Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 99-118) Also available online.

Made available in DSpace on 2015-07-22T20:50:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-29. Added 1 bitstream(s) on 2015-08-11T03:26:07Z : No. of bitstreams: 1 Publico-12659.pdf: 1883382 bytes, checksum: 33bb0f719078a000b15563f9e2debf90 (MD5) / A familia de genes Wnt esta envolvida na carcinogenese de diversos tecidos e na embriogenese. Para avaliar as vias Wnt canonica e nao-canonica no endometrio atrofico e no cancer de endometrio, avaliamos a expressao imuno-histoquimica do Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) e ƒÀ-catenina. O tecido endometrial foi retirado de pecas cirurgicas de pacientes operadas entre 1995 e 2005 na Escola Paulista de Medicina . UNIFESP e foram divididas em dois grupos: grupo A, endometrio atrofico (n=15) e grupo B, adenocarcinoma de endometrio (n=45). A imunorreacao do Wnt1, FZD1, Wnt5a, FZD5 and ƒÀ-catenina foi analisada em escores em cada grupo, individualmente. A expressao do Wnt1, FZD1 e Wnt5a nao teve associacao significante entre os grupos. Associacao significante foi observada entre os grupos para a expressao do FZD5 (p = 0,001). A proporcao do FZD5 positivo foi significativamente maior no grupo A (80,0%) comparado ao grupo B (31,1%). Analisando a curva de sobrevida para o FZD5 no grupo B, nao encontramos associacao significante entre as mulheres positivas e negativas. A expressao da scatenina nao foi significante, sendo a expressao para os grupos A e B 100% e 95.6%, respectivamente (p = 1,000). FZD5 tem menor expressao no adenocarcinoma de endometrio tipo I quando comparado ao endometrio atrofico. / The Wnt family is involved in tumorigenesis of several tissues as well in embriogenesis. In order to analize the canonical and noncanonical Wnt pathway in atrophic endometrium and endometrial adenocarcinoma, we evaluated the immunohistochemical expression of Wnt1, Frizzled-1 (FZD1), Wnt5a, Frizzled-5 (FZD5) and ƒÀ-catenin. Endometrial specimens were obtained from surgeries performed between 1995 and 2005 and the patients were divided in two groups: Group A, atrophic endometrium (N = 15); Group B, endometrial adenocarcinoma (N = 45). Immunoreactivity for Wnt1, FZD1, Wnt5a, FZD5 and ƒÀ-catenin was scored for each group. For the expression of Wnt1, FZD1 and Wnt5a, no significant association was observed between the groups. A significant association was observed between the groups for the FZD5 expression (p = 0.001). The proportion of FZD5 positive women was significantly higher for group A (80.0%) compared to group B (31.1%). Regarding the survival curve for FZD5 at group B, we found no significant association between positive and negative women. No significant association was observed between s-catenin expression and the patient group since the expression for groups A and B were 100% and 95.6%, respectively (p = 1.000). FZD5 is downregulated in type I endometrial adenocarcinoma when compared to atrophic endometrium. / TEDE / BV UNIFESP: Teses e dissertações

According to the cancer stem cells (CSCs) hypothesis, CSCs are responsible for the treatment failures. CSCs are a subset of cells possessing stemness properties within the heterogeneous tumor mass. Therapeutic intervention on Wnt signaling is of our great interest because an aberrant Wnt signaling is an important driver to maintain the potency of CSCs. In nasopharyngeal carcinoma (NPC), deregulated expression of the Wnt signaling components is frequently observed. ICG-001 is a selective Wnt modulator (CBP antagonist) that specifically interrupts the interaction between β-catenin and CBP, thereby encourages the interaction between β-catenin and p300 and the subsequent differentiation and reduction of the CSCs subset. For this reason, the present study aimed to evaluate the therapeutic potential of ICG-001 in NPC. Results showed that ICG-001 inhibited both the migration of the NPC cells and the formation of tumor spheres. In the first part of the mechanistic studies (Chapter 3), ICG-001 was found to restore the expression of miR-150 in NPC cells. MiR-150 was further found to directly reduce CD44 expression and inhibit NPC cell migration. In the second part of the mechanistic studies (Chapter 4), ICG-001 was found to reduce the expression of Evi1 in NPC cells. The effect was accompanied with the inhibition of both the NPC cells migration and the tumor spheres formation. Two molecular axes, namely miR-96/Evi1/miR-449a and survivin/Evi1/miR-449a, were found to be involved in the inhibition of the tumor cell migration and spheroids formation. The therapeutic potential of using this CBP antagonist (ICG-001) in NPC, namely the in vitro and in vivo efficacy of ICG-001 combined with cisplatin, was examined (Chapter 5). Concurrent treatment of ICG-001 and cisplatin exhibited a synergistic inhibition on the in vitro growth and the tumor sphere forming capacity of NPC cells as well as the growth of NPC xenografts. Taken together, results presented in this thesis suggested that ICG-001 (PRI-724 is the analog of ICG-001 currently used in clinical trials) has a therapeutic potential in NPC.

Orientadores: Lucia Elvira Alvares, Jose Xavier Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T10:17:43Z (GMT). No. of bitstreams: 1 Sobreira_DeboraRodrigues_M.pdf: 2481929 bytes, checksum: 2cb1105ccc78322b5f11f4528108d2fc (MD5) Previous issue date: 2009 / Resumo: Splicing Alternativo é um mecanismo importante para expandir a diversidade protéica em eucariotos. Este processo permite a produção de diferentes mRNAs a partir de uma mesma molécula de pré-RNA e é freqüentemente utilizado pelos genes envolvidos no desenvolvimento embrionário. O gene Oapper1 (Opr1) é um importante modulador da via de sinalização Wnt, atuando em diversos processos como especificação do tecido neural, morfogênese cefálica e desenvolvimento do coração e olho. Entre seus parceiros estão as '1lOléculas Dishevelled, o fator de transcrição TCF-3 (ambas as moléculas envolvidas na sinalização Wnt) e Dbf-4 (regulador do ciclo celular). Considerando que Dpr1 possui uma estrutura modular e interage com diferentes parceiros moleculares através de diferentes domínios estruturais, esta molécula poderia utilizar a maquinaria de Splicing Alternativo para combinar diferentes domínios e conseqüentemente ampliar suas funções biológicas. Neste estudo, descrevemos uma nova Variante do gene Opr1, identificada inicialmente no transcriptoma de camundongo utilizando ferramentas de Bioinformática. Esta nova Variante é maior em 111 pb em relação à codificada pela seqüência referência de RNAm para Dpr1 RefSeq, as quais são denominadas, respectivamente, como Variante A e Variante B. Estes transcritos variantes são gerados por dois sítios aceptores de Splicing distintos presentes no início do exon 4. O segmento exclusivo da Variante A codifica 37 aminoácidos localizados na região onde Opr1 se associa ao fator transcricional TCF-3. Uma análise comparativa do lócus de Opr1 entre diversos vertebrados (peixe, anfíbio, galinha, camundongo e humano) revelou que ambos os sítios aceptores de Splicing são conservados nos tetrápodas, enquanto que em peixe apenas um sítio é encontrado. Ensaios de RT-PCR confirmaram nossos resultados obtidos em Bioinformática. Além disso, demonstramos que ambas as Variantes são co-expressas ao longo do desenvolvimento de galinha, sugerindo que a concentração relativa dessas moléculas pode ser importante para a sua função. Finalmente, análises de pressão seletiva foram realizadas para a molécula de Dpr1. Apesar de não se confirmar a presença de seleção positiva ao longo da proteína Dpr1, o exon 4 parece estar sob pressão seletiva mais relaxada quando comparado aos outros exons. Nossos resultados são consistentes com a hipótese de que o mecanismo de Splicing Alternativo atua acelerando a evolução, reduzindo a seleção negativa. / Abstract: Alternative splicing is an important mechanism to expand protein diversity in eukaryotes. This process allows the production of different mRNAs from a single coding sequence and is frequentfy used by genes involved in development. Oapper 1 (Opr1) is an important rnodulator of Wnt signalling, working in several developmental processes, such as neural tissue specification, head morphogenesis, heart and eye development. While its interaction with Oishevelled is known to modulate Wnt signalling both in vivo and in vitre, the interaction wrth other molecules is required to mediate its multiple biological functions. Considering that Dpr1 has a modular structure that mediates its interaction with different partners through different structural domains, this molecule could greatly benefit from alternative splicing in order to combine different domains and consequently amplify its biological functions. In the present study we describe a new Opr1 isoform that was initially identified in the mouse transcriptome using bioinformatic tools. This isoform is 111 pb longer than the one encoded by the RefSeq mRNA for Opr1, here named O and E isoforms, respectively. The variant transcripts are generated through two distinct acceptor splice sites in exon 4. The segment exclusive of the O isoform is in frame and encodes 37 residues located in a variable region of Oprl exon 4, known to be necessary for the interaction with the transcriptional factor Tcf3. comparative analysis of the Opr1 locus among fish, frog, chicken, mouse and human revealed that in tetrapods two acceptor splice sites are conserved in the beginning of the exon 4, while in fish a single acceptor splice site is found. RT-PCR using species-specific primers confirmed the expression of the O and E isoforms in tetrapods while in fish only the O isoform was detected. In addition, we showed that the Opr1 isoforms are coexpressed throughout chicken development, suggesting that the relative concentration of these molecules may be important for their functionality. Finally, even though no evidence of positive selection was detected for the entire Dpr1 protein, exon 4 seems to be under more relaxed selective pressure than the other exons. These results are consistent with the notion that alternative splicing can act as a mechanism for opening accelerated paths of evolution by reducing negative selection pressure. / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

O meduloblastoma (MB) é o tumor cerebral maligno mais comum na infância. A formação/progressão desta neoplasia foi associada a alterações moleculares, que inclui a desregulação da via de sinalização Wingless (Wnt), responsável pelo desenvolvimento embrionário. Além disso, as proteínas da família Aurora-quinases (A, B e C) têm sido amplamente estudadas, uma vez que a Aurora A e B foram encontrados hiperexpressas em diversas neoplasias, como o MB. Estudos recentes mostraram que existe uma associação entre a Via Wnt e as Aurora-quinases. No entanto, poucos trabalhos foram realizados para confirmar essa associação. Ademais, não existem trabalhos que relatem os efeitos do AMG900, um pan-inibidor de aurora-quinases, em MB, dando enfoque na regulação da via Wnt. Assim, o objetivo deste trabalho foi avaliar a modulação da via Wnt pelo inibidor AMG900 nas linhagens celulares de meduloblastoma pediátrico. Foram realizados os ensaios de PCR convencional, sequenciamento, qRT-PCR, transfecção transiente, ensaio clonogênico, Western Blot e ciclo celular. As linhagens celulares UW402, UW473 e ONS-76 não apresentaram mutações no éxon 3 do gene CTNNB1 (?-catenina) e no éxon 15 do gene APC. Não foi observada uma expressão significativa de CTNNB1, confirmando que as linhagens não possuíam a via Wnt ativa. Com isso foi necessário a transfecção transiente com a ?- catenina. Após este ensaio, houve um aumento da expressão de CTNNB1, Ciclina D1 e CMyc nas três linhagens, o que não ocorreu com as Auroras A e B. No ensaio clonogênico foi observado uma redução do número de colônias nas linhagens UW473 e ONS-76. Observou-se um aumento da expressão proteica da ?-catenina, da Aurora A e B na UW473, o que ocorreu somente com a ?-catenina na linhagem ONS-76. Após o tratamento com o AMG900 ocorreu uma diminuição da expressão proteica de ?-catenina, da Aurora A e B em ambas as linhagens. A transfecção não alterou o percentil celular em G2/M na UW402 e UW473. Já na ONS-76 houve um aumento significativo em G2/M, e o AMG900 potencializou esse bloqueio apenas nessa linhagem. Os resultados sugerem que pode haver alguma relação entre a inibição das proteínas Aurora-quinases e a expressão de proteínas da via Wnt. / Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Tumor formation/progression has been associated to molecular alterations that include dysregulation of signaling pathway Wingless (Wnt), responsible for embryonic development. In addition, cell cycle proteins Aurora-kinase (A, B and C) have been widely studied since Aurora A and B were found overexpressed in many cancers such as MB. Recent studies show that there is an association between Wnt pathway and Aurora kinase proteins. However, few studies have been conducted to confirm this association. Moreover, there are no studies reporting the effects of AMG900 in MB, by focusing on the regulation of the Wnt pathway. The aim of this study is to evaluate Wnt pathway modulation by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell lines. Conventional PCR, sequencing, qRT-PCR, transient transfection, clonogenic assay, Western Blot and cell cycle assays were performed. UW402, UW473 and ONS-76 cell lines did not present mutations in exon 3 of CTNNB1 gene and exon 15 of APC gene. There was no significant expression of CTNNB1 and their target genes in these cell lines, confirming that they did not have Wnt pathway activated. Considering this, transient transfection was necessary. After this trial, there was an increase in expression of CTNNB1 gene and its target genes Cyclin D1 and C-Myc in the three cell lines, which was not observed in Aurora kinases. Furthermore, in the clonogenic assay, a reduction in the number of colonies in UW473 and ONS-76 cell lines was observed. It was also observed an increase in ?-catenin protein, Aurora A and B in UW473 cell line, but not in ONS-76 cell line. However, after treatment there was a decrease in protein expression of ?-catenin, Aurora A and B in both cells. Transfection did not change the cellular percentile in G2 / M in UW402 and UW473. In ONS-76 there was a significant increase in G2 / M, and the treatment with AMG900 potentiated this block only in this cell line. Results suggest that there may be some relation between the inhibition of Aurora kinase protein and protein expression in Wnt pathway.

La voie de signalisation Wnt/β-caténine (Wnt canonique) est impliquée dans une grande variété de fonctions biologiques, entre autres dans l’établissement des axes embryonnaires, l’organogenèse et l’homéostasie de cellules souches adultes. En absence de signaux Wnt, un complexe multiprotéique comprenant le suppresseur de tumeur Adenomatous Polyposis Coli (APC) marque la β-caténine pour la dégradation protéasomique. L’activation de la voie Wnt canonique induit un arrêt de la dégradation de la β-caténine, qui s’accumule dans le noyau, où elle active l’expression de gènes cibles de Wnt. Au cours de la myogenèse embryonnaire, processus pendant lequel le muscle squelettique est formé, une partie des cellules pluripotentes du dermomyotome acquièrt l’identité musculaire, se différencie et forme les myofibres, les unités fonctionnelles du muscle squelettique. La myogenèse n’est pas confinée qu’à la période embryonnaire. En effet, elle peut être réactivée dans le muscle adulte suite à une lésion ou dans certaines conditions pathologiques. Dans ce contexte, les cellules souches du muscle squelettique, appelées cellules satellites, sortent de leur quiescence et génèrent des progéniteurs myogéniques qui prolifèrent et se différencient en formant de nouvelles myofibres pour réparer le tissu. De plus, une partie des progéniteurs myogéniques retournent à l’état quiescent, renouvelant ainsi la population de cellules souches résidentes. Le rôle de la voie de signalisation Wnt/β-caténine dans l’engagement des cellules pluripotentes du dermomyotome vers le destin myogénique demeure méconnu. De même, la fonction de la voie Wnt canonique dans les cellules satellites au cours de la régénération du muscle squelettique adulte reste à l’heure actuelle controversée, car différentes approches sont parvenues à des conclusions contradictoires. Grâce à des modèles génétiques murins, nous avons caractérisé le rôle précis de la voie Wnt canonique au cours de la myogenèse embryonnaire et adulte. Nous montrons in vivo que l’hyperactivation constitutive de la voie de signalisation Wnt/β-caténine induite par l’inactivation conditionnelle d’APC, le principal régulateur négatif de la cascade, se traduit par un défaut majeur de formation et de régénération du muscle squelettique. Nos résultats ex vivo et in vitro démontrent que l’hyperactivation de la voie Wnt canonique altère la progression du cycle cellulaire et entraîne la mort par apoptose. De plus, l’inactivation conditionnelle de la β-caténine n’affecte pas la prolifération des progéniteurs myogéniques mais perturbe leur différenciation. Globalement, nos résultats suggèrent deux rôles différents de la voie de signalisation Wnt/β-caténine dans le muscle squelettique. D’une part, l’inhibition de la voie Wnt canonique est nécessaire au cours de l’initiation de la myogenèse pour permettre l’engagement myogénique des cellules pluripotentes du dermomyotome et l’activation des cellules satellites. D’autre part, la voie de signalisation Wnt/β-caténine est requise à la fois dans les progéniteurs musculaires embryonnaires et adultes pour leur différenciation et la formation des myofibres. / The Wnt/β-catenin signaling pathway, also called canonical Wnt signaling, is implicated in a large variety of biological processes, including embryonic axis determination, organogenesis and adult stem cells homeostasis. Canonical Wnt signaling regulates the stability of β-catenin, a transcriptional co-activator that, in absence of Wnt ligands, is targeted to proteasomal degradation by a multiproteic complex comprising the Adenomatous Polyposis Coli (APC) tumor suppressor. Activation of canonical Wnt signaling blocks β-catenin degradation and results in its accumulation into the nucleus, where it induces the expression of Wnt target genes. During embryonic myogenesis, the process of skeletal muscle formation, a proportion of pluripotent dermomyotomal cells restrict their fate to acquire a myogenic identity and differentiate into contractile myofibers, the functional units of skeletal muscle. Myogenesis can take place also in adult skeletal muscle. Indeed, upon acute injury or in pathological conditions, quiescent muscle-specific stem cells, called satellite cells, become activated and give rise to myogenic progenitors that massively proliferate, differentiate and fuse to form new myofibers and restore tissue functionality. In addition, a proportion of proliferating progenitors returns back to quiescence and replenish the pool of satellite cells in order to maintain the regenerative potential of skeletal muscle. The role of canonical Wnt signaling in the cell fate choice that drives multipotent dermomyotomal cells toward the myogenic lineage remains elusive. Similarly, a possible involvement of the Wnt/β-catenin cascade has been hypothesized in satellite cells during adult skeletal muscle regeneration, but different approaches came to contradictory results. In this study, we use genetic mouse models to investigate the precise role of canonical Wnt signaling in embryonic and adult myogenesis. In vivo constitutive overactivation of Wnt/β-catenin signaling following conditional deletion of APC, the major intracellular negative regulator of the pathway, results in complete abrogation of skeletal muscle formation and regeneration. By combining ex vivo and in vitro approaches, we show that canonical Wnt signaling hyperactivation alters cell cycle progression and results in programmed cell death. Conversely, conditional inactivation of β-catenin does not perturb the proliferative ability of myogenic progenitors but rather affects their differentiation. Collectively, our results demonstrate at least two distinct roles of the Wnt/β-catenin cascade in skeletal muscle. First, during myogenic initiation, canonical Wnt signaling must be inhibited to allow proper activation of myogenesis, in particular to elicit myogenic commitment of dermomyotomal cells and activation of adult satellite cells. Second, in myogenic progression, the Wnt/β-catenin pathway is required in both embryonic and adult muscle progenitors for proper differentiation and myofibers formation.

La voie de signalisation WNT/PCP, couramment associée à la polarité planaire cellulaire, joue un rôle fondamental dans la morphogenèse chez les vertébrés. Parmi les différents composants protéiques de la voie WNT/PCP, on retrouve la protéine tyrosine kinase 7 (PTK7), dont les fonctions restent encore très peu décrites. Au cours de ma thèse, j’ai montré que PTK7 interagissait avec le récepteur tyrosine kinase ROR2. Ce complexe, après fixation du ligand WNT5A, induit la migration de fibroblastes embryonnaires murins via l’activation de JNK. Au cours du développement embryonnaire du xénope, Ptk7 interagit de manière fonctionnelle avec Ror2, et contrôle l’expression de la protocadhérine Papc ainsi que la morphogénèse. De plus, en utilisant une approche de Tissue MicroArray, réalisée sur des patients atteints de cancers colorectaux, j’ai pu montrer que PTK7 était retrouvé surexprimé chez 34% des patients, et que cette surexpression était un facteur de mauvais pronostic. Dans des lignées cellulaires issues de cancers colorectaux, la suppression de PTK7 par shRNA entraine une diminution de la migration des cellules tumorales, mais n’impacte pas leur prolifération et leur résistance aux drogues anticancéreuses. Dans un modèle de xénogreffe murin, la suppression de PTK7 induit une diminution du développement tumoral et l’expression de ce dernier, dans des cellules négative pour PTK7, entraine une augmentation de l’apparition de métastases chez les animaux injectés. Ce travail apporte de nouveaux éclaircissements sur le du récepteur PTK7 dans la voie de signalisation WNT/PCP, et le définit comme potentiel biomarqueur et cible thérapeutique dans le cancer colorectal. / The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. We show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells and physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a and that Wnt5a stimulates the release of the Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. Moreover, using a Tissue MicroArray produced from CRC patients we correlated PTK7 expression with pathological features and patient outcome. PTK7 was significantly up-regulated in CRC tissue, and its overexpression was found in 34% of patients. In CRC cell lines, shRNA PTK7 reduced migration, but did not affect cell proliferation and resistance to drugs. In a xenograft mouse model, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. This work reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements and define PTK7 expression as a potential prognostic biomarker and a novel therapeutic target in CRC.

Les premières étapes de l’évolution animale restent obscures mais peuvent toutefois être appréhendées par l’étude comparative du développement des animaux basaux comme les éponges, les cnidaires ou les cténophores. Une des innovations majeures dans l’évolution des formes animales est l’apparition de l’épithélium, classiquement considérée comme une synapomorphie des eumétazoaires. Les homoscléromorphes sont les seules éponges à partager avec Eumetazoa la présence d’un véritable épithélium avec notamment une membrane basale contenant du collagène de type IV. Dans ce clade, la recherche des mécanismes épithéliaux sous-tendant le développement a pour enjeu la meilleure compréhension de leur origine et de leur importance dans l’évolution animale. Le travail de cette thèse a consisté à caractériser chez Oscarella lobularis des outils moléculaires responsables des morphogenèses épithéliales. Trois d’entre eux ont été étudiés chez l’adulte. Le premier, la voie WNT canonique, est capable d’induire l’invagination de l’épithélium externe de l’éponge, ce qui n’est pas sans rappeler ce même rôle dans d’autres contextes épithéliaux d’eumétazoaires. Le deuxième est la voie WNT non canonique ou « voie PCP », qui, quand elle est bloquée, empêche l’invagination initiée par la voie canonique. Enfin, le troisième outil est un membre de la famille des gènes à boîte T, OlTbx qui s’exprime spécifiquement dans l’épithélium après l’invagination sus-mentionnée. Cette expression rappelle des expressions d’autres gènes Tbx dans le feuillet endomésodermique invaginé lors de la gastrulation des eumétazoaires. L’invagination semble utiliser une partie d’un même programme génétique dans la gastrula des Eumetazoa et dans l’adulte des Homoscleromorpha. Mes résultats ouvrent des perspectives intéressantes concernant l’éventuelle reconnaissance d’un stade gastrula chez les éponges, point de discorde de la zoologie classique. / The first steps of animal evolution remain obscure but, nevertheless can be better understood by comparative studies of the most basally branching animals, such as sponges, cnidarians and ctenophores. Epithelium is one of the major innovations in the evolution of animal forms and is generally considered as one of the synapomorphies of Eumetazoa. The homoscleromorphs are the only sponges, with Eumetazoa, to have a true epithelium with a basal membrane containing type IV collagen. In this clade, the investigation of epithelial processes underlying development would give insights into their origin and their importance in animal evolution. The aim of my work was to characterize molecular tools involved in epithelial morphogenesis in Oscarella lobularis. I was able to characterize three of those molecular tools. The first one is the canonical WNT pathway inducing invagination of the external epithelium of the sponge, reminiscent of the same function in other epithelial contexts in Eumetazoa. The second one is the non-canonical WNT pathway or “PCP pathway” which blocks invagination when it is inhibited. The third one is a member of the T-box genes family, OlTbx, specifically expressed in the epithelial layer formed by the above-mentioned invagination. Similarly, other Tbx genes are expressed in the endomesodermal layer during eumetazoan gastrulation. Invagination processes involved in both eumetazoan gastrula and homoscleromorph adult tissue seem to share a part (WNT/Tbx) of a common genetical program. My results provide new investigation prospects, in order to answer the difficult question of the origin of gastrulation in sponges.

Des mutations du gène STK11 furent initialement décrites comme responsable du syndrome Peutz-Jeghers, dont la gravité est lliée à une incidence accrue d’apparition de tumeurs. Le produit de ce gène, la sérine/thréonine kinase LKB1, a une expression ou une activité catalytique réduite, voir perdue, consécutivement à des mutations somatiques dans plusieurs types de cancer mais principalement du poumon (30% des NSCLC). Cette kinase est considérée de ce fait comme un suppresseur de tumeur d’importance. Les mécanismes moléculaires responsables de sa propriété suppresseur de tumeur restent à identifier. En effet, alors que sa fonction dans le métabolisme cellulaire, au travers de l’activation de la kinase AMPK, fut longtemps privilégiée, elle est actuellement remise en cause au profit de sa fonction de régulatrice de la signalisation Wnt canonique. Mes travaux de thèse confortent cette éventualité dans le cas des tumeurs pulmonaires (NSCLC). En effet, parmi les deux complexes fonctionnels que forme LKB1 avec les pseudokinases STRADα ou β, mes résultats démontrent que seul celui impliquant STRADβ intervient dans la régulation de la voie Wnt. Pour cela, le complexe LKB1/STRADβ se localise au niveau du cil primaire et participe à l’activation de la kinase MARK3. Ces résultats, étayés par un modèle murin invalidé pour STRADβ ainsi que l’analyse, a posteriori, de bases de données transcriptomiques adossées aux données cliniques de patients atteints de NSCLC, suggèrent que l’activité suppresseur de tumeur de LKB1 est associée à sa localisation et à sa fonction au niveau du cil primaire en participant à l’activation de MARK3 et à la régulation de la signalisation Wnt canonique. / STK11 gene mutations were originally identified as responsible for the Peutz-Jeghers syndrome of which severity is mainly related to an increase incidence of tumor development. The product of this gene the serine/threonine kinase LKB1 gets its activity or its expression reduced, sometimes even lost, following somatic mutations in several types of cancer such as pancreas, liver but mainly from lung. Indeed, almost 30% of non-small cell lung carcinoma (NSCLC) does not express anymore or only an inactive form, has led to consider this kinase as tumor suppressor of importance. While there is no doubt of the involvement of its catalytic activity molecular mechanisms responsible for its tumor suppressor properties remain to be identified. Indeed, whereas its function as regulator of cellular metabolism through AMPK has been favor for a while, it is currently re-assess to benefit to its regulator function on canonical Wnt signaling. My thesis work, reinforce this eventuality in NSCLC. Indeed, among the two functional complexes formed by LKB1 through its association with STRADα or β pseudokinases, my results show that only the complex related to STRADβ is involved in the canonical Wnt pathway regulation. For that, LKB1/STRADβ complex localizes at primary cilia and participates to MARK3 kinase activation. These results strengthened by a STRADβ knockout mouse model and an a posteriori transcriptomic analysis of lung adenocarcinoma patient datasets related to their clinical records, suggest that LKB1 tumour suppressor activity is associated with its localization and its function at primary cilia participating in the activation of MARK3 and thus regulation of canonical Wnt signaling.

Ces dernières années le rôle du stroma tumoral (microenvironnement) dans la progression tumorale. De même le rôle des cellules endothéliales et de la néo-angiogenèse a été illustré dans de multiples études conduisant à la mise en place des thérapeutiques anti-angiogéniques. Cependant il est possible qu’au delà de leur simple rôle comme transporteur d’oxygène et de nutriments les cellules endothéliales jouent un véritable rôle dans la biologie tumorale en sécrétant des substances bioactives (cytokines, microparticules…). Ces médiateurs sont les acteurs actifs d’un dialogue entre cellules tumorales et cellules du stroma. Dans ce travail de thèse nous nous sommes intéressés au rôle particulier des microparticules. Nous avons pu montrer que les microparticules des cellules endothéliales avaient un effet pro-tumoral sur les cellules des cancers de l’ovaire et du sein. Elles étaient capable d’induire une tradition epithélio-mésenchymateuse. Dans la seconde étude nous avons montré que les cellules tumorales sécrétaient des microparticules capable d’activer la voie de signalisation ont/beta-catenin dans les cellules endothéliales par le recrutement de Rac1 et PAK. / In our study, we showed that microparticles participate to a complex dialogue between cancer and ECs. Our main finding showed the ability of MPs mediated cross-talk between cancer and ECs to functionalize an activated angiocrine pro-tumoral endothelial niche. Using endothelial Akt activation as a readout, we were able to differentiate MPs from cells with mesenchymal from cells with epithelial traits. Our data showed that MPs from mesenchymal-like cell lines were able to promote an activation of ECs through Akt phosphorylation, compared to MPs from epithelial-like cell lines. The overexpression of Arf6 in activated ECs is associated with quantitative changes of EC-MPs. Additionally, we were interested in determining the mechanisms that derive the activation of ECs toward supporting tumor growth and expansion. Here we showed that ovarian cancer MPs trigger β-catenin activation in ECs by inducing the upregulation of Wnt/bcatenin target genes and increasing the angiogenic proprieties. Interestingly, the activation of bcatenin in ECs was Wnt/Frizzled independent; but dependent on VE-cadherin localization disruption, bcatenin integrin activation and MMP activity.