency gives no advantage over those without MMR deficiency. Therefore better treatments for this subgroup of patients may be needed. The features of MMR deficiency in ovarian cancer were also characterized at the molecular level. After quantifying mRNA and protein expression of MMR genes in 19 ovarian cell lines, three cell lines (SKOV3, TOV21G and IGROV1) were found to have a defect in MLH1 expression at both the mRNA and protein level. Interestingly, the three cell lines also carried a defect in PMS2 expression at the protein level but not at the mRNA level, which is consistent with our clinical data demonstrating that MLH1 protein and PMS2 protein are paired in loss. In addition, across the 19 cell lines, MLH1 and PMS2 showed positive correlation at both the mRNA level (R=0.53, p=0.02) and protein level (R=0.72, p=0.0006). In order to study co-expression of MLH1 and PMS2, a plasmid encoding the cDNA for MLH1 was transfected into the three MLH1 deficient cell lines; and conversely siRNA targeting MLH1 was transfected into the MMR proficient cell line A2780 and expression of MLH1 protein and PMS2 protein was quantified. The results showed that re-introduction of MLH1 into MLH1 deficient cells resulted in increased expression of PMS2 protein, while knocking down MLH1 in MMR proficient cells leads to decreased PMS2 protein expression. This indicates that MLH1 may play a crucial role in regulating PMS2 protein expression. As the three MLH1 and PMS2 protein deficient cell lines all express PMS2 mRNA, the regulation of PMS2 expression by MLH1 is likely to be at the translational or post-translational level. However, the expression of PMS2 protein was not increased in the absence of MLH1, even when the proteasomal and lysosomal protein degradation pathways were blocked (as seen with SKOV3 cells), suggesting decreased PMS2 protein expression is not due to rapid degradation in the absence of MLH1. Therefore MLH1 may play a role in regulating the synthesis of PMS2 protein at the translational level, rather than preventing the degradation of PMS2. Thus, to investigate the mechanism by which PMS2 protein levels are regulated by MLH1, future work should focus on translational regulation of PMS2. In order to identify synthetic lethal strategies to target MMR deficiency in ovarian cancer, an isogenic cell line model of MMR deficiency was established by stable transfection of a plasmid for MLH1 and its corresponding empty vector into SKOV3 cells. The MLH1+ cell line SAC-1 and MLH1- cell line SN-5 were selected for drug screening based on their phenotype and growth rate. The AlamarBlue assay, with z’ above 0.5, was chosen for drug screening and a kinase inhibitor library containing 362 drugs of known target was screened. Two drugs with similar structures that targeted PLK1 showed greater growth inhibition of SN-5 compared with SAC-1. When the two cell lines were treated with another PLK1 inhibitor, BI2536, with different structure, a 2-fold difference in growth inhibition between SAC-1 and SN-5 was also observed, suggesting PLK1 is a potential synthetic lethal target for MLH1 deficiency in ovarian cancer. Together these data demonstrate that clinically, MMR deficiency is associated with non-serous subtypes of ovarian cancer and specific MMR proteins are paired in loss. While current standard therapy offers no selective benefit to ovarian cancer patients with MMR deficiency, inhibiting PLK1 activity may confer selective benefit.

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...