La transition épithélio-mésenchymateuse (TEM) est un processus physiologique décrit dans le développement embryonnaire et chez l'adulte au cours de la cicatrisation. La TEM est également détournée dans le contexte pathologique au cours de l'invasion tumorale et les mécanismes moléculaires qui la contrôlent font à ce jour l'objet d'intenses investigations. Cette étude décrit le rôle du gène de la lignée germinale NANOS-3 dans la régulation de l'invasion tumorale broncho-pulmonaire associée à la TEM. Nous démontrons que l'expression de Nanos-3 est corrélée à l'agressivité des cancers bronchiques non à petites cellules (CBNPC) humains in vivo et qu'il est surexprimé pendant la TEM induite in vitro. De plus, la surexpression de Nanos-3 dans les lignées tumorales bronchiques augmente leurs capacités invasives in vitro en induisant la TEM alors que son inhibition induit l'effet opposé et promeut la transition mésenchymo-épithéliale (TME). Au cours de cette étude, nous rapportons également des mécanismes à la fois transcriptionnels et post-transcriptionnels de régulation des cibles de Nanos-3. Ainsi, nous montrons que Nanos-3 réprime la transcription du gène CADHERINE-E indépendamment des facteurs de transcription des familles Snail et ZEB. Nous décrivons également que la protéine Nanos-3 co-immunoprécipite avec certains ARNm de ses cibles et, plus particulièrement, qu'elle est capable de réguler la longueur de la queue poly-(A) du transcrit codant pour une de ses cibles majeures : la Vimentine. En parallèle, par des méthodes d'études in silico et in vitro, nous démontrons une localisation à la fois cytoplasmique et nucléaire de Nanos-3 ainsi que son accumulation nucléolaire. Enfin, nous mettons en évidence que la réexpression ectopique de Nanos-3 dans le contexte tumoral pourrait être attribuée à une dérégulation des mécanismes épigénétiques physiologiquement mis en place dans les cellules somatiques adultes. Ainsi, cette étude démontre le rôle de Nanos-3 dans l'acquisition d'un phénotype invasif par les cellules tumorales bronchiques et décrit un nouveau mécanisme de régulation de la TEM dépendant de la longueur de la queue poly-(A) de certains ARNm. / The Epithelial-Mesenchymal Transition (EMT) is a basic cellular process used by embryo to generate different tissues or in adult during wound healing. EMT is also misappropriated by cancer cells during the first step towards metastasis. Molecular mechanisms driving EMT during tumor progression are extensively studied and post-transcriptional regulations of EMT-associated genes emerge as major and promising field in oncology. Here we report a dual post-transcriptional and transcriptional regulation of EMT-associated genes by the NANOS-3 germline gene during lung tumor invasion. We show that the Nanos-3 expression in vivo correlates with aggressiveness of human non-small cell lung carcinomas (NSCLC) and that Nanos-3 is upregulated in cells which undergo an EMT in our in vitro EMT-inducible models. Moreover, Nanos-3 overexpression in human NSCLC cell lines enhances their invasive abilities by EMT regulation while its silencing induces the opposite effect leading to a Mesenchymal-Epithelial Transition (MET). Molecular investigations indicate that Nanos-3 controls its targets by either transcriptional or post-transcriptional mechanisms. We show that Nanos-3 represses E-CADHERIN transcription independently of Snail and ZEB transcription factor families. Moreover, we also find that mRNAs of post-transcriptionally regulated targets are co-immunoprecipitated with the Nanos-3 protein and that Nanos-3 regulates the length of the 3' poly-A tail of VIMENTIN mRNA. This dual mechanism of EMT regulation by Nanos-3 is to be related to the specific subcellular localization of Nanos-3 in both cytoplasm and nucleus associated with a nucleolus accumulation as shown by in vitro and in silico experiments. Finally, we demonstrate an epigenetic regulation of NANOS-3 gene expression in lung cell lines, thus supporting that its ectopic expression could be attributed to an epigenetic machinery deregulation in cancer cells.Thus, here we demonstrate a new innovative role for Nanos-3 in the acquisition of an invasive phenotype by lung tumor cells and we describe a novel mechanism of post-transcriptional regulation of EMT via the control of the mRNA poly-A tail length.

O câncer de pâncreas é considerado um dos adenocarcinomas mais agressivos, ocupando o quarto lugar de mortes devidas a câncer, isto é dado principalmente a seu desenvolvimento silencioso e a sua complexidade genética, tornando-o de difícil detecção. Consequentemente, o diagnóstico desta doença ocorre apenas em fase tardia, quando o tratamento é apenas para fins paliativos. As anomalias genéticas mais frequentes no câncer de pâncreas invasivo estão relacionadas à ativação por mutações do gene KRAS e à inativação dos genes supressores de tumor CDKN2A, TP53, SMAD4 e BRCA2. Além destas conhecidas vias de sinalização, fatores de crescimento como o TGF-? e EGF também apresentam papel fundamental na progressão e metástase do câncer de pâncreas. Interessantemente, TGF-? e EGF são também indutores do processo denominado transição epitelial-mesenquimal (EMT), onde células epiteliais normais, durante a embriogênese, ou células cancerosas durante a progressão tumoral e metástase, perdem seus contatos intracelulares e adquirem caráter migratório. Desta forma a EMT, induzida por altos níveis de TGF-? e/ou EGF, é considerada como um dos mecanismos de progressão tumoral em adenocarcinomas. No presente estudo, foram estudados os proteomas e o fosfoproteoma da EMT do PanCa. Assim, células de câncer de pâncreas PANC- 1 foram induzidas à EMT em cultura com os fatores de crescimento TGF-?1 ou TGF-?2 e EGF, e após a indução, marcadores moleculares e propriedades funcionais de migração e invasão foram confirmados. Duas condições de indução da EMT foram estabelecidas, para as quais foram desenvolvidas as análises proteômicas quantitativas. A abordagem foi baseada em marcação isotópica de células em cultura (SILAC), em conjunto com fracionamento celular e de proteínas intactas, e cromatografia líquida acoplada à espectrometria de massas para identificação de proteínas em larga escala. No total, aproximadamente 5.000 proteínas foram identificadas, e a maioria delas quantificadas com precisão nas duplicatas experimentais. Foram selecionadas 37 proteínas com expressão diferencial estatisticamente significativa nos experimentos proteômicos, as quais participam principalmente em processos de biogênese, adesão e apoptóticos. A análise de redes de interação revelou que as proteínas alteradas estavam principalmente localizadas em vias de sinalização que controlam processos de organização da matriz extracelular, splicing alternativo e regulação da apoptose. A análise do fosfoproteoma foi feita usando TGF-?1 como agente indutor da EMT nas células PANC-1, usando a estratégia ERLIC para o enriquecimento de fosfopeptídeos. No total, foram identificados 5.965 fosfopeptídeos não redundantes, correspondendo a um total de 2.250 fosfoproteínas analisadas, sendo quantificadas 2.053 ao menos em duas replicatas, e destas foram identificados 61 fosfopeptídeos regulados pertencentes a 55 fosfoproteínas, relacionados com processos de regulação do mRNA e vias de sinalização ligadas à adesão celular. Em conclusão, nosso estudo elucida potenciais novos alvos para inibição da EMT, controle da metástase, ou para auxiliar no diagnóstico da doença, quando devidamente validada. / Pancreatic cancer kills more than 200 thousand people worldwide every year. Also, pancreatic cancer is considered one of the most aggressive adenocarcinomas and difficult to diagnose since it develops silently and presents a high genetic complexity. Consequently, the diagnostic is often late, when the pancreatic cancer has already metastasized and the treatment has only palliative purposes. The most frequent genetic alterations observed in pancreatic cancer are related to mutations in KRAS oncogene and CDKN2A, TP53, SMAD4 and BRCA2 tumor suppressor genes. In addition to these known frequent mutations, growth factors such as TGB- ? and EGF play important roles in pancreatic cancer progression and metastasis. Interestingly, TGB- ? and EGF are also inducers of the Epithelial to Mesenchymal Transition (EMT), in which epithelial cells lose their intracellular contacts and acquire migratory capacities. Therefore, EMT is considered one of the mechanisms responsible for tumor progression and metastasis in adenocarcinomas in addition of being correlated to the process of generating cancer stem cells. In our present study, pancreatic cancer cell line PANC-1 was induced to EMT by using growth factors TGF-?1 or TGF-?2 and EGF. Both molecular and functional properties, such as invasion and migration, were evaluated in PANC-1 cells undergoing EMT and confirm the induction. Two conditions of EMT induction were properly established and in-depth quantitative proteomic analysis based on stable isotope labeling in cell culture (SILAC) followed by cellular and protein fractionation were assessed. In total, 5.000 proteins were identified and most of them were accurately quantified in duplicate experiments. Thirty-seven proteins were selected as differentially expressed with statistical significance, and were related mainly with biogenesis, adhesion and apoptosis processes. Interaction network analysis showed that regulated proteins were predominantly participating in signaling pathways linked to extracellular matrix organization, alternative splicing and apoptosis regulation. Phosphoproteome analysis was done using TGF-?1 as EMT-inductor agent on PANC-1 cells and ERLIC strategy for phosphopeptides enrichment. In total, were identified 5.965 non-redundant phosphopeptides corresponding to approximately 2.250 analyzed phosphoproteins, thereof 2.053 were quantified in at least two replicates. At comparison, were identified 61 regulated phosphopeptides belonging to 55 phosphoproteins, which were related with mRNA regulation processes and signialing pathways linked to cellular adhesion. In conclusion, our study highlighted potential new targets for EMT inhibition, metastasis control or to help in pancreatic cancer diagnosis, when careful validated.

As neoplasias mamárias apresentam um grande desafio tanto para a medicina humana, quanto para a medicina veterinária. Esses tumores apresentam ampla heterogeneidade intertumoral e intratumoral, dificultando assim a busca por tratamentos eficazes. Recentemente, pesquisadores tem voltado sua atenção para uma população de células que apresentam características muito semelhantes as células-tronco. São as chamadas células iniciadoras de tumores (CITs). Estas são descritas como as principais responsáveis por falhas nas quimioterapias e no surgimento de recidivas tumorais, devido ao seu potencial tumorigênico, de auto-renovação e de resistência a drogas antineoplásicas. Entretanto, o estudo dessas células é limitado pelas dificuldades no isolamento e na caracterização pós-enriquecimento dessas células, devido à perda do fenótipo em modelos in vitro. Sendo assim, vários estudos estão buscando maneiras alternativas de enriquecer essa população. Uma das maneiras mais utilizadas, baseia-se na indução do processo de transição epitélio-mesenquimal, através da superexpressão de fatores de transcrição como SNAI1, SLUG, ZEB1 e ZEB2. Sendo assim, nós objetivamos expressar de maneira exógena os fatores de transcrição SLUG e ZEB1 em células oriundas de carcinomas mamários caninos, caracterizar seus efeitos nessas células e observar se esses fatores de transcrição seriam capazes de induzir o fenótipo de CIT. Primeiramente, quatro amostras de carcinomas mamários caninos foram analisados quanto sua morfologia e os níveis de expressão gênica de quatro fatores de transcrição associados a transição epitélio-mesenquimal: SLUG, STAT3, ZEB1 e ZEB2. Após, nós selecionamos duas dessas amostras (CC-20E e CL-28E), que apresentavam morfologia típica de células epiteliais e baixa expressão dos fatores de transcrição citados acima e expressamos de maneira exógena e de forma estável os fatores de transcrição SLUG e ZEB1, através do processo de transdução lentiviral. Entretanto, apenas a transdução com os plasmídeos contendo a região codificante de SLUG foi eficiente. Sendo assim, nós avaliamos os efeitos da expressão exógena de SLUG nas células CC-20E e CL-28E, quanto a alteração de morfologia e expressão de filamentos intermediários como citoqueratina, vimentina e actina. Além disso, nós avaliamos se a expressão exógena de SLUG poderia regular a expressão de outros genes associados a EMT, além de genes supressores de tumores, alvos de SLUG. Por fim, nós avaliamos se a expressão exógena de SLUG poderia induzir ao fenótipo de CITs, verificando se havia alteração na sensibilidade das células aos quimioterápicos doxorrubicina e paclitaxel, além de avaliar o potencial tumorigênico e de auto-renovação dessas células em cultivos de baixa aderência. A expressão exógena de SLUG nas células CC-20E e CL-28E, não induziu a alterações na morfologia epitelial das células. Entretanto, as células alteraram sua disposição em monocamada no cultivo, formando tipos de túbulos semidiferenciados, característicos do processo de EMT híbrido ou parcial. Além, disso, houve um equilíbrio entre a expressão dos filamentos intermediários de citoqueratina e vimentina nas células, além do aumento na expressão dos genes CDH1 (E-caderina) e CDH2 (N-caderina), resultado que sustentou a indução de EMT parcial. O processo de EMT parcial induziu maior resistência ao quimioterápico paclitaxel, além de potencializar a tumorigenecidade e a capacidade de auto-renovação das células em cultivos de baixa aderência. Sendo assim, no presente estudo, nós obtivemos um cultivo com características que mimetizam as CITs, demonstrando que os processos que induzem esse fenótipo são semelhantes tanto na espécie canina, quanto em humanos, sustentando a hipótese de que neoplasias mamárias caninas podem servir como modelo para o estudo das CITs e, consequentemente, do desenvolvimento neoplásico de tumores sólidos. / Mammary neoplasms present a major challenge for both human and veterinary medicine, due to intertumoral and intratumoral heterogeneity, hindering the search for effective treatments. Recently, researchers has highlighted a population of cells with features very similar to stem cells. Known as Tumor-Initiating Cells (TICs), they are described as the main responsible for chemotherapy failures and tumor recurrence, due to their tumorigenic potential, self-renewal ability and drug resistance. The study of TICs is limited mainly by their difficult isolation owing to specific markers absence, and furthermore, cells lose their phenotype when placed in vitro. Therefore, several studies are seeking for alternatives to enrich this population in regular cultures. One way is based on the epithelial-mesenchymal transition induction through of transcription factors overexpression, such as SNAI1, SLUG, ZEB1 e ZEB2. So, the aim of this study was to overexpresse the SLUG and ZEB1 transcription factors in a cell culture derived from canine mammary carcinomas, evaluate its effects and observe whether these transcription factors would be capable of inducing the TIC phenotype. First, four canine mammary carcinomas cell cultures were analyzed for their morphology and gene expression levels of four transcription factors associated with epithelial-mesenchymal transition: SLUG, STAT3, and ZEB1 ZEB2. After, we selected two samples (CC-20E and CL-28E) with typical morphology of epithelial cells and low expression of the transcription factors mentioned above. We then overexpress, stably, the transcription factors SLUG and ZEB1 by lentiviral transduction, However, only SLUG transduction was efficient. Then, we evaluated the effects of SLUG overexpression in CC-20E and CL-28E cells as the change of morphology, expression of intermediate filaments as cytokeratin, vimentin and actin. In addition, we evaluated whether SLUG overexpression could regulate the expression of other EMT-associated genes as well as tumor suppressor genes, and assessed evaluated the tumorigenic potential and self-renewal of these cells in low adherence cultures. Finally, we assessed whether SLUG overexpression could induce drug resistance through doxorubicin and paclitaxel sensivity assay. The SLUG overexpression did not induce modification in epithelial cell morphology, however, cells changed their arrangement in monolayer culture, inducing the semidifferentiated tubules, typical of hybrid or partial EMT process. In, addition, there was a balanced expression between cytokeratin and vimentin, possibly explained by an increase in CDH1 expression (E-cadherin) and CDH2 (N-cadherin) typical of partial EMT. Furthermore, the partial EMT generated cells presenting paclitaxel resistance, and enhanced the tumorigenic potential and self-renewal capacity of the cells on low adherent plates. Thus, in this study, we obtained a cell culture exhibiting features that mimics the TICs, demonstrating the mechanisms which regulate this phenotype are similar in dogs and humans, supporting the hypothesis that canine mammary carcinomas are a great model for the study of TICs and solid tumors development.

L’ensemble des tissus et des organismes vivants sont constitués de cellules dans lesquelles un certain nombre de décisions phénotypiques sont prises : division, différentiation, apoptose ou encore transformation. La biologie cellulaire s’est principalement concentrée sur la compréhension des déterminants moléculaires internes de ces décisions, mais il est important de considérer aussi l’existence de déterminants externes provenant des interactions intercellulaires qui sont essentielles à l’émergence de systèmes multicellulaires coordonnés. La compétition entre les déterminants internes et les déterminants externes est un aspect fondamental de la sociologie des communautés cellulaire menant à de possibles situations hautement individualisées ou, au contraire, à un effet collectif dominant. Ce travail de thèse a eu pour but de mettre en place une méthode permettant de mesurer la contribution relative de ces deux types de déterminants en les mettant en opposition. Pour cela, la stabilité collective d’un épithélium in vitro a été mise à l’épreuve grâce à l’induction hétérogène de la transition épithelio-mesenchymateuse (EMT) par le biais de la photoactivation du facteur de transcription Snail1. Les résultats principaux montrent que la réponse transcriptionelle de cellules induites à l’EMT dépend de la présence, ou non, de cellules avoisinantes non-induites. De la même manière, les cellules non-induites répondent de façon transcriptionelle à la présence de cellules induites. Ces effets de control mutuels introduisent la notion que la géométrie de la distribution d’une cause moléculaire donnée peut influencer la conséquence de cette même cause. Notre travail ouvre de nouvelle possibilités pour l’étude de la sociologie de communautés cellulaires hétérogènes, et une meilleure compréhension de phénomènes importants tel la suppression phénotypique ou encore les premiers instants de la carcinogenèse. / Tissues and organisms are built from cells in which important phenotype decisions are made: division, differentiation, apoptosis, and transformation. Cell biology has strongly focused on deciphering the internal molecular determinants of these decisions, but external information originating from intercellular interactions are key elements to coordinate multicellular physiology. The extent to which internal determinants dominate over external determinants or vice versa, is an essential feature of the sociology of cell communities, with possibly strong individualistic situations, or dominant collective effect. The present work was designed to set-up a method for assessing the relative contribution of internal vs. external determinant, by opposing these two classes of inputs. This is achieved by challenging the collective stability of an in vitro epithelium using the heterogeneous induction of the epithelial-to-mesenchymal transition (EMT) via the photoactivation of Snail1. The key results show that the transcriptional response of EMT-induced cells depends on the presence of non-induced cells in the culture. Conversely non-induced cells respond to the presence of induced cells. These mutual control effects lead to the notion that the geometry underlying the distribution of a given molecular cause strongly influences its consequence. Our work opens new perspectives for studying the sociology of heterogeneous cell communities, and better understand important phenomena such as phenotype suppression and or the onset of carcinogenesis.

Lors du développement d’une tumeur au sein d’un tissu, les cellules cancéreuses se retrouvent entourées par les cellules saines. Les interactions entre ces deux types cellulaires, transformé et normal, jouent un rôle important dans le devenir de la tumeur, mais restent à ce jour mal comprises. L’objectif de cette thèse a été de mettre en place des systèmes in vitro qui permettent d’étudier les interactions entre une population de cellules normales et une population de cellules transformées.Nous avons tiré profit d’une lignée de cellules épithéliales sensibles à la lumière, élaborée par Olivier Destaing (IAB, Grenoble). Lorsqu’elles sont exposées à la lumière bleue, ces cellules suractivent la protéine Src, connue pour être surexprimée dans de nombreux cancers. Sinon, elles gardent un phénotype normal. L’utilisation de ces cellules, appelées « OptoSrc », combinée à un dispositif optique, permet de créer des tissus mosaïques dans lesquels le motif des cellules mutées est déterminé par le motif d’illumination bleue. Notre système présente plusieurs avantages : le contrôle dans le temps et dans l’espace du motif de cellules transformées, mais aussi l’activation graduelle et réversible de l’oncoprotéine.Nous avons montré qu’en illuminant dans le bleu un îlot circulaire de cellules au sein d’une monocouche OptoSrc, les cellules activées s’extrudent collectivement, donnant naissance à un agrégat tri-dimensionnel cohésif surplombant la monocouche. Nous pouvons contrôler la taille et le temps d’apparition de ce sphéroïde en ajustant respectivement l’aire éclairée et la fréquence d’illumination. De plus, ce phénomène d’extrusion collective est réversible lorsque le stimulus de lumière bleue s’arrête. Finalement, nous avons montré que la formation de cet agrégat s’accompagne d’une diminution des E-cadhérines à la membrane, et de l’apparition de la vimentine, pour les cellules éclairées. Nos résultats suggèrent qu'un groupe de cellules surexprimant la protéine Src, au sein d’une monocouche de cellules normales, subit une transition epithéliale- mesenchymateuse partielle. / During the development of a tumour in a tissue, the cancer cells are surrounded by healthy cells. The interactions between these two cell types, transformed and normal, play an important role in the tumour stability, but remain to this day poorly understood. The aim of this thesis was to establish in vitro assays to study the interactions between populations of normal and transformed cells.We benefited from a light-sensitive cell line, constructed by Olivier Destaing (IAB, Grenoble). When they are exposed to blue light, these cells overactivate the protein Src, which is known to be overexpressed in many cancers. Otherwise, they keep a normal phenotype. Using these cells, called “OptoSrc”, in combination with an optical setup, we are able to create mosaic tissues in which the pattern of mutated cells is determined by the blue illumination pattern. Our system has several advantages: a selective control in time and space of the group of transformed cells, and a gradual and reversible activation of the oncoprotein.We have shown that when we illuminate a circular islet of cells from a monolayer of OptoSrc cells, the activated cells were collectively extruded, resulting in a cohesive three-dimensional aggregate on top of the monolayer. We can control the size and appearance time of this spheroid by tuning, respectively, the area and frequency of illumination. Besides, this collective extrusion is reversible when the blue light stimulation is stopped. Finally, we have shown that the formation of this three-dimensional aggregate coincides with the loss of E-cadherin at the membrane, as well as the apparition of vimentin, for the illuminated OptoSrc cells. Our results suggest that a group of cells overexpressing the protein Src, in a monolayer of normal cells, undergoes a partial epithelial-to-mesenchymal transition.

Objective: Tumor metastasis and resistance to chemotherapy are two critical factors that contribute to the high death rate of colorectal cancer (CRC) patients. Metastasis is facilitated by the epithelial-mesenchymal transition (EMT) of tumor cells, which has emerged not only as a fundamental process during metastasis, but is also a key process leading to chemoresistance of cancer cells. However, the underlying mechanisms of EMT in CRC cell remain unknown. Here, we aim to assess the role of dual serine/threonine and tyrosine protein kinase (DSTYK) in CRC metastasis and chemoresistance. Methods: To study the role of DSTYK in TGF-β-induced EMT, we employed techniques including Crispr/Cas9 knockout (KO) to generate DSTYK KO cell lines, RT-PCR to detect the mRNA expression, immunofluorescence analyses, and western blots to detect protein levels of DSTYK in the following 4 cell lines: control LS411N-TβRII and LS411N-TβRII/DSTYK KO, control LS513 and LS513/DSTYK KO cells, treated with/without TGF-β. The effects of DSTYK on apoptosis were investigated by MTT assays, flow cytometry assays, and TUNEL assays. The expression of DSTYK in CRC patients and its correlation with EMT markers were determined by bioinformatics analysis. For in vivo analysis, both xenograft and orthotopic tumor mouse models were employed to investigate the function of DSTYK in chemoresistance and metastasis of tumors. Results: In this study, we demonstrate that the novel kinase DSTYK promotes both TGF-β-induced EMT and the subsequent chemoresistance in CRC cells. DSTYK KO significantly attenuates TGF-β–induced EMT and chemoresistance in CRC cells. According to the Gene Expression Omnibus (GEO) database, the expression of DSTYK is not only positively correlated to the expression of TGF-β, but proportional to the death rate of CRC patients as well. Evidently, the expression of DSTYK in the metastatic colorectal cancer samples from patients was significantly higher than that of primary colorectal cancer samples. Further, we demonstrate in mouse models that chemotherapeutic drug treatment suppresses the growth of DSTYK KO tumors more effectively than control tumors. Conclusion: Our findings identify DSTYK as a novel protein kinase in regulating TGF-β–mediated EMT and chemoresistance in CRC cells, which defines DSTYK as a potential therapeutic target for CRC therapy.

L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires. / The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.

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No description available.

<p>Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes <i>in vivo. </i>Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis.<i> </i>Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.<br></p>