According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 68-77.

Malignant brain tumors are diseases with poor prognosis and/or severe long-term side effects of treatment. This thesis aimed to discover novel regulators in brain tumor development, based on studying neural stem cell and progenitor cell (NSPC) differentiation and using animal models to introduce new insights to mechanisms of human brain tumors. The enzyme heparanase (HPSE) that degrades heparan sulfate (HS) is active in cell signaling and ECM remodeling. In paper I, we found an enhanced differentiation to oligodendrocytes in ES cell-derived NSPCs overexpressing HPSE. Further analysis suggested that this enhanced formation of oligodendrocytes was associated with alterations in receptor tyrosine kinase signaling, and that HPSE might also exert anti-apoptotic functions. Subsequently, in paper II we studied the involvement of HPSE in glioma development. We observed that high HPSE levels associated with poor survival in glioma patients. In experimental models, we found that HPSE promoted glioma growth, and that an inhibitor of HPSE reduced glioma progression both in vitro and in vivo. We hypothesize that regulators in NSPC differentiation could have a potential role in brain tumor development. In paper III, we explored the function of NRBP2, a pseudokinase that is up-regulated during NSPC differentiation. We found low expression of NRBP2 in brain tumors, in comparison to normal brain. In medulloblastoma, in particular, low NRBP2 expression is linked to poor prognosis. Overexpression of NRBP2 in medulloblastoma cells led to impaired cell growth and migration, concomitant with an increased cell death. In paper IV, we searched for novel glioma susceptibility genes by sequencing dog breeds from the same ancestor but with different glioma incidence. In this way we identified three new glioma-associated genes. Two of these are significantly regulated in human glioma and one of those might have a role in glioblastoma stem cell differentiation.

O meduloblastoma (MB) é um câncer do sistema nervoso central, de origem embrionária, que surge no cerebelo. É o tumor maligno cerebral mais frequente na infância e corresponde a aproximadamente 20% de todos os tumores intracranianos pediátricos. Atualmente, o tratamento é realizado com cirurgia, quimioterapia e radioterapia e está relacionado com diversos efeitos colaterais em médio e longo prazo. Diversos fatores contribuem para o seu desenvolvimento e progressão, entre estes, alterações nas vias de sinalização, como a Sonic Hedgehog (SHH) e Wingless. As modificações nos padrões epigenéticos, como a metilação do DNA, tem também um papel central na biologia deste tumor. Tais alterações comprometem funções básicas da célula como o controle da proliferação, sobrevivência celular e apoptose. Drogas epigenéticas como os inibidores de DNA metiltransferases (DNMTs) têm demonstrado efeitos antineoplásicos e resultados promissores para terapia do câncer. A Zebularina é um inibidor de DNMTs, que consequentemente reduz a metilação do DNA, e tem se mostrado uma importante droga antitumoral, com baixa toxicidade e atividade adjuvante à quimioterapia em tumores quimio-resistentes. Diversos estudos têm descrito seus efeitos em diferentes tipos de neoplasias, entretanto, não há relatos da sua ação em MB. Sendo assim, o presente trabalho teve como objetivo analisar os potenciais efeitos antineoplásicos da Zebularina em quatro linhagens de MB pediátrico (DAOY, ONS-76, UW402 e UW473). Foi observado que o tratamento com a Zebularina promoveu inibição da proliferação celular e da capacidade clonogênica, aumentou o número de células apoptóticas e células na fase S do ciclo celular (p<0,05). Adicionalmente, o tratamento induziu um aumento na expressão proteica de p53, p21 e Bax e uma diminuição da ciclina A, Bcl-2 e Survivina. Além disso, quando combinada com o quimioterápico vincristina agiu de modo sinérgico; e de modo antagônico quando combinada com a cisplatina. Através de análises de expressão gênica em larga escala (plataforma Agilent de microarray), foi encontrada diferentes vias moduladas pela droga, incluindo a dos Receptores Toll-Like e o aumento dos genes SUFU e BATF2. Aqui, foi encontrado que a Zebularina pode modular a ativação da via SHH, reduzindo os níveis de SMO, de GLI1 e de um de seus alvos, o PTCH1; contudo sem alterar os níveis de SUFU. Confirmou-se que o gene BATF2 é induzido pela Zebularina e possui regiões ricamente metiladas. Além disso, a baixa expressão do gene BATF2 está associada à um pior prognóstico em MB. Todos esses dados sugerem que a Zebularina pode ser uma droga em potencial para o tratamento adjuvante do MB / Medulloblastoma (MB) is an embryonal cerebellum tumor. It is the most common brain malignancy in children and accounts for approximately 20% of all pediatric intracranial tumors. Currently, treatment consists of surgery, chemotherapy and radiation and is associated to medium- and long-term side effects. Several factors contribute to the development and progression of MB, for instance, alterations in signaling pathways, such as Sonic Hedgehog (SHH) and Wingless. Epigenetic changes in DNA methylation patterns also play a central role in the biology of this tumor. Such changes are able to alter basic cell functions, controlling cell proliferation, survival and apoptosis. Epigenetic drugs as DNA methyltransferases (DNMTs) inhibitors have shown anticancer effects and promising results for cancer therapy. Zebularine is a low toxicity DNMTs inhibitor that induces DNA demethylation and has been reported as an important antitumor drug with adjuvant activity to chemotherapy in chemoresistant tumors. Studies have described its effects on different types of cancer, however, there are not data concerning its action in MB. Therefore, this study aimed to analyze the potential anticancer effects of Zebularine in four pediatric MB lines (UW402, UW473, ONS- 76 and DAOY). It was observed that treatment with Zebularine promoted inhibition of cell proliferation and clonogenic capacity, increased the number of apoptosis rate and cells in S phase of the cycle (p <0.05). In addition, the treatment induced an increasing in the protein expression of p53, p21 and Bax and a decreasing in cyclin A, Survivin and Bcl-2. Also, when combined with the chemotherapeutic agent vincristine acted synergistically but resulted in antagonism when combined with cisplatin. Through large-scale gene expression analysis (Agilent microarray platform), it was found different pathways modulated by Zebularine, including the Toll-Like Receptors pathway and the overexpression of SUFU and BATF2 genes. Zebularine was able to modulate SHH pathway activation, by reducing levels of SMO, GLI1 and one of its targets, PTCH1, whereas there were no changes in SUFU levels. It was confirmed that the gene BATF2 is induced by Zebularine and contains regions richly methylated. In addition, BATF2 low expression is associated with a worse prognosis in MB. All these data suggest that Zebularine may be a potential drug for the adjuvant treatment of MB

O meduloblastoma (MB) é um tumor embrionário que se origina de alterações genéticas em vias importantes para neurôgenese do cerebelo como Sonic hedgehog (Shh) e Wingless (Wnt). Alterações específicas nessas vias permitem a classificação do MB pelo perfil de expressão e mutacional em quatro subgrupos: SHH, WNT, grupo 3 e grupo 4. A ativação dessas vias pode estar relacionada à hipermetilação de reguladores negativos. A dinâmica da hidroximetilação foi descrita durante o desenvolvimento cerebelar, mas não há relatos na literatura sobre os níveis da hidroximetilação em amostras de MB. Os principais objetivos desse trabalho foram: avaliar os níveis de expressão dos genes TETs e IDHs em MB por qPCR; investigar os níveis de hidroximetilação por meio de imuno-histoquímica e dot-blot; avaliar mutações no éxon 4 de IDH1 e IDH2 por sequenciamento; analisar a metilação e hidroximetilação em genes reguladores negativos das vias SHH, WNT, NOTCH, BMP; modular a atividade dos genes TETs por meio do ácido ascórbico e verificar sua influência funcional e epigenética. Foi observada diminuição na expressão dos genes TETs e IDHs em amostras de MB apenas em comparação com cerebelos fetais, mas não em relação aos cerebelos não-fetais. As linhagens celulares de MB apresentaram expressão diminuída em comparação aos dois grupos controles. A classificação das amostras de MB permitiu verificar uma expressão gênica subgrupo específica. A expressão de TET3 apresentou associação com status da doença; e maiores níveis de IDH2 foram associados à metástase. Não foram encontradas mutações no éxon 4 de IDH1, ou no éxon 4 de IDH2 em MB. Os níveis de hidroximetilação global estão diminuídos em amostras de MB e linhagens celulares em comparação aos cerebelos não-neoplásicos; porém não estão associados com características clínicas dos pacientes. Não foram encontrados níveis detectáveis de hidroximetilação nos genes estudados. Os efeitos do ácido ascórbico foram linhagem-específicos, não ocorreu aumento nos níveis de hidroximetilação, mas alterações na expressão do gene TET3. Em conclusão, níveis de hidroximetilação e expressão dos genes TETs e IDHs são importantes para o MB. No entanto, estudos funcionais direcionados à manipulação desses genes são necessários para elucidar suas funções nesse tumor. / Medulloblastoma (MB) is an embryonic tumor that originates from genetic alterations in pathways that are important to the neurogenesis of cerebellum, such as Sonic hedgehog (Shh) e Wingless (Wnt). These alterations allow us to classify MB based in expression and mutational profile in four subgroups: SHH, WNT, group 3 and group 4. The activation of these pathways could be related to hypermethylation of negative regulators. Hydroxymethylation dynamics was described during cerebellum development, but there are not reports in the literature about hydroxymethylation levels in MB samples. The main aims of this study were: to evaluate TET and IDH genes expression in MB using qPCR; to investigate hydroxymethylation levels using immunohistochemistry and dot-blot; to evaluate mutations in exon 4 of IDH1 and IDH2 genes through sequencing analysis; to analyze methylation and hydroxymethylation levels in genes that regulate SHH, WNT, NOTCH and BMP pathways; to modulate TET genes activity through ascorbic acid and verify its functional and epigenetic influence in MB cell lines. We observed a decrease in TET and IDH genes expression in MB samples compared to fetal cerebellum, but not according to non-fetal cerebellum. MB cell lines presented a decrease when compared to both control groups. The classification of MB samples allowed us to verify a subgroup-specific gene expression. TET3 expression was associated with disease status; and higher levels of IDH2 gene expression were associated with metastasis. We did not find mutations in exon 4 of IDH1 and exon 4 of IDH2 genes in MB samples. Hydroxymethylation levels were decreased in MB samples and cell lines when compared to non-neoplastic cerebellum; however, they were not associated with clinical characteristics of the patients. We did not detect hydroxymethylation levels in the studied genes. Ascorbic acid effects are cell linespecific: we did not observe increase in hydroxymethylation levels, but alterations in TET3 gene expression. In conclusion, hydroxymethylation and expression levels of TET and IDH genes are important for MB. Though, functional assays that target these genes are required to elucidate their function in MB.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

O meduloblastoma é o tumor maligno do sistema nervoso central mais frequente na infância e adolescência. A expressão de genes tipicamente expressos em células-tronco está correlacionada com pior prognóstico em pacientes com meduloblastoma e a expressão de POU5F1 se mostrou capaz de distinguir pacientes com desfecho clínico desfavorável e pior sobrevida. Apesar do seu valor prognóstico, não há evidências diretas da contribuição de OCT4 para a aquisição de fenótipos mais agressivos em meduloblastoma. Nesse contexto, o presente trabalho investigou o papel da isoforma OCT4A em características pró-tumorigênicas de meduloblastoma in vitro e in vivo, e também avaliou as alterações moleculares que podem ser responsáveis pela aquisição de fenótipo mais agressivo em células de meduloblastoma humano. Para tanto, foi realizada a superexpressão de OCT4A mediada por retrovírus em três linhagens celulares de meduloblastoma (Daoy, D283Med e USP-13-Med). As células de meduloblastoma com superexpressão de OCT4A exibiram maior proliferação e alterações no ciclo celular. Foram observados também aumentos na atividade clonogênica, geração de esferas tumorais e desenvolvimento tumoral em modelo subcutâneo, sendo esses efeitos dependentes dos níveis de OCT4A. A avaliação da mobilidade celular in vitro demonstrou diminuição na adesão celular e aumento da invasão celular de esferoide 3D. Em modelo ortotópico de meduloblastoma, as células com superexpressão de OCT4A geraram tumores mais desenvolvidos, com fenótipos mais agressivos, infiltrativos e metastáticos. A superexpressão de OCT4A foi associada a maior instabilidade genômica, entretanto, as aberrações em números de cópias variaram em frequência e tipo de alteração dependendo da linhagem celular, e sendo pouco associada com os genes diferencialmente expressos. De forma interessante, uma relevante expressão diferencial de RNAs não-codificadores de proteínas foi observada em células de meduloblastoma com superexpressão de OCT4A, incluindo os recém descobertos e pouco caracterizados RNAs não codificadores longos, além de múltiplos RNAs pequenos nucleolares. Assim, os resultados aqui apresentados fundamentam a relevância de fatores envolvidos em pluripotência para o agravamento de traços associados com desfecho clínico desfavorável em meduloblastoma e destacam o valor prognóstico e terapêutico de OCT4A neste tumor pediátrico do sistema nervoso central / Medulloblastoma is the most common malignant brain tumor in infants. The expression of typical pluripotency genes is correlated with poor prognosis in medulloblastoma and POU5F1 expression was shown capable of discriminating patients with poor survival outcome. Despite this prognostic value, direct evidences of OCT4 contribution to more aggressive traits in medulloblastoma are missing. In this context, we investigated the role of OCT4A isoform on pro-tumorigenic features of medulloblastoma in vitro and in vivo and evaluated molecular alterations that could be responsible for acquisition of a more aggressive phenotype in medulloblastoma cells. Retroviral-mediated overexpression of OCT4A were performed in three medulloblastoma cell lines (Daoy, D283Med and USP-13-Med). Medulloblastoma cells overexpressing OCT4A displayed enhanced cell proliferation and cell cycle alterations. Increased clonogenic activity, tumorsphere generation capability and subcutaneous tumor development were also observed, and these effects were OCT4A expression level-dependent. Evaluation of cell mobility in vitro showed loss of cell adhesion and greater 3D-spheroid invasion. In an orthotopic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive, infiltrative and metastatic tumors. OCT4A overexpression was associated with chromosomal instability but copy number aberrations varied in frequency and type according to the cell line, with little association with differently expressed genes. Interestingly, marked differential expression of non-coding RNAs, including newly discovered, still poorly characterized, long non-coding RNAs and multiple small nucleolar RNAs were observed in medulloblastoma cells with OCT4A overexpression. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24784号 / 医博第4976号 / 新制||医||1066(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 藤田 恭之, 教授 髙折 晃史, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tumors are composed of proliferating cells that invade healthy tissue and grow over time. Even though it is still unclear, it is a common opinion that the cells of origin should possess a proliferative capacity (Blanpain, 2013; Visvader, 2011). Particularly for brain cancers, the transition of neural progenitors to differentiated postmitotic neurons is considered irreversible in physiological and pathological conditions. Therefore, postmitotic neurons have not been considered as suitable cell of origin for brain cancer. Here, we show that neurons reprograming may occur upon Shh activation leading to medulloblastoma (MB) formation in vivo. Human SHH medulloblastoma (MB) is a brain tumor affecting adults and infants that is thought to originate from cerebellar granule neuron progenitors. Notably, several groups have shown that Shh pathway activation (SmoM2 overexpression) in mouse granule neuron progenitors is able to induce Shh MB (Schuller et al., 2008; Z.-J. Yang et al., 2008). These progenitors are present in infants and newborn mice, but they seem to be absent in adult humans and mice (Biran, Verney, &amp; Ferriero, 2012; Marzban et al., 2014; Z.-J. Yang et al., 2008). Furthermore, it was recently discovered that the two different forms of SHH MB are distinguished by different transcriptome/methylome levels suggesting that the adult SHH MB may originate from a different cell of origin (Cavalli et al., 2017; Kool et al., 2014). Relying on these data, we take advantage of a conditional Cre-Lox recombination system to recapitulate the human adult medulloblastoma pathogenesis in mice, demonstrating that cerebellar post-migratory mature granule neurons upon SmoM2 overexpression can dedifferentiate and give rise to SHH MB in vivo. Moreover, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Notably, gene expression profiling on our samples allowed to associate “cerebellar granule progenitors-derived MB” with the human infant form while “truncated BRPF1-induced tumors” clustered with human adult SHH MB. Furthermore, as previously described by Kool et al., 2014, human adult SHH MB is characterised by the copresence of p-AKT and p-S6, compared to the human infant SHH MB that are positive for either p- AKT or p-S6 and always in a mutually exclusive way. Truncated BRPF1-induced tumors are double positive for p-AKT and p-S6, similarly to adult patients, while cerebellar granule progenitors derived MB present only p-S6. Furthermore, to define the contribution of chromatin changes in granule neurons dedifferentiation in response to Shh activation, we profiled changes in chromatin accessibility by ATAC-seq analysis on mice cerebella. SmoM2 overexpression changed the epigenetic landscape of granule neurons, enriching the number of open chromatin regions 12 associated with stem/progenitor-like genes. Moreover, the cooperation between truncated BRPF1 and SmoM2 in reshaping the chromatin arrangement of granule neurons was explored applying ATAC-seq on differentiated human cerebellar neurons derived from neuroepithelial cells. ATAC-seq analysis pointed out a synergistic mechanism between SmoM2 and truncated BRPF1 in modifying the epigenetic landscape of postmitotic neurons, increasing the chromatin accessibility of super-enhancers, associated with stemness and chromatin organization/modification genes. Our novel model of cancer development could explain the human SHH medulloblastoma onset in adult individuals where granule neuron progenitors are no more present. For these reasons, we strongly believe that our model configures as an important starting point for a new field in cancer and stem cell biology focusing on the study of mechanisms driving tumorigenesis in postmitotic cells.

No description available.