In the intestinal epithelium, Wnt signaling drives the expression of the genes encoding tyrosine kinase receptors EphB2 and EphB3 and represses the expression of their membrane-tethered ligands, ephrin-Bs. Eph-ephrin interactions result in cellular repulsion and are involved in boundary formation. The project of this thesis is to understand the mechanism by which EphB−ephrin-B signals restrict cell positioning of cell types (cell sorting) in the normal intestinal epithelium and suppress colorectal cancer (CRC) progression beyond the earliest stages. We have demonstrated that at the onset of CRC EphB receptors impair the expansion of tumor cells through a mechanism dependent on E-cadherin–mediated adhesion. We show that EphB-mediated compartmentalization restricts the spreading of EphB+ tumor cells into ephrin-B1+ territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1+ intestinal cells at the onset of tumorigenesis. We have discovered that cell sorting is the outcome of two integrated mechanisms: cell contraction/repulsion and differential cell adhesion. The latter is the driving force to induce EphB/ephrin-B−mediated cell compartmentalization. We have developed in vitro models to analyze the mechanisms that induce E-cadherin remodeling upon EphB activation. We found RhoA, p120-catenin and the metalloproteinase ADAM10 as downstream effectors of EphB signaling involved in the control of cell sorting in CRC cells. / A l'epiteli intestinal, la ruta de senyalització Wnt indueix l'expressió dels gens que codifiquen per als receptors tirosina kinasa EphB2 i EphB3 i reprimeixen la dels seus lligands transmembrana, efrines de tipus B. Les interaccions Eph-efrina causen repulsió cel·lular i estan implicades en la formació de fronteres entre compartiments. La finalitat d'aquesta tesi és entendre el mecanisme pel qual la senyalització per EphB−efrina-B restringeix el posicionament dels diferents tipus cel·lulars a l'epiteli intestinal normal i suprimeix la progressió del càncer colorectal (CRC) en els primer estadis. Hem demostrat que, a l’inici del CRC, els receptors EphB restringeixen l'expansió de les cèl·lules tumorals a través d'un mecanisme depenent d'adhesió intercel·lular a través d’E-cadherina. En aquest treball es mostra in vitro i in vivo que la compartimentalització mitjançada per la senyalització dels receptors EphB restringeix l’invasió de les cèl·lules tumorals EphB+ als territoris efrina-B+. Aquests resultats indiquen que les cèl·lules de CRC han de silenciar l’expressió d'EphB per evitar les interaccions repulsives imposades per les cèl·lules intestinals normals efrina-B+ circumdants al començament del procés de tumorigènesi. Hem pogut discernir que el reordenament cel·lular per senyals EphB−efrina-B és el resultat de dos mecanismes integrats: la contracció/repulsió intercel·lular i l’adhesió diferencial entre diferents poblacions cel·lulars. Aquesta última és la força principal que condueix a la compartimentalització cel·lular mitjançada per EphB−efrina-B. Hem desenvolupat models in vitro per analitzar els mecanismes que provoquen el remodelament de la E-cadherina sota la senyalització per EphB. Presentem RhoA, p120-catenina i ADAM10 com a efectors de la senyalització de la ruta EphB implicats en el control de la compartimentalització cel·lular en el CRC.

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-17Bitstream added on 2014-06-13T18:08:59Z : No. of bitstreams: 1 ishii_pl_me_rcla.pdf: 341469 bytes, checksum: 30957c71a427ca667e0c86edb9d40228 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Nutrigenômica é definida como o efeito da dieta na expressão gênica, e a extensão pela qual as diferenças genéticas entre os indivíduos influenciam a resposta a um padrão específico de dieta, à ingestão de alimentos funcionais e à suplementação de micronutrientes, em termos de um resultado para a saúde humana. A α-tomatina é um glicoalcalóide encontrado no tomate (Lycopersicon esculentum) que possui funções biológicas importantes como a redução dos níveis de colesterol LDL, inibição do crescimento de células cancerosas, estimulação do sistema imune e efeito antimetastático. O objetivo deste estudo foi avaliar a citotoxicidade da α-tomatina, os seus efeitos na proliferação celular, na indução de apoptose e expressão de RNAm dos genes APC, Ciclina A2, Catenina, CASP9, BAK, BAX e BCL-XL em células HT29. As células foram cultivadas em meio de cultura DMEM, suplementado com 10% de soro bovino fetal, e tratadas nas concentrações de 0,1, 1 e 10 μg/mL para o ensaio do MTT e proliferação celular. Na análise de apoptose morfológica utilizou-se as concentrações de 0,1, 1 e 2 μg/mL. Já para a avaliação da expressão gênica utilizou-se a concentração de 1 μg/mL. Após 12 horas de tratamento, o RNA das células foi extraído e a expressão dos genes foi avaliada através do método de PCR em tempo real. O gene GPDH foi utilizado como normalizador. A análise estatística foi realizada por ANOVA/Tukey para o ensaio do MTT. Os resultados do ensaio de cinética de proliferação celular, viabilidade celular e avaliação da indução de apoptose foram analisados estatisticamente através de ANOVA/Dunnet, e para a análise da expressão gênica utilizou-se o método de Pfaffl et al. (2002), através do cálculo estimado pelo método ΔΔCt. Os estudos experimentais indicaram que a α-tomatina foi citotóxica apenas na concentração de 10 μg/mL... / Nutrigenomics is defined as the effect of diet on gene expression, and the extent to which genetic differences between individuals influence the response to a specific pattern of diet, intake of functional foods and micronutrient supplementation, in terms of a result for human health. The α- tomatine is highlighted as a glycoalkaloid found in tomato (Lycopersicon esculentum) that has important biological functions such as reducing levels of LDL cholesterol, inhibit cancer cell growth, stimulation of the immune system and antimetastátic effect. In view of these considerations, the objective of this study was to evaluate the cytotoxicity of α-tomatine, their effects on cell proliferation, induction of apoptosis and morphological expression of mRNA of APC gene, Cyclin A2, β-Catenin, CASP9, BAK, BAX and BCL-XL in HT29 cells. The cells were grown in DMEM culture medium supplemented with 10% fetal bovine serum, and treated at concentrations of 0.1, 1 and 10μg/mL for the MTT assay and cell proliferation. In the morphological analysis of apoptosis, we used concentrations of 0.1, 1 and 2μg/mL. As for the evaluation of gene expression we used a concentration of 1 mg/mL. After 12 hours of treatment, the RNA from cells was extracted and gene expression was evaluated by the method of real-time PCR. The gene GPDH was used as normalizer. Statistical analysis was performed by ANOVA / Tukey test for MTT. The test results of kinetics of cell proliferation, cell viability and assessment of apoptosis were analyzed with ANOVA / Dunnet, and for analysis of gene expression we used the method of Pfaffl et al. (2002), by calculating estimated by ΔΔCt. Experimental studies suggested that α-tomatine was cytotoxic only at concentration of 10μg/mL. In the evaluation of cell proliferation were no significant differences in the treatments with α-tomatine, except that for the concentration... (Complete abstract click electronic access below)

Made available in DSpace on 2014-07-29T14:46:49Z (GMT). No. of bitstreams: 1 Dissertacao Aline Machado Rapello do Nascimento.pdf: 1403008 bytes, checksum: d5736b2cde9e7188a689131927c1127c (MD5) Previous issue date: 2007-12-31 / To evaluate whether complete arteriovenous ischemia followed by reperfusion of mesenteric blood flow aggravates lesions involving small intestine of horses, β--catenin and occludin expressions were measured in jejune epithelia of seven equines, without definite breed. The animals had been submitted to aseptic ventral midline celiotomy and five jejune segments collected corresponding to a baseline segment, a five minutes segment subjected to ischemia, and three segments collected after being subjected to five, sixty and one hundred and twenty minutes of tecidual reperfusion, respectively. Experimentally induced lesions created by total arteriovenous occlusion followed by reperfusion were similar to naturally acquired lesions, as much in the period of ischemia as in the aggravation of the injuries observed during reperfusion. Changes that occur when ischemic intestine is reperfused are progressive, and include mucosal, submucosal, and serosal edema; polymorphonuclear and mononuclear cell infiltrates; and alterations in vascular patterns. Imunohistochemistry analyses have shown strong staining baseline segments for β-catenin in all animals and its progressive lack of staining during ischemia and reperfusion treatments. That indicates progressive lack of intercellular adherence. Similar results of specific decreases of total expression of Occludin, a tight junction protein, were observed. This protein is involved in regulating the movement of solutes in the paracellular pathway and also plays an important role in the maintenance of cell polarity. The reduction of expressions of both intercellular proteins measured directly demonstrates β-catenin injury due to progression of edema during ischemia and its aggravation during reperfusion and also shows an increase of the permeability of paracelular pathways and its related consequences to degradation of Ocludina. / Realizou-se o estudo da patogenia da lesão por isquemia arteriovenosa completa dos vasos mesentéricos e reperfusão jejunal em eqüinos por meio da avaliação da expressão das proteínas β-catenina e ocludina presentes neste epitélio, em sete eqüinos, sem raça definida. Os animais foram submetidos á celiotomia asséptica na linha alba e coleta de cinco segmentos jejunais correspondestes á um segmento controle, um segmento tratado com isquemia por cinco minutos, e três segmentos coletados durante o período de reperfusão tecidual aos cinco, sessenta e cento e vinte minutos, respectivamente. As lesões induzidas experimentalmente por oclusão arteriovenosa total seguida por reperfusão foram similares às lesões adquiridas naturalmente tanto no período de isquemia quanto no agravamento das lesões observado nos tempos de reperfusão. As mudanças que ocorrem quando um intestino isquêmico sofre reperfusão são progressivas e incluem edema de mucosa, submucosa e serosa, infiltração polimorfonuclear e mononuclear e alterações nos padrões vasculares. A análise imunoistoquímica revelou forte expressão da β- catenina em todos os animais e a sua progressiva perda da expressão quando submetidos à isquemia e reperfusão. Isso indica perda progressiva da adesividade intercelular. Resultados similares de redução do total da expressão da Ocludina, uma proteína de junção de oclusão, foram observados. Esta proteína está envolvida na regulação dos movimentos dos solutos na via paracelular e também desempenha um importante papel na manutenção da polaridade celular. A diminuição da expressão de ambas as proteínas intercelulares demonstra a lesão à β-catenina devido à progressão do edema durante isquemia e o agravamento durante a reperfusão tecidual e também mostra um aumento na permeabilidade da via paracelular e suas conseqüências relacionadas à degradação da Ocludina.

INTRODUÇÃO: A molécula HIG2 pode atuar como agonista da via Wnt/beta-catenina, pois se liga ao receptor Frizzled 10 e induz a expressão de genes da mesma. Dados recentes do nosso grupo mostraram expressão diferencial do gene HIG2 em células mononucleares do sangue periférico e em especial linfócitos T CD4+ naïve, mas não em células diferenciadas de memória em indivíduos sadios. Também observamos in vitro em linfócitos T CD4+ de indivíduos saudáveis que o peptídeo sintético HIG2 induziu a ativação da via Wnt/beta-catenina, produção de HIG2 e outros produtos da via, além da proliferação de células T CD4+ naïve sugerindo um papel do HIG2 na proliferação homeostática de linfócitos T CD4+. HIPÓTESE: Como as células T CD4+ naïve são diretamente exportadas pelo timo, os níveis aumentados de HIG2 neste tipo celular sejam decorrentes da ativação da via Wnt/?-catenina nos estágios tardios da diferenciação de timócitos. Portanto, as células T CD4+ naïve e timócitos simples positivos para CD4 (SP CD4) apresentariam perfil semelhante de expressão de HIG2 e genes da via Wnt/beta-catenina, incluindo receptores, fatores de transcrição, genes estruturais da via e alvos quando comparadas as demais populações celulares. OBJETIVO: Avaliar a expressão de HIG2 e outros genes da via Wnt/beta-catenina em timócitos e linfócitos T CD4+ naïve e memória de camundongos. MÉTODOS: Isolamos timócitos duplo negativos (DN), timócitos duplo positivos (DP), simples positivos para CD4 e CD8 (SP CD4 e SP CD8) de timo e também células T CD4+ naïve e memória do baço dos mesmos camundongos pelo procedimento de citometria de fluxo. Analisamos a expressão de vários genes da via Wnt/beta-catenina por PCR em tempo real. RESULTADOS: Em timócitos DN há expressão significativa dos genes que codificam para Frizzled 6, LRP5, TCF-1 e TCF-4 em relação as outras populações celulares. Nos timócitos DP há maior expressão dos genes que codificam para LRP5, LRP6, beta-catenina, GSK-3beta, TCF-1 e Bcl-XL em relação às demais populações. Em timócitos SP CD4 foi detectada expressão diferencial de genes que codificam para Frizzled 10, LRP6, beta-catenina, LEF-1 e HIG2 enquanto que na população de timócitos SP CD8 não observamos expressão significativa de nenhum gene da via Wnt/beta-catenina. Nas células T CD4+ naïve há expressão significativa de Frizzled 5 e Frizzled 10 quando comparadas a timócitos SP CD8 e células T CD4+ de memória . Já nos linfócitos T CD4+ de memória, detectamos maior expressão de Frizzled 6, TCF-4, Bcl-XL e ciclina D1 em relação as demais populações. CONCLUSÃO: Cada população apresenta um perfil distinto de expressão gênica. As maiores semelhanças ocorrem entre os timócitos DN e DP onde as principais diferenças são a expressão de Frizzled 6 e Ciclina D1.Os timócitos SP CD4 e as células T CD4+ naïve não apresentaram níveis semelhantes de expressão gênica de elementos da via Wnt canônica, o que não corrobora a hipótese de que o perfil transcripcional de timócitos SP CD4 e linfócitos T CD4+ naïve é semelhante. Ainda, não observamos expressão aumentada de HIG2 em linfócitos T CD4+ naïve comparados aos de memória, o que contrasta com os resultados obtidos anteriormente por nosso grupo com amostras humanas sugerindo que camundongos não regulam a expressão de HIG2 em linfócitos T CD4+ como os seres humanos / INTRODUCTION: HIG2 molecule can act as an agonist of Wnt/?-catenin pathway, because it able to bind to Frizzled 10 receptor and induce the expression of the genes related to this pathway. Recent data from our group have shown differential expression of the HIG2 gene in peripheral blood mononuclear cells, and particularly in naive CD4 + T cells, but not in memory T cells in healthy individuals. We have also observed that inducing the CD4 + T lymphocytes from healthy individuals with HIG2 synthetic peptide in vitro, led to the activation of Wnt/beta-catenin pathway, HIG2 production and expression of other target genes of this pathway and the proliferation of naïve CD4 + T cells, suggesting that HIG2 may play a role in homeostatic proliferation of CD4+ T cells. HYPOTHESIS: As naïve CD4 + T cells are directly exported from the thymus, we have hypothesized that increased levels of HIG2 in this cell type is due to the activation of Wnt/beta-catenin pathway in the later stages of thymocyte differentiation. Therefore, naïve CD4 + T cells and CD4 single-positive thymocytes (CD4 SP) may share a similar pattern of gene expression of HIG2 and Wnt/beta-catenin genes (genes that encodes receptors and co-receptors, transcription factors, structural and target genes) when compared to other cell populations. AIM: our major aim is to evaluate the expression of HIG2 and other genes of the Wnt/beta-catenin in thymocytes, naïve CD4 + T lymphocytes and memory CD4+ T cells from mice. METHODS: We have isolated thymocytes double negative (DN) T cells, positive double positive (DP) T cells, CD4 and CD8 single-positive thymocytes (CD4 SP and CD8 SP) of thymus from BALB/c mice and we have also isolated naïve CD4 + T cells and memory CD4+ T cells of the spleen from the same mice we have used the thymus. We have analysed the expression of several genes of Wnt/beta-catenin by real time PCR RESULTS: In DN cells there was expression of the Frizzled 6, LRP5, TCF-1 and TCF-4 genes compared to other cell populations. In DP thymocytes it could be observed a greater expression of LRP5, LRP6, beta-catenin, GSK-3beta, TCF-1 and Bcl-XL genes compared to other populations. In CD4 SP thymocytes, it was detected differential expression of the Frizzled 10, LRP6, beta-catenin, LEF-1 HIG2 genes and in CD8 SP cells we could not observe significant expression of any gene of Wnt/?-catenin pathway. In naïve CD4 + T cells there was a significant expression of Frizzled5 and Frizzled 10 genes when compared to all the samples. In memory CD4 + T cells, we have detected higher expression of Frizzled 6, TCF-4, Bcl-XL and cyclin D1 genes than in any other populations. CONCLUSION: Each population has a distinct gene expression pattern. The biggest similarities occur between DN and DP thymocytes where the main differences are the expression of Frizzled 6 and cyclin D1.However, the pattern of gene expression in SP thymocytes is not similar to those presented by naïve CD4+ T cells. Moreover, we have not observed increased expression of HIG2 in naïve CD4 + lymphocytes compared to memory CD4+ T cells, which contrasts the results obtained previously by our group with human samples suggesting that mice might not regulate the HIG2 expression in CD4 + T lymphocytes as human beings do

Introdução: O câncer de laringe é a segunda neoplasia maligna mais comum no segmento cervicofacial. As caderinas epiteliais (E-caderinas) em conjunto com cateninas formam o complexo E-caderina-catenina que atuam na adesão célula-célula. A perda dessa molécula pode levar à redução ou até mesmo ausência de expressão de E-caderina na membrana celular, acúmulo citoplasmático de beta-catenina e sua translocação para o núcleo, contribuindo para eventos carcinogênicos. Objetivo: Avaliar a expressão de E-caderina e beta-catenina em pacientes com tumor laríngeo precoce ou avançado e na presença ou ausência de metástase cervical. Métodos: Realizou-se estudo retrospectivo de 52 pacientes portadores de carcinoma epidermoide glótico ou supraglótico, tratados entre 1998 a 2011 e avaliados de acordo com o sítio de localização tumoral, grau de diferenciação histológica, estádio TNM, dados de sobrevida e confrontados com a expressão imunoistoquímica de E-caderina e beta-catenina. Resultados: Observou-se associação com significância estatística entre a queda da expressão de E-caderina com a localização supraglótica da lesão, a presença de metástase cervical, em tumores pouco diferenciados e em tumores localmente avançados quando em topografia glótica. Com relação à expressão de beta-catenina, também foi encontrada significância estatística ao relacionar a presença de metástase cervical e tumor de baixa diferenciação com a diminuição de expressão deste marcador. Quanto à análise de sobrevida, a baixa expressão de beta-catenina está relacionada a pior sobrevida global e a redução da expressão de ambos os marcadores a pior sobrevida livre de doença. Conclusão: A expressão anômala dos marcadores estudados levam a impacto prognóstico por poder propiciar tumores com maior agressividade local e presença de metástase cervical / Introduction: Larynx cancer is the second most common malignant neoplasm in the cervicofacial segment. Epithelial cadherins (E-cadherins) together with catenins form the E-cadherin-catenin complex that acts on cell-to-cell adhesion. The loss of this molecule may lead to the reduction or even absence of E-cadherin expression in the cell membrane, cytoplasmic accumulation of beta-catenin and its translocation to the nucleus, contributing to carcinogenic events. Objective: To evaluate the expression of E-cadherin and beta-catenin in patients with laryngeal tumor in different locoregional situations. Methods: A study retrospective of 52 patients with glottic or supraglottic squamous cell carcinoma treated between 1998 and 2011 was conducted, evaluated according to the tumor localization site, the degree of histological differentiation, TNM stage and survival analysis. These data were confronted with the immunohistochemical expression of E-cadherin and ?-catenin. Results: We observed statistically significant association between the fall of E-cadherin expression and the supraglottic localization of the lesion, the presence of cervical metastasis, poorly differentiated tumors and locally advanced tumors when in glottic topography. Related to the expression of beta-catenin, statistical significance was also found for the presence of cervical metastasis and tumor of low differentiation with the decreased expression of this marker. Regarding survival analysis, the low expression of beta-catenin is related to worse overall survival and the reduction of expression of both markers to worse disease-free survival. Conclusion: The anomalous expression of the markers studied leads to a prognostic impact in order to provide tumors with greater local aggressiveness and presence of cervical metastasis

Introdução: Agregação familial é observada em cerca de 10% dos casos de câncer e 1 a 3% é hereditário. O tipo difuso pode estar relacionado à agregação familial e a alterações genéticas no gene CDH1, que codifica a proteína E-caderina. Alterações na imunoexpressão de Beta-catenina e p53 também são observadas. Objetivos: Analisar a imunoexpressão da E-caderina, Beta-catenina e TP53 em adenocarcinomas gástricos de pacientes com câncer gástrico familial e comparar com os dados clinicopatológicos, além dos achados das alterações genéticas destes pacientes, estudadas previamente nesta Instituição. Casuística e Métodos: Vinte e seis casos de adenocarcinoma gástrico em blocos de parafina de pacientes do HC-FMUSP foram submetidos ao estudo imunoistoquímico para detecção e análise do padrão de imunoexpressão da E-caderina, Beta-catenina e TP53 através do método da streptavidina-biotina-peroxidase. A análise da imunoexpressão dos marcadores foi classificada segundo escala de intensidade e distribuição e os testes estatísticos utilizados foram o Teste t de Student e Exato de Fisher. Resultados: A localização predominante do tumor foi no antro (61,5%). 11 (42,3%) casos alterados para a imunoexpressão da E-caderina, sendo todos do tipo difuso; 15 (57,7%) casos normais, sendo 9 do tipo difuso e 6 do tipo intestinal (p=0,02). Em estudo prévio realizado nesta instituição, uma mutação missense no exon 12 do gene CDH1, códon 617, nucleotídeo 1849 G>A foi encontrada no mesmo caso em que foi observada ausência de imunorreatividade da E-caderina. 11 (42,3%) casos alterados para a imunoexpressão de Beta-catenina e 46,2% de imunorreatividade nuclear positiva para TP53. Conclusões: 1) O tipo difuso de Laurén está associado à alteração da imunoexpressão da E-caderina no Câncer Gástrico Familial; 2) Não houve associação entre a imunoexpressão da E-caderina, idade, gênero e localização do tumor; tampouco houve associação entre a imunoexpressão da Beta-catenina e os dados clínico-patológicos; houve associação inversa entre a imunoexpressão da E-caderina e TP53; 3) Nos casos em que foram detectadas alterações na imunoexpressão, parece haver duas rotas distintas de carcinogênese envolvidas no CGF. / Introduction: Familial clustering is observed in about 10% of the gastric cancer cases and 1-3% is hereditary. Diffuse type gastric cancer is related to genetic alterations in CDH1 gene, which translates the E-cadherin protein. The abnormal expression of E-cadherin is characterized by low expression of cytoplasmatic staining, or loss of membranous immunoreactivity. Aim: to analyze the immunoexpression of E-cadherin, Beta-catenin and TP53 in gastric adenocarcinomas in patients with Familial Gastric Cancer and compare with clinical-pathologic data, including the genetic alterations of these patients, found previously on this institution. Methods: 26 cases of paraffin-embedded gastric adenocarcinoma tissue of patients of Hospital das Clinicas - School of Medicine of University of Sao Paulo underwent immunostaining to detect the presence and to analyze the pattern of immunoexpression of E-cadherin, Beta-catenin and TP53 using Streptavidine-Biotine-Peroxidade technique. The immunoexpression evaluation was performed utilizing a semiquantitative scale for intensity and distribution. The statistical analysis was done through Students t test and Fishers Exact test. Results: E-cadherin immunoexpression was negative in 11 cases (42.3%), and all of them were diffuse type of Laurén. 15 cases (57.7%) were positive for E-cadherin, from which 9 were of the diffuse type and 6 of intestinal type (p=0.02). In previous study performed on this institution, one missense mutation in exon 12 of CDH1 gene, codon 617, nucleotide 1849 G>A was found on the same case that absence of E-cadherin immunostaining was observed. 61.5% of the tumors were located in the antrum. Beta-catenin immunoexpression was altered in 43.2% and TP53 nuclear immunoreactivity was positive in 46.2% of the tumors. TP53 was solely detected in 12 (46.2%) of the tumors, while E-cadherin was altered in 10/26 (38.5%) negative TP53 tumors, p=0.01. Conclusions: 1) Diffuse type of Laurén is associated to E-cadherin immunoexpression alteration in Familial Gastric Cancer; 2) There was no association between E-cadherin immunoexpression and age, gender or tumor location, as well as there was no association between Beta-catenin and the clinical-pathologic data; there was an inverse association between immunoexpression of TP53 and E-cadherin; 3) There may be two distinct carcinogenesis pathways on familial gastric cancer cases that imunoexpression alterations were detected.

Le carcinome hépatocellulaire (CHC) présente des mutations génétiques qui altèrent les principales voies de signalisation, notamment la voie Wnt/β-caténine. En absence de mutation génétique, certains CHC peuvent montrer une activité Wnt exacerbée suite à une inactivation épigénétique d’inhibiteurs ou à une surexpression de ligands Wnts ou de ses récepteurs. De plus, le remodelage de la matrice extracellulaire favorise la progression du CHC. Nous avons montré une association entre l’activation du signal Wnt et le remodelage de la matrice extracellulaire (MEC) dans les cirrhoses et le CHC. Puis modélisé in vitro, les effets des stimuli Wnt extracellulaires sur le phénotype de cellules hépatiques, en absence de mutation de la β-caténine. En effet, les cellules HepaRG ne présentent pas de mutations de la β-caténine, de l’axine et de p53. Ainsi, la stimulation Wnt3a des cellules HepaRG induisait la formation de palissades de cellules fusiformes. De plus, les cellules traitées exprimaient des taux élevés de αSMA, COLIV, c-MYC, CK19 et LGR5 suggérant un phénotype myofibroblastique, en accord avec l’expression des marqueurs de transition épithélio-mésenchymateuse (TEM), SNAIL et TWIST. Ces données sont en faveur du rôle déterminant du microenvironnement Wnt activé dans la progression du CHC entrainant les cellules vers un phénotype progéniteur plus agressif via une TEM. En outre, l'analyse in silico de la signature transcriptomique de l'activation non mutationnelle de la voie Wnt a révélé un réseau de gènes impliqués dans le remodelage de la MEC, la TEM et la différenciation cellulaire. Les résultats suggèrent le rôle de HAPLN1 qui affecterait la migration cellulaire et l'expression des gènes de la MEC. De plus, LGR5 semble favoriser la dédifférenciation des hépatocytes. Au total, 8 gènes marqueurs obtenus in vitro ont été validés in vivo dans une série de 81 CHC humains, par qPCR et immunohistochimie en utilisant des tissus micro-array (78 CHC et 5 foies contrôles). Au total, l'ensemble des données suggère que HAPLN1 a une valeur pronostique sur la récidive et la survie globale du CHC. HAPLN1semble être indépendant du statut mutationnel la β-caténine et des variables cliniques. De plus, sa valeur pronostique est additive avec celle de CK19 + EpCAM et il semble agir en synergie avec NOG. / Hepatocellular carcinoma (HCC) displays signaling pathway disorders, including Wnt/β-catenin. Up-regulation of extracellular Wnt pathway agonists and down-regulation of extracellular Wnt pathway inhibitors result in non-mutational activation of Wnt signaling. In addition, increased extracellular matrix remodeling fosters HCC progression. Thus, we showed that enhanced Wnt signaling is associated with extracellular matrix remodeling in human cirrhosis and cancer. To further investigate non-mutational Wnt pathway activation, we established a model of Wnt activation in HepaRG human HCC progenitor cells carrying wild-type β-catenin, axin and p53. HepaRG progenitor cells treated with Wnt3a became fusiform and grew in palisades with enhanced expression of αSMA, COLIV, CK19, c-MYC, LGR5, SNAIL and TWIST, suggesting that enhanced extracellular Wnt signaling may drive HCC cells toward a more aggressive progenitor and epithelial mesenchymal transition (EMT) phenotype. Moreover, in silico analysis of the transcriptomic signature of non-mutational Wnt activation revealed a gene network involved in ECM remodeling, EMT and cell fate. Results suggest a role of HAPLN1, affecting extracellular matrix gene expression and cell migration and of LGR5 in hepatocyte dedifferentiation. Eight genes among the HepaRG gene expression dataset were validated in vivo in a collection of 81 human HCC samples and controls by qPCR and immunohistochemistry using tissue micro-arrays (78 HCC samples and 5 normal livers) in the light of β-catenin activation and mutational status. In conclusion, data suggest that HAPLN1 has a prognostic value on overall survival and recurrence of HCC. HAPLN1 appears to be independent of clinical features and β-catenin mutationnal status. Moreover, HAPLN1 appears to have an additive prognostic value with CK19 + EpCAM and act synergistically with NOG.

Abstract Colorectal cancer is the second most common malignancy in the Western World. The overall 5-year survival is still only 50–60%. Thus, better prognostic markers are needed to improve survival of the disease. Most colorectal cancers develop from pre-existing adenomas including conventional, flat and serrated adenomas. The most important prognostic factors include tumour stage, histologic subtype and poor differentiation. The prognosis of colorectal cancer depends mainly on tumour stage. The growth of colorectal cancer is determined by cell proliferation, differentation and apoptosis. The progression of colorectal cancer is associated with the growth pattern of colorectal cancer and its invasive margin. Cancer cell budding means the presence of cells scattered in the stroma at the invasive margin, and is associated with β-catenin, an adhesion protein involved in the nuclear Wnt/β-catenin pathway. Hormones may be directly involved in the growth of a cancer, for example sex hormones play an important role in the development of most gynaecological cancers. The knowledge about the dependency of cancers on other hormones, such as thyroid hormones, is limited. This thesis focuses on factors affecting growth and prognosis in colorectal cancer. Antibodies for Ki-67, caspase cleavage site for keratin 18, β-catenin and TRβ1 were used to determine their possible associations with colorectal cancer growth patterns and the characteristics of the invasive margin. Apoptosis and proliferation were decreased at the invasive margin, particularly in serrated adenocarcinomas. The invasive margin showed a presence of budding cell clusters in 24.0% of the cases and this predicted a very poor 5-year-survival (15.4%, P < 0.00001), but nuclear β-catenin accumulation did not predict budding. Thyroid hormone receptor TRβ1 was associated with polypoid growth, presence of KRAS mutations and also with a higher WHO histological grade and advanced Dukes' stage, and in in vitro analysis, thyroid hormone T3 had a modulatory effect on colorectal cancer cell protein synthesis and apoptosis. In conclusion, the growth type of colorectal cancer, i.e. conventional polypoid, flat or serrated, has an association with the characteristics of the invasive margin. Budding margin is associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Association of TRβ1 expression with polypoid growth pattern and the presence of KRAS mutations suggest that abnormalities in thyroid hormone signalling involving TRβ1 play a role in the development of some types of colorectal adenocarcinomas.

Fra-1 appartient à la famille des facteurs de transcription AP-1. Son expression est particulièrement élevée dans les cellules de cancer du sein qui n'expriment pas le récepteur aux œstrogènes (RE-), c'est-à-dire les cellules les plus agressives. L'inhibition de Fra-1 dans ces cellules entraîne une diminution de la motilité, de l'invasion et de la prolifération, mais elle entraîne aussi de profonds changements de morphologie. Les cellules RE-, qui présentent un phénotype mésenchymateux s'arrondissent et établissent un plus grand nombre de contacts cellule-cellule après l'inhibition de Fra-1. Dans les cellules RE-, la β-caténine est localisée au noyau ou dans le cytoplasme, ce qui est un marqueur de mauvais pronostic. Au cours de cette thèse, j'ai montré que Fra-1 régule la localisation nucléaire de la β-caténine et ainsi régule son activité transcriptionelle en agissant très tardivement sur la voie Wnt. J'ai également mis en évidence une interaction physique directe entre Fra-1 et la β-caténine qui pourrait être responsable de cet effet. De plus, l'analyse de microarrays par RT-QPCR a révélé la régulation d'autres gènes comme la mœsine, la fibronectine et l'extracellular matrix protein 1, qui pourraient également jouer un rôle dans la régulation de l'agressivité tumorale par Fra-1. Par ailleurs, Fra-1 est une protéine instable et nous avons montré qu'elle est phosphorylée et stabilisée par PKCθ. Fra-1 est d'ailleurs nécessaire à l'effet de la kinase sur la motilité cellulaire. / Fra-1 is a member of the AP-1 transcription factor family. It is aberrantly expressed in breast cancer cells lacking Estrogen Receptor (ER-) expression, which are the most aggressive ones. Fra-1 inhibition in these cells leads to a decreased in motility, invasion and proliferation, but also to deep morphologic changes. ER- cells, which present a mesenchymal phenotype, become rounder and establish a greater number of cell-cell contacts after Fra-1 inhibition. In ER- cells, β-catenin is nuclear or cytoplasmic, which is considering as a poor prognosis marker. During this PhD, I demonstrate that Fra-1, which acts very downstream in the Wnt/β-catenin signaling pathway, regulates the nuclear localization of β-catenin leading to up-regulation transcriptional activity of β-catenin. I also found that Fra-1 directly interacts with β-catenin. In addition, RT-QPCR microarrays analysis has revealed the regulation of other genes such as mœsin, fibronectin and extracellular matrix protein 1, which might also take part in the tumoral aggressiveness regulated by Fra-1. Moreover, we show that Fra-1, which is an unstable protein, is phosphorylated and stabilized by PKCθ. Furthermore, Fra-1 is necessary to mediate the kinase effect on cell motility.

Les travaux réalisés au cours de cette Thèse avaient pour but de caractériser au niveau fonctionnel la protéine FAM110B, identifiée au laboratoire il y a plusieurs années par une méthode de sélection d’éléments génétiques suppresseurs destinée à rechercher de nouveaux gènes impliqués dans la sensibilité à un inhibiteur de Topoisomérase II, la 9-hydroxyéllipticine. Localisée au niveau cytoplasmique et très conservée chez les mammifères, FAM110B est essentielle à la survie comme le montre le blocage en phase S des cellules dans lesquelles son expression est transitoirement diminuée. Sa répression conduit d’ailleurs à l’inhibition de plusieurs voies impliquées dans la prolifération cellulaire comme les voies Wnt, Notch ou TGF-. Les résultats que nous avons obtenus suggèrent que ce rôle dans la prolifération peut être régulé par l’interaction de FAM110B avec la β-caténine. Cette interaction régule le niveau d’expression de la β-caténine et/ou sa localisation, ce qui a pour conséquence directe de moduler l’expression de ses gènes cibles impliqués dans la prolifération cellulaire. Nous avons également démontré que FAM110B intervient dans les processus de migration cellulaire en régulant directement ou indirectement l’expression de la E-cadhérine par la modulation sélective de l’expression d’un de ses répresseurs, Slug. L’augmentation de l’expression de la E-cadhérine dans des cellules sousexprimant FAM110B est accompagnée d’une diminution de l’expression de la N-cadhérine, un phénomène qui est fréquemment observé lors de la reverse EMT, passage d’un stade mésenchymateux à un stade épithélial au cours duquel, des cellules à caractère invasif et métastatique acquièrent des propriétés adhésives associées à une perte de leur propriétés de migration et d’invasion. Enfin, nous avons pu démontrer que FAM110B est également impliquée dans la sensibilité cellulaire à divers agents anticancéreux. Sa répression induit une sensibilisation à la camptothécine et au cisplatine alors qu’elle confère une résistance aux poisons de tubuline (taxol et vincritine) et aux inhibiteurs de Topoisomérases II par diminution du nombre de complexes de clivage ADN-enzyme associée à une réduction du niveau de Topo2 dont on ne connait pas encore l’origine. L’ensemble de nos résultats confirment l’importance de FAM110B dans la migration et la prolifération cellulaire ainsi que dans la réponse aux stress induits par diverses classes d’agents anticancéreux. De ce fait, FAM110B peut être considérée comme une nouvelle cible potentielle en cancérologie et son inhibition être utilisée pour potentialiser l’action de thérapeutiques existantes tels que les dérivés de la camptothécine ou les dérivés du platine qui sont largement utilisés en clinique. / FAM110B is a new protein that was identified several years ago in our laboratory by a functional screen, the selection of genetic suppressor elements (GSEs), which goal was to identify new genes involved in the cellular sensitivity to the topoisomerase II inhibitor 9-hydroxyellipticine. FAM110B is localized in the cytoplasm and is extremely conserved across mammals. We found that FAM110B is essential for cell survival, as its transient repression induces a blockage in the S phase of the cell cycle. Its repression also induces the inhibition of various pathways involved in the regulation of cell proliferation, such as Wnt, Notch or TGF-. Our results suggest that its role in cell proliferation relies on FAM110B interaction with β-catenin. This interaction regulates β-catenin expression and its subcellular localization, which directly impacts on the expression of its target genes involved in cell proliferation. We have also demonstrated that FAM110B is involved in cell migration by regulating the expression of E-cadherin via the specific modulation of one of its repressors, Slug. Increase in E-cadherin expression in cells with downregulated FAM110B is accompanied by a decrease in N-cadherin expression, a phenomenon which is reminiscent of a reverse EMT i.e. a mesenchymal to epithelial transition which is characterized by a loss of invasiveness and metastatic potential. Finally, we also showed that FAM110B is involved in the regulation of the cellular sensitivity to various anticancer agents. Transient repression of FAM110B sensitizes cells to camptothecin and cisplatin, whereas it confers a resistant phenotype to tubuline poisons (taxol and vincristine) and Top2 inhibitors. This latter effect is accompanied by a reduction in DNA-Topo2 cleavage complexes due to a reduction in Topo2 levels by a mechanism which is not fully elucidated. Together, our results confirm the importance of FAM110B in essential processes such as migration, cell proliferation, and cell response to various stresses induced by chemotherapeutic agents. Therefore, FAM110B can be considered as a new potential target for cancer treatment and its inhibition can also be used to potentiate existing treatments such as camptothecin derivatives and platinum compounds that are widely used in the clinic.