A transição endotélio-mesenquimal (EndMT) é uma forma especializada da transição epitéliomesenquimal (EMT) e é caracterizada pela alteração da morfologia celular para um formato fibroblastoide, perda da expressão dos marcadores endoteliais e ganho da expressão dos marcadores mesenquimais, bem como a aquisição de propriedades invasivas e migratórias. Entretanto, o mecanismo molecular envolvido nesse processo ainda não está totalmente elucidado. O objetivo desse trabalho foi avaliar os fatores indutores da EMT em células endoteliais (CEs) de fontes distintas por meio da superexpressão do fator de transcrição SNAIL e do tratamento com TGF-?2, bem como identificar os mecanismos moleculares envolvidos nesse processo. Para tal, as linhagens de CE da artéria pulmonar (HPAEC), pool de CE primária de veia de cordão umbilical (PHUVEC), CE da aorta (PAEC) e CE da artéria coronária (CAEC) foram induzidas em três condições distintas: I) TGF-?2; II) superexpressão do fator de transcrição SNAIL; III) superexpressão do fator de transcrição SNAIL associado ao tratamento com TGF-?2 (SNAIL+TGF-?2). Após a indução, a expressão dos genes relacionados com a EndMT foi analisada por PCR em tempo real (qPCR) e as CAECs foram as células que apresentaram maior mudança no perfil de expressão gênica, no qual o grupo SNAIL+TGF-?2 apresentou um aumento dos marcadores mesenquimal FN1, SM22, CNN1 e CD90. O grupo SNAIL+TGF-?2 também mostrou uma diminuição dos marcadores endoteliais CD31 e CDH5 por Western blot. Em seguida, a técnica de microarray foi realizada nas CAECs induzidas à EndMT e as análises revelaram um dendrograma cujo perfil mostrou que SNAIL e SNAIL+TGF-?2 se agrupam separadamente das outras condições. Os dados de microarray resultaram em uma rede na qual os genes mesenquimais COL1A1, COL1A2, FN1 e CNN1 estavam aumentados no grupo SNAIL+TGF-?2 comparado com o grupo controle. Os genes diferencialmente expressos entre a análise CT vs. SNAIL+TGF-?2 foram analisados quanto a participação em vias canônicas e a via de regulação da EMT foi uma das mais representadas, a qual inclui a via de sinalização Notch e Wnt. Nos dados de microarray, NOTCH3 e WNT5B estavam superexpressos no grupo SNAIL+TGF-?2 comparado com o controle. Sabendo que Wnt5b pode inibir a via ?-catenina, a expressão de NOTCH3, WNT5B e ?-CATENINA foi avaliada por qPCR e a expressão de NOTCH3 e WNT5B confirmou os dados do microarray e nenhuma diferença estatística foi observada na expressão de ?- CATENINA. Ainda, as CAECs induzidas foram submetidas ao ensaio de migração e de capacidade de formação de estruturas semelhantes a capilares. Foi observado que as CAECSNAIL+ TGF-?2 migraram significativamente comparadas com as outras condições e nenhuma das células induzidas (TGF-?2, SNAIL e SNAIL+TGF-?2) foram capazes de formar estruturas semelhantes a capilares. Alguns microRNAs foram selecionados e avaliados por qPCR. O miR-let7a foi significativamente expresso no grupo SNAIL e SNAIL+TGF-?2. O ensaio de perda e ganho de função do miR-let7a foi realizado, entretanto, a repressão ou a indução do miR-let7a não alterou a EndMT. Esses resultados sugerem que as CEs de fontes anatômicas distintas apresentam respostas diferentes quando estimuladas a sofrerem EndMT. Ademais, a associação entre SNAIL+TGF-?2 é um potente indutor para EndMT e essa indução pode ser mediada pelas vias de sinalização Notch e Wnt não canônica. / Endothelial-mesenchymal transition (EndMT) is a specialized form of epithelialmesenchymal transition (EMT) which is characterized by changes in cell morphology as a fibroblastoid conversion, expression of endothelial markers decreased, expression of mesenchymal markers increased and acquirement of invasive and migratory properties. However, the molecular mechanism associated with this process is not completely elucidated. The aim of this study was to evaluate the EMT-inducing factors in the endothelial cells (ECs) from different sources through the overexpression of the transcription factor SNAIL and through the treatment with TGF-?2, as well as to identify the molecular mechanisms involved in EndMT. For this purpose, primary pulmonary artery EC (HPAEC), primary pooled umbilical vein EC (PHUVEC), primary aortic EC (PAEC), primary coronary artery EC (CAEC) lineages were induced under three distinct conditions: I) TGF-?2; II) ectopic expression of SNAIL; III) ectopic expression of SNAIL associated with TGF-?2 (SNAIL+TGF- ?2). After the EndMT induction, the expression of the genes associated with EndMT was analyzed by Real time PCR (qPCR) and CAECs showed the most prominent alterations on their gene expression profile which showed that SNAIL+TGF-?2 group presented an increase of mesenchymal markers FN1, SM22, CNN1, and CD90 expression. CAEC-SNAIL+TGF-?2 group also showed a decrease of endothelial markers CD31 and CDH5 by western blot. Then, microarray was performed in CAECs after EndMT induction and hierarchical clustering analysis showed that the ectopic expression of SNAIL and SNAIL+TGF-?2 clustered separately from the other conditions. Microarray data resulted in a network which presented an upregulation of the mesenchymal genes such as COL1A1, COL1A2, FN1, and CNN1 in the CAEC-SNAIL+TGF-?2 compared to control cells. We analyzed the canonical pathways related to the differentially regulated genes between CAEC- SNAIL+TGF-?2 and control cells and the regulation of EMT pathways was the most represented, which includes Notch and Wnt signaling pathway. In the microarray data, NOTCH3 and WNT5B were overexpressed in CAEC-SNAIL+TGF-?2 compared to control. It is known that Wnt5b might inhibit the ?- catenin pathway. Therefore, NOTCH3, WNT5B and ?-CATENIN gene expression were analyzed by qPCR. NOTCH3 and WNT5B gene expression confirmed the microarray data and no statistical difference were observed in ?-CATENIN expression. Moreover, all the CAECs conditions were subjected to scratch migration assay and the formation of capillary-like structures assay. CAEC-SNAIL+TGF-?2 had a significant migration compared to other conditions and the three EndMT inductions (TGF-?2, SNAIL, and SNAIL+TGF-?2) were not able to form capillary-like structures. Some microRNAs were selected and evaluated by qPCR. The miR-let7a was significantly expressed in the SNAIL and SNAIL+TGF-?2 groups. The assay of gain or loss of function of miR-let7a was realized; however, the repression or induction of miR-let7a did not change the EndMT. These results suggest that endothelial cells from distinct anatomical sources have different responses when stimulated to undergo the EndMT. Moreover, the association between SNAIL+TGF-?2 is a potent inductor for EndMT and this induction can be mediated by Notch and non-canonical Wnt signaling pathway activation.

Les phéochromocytomes (PCC) et les paragangliomes (PGL) sont des tumeurs neuroendocrines rares, pour lesquelles le déterminisme génétique est très important, avec 16 gènes de prédisposition identifiés à ce jour. Au cours de ce travail de thèse, je me suis plus particulièrement intéressée aux conséquences des mutations du gène SDHB, car il avait été préalablement démontré qu’elles constituaient un facteur de risque de mauvais pronostic, associé à un phénotype métastatique et à une réduction de la survie des patients. Grâce à l’analyse du transcriptome d’une large cohorte de 188 échantillons de PCC/PGL humains, j’ai pu mettre en évidence que la voie de la transition épithélio-mésenchymateuse (EMT) était spécifiquement activée dans les tumeurs métastatiques SDHB-dépendantes. En effet, j’ai observé, dans ce sous-groupe de tumeurs, une surexpression de facteurs de transcription et de régulateurs précoces de l’EMT, comme TWIST1, TFC3, ou LOXL2 ; une perte d’expression de marqueurs de jonctions cellulaires, comme CDH2 et KRT19 ; ou encore une induction de gènes codant pour des enzymes pro-invasives, comme MMP1 et MMP2. Dans ces tumeurs, j’ai par ailleurs validé l’induction transcriptionnelle de l’EMT en mettant en évidence la rétention nucléaire de la protéine SNAIL (un facteur de transcription clé de l’EMT) sur des coupes de tissus. Dans cette même cohorte, l’analyse des données de méthylation globale de l’ADN nous a permis d’observer un phénotype hyperméthylateur dans les tumeurs SDHx, qui est expliqué par une inhibition de déméthylases de l’ADN et des histones par le succinate, qui s’accumule dans ces tumeurs où la succinate déshydrogénase est inactivée. Nous avons ainsi pu démontrer que le succinate est un oncométabolite qui induit des modifications épigénétiques impliquées dans l’extinction de nombreux gènes, et en particulier des gènes associés à l’EMT, comme le gène KRT19 (l’un des plus hyperméthylés dans les tumeurs SDHB-malignes, comparativement aux autres). J’ai ensuite caractérisé le premier modèle cellulaire de PCC/PGL porteur d’une inactivation complète du gène Sdhb, dans lequel j’ai confirmé l’activation de l’EMT, au niveau transcriptionnel et au niveau protéique. La caractérisation fonctionnelle de ces cellules m’a ensuite permis de mettre en évidence des propriétés migratoires, invasives, et adhésives spécifiques des cellules chromaffines Sdhb -/-. J’ai par la suite focalisé mon étude sur le gène Krt19, qui code pour une protéine du cytosquelette, la kératine 19 et qui est éteint dans les tumeurs et dans les cellules Sdhb -/-. La réintroduction de ce gène dans les cellules Sdhb -/- et son inhibition dans les cellules sauvages m’ont permis de conclure à l’implication de la kératine 19 dans les processus d’adhésion, de migration et d’invasion. Cependant je n’ai pas pu expliquer l’ensemble du phénotype par la seule modulation de ce gène, ce qui démontre l’implication d’autres acteurs dans la mise en place du phénotype invasif décrit. Mes travaux de thèse ont ainsi permis de démontrer que l’activation de l’EMT est responsable du caractère métastatique et invasif observé dans les tumeurs porteuses d'une mutation sur le gène SDHB et que cette activation est notamment secondaire aux modifications épigénétiques induites par l’inactivation de la succinate déshydrogénase. / Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors for which there is a major impact of genetic determinism with 16 identified predisposition genes to date. During this PhD training, I was particularly interested in the consequences of SDHB gene mutations because it was previously demonstrated that they constitute a high risk factor of poor prognosis, associated with metastatic phenotype and reduced survival. During my thesis, thanks to the analysis of transcriptomic data of a large cohort of 188 human PCC/PGL samples, I identified epithelial to mesenchymal transition (EMT) as activated in SDHB-metastatic tumors. Indeed, I observed a transcriptional induction of TWIST1, TCF3 and LOXL2, which are transcription factors or early regulators of EMT; a loss of expression of cellular junction components such as CDH2 and KRT19; and an up regulation of genes encoding pro-invasive proteases, such as MMP1 and MMP2. In these tumors, I validated the transcriptional induction of EMT by showing a nuclear retention of SNAIL protein, the master EMT transcription factor. In the same cohort, global DNA methylation data allowed us to describe a hypermethylator phenotype in SDHx related tumors, explained by the inhibition of DNA and histones déméthylases by succinate, which accumulates massively in cells inactivated for succinate dehydrogenase. We thus demonstrated that succinate is an oncometabolite responsible for epigenetic modifications implicated in gene extinction, including EMT-related genes such as KRT19 (one of the most hypermethylated genes in SDHB-metastatic samples). Following these observations, I characterized the first PCC/PGL cellular model displaying a complete Sdhb inactivation, in which I confirmed EMT activation, at transcriptional and protein levels. Functional characterization of these cells allowed me to show Sdhb-specific migratory, invasive and adhesive properties. I thus focused my study on Krt19, which encodes keratin 19, a cytoskeleton protein, lost in Sdhb -/- cells and in human SDHB-related tumors. Krt19 rescue in Sdhb -/- cells or Krt19 inhibition in wild-type cells allowed me to conclude that keratin 19 is implicated in adhesion, migration and invasion. However I was not able to explain the whole phenotype observed, demonstrating the implication of other actors in the establishment of the invasive phenotype. The main result of my PhD work is the discovery that EMT is a molecular pathway responsible for the metastatic and invasive phenotype observed in SDHB-mutated tumors as a result of epigenetic alterations due to succinate dehydrogenase inactivation.

O câncer de ovário (OvCa) se destaca dentre as neoplasias ginecológicas por ser um dos mais letais e de difícil diagnóstico. O OvCa ocorre devido ao acúmulo de alterações celulares progressivas promovidas por mutações no genoma de uma célula que, consequentemente, alteram as complexas vias de regulação celular que respondem a fatores internos, como reprogramação genética, ou externos, como a resposta a fatores de crescimento, que juntamente com outras alterações moleculares favorecem a progressão e a metástase. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem um caráter mais invasivo e migratório, além de se tornarem mais resistentes às drogas. A desregulação de fatores de transcrição como ZEB1, TWIST e SNAI1, vias de sinalização, microRNAs e fatores de crescimento incluindo EGF, TGF? e HGF podem desencadear a EMT. Após a eficiente indução da EMT com EGF na linhagem epitelial de adenocarcinoma de ovário humano Caov-3, foi realizada a análise proteômica quantitativa detalhada, baseada na análise de frações subcelulares enriquecidas em proteínas de membrana, citosol e núcleo, obtidas por centrifugação diferencial e subsequente fracionamento de proteínas por SDS-PAGE, a fim de compreender mais profundamente os mecanismos moleculares modulados pela EMT no OvCa. A partir da análise dos dados coletados em um sistema de espectrometria de massas de alta resolução acoplados a cromatografia líquida (LCMS/MS) e com o auxílio da bioinformática foram identificadas redes de interação proteína-proteína diferencialmente expressas, relacionadas principalmente com a regulação do ciclo celular e do metabolismo. A indução da EMT por EGF resultou na ativação de importantes vias de sinalização, tais como PI3K/Akt/mTOR e Ras/MAPK Erk, além da parada do ciclo celular na fase G1 regulada pelo aumento dos níveis de p21Waf1/Cip1, independentemente de p53, e diminuição de proteínas checkpoint. Através da proteômica dirigida, o monitoramento de reações múltiplas (MRM) revelou que, após a indução da EMT por EGF, o metabolismo das células Caov-3 foi alterado de uma maneira bastante peculiar. O estudo proteômico descrito permitiu a correlação entre processo da EMT induzido por EGF com o controle translacional, a regulação do ciclo celular e a alteração do metabolismo energético. / Ovarian cancer (OvCa) stands out among gynecological malignancies for being one of the most lethal and difficult to diagnose. OvCa occurs due to the accumulation of progressive cell changes promoted by mutations in the cell genome which, consequently, alter the complex cellular regulation pathways that respond to internal factors, such as genetic reprogramming, or external, such as response to growth factors, which together with other molecular changes favor the progression and metastasis. An important step of the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of epithelial phenotype and acquisition of mesenchymal phenotype by tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. Deregulation of transcription factors such as ZEB1, TWIST and SNAI1, signaling pathways, microRNAs and growth factors including EGF, TGF? and HGF can trigger EMT. After an efficient EMT induction by EGF in the epithelial cell line of human adenocarcinoma ovarian Caov-3, detailed quantitative proteomic analysis was performed based on analysis of subcellular fractions enriched in proteins from membrane, cytosol and nucleus, obtained by differential centrifugation and subsequent fractionation of proteins by SDS-PAGE, in order to understand deeply the molecular mechanisms modulated by EMT in OvCa. From the analysis of data collected in a highresolution mass spectrometry system coupled to liquid chromatography (LC-MS/MS) and with the aid of bioinformatics were identified protein-protein interaction networks differentially expressed, mainly related to regulation cell cycle and metabolism. EGF induced-EMT resulted in the activation of major signaling pathways such as PI3K/Akt/mTOR and Ras/MAPK Erk, in addition to G1 phase cell cycle arrest regulated by increased levels of p21Waf1/Cip1, regardless of p53, and reduction of checkpoint proteins. Through the targeted proteomics, multiple reaction monitoring (MRM) showed that after EGF induced-EMT, Caov-3 cells metabolism was changed in a very particular way. The proteomic study described allowed the correlation between EMT process induced by EGF with translational control, regulation of cell cycle and the change in the energy metabolism.

O desenvolvimento tumoral é um processo que compreende diversas etapas e consiste no desenvolvimento progressivo de células normais para um estado neoplásico através de diversas mudanças bioquímicas e fenotípicas. Entre as principais marcas do câncer estão a capacidade de invasão tecidual e metástase. A metástase é responsável por, aproximadamente, 90% das mortes causadas por câncer. Assim, os métodos mais efetivos para a melhoria dos índices de morbidade e mortalidade relacionados ao câncer são a detecção precoce, a prevenção e o tratamento da metástase. O processo de EMT, que ocorre naturalmente durante a embriogênese e reparo tecidual, está envolvido também na progressão e metástase do câncer. A EMT induz alterações celulares e microambientais complexas que resultam na aquisição de um fenótipo mesenquimal pelas células epiteliais, juntamente com um aumento das capacidades migratórias e invasivas celulares. A EMT pode ser induzida por diversos fatores extracelulares, como os fatores de crescimento TGF?, EGF, HGF e PDGF. Além disso, a superexpressão de fatores de transcrição como SNAIL, SLUG, ZEB1 e TWIST1 também é capaz de induzir a EMT in vitro. A fim de ampliar o conhecimento dos mecanismos envolvidos no processo de EMT a nível de proteínas, foram realizadas neste trabalho análises de alterações proteômicas em diferentes modelos de indução da EMT na linhagem de adenocarcinoma de mama MCF7, sendo eles a superexpressão do FT SNAIL, o tratamento com o inibidor de histonas deacetilases SAHA e o tratamento com o fator de crescimento EGF. A análise proteômica detalhada por LC-MS/MS das frações subcelulares de núcleo, citoplasma e membrana das células superexpressando SNAIL gerou uma lista de proteínas reguladas relacionadas com o processo de EMT e que foram avaliadas nos demais modelos de indução. Entre essas, a proteína HDAC1, que teve seus níveis diminuídos pela superexpressão de SNAIL. O tratamento da linhagem MCF7 com o inibidor de histonas deacetilases SAHA demonstrou uma correlação positiva com o aumento dos níveis de SNAIL nas células MCF7, sugerindo um cross-talk entre ambas as proteínas. Além disso, otratamento com SAHA induziu alterações celulares e proteicas que também sugerem a indução do processo de EMT nas células MCF7. Por fim, o tratamento com o fator de crescimento EGF também foi capaz de induzir a EMT nas células MCF7 e apresentou envolvimento na regulação do ciclo celular, alterações de proteínas em comum com os demais tratamentos e regulação diferencial de proteínas entre os subcompartimentos, indicando similaridades entre os processos e potenciais mecanismos de translocação subcelular. Em conclusão, este estudo relevou proteínas alvo relacionadas à EMT, abrindo possibilidades para tentar alterar processos relacionados à progressão tumoral e ao processo metastático. / Tumor development is a process comprising several steps and consists in the progressive development of normal cells into a neoplastic state through various biochemical and phenotypic changes. Among the major marks of cancer are the capacity for tissue invasion and metastasis. Metastasis accounts for approximately 90% of cancer deaths. Thus, the most effective methods for improving cancer-related morbidity and mortality rates are early detection, prevention and treatment of metastasis. The EMT process, which occurs naturally during embryogenesis and tissue repair, is also involved in cancer progression and metastasis. EMT induces complex cellular and microenvironmental changes that result in the acquisition of a mesenchymal phenotype by epithelial cells, together with an increase in migratory and invasive cellular capacities. EMT can be induced by various extracellular factors, such as TGF?, EGF, HGF and PDGF. In addition, overexpression of some transcription factors such as SNAIL, SLUG, ZEB1 and TWIST1 is also capable of inducing EMT in vitro. In order to increase the knowledge of the mechanisms involved in the EMT process, we performed proteomic analysis in different models of EMT induction in the MCF7 breast adenocarcinoma cell line, which were the overexpression of SNAIL, treatment with the histone deacetylase inhibitor SAHA and treatment with the growth factor EGF. The detailed proteomic analysis by LC-MS/MS of the subcellular fractions of nucleus, cytoplasm and membrane of the overexpressing SNAIL cells generated a list of regulated proteins related to the EMT process and that were evaluated in the other models of induction. Among these, the HDAC1 protein, which had its levels decreased by SNAIL overexpression. Treatment of the MCF7 cell line with the histone deacetylase inhibitor SAHA showed a positive correlation with the increase of SNAIL levels, suggesting a cross-talk between both proteins. In addition, SAHA treatment induced cellular and protein alterations that also suggest the induction of the EMT process in MCF7 cells. Finally, the treatment with the growth factor EGF was also able to induce the EMT in MCF7 cells and showed involvement in the regulation of the cell cycle, changes in proteins in common with the other treatments and differential regulation of proteins among thesubcompartiments, indicating similarities between the processes and potential mechanisms of subcellular translocation. In conclusion, this study revealed target proteins related to EMT, opening possibilities to try to alter processes related to tumor progression and metastatic process.

O câncer de ovário (OvCa) se destaca dentre as neoplasias ginecológicas por ser um dos mais letais e de difícil diagnóstico. O OvCa ocorre devido ao acúmulo de alterações celulares progressivas promovidas por mutações no genoma de uma célula que, consequentemente, alteram as complexas vias de regulação celular que respondem a fatores internos, como reprogramação genética, ou externos, como a resposta a fatores de crescimento, que juntamente com outras alterações moleculares favorecem a progressão e a metástase. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem um caráter mais invasivo e migratório, além de se tornarem mais resistentes às drogas. A desregulação de fatores de transcrição como ZEB1, TWIST e SNAI1, vias de sinalização, microRNAs e fatores de crescimento incluindo EGF, TGF? e HGF podem desencadear a EMT. Após a eficiente indução da EMT com EGF na linhagem epitelial de adenocarcinoma de ovário humano Caov-3, foi realizada a análise proteômica quantitativa detalhada, baseada na análise de frações subcelulares enriquecidas em proteínas de membrana, citosol e núcleo, obtidas por centrifugação diferencial e subsequente fracionamento de proteínas por SDS-PAGE, a fim de compreender mais profundamente os mecanismos moleculares modulados pela EMT no OvCa. A partir da análise dos dados coletados em um sistema de espectrometria de massas de alta resolução acoplados a cromatografia líquida (LCMS/MS) e com o auxílio da bioinformática foram identificadas redes de interação proteína-proteína diferencialmente expressas, relacionadas principalmente com a regulação do ciclo celular e do metabolismo. A indução da EMT por EGF resultou na ativação de importantes vias de sinalização, tais como PI3K/Akt/mTOR e Ras/MAPK Erk, além da parada do ciclo celular na fase G1 regulada pelo aumento dos níveis de p21Waf1/Cip1, independentemente de p53, e diminuição de proteínas checkpoint. Através da proteômica dirigida, o monitoramento de reações múltiplas (MRM) revelou que, após a indução da EMT por EGF, o metabolismo das células Caov-3 foi alterado de uma maneira bastante peculiar. O estudo proteômico descrito permitiu a correlação entre processo da EMT induzido por EGF com o controle translacional, a regulação do ciclo celular e a alteração do metabolismo energético. / Ovarian cancer (OvCa) stands out among gynecological malignancies for being one of the most lethal and difficult to diagnose. OvCa occurs due to the accumulation of progressive cell changes promoted by mutations in the cell genome which, consequently, alter the complex cellular regulation pathways that respond to internal factors, such as genetic reprogramming, or external, such as response to growth factors, which together with other molecular changes favor the progression and metastasis. An important step of the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of epithelial phenotype and acquisition of mesenchymal phenotype by tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. Deregulation of transcription factors such as ZEB1, TWIST and SNAI1, signaling pathways, microRNAs and growth factors including EGF, TGF? and HGF can trigger EMT. After an efficient EMT induction by EGF in the epithelial cell line of human adenocarcinoma ovarian Caov-3, detailed quantitative proteomic analysis was performed based on analysis of subcellular fractions enriched in proteins from membrane, cytosol and nucleus, obtained by differential centrifugation and subsequent fractionation of proteins by SDS-PAGE, in order to understand deeply the molecular mechanisms modulated by EMT in OvCa. From the analysis of data collected in a highresolution mass spectrometry system coupled to liquid chromatography (LC-MS/MS) and with the aid of bioinformatics were identified protein-protein interaction networks differentially expressed, mainly related to regulation cell cycle and metabolism. EGF induced-EMT resulted in the activation of major signaling pathways such as PI3K/Akt/mTOR and Ras/MAPK Erk, in addition to G1 phase cell cycle arrest regulated by increased levels of p21Waf1/Cip1, regardless of p53, and reduction of checkpoint proteins. Through the targeted proteomics, multiple reaction monitoring (MRM) showed that after EGF induced-EMT, Caov-3 cells metabolism was changed in a very particular way. The proteomic study described allowed the correlation between EMT process induced by EGF with translational control, regulation of cell cycle and the change in the energy metabolism.

Au cours de ma thèse, j’ai étudié la contribution de miR-205 dans le développement du cancer du sein. MiR-205 a été choisi suite à l'analyse comparative de l'expression du miRome entre la lignée « normale » MCF10A et une lignée cancéreuse dérivée MCF10A-CA1a. J’ai démontré que l’expression de miR-205 augmente durant la tumorigenèse tandis que miR-205 est non détectable dans la lignée cellulaire ayant un potentiel métastatique. De plus, j’ai montré que les cellules souches du cancer du sein expriment miR-205, contrairement à la population non souche. En utilisant des cultures de cellules épithéliales 3D, j’ai corrélé la fonction tumorigène de miR-205 à la répression de l'apoptose et non à une prolifération accrue. De plus, le niveau d'expression de la E-Cadhérine dépend de la quantité de miR-205 dans les différentes lignées cellulaires de MCF10A. Les études de perte de fonction suggèrent que la E-Cadhérine est impliquée dans le phénotype acini miR-205-Dépendant, en corrélation avec la transformation de cellules épithéliales du sein. L’ensemble de ces résultats met en lumière la complexité et la duplicité des miRNA durant le processus de cancérisation. Ce type d’étude ouvre des perspectives d’utilisation des miRNA dans le cadre des diagnostics et/ou thérapeutiques. / During the time I was working on my thesis, I aimed to understand the role of miR-205 in breast cancer development. MiR-205 was chosen from the comparative analysis of total micro-RNAs expression in non-Transformed and tumorigenic cell lines of the MCF10A breast epithelial cell model. I demonstrated the complexity of miR-205 functions during breast epithelial cell transformation by showing miR-205 overexpression in transformed non-Invasive cell lines and miR-205 down-Regulation in cell line with metastatic potential. Moreover, we demonstrated increased level of miR-205 expression in breast cancer stem cells in comparison with non-Stem cells. Using 3D cultures of breast epithelial cells, I succeeded to correlate the tumorigenic function of miR-205 with its role in modulation of acinar size, and to attribute it to the apoptosis repression but not increased proliferation. Further, I was able to show that miR-205 exercises its oncogenic functions via targeting ZEB1, an inhibitor of E-Cadherin. Indeed, E-Cadherin expression level depends on the amount of miR-205 in different MCF10A cell lines. Downregulating E-Cadherin restored normal acinar morphology in miR-205 expressing cells, consistent with E-Cadherin being involved in the miR-205-Dependent acini phenotype that correlates with tumorigenic breast epithelial cell transformation.

O desenvolvimento tumoral é um processo que compreende diversas etapas e consiste no desenvolvimento progressivo de células normais para um estado neoplásico através de diversas mudanças bioquímicas e fenotípicas. Entre as principais marcas do câncer estão a capacidade de invasão tecidual e metástase. A metástase é responsável por, aproximadamente, 90% das mortes causadas por câncer. Assim, os métodos mais efetivos para a melhoria dos índices de morbidade e mortalidade relacionados ao câncer são a detecção precoce, a prevenção e o tratamento da metástase. O processo de EMT, que ocorre naturalmente durante a embriogênese e reparo tecidual, está envolvido também na progressão e metástase do câncer. A EMT induz alterações celulares e microambientais complexas que resultam na aquisição de um fenótipo mesenquimal pelas células epiteliais, juntamente com um aumento das capacidades migratórias e invasivas celulares. A EMT pode ser induzida por diversos fatores extracelulares, como os fatores de crescimento TGF?, EGF, HGF e PDGF. Além disso, a superexpressão de fatores de transcrição como SNAIL, SLUG, ZEB1 e TWIST1 também é capaz de induzir a EMT in vitro. A fim de ampliar o conhecimento dos mecanismos envolvidos no processo de EMT a nível de proteínas, foram realizadas neste trabalho análises de alterações proteômicas em diferentes modelos de indução da EMT na linhagem de adenocarcinoma de mama MCF7, sendo eles a superexpressão do FT SNAIL, o tratamento com o inibidor de histonas deacetilases SAHA e o tratamento com o fator de crescimento EGF. A análise proteômica detalhada por LC-MS/MS das frações subcelulares de núcleo, citoplasma e membrana das células superexpressando SNAIL gerou uma lista de proteínas reguladas relacionadas com o processo de EMT e que foram avaliadas nos demais modelos de indução. Entre essas, a proteína HDAC1, que teve seus níveis diminuídos pela superexpressão de SNAIL. O tratamento da linhagem MCF7 com o inibidor de histonas deacetilases SAHA demonstrou uma correlação positiva com o aumento dos níveis de SNAIL nas células MCF7, sugerindo um cross-talk entre ambas as proteínas. Além disso, otratamento com SAHA induziu alterações celulares e proteicas que também sugerem a indução do processo de EMT nas células MCF7. Por fim, o tratamento com o fator de crescimento EGF também foi capaz de induzir a EMT nas células MCF7 e apresentou envolvimento na regulação do ciclo celular, alterações de proteínas em comum com os demais tratamentos e regulação diferencial de proteínas entre os subcompartimentos, indicando similaridades entre os processos e potenciais mecanismos de translocação subcelular. Em conclusão, este estudo relevou proteínas alvo relacionadas à EMT, abrindo possibilidades para tentar alterar processos relacionados à progressão tumoral e ao processo metastático. / Tumor development is a process comprising several steps and consists in the progressive development of normal cells into a neoplastic state through various biochemical and phenotypic changes. Among the major marks of cancer are the capacity for tissue invasion and metastasis. Metastasis accounts for approximately 90% of cancer deaths. Thus, the most effective methods for improving cancer-related morbidity and mortality rates are early detection, prevention and treatment of metastasis. The EMT process, which occurs naturally during embryogenesis and tissue repair, is also involved in cancer progression and metastasis. EMT induces complex cellular and microenvironmental changes that result in the acquisition of a mesenchymal phenotype by epithelial cells, together with an increase in migratory and invasive cellular capacities. EMT can be induced by various extracellular factors, such as TGF?, EGF, HGF and PDGF. In addition, overexpression of some transcription factors such as SNAIL, SLUG, ZEB1 and TWIST1 is also capable of inducing EMT in vitro. In order to increase the knowledge of the mechanisms involved in the EMT process, we performed proteomic analysis in different models of EMT induction in the MCF7 breast adenocarcinoma cell line, which were the overexpression of SNAIL, treatment with the histone deacetylase inhibitor SAHA and treatment with the growth factor EGF. The detailed proteomic analysis by LC-MS/MS of the subcellular fractions of nucleus, cytoplasm and membrane of the overexpressing SNAIL cells generated a list of regulated proteins related to the EMT process and that were evaluated in the other models of induction. Among these, the HDAC1 protein, which had its levels decreased by SNAIL overexpression. Treatment of the MCF7 cell line with the histone deacetylase inhibitor SAHA showed a positive correlation with the increase of SNAIL levels, suggesting a cross-talk between both proteins. In addition, SAHA treatment induced cellular and protein alterations that also suggest the induction of the EMT process in MCF7 cells. Finally, the treatment with the growth factor EGF was also able to induce the EMT in MCF7 cells and showed involvement in the regulation of the cell cycle, changes in proteins in common with the other treatments and differential regulation of proteins among thesubcompartiments, indicating similarities between the processes and potential mechanisms of subcellular translocation. In conclusion, this study revealed target proteins related to EMT, opening possibilities to try to alter processes related to tumor progression and metastatic process.

Environ 8% du génome humain est constitué de rétrovirus endogènes (HERV). La famille de bétarétrovirus HERV-K(HML2), l'une des plus actives chez l'homme, est entrée il y a 45 millions d'années dans le génome des primates et s'est amplifiée efficacement depuis, et ce malgré l'existence de nombreuses protéines cellulaires, appelées facteurs de restriction, qui s'opposent à la réplication du virus dans la cellule hôte. La Tetherin/BST2, l'un d'entre eux, est une protéine membranaire capable de bloquer le relargage des virions dans le milieu extracellulaire et est active sur la plupart des virus enveloppés testés jusqu'à présent, en particulier HERV-K(HML2). Nous avons tout d'abord mis en évidence que l'enveloppe (Env) de la famille HML2 est un antagoniste de la Tetherin, propriété qui a pu contribuer au succès de l'amplification de la famille HERV¬K(HML2) dans les génomes. Plusieurs domaines de l'enveloppe coopèrent pour s'opposer à l'action du facteur de restriction : la SU (domaine d'interaction), ainsi que la partie transmembranaire, alors que la queue cytoplasmique n'est pas indispensable. Le mécanisme de cette inhibition n'a pas été encore complètement élucidé, mais l'on sait, comme pour la glycoprotéine d'Ebola, que l'Env HERV-K(HML2) n'induit ni relocalisation, ni dégradation de la Tetherin. Etant donné le grand polymorphisme insertionnel de la famille HERV-K(1-IML2), il est très probable que cette activité anti-Tetherin endogène soit variable entre les individus, ce qui pourrait avoir des conséquences dans les pathologies où les éléments HERV-K(HML2) sont spécifiquement induits. Parmi ces pathologies, les cancers de la peau, du sein et de la lignée germinale présentent une association particulièrement forte avec l'expression de l'Env HERV-K(HML2), que nous avons voulu mieux comprendre dans la suite de ces travaux de thèse. Nous avons dans un premier temps montré que l'expression de l'Env dans des cellules humaines non transformées de l'épithélium de sein (MCF10A), induit la transition vers un phénotype mésenchymateux (EMT, transition épithélio-mésenchymateuse), caractéristique de l'apparition de métastases dans les cancers. Cette transition est associée à une augmentation de la mobilité des cellules (mise en évidence dans des tests Transwell), à un changement de morphologie des cellules et à une modification du profil d'expression de quelques marqueurs moléculaires caractéristiques (E-cadherin, N-cadherin, vimentin, fibronectin). Grâce à une étude transcriptomique en cellules 293T, nous avons mis en évidence que l'expression de l'Env HERV-K induit fortement plusieurs facteurs de transcription : ETV4, ETVS, ainsi que EGR1, qui ont été identifiés comme des marqueurs du processus de tumorigénèse dans différents modèles. Nous avons également montré que l'Env HERV-K active la voie des MAP kinases via ERK 1/2 —dérégulée dans un grand nombre de cancers- en amont de la kinase Raf. Ces phénomènes d'induction de la transduction de signal requièrent la présence de la queue cytoplasmique de l'enveloppe. De façon remarquable, seule l'enveloppe du bétarétrovirus de mouton JSRV, oncogénique in vivo, est capable d'activer les mêmes voies de signalisation, ce qui renforce l'hypothèse d'une implication de l'Env HERV-K(HML2) dans la tumorigenèse. / Human endogenous retroviruses (HERV) represent about 8% of our genomic content. HERV-K(HML2) betaretroviral family is one of the most active in humans. Although it entered 45 million years ago in the primate genomes, its members have amplified quite recently despite the existence of restriction factors, which are host proteins blocking viral replication in cells. Tetherin/BST2 is one of them and acts by keeping the viral particles attached to the cell surface. It targets most enveloped viruses tested so far including HERV-K(HML2). We show that the envelope protein (Env) of HML2 family is an antagonist of Tetherin retriction, property that probably helped the endogenous retrovirus to efficiently amplify in the genomes. We mapped several domains required for antagonism : the surface subunit of Env (SU), which interacts with Tetherin, and the transmembrane. We also show that the cytoplasmic tail is dispensable for counteraction. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate the restriction factor. Due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. This could have functional consequences in pathologies where HERV-K(HML2) is specifically induced. Among them, melanomas, breast cancers and germ line tumors display a strong association with HML2 Env expression, that we wanted to better analyse. We first show that Env expression in a model of epithelial human breast cancer cells induces the so-called EMT (epithelial mesenchymal transition), critical for cancer progression and the process of metastasis. This includes enhanced migratory capacities (shown by transwell assays), changes in cell morphology and characteristic modifications in a set of molecular markers (e.g. E-cadherin, N-cadherin, vimentin, fibronectin). Microarray experiments performed in 293T cells revealed that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETVS and EGRI, which have been associated with cellular transformation. Importantly, we also show that HERV-K(HML2) Env activates the MAP kinase pathway via ERK 1/2, key player in numerous cancers. This induction occurs upstream of the kinase Raf and involves the cytoplasmic tail of HERV-K(HML2) Env. In addition, this phenomenon is very specific, being absent with every other Env tested, except for JSRV Env which is already known to have transforming properties in vivo.

A transição endotélio-mesenquimal (EndMT) é uma forma especializada da transição epitéliomesenquimal (EMT) e é caracterizada pela alteração da morfologia celular para um formato fibroblastoide, perda da expressão dos marcadores endoteliais e ganho da expressão dos marcadores mesenquimais, bem como a aquisição de propriedades invasivas e migratórias. Entretanto, o mecanismo molecular envolvido nesse processo ainda não está totalmente elucidado. O objetivo desse trabalho foi avaliar os fatores indutores da EMT em células endoteliais (CEs) de fontes distintas por meio da superexpressão do fator de transcrição SNAIL e do tratamento com TGF-?2, bem como identificar os mecanismos moleculares envolvidos nesse processo. Para tal, as linhagens de CE da artéria pulmonar (HPAEC), pool de CE primária de veia de cordão umbilical (PHUVEC), CE da aorta (PAEC) e CE da artéria coronária (CAEC) foram induzidas em três condições distintas: I) TGF-?2; II) superexpressão do fator de transcrição SNAIL; III) superexpressão do fator de transcrição SNAIL associado ao tratamento com TGF-?2 (SNAIL+TGF-?2). Após a indução, a expressão dos genes relacionados com a EndMT foi analisada por PCR em tempo real (qPCR) e as CAECs foram as células que apresentaram maior mudança no perfil de expressão gênica, no qual o grupo SNAIL+TGF-?2 apresentou um aumento dos marcadores mesenquimal FN1, SM22, CNN1 e CD90. O grupo SNAIL+TGF-?2 também mostrou uma diminuição dos marcadores endoteliais CD31 e CDH5 por Western blot. Em seguida, a técnica de microarray foi realizada nas CAECs induzidas à EndMT e as análises revelaram um dendrograma cujo perfil mostrou que SNAIL e SNAIL+TGF-?2 se agrupam separadamente das outras condições. Os dados de microarray resultaram em uma rede na qual os genes mesenquimais COL1A1, COL1A2, FN1 e CNN1 estavam aumentados no grupo SNAIL+TGF-?2 comparado com o grupo controle. Os genes diferencialmente expressos entre a análise CT vs. SNAIL+TGF-?2 foram analisados quanto a participação em vias canônicas e a via de regulação da EMT foi uma das mais representadas, a qual inclui a via de sinalização Notch e Wnt. Nos dados de microarray, NOTCH3 e WNT5B estavam superexpressos no grupo SNAIL+TGF-?2 comparado com o controle. Sabendo que Wnt5b pode inibir a via ?-catenina, a expressão de NOTCH3, WNT5B e ?-CATENINA foi avaliada por qPCR e a expressão de NOTCH3 e WNT5B confirmou os dados do microarray e nenhuma diferença estatística foi observada na expressão de ?- CATENINA. Ainda, as CAECs induzidas foram submetidas ao ensaio de migração e de capacidade de formação de estruturas semelhantes a capilares. Foi observado que as CAECSNAIL+ TGF-?2 migraram significativamente comparadas com as outras condições e nenhuma das células induzidas (TGF-?2, SNAIL e SNAIL+TGF-?2) foram capazes de formar estruturas semelhantes a capilares. Alguns microRNAs foram selecionados e avaliados por qPCR. O miR-let7a foi significativamente expresso no grupo SNAIL e SNAIL+TGF-?2. O ensaio de perda e ganho de função do miR-let7a foi realizado, entretanto, a repressão ou a indução do miR-let7a não alterou a EndMT. Esses resultados sugerem que as CEs de fontes anatômicas distintas apresentam respostas diferentes quando estimuladas a sofrerem EndMT. Ademais, a associação entre SNAIL+TGF-?2 é um potente indutor para EndMT e essa indução pode ser mediada pelas vias de sinalização Notch e Wnt não canônica. / Endothelial-mesenchymal transition (EndMT) is a specialized form of epithelialmesenchymal transition (EMT) which is characterized by changes in cell morphology as a fibroblastoid conversion, expression of endothelial markers decreased, expression of mesenchymal markers increased and acquirement of invasive and migratory properties. However, the molecular mechanism associated with this process is not completely elucidated. The aim of this study was to evaluate the EMT-inducing factors in the endothelial cells (ECs) from different sources through the overexpression of the transcription factor SNAIL and through the treatment with TGF-?2, as well as to identify the molecular mechanisms involved in EndMT. For this purpose, primary pulmonary artery EC (HPAEC), primary pooled umbilical vein EC (PHUVEC), primary aortic EC (PAEC), primary coronary artery EC (CAEC) lineages were induced under three distinct conditions: I) TGF-?2; II) ectopic expression of SNAIL; III) ectopic expression of SNAIL associated with TGF-?2 (SNAIL+TGF- ?2). After the EndMT induction, the expression of the genes associated with EndMT was analyzed by Real time PCR (qPCR) and CAECs showed the most prominent alterations on their gene expression profile which showed that SNAIL+TGF-?2 group presented an increase of mesenchymal markers FN1, SM22, CNN1, and CD90 expression. CAEC-SNAIL+TGF-?2 group also showed a decrease of endothelial markers CD31 and CDH5 by western blot. Then, microarray was performed in CAECs after EndMT induction and hierarchical clustering analysis showed that the ectopic expression of SNAIL and SNAIL+TGF-?2 clustered separately from the other conditions. Microarray data resulted in a network which presented an upregulation of the mesenchymal genes such as COL1A1, COL1A2, FN1, and CNN1 in the CAEC-SNAIL+TGF-?2 compared to control cells. We analyzed the canonical pathways related to the differentially regulated genes between CAEC- SNAIL+TGF-?2 and control cells and the regulation of EMT pathways was the most represented, which includes Notch and Wnt signaling pathway. In the microarray data, NOTCH3 and WNT5B were overexpressed in CAEC-SNAIL+TGF-?2 compared to control. It is known that Wnt5b might inhibit the ?- catenin pathway. Therefore, NOTCH3, WNT5B and ?-CATENIN gene expression were analyzed by qPCR. NOTCH3 and WNT5B gene expression confirmed the microarray data and no statistical difference were observed in ?-CATENIN expression. Moreover, all the CAECs conditions were subjected to scratch migration assay and the formation of capillary-like structures assay. CAEC-SNAIL+TGF-?2 had a significant migration compared to other conditions and the three EndMT inductions (TGF-?2, SNAIL, and SNAIL+TGF-?2) were not able to form capillary-like structures. Some microRNAs were selected and evaluated by qPCR. The miR-let7a was significantly expressed in the SNAIL and SNAIL+TGF-?2 groups. The assay of gain or loss of function of miR-let7a was realized; however, the repression or induction of miR-let7a did not change the EndMT. These results suggest that endothelial cells from distinct anatomical sources have different responses when stimulated to undergo the EndMT. Moreover, the association between SNAIL+TGF-?2 is a potent inductor for EndMT and this induction can be mediated by Notch and non-canonical Wnt signaling pathway activation.

Le cancer épithélial de l'ovaire (CEO) est létal à cause de son potentiel d'invasion, sa progression insidieuse et sa capacité de métastase à travers la cavité péritonéale. L'antigène tumoral CA125 (MUC16) est le marqueur clinique du cancer de l'ovaire le plus important. Il est utilisé pour surveiller la progression de la maladie. L'antigène tumoral CA125 (MUC16) est une glycoprotéine de grand poids moléculaire (200-2000 kDa) de la famille des mucines transmembranaires. Il est encodé par le gène MUC16 à partir d'un long ARNm de 66kb. L'expression de CA125 (MUC16) est retrouvée dans la majorité des cancers épithéliaux de l'ovaire (CEO) mais il n'est pas détecté dans l'épithélium normal des ovaires. Cependant, sa contribution au développement du CEO reste pratiquement inconnue. Il y a des évidences croissantes que les mucines transmembranaires sont impliquées dans plusieurs voies de signalisation intracellulaire en régulant l'expansion cellulaire, l'adhésion cellulaire, la migration et la mort cellulaire. Il est envisageable que CA125 (MUC16) pourrait exercer certaines de ces fonctions. Nos données supportent ce concept. Pour étudier les fonctions de CA125, nous avons initialement développé un inhibiteur spécifique de CA125 qui consiste en un minianticorps de chaîne simple anti-CA125 (scFv) qui est retenu dans le réticulum endoplasmique et qui prévient la localisation de CA125 à la surface cellulaire, en imitant, par conséquence, un knockdown de son expression. Des transfectants stables exprimant ce scFv ont été dérivés à partir de la lignée parentale NIH: OVCAR-3. Nous avons démontré que le knockdown de CA125 altère la morphologie cellulaire, inhibe l'agrégation cellulaire homotypique et de façon dramatique, il diminue la croissance cellulaire indépendante d'ancrage et la tumorigénicité chez les souris nues. Nous avons déterminé que CA125 interagit avec E-cadhérine, [béta]-caténine, [alpha]-actinine, [béta]-actine. Plus tard, nous avons observé par immunofluorescence et par microscopie électronique que les cellules CA125 knockdown présentent des jonctions intercellulaires détériorées et un réarrangement des filaments d'actine, ce qui nous suggère que CA125 pourrait être associé aux jonctions intercellulaires et impliqué dans l'organisation du cytosquelette. La formation et la dissolution dynamique des complexes de jonction sont des processus centraux pendant la transition épithélio-mésenchymateuse.Le knockdown de CA125 augmente aussi la migration des cellules OVCAR-3. Nous avons constaté que le maintien de cette migration est dépendant de la présence du EGF dans le milieu de culture et qu'elle est presque exclusivement due à l'effet de ce facteur de croissance. L'ajout de différents inhibiteurs du récepteur d'EGF (EGFR) démontre une abolition presque complète de la migration cellulaire de clones CA125 knockdown. En plus, l'expression d'E-cadhérine et de claudine-7 est significativement diminuée chez les cellules CA125 knockdown, tandis que l'expression de la vimentine et de N-cadhérine est augmentée, nous suggérant que la perte de CA125 cause une EMT chez ces cellules. De façon intéressante, l'expression ectopique du domaine C-terminal (CTD) de CA125 chez les cellules de cancer de l'ovaire SKOV-3 confère aux cellules les mêmes phénotypes indiquant que ce domaine peut être suffisant pour moduler ces comportements cellulaires.Le domaine C-terminal pourrait représenter ce qui reste de CA125 à la surface cellulaire après le clivage protéolytique causant le relâchement de la majorité de son domaine extracellulaire. Cependant, l'expression ectopique des domaines extracellulaires de CA125 (domaines transmembranaire et unique (TMU) ou transmembranaire-unique plus une des répétitions (TMU+1R)) ne confère pas d'effet sur les jonctions intercellulaires ni sur la migration évoquant que l'expression isolée de ces domaines ne donne pas un avantage et que d'autres portions et/ou répétitions de la molécule sont requises. Si nous prenons l'ensemble de nos données, ces résultats nous permettent de proposer que CA125 régule la tumorigénicité et le potentiel EMT/métastatique en modulant les complexes de jonction et l'organisation du cytosquelette. Ce projet a permis de déterminer comment CA125 module ces comportements. Les effets du knockdown de CA125 sur les jonctions intercellulaires, l'adhésion cellulaire et la migration ont été comparés aux effets produits par l'expression forcée des différents domaines de CA125 (C-terminal, transmembranaire et une des répétitions). Une fois que les domaines ont été identifiés et liés à des phénotypes nous avons considéré qu'il était important de commencer à déterminer les voies de signalisation impliquées et modulées entre nos systèmes cellulaires. La présence du EGF dans le milieu de culture a donc été identifié comme un facteur essentiel permettant la migration de cellules CA 125 knockdown. Ces études contribuent de façon significative à notre compréhension des fonctions biologiques de CA125, son rôle dans le développement et progression du carcinome épithélial de l'ovaire et suggèrent que CA125 pourrait éventuellement être une nouvelle cible moléculaire pour le traitement de cette maladie.