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The Hippo signaling pathway was first discovered in Drosophila melanogaster and is involved in organ size control by regulating cell proliferation and apoptosis. This well conserved pathway is activated by various signal inputs, including cell-cell contact, mechanotransduction, and G-protein coupled receptors, with signals converging on the downstream effector protein Yap and its homologue Taz, which are transcriptional co-activators. When the Hippo pathway is activated, Yap/Taz are phosphorylated, leading to cytoplasmic retention and degradation, and diminishing their transcriptional activity. Yap has also been recently implicated as a potential oncogene, as it is upregulated and transcriptionally active in several tumor types. Furthermore, inhibiting Yap activity in various cancer models has been shown to revert cancer cells to a normal phenotype. Although the role of Yap has been described in several organ systems, there is a paucity of information about the function of Yap in the central nervous system. I investigated the function of Yap/Taz in the murine cerebellum to determine its significance during normal development and a potential role for Yap/Taz in medulloblastoma, a tumor that arises in the cerebellum. In Chapter 2, I describe the expression pattern of Yap from embryonic through adult stages in mice, and demonstrate the functional significance of Yap/Taz in different cell populations using conditional knockout mouse models. I show that Yap plays a significant role in cell fate determination as well as in cerebellar foliation: Yap is highly expressed in the ventricular zone and is required for the proper formation of ependymal cells, and is also strongly expressed in Bergmann glia (BG) during early developmental stages, where Yap, together with Taz, plays a significant role in cerebellar foliation. Furthermore, Yap/Taz-deficient BG exhibit migrational defects, as their cell bodies can be found mislocalized to the molecular layer (ML), rather than remaining tightly associated with Purkinje Cells (PCs) in the PC layer. BG support the health of PCs, and severely defective BG positioning eventually leads to a loss of PCs. However, although Yap is highly expressed in granule neuron progenitors (GNPs) during the rapid postnatal expansion stage, it does not appear to play a major role in proliferation of these cells as conditionally knocking-out Yap/Taz in GNPs does not alter their proliferative capacity. Our observations demonstrate that in the cerebellum, Yap has a novel function in glia that is required for the development of normal foliation and organization, but plays a minimal role in GNP proliferation. Importantly, I also show that the reduction of sphingosine-1-phosphate G-protein-coupled receptor (S1P1) signal transduction activates the upstream kinase Lats with concomitant increases of phosphorylated Yap as well as a reduction of the known Yap target connective tissue growth factor (CTGF). This study identifies a novel function of Yap/Taz in cerebellar glia that is required for the development of normal foliation and laminar organization with sphingosine-1-phosphate (S1P) signaling as a potential extracellular cue regulating Yap activity during cerebellar development. In Chapter 3, I present further support for the finding that Yap/Taz are not required for GNP proliferation in vivo by discussing the failure of Yap/Taz loss to rescue the Sonic-hedgehog (Shh) mediated medulloblastoma phenotype, in which GNPs are considered to be the tumor cell of origin. Furthermore, I provide evidence suggestive of a tumor suppressive function of Yap/Taz in the cerebellum. Together, previously unknown functions of Yap in the developing and malignant cerebellum are described, providing a foundation for future studies of Yap in the central nervous system (CNS). / Biomedical Sciences

Brain tumors represent a leading cause of cancer mortality, of which medulloblastoma (MB) and glioblastoma (GBM) represent the most frequent malignant pediatric and adult brain tumors, respectively. The identification of a rare clonal population of cells, termed cancer stem cells (CSCs) or brain tumor-initiating cells (BTICs), as having the ability to initiate, proliferate, and maintain tumor growth has offered a developmental framework for studying MB and GBM. Evidence in support of cell signaling programs carried forward from brain development into oncogenesis have provided opportunities for BTIC-directed therapies targeting the key BTIC property of self-renewal. Given that neural stem cells (NSCs) must maintain a relative balance between self-renewal and differentiation, brain tumorigenesis may be conceptualized as a disease of unregulated BTIC self-renewal. In this work, I aim to demonstrate the re-emergence of self-renewal genes that regulate NSCs in BTICs, use the Wnt pathway as a model by which these genes may be regulated in a context-specific manner, and identify clinically tractable therapies directed at the overall BTIC self-renewal signaling machinery. Specifically, in Chapter 2, I describe the presence of a shared signaling program between NSCs and MB BTICs consisting of Bmi1 and FoxG1. In Chapter 3, I provide evidence in support of a context-specific tumor suppressive function for activated Wnt/β-catenin signaling in MB. Lastly, in Chapter 4, I demonstrate a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation in GBM. Overall, the body of this thesis offers a mechanistic model by which BTICs may be regulated and targeted to impair tumor growth and improve overall survivorship in childhood MB and adult GBM. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25195号 / 医博第5081号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 実, 教授 椛島 健治, 教授 中本 裕士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The aim of this master thesis is to elaborate the issue of the PEDI-CAT test and to evaluate its contribution in the proces of occupational therapy in patients with medulloblastoma. The theoretical part deals with the test itself, its origin, process of standardization and comparison with the older version of the test. The theoretical part further informs about the possibility of using the test in different areas and in different diagnoses. The last part of the theory analyzes the area of occupational therapy in pediatric patients with diagnosis of medulloblastoma. The practical part contains three case reports of pediatric patients diagnosed with medulloblastoma, which make it described the benefits of practical use of the PEDI-CAT test in occupational therapy in this diagnosis. In the appendices of the thesis is a brief guide to the test, translated by the author of the thesis, which can serve as a guide to facilitate the work with the test, for those who will take the test in practice. The contribution of this thesis is to introduce the PEDI-CAT to the professional public in the Czech Republic. Putting the PEDI-CAT into practice will help facilitate testing and evaluating the outcome of occupational therapy in pediatric patients. Furthermore, the contribution of this thesis is to evaluate the...

Le médulloblastome (MB) est la plus fréquente des tumeurs cérébrales malignes pédiatriques qui représentent la première cause de mortalité par cancer chez l’enfant. Malgré les avancées des nouveaux traitements, les risques de récidive, séquelles et décès après traitement restent importants. Le récepteur de neuropiline-1 (NRP-1) a été récemment impliqué dans la progression tumorale des MBs et semble jouer un rôle important dans le phénotype des cellules souches cancéreuses (CSCs). Le ciblage de cette molécule pourrait ainsi présenter un intérêt thérapeutique dans le traitement des MBs. Nous avons sélectionné des cellules souches de MB capables de former des médullosphères (MS) à partir de 3 lignées cellulaires (DAOY, D283-Med et D341-Med). Ces modèles ont été caractérisés par l’expression de neuropilines (NRP-1 and NRP-2) et de marqueurs phénotypiques (CD133，CD15 et NF-M). Les résultats ont montré une augmentation significative de l’expression de NRP-1 par les cellules cultivées en médullosphères confortant notre stratégie de ciblage. L’impact du traitement de ces cellules par un composé innovant ciblant spécifiquement NRP-1, le MR438, a été ensuite évalué in vitro seul et association avec la radiothérapie notamment sur l’étude de la capacité d’auto-renouvellement des CSCs de MBs. Nous avons mis en évidence une diminution de la capacité d’autorenouvellement des cellules souches de MBs après exposition au MR438 avec une radiosensibilité augmentée pour les 3 modèles cellulaires. In vivo, le composé MR438 a été evalué sur des modèles de xénogreffes hétérotopiques chez la souris nude et montre un effet radiopotentialisant significatif pour les tumeurs issues de la lignée Daoy avec une tendance à la diminution de la progression tumorale pour les 2 autres lignées. De façon intéressante, le composé MR438 induit une diminution significative du nombre de cellules souches pour l’ensemble de nos modèles. Par conséquent, le composé semblerait induire les cellules souches vers un phénotype différencié au moins pour la lignée DAOY, même si les mécanismes n’ont pas pu être clairement élucidé. En conclusion, l’inhibition de NRP-1 via MR438 semble stimuler la différenciation des cellules souches cancéreuses pouvant à terme réduire la progression du MB et apporter un bénéfice en association avec la radiothérapie. L’evaluation du composé sur des modèles orthotopiques de MB permettrait d’obtenir des informations quant à son efficacité sur des modèles plus proche de la physiopathologie tenant compte de sa distribution au niveau cérébral / Medulloblastoma (MB) is the most common malignant pediatric brain tumors which is the leading cause of cancer death in children. Despite the progress of new treatments, the risk of recurrence, morbidity, and death after treatment remain important. The neuropilin-1 receptor (NRP-1) has recently been implicated in tumor progression of MBs, which seems to play an important role in the phenotype of cancer stem cells (CSCs). Targeting this molecule could thus present an interesting therapeutic value in the treatment of MB. We have selected cancer stem like cells of MBs in the form of medullospheres (MSs) from 3 cell lines (DAOY, D283-Med and Med-D341). These models were characterized by expression of neuropilins (NRP-1 and NRP-2) and phenotypic markers (CD133, CD15 and NF-M). Results showed a significant increase of the expression of NRP-1 by our CSCs models cultured in MSs that confirms our targeting strategy. The impact of the treatment of these cells with an innovative compound specifically targeting NRP-1, MR438, was then evaluated in vitro alone and in association with radiotherapy, especially on the study of the capacity for self-renewal. A decrease of self-renewal capacity for MB stem cells after exposition of MR438 with an increase of radiosensitivity for the 3 cell models in vitro was demonstrated. In vivo, MR438 was evaluated on heterotopic xenograft models in nude mice and showed a significant augmentation of radiosensitivity for DAOY tumors with a tendency to decrease tumor progression for the other 2 cell lines. Interestingly, the compound MR438 induced a significant decrease in the number of stem cells for all of our models. The compound appeared to induce CSCs to a differentiated phenotype at least for the DAOY cells, although mechanisms could not be clearly elucidated. In conclusion, inhibition of NRP-1 via MR438 seems to stimulate the differentiation of CSCs that may eventually reduce the progression of MB and bring a benefit in association with radiotherapy. Evaluation of this compound on orthotopic models of MB would provide information on its effectiveness on models closer to the physiopathology taking into account its distribution at the cerebral level

Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

Les médulloblastomes (MB) sont les tumeurs cérébrales les plus fréquentes en pédiatrie. Ils apparaissent le plus souvent au niveau du cervelet. Ils peuvent être stratifiés en quatre groupes : les groupes WNT et SHH, où ces voies de signalisation sont altérées, et les groupes 3 et 4, présentant des anomalies chromosomiques et amplifications multiples, dont c-Myc (groupe 3) et N-Myc (groupe 4). L’une des altérations génétiques les plus retrouvées dans les MB est la surexpression du facteur de transcription OTX2. Ce facteur est exprimé dans les précurseurs des cellules granulaires (GCP) du cervelet, cellules d’origine de la majorité des MB. Pendant la période périnatale, les GCP subissent une phase de prolifération très intense en réponse au mitogène Sonic Hedgehog (SHH), ce qui les rendrait particulièrement sensibles à la tumorigenèse. Au cours de cette thèse, nous nous sommes intéressé à la fonction d’Otx2 dans ces GCP. Nous avons montré que l’ablation conditionnelle d’Otx2 conduit à un défaut de prolifération des ces cellules. L’analyse approfondie de ce phénotype a permis de révéler qu’Otx2 stimule la prolifération des GCP parallèlement à la voie de signalisation Shh. Par ailleurs, l’ablation d’Otx2 dans un modèle murin mimant la formation de MB Shh-dépendants a montré qu’Otx2 s’avère indispensable pour leur maintien à long terme. En parallèle, nous avons tenté de créer un nouveau modèle murin mimant la formation de MB de groupe 3 en induisant l’expression, pendant la période postnatale, d’un dominant actif de c-Myc dans les cellules exprimant Otx2. Cette approche a donné des résultats inattendus : des carcinomes de plexus choroïdes, et non des MB, ont été obtenus. / Medulloblastomas (MB) are the most common brain tumors in paediatrics. They appear during development in the posterior part of the brain, mainly in cerebellum. MB can be stratified in four groups: the WNT and SHH groups, where these signalling pathways are aberrantly activated, and the groups 3 and 4, which display chromosomal abnormalities and multiple amplifications, including c-Myc (group 3) and N-Myc (group 4). One of the most frequent genetic alterations in MB is the overexpression of the Otx2 transcription factor (in 75% of cases). This factor, which is essential for central nervous system development, is expressed in granule cell precursors (GCP) of the cerebellum, which represent the cell of origin of the majority of MB. During the perinatal period, GCPs undergo intense proliferation in response to Sonic Hedgegog (SHH), making them particularly susceptible to tumorigenesis. During this thesis, we were interested in examining the function of Otx2 in GCPs. We have shown that conditional ablation of Otx2 leads to a GCP proliferation defect and that Otx2 stimulates the proliferation of these cells independently of the Shh signaling pathway. Moreover, ablation of Otx2 in a mice model of Shh-dependent medulloblastomas yielded very interesting results: while Otx2 does not seem to be required for the initiation of these tumors, it is essential for their long-term maintenance. In parallel, we tried to create a new murine model for the MB group 3 by inducing the expression, during the postnatal period, of an active dominant of c-Myc in cells expressing Otx2. This approach yielded unexpected results: choroid plexus carcinomas, instead of MB, were obtained.