Le récepteur à activité tyrosine kinase TrkC est un récepteur aux neurotrophines qui contrôle la mise en place des neurones proprioceptifs au cours du développement du système nerveux. En présence de son ligand, la neurotrophine‐3 (NT‐3), TrkC transmet un signal de survie, tandis qu’en absence des facteurs trophiques les neurones vont mourir via l’apoptose. Cependant, les mécanismes moléculaires de ce programme de mort ne sont pas connus. D'autre part, TrkC peut également accomplir un rôle proapoptotique dans le cancer. En effet, son expression est un facteur de bon pronostic pour le neuroblastome, la tumeur solide extra crânienne la plus courante chez l'enfant. Ce paradoxe apparent pourrait s’expliquer par la notion de récepteur à dépendance : ces récepteurs induisent la mort cellulaire en situation d’absence de ligand, alors que la présence de leur ligandinhibe leur activité proapoptotique. Nous avons fourni la preuve que parmi les récepteurs aux neurotrophines, seul TrkC est un récepteur à dépendance. Son activité nécessite un clivage par des caspases, générant un fragment proapoptotique. Par ailleurs, nous avons mis en évidence la surproduction autocrine de NT‐3 dans une fraction de neuroblastomes à haut risque, permettant aux cellules malignes de contourner le mécanisme de contrôle antitumoral de TrkC. De plus, nous avons démontré que la perturbation de l'interaction TrkC/NT‐3 induit la mort des cellules de neuroblastomein vitro et empêche la croissance tumorale et les métastases dans des modèles aviaires et murins. Ces travaux établissent les bases d’une potentielle stratégie thérapeutique s'appuyant sur la restauration d'une voie d’apoptose fonctionnelle induite par TrkC dépourvu du ligand / The tyrosine kinase receptor TrkC is a neurotrophin receptor that assures an adequate establishment of proprioceptive neurons during nervous system development. Upon neurotrophin‐3 (NT‐3) binding TrkC transduces a classic survival signal, while in absence of trophic support a program of apoptotic cell death will take place. However the molecular mechanisms leading to neuron cell death are not understood. On the other hand, although TrkC was first identified as an oncogene, it may also accomplish a proapoptotic role in cancer. Indeed its expression has been correlated with a good prognosis of Neuroblastoma, the most common extracranial solid tumor of early childhood. Thisapparent paradox could be explained by the dependence receptor notion: these receptors induce apoptotic cell death in settings of absence of ligand, whereas the presence of their ligand inhibits this proapoptotic activity. We provide evidence here that among neurotrophin receptors, only TrkC is a dependence receptor and its activity relies on the caspase‐mediated cleavage of its intracellular domain, which allows the release of a proapoptotic fragment. Moreover, we show that an autocrine production of NT‐3 concerns a large fraction of aggressive neuroblastoma and provides a selective advantage by allowing malignant cells to overcome with TrkC antitumoral control. We demonstrate that the disruption of the TrkC/NT‐3 interaction triggers neuroblastoma cell death in vitro andprevents tumor growth and metastasis in avian and murine models. This work set the basis for analternative anticancer therapeutic strategy relying on the reengagement of a cell death program mediated by unbound TrkC

ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.

Evidências sugerem que o transtorno bipolar esteja associado a uma toxicidade sistêmica, representada por alterações periféricas em marcadores de inflamação, estresse oxidativo e neurotrofinas, a qual parece estar associada aos episódios de humor e à progressão da doença levando a prejuízos sistêmicos e na neuroplasticidade. Os trabalhos apresentados nesta tese tiveram como objetivo revisar estas alterações e explorar possíveis mecanismos responsáveis por estes achados, com enfoque em uma desregulação transcricional no transtorno bipolar. No primeiro capítulo, revisamos os biomarcadores periféricos associados aos episódios de humor, a relação destes com a toxicidade sistêmica e os possíveis mecanismos subjacentes a esta toxicidade. Seguimos ilustrando, no capítulo 2, um exemplo de alterações estruturais cerebrais em um paciente bipolar com experiência de múltiplos episódios, como um possível exemplo da neuroprogressão no transtorno bipolar. Em seguida, buscamos por vias de regulação transcricional disfuncionais no córtex pré-frontal de pacientes que poderiam estar associadas a essas alterações e neuroplasticidade prejudicada. A partir de abordagem inovadora de bioinformática, no capítulo 3, identificamos algumas unidades regulatórias (regulons) associadas com as duas assinaturas gênicas do transtorno bipolar avaliadas, obtidas a partir de bancos de dados de microarranjo de pré-frontal postmortem. Em uma análise mais rigorosa, identificamos apenas o regulon do gene early growth response 3 (EGR3) enriquecido nas duas assinaturas da doença em duas redes transcricionais do pré-frontal, estando reprimido no fenótipo bipolar. Nossos resultados sugerem o regulon do EGR3 como um alvo importante no transtorno bipolar. Considerando seu papel fundamental na resposta ao estresse e na translação de estímulos ambientas em mudanças na expressão gênica neuronal, propomos que uma disfunção em vias biológicas envolvendo EGR3 poderia levar a uma resposta prejudicada ao estresse e influenciar no risco para o transtorno bipolar. No quarto capítulo, então, caracterizamos o perfil de expressão gênica do modelo de células SH-SY5Y diferenciadas e avaliamos o comportamento do regulon do EGR3 de acordo com o protocolo de diferenciação. Além disso, identificamos moléculas com potencial de modular os regulons enriquecidos no transtorno bipolar visando testar o efeito destas drogas no referido modelo celular. Nossos resultados reforçam o fenótipo neuronal deste modelo in vitro e demonstram que o regulon do EGR3 está enriquecido nas células diferenciadas, sugerindo que ele é importante nesse processo e que este modelo experimental é adequado para estudar este regulon e as moléculas selecionadas pela análise de mapa de conectividade. Nós ainda avaliamos nas células SH-SY5Y o efeito do soro de pacientes bipolares, para investigar o papel da toxicidade sistêmica em células neuronais. O soro de pacientes, especialmente em estágio tardio da doença, causou toxicidade às células, reduzindo a densidade de neuritos e a viabilidade celular. Esses achados representam uma forma de desafio celular relacionado à toxicidade sistêmica do transtorno bipolar, propondo as células SH-SY5Y diferenciadas como um modelo in vitro para estudo desta doença. Em suma, os resultados desta tese sugerem que a toxicidade sistêmica relacionada aos episódios recorrentes de humor pode influenciar nas alterações anatômicas cerebrais associadas com a progressão do transtorno bipolar, e a disfunção no regulon do EGR3 poderia estar envolvida com aspectos desta neuroprogressão considerando o papel de EGR3 na resposta ao estresse e na neuroplasticidade. Estas hipóteses, que também sugerem alvos interessantes para o desenvolvimento de novos tratamentos, devem ser apropriadamente validadas em modelos experimentais, como o modelo de células SH-SY5Y diferenciadas estudado neste trabalho. / Evidence suggests that bipolar disorder is associated with a systemic toxicity, represented by peripheral changes in markers of inflammation, oxidative stress and neurotrophins, which appears to be associated with mood episodes and illness progression leading to systemic damage and impaired neuroplasticity. The work presented in this thesis aimed to review these changes and explore possible mechanisms responsible for these findings, focusing on transcriptional regulation in bipolar disorder. In the first chapter, we review the peripheral biomarkers associated with mood episodes, their relationship with systemic toxicity and the possible mechanisms underlying this toxicity. We have shown, in Chapter 2, an example of structural brain changes in a bipolar patient with multiple episodes experience, as a possible example of ‘neuroprogression’ in bipolar disorder. Then we investigated dysfunctional transcriptional regulatory pathways in the prefrontal cortex of patients that could be associated with these changes and impaired neuroplasticity. Using innovative bioinformatics approaches, in Chapter 3, we identified some regulatory units (regulons) associated with the two gene signatures of bipolar disorder evaluated, obtained from microarray data sets from prefrontal postmortem studies. With a more rigorous analysis, we only identified the regulon of early growth response 3 gene (EGR3) enriched in the two bipolar signatures in the two transcriptional prefrontal networks evaluated, being EGR3 repressed in bipolar phenotype. Our results suggest the EGR3 regulon as an important target in bipolar disorder. Considering its key role in response to stress and translation of environmental stimuli into long-term changes in neuronal gene expression, we propose that a dysfunction in biological pathways involving EGR3 could lead to an impaired response to stress and influence on the risk for bipolar disorder. Then, in Chapter 4, we characterized the gene expression profile of differentiated SHSY5Y cells and evaluated the EGR3 regulon according to the differentiation protocol. Furthermore, we have identified molecules with potential to modulate the regulons enriched in bipolar disorder, using connectivity map analysis, in order to test the effect of these drugs on this cellular model in future studies. Our results consolidated the neuronal phenotype of this in vitro model and demonstrated that the EGR3 regulon is enriched in differentiated cells, suggesting that it is important in this process and that this experimental model is suitable for studying this regulon and molecules selected by connectivity map analysis. Moreover, in Chapter 5, we evaluated the effect of serum of bipolar patients on differentiated SH-SY5Y cells to investigate the role of systemic toxicity in neuronal cells. The serum of patients, especially at late stages of illness, caused toxicity to cells, reducing neurite density and cell viability. These findings represent a strategy of challenging cells related to the systemic toxicity of bipolar disorder, proposing differentiated SH-SY5Y cells as an in vitro model to study this disorder. Therefore, the results of this thesis suggest that systemic toxicity related to recurrent mood episodes may influence on the brain anatomical changes associated with the bipolar disorder progression, and dysfunction in EGR3 regulon could be involved with aspects of ‘neuroprogression’ considering the role of EGR3 in response to stress and neuroplasticity. These hypotheses, which also suggest interesting targets for the development of new treatments, should be further properly validated in experimental models such as the differentiated SH-SY5Y cells model studied in this work.

O transtorno bipolar (TB) é caracterizado como um grave transtorno psiquiátrico, que apresenta curso crônico com notável prejuízo cognitivo e funcional nos pacientes. Nesta tese, através de duas revisões avaliamos as alterações de marcadores periféricos de estresse oxidativo, neurotrofinas e inflamação presentes em pacientes com TB e discutimos o envolvimento destes na toxicidade sistêmica, bem como uma possível associação destas alterações com as disfunções cognitivas e funcionais apresentadas pelos pacientes ao longo do transtorno. Neste sentido, muitos estudos têm sido realizados buscando compreender a fisiopatologia do TB, no entanto, para o estabelecimento de um modelo in vitro é necessário ter um modelo celular adequado e um desafio que possa mimetizar a fisiopatologia da doença. Neste contexto, não há na literatura, até o momento, um modelo adequado que compreenda toda a complexidade dos sintomas do TB, por isso o objetivo geral desta tese consistiu na caracterização gênica do modelo in vitro de diferenciação celular da linhagem de neuroblastoma humano SH-SY5Y, induzido por ácido retinóico, e a busca por um modelo experimental para a avaliação da toxicidade sistêmica apresentada pelos pacientes com TB. O modelo de diferenciação foi avaliado através da técnica de microarranjo, onde verificamos que nas células diferenciadas há maior expressão de processos biológicos envolvidos no desenvolvimento neuronal, enquanto nas células indiferenciadas observamos maior expressão de processos biológicos relacionados a proliferação e manutenção celular. Ainda, genes relacionados a função sináptica e a síntese dopaminérgica estavam mais expressos nas células diferenciadas. Estes achados contribuem para a validação de um modelo de origem humana, de fácil manuseio e que apresenta perfil neuronal; auxiliando assim no estudo de doenças que acometem o sistema nervoso central, dentre elas o TB. Para avaliar o perfil de toxicidade no soro de pacientes bipolares, tratamos as células SH-SY5Y diferenciadas com soro de pacientes com TB, tanto em estágio inicial quanto avançado do transtorno. Como resultado, verificamos que o soro dos pacientes em estágio avançado apresenta maior toxicidade quando comparado ao soro de indivíduos controles por causar uma diminuição da viabilidade celular e uma diminuição na densidade de neuritos. Analisados em conjunto, os achados desta tese apontam para um novo modelo in vitro para analisar a fisiopatologia do TB, bem como o efeito de medicações e vias metabólicas envolvidas. Além disso, corroboram com dados prévios da literatura de que perifericamente os pacientes apresentam um índice de toxicidade relevante quando comparado a indivíduos controles e que este índice estaria relacionado a progressão do transtorno. / Bipolar disorder (BD) is characterized as a serious psychiatric disorder that presents chronic course with remarkable cognitive and functional impairment. In this thesis, we analyzed in two reviews the changes in peripheral markers of oxidative stress, neurotrophins and inflammation, discussing their involvement in systemic toxicity, as well as a possible association of these changes with the cognitive and functional impairment presented by BD patients throughout the disorder. In this sense, many studies have been performed seeking to understand the pathophysiology of this illness. Moreover, research on BD and drug development is hampered by the lack of suitable in vitro models. To counteract this, many attempts to explore patient-derived samples have been undertaken, resulting in partial reproduction of disease aspects. However, still does not exist in the literature a suitable model to understand the complexity of the BD symptoms. Therefore, the aims of this thesis was to evaluate the differential gene expression of the cell differentiation model of human neuroblastoma cell line SH-SY5Y, induced by retinoic acid, and the search for an experimental model for the evaluation of systemic toxicity in patients with BD. The differentiation model was assessed by microarray analysis and we found that the differentiated cells had increased expression of biological processes involved in neuronal development, while undifferentiated cells showed higher expression of biological processes related to cellular proliferation and maintenance. Also, genes related to the synaptic function and dopaminergic synthesis were more expressed in differentiated cells. These findings contribute to the validation of a cellular model from human source, easy handling and featuring neuronal profile; thereby aiding in the study of diseases that affect the central nervous system, including BD. In order to evaluate the toxicity profile in serum of bipolar patients, differentiated SH-SY5Y cells were treated with sera of BD patients in early and late stages of the disorder. As a result, for the first time in the literature, it has been verified the potential neurotoxicity of bipolar patients serum directly in human cells with a neuronal profile and we found that the serum of patients at late stage would present higher toxicity when compared to the control sera, causing a decrease in cell viability and a reduced neurite outgrowth density. Taken together, the findings of this thesis point to a new in vitro model to analyze the pathophysiology of BD, as well as the effect of medications and metabolic pathways involved in this disorder. Moreover, corroborate previous peripherally data found in the literature where patients would have a toxicity index compared to control subjects and that this index would be related to the progression of the disorder.

La télomérase est une ribonucléoprotéine, constituée d’un composant ARN (hTR) qui sert dematrice à l’addition des séquences télomériques aux extrémités des chromosomes et d’un composantprotéique catalytique à activité de transcriptase inverse (hTERT). La réactivation de la télomérasedans 90% des cancers compense le raccourcissement des télomères, permettant ainsil’immortalisation et la survie des cellules tumorales. Ce rôle canonique de la télomérase estaujourd’hui bien documenté. Cependant des travaux récents suggèrent que la télomérase pourraitavoir d'autres fonctions indépendantes de son rôle sur le maintien de la longueur des télomères dansla tumorigénèse et/ou la progression tumorale.Dans les neuroblastomes (NB), l’augmentation du niveau d’activité télomérase (AT) estassociée à un stade avancé de la maladie et à un mauvais pronostic. En effet, plusieurs études ontmontré que les neuroblastomes agressifs ont un niveau élevé d’AT alors que les tumeurs de bonpronostic, ont peu ou pas d’AT. En effet, les NB de stade 4S ayant la capacité de régresserspontanément ont un très faible niveau d'AT. Ces observations suggèrent que la télomérase peutjouer un rôle crucial dans le développement des NB.Afin de mieux comprendre l’implication de la télomérase dans le phénotype agressif desneuroblastes malins et dans la chimiorésistance, nous avons caractérisé les modificationsphénotypiques et génotypiques induites par l’inhibition de la télomérase via l’expression ectopiqued’un mutant dominant négatif catalytiquement inactif (DN-hTERT) dans la lignée IGR-N-91 induit unedifférenciation cellulaire de type stromale et une sensibilisation à l’apoptose en réponse à trois agentscytotoxiques (cisplatine, staurosporine, TRAIL). Cette chimiosensibilisation n’est pas la conséquenced’un raccourcissement des télomères mais probablement celui d’une modulation de l’expression decertains gènes impliqués dans la réponse apoptotique (ré-expression de la caspase 8 et de p53sauvage), suggérant une fonction non canonique de la télomérase. De plus, nous avons montréqu’hTERT régule activement l’expression de N-Myc. En effet, l’expression ectopique du mutanthTERT-DN entraîne une perte des copies surnuméraires de N-Myc conduisant à l’extinction del'expression de la protéine alors que la surexpression d’hTERT sauvage augmente au contraire lenombre de copies du gène. Cette élimination de la protéine N-Myc pourrait être le signe d’une pertedu caractère agressif des cellules tumorales comme en témoigne la diminution de l’expression de laNSE (marqueur de mauvais pronostique des NB) et l’induction du CD44 dans les cellules hTERT-DN.L’ensemble de nos résultats démontre donc un nouveau rôle majeur de la télomérase,indépendant de sa fonction canonique d’élongation des télomères, dans l’acquisition du phénotypemalin et dans la chimiorésistance des NB. Ces résultats sont importants en termes de connaissancede la biologie du NB et des possibilités thérapeutiques. En effet, ces résultats suggèrent quel’inhibition de la télomérase comme stratégie anti-cancéreuse est une approche qui présente un intérêttout particulier dans les cas de NB de stade 4 dans lesquels le taux de survie des patients reste trèsinsuffisant malgré les thérapeutiques les plus intensives. / Telomerase is a ribonucleoprotein consisting of an RNA component (hTR) that serves as atemplate for the addition of telomeric sequences at the ends of chromosomes and a proteincomponent catalytic activity of reverse transcriptase (hTERT). The reactivation of telomerase in 90%of cancers compensates the shortening of telomeres, allowing the immortalization and survival oftumor cells. This canonical role of telomerase is now well documented. However recent studiessuggest that telomerase may have other functions beyond its role in maintaining telomere length intumorigenesis and / or tumor progression.In neuroblastoma (NB), increased levels of telomerase activity (TA) is associated withadvanced disease and poor prognosis. Indeed, several studies have shown that aggressiveneuroblastomas have a high level of TA while favourable tumors, have little or no TA. Therefore, lowtelomerase activity appears to be linked with regression or maturation of NB as it can be seen in theparticular group of 4S stage neuroblastoma. These observations suggest that telomerase may play acrucial role in the development of NB.To better understand the involvement of telomerase in the aggressive phenotype of malignantneuroblasts and drug resistance, we characterized the phenotypic and genotypic changes induced byinhibition of telomerase via ectopic expression of a mutant dominant negative catalytically inactive(DN-hTERT) in a metastatic chemoresistant NB cell line IGR-N-9. Our results show that theexpression of this mutant induces a stromal-type cell differentiation a sensitization to apoptosis inresponse to three cytotoxic agents (cisplatin, staurosporine, TRAIL). The chemosensitization is not theresult of telomere shortening but probably f a modulation of the expression of certain genes involved inthe apoptotic response (re-expression of caspase 8 and wild-type p53), suggesting a noncanonicalfunction of telomerase Furthermore, we showed that hTERT actively regulates the expression ofMYCN. Indeed, ectopic expression of the inactive mutant causes a loss of supernumerary copies ofMYCN leads to the extinction of the expression of the protein, whereas overexpression of wild hTERTincreases the number of copies of the MYCN gene. The elimination of MYCN protein could be a signof a loss of the aggressiveness of the tumor cells as evidenced by the decreased expression of NSE(a marker of poor prognosis of NB) and induction of CD44 in DN-hTERT cells.Overall, our findings thus demonstrate a new role of telomerase independent of its canonicalfunction of telomere elongation in the acquisition of the malignant phenotype and drug resistance inNB. These results are important in terms of knowledge of the biology of NB and therapeuticpossibilities. Indeed, our data suggest that inhibition of telomerase as an anticancer strategy is anapproach that has a particular interest in cases of stage 4 NB in which the survival rate of patientsremains very inadequate despite the therapeutic more intensive.

Depuis la formulation de la théorie de l’immuno-surveillance en 1957 par Burnet et Thomas, le monde scientifique s’est efforcé d’identifier les cellules immunitaires impliquées dans ce processus. Les lymphocytes Natural Killer (NK) constituent une composant majeure de l’immuno-surveillance innée dans plusieurs cancers hématologiques et solides. L’activité des lymphocytes NK passe principalement par une grande variété de récepteurs avec un rôle activateur ou inhibiteur. Parmi les récepteurs activateurs présents à la surface des lymphocytes NK, le récepteur NCR3/NKp30 a un rôle majeur dans la toxicité directe contre la cellule cible et dans l’activation des cellules dendritiques.Les tumeurs stromales gastrointestinales (GIST) et le Neuroblastome (NB) sont deux tumeurs sensibles à l’immuno-surveillance par les lymphocytes NK. Dans une étude récente notre équipe a démontré que l’épissage alternatif du gène NCR3/NKp30 peut être déterminant dans la fonction NK et dans la survie des patients atteints de GIST.Afin de caractériser les lymphocytes infiltrant le GIST, nous avons effectué une recherche visant à analyser l’infiltrat des lymphocytes CD3+, des lymphocytes T régulateurs (Treg) et des lymphocytes NK dans des tumeurs GIST localisés, et corréler ces résultats à la survie des patients. Nous avons mis en évidence que, avant traitement, les lymphocytes NK sont surtout localisés au niveau des fibres trabéculaires qui entourent la tumeur, alors que les lymphocytes T sont localisé à l’intérieur de la tumeur en contact avec les cellules tumorales qui expriment HLA-I.Nous avons aussi observé que les cellules NK ont un phénotype plutôt CD56bright et migrent à l’intérieur de la tumeur après traitement par Imatinib. L’analyse de survie a mis en évidence que les lymphocytes NK et T peuvent prédire la survie sans progression (PFS). Ces résultats mettent en évidence l’importance de l’infiltrat immunitaire dans la prédiction du risque de rechute dans le GIST et surlignent l’importance de viser une réponse immunitaire dans les protocoles thérapeutiques.Nous avons ensuite déterminé la proportion de lymphocytes NK dans le sang périphérique et dans la moelle dans une cohorte de Neuroblastome (NB) localisé et métastatique : une infiltration plus important par les NK CD56bright a été observé chez les patients présentant une maladie métastatique et chez les patients avec une réponse mineure au traitement d’induction. De plus, les NK présents dans les échantillons de moelle osseuse infiltrés par les neuroblastes, présentaient une expression plus basse du récepteur NKp30. L’expression du ligand de NKp30, B7-H6, a été mise en évidence sur les neuroblastes infiltrant la moelle osseuse, et sa forme soluble, sB7-H6, a été retrouvée être positivement corrélée à l’extension de maladie et inversement à la réponse au traitement d’induction. L’analyse de l’épissage alternatif du gène NCR3/NKp30 a permis de mettre en évidence l’impact des isoformes NKp30 sur la survie sans progression chez les patients atteints de NB de haut risque en maladie minimale résiduelle après chimiothérapie d’induction. En particulier, les patients présentant un taux élevé de l’isoforme pro-inflammatoire (NKp30b) par rapport à l’isoforme immunosuppressive (NKp30c), présentent une meilleure survie sans évènement. Nous avons aussi démontré le rôle des monocytes dans l’amplification de la réponse NKp30 dépendant. Les résultats de notre recherche dans le GIST et dans le NB, deux maladies différentes mais toutes les deux sensibles aux lymphocytes NK, surlignent l’importance d’intégrer de nouvelles options thérapeutiques aptes à cibler le système immunitaire. / Since Burnet and Thomas formulated in 1957 the cancer immunosurveillance theory, the scientific world has made tremendous progress to identify the immune cells involved in this process. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of several hematological and solid malignancies. The activity of NK-cells is mainly mediated through their wide variety of receptors with activating and inhibitory functions. Among the versatile receptors present on NK cells, the activating receptor NCR3/NKp30 is a major receptor involved in both direct killing of target cells and mutual NK and dendritic cell activation.Gastrointestinal stromal tumors (GIST) and Neuroblastoma (NB) are known to be tumors sensitive to NK immunosurveillance. In a recent study we showed that alternative splicing of NCR3/NKp30 gene can affect NK cell function and GIST patient’s outcome.In order to better characterize the GIST tumor-infiltrating lymphocytes, we analyzed the CD3+, T regulatory (Treg) and NK lymphocytes infiltration within primary localized GIST tumors and we determined their prognostic value. We described that, before treatment, NK cells are mainly localized in fibrous trabeculae while T lymphocytes are in the tumor nests in HLA-I positive tumor cells contact. Moreover infiltrating NK cells displayed a secreting CD56bright phenotype, and accumulate in tumor nests after Imatinib (IM) treatment. Importantly CD3+ and NK lymphocytes independently predicted progression free survival (PFS). These results highlight the importance of the immune infiltrate in re-define the GIST risk stratification and allow enhancing the immune response in the therapeutic decisions.We next investigated the proportions of NK cells in blood and bone marrow (BM) in a cohort of localized and metastatic NB; a high proportion of CD56bright NK cells was associated with metastatic NB and with poor response to induction treatment within the metastatic NB. Moreover, infiltrated BM presented NKp30 down regulation. The expression of the NKp30 ligand, B7-H6, was found on BM neuroblasts, while the soluble protein, sB7-H6 correlated with resistance to treatment. Furthermore the transcriptional status of NKp30/NCR3 dictated the event-free survival rates of HR-NBs with minimal residual disease post-induction chemotherapy: in particular patients presenting a high proportion of the immunosuppressive isoform (NKp30c) compared to the pro-inflammatory isoform (NKp30b), presented a worse outcome. We further demonstrated the significant role of monocytes to amplify the NKp30 activation response.These researches in GIST and NB, two different but at the meantime NK-sensitive diseases support the effort to define new immunological therapeutic approaches and to determine their optimal use.

Le neuroblastome, tumeur embryonnaire d’origine neuro‐ectodermique, représente, après les tumeurs cérébrales, la tumeur maligne la plus préoccupante de l’oncologie pédiatrique. L’extrême hétérogénéité des tumeurs neuroblastiques conduit d’une part, à rechercher les mécanismes de son oncogenèse, d’autre part, à améliorer la prédiction du risque de gravité, au diagnostic de la maladie.Le travail de thèse a consisté, à l’aide d’une cohorte tumorale de patients et de lignées de neuroblastome, à rechercher les microARN impliqués dans la progression tumorale. En comparant des tumeurs de bas risque à celles de haut risque, plusieurs microARN du cluster C14MC, situés au locus 14q32.31, ont été identifiés. L’expression de ces microARN corrèle le pronostic ; les tumeurs de haut risque présentant une perte d’expression différentielle. Ainsi, l’expression de miR‐487b et miR‐410 s’est révélée être un facteur pronostique supérieur à l’algorithme de risque standard actuel (âge, stade, statut de l’amplification de l’oncogène N‐MYC). Le contexte d’empreinte génomique parentale du cluster C14MC a conduit à rechercher d’autres microARN d’intérêt sur le second cluster de microARN du génome, C19MC, lui aussi soumis à empreinte. Dans les tumeurs de haut risque, une hyper‐expression relative du miR‐516a‐5p est significativement associée au pronostic. La combinaison des niveaux d’expression de miR‐487b et miR‐516a‐5p se révèle être un facteur pronostique supérieur aux seuls microARN du cluster C14MC : elle offre une nouvelle stratification de risque.Dans les tumeurs neuroblastiques, la dérégulation d’expression serait circonscrite aux microARN des deux clusters C14MC et C19MC ainsi qu’aux gènes vicinaux DLK1 et MEG3 du locus 14q 32.31, elle résulterait d’anomalies de méthylation. Le traitement de lignées de neuroblastome de phénotype neuronal par des modulateurs de l’épigénome (5‐Azacytidine et acide phényl‐butyrique) lève l’expression des microARN du C14MC et des gènes DLK1 et MEG3. Quant aux gènes cibles des miR‐487b et miR‐516a‐5p, les recherches désignent les gènes N‐MYC, TWIST1 et TWIST2 comme candidats directs ou indirects. Ces résultats et la littérature – rapportant, dans les formes agressives de plusieurs types de cancers de l’adulte, des anomalies d’expression des microARN des clusters C14MC (hypo‐expression) et C19MC (hyperexpression) – suggèrent très fortement l’implication de ces deux clusters dans la carcinogenèse humaine. / Neuroblastoma, an embryonal tumour of neuro‐ectodermal origin, stands up with brain tumours as the most worrying cancer of paediatric oncology. The huge heterogeneity of neuroblastic tumours has led i) to find oncogenic mechanisms, and ii) to refine risk stratification of the disease. In using a tumour cohort of patients as well as human NB lines, we sought for microRNA involved in neuroblastoma tumour progression. Comparison of tumours of low‐risk to those of high‐risk resulted to identifying several microRNA composing the C14MC cluster (located within the 14q32.31 locus), whose expression was associated with prognosis; high risk tumours having a differential lower transcript level.Expression of miR‐487b and miR‐410 was a better prognostic factor than the standard algorithm based on age, stage, and N‐MYC genomic content status. As the C14MC cluster belongs to a imprinted locus, the second cluster of microRNAs so far described in the human genome as imprinted, i.e., the C19MC, was analysed: in high‐risk neuroblastoma, miR‐516a‐5p transcript level was differentially up‐regulated (contrasting to microRNAs from C14MC) and was also associated with prognosis. Combination of transcript levels of miR‐487b and miR‐516a‐5p provides a powerful prognostic factor better than only miR expression from C14MC. Therefore, new risk stratification has been proposed.In neuroblastoma, tumour expression deregulation found to be restricted to C14MC and C19MC as well as the DLK1 et MEG3 harboured by the 14q32.31 locus, should result from methylation anomalies. Epigenetic modulators (5‐AZA and PBA) resulted in a significant increase of miR from C14MC as well as DLK1 and MEG3 genes. With regards to target genes, our results point out N‐MYC, TWIST1 and TWIST2 as direct or indirect targets of miR‐487b & miR‐516a‐5p. Our data and literature – indicating relative underexpression of C14MC microRNAs and hyper‐expression of C19MC microRNAs in aggressive forms of various adult cancers – thus stress the potential involvement of the two clusters in human carcinogenesis.

Le neuroblastome (NB) est une tumeur pédiatrique du système nerveux sympathique. Des mutations activatrices du gène ALK (Anaplastic Lymphoma Kinase) ont été identifiées dans 8 % des formes sporadiques et dans des formes familiales de NB. Le gène ALK code pour un récepteur tyrosine kinase appartenant à la famille des récepteurs à l’insuline, principalement exprimé dans le système nerveux central et périphérique. Le récepteur ALK représente une cible thérapeutique pertinente dans ce cancer. Des mutations de novo du gène ALK ont également été rapportées dans une forme syndromique associant NB congénital et encéphalopathie sévère avec dysmorphie du tronc cérébral, suggérant un rôle développemental du gène ALK en plus de son implication dans l’oncogenèse.Dans ce contexte, mon projet de thèse avait pour but de déterminer le rôle du récepteur ALK muté dans l’oncogenèse du NB et le développement, principalement à l’aide de modèles murins originaux obtenus au laboratoire. J’ai ainsi largement caractérisé deux lignées de souris KI (Knock-In) Alk pour les deux mutations les plus fréquemment observées dans le NB: F1174L et R1275Q chez l’homme, correspondant à F1178L et R1279Q chez la souris.Une analyse détaillée de ces deux lignées de souris n’a pas révélé de phénotype majeur chez les souris KI AlkR1279Q hétérozygotes et homozygotes ainsi que chez les hétérozygotes KI AlkF1178L. Par contre, nous avons documenté une forte létalité post-natale des animaux KI AlkF1178L homozygotes et montré que ces nouveaux-nés présentent des troubles majeurs d’alimentation. Les homozygotes KI AlkF1178L phénocopient donc partiellement les patients encéphalopathes. La différence d’effet observé entre les animaux hétérozygotes et homozygotes suggère fortement qu’il existe un seuil d’activation du récepteur Alk compatible avec la survie.Nous avons ensuite exploré le rôle du récepteur ALK muté dans le système nerveux sympathique des souris KI Alkmut. Cette analyse a montré que l’activation du récepteur induit un excès de prolifération des neurones sympathiques de E14.5 à la naissance. Néanmoins, nous n’avons pas observé de NB chez ces animaux. En croisant ces souris avec la lignée TH-MYCN, nous avons documenté une coopération des mutations Alk avec l’oncogène MYCN pour le développement de NB. La comparaison des profils transcriptomiques des tumeurs murines MYCN et MYCN/Alkmut a révélé que l’expression de l’oncogène Ret (codant également un récepteur à activité tyrosine kinase) était fortement induite par l’activation du récepteur Alk. Le traitement des souris par un inhibiteur de l’activité kinase du récepteur Ret a montré une diminution de la taille des tumeurs suggérant que le gène Ret joue un rôle majeur dans l’oncogenèse induite par le récepteur Alk muté. Par ailleurs, l’induction de l’expression du gène RET par le récepteur ALK muté dans les NB a été confirmée dans des lignées et des tumeurs humaines.Afin de déterminer le mécanisme par lequel l’activation du récepteur ALK aboutit à la régulation de l’expression du gène RET des expériences ont été effectuées sur des lignées humaines de NB dans lesquelles le récepteur ALK peut être activé ou inactivé. Ce travail a montré que l’expression du gène RET est dépendante de l’axe ALK-ERK-ETV5. En effet, la modulation de l’activité du récepteur ALK affecte l’expression des gènes ETV5 et RET. Cet effet est dépendant de l’activation de la voie MEK/ERK. Par ailleurs, ETV5 active l’expression du gène RET. Afin de confirmer le rôle de Ret dans l’oncogenèse dépendante du récepteur Alk, nous avons croisé des souris portant une mutation activatrice de Ret avec les souris TH-MYCN. Nous avons ainsi mis en évidence que le récepteur Ret activé coopère avec l’oncogène MYCN dans le développement de tumeurs et que ces tumeurs sont des NB présentant des caractéristiques très semblables à celles des tumeurs MYCN/Alkmut. Le gène Ret apparaît donc comme une cible essentielle du récepteur Alk muté dans l’oncogenèse du NB. / Neuroblastoma (NB) is a pediatric tumor arising from the sympathetic nervous system. Activating mutations of the ALK gene have been observed in around 8 % of sporadic neuroblastoma as well as in familial cases. The ALK gene encodes a tyrosine kinase receptor of the insulin receptor super-family. It is mainly expressed in the central and peripheral nervous system. The ALK receptor represents a therapeutic target in this cancer. De novo ALK mutations have also been reported in a syndrome associating congenital NB and severe encephalopathy with abnormal shape of the brainstem, suggesting a developmental role for the ALK gene in addition to its implication in oncogenesis.In this context, my PhD project was to determine the role of the mutated ALK receptor in NB oncogenesis and in development, mainly with original mouse models obtained in the laboratory. I extensively characterized two knock-in (KI) Alk mouse lines with the two mutations that are most frequently observed in NB: F1174L and R1275Q in human and F1178L and R1279Q in mouse.A detailed analysis of these two mouse lines showed that the KI AlkR179Q heterozygous and homozygous mice as well as the KI AlkF1178L heterozygous mice do not show striking clinical signs. On the contrary, we documented a high postnatal lethality for KI AlkF1178L homozygous mice and showed that these pups presented with a dramatic reduced milk intake. Thus, the KI AlkF1178L homozygous mice partially phenocopy the human patients with encephalopathy. The difference of phenotype between the heterozygous and the homozygous KI AlkF1178L mice highly suggest a threshold of activity of the Alk receptor compatible with survival.We then explored the role of the mutated ALK receptor in the sympathetic nervous system of the KI Alkmut mice. This analysis showed that the activation of the receptor induces an excess of proliferation in sympathetic neurons from E14.5 to birth. However, we could not observe NB in these animals. We next bread these mice with the transgenic TH-MYCN line. We documented cooperation between Alk mutations and the MYCN oncogene to induce NB. Comparison of transcriptomic profiles of MYCN vs MYCN/Alkmut tumors revealed that the expression of the Ret oncogene (encoding a tyrosine kinase receptor) was strongly induced by the activation of the Alk receptor. Besides, the induction of the expression of the RET gene by the mutated ALK receptor in NB was confirmed in human cell lines and tumors.In order to determine the mechanism by which the activation of the ALK receptor regulates RET gene expression, experiments were done on human NB cell lines in which the ALK receptor can be activated or inactivated. This work showed that RET gene expression is dependent of the ALK-ERK-ETV5 axis. Indeed, the modulation of the ALK receptor activity affects gene expression of ETV5 and RET. This effect is dependent of the activation of the MEK/ERK pathway. Besides, ETV5 increases RET gene expression. In order to confirm the role of the Ret receptor in oncogenesis driven by the mutated Alk receptor, we bread mice bearing an activating mutation of the Ret gene with the TH-MYCN mice. We showed that the activated Ret receptor cooperates with the MYCN oncogene in tumor formation and that these tumors are NB presenting with characteristics very close to MYCN/Alkmut tumors. Thus, the Ret gene appears to be an essential target of the mutated Alk receptor in NB oncogenesis.

The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-10-30T11:55:11Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_EfeitoAntineoplasicoComposto.pdf: 1843144 bytes, checksum: 2c1a12d9e05779f464daf3de28979f2c (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-10-30T13:57:12Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_EfeitoAntineoplasicoComposto.pdf: 1843144 bytes, checksum: 2c1a12d9e05779f464daf3de28979f2c (MD5) / Made available in DSpace on 2014-10-30T13:57:12Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_EfeitoAntineoplasicoComposto.pdf: 1843144 bytes, checksum: 2c1a12d9e05779f464daf3de28979f2c (MD5) Previous issue date: 2014 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neuroblastoma é a neoplasia mais frequentemente diagnosticada na infância. O termo é comumente usado para se referir a uma ampla variedade de tumores neuroblásticos, incluindo os neuroblastomas, ganglioneuroblastomas e ganglioneuromas. Estimativas mostram que 8 milhões de crianças até 15 anos de idade por ano são atingidas por esta neoplasia, onde 80% dos casos são acometidos em até 4 anos de idade, o tumor é derivado de células malignas embrionárias advindas de células neuronais primordiais, desde gânglios simpáticos até medula adrenal e outros pontos. Neste estudo, foi avaliado o potencial citotóxico do composto 4,2´,3´,4´ tetrametoxi chalcona em modelo in vitro de neuroblastoma B103 de rato. Foram preparadas soluções estoques da droga a 50mM em dimetilsulfóxido (DMSO) e armazenadas a -20ºC para o preparo de novas concentrações (150μM, 100 μM, 75 μM e 50 μM). A viabilidade celular foi testada a partir de cultura de células da glia do córtex de rato e de neuroblastoma b103. Ensaios de migração celular e formação de colônias também foram realizados. Para a análise estatística foi realizado a análise de variância um critério (ANOVA) seguido pelo teste de Tukey, utilizando-se o programa BioEstat 5.0. Na avaliação do efeito citotóxico das chalconas, foi observado que o tratamento com o composto 4,2`3`4´- tetrametoxi chalcona não demonstrou nenhum efeito citotóxico contra células normais do córtex de rato para as concentrações testadas, enquanto que em culturas de células de neuroblastoma B103 foi demonstrado que esta droga promove a morte celular de forma significativa. / Neuroblastoma is the most frequently diagnosed malignancy in childhood. The term is commonly used to refer to a wide variety of neuroblastic tumors, including neuroblastomas, ganglioneuromas and ganglioneuroblastomas. Estimates show that 8 million children under 15 years of age per year are affected by this cancer, where 80% of cases are affected in up to 4 years of age, the tumor is malignant cells derived from embryonic arising from primary neuronal cells, since nodes adrenal medulla and sympathetic to other points. In this study, we assessed the cytotoxic potential of the compound 4,2 ', 3', 4'- tetrametoxchalcone in vitro model B103 rat neuroblastoma. Drug stock solutions were prepared at 50mM in dimethylsulphoxide (DMSO) and stored at - 20 ° C for the preparation of new concentrations (150μM, 100 mM, 75 mM and 50 mM). Cell viability was assayed from culture of glial cells from rat cortex. Cell migration assays and colony formation were also conducted. For statistical analysis, analysis of variance criterion (ANOVA) followed by Tukey test using the BioEstat 5.0 program was conducted. In the evaluation of cytotoxic effect of chalcones, it was observed that treatment with the compound 4,2’,3’,4’- tetrametoxchalcone showed no cytotoxic effect against normal cells of rat cortex for the concentrations tested, whereas in cell cultures neuroblastoma B103 was shown that the drug promotes cell death significantly.