Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC. / Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC.

Première partie: Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes qui se développent le plus souvent chez la femme jeune suite à la prise de contraceptifs oraux. Ils sont classés, sur le plan moléculaire, en: 1) AHC inflammatoires (AHCI), caractérisés par des mutations de différents gènes (IL6ST, GNAS ou STAT3) entrainant une activation de la voie de signalisation de l'interleukine 6; 2) les AHC avec mutations biallèliques inactivatrices d'HNF1A (H-AHC); 3) les AHC mutés pour CTNNB1 (bAHC) qui présentent un risque accru de transformation maligne et 4) les AHC non classés (UAHC). La glycogénose de type I (GSD I) est une maladie métabolique héréditaire rare qui prédispose au développement des AHC. Cependant les principales études moléculaires des AHC ont été réalisées sur des tumeurs sporadiques, et le profil moléculaire des AHC associée à la GSDI reste à déterminer.L'objectif de notre a été de caractériser, par des techniques de séquencage et de PCR quantitative, les altérations moléculaires d'une série d'AHC développés chez les patients atteints de GSD. Nous avons mis en évidence que le profil des AHC développés dans un contexte de GSDI était différent de celui des AHC sporadiques, avec aucun H-AHC et une tendance vers une augmentation de la fréquence des mutations de CTNNB1. Nous avons également observé que les foies non tumoraux des patients atteints de GSDI présentaient différentes anomalies métaboliques (répression de la néoglucogenèse et activation de la glycolyse et de la synthèse des acides gras) également retrouvées dans les H-AHC sporadiques. Ces anomalies sont d'autre connues pour favoriser la cancérogénèse. Notre étude a ainsi démontré que la GSDI prédisposait à des AHC de sous type moléculaire particulier, elle a également identifié diverses anomalies métaboliques observées dans les foies non tumoraux des patients atteints de GSDI qui pourraient contribuer au développement des tumeurs. Deuxième partie: Le carcinome hépatocellulaire (CHC), développé à partir des hépatocytes, est la tumeur maligne hépatique primitive la plus fréquente. Il représente le cinquième cancer le plus fréquent dans le monde et la troisième cause de mortalité liée au cancer. Il se développe dans environ 80% des cas dans un contexte d'hépatopathie chronique, sur parenchyme cirrhotique. Le processus de transformation maligne de la cirrhose en CHC est séquentiel, et passe par différentes étapes prénéoplasiques, comme les macronodules de bas ou haut grade. Il a récemment été mis en évidence que l'anomalie moléculaire la plus fréquente dans le CHC était les mutations du promoteur de TERT, gène codant pour la télomérase. Le but de notre étude a été d'étudier si ces mutations étaient également présente dans une série de 96 nodules développés sur cirrhose. Dans 31 cas, nous avons également déterminer le statut mutationnel de 10 gènes fréquemment mutés dans le CHC. L'ensemble des nodules a fait l'objet d'une relecture par 6 anatomopathologistes spécialisés en pathologie hépatique, et l'expression de marqueurs immunohistochimiques de malignité (Glypican 3, heat Shock protein 70, Glutamine Synthetase) a été évaluée. Nous avons identifié des mutations dans 6% des macronodules de bas grade, 20% des macronodules de haut grade, et dans environ 50% des nodules transformés en CHC. L’existence de mutations était corrélée à plusieurs critères morphologiques et immunohistochimiques de malignité. Aucune mutation dans un des 10 autres gènes fréquemment mutés dans le CHC n’a été retrouvée. Nos résultats démontrent que la fréquence des mutations du promoteur de TERT augmente au cours de la carcinogénèse hépatique, et qu’il s’agit d’une anomalie très précoce qui pourrait constituer un biomarqueur d'évaluation du risque de transformation des lésions prénéoplasiques développées sur cirrhose. / First Part: Hepatocellular adenomas (HCA) are benign tumors which most often develop in young women taking oral contraceptives. They are classified as : 1) inflammatory HCA (IHCA), characterized by mutations of genes (IL6ST, GNAS, STAT3) involved in the interleukin 6 pathway; 2) HCA with biallelic inactivating mutations of HNF1A (H-HCA) ; 3) CTNNB1 mutated HCA (bAHC), which harbour a high risk of malignant transformation and 4) unclassified HCA (UHCC). Glycogen storage disease type 1 (GSD1) is a rare hereditary metabolic disease that predispose to HCE development. The main molecular studies of HCA were pervformed on sporadic cases, and the molecular profile of HCA associated to GSDI remains to be investigated. The aim of our study was to characterize, by gene sequencing and gene expression profiling, a series of HCA developed in patients with GSD1. The molecular profile of GSD1 HCA was different to that of sporadic HCA, with a lack of H-HCA and a high frequency of b-HCA. We also observed that non tumoral livers of GSD1 patients featured several metabolic alterations (gluconeogenesis repression, glycolysis and fatty acid synthesis activation) that were also observed in sporadic H-HCA and may favor carcinogenesis. Alltogether, our study demonstrated that GSD1 predispose to particular HCA subtypes and the metabolic alterations observed in non tumorl liver of GSD1 patients may contribute to tumor formation. Second Part: Hepatocellular carcinoma (HCCIt is the fifth most frequent cancer and the third cause of cancer related death worldwide. If TERT promoter mutations are, so far, the most frequent recurrent molecular alterations of HCC, genetic determinants of the early steps of carcinogenesis on cirrhosis are still poorly understood. We aimed to evaluate the occurrence of telomerase reverse transcriptase (TERT) promoter mutations in a series of 96 macrondouels developed in a cirrhotic background. For 30 cases, 10 genes frequently mutated in HCC were also screeened. Six liver pathologists reviewed all the samples, and mmunohistochemistry (IHC) analyses were performed for glypican 3, glutamine synthase, and heat shock protein 70. TERT promoter mutations were highly related to the step-wise hepatocarcinogenesis because mutations were identified in 6% of low grade dysplastic nodules, 19% of high grade dysplastic nodules, 61% of high grade nodules with foci of transformation into HCC, and 42% of small HCC. Mutations in the 10 genes recurrently mutated in HCC were only identified in 28% of the small HCC. In conclusion, Frequency of TERT promoter mutations rapidly increases during the different steps of the transformation of premalignant lesions into HCC on cirrhosis. Consequently, somatic TERT promoter mutation is a new biomarker predictive of transformation of premalignant lesions into HCC.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a malignant liver tumor which is thought to be a variant of conventional hepatocellular carcinoma (HCC). It accounts for a small proportion of HCC cases and occurs in a distinctly different group of patients which are young and usually not in the setting of chronic liver disease. The diagnosis of FL-HCC requires the integration of clinical information, imaging studies, and histology. In terms of the treatment options, the only potentially curative treatment option for patients who have resectable disease is surgery either liver resection (LR) or liver transplantation (LT). When performed in a context of aggressive therapy, long-term outcomes after surgery, particularly liver resection for FL-HCC, were favorable. The clinical outcome of patients with unresectable disease is suboptimal with median survival of less than 12 months. The aim of this review is to update the available evidence on diagnosis, treatment options, outcome predictors, and recent developments of patients with this rare disease and to provide a summarized overview of the available literature.

Introduction: The development of liver fibrosis is characterised by dramatic changes in the biomechanical composition and mechanical properties of the extracellular matrix (ECM). Increases in matrix stiffness associated with inflammation and fibrosis are implicated in promoting cancer development. Clinical studies have demonstrated a close association between increases in liver stiffness and the incidence of hepatocellular carcinoma (HCC). The effect of changes in matrix stiffness on tissue-resident hepatic progenitor cells (HPC) is unknown. Aberrant HPC proliferation has been implicated in the pathogenesis of HCC. It was hypothesised that changes in the stiffness of the cellular microenvironment are important in regulating the behaviour of liver-resident cell populations and may promote the development of HCC. Aims: i) to determine how changes in the stiffness of the cancer cell niche might regulate proliferation, differentiation and chemotherapeutic resistance in HCC; ii) to determine the relationship between changes in liver stiffness and hepatic progenitor cell (HPC) response in rodent models of chronic liver disease; and iii) to determine whether changes in the stiffness of the HPC niche regulate proliferation and differentiation in these cells. A secondary aim of the thesis was to characterise the pattern of histological changes observed in rodent models of chronic hepatic congestion and whether this might provide insight into the effect of oedema and congestion on the development of liver fibrosis. Methods: Cell culture experiments in HCC (Huh7/ HepG2) and HPC cell lines were performed using a system of ligand-coated polyacrylamide (PA) gel supports of variable stiffness. The stiffness of the PA supports (expressed as shear modulus) was altered across a physiological change (1-12kPa) corresponding to values encountered in normal and fibrotic livers. Thiacetamide and carbon tetrachloride (CCl4) models of liver fibrosis were used to investigate the relationship between increasing liver fibrosis, changes in matrix stiffness and HPC response. The pattern of histological changes in the liver in response to hepatic congestion was assessed in two unrelated murine models of dilated cardiomyopathy; the python and CREB S133A mice. Results: Increases in matrix stiffness, as would be encountered in liver fibrosis, promote HCC cell proliferation. Increasing matrix stiffness is associated with enhanced basal and hepatocyte growth factor-mediated signalling though ERK, PKB/ Akt and STAT3. Stiffness-dependent HCC cell proliferation is modulated by β1-integrin and focal adhesion kinase. Increasing matrix stiffness is associated with a reduction in chemotherapy-induced apoptosis in HCC cells. However, following chemotherapy there was an increase in the frequency of clone-initiating cells for cells maintained in a low stiffness environment. Flow cytometry in HepG2 cells demonstrated that culture in a low stiffness environment was associated with an increase in the frequency of the stem cell markers CD44, CD133 and CXCR-4. This effect was further enhanced in the presence of chemotherapy. There is a close association between HPC numbers and liver stiffness measurements in a rat CCl4 model of chronic liver fibrosis. The major expansion in HPC numbers in this model coincides with a similarly large increase in fibrous tissue deposition. In vitro experiments using PA supports demonstrate that increasing matrix stiffness promotes the proliferation of both primary murine HPCs and an immortalised HPC line (BMOL). Changes in matrix stiffness regulate the expression of hepatocyte and biliary markers in BMOL cells. Histological studies in both the Python and CREB S133A models reveal findings consistent with acute on chronic cardiac hepatopathy (ischaemic hepatitis). Features of chronic passive congestion and centrilobular necrosis are present concurrently and develop rapidly. Bridging fibrosis and cirrhosis are not present. Conclusions: Physiologically-relevant changes in matrix stiffness regulate proliferation, differentiation, chemotherapeutic-resistance and stem cell marker expression in HCC cells. Similarly, increases in matrix stiffness are closely correlated to HPC response in vivo and regulate HPC proliferation and differentiation in vitro.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine degradation pathway. The defective fumarylacetoacetate hydrolase enzyme causes the accumulation of upstream metabolites such as fumarylacetoacetate (FAA), maleylacetoacetate (MAA), succinylacetone (SA) and p-hydroxyphenylpyruvic acid (pHPPA). In vitro and in vivo studies showed that the accumulation of these metabolites are detrimental to cell homeostasis, by inducing cell cycle arrest, apoptosis, and endoplasmic reticulum stress, depleting GSH, inhibiting DNA ligase, causing chromosomal instability, etc. For in vivo studies different models of HT1 were developed. Most notably was the fah deficient mouse, whose neonatally lethal phenotype is rescued by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Although, this model most closely resembles the human phenotype with elevated tyrosine levels and the development of hepatocellular carcinoma (HCC), the model is not human genome based. Both the in vitro and in vivo studies suggested that DNA repair is affected in HT1. However, it is not yet clear which DNA repair mechanisms are affected and if only protein functionality is affected, or if expression of DNA repair proteins are also affected. Characteristic of HT1 is the high prevalence of HCC and the presence of liver mosaicism. The liver mosaicism observed in HT1 patients are the result of reversion of the inherited mutation to wild-type. The general consensus is that the reversion is the result of a true back mutation. However, the mechanism underlying the back mutation is still unresolved. It was suggested that cancer develops either through a chromosomal instability mutator phenotype, a microsatellite instability mutator phenotype, or a point mutation instability mutator phenotype. In HT1 only chromosomal instability was reported. The aims of this study were to contribute to the understanding of the molecular basis of the genetic mosaicism in hereditary tyrosinemia type 1. More specifically, determine whether baseand nucleotide DNA repair mechanisms are affected and to what extent, and to determine if microsatellite instability is found in HT1. To achieve these aims, a parallel approach was followed: i.e. to develop a HT1 hepatic cell model and to use HT1 related models and HT1 patient material. To assess the molecular basis of the genetic mosaicism in HT1, the comet assay, gene expression assays, microsatellite instability assays, high resolution melting and dideoxy sequencing techniques were employed. Results from the comet assay showed that the HT1 accumulating metabolites, SA and pHPPA, decreased the capacity of cells for base- and nucleotide excision repair. Gene expression assays showed that short term exposure to SA and/or pHPPA do not affect expression of hOGG1 or ERCC1. The expression of these genes were, however, low in HT1 patient samples. Microsatellite instability assays showed allelic imbalance on chromosome 7 of the mouse genome, and microsatellite instability in the lymphocytes of HT1 patients. Although high resolution melt and sequencing results did not reveal any de novo mutations in fah or hprt1, the appearance of de novo mutations on other parts of the genome can not be ruled out. To conclude, results presented in this thesis, for the first time show that in HT1 the initiating proteins of the base- and nucleotide repair mechanisms are affected, the gene expression of DNA repair proteins are low, and microsatellite instability is found in HT1. By contributing to the elucidation of the mechanism underlying the development of HT1-associated HCC, and providing evidence for the development of a mutator phenotype, the results presented in this thesis contributes to the understanding of the molecular mechanisms underlying the genetic mosaicism in HT1. In addition to these contributions, a hypothesis is posited, which suggests that a point mutation instability (PIN) mutator phenotype is the mechanism underlying the mutation reversions seen in HT1. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

HuR est une protéine liant l’ARN, qui contrôle l’expression des gènes au niveau post-transcriptionnel. Dans le cytoplasme, HuR module la stabilité et la capacité de traduction des ARNm sur lesquels elle se fixe. Nos résultats montrent que HuR est surexprimée dans le carcinome hépatocellulaire (CHC) humain et dans des lignées de CHC en culture. HuR est anormalement retrouvée dans le cytoplasme des cellules hépatiques tumorales, et participe à leur prolifération. En combinant l’analyse globale des gènes régulés par l’extinction d’HuR, celle des ARNm liés à HuR et celle du transcriptome des CHC humains, nous avons identifié 2 gènes dont l’expression est régulée par HuR. Ces gènes sont sous-exprimés dans les tissus de CHC et participent à la mise en place du phénotype cancéreux (résistance à l’apoptose, prolifération cellulaire, invasion,...). / HuR is a RNA binding protein that controls gene expression at post-transcriptional level. In the cytoplasm, HuR modulates the stability and capacity of mRNA translation upon which it binds. Our results show that HuR is overexpressed in hepatocellular carcinoma (HCC) and in human HCC cell lines in culture. HuR is abnormally found in the cytoplasm of liver tumor cells, and contribute to their proliferation. By combining the global analysis of genes regulated by the extinction of HuR, the mRNAs associated with HuR and the transcriptome of human HCC, we identified two genes whose expression is regulated by HuR. These genes are under-expressed in HCC tissues and participate in the development of cancerous phenotype (resistance to apoptosis, cell proliferation, invasion ,...).

La protéine liant les ARNs HuR/ELAVL1 est impliquée dans la stabilisation d’ARNm dont la région 3’UTR présente des motifs riches en A et U (« AU-rich element », ARE). Ces ARNm codent majoritairement des protéines dont la dérégulation favorise l’émergence de cancers (oncogènes, cyclines, facteurs de croissance…). Notamment, HuR est surexprimée dans le carcinome hépatocellulaire (CHC) humain et dans des lignées de CHC en culture et est anormalement retrouvée dans le cytoplasme des cellules hépatiques tumorales participant à leur prolifération. De plus, les modifications post-traductionnelles des protéines liant les ARNs modulent leur localisation subcellulaire ainsi que leur capacité à lier et contrôler le devenir de leurs cibles. Plusieurs serine/thréonine kinases identifiées sont capables de moduler la fonction de l’AUBP (« AU-binding protein ») HuR. Via des études in vitro, nous avons identifié la Y200 comme étant une cible (probablement la seule) de la « tyrosine kinase non-récepteur » Abelson sur HuR. Cette tyrosine kinase est également surexprimée dans le CHC humain et dans des lignées de CHC en culture (comme les cellules HuH7). L’inhibition d’Abl par le Nilotinib influence le profil acido-basique de la protéine HuR, en gel bidimensionnel, dans les cellules HuH7, suggérant un rôle d’Abl dans les fonctions d’HuR sur ses cibles ARNm. / The RNA binding proteins (RBPs) HuR/ELAVL1 is involved in the stabilization of mRNAs whose 3'UTR contains motifs enriched in A and U (« AU-rich element », ARE). These mRNAs encode mainly proteins whose deregulation promotes the emergence of cancer (oncogenes, cyclins, growth factors...). In particular, HuR is overexpressed in hepatocellular carcinoma (HCC) and in human HCC cell lines in culture and is abnormally found in the cytoplasm of liver tumor cells contributing to their proliferation. Furthermore, the Posttranslational modifications of RNA binding proteins (RBPs) modulate their subcellular localization and their ability to bind and control the fate of their targeted mRNAs. Some sérine/thréonine identified kinase are capable of modulating the function of the AUBP (« AU-binding protein ») HuR. By in vitro studies, we identified Y200 as a target (probably the only one) of the « non-receptor tyrosine kinase » Abelson on HuR. This kinase is also overexpressed in hepatocellular carcinoma (HCC) and in human HCC cell lines in culture (as cells HuH7). The inhibition of Abl by Nilotinib (one inhibitor) influences the acido-basic profile of the protein HuR, on Gel 2D, in cells HuH7, suggesting a role of Abl in the functions of HuR on its targets ARNm.

Le carcinome hépatocellulaire (CHC) est un cancer de très mauvais pronostic, disposant de peu d’options thérapeutiques. Il est urgent de développer de nouvelles stratégies thérapeutiques. Notre équipe a montré antérieurement que l’expression du récepteur Frizzled-7 (FZD7) dans le CHC jouait un rôle dans le contrôle du phénotype cancéreux. Le but de ma thèse était de développer une stratégie pour inhiber le signal FZD7 et d’étudier l’effet anti-tumoral potentiel d’une telle inhibition. A l’aide d’une approche peptidique inhibitrice de la liaison du récepteur avec son adaptateur cytoplasmique (Dishevelled), j’ai pu démontrer que l’inhibition du signal FZD7 exerce un effet anti-tumoral sur des lignées cellulaires de CHC, mais également in vivo, dans un modèle murin transgénique de CHC. J’ai élucidé une partie des mécanismes de cet effet anti-tumoral en étudiant l’impact du peptide sur les voies qui sont potentiellement activées par FZD7 dans des lignées cellulaires de CHC. Cette approche peptidique m’a permis de valider l’inhibition de FZD7 comme cible thérapeutique pour le CHC. Parallèlement, nous avons développé une collaboration avec l’entreprise IMAXIO qui a mis au point un test double-hybride permettant de cribler des molécules inhibitrices d’une interaction. Nous avons identifié 8 molécules chimiques qui inhibent l’interaction FZD7/Dishevelled. La poursuite de ce projet va permettre d’identifier, parmi ces 8 molécules, celles qui ont un potentiel anti-tumoral comparable à l’approche peptidique que j’ai développée, grâce aux modèles cellulaires et murins disponibles au laboratoire / Hepatocellular carcinoma (HCC) is a very bad prognostic cancer, with few therapeutic options. The development of new therapeutic strategies is an emergency. In previous studies, our team showed that overexpression of Frizzled-7 receptor (FZD7) in HCC plays a role in the control of cancer phenotype. The aim of my thesis was to develop a strategy to inhibit the FZD7 signal and study the potential antitumor effect of such an inhibition. I used a small-peptide approach to inhibit the binding between FZD7 and its cytoplasmic adaptator, Dishevelled, and showed that the inhibition of FZD7 signal display antitumor effects in vitro on HCC cell lines, as well as in vivo, in a murine transgenic model of HCC. I explored the molecular mechanisms of this antitumor effect on a HCC cell line. Thanks to this small-peptide approach, I validated the inhibition of FZD7 signal as a target for HCC therapy. We also developed a two-hybrid high throughput screening with IMAXIO (Lyon) to identify chemicals able to inhibit the FZD/Dvl interaction and we identified 8 compounds. Prospects of this study are to test the potential anti-tumor effect of these compounds on HCC cell lines and HCC in vivo models.

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.