Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

La voie de signalisation Notch contient quatre récepteurs transmembranaires qui sont activés de façon ligand dépendant. Les contacts cellulaires permettent l'activation des récepteurs et la relâche entre autres du fragment actif Notch 1 clivé (NIC1). Ensuite, il transloque au noyau pour s'associer à son co-activateur, CSL afin de permettre la formation d'un complexe transcriptionnel actif. La signalisation Notch est régulée à divers niveaux cellulaires. La voie Notch est très engagée durant le développement du pancréas. De plus, différents modèles ont été générés afin de mieux comprendre l'implication de la voie Notch dans le phénomène de la prolifération des cellules pancréatiques Ainsi, la voie Notch est active dans le pancréas immature et est réprimée dans le tissu adulte. La signalisation Notch est très importante pendant la carcinogenèse pancréatique. Il a été démontré que l'inhibition de la voie Notch induit une répression de la croissance cellulaire et bloque la tumorigenèse pancréatique. Une autre voie de signalisation très impliquée dans le cancer du pancréas est la voie KRas qui est mutée dans 90% des adénocarcinomes. Une coopération entre les voies Notch et KRas a été reportée dans différents types de carcinogenèse. De plus, il a été suggéré que la collaboration Notch et KRas passe spécifiquement par la cascade Mek/Erk en aval de KRas. De plus, l'inhibition de la voie Mek/Erk mène à l'inhibition de l'expression d'Hes1, une cible de la voie Notch. Ainsi, nous avions supposé que la cascade KRas/Raf/Mek/Erk favorise la transcription induite par la voie de signalisation Notch. Pour y répondre, il était donc important de déterminer l'impact de la voie Mek/Erk sur l'expression de Notch 1 clivé (NIC1) et de clarifier l'effet de cette voie sur son activité transcriptionnelle. Nous avons observé que la suractivation de la voie Mek/Erk induit un retard de migration de la protéine NIC1 qui est complètement aboli par l'inhibition de la voie Mek/Erk. Des études plus approfondies ont révélé que ces formes de plus haut poids moléculaires étaient des formes phosphorylées de NIC 1 qui pouvaient être associées directement à l'action des Erk 1. Nous avons découvert que ces modifications permettent de réguler l'activité transcriptionnelle de Notch. Ainsi, la phosphorylation de NIC1 induite par la voie Mek/Erk est associée à une augmentation de l'activité transcriptionnelle dépendante de Notch qui mène à l'expression du gène cible, Hes1. Il semble aussi que le domaine PEST de NIC 1 est essentiel afin de permettre les phosphorylations Erk-dépendantes. D'autres effets spécifiques de la voie Mek/Erk sur Notch restent à être découverts. Enfin, le potentiel de cette collaboration entre les voies Notch et KRas/Raf/Mek/Erk pourrait être impliquée dans divers processus cellulaires.

No description available.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

Universiẗat, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

Thèse (M.Sc.)--Université Laval, 2003. / Titre de l'écran-titre (visionné le 26 mars 2004). Bibliogr.

We identified four metabolic genes as direct targets of Notch signaling pathway both in vitro and in vivo and investigated the hypothesis that Notch directed metabolic changes support the growth of the imaginal wing dics. Vice versa, we observed the influence of metabolic changes on the activity of Notch signaling pathway and we identified Sirt1 as a metabolic sensor for the Notch pathway that helps to elicit an efficient response to Notch signal, in a metabolism sensitive manner.