Die Entwicklung von Fernmetastasen ist die Haupttodesursache bei Tumorerkrankten und der entscheidende klinisch relevante Schritt während der Tumorprogression. Die Seed and Soil Theorie von Stephen Paget besagt, dass verschiedene Tumorzellen spezifische Zielorgane während der Metatasierung bevorzugen. Während das häufigste Target der kolorektalen Karzinome die Leber ist, hat der triple-negative molekulare Subtyp von Brustkrebs die Neigung, in das Gehirn zu metastasieren. Interessanterweise spielen sowohl deregulierte EGFR (Epithelial growth factor receptor) als auch WNT Signalwege in diesen beiden Entitäten eine entscheidende Rolle. Das Ziel der Arbeit ist, die Rolle der beiden Signalwege in soliden Tumorzelllinien in Bezug auf die Tumorprogression und Kolonisation zu untersuchen. Im Rahmen der molekularen Charakterisierung der Zelllinien zeigten sich die Mammakarzinomzelllinie 410.4 und die kolorektale Tumorzelllinie CMT-93 als passende Modellsysteme für unsere Fragestellung. Anschließend wurden der EGFR und der WNT Signalweg in diesen Zellen im Sinne von gain of function und loss of function moduliert und die Auswirkungen auf Aspekte der Tumorprogression analysiert. In CMT-93 Zellen wurde ein EGFR Knockdown etabliert. Während der Knockdown keinen Einfluss auf die Proliferation hat, vermindert er die Invasion der Zellen. Somit konnte dem effizienten Knockdown eine funktionelle Wirksamkeit zugeschrieben werden. Eine EGFR Überexpression konnte sowohl in 410.4 als auch in CMT-93 Zellen etabliert werden. Die Analyse der jeweiligen Signalkaskadenweiterleitung ergab zwar Änderungen und somit eine funktionelle Relevanz, dies blieb jedoch ohne Auswirkungen auf das Invasionspotential der Zelllinien. Ein Knockdown von β-Catenin konnte in 410.4 zwar etabliert werden, blieb jedoch ohne funktionelle Auswirkungen. Eine stabile Überexpression von β-Catenin war nicht erfolgreich, da dies offenbar mit der Viabilität der Zellen interferierte. Die Relevanz des β-Catenin-abhängigen WNT Signalwegs in den beiden gewählten Zelllinien konnte somit nicht abschließend geklärt werden. Des Weiteren wurde die Bedeutung des nicht-kanonischen WNT Signalwegs via ROR2 und WNT11 untersucht. Dabei ergab sich, dass die Überexpression von WNT11 und ROR2 in 410.4 Zellen deren Invasion durch einen RHOA-abhängigen Mechnismus steigert und einen Einfluss auf den PI3K Signalweg hat. Es ist anzunehmen, dass WNT11 als downstream Target über ROR2 induziert wird und über einen positiven Feedback-Loop via ROR2 eine autokrine Stimulation ausübt.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

No description available.

<p>Brain tumors are an incredibly diverse group of neoplasms, as evidenced by their varied locations in the brain, histological characteristics, and genetic alterations. Brain tumor heterogeneity can be potentially explained by distinct oncogenic events or cells-of-origin, or by region-specific intrinsic or extrinsic factors. Brainstem Glioma (BSG) is a particularly deadly brain tumor, afflicting 200-300 children in the United States each year. High-grade BSG (also known as Diffuse Intrinsic Pontine Glioma, DIPG) cannot be surgically removed, and the standard treatment of radiation therapy provides only temporary relief from symptoms. The past 5 years has witnessed a dramatic increase in knowledge regarding the biological basis of this disease along with the realization that BSG is distinct from other more common types of glioma, such as cerebral cortex glioma (CG). It was the goal of this study to investigate the regional differences in gliomas arising in the brainstem versus the cerebral cortex, using mice as a model system, and to begin to understand the contributions of the various possible sources of heterogeneity.</p><p> </p><p>In doing so, we have uncovered region-specific gene expression patterns in these two types of pediatric gliomas that are apparent even when the initiating genetic alterations and cell-of-origin are kept constant. Focusing on the <italic>paired box 3</italic> (Pax3) gene, which is expressed at higher levels in BSG than CG, we have found that Pax3 expression not only characterizes mouse BSGs driven by PDGF signaling, Ink4aARF-loss, p53-loss, and H3.3-K27M expression, but also identifies a novel subset of human BSGs that are associated with <italic>PDGFRA</italic> alterations and wild type <italic>ACVR1</italic> and that commonly harbor <italic>TP53</italic> alterations and the H3.3-K27M mutation. </p><p>As Pax3 plays a pro-tumorigenic role in other types of cancer, we hypothesized that Pax3 expression contributes to the brainstem gliomagenesis process as well. By utilizing mouse models, we found that Pax3 inhibits apoptosis and promotes proliferation of Nestin-expressing brainstem progenitor cells <italic>in vitro</italic> and enhances PDGF-B-driven BSG <italic>in vivo</italic>. Furthermore, we speculate that Pax3 expression may be a marker for Wnt pathway activation in BSG, which is targetable via pharmacologic agents. Indeed, a subset of Wnt inhibitors tested effectively slowed the growth of BSG cells <italic>in vitro</italic>, however cross talk with the Shh pathway might indicate that dual Wnt and Shh inhibition is necessary.</p><p>In addition, the regional expression pattern of Pax3 in gliomas correlates with its expression in normal murine brain development, leading us to hypothesize that Pax3 progenitor cells in the neonatal brainstem can serve as a cell-of-origin for BSG. We discovered that targeting Pax3 progenitors with PDGF-B overexpression and Ink4aARF- or p53-loss induces high-grade BSG that physiologically resemble the human disease. This novel and distinct model of BSG may be utilized in the future for preclinical studies.</p><p>The identification of Pax3 as a regional marker of mouse and human BSG has led to the discovery of a novel subset of the human disease, the identification of a novel oncogene contributing to pathogenesis, and the characterization of a novel cell-of-origin with the potential to give rise to the disease. This information contributes significantly to the current understanding of the mechanisms and cellular origins of BSG, and will hopefully instruct future investigations into how to better treat this disease.</p> / Dissertation

Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Shp-1 (Src homology 2 domain-containing phosphatase 1) est une tyrosine phosphatase retrouvée principalement chez les cellules hématopoïétiques, mais également chez les cellules épithéliales. Bien que Shp-1 soit reconnue comme étant un régulateur négatif de plusieurs voies de signalisation intracellulaire chez les cellules hématopoïétiques, son rôle dans les cellules épithéliales a été jusqu’ici très peu étudié. Afin de mieux comprendre son rôle dans les cellules épithéliales intestinales, nous avons généré un modèle murin de délétion conditionnelle de Shp-1 spécifiquement dans l’épithélium intestinal (Shp-1CEI-KO). De manière intéressante, dès l’âge de 6 semaines, les souris expérimentales présentent une intestinalomégalie associée à une légère augmentation de la prolifération cryptale. La taille des cellules épithéliales est également augmentée, suggérant de l’hypertrophie cellulaire chez les souris invalidées pour Shp-1. Parallèlement, la voie de signalisation PI3K/Akt/mTor est activée dans l’épithélium des souris mutantes. Nous avons également noté une production accrue de cellules caliciformes et de leurs précurseures, les cellules intermédiaires, en absence de Shp-1. Par contre, la maturation des cellules de Paneth semble grandement compromise vu la baisse importante d’expression du lysozyme et des RegIIIβ et RegIIIγ, de même que la faible densité de leurs granules de sécrétion. La comparaison du phénotype intestinal des souris Shp-1CEI-KO avec celui des souris PtenCEI-KO suggère que l’hyperactivation de la voie PI3K/Akt/mTor est responsable en partie des altérations phénotypiques observées chez la souris invalidée pour Shp-1. En conclusion, nos résultats montrent que la tyrosine phosphatase Shp-1 est un régulateur important de l’homéostasie de l’épithélium intestinal en contrôlant notamment la croissance cellulaire et la différenciation des cellules de la lignée sécrétrice.

Wnt signalling belongs to conserved pathways and mediates cell fate decision, development, regeneration and adult tissue homeostasis. Disruption or misregulation of Wnt signalling pathway often leads to disease. Wnt proteins are hydrophobic glycoproteins which need a special receptor for transport from Golgi Apparatus to cell surface, which is called MIG-14 in Caenorhabditis elegans and Wntless (Wls) in mammals. In this study, I focus on understanding mechanisms that regulate MTM-6 protein activity. MTM-6, a lipid phosphatase associated with endosomal membrane, has been recently identified as a regulator of MIG-14/Wls trafficking in Caenorhabditis elegans. Silencing of mtm-6 leads to misregulation of some Wnt-directed processes, such as migration of Q neuroblasts progeny. This study reports identification of novel mtm-6 genetic interactors that have been found to influence migration of Q neuroblasts progeny through Wnt signalling. New knowledge about mtm-6 genetic interactions bring us near to understanding of Wnt signalling regulation. Keywords: Caenorhabditis elegans, MTM-6, SEL-5, Wntless, Wnt, endosomes, phosphoinositides, retromer

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.

Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.