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The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase to anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity. With that aim and considering the limitations of conventional 2D cell culture in mimicking the tumor environment and predicting drug responses in animal models and humans, we also established 3D organoid cultures of PCa cells and immortalized human prostate epithelial cells (hPrEC) in Matrigel. This allowed us to explore cell to extracellular matrix (ECM) interactions. PC3 cells initially form round organoids in Matrigel, followed by an invasive switch to where cell protrusions invade the surrounding ECM. Strikingly, B55α reconstitution, dramatically suppressed rupture of the basement lamina and ECM invasion, while proliferation appeared not affected. Tracking organoid growth at defined time points or using live imaging, shows that protrusions in PC3 organoids are very dynamic and resemble invadopodia. Interestingly, reconstitution of B55α in PC3 organoids just prior the invasive switch results in reduction of invasive leads and those protrusions that appear to initiate keep forming and collapsing, with most organoids remain round. Our previous phosphoproteomics data in 2D culture suggests that cell-to-ECM signaling is likely altered with B55α reconstitution, identifying potential B55α/PP2A substrates among key mediators of integrin signaling. In sum, reconstitution of B55α suppresses invasion in PC3 organoids, possibly by regulating potential B55α substrates in focal adhesion signaling, such as Paxillin and/or Talin. Alternatively, centrosomal defects due to dephosphorylation of B55α substrates (e.g. HAUS6, NEDD1) may cause microtubule defects that preclude invasion. Further studies are required to identify the mechanism. Moreover, because our studies presented above are based on prostate cancer cell lines with undefined genetic alterations, we have immortalized primary human PrEC by a novel approach to generate a cell model to study cooperation of PPP2R2A loss with step-wise introduction of specific oncogenes and/or tumor suppressor gene alterations in transformation, tumorigenicity and invasion. Our newly develop method combines expression of hTERT, which appears insufficient for immortalization of hPrEC with CDKN2A knockout, which we predicted will prevent stress-induced replicative senescence. We have obtained two independent immortalized clones (hPrEC-T-ΔN2A) using this method and confirmed their identity using PCR and western blot analyses. Although cytogenetic analysis showed these two clones are of mixed population with minor alterations in karyotype, 4 out of 11 cells examined in clone 1 appear completely normal. We also find that the clones exit the cell cycle upon contact inhibition and induce p53 expression when treated with flavopiridol, further supporting that hPrEC-T-ΔN2A clones exhibit the features of normal cells. Characterization in 3D culture reveals that the clones are likely of basal epithelial origin. Finally, soft agar and clonogenic assays show hPrEC-T-ΔN2A clones are highly proliferative but not transformed. We are using these cell models to dissect the role of PPP2R2A depletion in step-wise transformation of immortalized PrEC and hope to develop a defined 3D organoid system to study invasion, which could also be suitable for drug screens. Altogether our work has very significantly advanced our understanding of B55α in suppressing transformation in prostate cancer cells and has developed novel tools for further mechanistic characterization of PPP2R2A haploinsufficiency and the development of potential pharmacologic therapeutic agents. / Biomedical Sciences

Tetraploidy – the presence of four copies of the haploid chromosome complement – is common in cancer. There is evidence that ~40% of tumors pass through a tetraploid stage at some point during their development, and tetraploid cells injected in mice are more tumorigenic than their diploid counterparts. However, the reason for this increased tumorigenicity of tetraploid cells is not well established. Most routes by which cells may become tetraploid also confer cells with double the number of centrosomes, the small membraneless organelle that organizes the cell's microtubule cytoskeleton and mitotic spindle apparatus. Centrosome number homeostasis is crucial for health, and recent studies have shown inducing extra centrosomes in cells can induce tumor formation in mice. This has led some researchers to propose that the extra centrosomes that arise together with tetraploidy may be the reason that tetraploid cells are more tumorigenic. However, several anecdotal reports have found that tetraploid clones generated and grown in vitro appear to lose their extra centrosomes. Here, I investigate the population dynamics of the loss of extra centrosomes in newly formed tetraploid cells generated via cytokinesis failure. I uncover the mechanism driving the process and build a mathematical model that captures the experimentally observed dynamics. Next, I investigate karyotypic heterogeneity in newly formed tetraploid cells and their counterparts that are grown for 12 days under standard culture conditions and find that karyotypic heterogeneity has increased after 12 days of growth after tetraploidization. The day 12 'evolved' population with increased heterogeneity formed larger colonies in soft agar than newly formed tetraploid cells or diploid parental precursors and karyotype analysis of the largest soft agar colonies revealed recurrent aneuploidies shared by a subset of colonies. Finally, I investigate the effects of different culture conditions - meant to mimic various conditions in the tumor microenvironment - on the evolution of centrosome and chromosome number in newly formed tetraploid cells and identify a small subset of conditions that altered centrosome homeostasis or the fitness of tetraploid cells. / Doctor of Philosophy / The genetic information in cancer cells is often drastically altered compared to normal cells in the body. As one important example of this, the number and structure of chromosomes - the DNA structures that hold the genetic information - is often abnormal in cancer cells. Abnormal chromosome number is closely linked with cancer development, but the details of why this leads to more cancer are not clear. One important kind of chromosome number change is when a cell undergoes incomplete cell division, and the resulting cell acquires double the number of chromosomes compared to a normal cell (known as tetraploidy). Tetraploidy occurs in close to 40% of cancers and is linked with the most aggressive cases. The abnormal cell divisions that cause tetraploidy also lead to other cellular changes. One important change is that tetraploid cells also acquire double the number of the structures that organizes cell division (centrosomes). The centrosome organizes the mitotic spindle, the major apparatus that is responsible for equally distributing chromosomes to two daughter cells during the cell division process. Extra centrosomes in cells are closely linked with cancer and can lead to additionally chromosome number changes. Researchers have believed that the extra centrosomes that are acquired with doubled chromosome number may be the major reason that tetraploid cells are linked to more aggressive cancer. However, recent studies have suggested that tetraploid cells may lose their extra centrosomes, calling into question the details of the relationship between tetraploidy and tumor formation. Here, I use human cells grown in culture to understand how extra centrosomes are lost from tetraploid cells. I find that extra centrosomes in newly formed tetraploid cells promote abnormal 'multipolar' cell divisions, in which chromosomes are segregated unevenly to three or more partitions. Such divisions are often fatal to daughter cells. In some cases, the extra centrosomes can cluster to form bipolar spindles that segregate chromosomes into two equal partitions (as is normal). When forming bipolar spindles, extra centrosomes can cluster asymmetrically (three centrosomes at one pole, one at the other) or symmetrically (two centrosomes at each pole). Tetraploid cells with a normal number of centrosomes emerge when extra centrosomes cluster asymmetrically in a bipolar spindle, yielding one tetraploid daughter cell with a normal number of centrosomes. Such cells have a fitness advantage over cells with extra centrosomes, which over time are very likely to undergo fatal multipolar divisions. Thus, cells with a normal centrosome number 'take over' the population. Next, I investigate the cancer-like properties of tetraploid cells without their extra centrosomes and find that they display increased tumor-like behavior even in the absence of extra centrosomes. Finally, I investigate whether changing the conditions in which cells are grown (in ways meant to mimic different conditions that may be experienced in the body) affects whether tetraploid cells lose their extra centrosomes. We identify a small number of conditions that do influence loss of extra centrosomes. Together, these studies illuminate important details of the relationship between tetraploidy and tumor formation. This work lays the foundation to further explore and understand the relative roles of tetraploidy, extra centrosomes, and tissue environment in cancer.

This dissertation unravels intricate biological dynamics in three distinct biological systems as the following. These studies combine mathematical models with experimental data to enhance our understanding of these complex processes. 1. Bipolar Spindle Assembly: Mitosis relies on the formation of a bipolar mitotic spindle, which ensures an even distribution of duplicated chromosomes to daughter cells. We address the issue of how the spindle can robustly recover bipolarity from the irregular forms caused by centrosome defects/perturbations. By developing a biophysical model based on experimental data, we uncover the mechanisms that guide the separation and/or clustering of centrosomes. Our model identifies key biophysical factors that play a critical role in achieving robust spindle bipolarization, when centrosomes initially organize a monopolar or multipolar spindle. These factors encompass force fluctuations between centrosomes, balance between repulsive and attractive inter-centrosomal forces, centrosome exclusion from the cell center, proper cell size and geometry, and limitation of the centrosome number. 2. Chromosome Oscillation: During mitotic metaphase, chromosomes align at the spindle equator in preparation for segregation, and form the metaphase plate. However, these chromosomes are not static; they exhibit continuous oscillations around the spindle equator. Notably, either increasing or decreasing centromeric stiffness in PtK1 cells can lead to prolonged metaphase chromosome oscillations. To understand this biphasic relationship, we employ a force-balance model to reveal how oscillation arises in the spindle, and how the amplitude and period of chromosome oscillations depend on the biological properties of spindle components, including centromeric stiffness. 3. Pattern Formation in Bacterial-Phage Systems: The coexistence of bacteriophages (phages) and their host bacteria is essential for maintaining microbial communities. In resource-limited environments, mobile bacteria actively move toward nutrient-rich areas, while phages, lacking mobility, infect these motile bacterial hosts and disperse spatially through them. We utilize a combination of experimental methods and mathematical modeling to explore the coexistence and co-propagation of lytic phages and their mobile host bacteria. Our mathematical model highlights the role of local nutrient depletion in shaping a sector-shaped lysis pattern in the 2D phage-bacteria system. Our model further shows that this pattern, characterized by straight radial boundaries, is a distinctive indicator of extended coexistence and co-propagation of bacteria and phages. Such patterns rely on a delicate balance among the intrinsic biological characteristics of phages and bacteria, which have likely arisen from the coevolution of cognate pairs of phages and bacteria. / Doctor of Philosophy / Mathematical modeling is a powerful tool for studying intricate biological dynamics, as modeling can provide a comprehensive and coherent picture about the system of interest that facilitates our understanding, and can provide ways to probe the system that are otherwise impossible through experiments. This dissertation includes three studies of biological dynamics using mathematical modeling: 1. Bipolar Spindle Assembly: Mitotic spindle is a bipolar subcellular structure that self-assembles during cell division. The spindle ensures an even distribution of duplicated chromosomes into two daughter cells. Certain perturbations can cause the spindle to assemble abnormally with one pole or more than two poles, which would cause the daughter cells to inherit incorrect number of chromosomes and die from the error. However, the cell is surprisingly good at correcting these spindle abnormalities and recovering the bipolar spindle. Here we build a model to explore how the cell achieves such recoveries and preferentially form a bipolar spindle to rescue itself. 2. Chromosome Oscillation: In mitotic metaphase, chromosomes are aligned at the spindle equator before they segregate. Interestingly, unlike the cartoon images in textbooks, the aligned chromosomes often move rhythmically around the spindle equator. We used a mathematical model to unravel how the chromosome oscillation arises and how it depends on the biological properties of the spindle components, such as stiffness of the centromere, the structure that connects the two halves of duplicated chromosomes. 3. Pattern Formation in Bacterial-Phage System: Phages are viruses that hijack their host bacteria for proliferation and spreading. In this study we developed a mathematical model to elucidate a common lysis pattern that forms when expanding host bacterial colony encounters phages. Interestingly, our model revealed that such a lysis pattern is a telltale sign that the bacterium-phage pair have achieved a delicate balance between each other and are capable of spreading together over a long period of time.

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le juste positionnement du noyau durant la formation musculaire semble important pour la fonction musculaire et des défauts ont été associés à plusieurs maladies musculaires. Le positionnement nucléaire dépend des microtubules (MTs), qui sont réorganisés depuis le centrosome, dans les myoblastes proliférants, vers l’enveloppe nucléaire (EN), dans les myotubes différenciés. Cette réorganisation s'accompagne de la redistribution des protéines centrosomales vers l’EN qui adopte le rôle de centre organisateur des microtubules (MTOC) lors de la différenciation myogénique. Néanmoins, les mécanismes sous-jacents restent inconnus. Ici, nous avons identifié les protéines Nesprin-1 et Sun1/2, localisées respectivement à la membrane nucléaire externe et interne, comme impliquées dans le recrutement de la fonction MTOC à l’EN. Les cellules déficientes en Nesprin-1 ou Sun1/2 ont montré une localisation altérée des protéines centrosomales dans le cytoplasme et l’absence des MTs depuis l’EN. De plus, Nesprin-1alpha, une myo-isoforme de Nesprin-1, s’associait aux protéines centrosomales Akap450, Pericentrin et Pcm1 dans les myotubes C2C12 et était suffisante pour corriger les défauts observés dans des cellules déplétées en Nesprin-1. Parmi les protéines centrosomales recrutées par Nesprin-1alpha, seule Akap450 semble nécessaire à la nucléation des MTs à l’EN. Ce processus, médié par Akap450 et Nesprin-1alpha, s’est avéré important pour le positionnement nucléaire lors du développement des myotubes. Ces résultats renforcent notre compréhension sur le lien causal entre des défauts lors de la formation du MTOC à l’EN et des défauts de positionnement nucléaire dans les dystrophies musculaires. / The accurate position of the nucleus during skeletal muscle formation seems to be important for muscle function, and defects have been associated with numerous muscle diseases. Nuclear positioning requires microtubules (MTs) which are reorganized from the centrosome in proliferating myoblasts to the nuclear envelope (NE) in differentiated myotubes. This dramatic MT reorganization is accompanied by a redistribution of proteins from the centrosome to the NE which thus takes over the function as a microtubule-organizing center (MTOC) during myogenic differentiation. However, the underlying mechanisms are still unknown. Here, we identified Nesprin-1 and Sun1/2, outer and inner nuclear membrane proteins, respectively, to be involved in the recruitment of MTOC function to the NE. Nesprin-1 or Sun1/2 deficient cells displayed mislocalization of centrosomal proteins to the cytoplasm and failed to regrow MTs from the NE. Moreover, the muscle-specific isoform of Nesprin-1, namely Nesprin-1alpha, was shown to be highly associated with the centrosomal proteins Akap450, Pericentrin and Pcm1 in C2C12 myotubes and to be sufficient to rescue the observed defects in Nesprin-1 depleted cells. Among the centrosomal proteins localizing at the NE during myogenic differentiation, solely Akap450 seemed to be required for MT nucleation. Akap450-Nesprin-1alpha-mediated MT nucleation from the NE was demonstrated to play an important role in nuclear positioning during myotube formation. These findings strengthen our understanding on how defects in MTOC formation at the NE can link to nuclear positioning defects in muscular dystrophies.

Les cellules déficientes pour la recombinaison homologue (RH) présentent un ralentissement des fourches de réplication, un nombre aberrant de centrosomes et une aneuploïdie même en absence de stress exogène (Bertrand P 2003, Daboussi F 2005, 2008, Deng 1999, 2002, Griffin 2000, Kraakman-van der Zwet 2002). De plus, la fréquence des mitoses présentant des chromosomes surnuméraires est plus élevée dans ces cellules.L’ensemble des ces résultats suggéraient que le ralentissement des fourches de réplication dans les cellules déficientes pour la RH pourrait avoir un impact direct sur la formation de centrosomes surnuméraires. De plus, nous voulions savoir si cela pouvait également influencer la ségrégation des chromosomes au cours de la mitose. Les résultats que nous avons obtenus sont rassemblés dans l’article intitulé : “Homologous Recombination protects against mitotic defects and unbalanced chromosome segregation caused by spontaneous replication stress”.Le traitement des cellules compétentes pour la RH avec 5µM d’hydroxyurée (HU), un inhibiteur de l’enzyme de synthèse des dNTPs, induit un ralentissement des fourches de réplication parfaitement comparable à celui observé dans les cellules déficientes pour la RH. Après traitement à l’HU des cellules compétentes pour la RH, la fréquence de mitoses présentant des chromosomes surnuméraires augmente et devient similaire à la fréquence de mitoses avec des chromosomes surnuméraires pour les cellules déficientes pour la HR non traitées à l’HU. Nous avons mesuré l’impact de l’HU sur l’apparition des ponts anaphasiques et sur des défauts de ségrégation des chromosomes lors de la mitose. En l’absence de traitement, nous observons une fréquence plus élevée de ponts anaphasiques et de défauts de ségrégation dans des cellules déficientes pour la RH. Des traitements avec 5µM d’HU augmentent la fréquence des ponts anaphasiques et des erreurs de ségrégation dans les cellules compétentes pour la RH, pour atteindre un niveau comparable aux cellules déficientes pour la RH. Ainsi, une altération de la dynamique de réplication consécutive à une déficience de RH ou à un traitment avec de faibles doses d’HU induit des défauts au cours de la mitose. Un lien direct entre une dynamique de réplication anormale et l’apparition d’un nombre aberrant de centrosomes pourrait être la persistance en mitose d’ADN non répliqué ou endommagé.  Comme l’ADN non répliqué ou les fourches bloquées induisent la formation d’ADN simple brin couvert par RPA, nous avons compté le nombre de cellules en G2/M présentant des foyers de RPA, et observé que la fraction de cellules ayant plus de 5 foyers de RPA augmente dans des cellules déficientes pour Brca2. En conclusion, nous proposons un lien direct entre des altérations de la cinétique de réplication, l’apparition de centrosomes surnuméraires et des défauts de ségrégation des chromosomes en mitose dans les cellules déficientes pour la RH même non soumises à un stress exogène. L’utilisation de faibles doses d’HU dans des cellules compétentes pour la RH mime les phénotypes observés dans les cellules déficientes pour la RH et confirme notre modèle.Nous avons également cherché à comprendre les causes du ralentissement des fourches de réplication observé dans les cellules déficientes pour la RH. Il est ainsi possible que les arrêts de fourches spontanés soient une conséquence d’un stress oxydatif endogène. Dans les cellules compétentes pour la RH, le redémarrage des fourches bloquées est possible et assure une progression normale de la réplication de l’ADN. Ceci favorise une ségrégation équilibrée des chromosomes, le maintien de la diploïdie et la stabilité du génome. Dans des cellules déficientes pour la RH, les blocages de fourches devraient être délétères puisque les principaux mécanismes de redémarrage des fourches ne sont pas fonctionnels. De plus, l’arrêt prolongé des fourches ainsi que les cassures double brin générées par l’effondrement des fourches devraient activer des voies de signalisation. Nous avons néanmoins observé que les cellules ne sont pas bloquées dans le cycle cellulaire, ce qui suggère qu’un seuil supérieur de dommages doive être atteint pour induire l’arrêt du cycle. Les stress endogènes ne semblent donc pas suffisamment élevés pour atteindre ce seuil : même si l’ensemble des fourches parcourant le génome sont ralenties, l’activation d’origines cryptiques permet de compenser et ainsi de maintenir la progression dans le cycle. Mais puisque les cellules ne sont pas arrêtées dans le cycle, des fourches bloquées, de l’ADN endommagé ou non répliqué pourraient persister jusqu’à la transition G2/M et in fine perturber le déroulement de la mitose. Des centrosomes multipolaires provoquent la formation de fuseaux multipolaires et favorisent la ségrégation déséquilibrée des chromosomes, entraînant l’aneuploïdie, la déstabilisation du génome et le développement de cancers. / HR deficient cells show slow replication kinetics, aberrant centrosome number and aneuploidy even in the absence of any exogenous stress (Bertrand P 2003, Daboussi F 2005, 2008, Deng 1999, 2002, Griffin 2000, Kraakman-van der Zwet 2002). Frequency of mitosis with extra centrosomes is elevated and replication kinetics decreased in HR deficient compared to HR proficient cells, in the absence of exogenous stress. Thus the question arose, if replication slowing down in HR deficient cells has direct impact on the appearance of supernumerary centrosomes. Furthermore we wanted to know if this might directly impact chromosome segregation. The results we gained are brought together in the paper “Homologous recombination suppression causes spontaneous mitotic alterations through endogenous replication stress”. By treating our HR proficient cells with 5µM HU we found the perfect concentration to mimic replication dynamics of HR deficient cells in an HR proficient background. This concentration was applied to HR proficient cells. After HU treatment the frequency of mitosis with extra centrosomes was elevated in HR proficient cells. Now they showed the same frequency of mitosis with extra centrosomes, than unchallenged HR deficient cells. We measured the impact of HU treatment on occurrence of anaphase bridges or aberrant mitotic segregation. In the absence of treatment higher frequency of anaphase bridges and aberrant mitotic segregation was detected for HR deficient cells. With 5µM HU the frequency of anaphase bridges and aberrant mitosis could be elevated in HR proficient cells. Now they showed aberrant mitotic features with the same frequency than unchallenged HR deficient cells. A direct link between abnormal replication kinetics and aberrant centrosomes might be unreplicated or damaged DNA, that enter mitosis. Unreplicated or blocked DNA might harbour ss DNA bound RPA. Thus we counted G2/M cells with RPA foci. Indeed the fraction of cells that harbour more than 5 RPA foci was elevated in Brca2 deficient in comparison to Brca2 proficient cells. In conclusion we propose a direct link between delayed replication, supernumerary centrosomes and aberrant chromosome segregation in unchallenged HR deficient cells. If we mimicked replication kinetics of HR deficient cells in an HR proficient background, we also mimicked frequency of mitosis with extra centrosome number and aberrant chromosome segregation. Furthermore we investigated the causes of replication slowing down in HR deficient cells. It can be hypothesized that endogenous oxidative stress is implicated in spontaneous fork arrest. In HR proficient cells, reactivation of stalled replication forks and therefore normal replication progression is assured. This favours balanced chromosome segregation, diploidy and genetic stability.In HR deficient cells, replication fork blockage might be detrimental as the main restart mechanism for blocked forks is absent. Prolonged fork blockage or DSB’s arising by fork collapse or resolution of blocked replication forks might activate signalling pathways. However cells are not arrested in cell cycle progression, suggesting that a threshold should be reached to activate cell cycle arrest. Endogenous stress is not sufficient high to reach this threshold. Replication is genome wide slowed down. In this context, the activation of cryptic origins compensates at least partly the slow replication velocity. However, because cells were not arrested in cell cycle progression, some blocked replication forks and damaged or unreplicated DNA regions might persist until G2/M phase and affect centrosome duplication and chromosome segregation. Multipolar centrosomes cause multipolar spindles and favour unbalanced chromosome segregation leading to aneuploidy, genetic instability and cancer development.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal