Conselho Nacional de Desenvolvimento Científico e Tecnológico / Despite extensive research on an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Moreover, patients who survive AKI are at high risk for chronic progressive kidney disease. Mesenchymal stromal cells derived from human amniotic fluid (hAFSCs) are a new source of stem cells which express renal progenitor markers (CD24). The possibility of combining gene and cell therapy allows stem cells to be manipulated to overexpress vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), two of the most important growth factors for kidney regeneration. Therefore, the aim of this study was to evaluate whether hAFSCs overexpressing VEGF and HGF demonstrate a nephroprotective effect due to their mitogenic and anti-inflammatory effect, leading to a long term inhibition of fibrosis. In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples, characterized their immunophenotypic properties and differentiation capacity and selected a clonal lineage which expresses CD24 and CD117 markers. This lineage was subqequently transduced with lentiviral vectors (LV) encoding VEGF and HGF. In a second phase, renal ischemia and reperfusion (IR) injury was induced in a rat model by clamping the renal pedicle for 50 minutes in 50 male Wistar rats. Treatment groups (n = 10 per group) were assigned as follows: a control group treated with Chang Medium only; a group which received non-transduced AFSC (1x106 cells/animal); a group which received AFSC transduced with LV-VEGF (1x106 cells/animal); a group which received AFSC transduced with LV-HGF (1x106 cells/animal); and a group treated with AFSC transduced both with LV-VEGF (0,5x106 cells/animal) and AFSC transduced with LV-HGF (0,5x106 cells/animal). Serum creatinine was measured at 24 hours, 48 hours and 2 months after IR injury and histological analysis was performed to analyze following parameters: tubular necrosis and hyaline cast formation by PAS and H&E staining at 48 hours, and interstitial fibrosis by Masson s Trichrome and Picrosirius Red staining at 2 months. Additionally, the expression of KI-67, α-SMA and TGF-β1 was assessed by immunohistochemistry. The results showed a beneficial effect of AFSCs delivered to rats with IR injury, which was characterized by a faster improvement in renal function and a lower fibrotic index. However, administration of hAFSCs overexpressing VEGF and HGF resulted in an even better outcome compared to non-transduced AFSCs. As early as 24 hours after AFSC delivery, a nephroprotective effect was observed after both hAFSC and hAFSC VEGF + HGF treatment, which was characterized by significantly lower creatinine values compared to those of the control group. At 48 hours, all treatment groups still demonstrated a significant increase in creatinine values compared to sham animals, except in the hAFSC HGF + VEGF group. In hAFSC HGF + VEGF and hAFSC VEGF treatment groups, a significant increase in renal tubules proliferation was observed, measured by an increase in KI-67 expression, which is probably due to the effect of VEGF overexpression. Furthermore, we observed a decrease in α-SMA and TGF-β expression at 48 hours in the non-transduced hAFSC, hAFSC HGF and hAFSC VEGF + HGF groups. As TGF-β1 is involved in transdifferentiation of tubular epithelial cells to α-SMA-positive myofibroblasts which increases extracellular matrix deposition, the reduction in the expression of α-SMA and TGF-β indicates an inhibition of fibrosis. Although VEGF and HGF have both been described to have nephroprotective properties, we interestingly observed that the hAFSC expressing both VEGF and HGF resulted in more pronounced kidney damage compared to non-treated animals when treated with 1x106 cells/animal, which suggests that toxic side effects are possibly induced by high secretion levels of growth factors. In conclusion, cellular therapy using the combination of hAFSCs transduced with lentiviral vectors encoding VEGF and HGF, resulted in a stronger nephroprotective effect than non-transduced hAFSC delivered to rats with I/R injury, which was characterized by an increased mitosis index, an improved renal function and an inhibition of genes involved in fibrosis resulting in a lower fibrotic index at two months. / O tratamento eficaz para a lesão renal aguda tem melhorado nos últimos anos, sendo objeto de inúmeras pesquisas, no entanto a taxa de mortalidade desta patologia ainda permanece elevada. Além disso, pacientes que sobrevivem após evento isquêmico possuem altos riscos de doença renal crônica progressiva. As células mesenquimais estromais do líquido amniótico humano (hAFSC) são uma nova fonte alternativa de células-tronco que expressam marcadores progenitores renais (CD24). A possibilidade da associação das terapias gênica e celular permite a manipulação dessas para superexpressar o fator de crescimento vascular endotelial (VEGF) e o fator de crescimento de hepatócitos (HGF), dois dos fatores de crescimento mais importantes para a regeneração renal. Diante disso, o objetivo desse estudo foi avaliar se as hAFSCs transduzidas com VEGF e HGF possuem maior ação nefroprotetora, por meio de efeito mitótico e anti-inflamatório a curto prazo, levando a uma inibição da fibrose a longo prazo em modelos de isquemia e reperfusão renal. Na primeira fase do experimento, isolou-se, caracterizou-se as propriedades imunofenotípicas e a capacidade de diferenciação das hAFSCs e após selecionou-se uma linhagem clonal que expressasse os marcadores CD24 e CD117. Após essa linhagem foi transduzida com vetores lentivirais (VL) codificando VEGF e HGF. Na segunda fase, induziu-se lesão de isquemia e reperfusão (IR) renal pelo clampeamento do pedículo renal por 50 minutos, em 50 ratos Wistar, machos. O grupos de tratamento foram divididos como (n=10 por grupo): grupo controle, tratado somente com o meio Chang; grupo tratado com hAFSC não transduzidas (1x106/rato); grupo tratado com hAFSC transduzidas com LV-VEGF (1x106/rato); grupo tratado com hAFSC transduzidas com LV-HGF (1x106/rato) e o grupo tratado tanto com hAFSC transduzidas com LV-VEGF (0,5x106/rato) quanto LV-HGF (0,5x106/rato). A creatinina sérica foi mensurada em 24 horas, 48 horas e 2 meses após a lesão IR e as análises histológicas foram realizadas para avaliar os seguintes parâmetros: necrose tubular e formação de cilíndros hialinos pelas colorações de PAS e H&E em 48 horas e fibrose intersticial pelas colorações Tricrômico de Masson e Picrosirius Red em 2 meses Adicionalmente, a expressão de KI-67, -SMA e TGF- foram analisadas por imunoistoquímica em 48 horas. Os resultados permitiram observar um efeito benéfico da terapia com hAFSCs em lesões de IR pela melhora mais rápida da função renal e menor índice fibrótico a longo prazo, no entanto obteve-se um efeito ainda melhor quando associaram-se as hAFSCs. transduzidas com VEGF e HGF comparado com as hAFSC não transduzidas. Já em 24 h observou-se o efeito renoprotetor nos grupos hAFSC e hAFSC VEGF + HGF pelo valor significativamente menor da creatinina comparado com o controle. Em 48h todos os grupos ainda apresentavam valores significativamente elevados de creatinina comparado com os ratos sham, exceto o grupo hAFSC HGF + VEGF. Observou-se também que os grupos hAFSC VEGF +HGF e hAFSC VEGF tiveram um aumento significativo na proliferação tubular renal, provavelmente pelo efeito da superexpressão de VEGF. Além disso, observou-se redução da expressão de α-SMA e TGF-β em 48h nos grupos hAFSCs não-transduzidas, hAFSC VEGF+HGF e hAFSC HGF. Como o TGF- β1 está envolvido na transdiferenciação de células epiteliais tubulares em miofibroblastos α-SMA-positivos, o qual aumenta a deposição de matriz extracelular, a redução na expressão de α-SMA e TGF-β são indicadoras de inibição da fibrose. Apesar de serem descritos diversos benefícios nefroprotetores do VEGF e do HGF observou-se nesse estudo uma lesão renal mais pronunciada do que o controle nos grupos hAFSC VEGF e hAFSC HGF, tanto em relação à função renal quanto à necrose tubular, o que sugere um efeito tóxico causado pela alta concentração de secreção desses fatores de crescimento. A terapia celular utilizando a combinação de hAFSCs transduzida com vetores lentivirais codificando VEGF e HGF resultou em efeito nefroprotetor ainda maior do que sua forma não transduzida após evento isquêmico renal, o qual foi caracterizado pelo aumento do índice mitogênico, melhor função renal e inibição de genes fibrogênicos, levando a um menor índice fibrótico em dois meses.

Il est admis que le Transforming Growth Factor-bêta (TGF-ß) est athéroprotecteur. Son apport bénéfique dans l'athérosclérose semble être contrarié par l'Interleukine-1-bêta (IL-1ß) qui est l'archétype des cytokines pro-inflammatoires. Notre but est, d'une part, de comprendre la régulation transcriptionnelle du TGF-ß dans les cellules musculaires lisses vasculaires (CMLv) humaines, dans l'athérome carotidien humain à partir des données du transcriptome obtenues sur 32 patients à la fois dans les plaques athéromateuses (ATH) et dans le tissu avoisinant macroscopiquement sain (TMS) et, d'autre part, d'étudier l'antagonisme des effets du TGF-ß et de l'IL-1ß sur des CMLv en culture provenant du TMS. Nous avons abordé nos problématiques par deux approches : (1) une analyse bioinformatique de données transcriptomiques issues de biopuces. (2) une étude in vitro sur des CMLv de la carotide humaine. in vitro. Grace à l'analyse in silico nous avons montré que l'implication de KLF6 dans la transcription du TGFB1 était assez spécifique des cellules musculaires lises vasculaires (CMLv). Nos résultats permettent également de proposer de nouveaux FT potentiels, spécifiques des CMLv (SLC2A4RG, GABP et SALL2), qui pourraient favoriser le rôle athéroprotecteur de TGFB1 dans les CMLv de la carotide. Enfin, il semble qu'il existe un équilibre entre les FT activateurs ou inhibiteurs de l'expression de TGFB1 qui permet d'en moduler finement la transcription. Notre approche in vitro, à montre que l'IL-1ß induisait un phénotype inflammatoire associé à une activité élastolytique consécutive, pour l'essentiel, à l'augmentation de l'expression de la cathépsine S (CTSS). La neutralisation du TGF-ß endogène dé-réprime l'expression de la CTSS et exerce un effet additif à celui de l'IL-1ß sur l'expression de l'enzyme. En conclusion, l'expression du TGFB1 dans les CMLv étant soumise à un contrôle transcriptionnel spécifique du type cellulaire, il est envisageable de développer des approches pharmacologiques qui permettent de maintenir l'expression du TGFB1 dans la paroi artérielle au niveau requis pour qu'il y exerce ses effets athéroprotecteurs / It is accepted that the TGF-β is atheroprotective. Its beneficial contribution atherosclerosis seems to be opposed by IL -1β that is the archetype of the pro-inflammatory cytokines. Our goal is, first, to understand the transcriptional regulation of TGF-β in human vSMCs in carotid atherosclerosis from the transcriptomic data obtained from 32 patients in both atherosclerotic plaques (ATM) and in the adjacent macroscopically intact tissue (MIT) and, secondly, to study the antagonism of the effects of TGF-β and IL-1β on vSMCs cultured from MIT samples. We followed two approaches: (1) a bioinformatic analysis of transcriptomic data from the microarrays; (2) an in vitro study of the human vSMCs. In silico, we have shown that the involvement of KLF6 in the transcription of TGFB1 was specific to the vSMCs. Our results also identify potential new transcription factors (SLC2A4RG, GABP and SALL2) that are vSMC-specific and could promote the atheroprotective role of TGFB1 in carotid vSMCs. The balance between the FT activating or inhibiting the expression of TGFB1 allows the fine tuning of its transcription. Our in vitro approach showed that IL-1β induces in the vSMCs an inflammatory phenotype associated with an elastolytic activity resulting mainly from the increase in the expression of cathepsin S (CTSS). Neutralization of endogenous TGF-β in the vSMCs de-represses the expression of the CTSS and exerts an additive effect to that of IL-1β on the expression of the enzyme. In conclusion, the expression of TGFB1 in the vSMCs is submitted to a cell-specific transcriptional control. It is possible to develop pharmacological approaches that maintain the expression the expression of TGFB1 in the arterial wall at the level required allowing TGF-β1 to exert its atheroprotective effects

Les invadosomes sont des structures d’invasion plastiques et dynamiques qui interagissent avec leur microenvironnement. Ils possèdent différentes fonctions telles que l’adhésion, la mécanotransduction ou encore la dégradation de la matrice extracellulaire (MEC). Mon travail de thèse s’est concentré sur i) l’étude globale de la composition des invadosomes par spectrométrie de masse et ii) sur l’impact d’éléments du microenvironnement dans la formation de ces structures d’invasion.i) Les invadosomes sont des complexes multi-protéiques dont tous les partenaires ne sont pas encore totalement identifiés. Au laboratoire, une nouvelle approche combinant la microdissection laser suivie d’une analyse par spectrométrie de masse, a été développée. Cette technique a été appliquée à l’étude des invadosomes rosettes. Nous avons ainsi mis en évidence une nouvelle fonction associée aux invadosomes, en les définissants comme des sites actifs de traduction protéique. Les invadosomes cependant, sont des structures plastiques dont la formation et la morphologie sont modulées par différents éléments de l’environnement. Nous souhaitons à présent déterminer les partenaires communs et spécifiques entre les différentes organisations des invadosomes afin d’identifier les molécules impliquées dans cette plasticité.ii) La formation des invadosomes peut être induite par différents éléments du microenvironnement comme des facteurs de croissance ou encore la composition et la rigidité de la MEC. Le TGF-β est un facteur de croissance impliqué dans la formation des invadosomes, dans la promotion de la rigidité de la MEC et dans la fibrose hépatique pouvant mener au développement du carcinome hépatocellulaire. Nous avons alors étudié l’impact du TGF-β dans la formation des invadosomes linéaires en contexte de collagène de type I. Nous montrons que le TGF-β module la machinerie moléculaire associée aux invadosomes linéaires en induisant l’expression de DDR1 et MT1-MMP, ainsi que des éléments impliqués dans leur formation tels que le collagène I. Ces modulations sont dépendantes de la voie de signalisation canonique du TGF-β passant par Smad4 et favorisent la formation et l’activité des invadosomes linéaires. De plus, le TGF-β induit une surexpression de la LOXL2 qui est une enzyme de réticulation du collagène, augmentant la rigidité de la matrice ce qui favorise la formation des invadosomes.Les résultats obtenus durant ma thèse auront permis de mieux définir les éléments impliqués dans la composition et la formation des invadosomes. / Invadosomes are plastic and dynamic invasive structures interacting with the microenvironement. Those structures are involved in several functions as adhesion, mecanotransduction and degradation of the extracellular matrix (ECM). My PhD work focuses on i) the study of the invadosomes composition by mass spectrometry and ii) on the impact of microenvironmental elements on the formation of those invasive structures.i) Invadosomes are multi-protein complexes in which all partners are not yet fully identified. In the laboratory, a new approach combining laser micordissection followed by mass spectrometry analysis was developed. This technique has been applied to the study of invadosome rosettes. We have demontrasted a new function associated with indosomes, defining them as active sites of protein translation. Invadosomes, however, are plastic structures whose formation and morphology are modulated by different elements of the environment. We now wish to determine the common and specific partners between the different invadosomes organizations in order to identify the molecules involved in their plasticity.The invadosomes formation can be induced by different elements of the microenvironment such as growth factors or the composition and rigidity of the ECM. TGF-β is a growth factor involved in the invadosomes formation, in the ECM rigidity and in liver fibrosis that can lead to the development of hepatocellular carcinoma. We have studied the impact of TGF-β in the formation of linear invadosomes in the context of type I collagen. We show that TGF-β modulates the molecular machinery associated with linear invadosomes by inducing the expression of DDR1 and MT1-MMP, as well as elements involved in their formation, such as collagen I. These modulations are dependent on the TGF-β canonical signaling pathway through Smad4 and promote the formation and activity of linear invadosomes. In addition, TGF-β induces an overexpression of LOXL2, which is a collagen cross-linking enzyme, increasing the matrix stiffness and promotes the formation of these structures.Taken together, these results enabled us to better define the elements involved in the composition and formation of invadosomes.

La survie des patients atteints de cancer et traités avec des thérapies conventionnelles reste faible dans plusieurs types de tumeurs. Récemment, une nouvelle approche immunothérapeutique a été développée pour cibler le système immunitaire au lieu de la tumeur elle-même, afin de restaurer la fonctionnalité des cellules immunitaires et la destruction des cellules cancéreuses. L’immunothérapie ciblant le récepteur inhibiteur PD-1 occupe une place privilégiée dans les thérapies anticancéreuses en raison de sa haute spécificité et de sa faible toxicité par rapport aux thérapies conventionnelles. Cependant, le taux de réponse reste faible avec seulement 20 à 25% de patients répondant à une immunothérapie anti-PD-1. Il est donc important de comprendre les mécanismes associés à la résistance à ces thérapies et d’identifier des biomarqueurs prédictifs de réponse. L'expression du ligand de PD-1, PD-L1, sur les cellules tumorales, la charge mutationnelle tumorale et l'infiltration tumorale par les lymphocytes ont déjà été décrits, mais de nouveaux biomarqueurs sont nécessaires pour mieux déterminer la sous-population de patients susceptible de bénéficier de ces traitements. Au cours de ce travail, nous avons établi une cohorte de 118 patients atteints d'un cancer du poumon non à petites cellules (CBNPC) traités avec une immunothérapie anti-PD-1/PD-L1, et nous avons étudié l'expression de plusieurs biomarqueurs potentiels, en particulier les cellules T mémoires résidentes dans le tissu (TRM) CD8+CD103+. Ces cellules constituent un candidat potentiel car elles représentent une population privilégiée de lymphocytes T CD8 grâce à l’expression de PD-1 et une forte capacité cytotoxique vis-à-vis des cellules tumorales autologues suite à la neutralisation de l’interaction de PD-1 avec PD-L1. Nous montrons qu’une forte infiltration de tumeurs de CBNPC avec des cellules TRM corrèle à une survie sans progression plus élevée (PFS) et une réponse plus efficace à anti-PD-1 que les tumeurs avec une faible infiltration par des TRM. De plus, les tumeurs qui expriment fortement ICAM-1, un ligand de l’intégrine LFA-1 exprimée sur les lymphocytes T CD8, sont hautement infiltrées par des TRM. Par ailleurs, il est bien connu que le signal TGF-β est crucial pour l’induction de CD103 et la formation de TRM CD8+CD103+. Je me suis donc intéressée à l'activation du TGF-β par les intégrines αV exprimée par les cellules tumorales humaines et murines. À l'aide des modèles in vitro et in vivo, nous montrons que les cellules tumorales exprimant les intégrines αV activent le TGF-β et induisent l'expression de CD103 à la fois par les cellules T CD8+ provenant de cellules mononucléées du sang périphérique (PBMC) et de lymphocytes infiltrant la tumeur (TIL). L’expression plus faible de CD103 par les TIL CD8+ de souris greffées avec des tumeurs déficientes pour l’expression d'αV n'a pas d'effet sur le contrôle de la croissance tumorale. De manière intéressante, nous montrons dans des modèles de tumeurs déficientes pour l’expression d’αV, que le traitement avec des anticorps anti-PD-1 bloquants corrèle avec un meilleur contrôle de la croissance tumorale et une meilleure réponse à l'immunothérapie anti-PD-1 qui sont associés à une infiltration plus forte de TIL et un état d'activation plus élevé des TIL CD8+ exerçant une activité cytotoxique spécifique. De plus, une expression élevée de l'intégrine αV dans les tumeurs corrèle avec une réponse plus faible des patients atteints de CBNPC à une immunothérapie anti-PD-1/PD-L1. Ces données montrent comment trois marqueurs distincts, cellules TRM, ICAM-1 et les intégrines αV, régulent le microenvironnement tumoral et l’immunité T CD8 avec des implications potentielles pour potentialiser les réponses aux immunothérapies. / The survival of cancer patients treated with conventional therapies remains low in multiple cancers. Recently, a new immunotherapeutic approach has been developed to target the immune system instead of the tumor itself, in order to restore immune cell functions in cancer destruction. Immunotherapy targeting the T cell inhibitory receptor PD-1 occupies a privileged place in cancer therapy thanks to its high specificity and low toxicity compared to chemotherapies. However, the response rate remains low with only 20-25% of patients responding to anti-PD-1 immunotherapy. An important issue is therefore to understand the mechanisms associated with resistance to these therapies and to identify the predictive biomarkers of response. The expression of the PD-1 ligand, PD-L1, on tumor cells, tumor mutational burden (TMB) and tumor infiltration by lymphocytes have been described to predict the response to immune checkpoint blockade (ICB). However, new biomarkers are needed to better determine patient subpopulation which could benefit from this treatment. To address this question, we established a cohort of 118 non-small cell lung cancer (NSCLC) patients treated with anti-PD-1/PD-L1 immunotherapy and studied the expression of several potential biomarkers. Tissue-resident memory T (TRM) cells are a potential candidate because they represent a distinct population of CD8+ T cells highly expressing integrin αEβ7 (CD103) and PD-1; and showing strong cytotoxic capacity towards autologous tumor cells upon neutralisation of PD-1/PD-L1 interaction. Results from the present study show that high infiltration of TRM cells in NSCLC tumors correlates with higher progression-free survival (PFS) and a better response to anti-PD-1/PD-L1 immunotherapy. Moreover, tumors with high expression levels of ICAM-1, the ligand of integrin LFA-1 expressed on T cells, show higher TRM infiltration. TGF-β is a cytokine directly involved in CD103 induction on activated tumor-specific T cells. Therefore, I also investigated the role of αV integrins in activating TGF-β and thereby in controlling TRM differentiation and anti-tumor T cell immunity. Using human and mouse models, we show that tumor cells expressing αV integrins activate TGF-β, which can in turn induce expression of CD103 on CD8+ T cells in vitro on peripheral blood mononuclear cells (PBMCs) and in vivo on tumor infiltrating lymphocytes (TIL). However, lower CD103 expression on CD8+ TIL and thus CD103+ TRM cell formation in C57BL/6 mice engrafted with αV-lacking cancer cells had no effect on tumor growth control. Remarkably, αV-deficient tumors responded more effectively to anti-PD-1 immunotherapy than αV-efficient tumors and this response correlates with higher tumor infiltration by activated CD8+ T cells and stronger cytotoxic activity toward autologous cancer cells. Moreover, high expression of αV integrins in NSCLC tumors correlates with worse response to anti-PD-1/PD-L1 immunotherapy. These data show how three distinct markers, TRM cells, ICAM-1, and αV integrins regulate the tumor microenvironment and CD8+ T cell immunity, with potential implications in improving response to ICB immunotherapies.

Rôle de la NADPH oxydase NOX4 dans la régulation de l'expression du symporteur sodium/iode (NIS) dans le cas du cancer papillaire de la thyroïde (PTC). L’activation autocrine de la voie TGF-β induite par BRAFV600E régule négativement l’expression du symporteur sodium/iode (NIS) via une production de ROS dépendante de la NOX4 dans le cancer papillaire de la thyroïde. Résumé : Le cancer papillaire de la thyroïde (PTC) est la pathologie thyroïdienne la plus répandue. Les mutations ponctuelles de BRAF sont retrouvées dans 40 à 60 % des cas de PTC. La transversion BRAFT1799A est la mutation de BRAF la plus fréquente. Les tumeurs porteuses de la mutation BRAFV600E sont souvent associées avec une diminution significative de l’expression du transporteur sodium/iode (NIS). Les résultats cliniques sur les patients atteints d’un cancer de la thyroïde porteur de la mutation BRAFV600E ont montré que l’inhibition de la voie MAPK ne permet pas de rétablir de manière assez importante l’expression du NIS induite par BRAFV600E. L’expression de BRAFV600E induit la sécrétion de TGF-β fonctionnel, qui inhibe l’expression des protéines thyroïdiennes impliquées dans le métabolisme de l’iode, et particulièrement le NIS. La NOX4 est surexprimée dans un nombre croissant de tumeurs, et particulièrement dans les cas de PTC. Dans le cas du cancer du sein, les mécanismes critiques pour le développement du cancer impliquent la régulation par le TGF-β de la NOX4 au niveau transcriptionnel via le facteur de transcription Smad3. Ces données nous mènent à considérer la NOX4 comme un candidat sérieux pour le rôle de système générateur de ROS contrôlé par la boucle autocrine TGF-β induite par BRAFV600E. Dans cette étude, nous avons tout d’abord observé une corrélation entre la présence de l’oncogène BRAFV600E, la surexpression de la NOX4 et l’inhibition de l’expression du NIS dans les cancers papillaires de la thyroïde. Puis, en utilisant la lignée BCPAP comme modèle in vitro de PTC, nous avons démontré BRAFV600E contrôle l’expression de la NOX4 et de la p22phox par l’intermédiaire de la signalisation TGF-β/Smads. La boucle TGF-β induite par BRAFV600E induit l’expression de la NOX4 et de la p22phox au niveau transcriptionnel via phosphorylated SMAD3. L’expression constitutive de la NOX4 et de la p22phox, qui forment ensemble un complexe NADPH oxydase fonctionnel, contribue au stress oxydatif observé dans les cellules BCPAP. Le traitement des cellules BCPAP par des scavengers de ROS comme le N-acetyl cysteine (NAC) et le Tiron permettent d’augmenter l’expression du NIS au niveau transcriptionnel et de rétablir l’expression d’une protéine fonctionnelle permettant la captation d’iode, ce qui indique que les ROS sont impliqués dans l’inhibition de l’expression du NIS. L’inhibition spécifique de la NOX4 par siRNA permet de réinduire l’expression de l’ARN messager et de la protéine NIS. Ces résultats montrent pour la première fois que les ROS produits par la NOX4 jouent un rôle critique dans l’inhibition de l’expression du NIS induite par BRAFV600E via la signalisation TGF-β/SMAD3. / BRAFV600E induced-TGF-β secretion down-regulates sodium iodide symporter (NIS) expression via NOX4-dependent ROS generation in papillary thyroid carcinoma. Abstract : Papillary thyroid cancer (PTC) is the most common thyroid pathology and BRAF point mutations account for 40-60% of tumors. BRAFT1799A is the most frequent BRAF mutation and BRAFV600E positive tumors are often associated with a significant loss of sodium/iodide symporter (NIS) expression. Clinical results on patients harboring thyroid cancer with BRAF mutation have recently shown that MAPK pathway inhibition does not fully reverts the BRAF-induced NIS repression. BRAFV600E expression induces secretion of functional TGF-β which is a repressor of thyroid specific genes such as NIS. Importantly, NOX4 has been shown to be prominently expressed in an increasing number of tumors, in particular in PTCs. In breast cancer cells, a critical mechanism for cancer development involves the transcriptional regulation of NOX4 by TGF-β. This result prompted us to test NOX4 as a ROS-producing candidate induced by BRAF-induced TGF-β. In this report, we first show in PTCs a correlation between BRAFV600E status, NOX4 overexpression, and low NIS expression level. Then, using BCPAP cells as an in vitro PTC model, we demonstrate that BRAFV600E controls NOX4 and p22phox expression via TGF-β signalling. The TGF-β autocrine loop activated by BRAFV600E induces NOX4 and p22phox expression at the transcriptional level via phosphorylated SMAD3. Both constitutively expressed proteins form a functional NADPH oxidase which produces high intracellular ROS levels. ROS scavengers increase the NIS expression at both mRNA and protein levels, and rescue a functional NIS, indicating that ROS are involved in the repression of NIS. Knocking down NOX4 with specific siRNAs reinduces NIS expression at both mRNA and protein levels. Altogether, these results show for the first time that NOX4-dependent ROS generation has a critical role in BRAF-induced NIS repression via the TGF-β/SMAD3 oncogenic signalling.

Le syndrome von Hippel Lindau (VHL) prédispose au développement de multiples tumeurs hautement vascularisées, telles que des hémangioblastomes rétiniens ou du système nerveux central, des phéochromocytomes et des carcinomes rénaux à cellules claires (CCRCC). Les patients atteints de ce syndrome sont porteurs d’une mutation du gène VHL. Ce gène, composé de trois exons, est transcrit en deux ARN messagers par épissage alternatif de l’exon 2. L’ARNm composé des 3 exons (variant #1) est la forme majoritairement exprimée par rapport à l’ARNm dépourvu de l’exon 2 (variant #2). Toutefois, une diminution du ratio variant #1/variant #2 a été essentiellement décrite dans deux situations : (i) dans les tissus embryonnaires humains et en particulier le rein, et (ii) dans certains CCRCC. Ces données suggèrent un rôle potentiel de ce variant #2 dans la tumorigenèse rénale. Deux protéines, pVHL213 et pVHL160, sont produites à partir du variant #1 et elles agissent comme suppresseurs de tumeur. Au début de ce travail, l’expression de l’isoforme protéique pVHL172 produite à partir du variant #2 restait à démontrer et sa fonction était inconnue. Les travaux effectués au cours de cette thèse ont permis de mettre en évidence l’expression de pVHL172 dans des lignées cellulaires et dans des tissus tumoraux grâce à un nouvel anticorps monoclonal de souris dirigé contre les trois isoformes protéiques humaines de pVHL. Pour savoir si l’isoforme pVHL172 a un rôle de suppresseur de tumeur, des lignées cellulaires tumorales rénales exprimant stablement cette protéine ont été établies puis des expériences de xénogreffes de ces cellules chez la souris ont été réalisées. Non seulement pVHL172 n’inhibe pas la formation de tumeurs mais son expression induit un phénotype tumoral plus agressif avec une composante sarcomatoïde plus importante ainsi qu’une vascularisation immature plus conséquente que dans les tumeurs contrôles (n’exprimant pas pVHL). De plus, pVHL172 augmente l’expression des métalloprotéases de matrice MMP1 et MMP13, en partie via l’activation de la voie de signalisation Smad-dépendante du TGF-β. Par ailleurs, des partenaires protéiques de cette protéine ont été recherchés par une analyse protéomique différentielle. Les réseaux d’interaction réalisés à partir des protéines identifiées concernent entre autres la régulation de la matrice extracellulaire et le contrôle qualité des protéines. En conclusion, ce travail a montré que le gène VHL produit des isoformes protéiques avec des fonctions distinctes voire antagonistes, ce qui implique que la balance de leur expression influencerait la progression tumorale rénale. Chez certains patients, une augmentation de l’expression de pVHL172 pourrait être corrélée à une pathologie plus sévère. Ce travail montre l’intérêt de poursuivre l’étude des fonctions de cette protéine pour une meilleure compréhension de son implication dans le cancer du rein et dans la maladie VHL afin d’envisager de nouvelles approches thérapeutiques. / VHL disease predisposes to the development of multiple and highly vascularized tumors, including central nervous system and retinal haemangioblastomas, phaeochromocytomas and clear cell renal cell carcinomas (ccRCCs). Patients with VHL disease harbor a mutant allele of the VHL gene. This gene is transcribed into two mRNAs by alternative splicing of the exon 2. The mRNA variant #1 composed of 3 exons usually predominates over the mRNA variant #2 lacking exon 2. A decrease of the variant #1/variant #2 ratio was however described in 2 situations: (i) in embryonic tissues, particularly in the kidney, and (ii) in some ccRCCs. These data suggest a potential role for the variant #2 in kidney tumorigenesis. pVHL213 and pVHL160 are the two proteins encoded by the mRNA variant #1 and act as tumor suppressors. At the beginning of this Ph.D. project, the expression of pVHL172 isoform encoded by the mRNA variant #2 remained to be established and its function was unknown. The experiments performed during this Ph.D. shed light on pVHL172 expression in cell lines and in tumor tissues using a newly produced mouse monoclonal antibody recognizing the three human pVHL isoforms. To examine if pVHL172 had a tumor suppressor function, human kidney tumor cell lines stably expressing this isoform were established, characterized and then grafted in mice. pVHL172 not only inhibits tumor formation, but its expression also induces a more aggressive phenotype with a higher sarcomatoid component and a more immature vasculature compared to control tumors (that do not express any pVHL). Moreover, pVHL172 increases the matrix metalloproteases MMP1 and MMP13 expression, partly by the activation of the Smad-dependent TGF-β signalling pathway. Besides, we looked for protein partners of pVHL172 by a differential proteomic analysis and showed that interaction networks obtained with the identified proteins are related to extracellular matrix regulation and protein quality control. To conclude, this work demonstrated that the VHL gene encodes protein isoforms with distinct and even antagonistic functions. The balance of expression of these isoforms is likely to influence kidney tumor progression. For some patients, an increase of pVHL172 expression could be correlated with a more severe pathology. This work shows the importance of further studying this isoform’s functions to better understand its involvement in kidney cancer and in VHL disease, so that new therapeutic approaches could be developed.

No description available.

Systemic sclerosis (SSc) is immune-mediated fibrotic disease of unknown aetiology. Among the dominant pathogenic manifestations of SSc belong vascular changes, production of autoantibodies, activation of innate and adaptive immune responses and fibrotic processes. Transforming growth factor beta (TGF-β) has been identified as a central profibrotic factor stimulating fibroblasts to produce collagen. There are, however, a number of other mediators involved in the pathogenesis of SSc. Mutual activation and amplification of these molecules and their cascades may be a central mechanism of the SSc pathogenesis. Hedgehog (Hh) canonical signalling pathway plays an important role in the development and progression of fibrotic diseases. Expression of Hh target genes can be regulated through a canonical or non-canonical signalling cascade. The non-canonical activation of GLI transcription factors by TGF-β has not yet been investigated in SSc. The substantial part of this thesis is focused on the study of the mutual interaction of TGF-β and Hh signalling pathway. In vitro analysis confirmed TGF- β/SMAD3 dependent activation of GLI2 in dermal fibroblasts. Fibroblasts specific knockout of GLI2 prevented the development of experimental fibrosis in vivo. Combined targeting of canonical and non-canonical Hh...

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-25T15:45:53Z No. of bitstreams: 2 Dissertação - Diego Antônio Costa Arantes - 2015.pdf: 5243498 bytes, checksum: 85643aafc3a6a4e6090981926b875c7f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-25T15:48:09Z (GMT) No. of bitstreams: 2 Dissertação - Diego Antônio Costa Arantes - 2015.pdf: 5243498 bytes, checksum: 85643aafc3a6a4e6090981926b875c7f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-05-25T15:48:09Z (GMT). No. of bitstreams: 2 Dissertação - Diego Antônio Costa Arantes - 2015.pdf: 5243498 bytes, checksum: 85643aafc3a6a4e6090981926b875c7f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The transforming growth factor β (TGF-β) and interleukin 10 (IL-10) are immunosuppressive cytokines which promote failure of the local anti-tumor immune response and, therefore, influence the proliferation and prognosis of malignant neoplasms. The aim of this study was to investigate the tissue and salivary expression of TGF-β and IL-10 in patients with oral squamous cell carcinoma (OSCC) and compare it with that of healthy subjects (Control). The association of these cytokines with clinical parameters of prognosis (staging, metastasis and survival) and histological grade of malignancy (WHO grading) was also investigated. Cytokines in the tissue (OSCC, n = 65; Control, n = 30) were identified using the immunohistochemistry technique (IHC) and in the saliva (OSCC, n = 22; Control, n = 23) the Enzyme-linked Immunosorbent Assay (ELISA) was used. The tissue expression of TGF-β and IL-10 in metastatic lymph nodes (n = 23) of OSCC patients was investigated. The expression of TGF-β and IL-10 in the tissue was measured using a semi-quantitative method in conjunction with staining intensity. Our findings demonstrated a high tissue expression of IL-10 and TGF-β2, and a low or absent expression of TGF-β1, in the majority of OSCC samples when compared to the group with clinically healthy oral mucosa (Control) (p < 0.05 for IL-10 and TGF-β2). The salivary concentration of IL-10 was also high, and distinguished the OSCC patients from their healthy counterparts (p = 0.04), while the salivary concentration of TGF-β1 was similar for both the OSCC and control groups (p = 0.97). The relationship between the cytokine expression and clinical and microscopic prognostic factors showed that the expression of IL-10 and TGF-β2 in neoplastic cells of the primary tumor was maintained by the metastatic neoplastic cells in the cervical lymph nodes. The expression of TGF-β1 remained low or absent in the metastatic lymph nodes. It was shown that there was an association between the high expression of IL-10 by tumor cells and the advanced clinical stages (T3-T4) of patients (p = 0.02). Although not statistically significant, the expression of TGF-β2 was higher in tumors at more advanced stages (p > 0.05). These findings demonstrate that OSCC provides an immunosuppressive environment conducive to tumor proliferation, with high expression of IL-10 and TGF-β2, which contributes to a worse clinical prognosis. In addition, of the immunosuppressive cytokines investigated, IL-10 has greater potential for becoming salivary biomarker when associated with an unfavorable clinical prognosis of OSCC patients. / Fator de crescimento transformador β (TGF-β) e interleucina 10 (IL-10) são citocinas imunossupressoras que propiciam a falha da resposta imune anti-tumoral local e influenciam, assim, na progressão e prognóstico de neoplasias malignas. O objetivo deste estudo foi investigar a expressão tecidual e salivar de TGF-β e IL-10 em pacientes com carcinoma espinocelular de cavidade oral (CECO) e comparar essa expressão com aquela dos indivíduos saudáveis (Controle). A associação dessas citocinas com parâmetros clínicos de prognóstico (estadiamento, metástase e sobrevida) e grau histológico de malignidade (segundo a OMS) também foi investigado. A identificação das citocinas no tecido (CECO - n= 65 e Controle - n=30) foi feita pela técnica de imunohistoquímica (IHC) e na saliva (CECO - n=22 e Controle - n=23) pelo ensaio imunoenzimático (ELISA). A expressão tecidual de TGF-β e IL-10 em linfonodos metastáticos (n= 23) de pacientes com CECO foi investigada. No tecido, a expressão de TGF-β e IL-10 foi mensurada por método semi-quantitativo associada à intensidade de marcação. Nossos achados demonstraram uma alta expressão tecidual de IL-10 e TGF- β2 e, baixa ou ausente expressão de TGF-β1, na maioria das amostras de CECO se comparada ao grupo de mucosa oral clinicamente saudável (Controle) (P<0,05 para IL-10 e TGF-β2). A concentração salivar de IL-10 também foi elevada e distinguiu o paciente com CECO dos indivíduos saudáveis (P= 0,04). Já a concentração salivar de TGF-β1 foi similar entre o grupo de paciente com CECO e o controle (P= 0,97). A relação da expressão das citocinas com fatores clínicos e microscópicos de prognóstico revelou que a expressão de IL-10 e TGF-β2 nas células neoplásicas do tumor primário foi mantida pelas células neoplásicas metastáticas nos linfonodos cervicais. A expressão de TGF-β1 se manteve baixa ou ausente em linfonodos mestastáticos. Associação entre alta expressão de IL-10 pelas células neoplásicas e o estadiamento clínico avançado (T3-T4) dos pacientes foi evidenciada (P=0,02). Embora sem diferença estatística, a expressão de TGF-β2 foi maior nos tumores em estágios mais avançados (P>0,05). Esses achados demonstram que o CCEO possui um ambiente imunossupressor propício para progressão tumoral, com elevada expressão de IL-10 e TGF-β2, o qual contribui para um pior prognóstico clínico dos pacientes. Além disso, das citocinas imunosupressoras investigadas, a IL-10 apresenta maior potencial para se tornar um biomarcador salivar associado ao prognóstico clínico desfavorável do paciente com CECO.

Der Skelettmuskel ist ein dynamisches Gewebe, das seine Funktionalität durch Anpassung des Gleichgewichts zwischen Proteinabbau und Proteinsynthese aufrechterhält. Kritisch kranke septische Patienten in der Intensivstation erleiden häufig eine dort erworbene tiefgreifende Muskelschwäche und –atrophie (intensive care unit acquired weakness, ICUAW). Es gibt Hinweise darauf, dass die Regenerationsfähigkeit des Muskels bei kritisch kranken Patienten beeinträchtigt ist. Die Pathogenese der ICUAW ist nur unzureichend verstanden, jedoch werden Sepsis und Entzündungen als führende Risikofaktoren angesehen. In früheren RNA-Sequenzierungsanalysen aus Muskeln septischer Mäuse wurde eine Herunterregulierung des Transforming Growth Factor beta (TGF-β)-Signals und eine erhöhte Genexpression von SPRY domain and SOCS-box containing protein 1 (SPSB1) gefunden. Ich stellte also die Hypothese auf, dass SPSB1 die Proteinhomöostase im Muskel beeinträchtigt, indem es den TGF-β/TβRII-Signalweg beeinflusst und eine entzündungsinduzierte Muskelatrophie verursacht. Die quantitative Echtzeit-Polymerase-Kettenreaktion verifizierte eine erhöhte Spsb1/SPSB1-Expression im Skelettmuskel von septischen Mäusen und ICUAW-Patienten. Die inflammatorischen Zytokine IL-6, IL-1β und Tumor-Nekrose-Faktor induzierten Spsb1 in C2C12-Myozyten in vitro. Die Überexpression von SPSB1 hemmte den TGF-β-Akt-Signalweg durch Destabilisierung von TβRII, was zu einer reduzierten Proteinsynthese in Myozyten führte. Als Konsequenz beeinträchtigte die SPSB1-Überexpression die Myogenese von C2C12-Myoblasten, gemessen an reduzierten Differenzierungs- und Fusionsindizes, sowie einer verminderten Protein- und mRNA-Expression der Differenzierungsfaktoren Mymk, Mymx, Myog, Myh1, 3 und 7. Zusammengenommen hemmt SPSB1 den TβRII-Signalweg im entzündeten Skelettmuskel, was die Myogenese beeinträchtigt. Daher könnte die Hemmung von SPSB1 hilfreich sein, um entzündungsinduziertes Muskelversagen zu verhindern. / Skeletal muscle is a dynamic tissue which maintains its functionality by adapting the balance between protein degradation and protein synthesis. Critically ill septic patients often develop intensive care unit acquired weakness (ICUAW), characterized by profound muscle weakness and atrophy. Emerging evidence suggests that the regenerative ability is impaired in patients with ICUAW. However, the pathogenesis of this disease is poorly understood. Sepsis and inflammation are considered as leading risk factors for ICUAW. In previous RNA sequencing analyses from muscle of septic mice, downregulation of transforming growth factor beta (TGF-β)-signaling and an increased gene expression of SPRY domain and SOCS-box containing protein 1 (SPSB1) have been found. If SPSB1 and TGF-β signaling play a role in inflammation-induced muscle atrophy was unknown. I hypothesized that SPSB1 impairs protein homeostasis in muscle by affecting TGF-β/TβRII signaling and causes inflammation-induced muscle atrophy. Quantitative real-time polymerase chain reaction verified increased Spsb1/SPSB1 expression in skeletal muscle of septic mice and ICUAW patients. The inflammatory cytokines IL-6, IL-1β and tumor necrosis factor induced Spsb1 in C2C12 myocytes in vitro. Overexpression of SPSB1 inhibited the TGF-β-Akt signaling pathway by destabilization of TβRII, leading to reduced protein synthesis in myocytes. These effects on TβRII signaling were mediated by the SPRY- and SOCS-box domains of SPSB1. As a consequence, SPSB1 overexpression impaired myogenesis of C2C12 myoblasts as measured by reduced differentiation and fusion indices, decreased protein and mRNA expression of the differentiation factors Mymk, Mymx, Myog, Myh1, 3 and 7. Taken together, SPSB1 binds and inhibits TβRII signaling in the inflammatory skeletal muscle resulting in impaired myogenesis. Therefore, inhibition of SPSB1 could be useful to prevent inflammation-induced muscle failure.