Hedgehog-signalling in malignant gliomas The Hedgehog signalling pathway is important for the development of the central nervous system. On the other hand, aberrant induction is observed in different tumors. Immunofluorescence and real-time qRT-PCR confirmed that in some gliomas, specifically in Glioblastoma multiforme (GBM), Gli1, a transcription factor activated by signalling, is present. In general, the hedgehog pathway is initiated by binding of extracellular ligands to the transmembrane receptor Patched and leads finally to the activation of the transcription factors Gli1, Gli2, Gli3 and Gli4. Whereas Gli1 acts as an activator, Gli2 appears to be an activator but retains some repressor activities and Gli3 and Gli4 are believed to act only as inhibitors. Therefore, the determination of hedgehog activity at the level of transcription requires additional experiments measuring gene activation. For that reason, cells isolated from 13 tumors of patients with glioblastoma (WHO Grade IV) and cells from two different glioma cell lines were transfected with reporter genes. These reporter genes carried the luciferase gene from Gaussia princeps under the control of two promoters (pT109 and pT81) conjugated to Gli binding sites. The activity of the reporter genes was compared to a control plasmid with mutant Gli-binding sites. In addition reporter gene activity was analysed in the absence and presence of the hedgehog signalling inhibitor cyclopamine and the effect of cyclopamine on cellular metabolism was studied. The analysis revealed that the two cell lines and cells from 6 glioblastomas exhibited enhanced reporter gene activity compared to the activity mutant control. This points towards an enhanced expression of Gli1. In three cultures a repression was detected suggesting that Gli3 may be active in these cells. Four cultures did neither show activation nor repression. This could provide evidence that Gli1 and Gli3 effects cancel each other out or that there is no effect at all. Enhanced luciferase activity in cells from the line T98G and in cells from four primary cultures was not influenced by the hedgehog inhibitor cyclopamine, whereas one cell line significantly responded to its presence with a decreased activity. Interestingly, ATP level was suppressed by cyclopamine in cells from the line T98G and also in cells from one primary culture that responded to the inhibitor. This may point towards an effect of cyclopamine independent of smo. Since cyclopamine is a potential new substance for the treatment of tumors, the observed effect of this inhibitor even in cells without an indication of hedgehog signalling activity should be investigated in further experiments in more detail. / Der Hedgehog (Hh) -Signalweg spielt während der Embryonalentwicklung eine wichtige Rolle, so auch bei der Entstehung des zentralen Nervensystems (Varjosalo & Taipale 2008). Andererseits führt seine unregulierte Aktivität zur Ausbildung verschiedenster Tumore (Bailey et al. 2009; Fiaschi et al. 2009; Shaw et al. 2009; Velcheti & Govindan 2007). Vorausgegangene Studien wiesen durch Immunfluoreszenz und real-time qRT-PCR nach, dass auch in Gliomen, speziell in Glioblastoma multiforme, dem agressivsten Hirntumor des Menschen, Effektoren des Signalweges (Gli1) überexprimiert werden (Wang et al. 2010). Die Aktivierung des Signalweges geschieht über Bindung des Hh-Liganden an den Rezeptor Ptch und endet mit der Aktiverung der Transkriptionsfaktoren der Gli Familie (Kinzler & Vogelstein 1990; Stone et al. 1996). Die aktuell bekannten Vertreter dieser Familie sind der Aktivator der Transkription Gli1, Gli2, der als Aktivator und Repressor agieren kann sowie Gli3 und Gli4, die die Transkription inhibieren (Marine et al. 1997; Ruppert et al. 1988). Ziel dieser Arbeit war es, herauszufinden, inwieweit die Transkriptionsfaktoren der Gli-Familie in Zellen von Glioblastoma multiforme aktiv sind. Dafür wurden Zellen aus Tumormaterial isoliert und daraus Primärkulturen hergestellt. In diese 13 Primärkulturen, wie auch in zwei Gliom-Zelllinien, wurden mittels transienter Transfektion Reporterplasmide eingebracht. Diese enthielten ein Gen der Gaussia-Luciferase, das unter der Kontrolle zweier verschiedener Promotoren (pT109 und pT81) mit Bindungsmotiven für die Transkriptionsfaktoren der Gli-Familie stand. Weiterhin wurde der Einfluss des Inhibitors des Hh-Signalweges Cyclopamin auf die Gli-Aktivität und die Metabolische Aktivität der Zellen untersucht. Die Beobachtungen ergaben, dass die zwei Zelllinien und sechs der primären Kulturen eine erhöhte Luciferaseaktivität und damit gesteigerte Aktivität von Gli1 zeigten. Weiterhin wiesen vier Kulturen eine verminderte Luciferaseaktivität auf. Dies ließ darauf schließen, dass in diesen Zellen Gli3 aktiv war. In den restlichen vier Kulturen zeigte sich keine Veränderung der Luciferaseaktiviät, was für einen Aufhebungseffekt von Gli1 und Gli3 oder gar keinen Effekt spricht. Weiterhin konnte gezeigt werden, dass die Luciferaseaktivität und damit die Aktivität von Gli1 in Zellen der Zelllinie T98G und von vier Primärkulturen nicht durch Cyclopamin beeinflusst wird. Lediglich eine Probe der Primärkulturen reagierte mit einer Abnahme der Luciferaseaktivität. Außerdem konnte Cyclopamin die ATP-Produktion sowohl in Zellen von T98G als auch in Zellen der Zelllinie, deren Gli-Aktivität durch Cyclopamin vermindert wurde, senken. Dies sprach für eine Smo unabhängige Wirkung des Cyclopamins. Da Cyclopamin ein potenzielles Pharmakon für die Antitumortherapie ist, bedarf dieser Umstand näherer Untersuchungen.

Le médulloblastome (MB), une tumeur formée à partir du cervelet en développement, est l’un des cancers pédiatriques malins les plus fréquents. Des profils d’expression géniques ont montré l’existence de quatre groupes distincts de MB qui présentent des profils moléculaires et des pronostics différents. Parmi ces groupes, l’un d’entre eux est caractérisé par une activation de la voie de signalisation Sonic Hedgehog (SHH). Ce groupe de MB provient des précurseurs de neurones en grain lors du développement cérébelleux. Les traitements actuels comprennent la chirurgie, la chimiothérapie ainsi que la radiothérapie, ce qui a pour effet d’altérer les capacités cognitives et sociales des survivants. Ainsi, des efforts considérables ont été mis en œuvre dans le but de trouver des cibles thérapeutiques afin de bloquer spécifiquement les mécanismes tumorigéniques sans affecter le développement normal. De récentes analyses à grande échelle ont révélé le rôle crucial de mécanismes épigénétiques, et en particulier dans le groupe SHH, dans lequel la perte de fonction d’un certain nombre de modificateurs chromatiniens a été identifiée. Ainsi, l'objectif principal de ma thèse est d'étudier l'implication de potentiels candidats modificateur chromatinien, à la fois au cours du développement cérébelleux et lors du MB SHH. Nous avons concentré notre étude sur plusieurs modificateurs de la chromatine qui ont été trouvés mutés dans les MB SHH humains. Nous avons commencé l’étude avec trois modificateurs chromatiniens sélectionnés selon (i) leur impact sur la survie, (ii) leur expression au cours du développement du cervelet, (iii) leur expression dans les MB SHH humains et nous sommes finalement concentré sur un.Afin d'étudier ce candidat, un objectif important de ma thèse a été de développer des outils fiables. Dans ce contexte, nous avons développé des modèles de souris knock-out conditionnelles et le système CRISPR-cas9 dans le développement cérébelleux postnatal afin d'étudier l'impact de la perte du candidat à la fois dans le développement du cervelet et dans le MB SHH. Ensuite, nous nous sommes intéressés aux mécanismes moléculaires contrôlés par ce modificateur de la chromatine. Plus précisément, nous avons défini (i) l’interactome, et (ii) des cibles transcriptionnelles spécifiques qui nous ont aidé à comprendre comment une protéine impliquée dans la modification de la chromatine peut favoriser l’état tumoral. En conclusion, ces travaux permettent de mettre en évidence comment la perte de la fonction de modificateur chromatinien spécifique peut différemment affecter le destin cellulaire dans le développement normal cérébelleux et dans le MB SHH et soulève la question d’une prise en charge plus personnalisée des patients atteints de MB SHH. / Medulloblastoma (MB), a tumor arising from the developing cerebellum, is one of the most common malignant pediatric brain tumors. Gene expression profiling showed the existence of four groups of MB with distinct molecular profiles and patient outcomes. Among these groups, one of them is associated with an activation of the Sonic Hedgehog (SHH) pathway.This specific group is thought to arise from cerebellar Granule Neuron Progenitors (GNPs) during cerebellar development. The actual treatment is heavy and consists of surgery, chemotherapy as well as radiotherapy impairing social and cognitive ability of survivors. Thus, considerable effort has been made in order to find drug targets that would specifically block tumorigenic mechanisms without affecting normal development.Recent large scale analysis revealed the crucial role of epigenetic mechanisms, and especially in the SHH group of MB in which loss of function mutation of several chromatin modifiers has been identified. Thus, the main goal of my PhD is to study the involvement of potential candidate chromatin modifiers both during cerebellar development and in SHH MB.We focused our study on several chromatin modifiers that were found mutated in human SHH MB. We began to study three chromatin modifiers that were selected according to (i) their impact on survival, (ii) their expression during cerebellar development, (iii) their expression in human SHH MB and finally we selected one for further functional validation.In order to study this candidate, one important goal of my PhD has been to develop reliable tools. In that context, we developed conditional knock-out mice models and the CRISPR-Cas9 system in postnatal cerebellar development in order to study the impact of the loss of this chromatin modifier both in cerebellar development and SHH MB initiation. Then, we investigated the molecular mechanisms controlled by this chromatin modifier. In particular, we defined (i) the interactome, and (ii) specific target genes that helped us understanding how a protein implicated in chromatin modification can favor tumors. In conclusion, this work provides insights into how the loss of function of a specific chromatin modifier can differentially affect cell fate in the context of normal cerebellar development and in SHH MB, stressing the question of a more personalized patient care.

Pendant le développement du cervelet, les progéniteurs des neurones granulaires (PNG) nécessitent la présence du cil primaire pour proliférer en réponse à Sonic Hedgehog (SHH). En effet, la prolifération dérégulée des PNGs peut conduire à la formation d'une tumeur pédiatrique maligne appelée SHH-médulloblastome (MB), de ce fait comprendre comment le cil primaire est régulé dans les PNGs est crucial.Nous montrons que le facteur de transcription Atoh1 contrôle la présence du cil primaire dans les PNGs in vitro et in vivo. En particulier, la suppression du cil primaire par l’inactivation génétique de gènes impliqués dans la ciliogenèse (par exemple, Kif3a ou Ift88) empêche Atoh1 de maintenir les PNGs en prolifération, ce qui indique qu’Atoh1 favorise l’expansion des PNGs en maintenant la présence du cil primaire. D’un point de vue moléculaire, Atoh1 contrôle la formation du cil primaire en régulant le bon positionnement peri-centrosomal des satellites centriolaires (SC), complexes protéiques essentiels pour la ciliogenèse. L'inactivation de Atoh1 dans les PNGs perturbe en effet la distribution subcellulaire des SCs, altérant ainsi inévitablement la ciliogenèse. Cette nouvelle fonction de Atoh1 est gouvernée par la régulation transcriptionnelle directe d'un composant clé des SCs, Cep131. L’expression ectopique de Cep131 dans les PNGs restore les effets liés à l'inactivation d'Atoh1, rétablissant la localisation correcte du SC et comme conséquence la présence d’un cil primaire.De plus, nous avons montré que cette voie Atoh1-SC-cil primaire-SHH contrôlant la prolifération des PNGs est également conservée dans le contexte du SHH-MB, où Atoh1 est surexprimée et essentielle pour sa formation et sa maintenance.Ces données révèlent un mécanisme par lequel la ciliogenèse est régulée dans des progéniteurs de neurones, offrant de nouvelles informations sur la neurogenèse dans le cervelet et sur la pathogenèse du SHH-MB. / Cerebellar granule neuron progenitors (GNPs) require the primary cilium to proliferate in response to Sonic Hedgehog (SHH) during cerebellar development. As aberrant proliferation of GNPs may lead to SHH-type medulloblastoma (SHH-MB), a pediatric brain tumor, understanding which mechanisms control ciliogenesis in GNPs represents a major interest in the field. Here, we show that the proneural bHLH transcription factor Atoh1 controls the presence of primary cilia in GNPs both in vitro and in vivo, thus maintaining GNPs responsive to the mitogenic effects of SHH. Indeed, loss of primary cilia induced via knockdown of specific ciliary components (e.g. Kif3a and Ift88) abolishes the ability of Atoh1 to keep GNPs in proliferation in vivo. Mechanistically, Atoh1 controls ciliogenesis by regulating the proper peri-centrosomal clustering of centriolar satellites (CS), large multiprotein complexes working as essential machineries for ciliogenesis. Knockdown of Atoh1 in GNPs perturbs CS subcellular distribution, leading to impairment of ciliogenesis. Luciferase reporter assays and chromatin immunoprecipitation experiments indicate that Atoh1 can directly regulate the expression of Cep131, a key CS core component. Importantly, ectopic expression of Cep131 in GNPs depleted of Atoh1, is sufficient to restore proper CS localization and consequent primary cilia formation, indicating that the Atoh1-Cep131-CS axis is responsible for ciliogenesis in GNPs.In addition, we further demonstrated that these functions of Atoh1 are conserved in the context of SHH-MB, where Atoh1 is typically overexpressed and acts as a lineage-dependent transcription factor.These data reveal a mechanism whereby ciliogenesis is regulated in neuron progenitors providing novel insights into cerebellar neurogenesis and pathogenesis of SHH-MB.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.

The Drosophila Lipoprotein particles bear lipid-linked morphogens on their surface and are required for long-range signaling activity of Wingless and Hedgehog. They also bind a wide variety of gpi-linked proteins. Whether any of these proteins affect morphogen signaling is unknown. Here, I show that the gpi-linked heparan sulfate proteoglycan Dally is released from cell membranes and binds to lipoprotein particles both with and without its lipid anchor. Hedgehog signaling efficiency is reduced in Dally mutant discs, but can be rescued non-autonomously by expression of non-gpi-modified Dally. This Dally isoform colocalizes with Hedgehog, Patched and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting Hedgehog distribution. These data show that Hedgehog signaling activity can be influenced by other Lipophorin-associated proteins, and suggest Lipoproteins provide a platform for regulation of morphogen signaling.

No description available.

<p>Lung squamous cell carcinoma (LSCC) comprises ~30% of non-small cell lung cancers, and currently lacks effective targeted therapies. Previous immunohistochemical and microarray studies reported overexpression of Hedgehog (HH)-GLI signaling components in LSCC. However, they addressed neither the tumor heterogeneity nor the requirement for HH-GLI signaling. Here, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI pathway suppression. </p><p>Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation of HH-GLI signaling. Four human LSCC cell lines were examined for HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. Xenografts in immunodeficient mice were used to determine the <italic>in vivo<italic> efficacy of GLI inhibitor GANT61. </p><p>In both patient cohorts, we found that activation of HH-GLI signaling was significantly associated with the classical subtype of LSCC. <italic>GLI2<italic> expression level was significantly higher than <italic>GLI1<italic>, and displayed strong positive correlations with the prominent markers for the classical subtype (<italic>SOX2<italic>, <italic>TP63<italic> and <italic>PIK3CA<italic>) on chromosome 3q. In cell lines, genetic knockdown of SMO produced minor effects on cell survival, while GLI2 knockdown significantly reduced proliferation and induced extensive apoptosis. Consistently, the SMO inhibitor GDC-0449 resulted in limited cytotoxicity in LSCC cells, whereas the GLI inhibitor GANT61 was very effective. Importantly, GANT61 demonstrated specific <italic>in vivo<italic> anti-tumor activity in xenograft models of GLI-positive cell lines. </p><p>Taken together, we report SMO-independent regulation of GLI in LSCC, and demonstrate an important role for GLI2 in LSCC. Different from standard-of-care chemotherapy or small molecule inhibition of kinase signaling cascades, we present a novel and potent strategy to treat a subset of LSCC patients by targeting the GLI transcriptional network.</p> / Dissertation

La voie de signalisation HH joue un rôle crucial dans le contrôle de la prolifération et la différenciation cellulaires. Le dérèglement de cette voie est responsable de nombreux cancers. J'ai ainsi mis en place les outils moléculaires nécessaires pour identifier des régions importantes dans les interactions protéines-protéines au sein du complexe intracellulaire de la voie qui vont permettre de mieux comprendre les mécanismes de régulation du facteur de transcription CI en fonction de l'état d'activation de la voie. Mes travaux ouvrent la voie à de nombreuses études structurales et fonctionnelles de ces protéines jusqu'ici peu étudiées.

During embryogenesis, airway epithelial cells possess primary cilia, and HH signaling guides lung development. As epithelial cells mature, they produce hundreds of motile cilia and continue to produce the sonic hedgehog (SHH) ligand, which is found apically in the thin layer of liquid covering airways. However, whether ciliated airway cells express apical HH signaling components and what their function might be have remained unknown. Here we show that motile cilia are enriched for HH signaling proteins, including patched 1 and smoothened. These cilia are also enriched for proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Surprisingly, SHH in differentiated airway epithelia did not elicit the canonical SHH signaling pathway that regulates transcription during development. But instead, activating HH signaling decreases intracellular levels of cAMP, which reduces ciliary beat frequency and airway surface liquid pH, similar to changes that have been observed in the airway of people with chronic obstructive pulmonary disease (COPD). Furthermore, we observed that significant increase of SHH ligand expression in differentiated airway epithelia with COPD, suggesting a potential role of SHH signaling in the pathogenesis of airway disease. Collectively, our study indicates that airway cilia detect apical SHH to mediate airway physiology through noncanonical HH signaling. SHH may dampen defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. This may suggest a potential role of SHH signaling in the pathogenesis of airway disease, such as COPD.