O hepatoblastoma é uma neoplasia embrionária hepática que ocorre na faixa pediátrica, rara, sendo bastante heterogênea devido aos seus diferentes componentes epiteliais e mesenquimais. Pouco ainda se sabe a respeito de sua patogênese. Utilizando um microscópio de captura a laser foram dissecadas áreas de componente epitelial do hepatoblastoma e áreas de tecido hepático adjacente não acometido. Destas amostras obtidas de pacientes não submetidos ao tratamento prévio, foram extraídos RNA e confeccionados cDNA microarrays, para análise comparativa da expressão gênica, seguida de validação por método imunohistoquímico de alguns genes selecionados. Comparando neoplasia e tecido hepático em duas amostras criopreservadas foram identificados 70 genes diferencialmente expressos, sendo 19 hiperexpressos e 51 hipoexpressos no tumor. A maioria dos genes encontrados foi mapeada nos cromossomos 1 e 2. Dos genes selecionados para validação por método imuno-histoquímico, destacaram-se o receptor de Insulina e o TFE3 (genes hipoexpressos no cDNA microarray). A imunomarcação para o receptor de insulina foi positiva tanto no tecido hepático não acometido quanto no componente epitelial fetal do hepatoblastoma , mas foi consistentemente negativa nas amostras de componente embrionário (9/9). A imunomarcação para o TFE3 foi positiva no tecido hepático não acometido, e nos componentes epitelial fetal e embrionário, em proporção variável das células com expressão mais intensa no componente embrionário. As reações imuno-histoquímicas para os outros genes selecionados não permitiram interpretação conclusiva. A alta proporção dos genes diferencialmente expressos localizados nos cromossomos 1 e 2 reflete os achados citogenéticos relatados na literatura relacionada ao hepatoblastoma . Achados de imunoexpressão de proteínas relacionadas aos genes TFE3 e receptor de insulina no tecido hepático e nos diferentes componentes do hepatoblastoma são inéditos e sugerem participação da via sinalizadora da insulina na patogênese destes tumores / Hepatoblastoma is a rare tumor, and little is known about its pathogenesis and genetic alterations. Using a laser capture microdissection microscope we sampled areas of epithelial component of hepatoblastoma prior chemotherapy and their normal liver counterpart in order to perform the comparative gene expression analysis followed by the validation of selected genes by immunohistochemistry. Comparing tumor and non-diseased liver in two frozen samples, 70 differentially expressed genes were found, 19 overexpressed and 51 underexpressed in the tumor. Most of the genes were located at chromosomes 1 and 2. Of the genes selected for validation by immunohistochemistry, the most interesting findings came from Insulin receptor and TFE3 (both underexpressed genes). Insulin receptor was positive in non diseased liver and in the fetal component of the Hepatoblastoma but was consistently negative in the embryonal component (9/9 cases). The TFE3 staining was positive in the normal liver and fetal and embryonal components of the tumor in variable proportion of the cells, more marked in the embryonal component. The higher proportion of genes located at chromosomes 1 and 2 corroborates the cytogenetics findings reported in the literature related to Hepatoblastoma . The immunohistochemistry findings of different expression of insulin receptor in the fetal and embryonal components of Hepatoblastoma and the positivity of TFE3 in normal liver and in the tumors epithelial components deserves further investigation regarding the role of these genes to the pathogenesis of Hepatoblastoma

O hepatoblastoma é uma neoplasia embrionária hepática que ocorre na faixa pediátrica, rara, sendo bastante heterogênea devido aos seus diferentes componentes epiteliais e mesenquimais. Pouco ainda se sabe a respeito de sua patogênese. Utilizando um microscópio de captura a laser foram dissecadas áreas de componente epitelial do hepatoblastoma e áreas de tecido hepático adjacente não acometido. Destas amostras obtidas de pacientes não submetidos ao tratamento prévio, foram extraídos RNA e confeccionados cDNA microarrays, para análise comparativa da expressão gênica, seguida de validação por método imunohistoquímico de alguns genes selecionados. Comparando neoplasia e tecido hepático em duas amostras criopreservadas foram identificados 70 genes diferencialmente expressos, sendo 19 hiperexpressos e 51 hipoexpressos no tumor. A maioria dos genes encontrados foi mapeada nos cromossomos 1 e 2. Dos genes selecionados para validação por método imuno-histoquímico, destacaram-se o receptor de Insulina e o TFE3 (genes hipoexpressos no cDNA microarray). A imunomarcação para o receptor de insulina foi positiva tanto no tecido hepático não acometido quanto no componente epitelial fetal do hepatoblastoma , mas foi consistentemente negativa nas amostras de componente embrionário (9/9). A imunomarcação para o TFE3 foi positiva no tecido hepático não acometido, e nos componentes epitelial fetal e embrionário, em proporção variável das células com expressão mais intensa no componente embrionário. As reações imuno-histoquímicas para os outros genes selecionados não permitiram interpretação conclusiva. A alta proporção dos genes diferencialmente expressos localizados nos cromossomos 1 e 2 reflete os achados citogenéticos relatados na literatura relacionada ao hepatoblastoma . Achados de imunoexpressão de proteínas relacionadas aos genes TFE3 e receptor de insulina no tecido hepático e nos diferentes componentes do hepatoblastoma são inéditos e sugerem participação da via sinalizadora da insulina na patogênese destes tumores / Hepatoblastoma is a rare tumor, and little is known about its pathogenesis and genetic alterations. Using a laser capture microdissection microscope we sampled areas of epithelial component of hepatoblastoma prior chemotherapy and their normal liver counterpart in order to perform the comparative gene expression analysis followed by the validation of selected genes by immunohistochemistry. Comparing tumor and non-diseased liver in two frozen samples, 70 differentially expressed genes were found, 19 overexpressed and 51 underexpressed in the tumor. Most of the genes were located at chromosomes 1 and 2. Of the genes selected for validation by immunohistochemistry, the most interesting findings came from Insulin receptor and TFE3 (both underexpressed genes). Insulin receptor was positive in non diseased liver and in the fetal component of the Hepatoblastoma but was consistently negative in the embryonal component (9/9 cases). The TFE3 staining was positive in the normal liver and fetal and embryonal components of the tumor in variable proportion of the cells, more marked in the embryonal component. The higher proportion of genes located at chromosomes 1 and 2 corroborates the cytogenetics findings reported in the literature related to Hepatoblastoma . The immunohistochemistry findings of different expression of insulin receptor in the fetal and embryonal components of Hepatoblastoma and the positivity of TFE3 in normal liver and in the tumors epithelial components deserves further investigation regarding the role of these genes to the pathogenesis of Hepatoblastoma

Os tumores malignos hepáticos são raros nas crianças, sendo o tumor embrionário hepatoblastoma a neoplasia hepática mais comum em crianças abaixo dos 5 anos e, ainda assim, responsável por apenas 1% dos tumores incidentes nessa faixa etária. Devido, entre outros fatores, à sua baixa incidência, o hepatoblastoma é um tumor ainda pouco caracterizado a nível molecular. O presente trabalho contemplou três frentes de análises moleculares em hepatoblastomas: análise de alterações em número de cópias (através da técnica array-CGH), investigação de mutações somáticas em regiões codificadoras (sequenciamento de exoma por next-generation) e delineamento de perfil global de metilação de DNA (beadarrays). Encontramos um baixo número de alterações em número de cópias, na maioria ganhos de grande extensão cromossômica, evidenciando uma baixa instabilidade cromossômica em comparação ao encontrado em outros tumores sólidos de adultos. Uma região em 2q24 previamente associada a um pior prognóstico em hepatoblastomas foi encontrada como ganho de alta amplitude (amplicon). A análise de expressão gênica na área destacou 5 dos 48 genes como super-expressos em nossas amostras de hepatoblastomas (DAPL1, ERMN, GALNT5, SCN1A e SCN3A). Foi observada também uma baixa ocorrência de mutações somáticas não-sinônimas, quando comparada à magnitude de variações encontradas em outros tumores sólidos de adultos. Descrevemos uma nova mutação não-sinônima danosa no exon 3 do gene CTNNB1 (beta-catenina), marcador molecular de destaque em hepatoblastomas, que provavelmente desenvolve um papel importante na tumorigênese deste tipo de neoplasia. Dentre a lista de alterações somáticas encontradas, cabe destacar o enriquecimento da via Wnt, via da beta-catenina, já bem estudada em hepatoblastomas e que apresenta correlação tanto com desenvolvimento embrionário como com o processo de carcinogênese. Destacamos uma lista de mutações somáticas não-sinônimas presentes com boa cobertura em nossos experimentos, e ausentes ou presentes em baixa frequência (< 1%) na população. O presente estudo é o pioneiro na análise de alterações globais no padrão de metilação de citosinas em hepatoblastomas, e revelou um padrão incomum de hipometilação global nos tumores, não associado à metilação de regiões repetitivas LINE-1; especificamente, hepatobastomas apresentam um nível intermediário entre o encontrado para os fígados maduros e aquele encontrado para os fígados fetais. Tal achado dá suporte ao modelo no qual a tumorigênese do hepatoblastoma recapitularia fases do desenvolvimento fetal do fígado. Foram destacados 11 genes que apresentaram padrão de metilação de DNA diferencial em seus promotores. Em suma, nossos achados revelam que o tumor embrionário hepatoblastoma apresenta relativa estabilidade genética, com uma frequência menor de alterações (alterações em número de cópias e mutações somáticas em regiões codificadoras) que tumores sólidos de adultos. Simultaneamente, foi detectado um padrão marcante de hipometilação global de DNA associado ao tumor, evidenciando a importância de aspectos epigenéticos em sua tumorigênese / Hepatic malignant tumors are rare in children, the embryonal tumor hepatoblastoma is the most common liver cancer in children under 5 years old but, in spite of that, accounts for only 1% of incident tumors in this age group. Due to its low incidence, among other factors, hepatoblastoma is a tumor poorly characterized at molecular level. This study included three approaches of molecular analyzes in hepatoblastomas: examination of copy number alterations (through array-CGH technique), investigation of somatic mutations in coding regions (next-generation exome sequencing) and determination of the global DNA methylation profile (bead arrays). We found a low frequency of copy number alterations, mostly consisting of large extent chromosomal gains, reflecting lower chromosomal instability than other solid adult tumors. A region at 2q24, previously associated with worse prognosis in hepatoblastomas, was found amplified in high amplitude (amplicon). Gene expression analysis of the 48 genes comprised in the amplicon segment highlighted five genes as overexpressed (DAPL1, ERMN, GALNT5, SCN1A and SCN3A). We also observed a low frequency of non-synonymous somatic mutations in our hepatoblastomas samples compared to the amount of variation found in other adult solid tumors. We described a predicted harmful non-synonymous mutation in exon 3 of CTNNB1 gene (beta-catenin), the best characterized molecular marker in hepatoblastomas. The list of somatic alterations points to a remarkable enrichment of genes from the Wnt pathway, which is well-studied in hepatoblastomas and is associated both with embryonic development and with the process of carcinogenesis. We highlighted a list of non-synonymous somatic mutations that presented high coverage in our experiments, and were absent or present at low frequency (<1%) in the general population. This study is the first to analyze global changes in cytosine methylation in hepatoblastomas, and revealed an unusual pattern of global hypomethylation in these tumors, not associated with LINE-1 repetitive regions; specifically, hepatobastomas exhibited an intermediate level of methylation, in between the patterns of mature and fetal livers. This finding supports the model in which the oncogenesis of hepatoblastoma recapitulates stages of fetal liver development. We highlighted 11 genes that showed differential pattern of DNA methylation in their promoters compared to differentiated liver. In summary, our findings show that the embryonal tumor hepatoblastoma are relatively genetically stable with lower frequency of alterations (changes in copy number and somatic mutations in coding regions) than most solid adult tumors. Simultaneously, a clear pattern of global hypomethylation of non-repetitive DNA was associated with the tumor, indicating the importance of epigenetic aspects in its tumorigenesis

Os tumores malignos hepáticos são raros nas crianças, sendo o tumor embrionário hepatoblastoma a neoplasia hepática mais comum em crianças abaixo dos 5 anos e, ainda assim, responsável por apenas 1% dos tumores incidentes nessa faixa etária. Devido, entre outros fatores, à sua baixa incidência, o hepatoblastoma é um tumor ainda pouco caracterizado a nível molecular. O presente trabalho contemplou três frentes de análises moleculares em hepatoblastomas: análise de alterações em número de cópias (através da técnica array-CGH), investigação de mutações somáticas em regiões codificadoras (sequenciamento de exoma por next-generation) e delineamento de perfil global de metilação de DNA (beadarrays). Encontramos um baixo número de alterações em número de cópias, na maioria ganhos de grande extensão cromossômica, evidenciando uma baixa instabilidade cromossômica em comparação ao encontrado em outros tumores sólidos de adultos. Uma região em 2q24 previamente associada a um pior prognóstico em hepatoblastomas foi encontrada como ganho de alta amplitude (amplicon). A análise de expressão gênica na área destacou 5 dos 48 genes como super-expressos em nossas amostras de hepatoblastomas (DAPL1, ERMN, GALNT5, SCN1A e SCN3A). Foi observada também uma baixa ocorrência de mutações somáticas não-sinônimas, quando comparada à magnitude de variações encontradas em outros tumores sólidos de adultos. Descrevemos uma nova mutação não-sinônima danosa no exon 3 do gene CTNNB1 (beta-catenina), marcador molecular de destaque em hepatoblastomas, que provavelmente desenvolve um papel importante na tumorigênese deste tipo de neoplasia. Dentre a lista de alterações somáticas encontradas, cabe destacar o enriquecimento da via Wnt, via da beta-catenina, já bem estudada em hepatoblastomas e que apresenta correlação tanto com desenvolvimento embrionário como com o processo de carcinogênese. Destacamos uma lista de mutações somáticas não-sinônimas presentes com boa cobertura em nossos experimentos, e ausentes ou presentes em baixa frequência (< 1%) na população. O presente estudo é o pioneiro na análise de alterações globais no padrão de metilação de citosinas em hepatoblastomas, e revelou um padrão incomum de hipometilação global nos tumores, não associado à metilação de regiões repetitivas LINE-1; especificamente, hepatobastomas apresentam um nível intermediário entre o encontrado para os fígados maduros e aquele encontrado para os fígados fetais. Tal achado dá suporte ao modelo no qual a tumorigênese do hepatoblastoma recapitularia fases do desenvolvimento fetal do fígado. Foram destacados 11 genes que apresentaram padrão de metilação de DNA diferencial em seus promotores. Em suma, nossos achados revelam que o tumor embrionário hepatoblastoma apresenta relativa estabilidade genética, com uma frequência menor de alterações (alterações em número de cópias e mutações somáticas em regiões codificadoras) que tumores sólidos de adultos. Simultaneamente, foi detectado um padrão marcante de hipometilação global de DNA associado ao tumor, evidenciando a importância de aspectos epigenéticos em sua tumorigênese / Hepatic malignant tumors are rare in children, the embryonal tumor hepatoblastoma is the most common liver cancer in children under 5 years old but, in spite of that, accounts for only 1% of incident tumors in this age group. Due to its low incidence, among other factors, hepatoblastoma is a tumor poorly characterized at molecular level. This study included three approaches of molecular analyzes in hepatoblastomas: examination of copy number alterations (through array-CGH technique), investigation of somatic mutations in coding regions (next-generation exome sequencing) and determination of the global DNA methylation profile (bead arrays). We found a low frequency of copy number alterations, mostly consisting of large extent chromosomal gains, reflecting lower chromosomal instability than other solid adult tumors. A region at 2q24, previously associated with worse prognosis in hepatoblastomas, was found amplified in high amplitude (amplicon). Gene expression analysis of the 48 genes comprised in the amplicon segment highlighted five genes as overexpressed (DAPL1, ERMN, GALNT5, SCN1A and SCN3A). We also observed a low frequency of non-synonymous somatic mutations in our hepatoblastomas samples compared to the amount of variation found in other adult solid tumors. We described a predicted harmful non-synonymous mutation in exon 3 of CTNNB1 gene (beta-catenin), the best characterized molecular marker in hepatoblastomas. The list of somatic alterations points to a remarkable enrichment of genes from the Wnt pathway, which is well-studied in hepatoblastomas and is associated both with embryonic development and with the process of carcinogenesis. We highlighted a list of non-synonymous somatic mutations that presented high coverage in our experiments, and were absent or present at low frequency (<1%) in the general population. This study is the first to analyze global changes in cytosine methylation in hepatoblastomas, and revealed an unusual pattern of global hypomethylation in these tumors, not associated with LINE-1 repetitive regions; specifically, hepatobastomas exhibited an intermediate level of methylation, in between the patterns of mature and fetal livers. This finding supports the model in which the oncogenesis of hepatoblastoma recapitulates stages of fetal liver development. We highlighted 11 genes that showed differential pattern of DNA methylation in their promoters compared to differentiated liver. In summary, our findings show that the embryonal tumor hepatoblastoma are relatively genetically stable with lower frequency of alterations (changes in copy number and somatic mutations in coding regions) than most solid adult tumors. Simultaneously, a clear pattern of global hypomethylation of non-repetitive DNA was associated with the tumor, indicating the importance of epigenetic aspects in its tumorigenesis

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

La phosphoinositide 3-kinase p110δ (PI3Kδ) est principalement exprimée dans les cellules hématopoïétiques et son inhibiteur, l'idélalisib est approuvé pour le traitement de la leucémie et du lymphome. Cependant, la fonction de cette isoforme dans les cellules non hématopoïétiques reste insaisissable. Dans cette étude, nous rapportons que la formation des canalicules biliaires est dépendante de l’activité de la PI3Kδ lors de la culture en 3D de cellules de carcinome hépatocellulaire. Une étude in-vitro reposant sur la différenciation de cellules souches embryonnaires humaines en hépatocytes révèle que la PI3Kδ est enrichie dans les cellules souches et que son expression diminue au fur et à mesure que les cellules se différencient. Lorsqu’elle est surexprimée, la PI3Kδ reprogramme ces cellules en cellules ressemblant à des cellules souches formant des rosettes polarisées et perdant des marqueurs d’hépatocytes pour acquérir des traits de cholangiocytes. Dans le foie murin, la réexpression de la PI3Kδ entraine des modifications de la morphologie de cellules jouxtant la veine porte, ainsi que l’augmentation de l’expression du gène codant pour EpCAM. Cette reprogrammation dépendante de la PI3Kδ est associée à l'activation de la voie de Notch et requiert l'activation de la protéine Src. Enfin, la PI3Kδ est exprimée dans les lignées cellulaires dérivées d’hépatoblastome humain et le traitement des souris par l’idélalisib diminue la taille des tumeurs formées par les PDX d’hépatoblastome. La PI3Kδ représente donc une cible thérapeutique prometteuse dans ce cancer pédiatrique avec des caractéristiques de cellules souches. / The phosphoinositide 3-kinase p110δ (PI3Kδ) is primarily expressed in the hematopoietic cells and its inhibitor, Idelalisib, is approved for leukemia and lymphoma treatments. Nevertheless, the function of PI3Kδ in the non-hematopoietic cells is still elusive. Here we report that the formation of bile canaliculi is dependent on the PI3Kδ activity using hepatocellular carcinoma (HCC) cells grown in a 3D culture. In-vitro study based on hepatocytes differentiation from human Embryonic Stem Cell (hESC) highlights that PI3Kδ is enriched in hESC and increasingly reduces over time while cells are differentiating. When PI3Kδ is overexpressed, it reprograms those cells into stem-like cells forming polarized rosette structures and losing hepatocyte markers to gain cholangiocyte characteristics. These changes were observed in mice liver overexpressing PI3Kδ. The aforementioned reprogramming, dependent on PI3Kδ, is associated with the Notch pathway activation and requires the Src protein activation. Finally,PI3Kδ is expressed in hepatoblastoma cell lines and idelalisib efficiently reduces tumor size formed by patient derived xenograft (PDX). Therefore, PI3Kδ represents a promising therapeutic target for this pediatric cancer with stem cell features.

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Whilst, several phase II drugs as well as endogenous & exogenous steroids/steroid metabolites have been identified as inducers of gene transcription, the cellular transcription factors controlling the expression of AKR1C1 are incompletely elucidated. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene that controls its transcription. The 5’ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5’-flanking, ~3.0 kb region of the human AKR1C1 gene identified the region between -128 to -88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Antioxidant response elements (ARE) have been shown to modulate the transcription ofv genes coding for phase II drug metabolizing enzymes. Cloning of the ARE upstream of the AKR1C1 proximal promoter resulted in increased transcription in human lung adenocarcinoma and liver hepatoblastoma cells but not in human ovarian carcinoma cells. Further, ARE increased the induction of the AKR1C1 gene in response to treatment with phase II drug inducers. However, ARE did not induce the transcription of AKR1C1 gene promoter in the presence of cisplatin in any of the cell lines. A computational analysis utilizing the Alibaba 2.0 on the proximal AKR1C1 gene promoter region was performed to identify potential transcription factor binding sites. Based on this analysis, a set of potential, putative transcription factor binding sites for Oct1, Sp1, Cp-1/NF-Y, CEBP, p40X, USF, NF1 and AP-2 were identified in the region -180 to -88 of the AKR1C1 gene promoter. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at -109ATTGG-105 of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Further, ectopic expression of NF-Y’s increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or knockdown of NF-YA by siRNA transfection, decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that was CCAAT box-dependent. These results indicate that NF-Y regulates basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and cisplatin-induced transcription in human ovarian carcinoma cells. / Pathology

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Nouveau modèle d’étude de l’hépatoblastome in vivo et identification de microARNs régulateurs de la β-caténineL’hépatoblastome (HBL) est le cancer du foie le plus fréquent chez l’enfant. Au niveau moléculaire, il est caractérisé par des mutations activatrices dans le gène de la β-caténine (CTNNB1) entrainant une accumulation de sa protéine et une activation anormale de la voie de signalisation Wnt, responsables de la transformation maligne des cellules hépatiques. Ainsi, la β-caténine est une cible thérapeutique majeure dans l’HBL.Ce manuscrit décrit les deux parties principales de mon projet de thèse, dont l’objectif était d’étudier le rôle de microARNs régulateurs de la β-caténine sur la carcinogenèse de l’HBL.La première partie présente le développement d’un nouveau modèle d’étude in vivo de l’HBL. Ce travail a consisté à réaliser des xénogreffes de cellules dérivées d’HBL sur la membrane chorioallantoidienne (CAM) de l’embryon de poulet, et à étudier le développement des tumeurs par des approches histologiques et moléculaires. Mes résultats montrent que les cellules d’HBL modifient leur phénotype et activent des mécanismes de survie et de prolifération pour former des nodules tumoraux structurés et vascularisés au sein de la CAM. Le traitement des cellules par le cisplatine conduit à un arrêt net de la progression tumorale.La seconde partie présente les résultats concernant la régulation de la β-caténine et des processus oncogéniques par les microARNs dans les cellules d’HBL. Un crible fonctionnel m’a permis d’identifier plusieurs microARNs ayant un effet inhibiteur sur la β-caténine. Quatre microARNs, sous-exprimés dans les tumeurs de patients, bloquent la prolifération cellulaire et l’activité de la voie Wnt in vitro. Le miARN le plus efficace interagit avec l’ARNm de la β-caténine via un site unique localisé dans sa région 3’ non traduite et bloque la croissance des tumeurs d’HBL sur la CAM.Durant ma thèse j’ai développé un nouveau modèle animal d’étude de l’HBL qui permet de modéliser la croissance tumorale et de tester l’impact de molécules thérapeutiques. Ce travail a également permis d’identifier de nouveaux microARNs sous-exprimés dans les tumeurs d’HBL, qui inhibent l’expression de la β-caténine et agissent comme des gènes suppresseurs de tumeur. Ces microARNs constituent de potentielles nouvelles molécules thérapeutiques dans le traitement de l’HBL et pourraient aussi être utilisés comme des biomarqueurs diagnostiques et pronostiques. / Hepatoblastoma (HBL) is the most common pediatric liver cancer. At molecular level it is characterized by activating mutations in β-catenin gene (CTNNB1) that cause protein accumulation and abnormal Wnt signaling pathway activation leading to malignant transformation of hepatic cells. Therefore, β-catenin is a key therapeutic target in HBL.This manuscript describes two major parts of my thesis project that aimed to understand the role of β-catenin-regulating microRNAs in HBL carcinogenesis.The first part presents the development of a new in vivo HBL model. This work consisted of making HBL-derived cells xenograft on chick embryo chorioallantoic membrane (CAM) and studying tumor development using histological and molecular approaches. My results show that HBL cells implanted on CAM modify their phenotype and activate survival and proliferation mechanisms to form organized and vascularized tumor nodules. Cell treatment with cisplatin leads to tumor progression arrest.The second part presents results concerning the regulation of β-catenin and oncogenic processes by microRNAs in HBL cells. A functional screening allowed me to identify several microRNAs with an inhibitory effect on β-catenin. Four microRNAs down-regulated in patient tumors inhibit cell proliferation and Wnt pathway activity in vitro. The most efficient microRNA directly interacts with β-catenin mRNA through a unique site localized in 3’ untranslated region and blocks HBL tumor growth on CAM.During my thesis I developed a new animal model to study HBL that allows modelling rapid tumor growth and testing the impact of therapeutic molecules. My work also led to the identification of new microRNAs down-regulated in HBL tumors that inhibit β-catenin expression and act as tumor suppressor genes. Those microRNAs represent new potential therapeutic molecules for HBL treatment and could be also used as diagnostic and prognostic biomarkers.

Background & Aims: Despite advances in surgical care and chemotherapeutic regimens, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and β-Catenin co-activation occurs in 80% of children’s HB; however, a lack of conditional genetic models precludes exploration of tumor maintenance and therapeutic targets. Thus, the clinical need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in children’s tumors, I sought to evaluate YAP1 as a therapeutic target in HB. Approach & Results: Herein, I engineered the first conditional murine model of HB using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A, constitutive β-CateninDelN90, and a luciferase reporter to murine liver. Tumor regression was evaluated using in vivo bioluminescent imaging, and tumor landscape characterized using RNA sequencing, ATAC sequencing and DNA foot-printing. Here I show that YAP1 withdrawal in mice mediates >90% tumor regression with survival for 230+ days. Mechanistically, YAP1 withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative “hbHep cells.” hbHep cells have hepatocyte-like morphology and partially restored mature hepatocyte gene expression. YAP1 withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and can rescue liver damage in mice. Conclusions: YAP1 withdrawal, without modulation of oncogenic β-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. Modulating YAP1 expression alone is sufficient to drive long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.