La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire. Son expression est fortement réprimée dans de nombreux cancers et l’invasion tumorale est accrue chez des souris TNX-/-. La TNX apparaît donc comme un répresseur potentiel du développement des tumeurs. L’objectif de notre travail est d’étudier cet effet présumé et d’en comprendre les mécanismes, en analysant in vitro le rôle de la TNX sur la croissance et la migration de cellules de fibrosarcome HT-1080 dans des modèles de culture bi- et surtout tridimensionnels, plus représentatifs de l’environnement cellulaire in vivo. Nos résultats montrent que la TNX inhibe la croissance des cellules tumorales, sans induire de mort apoptotique ou nécrotique. Des observations par microscopie confocale ont montré que la présence de TNX réduit l’étalement des cellules ainsi que leur efficacité de migration. Nous avons pu mettre en évidence que la TNX provoque un ralentissement de la migration des cellules tumorales ainsi qu’une diminution de la directionnalité de leurs trajectoires. L’observation de la protéolyse du collagène de type I par les cellules en migration montre qu’elle est inhibée en présence de TNX. Par ailleurs, la TNX réduit l’expression et l’activation des MMP 2, MMP-9, et MT1 MMP. Certaines voies de signalisation associées ont été étudiées : la TNX inhibe la phosphorylation de FAK sur sa tyrosine 397, ainsi que l’activation des GTPases RhoA et Rac, sans affecter celle de Cdc42. Par une régulation fine de ces molécules, qui sont impliquées dans le contrôle de la croissance et de la migration cellulaire, la TNX se caractérise comme un inhibiteur extracellulaire de l’invasion tumorale / Tenascin-X (TNX) is involved not only in the organisation of the extracellular matrix architecture but also in the regulation of cell behaviour. This matrix glycoprotein is down-regulated in many tumor types, while tumor invasion is promoted in TNX-deficient mice. In order to decipher the mechanisms by which TNX modulates tumor cell growth and migration, we compared the behaviour of HT1080 fibrosarcoma cells in conventionnal 2D culture model or embedded in 3D collagen gels, both containing or not recombinant TNX. Some experimentations have permit us to demonstrate that TNX inhibits tumor cells growth, without inducing apoptotic or necrotic cell death. Laser confocal microscopy observations demonstrated that the presence of TNX reduces cell spreading and migration efficency. Moreover, video time-lapse analysis showed that TNX reduces both velocity and directionnality of cell migration. This result is partly due to a decrease of pericellular proteolysis, as observed in situ using FITC-collagen-containing gels. Besides, we showed that TNX led to a decrease of MMP 2, MMP-9, MT1 MMP expression and activity. Then, we determined that both FAK phosphorylation on tyrosine 397 and activation of Rac1/2/3 and RhoA small GTPases were inhibited in TNX conditions. An inactivation of these small GTPases of the Rho family is known to deregulate cell cycle and highly decrease tumor cell spreading and migration efficiency in 3D environment. Taken together, these results indicate that TNX is an extracellular inhibitor of cell invasion, which acts by downregulating the main signalling pathways responsible for cell growth and motility in 3D-collagen gels

Au cours de l'invasion tumorale, la matrice extracellulaire du tissu broncho-pulmonaire, riche en élastine, subit de nombreux remaniements. La dégradation de cette élastine conduit à la production de peptides bioactifs. Ces peptides d'élastine (PE) possèdent un récepteur spécifique, le complexe récepteur de l'élastine (CRE), et peuvent également interagir avec l'intégrine alphavbeta3 et la galectine-3. Dans cette étude, nous avons étudié le rôle des PE et de leurs récepteurs dans la progression tumorale des carcinomes broncho-pulmonaires.Des cellules épithéliales bronchiques tumorales sont incubées in vitro avec un mélange de PE, la kappa-élastine (kE), ou avec des peptides synthétiques. Le traitement par les peptides entraine une augmentation de la capacité infiltrante des cellules invasives associée à un relargage précoce de MMP 2, MMP 9 et uPA mais n'a pas d'effet sur la prolifération et le phénotype cellulaire. Les niveaux d'ARNm des 3 protéases stimulées ne sont pas modifiés et ni l'actinomycine D, ni le cycloheximide ou la bréfeldine A ne sont capables d'inhiber les effets liés à la kE. Ces effets ne sont pas non plus inhibés par le lactose et les autres antagonistes des trois récepteurs. Enfin, les peptides VGVAPG et GRKRK, présentant les séquences spécifiques reconnues par les récepteurs, ne réussissent pas à reproduire les effets observés avec la kE, alors que des nonapeptides les reproduisent de façon quasi-identique.Ces résultats montrent que les PE régulent la capacité invasive des carcinomes broncho-pulmonaires, via le relargage d'enzymes protéolytiques. Cette modulation mettrait en jeu des mécanismes post-traductionnels et un récepteur lactose-insensible, différent du CRE, de l'intégrine alphavbeta3 et de la galectine-3, et reconnaissant des nonapeptides d'élastine. / Elastin-rich lung extra-cellular matrix is largely remodeled during tumor invasion. Elastin degradation produces peptides displaying a wide range of biological activities. These elastin derived peptides (EP) interact with the Elastin Receptor Complex (ERC) but also bind to alphaVbeta3 integrin and galectin-3. In this study, we explored the role of EP and their receptors in tumor progression of lung carcinomas.In vitro, lung tumor cells were incubated in presence of kappa-elastin (kE), a mix of EP or with synthetic elastin peptides. EP treatment induced an increase of invasive capacity of invasive cells with quickly increased levels of MMP-2, MMP 9 and uPA but had no effect on cell proliferation and phenotype. Interestingly, protease regulation was not observed at the mRNA level and actinomycin D, cycloheximide and brefeldin A were unable to inhibit kE effects. These effects could not be inhibited either by classical receptor antagonists including lactose or blocking antibodies. Finally, synthetic peptides VGVAPG and GRKRK, displaying receptor-specific sequences, failed to reproduce kE effects whereas nonapeptides partially mimicked them.These results demonstrate that treatment with EP up-regulates invasiveness of lung tumor cells via the release of proteolytic enzymes. This modulation involves post-translational mechanisms and a lactose-insensitive receptor, different from the ERC, alphaVbeta3 integrin and galectin-3 and recognizing nonapeptidic sequences.

3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (IK9) was recently reported to be a potent inhibitor of cancer cell invasion and angiogenesis. It markedly reduced in vitro invasion of human HT1080 fibrosarcoma cells through collagen-coated porous membranes (Boyden chamber assay) and in vivo tumour growth in athymic nude mice. It was furthermore able to decrease angiogenesis ex vivo in a rat aortic ring assay and in vivo in a choroidal neovascularisation mice model. It nevertheless presents some water solubility and stability problems, which should be taken into account for further investigations. In the first part of the project, we synthesized original IK9 derivatives, modulated at the 3- and 6-positions, by introducing functional groups able to improve water solubility and metabolic stability. Their anti-invasive potency was screened in the Boyden chamber assay and the generated results highlighted some structure-activity relationships. A second part of the project was devoted to the elucidation of the actually unknown mechanism of action of IK9. Anti-invasive or anti-proliferative effects against endothelial cells, main actors of the angiogenic process, were not emphasised. We showed that IK9 acts likely not as an inhibitor of receptor tyrosine kinases (EGFR, PDGFR and VEGFR). The compound generates a weak decrease of mRNA coding for metalloproteinases (MMPs) 2 and 9, and on the other hand a substantial diminution of MMP 2 and 9 secretions by HT1080 fibrosarcoma cells. In conclusion, the consideration of anti-invasive properties together with the worked out solubility and stability profiles highlights several series, notably 6-hydroxycoumarins, 6-hydroxymethylcoumarins and coumarin-3-sulfonamides, whose interest as potential successors to IK9 is undeniable. Le 6-acétoxyméthyl-2-oxo-2H-1-benzopyrane-3-carboxylate de 3-bromophényle (IK9) est un dérivé coumarinique décrit comme inhibiteur puissant de linvasion tumorale et de langiogenèse. Il inhibe linvasion des cellules HT1080 de fibrosarcome humain in vitro à travers une membrane poreuse recouverte dune couche de collagène (test en « chambres de Boyden ») et la croissance tumorale in vivo chez des souris athymiques nues. Il est par ailleurs capable de bloquer langiogenèse, à la fois dans un modèle ex vivo danneaux daorte de rats et dans un modèle in vivo de néovascularisation choroïdale chez la souris. Il présente pour autant une problématique dhydrosolubilité et de stabilité, dont il faudra tenir compte lors dinvestigations futures. Dans une première partie du projet, nous avons synthétisé des dérivés originaux de lIK9, modulés en position 3 et 6 du noyau coumarinique, en introduisant des fonctions susceptibles daugmenter lhydrosolubilité et la stabilité métabolique des molécules obtenues. Leur pouvoir anti-invasif a été évalué dans le test en « chambres de Boyden », ce qui nous a permis de mettre en évidence différentes relations structure-activité. Une deuxième partie du projet fut consacrée à létude du mécanisme daction de lIK9, qui est non identifié jusquà présent. Un effet anti-invasif ou anti-prolifératif envers les cellules endothéliales, actrices principales du processus dangiogenèse, na pu être observé. LIK9 nagit vraisemblablement pas en tant quinhibiteur de plusieurs récepteurs de type tyrosine kinase (EGFR, PDGFR et VEGFR). Le composé engendre une légère baisse de lexpression de lARNm codant pour les métalloprotéases matricielles (MMPs) 2 et 9 mais par contre entraîne une diminution substantielle de la sécrétion des MMPs 2 et 9 par les cellules HT1080 de fibrosarcome humain. En conclusion, la prise en compte simultanée de lactivité anti-invasive, de lhydrosolubilité et de la stabilité met en avant plusieurs séries de dérivés, notamment des 6-hydroxycoumarines, des 6-hydroxyméthylcoumarines et des coumarine-3-sulfonamides, dont lintérêt en tant que successeurs potentiels de lIK9 est indéniable.

Les môles hydatiformes complètes (MHC) sont des lésions bénignes précancéreuses du placenta qui dans 5% des cas évoluent vers un cancer très prolifératif dénommé, choriocarcinome (CC). Différents travaux ont rapporté une corrélation entre le développement des MHC récurrentes et les mutations du gène NLRP7. Ce gène code pour la protéine NLRP7 de l’inflammasome dont l’activation contribue à la production d’IL-1 et IL-18. La majorité des travaux publiés à ce jour se sont intéressés à l’étude des mutations de NLRP7. En revanche, aucune étude n’a caractérisé son rôle dans la placentation normale et tumorale. L’objectif de mon projet de thèse était de caractériser le rôle de cette protéine dans le placenta normal et tumoral. Trois approches ont été utilisées, i) Clinique, en collaboration avec le CHU de Casablanca ; le centre de référence Français des Maladies Gestationnelles Trophoblastiques (MGT) et l’Université Mc Gill pour l’accès aux tissus et sera collectés chez des patientes MHC ou CC; ii) In Vitro/Ex Vivo, pour la caractérisation du rôle du NLRP7 dans les processus clés du développement placentaire normal et tumoral dans des systèmes de cultures en 2D et 3D. Deux lignées trophoblastiques ont été utilisées, les cellules non tumorales HTR-8 Sv/Neo et la lignée JEG3 issue d’un CC humain ; iii) In Vivo, par l’injection orthotopique de cellules JEG3, invalidées ou non pour l’expression du gène NLRP7 (stratégie ShRNA), dans le placenta de souris gestantes. L’impact tumoral des JEG3 suite à leur injection dans la corne utérine et dans la veine de la queue de souris non gestantes a également été étudié.Mes travaux ont démontré que la protéine NLRP7 est abondamment exprimée dans le placenta normal au cours du premier trimestre de la grossesse, que son expression est régulée positivement par l’hypoxie, paramètre clé du développement du placenta et que cette protéine contrôle les processus clés du développement placentaire comme la prolifération et la différentiation trophoblastique. Par ailleurs, j’ai aussi démontré que NLRP7 joue un rôle compensatoire important dans la pathologie du retard de croissance intra-utérin. En relation avec le développement tumoral placentaire, mes travaux ont démontré que, i) la protéine NLRP7 est augmentée dans le placenta de patientes MHC et CC, et que les protéines de la machinerie des inflammasomes sont aussi dérégulées, ii) les cellules tumorales JEG3 surexpriment le NLRP7 comparé au HTR-8 Sv/Neo, iii) l’invalidation de NLRP7 dans les JEG3 induit une baisse de leur prolifération et augmentation de leur migration et invasion dans les systèmes de culture 2D et 3D. L’étude in vivo a démontré que l’invalidation de NLRP7 diminue le développement et la métastase du CC humain dans les trois modèles étudiés. Les analyses immunohistologiques, RNAseq et anticorps-array ont permis la caractérisation des mécanismes régulés par la protéine NLRP7 dans les JEG3. L’ensemble de mes travaux ont mis en évidence le rôle critique de la protéine NLRP7 dans le développement placentaire normal et tumoral et proposent la machinerie NLRP7 comme cible potentielle pour le traitement du CC. / Complete hydatidiform moles (CHM) are benign precancerous lesions of the placenta that evolve in 5% of cases into a highly proliferative cancer called choriocarcinoma (CC). Numerous studies have reported correlations between the development of recurrent CHM and mutations in the NLRP7 gene. NLRP7 protein belongs the NLRP7-inflammasome, whose activation contributes to the production of mature IL-1 and IL-18. Most of the work published on NLRP7 was focused on the study of NLRP7 mutations in CHM. Though, no study has characterized its role in normal and tumor placental development. The aim of my thesis project was to characterize the role of this protein in the normal and tumor placenta. Three approaches were used, i) A clinical approach, in collaboration with Casablanca University Hospital; the French reference center of Trophoblastic Gestational Disease and with McGill University. These collaborations allowed for tissue access from MHC and CC patients; ii) An in Vitro / Ex Vivo approach for the characterization of the role of NLRP7 in key processes of normal and tumor placental development using 2D and 3D culture systems. Two trophoblastic cell types were used, a non-tumor cell line, the HTR-8 Sv/Neo and the JEG3 cells, derived from human CC; iii) An In Vivo approach through orthotopic injection of JEG3 cells, invalidated or not for the expression of the NLRP7 gene (ShRNA strategy), in the placenta of gravid mice. The tumor impact of JEG3 following their injection into the uterine horn and the vein of the tail of non-pregnant mice was also examined.The first part of my work showed that NLRP7 protein is abundantly expressed in the normal placenta during the first trimester of pregnancy, that its expression is upregulated by hypoxia, a key parameter in placental development, and that this protein controls key processes of placental development such as proliferation and differentiation. Importantly, I have also demonstrated that NLRP7 plays an important compensatory role in the pathology of intrauterine growth retardation. The second part of my work concerning the role of NLRP7 protein in the placental tumor development demonstrated that i) NLRP7 protein levels are increased in the placenta of MHC and CC patients, and that components of the inflammasome machinery are also deregulated, ii) the JEG3 cells overexpress NLRP7 compared to HTR-8 Sv/Neo, iii) NLRP7 knock-down in JEG3 induced a significant decrease in their proliferation and an increase in their migration and invasion both in the 2D and 3D culture systems. The in vivo study demonstrated that the knock-down of NLRP7 decreased the development and metastasis of human CC in the three tested routes. Immunohistological, RNAseq and antibody-array analyses allowed the characterization of the pathways regulated by the NLRP7 protein in JEG3 cells.Altogether my PhD project characterized the critical role of the NLRP7 protein in normal and tumor placental development and proposes the NLRP7 machinery as a potential target for CC treatment.

Os gliomas apresentam comportamento amplamente infiltrativo e a invasão para o tecido cerebral sadio adjacente ao tumor o tornam um grande desafio para intervenções clínicas agressivas. Estudos recentes dos mecanismos de invasão de gliomas sugerem que as metaloproteinases (MMPs) desempenham papel crítico neste processo. As MMPs são super-expressas em quase todos os tumores humanos e vastos dados pré-clínicos as apontam como contribuintes do desenvolvimento maligno; portanto, a inibição de atividade de MMPs poderia ser adotada como uma estratégia de terapêutica anti-câncer. A atividade inibitória de MMP-2 e MMP-9 do extrato liofilizado da raiz de P. umbellata foi avaliada em estudos prévios de nosso laboratório por Ropke e colaboradores. Assim, esta dissertação teve como objetivo, primeiramente, explorar a citotoxicidade do extrato de P. umbellata e posteriormente, a avaliar o efeito do 4-neroridilcatecol (o princípio ativo isolado do extrato) quanto à citotoxicidade e potencial de inibição de MMPs durante o processo de invasão de duas linhagens de glioma humano, caracterizadas pelos diferentes níveis de invasividade. Os resultados revelam os índices de citotoxicidade do extrato bruto da raiz de P. umbellata e do composto 4-NC presente nesse extrato. Ensaios de invasão tumoral in vitro em concentrações não citotóxicas mostraram que há forte inibição desse processo pelo composto. Interessantemente, o 4-NC não alterou significativamente a atividade e expressões gênicas e protéicas de MMPs envolvidas na invasão deste tipo de tumor, nem de seus reguladores. O ensaio de fragmentação do DNA nos indica que a morte das células é, em parte, devido à apoptose, além da participação do processo autofágico. Ensaios analisando a expressão de proteínas e genes envolvidos na via de autofagia mostraram que há indução deste processo pelo composto estudado. Quando este processo é inibido farmacologicamente há incremento na morte celular causada pelo 4-NC. Considerando que em câncer, a autofagia é um processo essencial na modulação da resistência a quimioterápicos, descrevemos aqui, que 4-NC, a exemplo de seu poder citotóxico em melanomas, atua no processo invasivo de gliomas e induz morte celular além de desencadear o processo autofágico. / Gliomas present extensive invasive behavior and the invasion to healthy adjacent neural tissue makes this type of tumor a great challenge for aggressive clinical intervention. Recent studies concerning glioma invasion mechanisms suggest that metalloproteinases (MMPs) have a critical role in this process. MMPs are overexpressed in almost all types of human tumors and many pre-clinical data point to them as functional enhancers to malignant development; then, the inhibition of MMP activity could be adopted as an anti-cancer therapeutic strategy. P. umbellate root extract inhibitory activity over MMPs-2 and 9 was previously evaluated in studies from our group by Ropke et al. Thus, this dissertation has as an objective to evaluate the cytotoxic effect of 4-nerolidylcatechol (a compound purified from the P. umbellate extract), and its potential for MMP inhibition during the invasive process of two human glioma cell lines, characterized for their different invasive levels. Our results show the cytotoxic levels of the crude extract from the root of P. umbellate and from the 4-NC isolated from the extract. In vitro tumor invasion assay in non-cytotoxic drug concentrations indicate a strong inhibition of this process by the compound. Interestingly, 4-NC did not significantly alter enzyme activity, genetic or proteic expression of the MMPs involved in glioma invasion, or its regulators. The DNA fragmentation assay shows that cells die, in part, due to apoptosis, but autophagy pathway is also involved in cell death. Assays showing expression of proteins and genes involved in the autophagy pathway showed an increase of this process by the compound studied. Pharmacological inhibition of this process leads to an increase in cell death caused by 4-NC. Considering that in cancer, autophagy plays an essential role in the modulation of chemoresistance, we describe here that 4-NC, as described previously for melanomas, acts on glioma invasive process, induces cell death and is capable of up-regulating autophagic process.

OBJETIVOS: Estudar variáveis anatomopatológicas relacionadas à progressão tumoral em carcinomas uroteliais primários de bexiga e sua associação com a imunoexpressão de metaloproteinases de matriz (MMPs) - 2, -9 e -14 no epitélio e no estroma dos tumores primários e nas metástases linfonodais. MÉTODOS: Sessenta e um casos de carcinomas uroteliais musculoinvasivos ou localmente avançados primários da bexiga operados no Hospital das Clínicas da Faculdade de Medicina da USP e no Instituto do Câncer do Estado de São Paulo, sendo 34 casos com metástase para linfonodo regional, foram caracterizados quanto ao gênero, idade, tamanho, focalidade, grau histológico, tipo/configuração neoplásica, tipo papilífero da neoplasia, padrão arquitetural de invasão tumoral, grau de atipia nuclear, componente sarcomatoide, diferenciações escamosa e glandular, variante histológica, invasões linfovascular e perineural, carcinoma in situ, estádio do tumor primário, metástase para linfonodo regional, tamanho da metástase e extensão extranodal. Amostras teciduais de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica (IH) das enzimas MMP-2, MMP-9 e MMP-14. A expressão IH das MMPs foi graduada em uma escala semiquantitativa de 0 (ausência de expressão) até 20 (maior expressão). As associações entre a imunoexpressão das MMPs de forma global, no epitélio e estroma do tumor primário e na metástase linfonodal com as variáveis anatomopatológicas foram avaliadas através do teste do qui-quadrado de Pearson, sendo consideradas significativas ao nível de p<0,05. RESULTADOS: Trinta e seis, 57 e 60 casos do tumor primário foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. Nas metástases linfonodais, 20, 27 e 26 casos foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. A imunoexpressão global de MMP-2 no tumor primário mostrou-se associada com o padrão arquitetural de invasão (p=0,022) e sua expressão no estroma com o grau de atipia nuclear (p=0,032) e a porcentagem de componente sarcomatoide (p=0,003). A imunoexpressão global de MMP-9 no tumor primário mostrou-se associada com diferenciação escamosa (p=0,033). O padrão arquitetural de invasão relacionou-se com a expressão de MMP-9 no epitélio (p=0,043) e no estroma (p=0,044). A expressão de MMP-9 no estroma mostrou-se associada com o grau de atipia nuclear (p=0,031), componente sarcomatoide (p=0,036) e com a porcentagem desse componente no tumor primário (p=0,013). O estádio tumoral agrupado pT2+pT3 vs pT4 demonstrou associação com a MMP-9 expressa no epitélio (p=0,049). Para a MMP-14, o padrão arquitetural de invasão demonstrou associação significativa com a imunoexpressão global (p=0,022) e no epitélio tumoral (p=0,045). A porcentagem de componente sarcomatoide relacionou-se ainda com a expressão estromal de MMP-14 (p<0,001). Considerando a imunoexpressão de MMPs apenas na metástase linfonodal, houve associação significativa da MMP-9 com o tipo de variante histológica do tumor (p=0,021) e da expressão de MMP-14 com a porcentagem de componente sarcomatoide no tumor primário (p=0,017). CONCLUSÃO: O estudo da imunoexpressão de MMP-2, MMP-9 e MMP-14 em amostras organizadas em TMA, tanto no epitélio como no estroma dos carcinomas uroteliais de bexiga e nas metástases de linfonodos regionais, demonstrou associações estatísticas com características anatomopatológicas reconhecidas como de prognóstico ruim para essas neoplasias, indicando a ação dessas enzimas na transição epitélio-mesênquima e nas etapas de progressão e metástase dos carcinomas uroteliais / OBJECTIVES: To study morphological features related to tumor progression in urothelial carcinoma of the urinary bladder and its association with immunohistochemical (IHC) expression of matrix metalloproteinases (MMPs) -2, -9 and -14 in epithelial and stromal cells of primary tumor and regional lymph node metastases. METHODS: Sixty-one cases of muscle-invasive or locally advanced urothelial carcinomas of the bladder operated on Clinic\'s Hospital of Faculty Medicine Sao Paulo University and the Cancer Institute of the State of Sao Paulo, with 34 cases showing regional lymph nodes metastases, were characterized regarding gender, age, tumor size, multifocality, histological grade, neoplastic type/configuration, papillary type, architectural pattern of invasive tumor, nuclear atypia, sarcomatoid component, squamous and glandular diffentiation, histological variants, lymphovascular and perineural invasion, carcinoma in situ, tumor stage, metastases to regional lymph nodes, metastases size and extranodal extension. Tissue samples of 1.0 mm were arranged in tissue microarrays blocks (TMA) for IHC detection of MMP-2, MMP-9 and MMP-14. The grading of expression of MMPs was determined to a semiquantitative scale from 0 (absence) to 20 (higher expression). The associations between the IHC global expression of MMPs, in epithelium and in stromal cells of the primary tumor and in the lymph node metastases with the morphological features were obtained through Pearson\'s chi-square (significant at p<0.05). RESULTS: Thirty-six, 57 and 60 cases of primary tumor were positive for MMP-2, MMP-9 and MMP-14 respectively. In the lymph nodes metastases, 20, 27 and 26 cases were positive for MMP-2, MMP-9 and MMP-14 respectively. The global IHC expression of MMP-2 in primary tumor has been associated with the architectural pattern of invasion (p=0.022). The expression in stromal cells were correlated with the degree of nuclear atypia (p=0.032) and the percentage of sarcomatoid component (p=0.003). The IHC expression of MMP-9 in primary tumor has been associated with squamous differentiation (p=0.033). The architectural pattern of invasion was related to the expression of MMP-9 in epithelium (p=0.043) and in the stroma (p=0.044). Expression of MMP-9 in the stroma was associated with the degree of nuclear atypia (p=0.031), sarcomatoid component (p=0.036) and the percentage of this component in primary tumor (p=0.013). The grouped tumoral stage pT2+pT3 vs pT4 showed association with MMP-9 expressed in epithelium (p=0.049). For MMP-14, the architectural pattern of invasion showed significant association with global IHC expression (p=0.022) and tumor epithelium (p=0.045). The percentage of sarcomatoid component related to the estromal expression of MMP-14 (p<0.001). Considering the IHC expression of MMPs in lymph nodes metastases, there was a significant association between MMP-9 with the type of histological variants (p=0.021) and the expression of MMP-14 with the percentage of sarcomatoid component in primary tumor (p=0.017). CONCLUSION: The study of IHC expression of MMP-2, MMP-9 and MMP-14 in bladder carcinoma samples arranged in TMA, both in epithelium and in stromal cells and regional lymph nodes metastases, demonstrated significant association with morphological features recognized as prognostically important for these tumors. These findings herald the importance of action of these enzymes in epithelialmesenchymal transition, providing basis for the understanding of tumoral progression and metastases in urothelial carcinoma

Os gliomas apresentam comportamento amplamente infiltrativo e a invasão para o tecido cerebral sadio adjacente ao tumor o tornam um grande desafio para intervenções clínicas agressivas. Estudos recentes dos mecanismos de invasão de gliomas sugerem que as metaloproteinases (MMPs) desempenham papel crítico neste processo. As MMPs são super-expressas em quase todos os tumores humanos e vastos dados pré-clínicos as apontam como contribuintes do desenvolvimento maligno; portanto, a inibição de atividade de MMPs poderia ser adotada como uma estratégia de terapêutica anti-câncer. A atividade inibitória de MMP-2 e MMP-9 do extrato liofilizado da raiz de P. umbellata foi avaliada em estudos prévios de nosso laboratório por Ropke e colaboradores. Assim, esta dissertação teve como objetivo, primeiramente, explorar a citotoxicidade do extrato de P. umbellata e posteriormente, a avaliar o efeito do 4-neroridilcatecol (o princípio ativo isolado do extrato) quanto à citotoxicidade e potencial de inibição de MMPs durante o processo de invasão de duas linhagens de glioma humano, caracterizadas pelos diferentes níveis de invasividade. Os resultados revelam os índices de citotoxicidade do extrato bruto da raiz de P. umbellata e do composto 4-NC presente nesse extrato. Ensaios de invasão tumoral in vitro em concentrações não citotóxicas mostraram que há forte inibição desse processo pelo composto. Interessantemente, o 4-NC não alterou significativamente a atividade e expressões gênicas e protéicas de MMPs envolvidas na invasão deste tipo de tumor, nem de seus reguladores. O ensaio de fragmentação do DNA nos indica que a morte das células é, em parte, devido à apoptose, além da participação do processo autofágico. Ensaios analisando a expressão de proteínas e genes envolvidos na via de autofagia mostraram que há indução deste processo pelo composto estudado. Quando este processo é inibido farmacologicamente há incremento na morte celular causada pelo 4-NC. Considerando que em câncer, a autofagia é um processo essencial na modulação da resistência a quimioterápicos, descrevemos aqui, que 4-NC, a exemplo de seu poder citotóxico em melanomas, atua no processo invasivo de gliomas e induz morte celular além de desencadear o processo autofágico. / Gliomas present extensive invasive behavior and the invasion to healthy adjacent neural tissue makes this type of tumor a great challenge for aggressive clinical intervention. Recent studies concerning glioma invasion mechanisms suggest that metalloproteinases (MMPs) have a critical role in this process. MMPs are overexpressed in almost all types of human tumors and many pre-clinical data point to them as functional enhancers to malignant development; then, the inhibition of MMP activity could be adopted as an anti-cancer therapeutic strategy. P. umbellate root extract inhibitory activity over MMPs-2 and 9 was previously evaluated in studies from our group by Ropke et al. Thus, this dissertation has as an objective to evaluate the cytotoxic effect of 4-nerolidylcatechol (a compound purified from the P. umbellate extract), and its potential for MMP inhibition during the invasive process of two human glioma cell lines, characterized for their different invasive levels. Our results show the cytotoxic levels of the crude extract from the root of P. umbellate and from the 4-NC isolated from the extract. In vitro tumor invasion assay in non-cytotoxic drug concentrations indicate a strong inhibition of this process by the compound. Interestingly, 4-NC did not significantly alter enzyme activity, genetic or proteic expression of the MMPs involved in glioma invasion, or its regulators. The DNA fragmentation assay shows that cells die, in part, due to apoptosis, but autophagy pathway is also involved in cell death. Assays showing expression of proteins and genes involved in the autophagy pathway showed an increase of this process by the compound studied. Pharmacological inhibition of this process leads to an increase in cell death caused by 4-NC. Considering that in cancer, autophagy plays an essential role in the modulation of chemoresistance, we describe here that 4-NC, as described previously for melanomas, acts on glioma invasive process, induces cell death and is capable of up-regulating autophagic process.

OBJETIVOS: Estudar variáveis anatomopatológicas relacionadas à progressão tumoral em carcinomas uroteliais primários de bexiga e sua associação com a imunoexpressão de metaloproteinases de matriz (MMPs) - 2, -9 e -14 no epitélio e no estroma dos tumores primários e nas metástases linfonodais. MÉTODOS: Sessenta e um casos de carcinomas uroteliais musculoinvasivos ou localmente avançados primários da bexiga operados no Hospital das Clínicas da Faculdade de Medicina da USP e no Instituto do Câncer do Estado de São Paulo, sendo 34 casos com metástase para linfonodo regional, foram caracterizados quanto ao gênero, idade, tamanho, focalidade, grau histológico, tipo/configuração neoplásica, tipo papilífero da neoplasia, padrão arquitetural de invasão tumoral, grau de atipia nuclear, componente sarcomatoide, diferenciações escamosa e glandular, variante histológica, invasões linfovascular e perineural, carcinoma in situ, estádio do tumor primário, metástase para linfonodo regional, tamanho da metástase e extensão extranodal. Amostras teciduais de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica (IH) das enzimas MMP-2, MMP-9 e MMP-14. A expressão IH das MMPs foi graduada em uma escala semiquantitativa de 0 (ausência de expressão) até 20 (maior expressão). As associações entre a imunoexpressão das MMPs de forma global, no epitélio e estroma do tumor primário e na metástase linfonodal com as variáveis anatomopatológicas foram avaliadas através do teste do qui-quadrado de Pearson, sendo consideradas significativas ao nível de p<0,05. RESULTADOS: Trinta e seis, 57 e 60 casos do tumor primário foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. Nas metástases linfonodais, 20, 27 e 26 casos foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. A imunoexpressão global de MMP-2 no tumor primário mostrou-se associada com o padrão arquitetural de invasão (p=0,022) e sua expressão no estroma com o grau de atipia nuclear (p=0,032) e a porcentagem de componente sarcomatoide (p=0,003). A imunoexpressão global de MMP-9 no tumor primário mostrou-se associada com diferenciação escamosa (p=0,033). O padrão arquitetural de invasão relacionou-se com a expressão de MMP-9 no epitélio (p=0,043) e no estroma (p=0,044). A expressão de MMP-9 no estroma mostrou-se associada com o grau de atipia nuclear (p=0,031), componente sarcomatoide (p=0,036) e com a porcentagem desse componente no tumor primário (p=0,013). O estádio tumoral agrupado pT2+pT3 vs pT4 demonstrou associação com a MMP-9 expressa no epitélio (p=0,049). Para a MMP-14, o padrão arquitetural de invasão demonstrou associação significativa com a imunoexpressão global (p=0,022) e no epitélio tumoral (p=0,045). A porcentagem de componente sarcomatoide relacionou-se ainda com a expressão estromal de MMP-14 (p<0,001). Considerando a imunoexpressão de MMPs apenas na metástase linfonodal, houve associação significativa da MMP-9 com o tipo de variante histológica do tumor (p=0,021) e da expressão de MMP-14 com a porcentagem de componente sarcomatoide no tumor primário (p=0,017). CONCLUSÃO: O estudo da imunoexpressão de MMP-2, MMP-9 e MMP-14 em amostras organizadas em TMA, tanto no epitélio como no estroma dos carcinomas uroteliais de bexiga e nas metástases de linfonodos regionais, demonstrou associações estatísticas com características anatomopatológicas reconhecidas como de prognóstico ruim para essas neoplasias, indicando a ação dessas enzimas na transição epitélio-mesênquima e nas etapas de progressão e metástase dos carcinomas uroteliais / OBJECTIVES: To study morphological features related to tumor progression in urothelial carcinoma of the urinary bladder and its association with immunohistochemical (IHC) expression of matrix metalloproteinases (MMPs) -2, -9 and -14 in epithelial and stromal cells of primary tumor and regional lymph node metastases. METHODS: Sixty-one cases of muscle-invasive or locally advanced urothelial carcinomas of the bladder operated on Clinic\'s Hospital of Faculty Medicine Sao Paulo University and the Cancer Institute of the State of Sao Paulo, with 34 cases showing regional lymph nodes metastases, were characterized regarding gender, age, tumor size, multifocality, histological grade, neoplastic type/configuration, papillary type, architectural pattern of invasive tumor, nuclear atypia, sarcomatoid component, squamous and glandular diffentiation, histological variants, lymphovascular and perineural invasion, carcinoma in situ, tumor stage, metastases to regional lymph nodes, metastases size and extranodal extension. Tissue samples of 1.0 mm were arranged in tissue microarrays blocks (TMA) for IHC detection of MMP-2, MMP-9 and MMP-14. The grading of expression of MMPs was determined to a semiquantitative scale from 0 (absence) to 20 (higher expression). The associations between the IHC global expression of MMPs, in epithelium and in stromal cells of the primary tumor and in the lymph node metastases with the morphological features were obtained through Pearson\'s chi-square (significant at p<0.05). RESULTS: Thirty-six, 57 and 60 cases of primary tumor were positive for MMP-2, MMP-9 and MMP-14 respectively. In the lymph nodes metastases, 20, 27 and 26 cases were positive for MMP-2, MMP-9 and MMP-14 respectively. The global IHC expression of MMP-2 in primary tumor has been associated with the architectural pattern of invasion (p=0.022). The expression in stromal cells were correlated with the degree of nuclear atypia (p=0.032) and the percentage of sarcomatoid component (p=0.003). The IHC expression of MMP-9 in primary tumor has been associated with squamous differentiation (p=0.033). The architectural pattern of invasion was related to the expression of MMP-9 in epithelium (p=0.043) and in the stroma (p=0.044). Expression of MMP-9 in the stroma was associated with the degree of nuclear atypia (p=0.031), sarcomatoid component (p=0.036) and the percentage of this component in primary tumor (p=0.013). The grouped tumoral stage pT2+pT3 vs pT4 showed association with MMP-9 expressed in epithelium (p=0.049). For MMP-14, the architectural pattern of invasion showed significant association with global IHC expression (p=0.022) and tumor epithelium (p=0.045). The percentage of sarcomatoid component related to the estromal expression of MMP-14 (p<0.001). Considering the IHC expression of MMPs in lymph nodes metastases, there was a significant association between MMP-9 with the type of histological variants (p=0.021) and the expression of MMP-14 with the percentage of sarcomatoid component in primary tumor (p=0.017). CONCLUSION: The study of IHC expression of MMP-2, MMP-9 and MMP-14 in bladder carcinoma samples arranged in TMA, both in epithelium and in stromal cells and regional lymph nodes metastases, demonstrated significant association with morphological features recognized as prognostically important for these tumors. These findings herald the importance of action of these enzymes in epithelialmesenchymal transition, providing basis for the understanding of tumoral progression and metastases in urothelial carcinoma

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La spleen tyrosine kinase (Syk) est une protéine kinase cytoplasmique qui intervient dans la signalisation immunitaire. Notre équipe a montré pour la première fois que Syk est exprimée aussi dans les cellules épithéliales mammaires et que son expression est perdue au cours de l’acquisition d’un phénotype invasif/métastatique. Syk agit comme un suppresseur de tumeurs et de métastases dans des modèles de xénogreffes de cancer du sein. Ces observations ont été étayées par des études cliniques qui montrent que la perte d’expression de Syk correspond à un risque accru de développement de métastases (facteur de mauvais pronostic) dans le cancer du sein et d’autres carcinomes. Par une approche de phospho-protéomique quantitative (SILAC), nous avons pu identifier de nouveaux substrats potentiels de Syk dans les cellules de cancer du sein. De façon intéressante, ressortent de nombreuses protéines impliquées dans l'adhésion intercellulaire (E-cadhérine/caténines) et la polarisation épithéliale (eg ZO3, occludine, claudine-3). Ces protéines, qui se localisent aux jonctions d'adhésion et d'occlusion, sont connues comme composants plate-formes de signalisation et exercent souvent une fonction de suppresseur de tumeur.Dans ce travail de thèse je me suis focalisé principalement sur :(i) le rôle de l’activité kinase de Syk dans la régulation du complexe E-cadhérine/caténines et(ii) les conséquences de l’invalidation conditionnelle de Syk dans la glande mammaire murine (développement mammaire et tumorigenèse).Par une approche de phosphorylation in vitro, nous avons montré que la E-cadhérine et différentes caténines sont des substrats directs de Syk. Les résidus tyrosines phosphorylés dans ces protéines ont été identifiés par spectrométrie de masse et les anticorps phospho-spécifiques correspondants ont été générés. En immunofluorescence, Syk endogène colocalise avec la E-Cadhérine au niveau des jonctions adhérentes et la surexpression de Syk stimule la phosphorylation de la E cadhérine et différentes caténines au niveau des jonctions intercellulaires. Des expériences d’immunoprécipitation montrent que les protéines E-cadhérine et caténines phosphorylées restent associées dans un complexe au niveau des jonctions adhérentes. L’extinction de Syk par shRNA dans une lignée de cancer de sein inhibe partiellement la ré-agrégation intercellulaire (2D/3D) et augmente l’invasion et la migration cellulaires et la croissance en 3D dans le Matrigel. Inversement, la surexpression de Syk inhibe la migration et l’invasion et favorise l'adhérence intercellulaire. Syk semble par la phosphorylation du complexe E-cadhérine/caténines consolider leurs interactions renforçant ainsi les jonctions intercellulaires et l’intégrité de l’épithélium, ce qui pourrait révéler un mécanisme majeur responsable de son activité anti-invasive. Leurs mécanismes moléculaires ont été explorés.Ces modèles cellulaires in vitro ont ensuite été étendus vers un modèle intégré murin. L'invalidation homozygote du gène SYK étant létale, nous avons développé un modèle d’invalidation conditionnelle de Syk dans la glande mammaire (Syk-flox:WAP-Cre). Ce modèle nous a permis tout d’abord d’étudier le rôle de Syk dans le développement et la physiologie de la glande mammaire au cours de la lactation et de l’involution, les glandes Syk-négatives montrant des défauts de développement. Il permet à plus long terme également d’évaluer l'implication de Syk dans la formation et la progression du cancer du sein chez des souris cKO Syk, après croisement ou non avec des souris transgéniques exprimant l’oncogène MMTV-Neu/Her2.Déterminer si Syk est un bona fide suppresseur de tumeurs est crucial car un inhibiteur de Syk est en cours d’étude clinique pour le traitement de l’arthrite rhumatoïde. L’identification des voies de signalisation gouvernées par Syk pourrait ultérieurement déboucher sur le développement de nouvelles thérapies ciblant ces protéines et bloquant l'évolution cancéreuse. / The spleen tyrosine kinase (Syk) is a cytoplasmic protein kinase involved in immune-response signaling. Our team showed for the first time that Syk is also expressed in mammary epithelial cells and that its expression is lost during acquisition of an invasive/metastatic phenotype. Syk acts as a tumor and metastasis suppressor in breast cancer xenograft models. Clinical studies corroborated that loss of Syk expression is correlated with a decreased survival and an increased risk of metastasis development (poor prognosis) in breast cancer and other carcinomas. Using a quantitative phospho-proteomic SILAC approach in breast cancer cells, our group identified new potential Syk substrates. Interestingly, many proteins are involved in intercellular adhesion (E-cadherin/catenin) and epithelial polarization (eg ZO3, occludin, claudin-3). These proteins are localized at the adherens and tight junctions and are known as signaling platforms and components often presenting a tumor suppressor function.In this thesis I mainly focused on:(i) the role of the Syk kinase activity in the regulation of the E-cadherin/catenin complex and(ii) the consequences of the conditional Syk knockout in the mouse mammary gland on breast development and tumorigenesis.Using in vitro kinase assays, we demonstrated that E-cadherin (E-Cdh) and different catenins are direct Syk substrates. The phosphorylated tyrosine residues were identified by mass spectrometry and corresponding phospho-specific antibodies were generated. By immunofluorescence, we observed that endogenous Syk and E-Cdh colocalize at adherens junctions (AJ) and that Syk overexpression stimulates Syk-dependent phosphorylation of E-cadherin and different catenins at AJ. Immunoprecipitation experiments indicate phosphorylated E-cadherin and catenin proteins are associated in a complex. Using functional tests, Syk knockdown by shRNA in breast cancer cells partially inhibited intercellular re-aggregation (2D/3D) and increased cell invasion, migration and 3D-growth in Matrigel. Conversely, Syk overexpression inhibited migration and invasion and promoted intercellular adhesion. Thus, Syk seems to strengthen the intercellular junctions and the integrity of the epithelium via the phosphorylation of the E-cadherin/catenin complex of which its molecular mechanisms were explored. This could be a major mechanism responsible for its anti-invasive activity.These in vitro observations were subsequently extended to an integrated mouse model. As the homozygous SYK gene knockout is lethal; we developed a conditional Syk deletion model in the murine mammary gland (Syk-flox:WAP-Cre).This model allowed us to study the role of Syk in the development and physiology of the mammary gland during lactation and involution, the Syk-negative glands showing developmental defects. On a long-term basis, it also allows to assess the involvement of Syk in the formation and progression of breast cancer in aging cKO Syk mice, bred or not with transgenic mice expressing the MMTV-Neu / Her2 oncogene.Whether Syk is a bona fide tumor suppressor is a crucial issue as Syk inhibitors are being evaluated in clinical studies for the treatment of rheumatoid arthritis. Identification of the signaling pathways governed by Syk could lead to the development of new therapies targeting these proteins and blocking tumor development and progression.