Le carcinome hépatocellulaire (HCC) est une tumeur de mauvais pronostic caractérisée, comme la plupart de cancers, par l'association paradoxale de télomères courts et d'une importante activité télomérase. Cette attrition télomérique joue un rôle central dans l'instabilité chromosomique à l'origine de la promotion et de l'évolution tumorale. Les premières causes d'HCC correspondent aux infections chroniques par les virus hépatotropes : virus de l'hépatite B (VHB) et virus de l'hépatite C (VHC). Dans une première étape nous avons disséqué les dérégulations télomériques dans les HCC et les cirrhoses liées au VHB, VHC ou encore à l'alcool. Nous avons observé que ces dérégulations sont acquises tôt, au stade prétumoral, et persistent au stade tumoral. Ces dérégulations sont spécifiques d'un carcinogène donné. Aux stades tardifs et tumoraux de l'infection par le VHB, les cellules hépatiques expriment fréquemment une protéine tronquée en partie C-terminale d'HBx ainsi que des formes réarrangées du gène PreS/S telle que PreS2. Dans une seconde étape nous avons trouvé qu'à l'opposé de la forme sauvage, HBx tronquée réprime hTERT, diminue l'activité télomérase, raccourcit les télomères, augmente la proportion de ponts anaphasiques et déclenche la sénescence de cellules primaires. Sachant qu'au stade tumoral les cellules transformées ré-expriment hTERT nous avons testé l'effet d'une coexpression de PreS2 et d'HBx tronquée et avons pu montrer que PreS2 contrecarrait l'effet répresseur d'HBx sur hTERT. Néanmoins de façon étonnante, PreS2 ne parvenait pas à rallonger les télomères en présence d'HBx tronquée. Les facteurs protégeant les télomères coopèrent avec la télomérase pour l'élongation et plusieurs de ces protéines sont par ailleurs impliquées dans la réparation des dommages à l'ADN. Nous avons trouvé qu'HBx tronquée modifiait spécifiquement l'expression de la plupart des protéines du télosome selon un patron connu pour inhiber l'effet de la télomérase. Nous avons montré que des dommages à l'ADN télomérique liés à l'incubation de cellules primaires avec la néocarcinostatine inhibaient l'élongation des télomères par hTERT. Ayant trouvé que PreS2 et HBx tronquée induisaient des dommages à l'ADN, nous proposons que cet effet explique l'impossibilité pour PreS2 d'allonger les télomères de cellules exprimant HBx tronquée / Among the numerous genetic defects that underly with hepatocarcinogenesis, telomere abnormalities seem to play a role both in tumor promotion and maintenance. Telomeres, the chromosome extremities, are protected by specific proteins, the Shelterin complex and by additional factors. Besides telomerase dysregulation, changes in the expression of these telomere factors have been observed in cancers. Herewe first tested the hypothesis that such dysregulations might occur in HCC with patterns depending onthe cause of HCC. For HBV-, HCV- and alcool-dependant HCC we found that telomeric dysregulations appear to be carcinogen-specific and occur early during the course of the disease and are persistent in the tumor. At the late stage of HBV-dependent disease and corresponding tumors, hepatocytes produce 3’ deleted mutants of HBx (3’DM HBx) but also a rearranged form of the PreS/S gene: PreS2. We then found that, unlike WT HBx, 3’ DM HBx repress hTERT transcription, decrease telomerase activity, shorten telomere length, increase anaphase bridges and trigger senescence in transfected primary cells. It’s well known that hTERT it re-expressed in tumors, so we tested PreS2 and 3’DM HBx transfection. We show that PreS2 counteracts 3’DM HBx effect on hTERT transcription and telomerase activity. However surprisingly PreS2 wasn’t able to elongate telomeres in 3’DM HBx expressing cells. Telomeric factors interact with telomerase allowing telomere elongation. Moreover many of these factors are implicated in DNA damage repair systems. We found that all Shelterin’s and some other telomeric factors’s expression in dyregulated in 3’DM HBx expressing cells. Moreover we show that neocarcinostatin dependent DNA damage in MRC5 primary cell prevent hTERT-based telomere elongation. Also finding that PreS2 and 3’DM induce DNA damage, we suggest that 3’DM HBx prevents PreS2 and hTERT- based telomere elongation

Student Number : 9903082V - MSc (Med) dissertation - Faculty of Health Sciences / Chronic infection with the hepatitis B virus (HBV) is endemic to sub-Saharan Africa and south-east Asia where it is a major risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). Currently available therapy is only effective in a small subset of chronic carriers. The development of novel treatment modalities for the management of HBV therefore remains an important global medical objective. Sequence plasticity of the HBV genome is limited by its small size and the overlapping nature of its open reading frames (ORFs). These features make HBV an ideal target for therapy based on nucleic acid hybridization. The use of ribozymes (RNA enzymes) and antisense molecules to inhibit gene expression is well documented. The recent discovery of RNA interference (RNAi) has added to the arsenal of therapy based on nucleic acid hybridization. RNAi is the process whereby short RNA duplexes (called short interfering RNA or siRNA) mediate the sequence-specific post-transcriptional silencing of genes homologous in sequence to the siRNA. siRNA function by guiding a protein complex (RNA Induced Silencing Complex or RISC) to target mRNA for degradation or translational repression. The protein X ORF (HBx ORF) is a conserved region of the HBV genome and is common to all viral transcripts. HBx is required for infection by the virus and plays an important role in the establishment of chronic infections in vivo as well as in the development of HCC. RNAi targeted against the HBx ORF may therefore prove useful as treatment of chronic HBV infection. Plasmid based expression cassettes capable of endogenously generating short hairpin RNA (shRNA) targeted to the HBx ORF were constructed. The shRNA function as substrates for the RNAi machinery and are processed into siRNA. The ability of the expression cassettes to knockdown markers of HBV gene expression was tested in a human hepatoma cell line. A panel of 10 U6 promoter-driven shRNA expression vectors was generated. The U6 promoter (an RNA polymerase III promoter) is normally involved in the transcription of small nuclear RNA and as such is ideal for the generation of shRNA of precisely defined length. Three cytomegalovirus (CMV) promoter-driven shRNA expression cassettes incorporating ribozymes that produce defined hairpin sequences were also generated. The CMV promoter (an RNA polymerase II) promoter is involved in the transcription of large messenger RNA. Two hammerhead ribozymes lying 5’ and 3’ of the shRNA encoding sequence were incorporated into the cassette. Cis-cleavage by the ribozymes releases a shRNA of defined length thereby overcoming the limitations imposed by extraneous sequences from CMV promoter-driven transcription. U6 promoter-driven shRNA expression vectors efficiently knocked down markers of HBV replication in liver cells. The CMV promoter-driven expression vectors were incapable of inhibiting HBV gene expression; however shRNA generated in vitro from these vectors mediated efficient knockdown of HBV replication. shRNA-mediated inhibition of gene expression therefore holds promise as a novel treatment strategy for the management of HBV and other mobile genetic elements.

Introdução: A injeção percutânea de etanol (PEI) é um método de ablação local considerado curativo para carcinomas hepatocelulares (CHC) pequenos. Não há dados sobre PEI para tratamento do CHC na América Latina. Pacientes e métodos: Incluídos 100 pacientes cirróticos submetidos à PEI como terapia primária para o CHC no Serviço de Gastroenterologia do HCFMUSP entre setembro de 1997 e dezembro de 2005. Avaliados resposta ao tratamento e taxa de sobrevida. Resultados: Resposta completa ao tratamento ocorreu em 41% dos casos. A sobrevida foi de 83% em 1 ano, 49% em 3 anos e 29% em 5 anos, mas no grupo com tumores de até 2 cm, sem invasão vascular e com resposta completa ao tratamento, atingiu 89%, 70% e 70%, respectivamente. Conclusão: PEI apresentou boa sobrevida em pacientes com tumores 2 cm, sem invasão vascular e resposta completa ao tratamento. / Introduction: Percutaneous ethanol injection (PEI) is a curative local ablative method for treatment of small hepatocellular carcinoma (HCC). There is no data about PEI for HCC treatment in Latin America. Patients and Methods: A total of 100 consecutive cirrhotic patients were enrolled. All of them had been submitted to PEI as first-line therapy for the treatment of HCC at the Service of Gastroenterology of HCFMUSP in the period of September 1997 to December 2005. Response to treatment and survival rates were assessed. Results: Complete response to treatment was obtained in 41% of the patients. Survival rates were 83% in 1 year, 49% in 3 years and 29% in 5 years, but in patients with tumors up to 2 cm, no vascular invasion and complete response to treatment, were 89%, 70% and 70%, respectively. Conclusion: PEI is associated with long-term survival in patients with tumors 2 cm, absence of vascular invasion and complete response to treatment

O câncer é um dos principais problemas de saúde pública no mundo. Dentre as neoplasias primárias que acometem o fígado, o carcinoma hepatocelular (HCC) é a mais frequente. Diversos fatores de risco predispõem ao HCC, entre eles a doença hepática gordurosa não alcóolica (NAFLD). Segundo estudos prévios do grupo, a &#946-ionona (BI), apresenta potencial quimiopreventivo na hepatocarcinogênese, promovendo redução de lesões preneoplásicas (LPN). Assim pretendeu-se investigar se a NAFLD potencializaria o desenvolvimento de LPN em ratos Wistar submetidos ao modelo do hepatócito resistente (RH) na etapa de iniciação/promoção inicial da hepatocarcinogênese e se a BI apresenta efeito quimiopreventivo nesse contexto. Para tanto, os animais foram distribuídos em 4 grupos experimentais: grupo não-tratado (NT), grupo submetido ao RH (RH), grupo submetido ao RH e ao modelo de NAFLD, que consiste na administração de emulsão hiperlipídica, (AS) e grupo AS tratado com BI (AS + BI). Em uma primeira instância, 5 animais pertencentes aos grupos NT, AS,AS + BI foram eutanasiados após 6 semanas de administração de emulsão hiperlipídica, antes da aplicação do modelo RH, para se confirmar o desenvolvimento da NAFLD. Foi possível observar que a administração de emulsão hiperlipídica durante 6 semanas foi suficiente para o desenvolvimento de esteatose hepática. Após as 6 semanas foi introduzido o modelo do RH concomitante à administração da emulsão hipercalipídica até o fim do experimento na 13a semana . Após 13 semanas o grupo AS apresentou maiores (p<0,05) valores de focos de inflamação, hepatócitos balonizados e grau de esteatose hepática em relação ao grupo AS+BI, assim como maiores (p<0,05) níveis séricos de triacilgliceróis, colesterol, HDL, LDL, VLDL, e maior (p<0,05) valor de MDA em relação ao grupo RH embora sem diferença estatística. O grupo AS também apresentou maior (p<0,05) incidência, número total e multiplicidade de nódulos, além de maior (p<0,05) número e tamanho de LPN persistentes (pLPN) e índice de proliferação quando comparado aos grupos RH e AS+BI. O grupo AS + BI, por sua vez, demonstrou menores (p<0,05) valores de escore de células ovais e menores valores de comprimentos de cometa e danos no DNA quando comparado ao grupo AS, embora sem diferença estatista para este último parâmetro. Em relação à expressão gênica, o grupo AS apresentou menores (p<0,05) valores de expressão do gene Hmgcr em relação ao grupo RH e maiores (p<0,05) valores dos genes Insig 1 e Thy 1 quando comparados ao grupo AS+BI. Portanto, no contexto de esteatose hepática associada ao modelo do RH, a administração de BI durante a etapa de iniciação/promoção em ratos Wistar resultou em atividade quimiopreventiva que se deu pela diminuição de pLNP, redução da proliferação celular e do número de células ovais, consideradas potenciais alvos para o desenvolvimento da hepatocarcinogênese, entretanto os genes analisados parecem não serem modulados pela BI. / Cancer is a major public health problem in the world. Among the primary neoplasms affecting the liver, hepatocellular carcinoma (HCC) is the most frequent. Several risk factors predispose to HCC, including the nonalcoholic fatty liver disease (NAFLD). According to previous studies of the group, the &#946-ionone (BI), has potential chemopreventive in hepatocarcinogenesis, promoting reduction of preneoplastic lesions (LPN). Thus we investigated whether NAFLD would increase the development of LPN in Wistar rats resistant hepatocyte model (RH) at the stage of initiation / promotion of hepatocarcinogenesis and if BI has chemopreventive effect in this context. Therefore, the animals were divided into 4 groups: non-treated group (NT), the group submitted to HR (HR), the group submitted to HR and NAFLD model, consisting of the fatty emulsion administration (AS) and AS group treated with BI (AS + BI). In a first point, 5 animals belonging to the groups NT, AS, AS + BI were euthanized after 6 weeks of administration of fat emulsion prior to application of the HR model, to confirm the development of NAFLD. It was observed that the administration of fatty emulsion for 6 weeks was sufficient to the development of hepatic steatosis. After 6 weeks it was introduced into the model HR the concomitant administration of fatty emulsion until the end of the experiment at 13 weeks. In the endpoint, the AS group had higher (p <0.05) of serum triacylglycerols, cholesterol, HDL, LDL, VLDL although no statistical difference inrelation to RH group, and increased (p <0.05) amount of MDA in relation to the group RH. The AS group also had higher (p <0.05) incidence, multiplicity and total number of nodes and greater (p <0.05) number and size of persistent LPN (pLPN) and proliferation index when compared to HR groups and AS + BI. AS + BI group. It was observed in AS+BI group lower (p <0.05) cell oval score values compared to AS group. In addition the AS+BI group showed lower values of the comet length and DNA damage compared to the AS group, although no statistical differences. In relation to gene expression, the AS group showed lower(p <0.05) HMGCR gene expression values in relation to HR group and higher (p <0.05) expression of Insig genes 1 and Thy 1 compared to group AS + BI.Therefore, in the context of hepatic steatosis associated with HR model BI for administration to the stage of initiation / promotion in rats resulted in chemopreventive activity was due to decrease in area of pLNP, reducing cell proliferation, and the number of oval cells, as potential targets for the development of hepatocarcinogenesis, however the genes do not seem to be modulated analyzed by BI.

A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum e está associado com a segunda menor taxa de sobrevida em 5 anos de todos os tipos de tumores. A melatonina (Mel) é uma potente molécula antioxidante que se tem mostrado benéfica em diversas situações patológicas, incluindo o CHC. Objetivo: Avaliar o efeito da Mel sobre marcadores de estresse oxidativo e angiogênicos no tecido hepático de ratos Wistar no modelo experimental de carcinogênese hepática induzida por dietilnitrosamina (DEN) associado ao acetilaminofluoreno (2-AAF). Métodos: 32 ratos machos Wistar (150g) foram divididos em 4 grupos: Controle (CO); Controle+Mel (CO+Mel); DEN e DEN+Mel. O DEN (50mg/kg) foi administrado por via intraperitoneal duas/uma vez por semana, associado a uma única dose de 2-AAF (100mg/kg). A Mel foi administrada na água de beber dos animais na concentração final de 20 mg/L e o tratamento teve início na 12ª semana perdurando até o fim das 19 semanas de experimento. O sangue dos animais foi coletado para as análises de AST, ALT, FA, γ-GT e amostras de fígado utilizadas para avaliar a lipoperoxidação (LPO), a atividade das enzimas antioxidantes (CAT, GPx e GST), os níveis de GSH e de metabólitos do óxido nítrico, a análise histógica e as proteínas envolvidas na angiogênese tumoral (VEGF, PI3K, p-Akt e eNOS). Resultados: O dano tecidual e o processo fibrogênico presentes no parênquima hepático estavam diminuídos no grupo DEN+Mel, assim como o nível de TBARS e a atividade da enzima GST quando comparados ao grupo DEN. A atividade da CAT mostrou-se aumentada no grupo DEN+Mel quando comparada ao grupo DEN. No processo angiogênio, a expressão de VEGF, PI3K, p-Akt mostrou-se diminuída no grupo DEN+Mel enquanto a expressão da eNOS apresentou-se aumentada quando comparado ao grupo DEN. Conclusão: Constatamos que a Mel foi capaz de minimizar os danos no parênquima hepático, de diminuir a LPO, modular a atividade da CAT, além de mostrar-se eficaz na redução de VEGF e da via PI3K/Akt no modelo experimental de carcinogênese hepática. / Background: Hepatocellular carcinoma (CHC) is the most common primary liver cancer and is associated with the second lowest 5-year survival rate of all tumor types. Melatonin (Mel) is a powerful antioxidant molecule that has been demonstrated to be beneficial in various pathological conditions, including HCC. Objective: The aim of this study was to evaluate the effect of Mel on oxidative stress and angiogenic markers in liver tissue of Wistar rats in the experimental model of hepatic carcinogenesis induced by diethylnitrosamine (DEN) and acetylaminofluorene (2-AAF). Methods: 32 male Wistar rats (150g) were divided into 4 groups: Control (CO); Control+Mel (CO+Mel); DEN and DEN+Mel. DEN (50mg/kg) was administered intraperitoneally once or twice a week, associated with a single dose of 2-AAF (100mg/kg). Mel was given in drinking water at the final concentration of 20 mg/L and the treatment was started at 12th week and continued until the end of the 19 weeks of experiment. Blood samples were collected for AST, ALT, AP, γ-GT and liver samples were used to evaluate lipid peroxidation (LPO), activity of antioxidant enzymes (CAT, GPx and GST), levels of GSH and nitric oxide levels, histological analysis and expression of proteins involved in tumor angiogenesis (VEGF, PI3K, p-Akt and eNOS). Results: The tissue damage and the fibrogenic process present in the hepatic parenchyma were decreased as well as the levels of TBARS and the activity of GST in the group DEN+Mel when compared to DEN group. CAT activity was increased in DEN+Mel group when we compared with DEN group. The expression of VEGF, PI3K, p-Akt was decreased in DEN+Mel group while eNOS expression was increased when compared to DEN group. Conclusion: Mel was able to minimize damage in the hepatic parenchyma, reduce LPO, modulate the activity of CAT and reduce VEGF and the PI3K/Akt pathway in a experimental model of hepatic carcinogenesis.

Racional: O carcinoma hepatocelular é uma neoplasia maligna agressiva com elevada morbidade e mortalidade. Objetivo: Revisão da literatura sobre o diagnóstico e o manejo do carcinoma hepatocelular nos vários estágios da doença. Método: Revisão da literatura utilizando a base Medline/PubMed e literatura adicional. Resultados: O carcinoma hepatocelular é geralmente complicação da cirrose hepática. As hepatites virais crônicas B e C também são fatores de risco para o surgimento do carcinoma hepatocelular. Quando associado à cirrose hepática, o carcinoma hepatocelular geralmente surge a partir da evolução de um nódulo regenerativo hepatocitário que sofre degeneração maligna. O diagnóstico é efetuado através de tomografia computadorizada de abdome com contraste endovenoso (efeito wash in e wash out), e a ressonância magnética pode auxiliar nos casos que não possam ser definidos pela tomografia computadorizada. O único tratamento potencialmente curativo para o carcinoma hepatocelular é a ressecção do tumor, seja ela realizada através de hepatectomia parcial ou de transplante. Infelizmente, apenas cerca de 15% dos carcinomas hepatocelulares são passíveis de tratamento cirúrgico. Pacientes portadores de cirrose hepática estágio Child B e C não devem ser submetidos à ressecção hepática parcial. Para esses pacientes, as opções terapêuticas curativas restringem-se ao transplante de fígado, desde que selecionáveis para esse procedimento, o que na maioria dos países dá-se através dos Critérios de Milão (lesão única com até 5 cm de diâmetro ou até três lesões de até 3 cm de diâmetro). A sobrevida em 5 anos para pacientes transplantados para o carcinoma hepatocelular pode alcançar 70% Conclusão: Quando diagnosticado em seus estágios iniciais, o carcinoma hepatocelular é potencialmente curável. O conhecimento das estratégias de 17 diagnóstico e tratamento do carcinoma hepatocelular a fim propiciam sua identificação precoce e a indicação de tratamento apropriado. / Introduction: Hepatocellular carcinoma is an aggressive malignant tumor with high lethality. Aim: A literature review on diagnosis and management of hepatocellular carcinoma was performed. Methods: Literature review utilizing databases Medline/PubMed. Results: Hepatocellular carcinoma is a common complication of hepatic cirrhosis. Chronic viral hepatitis B and C also constitute as risk factors for development of hepatocellular carcinoma. In patients with cirrhosis, hepatocelular carcinoma usually develops from a malignant transformation of a dysplastic regenerative nodule. Diagnosis is confirmed through computed tomography scan with intravenous contrast (wash in and wash out effect), and magnetic resonance may be helpful in some instances. Curative treatment for hepatocellular carcinoma may be performed through partial liver resection or liver transplantation. Only 15% of all hepatocellular carcinomas are localized and amenable to operative treatment. Patients with Child C liver cirrhosis are not amenable to partial liver resections. The only curative treatment for hepatocellular carcinomas in patients with Child B or C cirrhosis is liver transplantation. In most countries, only patients with hepatocellular carcinoma under Milan Criteria (single tumor with up to 5 cm diameter or up to three nodules with a maximum diameter of 3 cm) are considered candidates for liver transplant. Five-year survival following liver transplantation for hepatocellular carcinoma may reach 70%. Conclusion: Hepatocellular carcinoma is a potentially curable neoplasm if discovered in its initial stages. Clinicians and surgeons should be familiar with strategies for early diagnosis and treatment of hepatocellular carcinoma as a way to decrease mortality associated with this malignant neoplasm.

Les carcinomes hépatocellulaires (CHC) mutés CTNNB1 ont des caractéristiques phénotypiques propres en termes de polarité et de métabolisme (absence de stéatose). Nous avons émis l’hypothèse que ce phénotype pouvait être secondaire à l’activation du gène suppresseur de tumeurs LKB1 qui code une Ser/Thr kinase multitâches.Nous avons tout d’abord montré qu’il existait effectivement un dialogue complexe entre les voies Wnt/β-Caténine et LKB1 dans le foie. Les mutations de CTNNB1 sont en effet capables d’induire l’expression protéique de LKB1 dans des lignées hépatomateuses humaines, et les CHC mutés CTNNB1 présentent une expression protéique accrue de LKB1 et une signature transcriptionnelle d’activation de LKB1. De plus, dans deux modèles murins d’invalidation hépatospécifique de Lkb1, LKB1 est apparu comme requis pour l’activation complète du programme transcriptionnel de β-Caténine mais de façon dépendante du stade de développement et du contexte nutritionnel. Enfin, la signalisation LKB1 est apparue comme nécessaire à la survie des hépatocytes activés pour β-Caténine dans deux modèles murins différents.Nous avons aussi caractérisé les rôles métaboliques de LKB1 dans le foie. L’invalidation hépatospécifique de Lkb1 induisait une augmentation progressive de la masse grasse corporelle avec utilisation préférentielle des glucides comme substrat énergétique. Il existait une activation de la néoglucogenèse hépatique avec hyperglycémie et une lipogenèse accrue avec accumulation hépatocytaire de lipides. Enfin, nous avons mis en évidence une activation paradoxale de la signalisation AKT dans les hépatocytes, même à jeun, et une dépendance énergétique aux acides aminés. Enfin, nous avons identifié une nouvelle isoforme protéique de LKB1 délétée de son domaine N-Terminal et d’une partie de son domaine kinase. D’expression tissulaire préférentiellement musculaire et myocardique, cette isoforme catalytiquement inactive se comportait comme dominant positif sur l’activation de l’AMPK par la forme conventionnelle mais comme dominant négatif dans l’activité polarisation induite par LKB1. Enfin, elle était capable d’induire, en l’absence de la forme conventionnelle, la prolifération cellulaire et la tumorigenèse chez la souris nude. Elle pourrait exercer des rôles métaboliques particuliers dans les tissus fortement oxydatifs et des rôles oncogéniques dans certains contextes. / CTNNB1-Mutated hepatocellular carcinomas (HCC) share a specific polarity and metabolic phenotype without steatosis. We hypothesized that such phenotype could imply the tumor suppressor gene LKB1 that encodes for a multi-Task Ser/Thr kinase.We first demonstrated that a complex crosstalk indeed exists in the liver between LKB1 and the Wnt/β-Catenin pathway. LKB1 proteic expression was controlled by mutant β-Catenin in hepatomatous cell line and CTNNB1-Mutated HCCs had an enhanced LKB1 proteic expression as well a transcriptomic signature of LKB1 activation. In two mouse model of liver-Specific invalidation of Lkb1, we showed that LKB1 was required for full activation of the β-Catenin transcriptomic program, but it depended on the developmental stage and nutritional context. At least, LKB1 appeared to be required for the survival of β-Catenin activated liver cells in two other mouse models.Then, we wanted to caracterize the metabolic roles of LKB1 in the liver. Liver-Specific invalidation of Lkb1 progressively raised the body fat mass and we observed that carbohydrates were preferred as whole-Body energetic fuel. In the liver, gluconeogenesis and lipogenesis were enhanced, resulting in mild hyperglycemia and lipid accumulation in the hepatocytes. At least, we identified an aberrant activation of the AKT signaling in the liver, even during fasting, and an energetic dependence towards amino acids.At least, we identified a novel LKB1 proteic isoform that is deleted of its N-Terminal domain and part of its kinase domain. Highly expressed in the muscle and in the heart, this catalytically inactive isoform however acted as a positive dominant towards AMPK activation by full length LKB1 but as a negative dominant towards LKB1-Induced cell polarization. This isoform is also able to enhance cell proliferation and to induce tumors in a xenograft model, even when expressed alone. It could play specific metabolic roles in oxidative tissues and could be oncogenic in some contexts.

La voie de signalisation WNT/β-caténine est impliquée dans de nombreuses processi cellulaires, du développement à la physiologie. Dans le foie adulte, elle est nécessaire pour établir et maintenir la zonation métabolique, condition préalable pour la fonctionnalité de l’organe, mais elle est aussi cause d’un pourcentage non négligeable (11-32%) de carcinomes hépatocellulaires (CHC), qui surviennent après mutation activatrice du gène codant la β-caténine. Mes travaux se sont inscrits dans la continuité de travaux de l’équipe auxquels j’ai participé , et ont eu pour principaux objectifs : (1) d’explorer le rôle du facteur de transcription Hnf4α dans la physiologie et les cancers du foie, en lien avec la signalisation β-caténine ; (2) de déterminer si une imagerie tumorale par tomographie par émission de positrons (TEP) spécifique de la captation de choline pouvait prédire les CHC mutés pour la β-caténine et si le métabolisme de la choline pouvait présenter une piste thérapeutique des cancers du foie.Pour ces deux projets, j’ai eu accès à des cohortes de patients atteints de cancers du foie, mais j’ai également pu bénéficier du modèle gain-de-fonction de la β-caténine développé au laboratoire, qui consiste en une perte du suppresseur de tumeur Apc, frein majeur de la voie β-caténine conduisant à des cancers du foie. Grâce à un modèle d’invalidation hepato-spécifique et conditionnelle du gène Hnf4α, j’ai pu prouver que la perte de Hnf4α mène à une augmentation de la prolifération, du stockage des lipides dans le foie et à une désorganisation de l’architecture zonale hépatique, en particulier celle de la triade portale. J’ai aussi démontré que dans un contexte de carcinome murin invalidé par Apc, le rôle suppresseur de tumeur d’Hnf4α était mineur.Une approche métabolomique avait montré qu’un signal β-caténine perturbait le métabolisme des phospholipides dérivant de la choline. Grâce à une étude parallèle réalisée chez des patients porteurs de CHC d’une part, et dans nos modèles murins d’autre part, nous avons pu mettre en évidence par TEP une fixation accrue de la F-choline dans les tumeurs activées β-caténine. Ce phénotype est spécifique d’une signalisation β-caténine active puisque cette captation accrue n’était pas présente chez les patients porteurs de carcinome hépatocellulaire non mutés ou chez les souris présentant une cancérogenèse indépendante de la β-caténine (modèle DEN). J’ai ensuite étudié le devenir intracellulaire de la choline. En utilisant de la choline radiomarquée j’ai montré in vitro qu’un signal β-caténine aberrant accroit l’incorporation de choline dans les phospholipides, et accroit également son rôle de donneur de groupements méthyles, participant à la méthylation de l’ADN. Cela pourrait expliquer pourquoi l’ADN est hyperméthylé chez les souris avec la perte d’Apc, puisque l’administration d’un régime sans choline et methionine à ces souris réverse le phénotype d’hyperméthylation. L’ensemble de ces résultats suggère que la choline pourrait jouer un rôle important dans la cancérogenèse liée à la β-caténine. Nous proposons que des TEP F-choline pourraient être utilisés pour diagnostiquer les CHC mutés β-caténine, et à terme des thérapies ciblées sur ce métabolisme pourraient être envisagées. / WNT/β-catenin is a pillar during development and in adult physiology. In particular in the adult liver it is a double-edged sword: it is necessary to establish the metabolic zonation, requirement for having a functional organ, but it is also involved in the onset of 11-32% of hepatocellular carcinoma (HCC). My thesis work has been based on the team previous results and it is focused on two main subjects: (1) the first aim was to decipher the role of Hnf4α both in physiology and in HCC development and its relationship with WNT/β-catenin signalling and (2) the second part explores the possible use of Fluoro-choline (FCh) positron emission tomography (PET) in the diagnosis of β-catenin-activated liver tumours.In this study I used cohorts of patients having HCC, but also inducible and hepatospecific knock-out mice for adenomatous polyposis coli (APC) gene (thereafter called ApcKO mice). Apc is the most important negative regulator of β-catenin, and it hepatic loss leads to aberrant activation of β-catenin, disrupting liver zonation and initiating long-term liver cancers. I generated also inducible hepatospecific Hnf4α knock-out mice and I demonstrated an increased proliferation, lipids accumulation and disorganization in the portal triad architecture, together with a mild distruption of liver zonation. Then, looking at cancer onset, I demonstrated that Hnf4α loss is not able per se to initiate liver cancer, and has no tumour suppressor role in β-catenin activated tumours onset and progression.We performed a metabolic analysis of ApcKO livers, showing that β-catenin is able to deregulate lipids metabolism, in particular that of phospholipids derived from choline. In collaboration with clinicians, I studied human patients who underwent FCh/PET, showing that β-catenin-mutated tumours had an increased uptaken of F-Choline whereas non-mutated β-catenin human HCC had not. Similar results were obtained with mice, either ApcKO β-catenin-activated HCC or β-catenin-independent mice HCC, obtained through a N-diethylinitrosamine (DEN) injection.Choline in cells splits in two main pathways: it is both a methyl-group donor and a precursor for phospholipids production. I tested this through radiolabeled fluxes in in vitro experiments. In β-catenin activated hepatocytes and tumours there are more phospholipids and more methyl groups in DNA derived from choline than in control mice. Moreover in ApcKO DNA is hypermethylated, and it is dependent on choline supply from diet.All these results together show the importance for β-catenin activated tumours to have a supply in choline, and so open a way not only in PET exploitation for having a precise diagnosis, but also in deciphering the importance of choline pathway, to possibly develop a targeted therapy.

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-10-25T16:54:50Z No. of bitstreams: 1 NathaliaTunissiolli_dissert.pdf: 12888654 bytes, checksum: 328d8640cfc58ce2cf35d21fabb3bc91 (MD5) / Made available in DSpace on 2018-10-25T16:54:50Z (GMT). No. of bitstreams: 1 NathaliaTunissiolli_dissert.pdf: 12888654 bytes, checksum: 328d8640cfc58ce2cf35d21fabb3bc91 (MD5) Previous issue date: 2017-09-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Hepatocellular Carcinoma (HCC) is the primary liver cancer with high incidence and mortality rates due to its late diagnosis. Its development results from the interaction between environmental and genetic factors. Thus, the objective of this study was to evaluate clinical, epidemiological and histopathological parameters of CHC patients submitted to liver transplant surgery from 2010 to 2016 at a University Reference Center. Materials and methods: It is a retrospective, descriptive and cross-sectional study. We evaluated all HCC patients submitted to liver transplantation from 2010 to 2016 from a University Reference Center in the Northwest of São Paulo. The variables analyzed were: age, gender, ethnicity, smoking, hepatitis B virus (HBV) infection, hepatitis C virus (HCV), cirrhosis, alcoholic liver disease (ALD), diabetes, non-alcoholic fatty liver disease (NAFLD), genetic disease, alpha-fetoprotein (AFP), survival and relapse rate. Results: Of the 60 patients included in this study, 48 (80%) were men with a mean age of 58.3± 10.6 years. Cirrhosis was present in 100% of cases. The etiologies identified for the development of hepatopathy were 56.6% HCV, 20% of which were associated with alcohol, 20% of HBV, 1.66% of patients with hemochromatosis, 50.9% of ALD and 25% of NAFLD. In addition, AFP levels were measured in 42 patients, with 88.09% presenting levels below 20 ng/mL and 7.14% having levels >150 ng/mL. Regarding the histological classification of Edmondson-Steiner, 58.5% of the patients were classified as grade ≤ II and 41.5% grade ≥ III. Conclusion: Predominant male patients, with a mean age of 58.3 years. In relation to the histological classification of Edmondson-Steiner the degree ≤ II is the most frequent. Cirrhosis was prevalent in the studied patients. HCV, ALD and NAFLD were the most common etiological agents found in the study. The high prevalence of NAFLD in the pre-transplanted underestimated sample is due to the fact that all patients present cirrhosis, masking the NAFLD signals. / O Carcinoma Hepatocelular (CHC) é o câncer primário do fígado com altas taxas de incidência e mortalidade devido ao seu diagnóstico tardio. Seu desenvolvimento resulta da interação entre fatores ambientais e genéticos. Assim o objetivo deste estudo foi avaliar parâmetros clínicos, epidemiológicos e histopatológicos de pacientes com CHC submetidos à cirurgia de transplante hepático no período de 2010 a 2016 em um Centro Universitário de Referência. Metodologia: Trata-se de um estudo retrospectivo, descritivo e transversal. Foram avaliados todos os pacientes com CHC submetidos ao transplante hepático no período de 2010 a 2016 de um Centro Universitário de Referência do Noroeste Paulista. As variáveis analisadas foram: idade, gênero, etilismo, tabagismo, infecção pelo vírus da hepatite B (VHB), vírus da hepatite C (VHC), cirrose, doença hepática alcoólica (DHA), diabetes, doença hepática gordurosa não alcoólica (DHGNA), portador de doença genética, dosagem de alfafetoproteína (AFP), sobrevida e índice de recidiva. Resultados: De 60 pacientes incluídos neste estudo, 48 (80%) eram homens com média de idade de 58,3 ± 10,6 anos. A cirrose estava presente em 100% dos casos. As etiologias identificadas para o desenvolvimento da hepatopatia foram 56,6% VHC das quais 20% o vírus estava associada ao álcool, 20% o VHB, 1,66% portador de doença genética, hemocromatose; 50,9% de DHA e 25% de DHGNA. Além disso, os níveis de AFP foram dosados em 42 pacientes, 88,09% apresentaram níveis inferiores a 20 ng/mL e 7,14% obtiveram níveis >150 ng/mL. Em relação à classificação histológica de Edmondson-Steiner 58,5% dos pacientes foram classificados grau ≤ II e 41,5% grau ≥ III. Conclusão: Predominaram-se pacientes do gênero masculino, com média de idade de 58,3 anos. Em relação à classificação histológica de Edmondson-Steiner o grau ≤ II é o mais frequente. A cirrose foi prevalente nos pacientes estudados. VHC, DHA e DHGNA foram os agentes etiológicos mais comuns encontrados no estudo. A alta prevalência de DHGNA na amostra estudada com diagnóstico subestimado pré-transplante, se deve ao fato de todos os pacientes apresentarem cirrose, mascarando os sinais de DHGNA.