TIF1γ est une protéine nucléaire de 1127 acides aminés possédant deux activités : une activité d'E3-ubiquitine ligase et des fonctions de régulateur transcriptionnel. TIF1γ exerce majoritairement ses fonctions dans les processus de développement embryonnaire et de différenciation cellulaire, notamment via son implication dans la voie de signalisation du TGFβ. Le rôle anti-tumoral de TIF1γ a été mis en évidence dans plusieurs modèles murins et son expression est diminuée dans de nombreuses tumeurs humaines de diverses origines tissulaires. Néanmoins, les mécanismes moléculaires et cellulaires par lesquels TIF1γ exerce ses fonctions oncosuppressives sont méconnus. Dans ces travaux, nous avons pu mettre en évidence le rôle inhibiteur de TIF1γ sur la transition épithélio-mésenchymateuse (EMT, Epithelial-to- Mesenchymal Transition) médiée par le TGFβ in vivo, permettant ainsi de limiter les propriétés agressives des cellules tumorales. De plus, nous avons décrit l'implication de TIF1γ dans la progression de la mitose et le point de contrôle du fuseau mitotique : les cellules n'exprimant plus TIF1γ présentent de nombreuses anomalies nucléaires ainsi qu'une forte aneuploïdie associée à une résistance aux agents ciblant les microtubules, molécules classiquement utilisées en chimiothérapie. De plus, nous avons pu corréler la faible expression de TIF1γ à une augmentation de l'instabilité chromosomique dans différentes tumeurs humaines. Ainsi, nos travaux ont permis de mettre en évidence le phénotype cellulaire induit par la perte de TIF1γ dans les cellules tumorales : instabilité chromosomique, résistance aux traitements chimiothérapeutiques et acquisition de propriétés invasives / TIF1γ / TRIM33 (Transcriptional Intermediary Factor 1γ / TRIpartite Motif-containing 33) is a 1,127 amino acids nuclear protein with two biochemical activities: an E3-ubiquitin ligase activity and transcriptional regulatory functions. TIF1γ is ubiquitously expressed in many organisms and exerts its functions mainly in the processes of embryonic development and cell differentiation, particularly through its involvement in the TGFβ signaling pathway. The oncosuppressive functions of TIF1γ have been demonstrated in several mouse models and its expression is reduced in many human tumors of various tissue origins. Nevertheless, the molecular and cellular mechanisms driving TIF1γ anti-tumoral activities are unknown. In this work, we highlight its inhibitory role on TGFβ-mediated EMT (Epithelial-to-Mesenchymal Transition) in vivo, thus limiting the aggressive properties of tumor cells. In addition, we describe TIF1γ involvement in mitotic progression and the Spindle Assembly Checkpoint (SAC): TIF1γ deleted cells display many nuclear abnormalities, aneuploidy and resistance to spindle microtubule-disrupting agents, which are drugs classically used in chemotherapeutic treatments. Finally, we correlated the low expression level of TIF1γ to an increased rate of chromosomal instability in different human tumors. Thus, our work has highlighted the tumor suppressor role of TIF1γ: its deletion in tumor cells induce chromosomal instability, resistance to chemotherapeutic treatments and acquisition of invasive properties

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No description available.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Das nur 16,5 kDa große Survivin ist ein bifunktionales Protein, welches eine bedeutende Rolle in zwei wichtigen zellulären Prozessen spielt, der Apoptose und der Mitose. Aufgrund seiner BIR Domäne wird es zu den Inhibitor der Apoptose Proteine (IAP) gezählt. Diese Gruppe an Proteinen interferiert negativ mit der Aktivierung der Caspasen und wirkt somit einer Induktion der Apoptose entgegen. Neben seiner anti-apoptotischen Funktion besitzt das Survivin zudem eine essentielle Rolle bei der Segregation der Chromosomen und während der Zytokinese. In der Mitose bildet Survivin mit Borealin, INCENP und der mitotische Aurora B Kinase den Chromosomalen Passenger Complex (CPC). Das Survivin besitzt zudem eine grosse medizinische Relevanz und gilt als Tumor-assoziertes Antigen, da es zu den Top vier Transkripten zählt, die in einer Vielzahl unterschiedlicher Tumorentitäten überexprimiert werden, aber nicht in Normalgewebe. Diese Überexpression geht einher mit einer erhöhten Resistenz der Tumore gegenüber Chemo- und Strahlentherapie und macht Survivin zu einem idealen molekularen Ziel einer Krebstherapie mittels RNA Interferenz oder spezifischer pharmakologischer Inhibitoren. In einer Vielzahl an Studien, in denen das Survivin-Protein mittels RNAi, dominant negativer Proteine oder „knock out“ des Survivin Genes (BIRC5) aus geschalten wurde, konnte eine Aktivierung des Tumorsuppressorproteins p53, einem wichtigen Mediator der Zellzyklusregulation, beobachtet werden. Bis heute ist es weitgehend unklar, wie eine Aktivierung von p53 nach einem Survivin Verlust erfolgen kann. Zudem stellte sich die Frage, ob eine therapeutische Intervention, welche die Ausschaltung des Survivin-Proteins zum Ziel hat, neben Tumorzellen auch normales Gewebe schädigen kann. Da Tumorzellen sich von normalen Zellen insbesondere dadurch unterscheiden, dass sie Defekte in p53-Signalwegen bzw. eine inaktivierende p53-Mutation oder Gendeletion besitzen, wurde die Auswirkung einer Survivin-Depletion auf p53-positive Tumorzellen und auf isogene Tumorzellen mit ausgeschalteten p53 untersucht. Zu diesem Zweck wurde p53 mittels RNAi in U87-MG und MCF-7 Zellen ausgeschalten und stabile p53-defiziente Zellen generiert. Insgesamt standen für die Untersuchungen mit HCT116, MCF-7 und U87-MG drei Zelllinien unterschiedlichen Ursprungs sowie ihre isogenetischen, aber p53-defizienten Derivate zur Verfügung. Survivin wurde in diesen Zellen durch einen retroviralen Vektor, der für eine shRNA (small hairpin RNA) gegen Survivin codiert, ausgeschalten. Der Verlust an Survivin führte dabei in Wildtyp- als auch in den p53-defizienten Zellen zu Polyploidie, einer gestörten Zytokinese und multipolaren Spindeln. Zusätzlich konnte eine Induktion an p53/p21waf/cip sowie eine erhöhte, p53- und Caspase 3-unabhängige Apoptose festgestelt werden. Es konnte gezeigt werden, dass die Expression an p21waf/cip in Wildtyp-Zellen sowie seines potentiellen Targets Cyclin D1 mit der Zunahme an Polyploidie nach Survivin RNAi korreliert. Allerdings führt die Expression des Cdk Inhbibitors p21waf/cip nur zu einem transienten Arrest der Zellen, da polyploide, Survivin-depletierte Zellen BrdU inkorporierten und dadurch proliferierten. Zudem wird zum ersten Mal eine ATM/ATR abhängige „DNA Damage Response“ (DDR) in Survivin-depletierten p53-defzienten und Wildtyp Zellen beschrieben, die zu einer Phosphorylierung und Stabilisierung von p53 führt. Sky-Analysen bestätigten numerische als auch schwere chromosomale Aberrationen wie Translokationen und dizentrische Chromosomen in Survivin-depletierten polyploiden Zellen. Die Inhibierung der Aurora B Kinase, einem weiteren Bestandteil des CPC, mittels eines chemischen Inhibitors zeigt analog das Auftreten von DNA Schäden, eine p53/p21waf/cip Aktivierung sowie eine Zunahme an Polyploidie, wie sie für Survivin beschrieben wurde. Diese Erkenntnisse zeigen deutlich auf, dass die DNA Schäden und der p53/p21waf/cip-abhängige G1 Arrest nach dem „knock down“ von Survivin aufgrund einer gestörten Mitose hervorgerufen wurde, während eine IAP-Funktion des Survivins unter den gewählten experimentellen Bedingungen nicht festzustellen war.

La kinase Aurora B appartient au complexe des protéines passagères. Ce complexe est impliqué dans la régulation de la condensation, constriction et ségrégation des chromosomes, ainsi que dans la cytokinèse. Son rôle est donc crucial pour prévenir la formation de cellules cancéreuses. Cependant, l'étude de la fonction précise d'Aurora B dans chacune des phases de la mitose est limitée par la durée de celle-ci, et par le manque de spécificité des inhibiteurs existants. Nous avons donc développé une stratégie basée sur la photo-inactivation de la kinase par le chromophore Killer-Red, fusionné à la protéine. L'émission locale de ROS après irradiation, permet alors la photo-inactivation spécifique et temporelle d'Aurora B. La photo-inactivation d'Aurora B avant anaphase aboutie soit à un arrêt de la mitose, soit à la régression du sillon de division, provoquée par l'entrée en anaphase en présence de chromosomes retardés. La photo-inactivation d'Aurora B en début d'anaphase a pour conséquence la régression du sillon de division en cytokinèse ; apportant la première indication directe de l'implication d'Aurora B dans le fuseau mitotique en cytodiérèse. De façon surprenante, la photo-inactivation de la kinase au niveau du corps résiduel, après constriction du sillon de division, n'affecte pas l'abscission. La photo-inactivation d'Aurora B n'affecte pas la localisation des autres membres du complexe des protéines passagères, indiquant que la kinase n'est pas impliquée dans la dynamique du complexe. Les résultats obtenus montrent sans aucun doute l'implication d'Aurora B dans chacune des phases de la mitose, suggérant que la phosphorylation par Aurora B de ses substrats permet le controle de la division cellulaire. / Aurora B is a mitotic kinase involved in chromosome condensation and segregation as well as cytokinesis. Aurora B together with INCENP, Survivin, TD60 and Borealin constitute the chromosome passenger protein complex (CPC), which localizes to the inner centromeres all through metaphase, transfers to the spindle midzone in anaphase and to the midbody in cytokinesis. In order to dissect the mitotic kinase functions of Aurora B as well as its role as an integral part of the CPC in a temporal manner, we have used chromophore assisted light inactivation (CALI) approach. We have combined miRNA ablation of endogenous Aurora B with ectopic expression of miRNA resistant Aurora B fused to the photosensitizer Killer Red (AurB-KR) in HeLa cells. Irradiation at distinct phases of mitosis led to photobleaching of the Killer Red protein, accompanied by emission of reactive oxygen species (ROS) resulting in the photoinactivation of the fused Aurora B. Photoinactivation before anaphase led to either mitotic arrest or cleavage furrow regression due to entry into anaphase with chromosome bridges. CALI at early anaphase also led to cytokinesis failure underlying the role of Aurora B in central spindle function. Consistent with the effects of dominant negative dead-kinase Aurora B, upon CALI the localisation of Incenp, Survivin and Borealin was not affected. Importantly, photoinactivation of Aurora B-KR following cleavage furrow constriction at the midbody had no effect on the completion of abscission. These data, demonstrate unequivocally the distinct roles Aurora B exerts at each phase of mitosis and in particular suggest that Aurora B substrate phosphorylation from metaphase to anaphase is implicated in the spatio-temporal control of cell division and cytokinesis.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

La division cellulaire est un processus biologique universel nécessaire à la reproduction, au développement, à la survie cellulaire ainsi qu’à la réparation des tissus. Une ségrégation chromosomique exacte pendant la mitose est essentielle pour une répartition égale des chromosomes répliqués entre les cellules filles. Des erreurs dans la ségrégation des chromosomes mènent à une condition appelée aneuploïdie, définie par un nombre inadéquat de chromosomes dans une cellule. L’aneuploïdie est associée à une altération de la santé cellulaire, la tumorigénèse, des malformations congénitales et l'infertilité. Contre toute attente, les embryons préimplantatoires de mammifères, dont les humains, consistent souvent en un mélange de cellules euploïdes et de cellules aneuploïdes. Ce mosaïcisme est inexorablement causé par des erreurs dans la ségrégation des chromosomes au cours des divisions mitotiques suivant la fécondation et est associé à un potentiel de développement réduit lors des traitements de fertilité. Malgré sa découverte il y a 25 ans, les mécanismes qui sous-tendent l’apparition de l'aneuploïdie mosaïque dans les embryons préimplantatoires sont toujours méconnus. Pour explorer les causes et les conséquences des erreurs de ségrégation chromosomique, des approches d'imagerie de fine pointe ont été utilisées sur des embryons préimplantatoires murins. L'analyse de la dynamique de la ségrégation des chromosomes via l’imagerie de cellules vivantes a permis d’identifier les chromosomes retardataires, lors de l’anaphase, comme la forme la plus répandue des erreurs de ségrégation. Ces chromosomes retardataires entraînent fréquemment une encapsulation de chromosome unique dans une structure appelée micronoyau. D'autres expériences d'imagerie par immunofluorescence sur des cellules vivantes ou fixées ont révélé que les chromosomes des micronoyaux subissent des dommages importants à l'ADN et sont mal répartis de manière récurrente lors des divisions cellulaires subséquentes dans la phase préimplantatoire. D’autres approches ont aussi permis d’examiner l'efficacité du mécanisme de contrôle de l’assemblage du fuseau mitotique, (SAC pour Spindle Assembly Checkpoint). Les résultats obtenus attestent que le SAC fonctionne, cependant la signalisation liée au SAC n’est pas efficace et ne permet pas de différer l'anaphase, malgré la présence de chromosomes retardataires et ce indépendamment de la taille des cellules. Les résultats présentés révèlent aussi qu’une inhibition partielle d’une cible du SAC, le complexe de promotion de l'anaphase (APC/C), cause une mitose prolongée et une réduction des erreurs de ségrégation. En outre, les études présentées démontrent que la fonction déficiente du SAC pendant le développement préimplantatoire est la cause principale d’une forte incidence de chromosomes retardataires qui entraînent une mauvaise ségrégation chromosomique répétée et qui causent une aneuploïdie mosaïque dans l’embryon. De plus, ce travail fournit la preuve que la modulation pharmacologique de la signalisation SAC-APC/C permet d’éviter les erreurs de ségrégation des chromosomes dans les embryons précoces. En conclusion, ces résultats apportent de nouvelles perspectives sur les causes et la nature des erreurs de ségrégation chromosomique dans les embryons. De plus, ce travail apporte de nouvelles explications mécanistiques sur l'apparition du mosaïcisme dans les embryons ce qui aura des implications importantes dans la détection et la prévention thérapeutique potentielle de l'aneuploïdie mosaïque dans les embryons préimplantatoires. / Cell division is a universal biological process necessary for reproduction, development, cell survival and the maintenance and repair of tissues. Accurate chromosome segregation during mitosis is essential to ensure replicated chromosomes are partitioned equally into daughter cells. Errors in chromosome segregation often result in cells with abnormal numbers of chromosomes, a condition termed aneuploidy, which is associated with impaired cellular health, tumorigenesis, congenital defects and infertility. Counterintuitively, preimplantation embryos from many mammalian species, including humans, often consist of a mixture euploid and aneuploid cells. Such mosaic aneuploidy in embryos is inexorably caused by errors in chromosome segregation during mitotic divisions following fertilization and has been associated with reduced developmental potential in fertility treatments. However, ever since its discovery 25 years ago, how and why mosaic aneuploidy arises in the preimplantation embryo has remained elusive. To explore the causes and consequences of embryonic chromosome segregation errors, advanced imaging approaches were employed in the mouse preimplantation embryo. Live cell imaging analysis of chromosome segregation dynamics identified lagging anaphase chromosomes as the most prevalent form of chromosome mis-segregation in embryos. Lagging chromosomes frequently result in the encapsulation of single chromosomes into micronuclei, which occur in embryos in vitro and in vivo. Further live imaging and immunofluorescence experiments revealed chromosomes within micronuclei are subject to extensive DNA damage and centromeric identity loss, failing to assemble functional kinetochores and being recurrently mis-segregated during ensuing cell divisions in preimplantation development. To uncover the underlying causes for the increased propensity for chromosome mis-segregation in embryos, live imaging and loss-of-function approaches were used to examine the effectiveness of the mitotic safeguard mechanism, the Spindle Assembly Checkpoint (SAC). These studies demonstrated that the SAC normally functions to prevent segregation errors during preimplantation development but SAC signaling at misaligned chromosomes fails to delay anaphase. Moreover, SAC failure in embryos is most evident during mid-preimplantation development, independent of cell size. Partial inhibition of SAC target, the Anaphase Promoting Complex (APC/C), extended mitosis and reduced chromosome segregation errors in embryos. These studies have uncovered deficient SAC function during preimplantation development as a major cause for the high incidence of lagging chromosomes in embryos, which result in repeated mis-segregation of single chromosomes in a manner that necessarily causes mosaic aneuploidy. Additionally, this work provides proof-of-principle demonstration that pharmacological modulation of SAC-APC/C signalling can avert chromosome segregation errors in the early embryo. Altogether, these findings present new insights into the causes and nature of chromosome mis-segregation in embryos, providing novel mechanistic explanations for the occurrence of mosaicism that will have substantial implications for the detection and potential therapeutic prevention of aneuploidy in preimplantation embryos.

The mitotic spindle is a bipolar microtubule-based structure that mediates proper cell division by segregating the genetic material and by positioning the cytokinesis cleavage plane. Spindle assembly is a complex process, involving the modulation of microtubule dynamics, microtubule focusing at spindle poles and the formation of stable microtubule attachments to chromosomes. The cellular events leading to spindle formation are highly regulated, and mitotic kinases have been implicated in many aspects of this process. However, little is known about their counteracting phosphatases. A screen for genes required for early embryonic cell divisions in C. elegans identified rsa-1 (for regulator of spindle assembly 1), a putative Protein Phosphatase 2A (PP2A) regulatory subunit whose silencing causes defects in spindle formation. Upon rsa-1(RNAi), spindle poles collapse onto each other and microtubule amounts are strongly reduced. My thesis work demonstrates that RSA-1 indeed functions as a PP2A regulatory subunit. RSA-1 associates with the PP2A enzyme and recruits it to centrosomes. The centrosome binding of PP2A furthermore requires the new protein RSA-2 as well as the core centrosomal protein SPD-5 and is based on a hierarchical protein-protein interaction pathway. When PP2A is lacking at centrosomes after rsa-1(RNAi), the centrosomal amounts of two critical mitotic effectors, the microtubule destabilizer KLP-7 and the kinetochore microtubule stabilizer TPXL-1, are altered. KLP-7 is increased, which may account for the reduction of microtubule outgrowth from centrosomes in rsa-1(RNAi) embryos. TPXL-1 is lost from centrosomes, which may explain why spindle poles collapse in the absence of RSA-1. TPXL-1 physically associates with RSA-1 and RSA-2, suggesting that it is a direct target of PP2A. In summary, this work defines the role of a novel PP2A complex in mitotic spindle assembly and suggests a model for how different microtubule re-organization steps might be coordinated during spindle formation.