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Simple Summary Gene therapy is a novel approach to treat diseases by introducing corrective genetic information into target cells. Adeno-associated virus vectors are the most frequently applied gene delivery tools for in vivo gene therapy and are also studied as part of innovative anticancer strategies. Here, we report on the natural preference of AAV2 vectors for hepatocellular carcinoma (HCC) compared to nonmalignant liver cells in mice and human tissue. This preference in transduction is due to the improved intracellular processing of AAV2 vectors in HCC, resulting in significantly more vector genomes serving as templates for transcription in the cell nucleus. Based on this natural tropism for HCC, novel therapeutic strategies can be designed or existing therapeutic approaches can be strengthened as they currently result in only a minor improvement of the poor prognosis for most liver cancer patients. Abstract Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

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Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). The virally encoded X antigen, HBx, contributes importantly to the development of HCC through its trans-activating role in various signal transduction pathways. Pathways implicated in stem cell self-renewal also contribute to carcinogenesis. Thus, experiments were designed to test if HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HBx expressing (HepG2X) and control (HepG2CAT) human cell lines were assayed for phenotypic and molecular characteristics of "stemness." Western blotting of protein extracts from HepG2X and HepG2CAT cells as well as immunohistochemical staining of HCC and adjacent liver tissue sections from HBV infected patients showed up-regulation of "stemness"-associated (EpCAM and beta-catenin) and "stemness" (Oct-4, Nanog, Klf-4) markers by HBx. Moreover, HBx stimulated cell migration and spheroid formation. HBx expression was also associated with depressed levels of E-cadherin and subsequent activation of beta-catenin and EpCAM. Results from ChIP-chip data performed previously in this lab suggest an associative link between HBx and the expression of epigenetic co-repressor, mSin3A, which is known to repress E-cadherin when complexed with histone deacetylases. Thus, experiments were also designed to test if HBx represses the E-cadherin gene (CDH1) through histone deacetylation by the mSin3A/HDAC complex. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was showed by immunofluorescent staining. Chromatin immunoprecipitation revealed that HBx mediated the recruitment of the mSin3A/HDAC complex to the CDH1 promoter. HDAC inhibition by Trichostatin A treatment restored E-cadherin expression. Thus, HBx-associated epigenetic repression of E-cadherin and up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristic of CSCs. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. / Biology

Chronic infection with hepatitis B virus (HBV) is a primary risk factor for the development of hepatocellular carcinoma (HCC). HCC is the fifth most common cancer type worldwide with few treatment options. The hepatitis B encoded x antigen (HBx) plays a crucial role in the pathogenesis of HCC through several mechanisms. HBx alters signaling pathways shown to promote carcinogenesis and mediates epigenetic changes that silence tumor suppressor genes and activate host oncogenes. Short chain fatty acids (SCFAs) are made by selected gut bacteria with largely anti-inflammatory properties. They alter gene expression by functioning as histone deacetylase inhibitors (HDACi) and can bind to G protein coupled receptors (GCPR) to stimulate signaling pathways. Due to the documented anti-cancer properties of SCFAs, experiments were designed to test the hypothesis that SCFAs delay the development of HCC in HBx transgenic (HBxTg) mice. A diet of SCFAs was fed to HBxTg for three months prior to the expected appearance of dysplastic nodules and HCC. The results showed a statistically significant reduction in the number of dysplastic nodules as well as the presence and frequency of HCC. The effect of SCFAs on tumor growth was also evaluated in nude mice subcutaneously injected with human HCC cells. Tumor size in SCFA-treated mice was statistically smaller compared to the controls. The effect of SCFAs on cell viability of cancer and primary human hepatocytes was evaluated. SCFAs were shown to reduce cell viability in cancer cells only, with no effect on primary hepatocytes. Proteomics was performed on SCFA-treated compared to control livers from HBxTg to investigate changes on the molecular level that are associated with reduced preneoplastic and neoplastic nodule formation. Pathway analysis showed a decrease in important cancer-promoting pathways altered by HBx in HCC, including inflammation, oxidative stress, PI3K, VEGF, EGF, and Ras. These pathways are involved in biological processes central to carcinogenesis such as cell proliferation, survival, and angiogenesis. The ability of SCFAs to decrease these pathways has never been demonstrated. Further investigation confirmed that Ras activity was decreased in 12-month old livers treated with SCFAs. Taken together, these results show that SCFAs are capable of delaying the rate of tumor growth and tumor frequency in two mouse models of HBV-associated HCC, as well as reduce cell viability in cancer cells specifically. This data suggests that SCFAs may be a novel treatment option for HBV-associated HCC. / Biology

Chronic Hepatitis B Virus (HBV) infection is a global health problem because of its connection to acute and chronic liver diseases as well as hepatocellular carcinoma (HCC). There is increasing evidence showing that HBV contributes to HCC due to persistently high levels of trans-activating protein---hepatitis B encoded x antigen (HBxAg). Studies have shown that the HBxAg affects and alters the activity of many different transcription factors and plays an essential role in several cytoplasmic signaling transduction pathways, such as Wnt signaling pathways. One of the upregulated genes, designated URG11, was found transactivated by HBxAg. URG11 could stimulate the ß-catenin promoter and hepatocellular growth and survival which suggest that URG11 may be a regulatory element in the ß-catenin signaling pathways. microRNA148a (miR148a) was identified from two miRNA microarrays as one of the up-regulated miRNAs in cells stably expressing HBxAg or over-expressing URG11. Moreover, the expression of miR148a was also elevated in HBV-mediated HCC patient tissue samples. To study the function of miR148a, HepG2 (hepatoblastoma) and Hep3B (hepatoma) cells stably expressing HBxAg or over-expressing URG11 were transduced by recombinant lentiviruses encoding anti-miR148a. anti-miR148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Further, introduction of anti-miR148a increased PTEN protein and mRNA expression, suggesting that PTEN was suppressed by miR148a. In addition, anti-miR148a blocked the stimulation of Akt signaling, resulting in decreased expression of ß-catenin. Thus, miR148a may play a central role in HBxAg/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. / Biology

Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus. / Ph. D.

Objective: The primary aim of this study was to assess patients’ preferences between radiofrequency ablation (RFA) versus hepatic resection (HR) for the treatment of small hepatocellular carcinomas (HCC). Methods: Decision analysis was performed by using probability trade-off (PTO) technique to elicit patients’ preferences and the strength of their decisions. Results: The vast majority of the study population preferred RFA over HR (70% vs. 30%, p=0.001). Their initial choice changed if 5-year survival benefit after surgery was at least 14% superior to RFA and if the 3-year disease-free survival advantage was at least 13% better than ablation. Conclusions: The results of this study suggest that fully informed cirrhotic patients would prefer RFA if diagnosed with early stage HCC even if able to undergo surgery.

Résumé : La répression fréquente du gène encodant pour le « suppressor of cytokine signaling 1 » (SOCS1) dans le carcinome hépatocellulaire (CHC) et la forte susceptibilité des souris déficientes pour SOCS1 à développer des tumeurs hépatiques expérimentales suggèrent que SOCS joue un rôle de suppresseur de tumeur. Cette notion est supportée par les études impliquant la répression de l’expression de SOCS1 via des évènements épigénétiques ou par les microARN dans plusieurs autres types de cancers. Les mécanismes moléculaires sous-jacents au rôle potentiel de suppresseur de tumeur de SOCS1 dans le foie demeurent à ce jour inconnus. Bien que les récepteurs à activité tyrosine kinase (RTK) sont reconnus pour induire l’expression de l’ARNm de SOCS1, le rôle et les mécanismes par lesquels SOCS1 peut réguler la signalisation des RTK sont incertains. Le RTK MET, qui a pour ligand le facteur de croissance des hépatocytes (HGF), régule plusieurs fonctions cellulaires normales. La dérégulation de la signalisation du récepteur MET joue des rôles importants dans la pathogenèse du CHC. Des études ont démontré que l’activation de MET promeut la prolifération, l’invasion et la migration des cellules cancéreuses du foie ainsi que leur dissémination métastatique. La signalisation aberrante de MET est un trait commun de plusieurs autres cancers et serait à l’origine de l’émergence de la résistance à la chimiothérapie. Dans ce projet, j’ai investigué les mécanismes moléculaires par lesquels SOCS1 régule l’activité du récepteur MET. Mes résultats indiquent que le foie des souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-] se régénère plus rapidement que celui des souris contrôles. Suivant une stimulation au HGF, les hépatocytes issus des souris Socs1[indice supérieur -/-] Ifng[indice supérieur -/-] présentent une augmentation de la signalisation de MET, de la migration et de la prolifération cellulaires. L’expression exogène de SOCS1 dans différentes lignées cellulaires d’hépatocarcinomes humains et murins inhibe la signalisation induite par HGF. De plus, SOCS1 diminue la prolifération, la croissance indépendante de l’anchrage et la migration dans ces lignées de CHC in cellulo et réduit de façon significative leur croissance dans les essais de xénogreffes chez les souris immunodéficientes. Mes résultats suggèrent que l’activation de la signalisation HGF-MET induit la transcription du gène SOCS1, suivi par une interaction physique entre SOCS1 et MET. L’analyse de divers mutants de SOCS1 révèle que cette interaction implique principalement les domaines SH2 et « kinase inhibitory region » (KIR) de SOCS1. L’activité kinasique de MET est requise pour cette interaction puisque l’interaction entre SOCS1 et un mutant kinase-inactif de MET est fortement réduite. SOCS1 est aussi phosphorylé en aval de MET sur quatre résidus tyrosine (Tyr). Quoique ces résidus Tyr représentent théoriquement des sites d’interaction pour des protéines adaptatrices possédant des domaines de liaison aux phospho-Tyr, elles ne semblent pas impliquées dans l’interaction de SOCS1 avec MET. Je démontre également que SOCS1 induit l’ubiquitination de MET via l’élongation de chaînes de polyubiquitine de type K48, conduisant à sa dégradation par le protéasome. Cette modulation négative de MET par SOCS1 dans les cellules CHC survient indépendamment de la voie de dégradation lysosomale de Cbl qui est partagée par plusieurs autres RTK. // Abstract : Frequent repression of the gene coding for the suppressor of cytokine signaling 1 (SOCS1) in hepatocellular carcinoma (HCC) and increased susceptibility of SOCS1 deficient mice to experimental hepatocarcinogenesis suggest a tumor suppressor role for SOCS1. This notion is supported by epigenetic and micro-RNA-mediated blockade of SOCS1 expression in several other cancers. Molecular mechanisms underlying the putative tumor suppressor function of SOCS1 in the liver have not been elucidated yet. Although receptor tyrosine kinases (RTK) can induce SOCS1 mRNA expression, the role and mechanisms of SOCS1 in regulating RTK signaling are not yet clear. c-Met is the RTK for hepatocyte growth factor (HGF) and mediates several normal cellular functions. HGF signaling and MET activation also play important roles in the pathogenesis of HCC. Experimental studies have shown that the activated MET promotes proliferation, invasion and migration of liver cancer cells and enhances metastasis. Aberrant MET signaling is a hallmark of many other cancers and underlies the emergence of chemoresistant clones. In this project, I investigated the molecular mechanisms by which SOCS1 regulates MET RTK activity. My results illustrate that the Socs1[superscript -/-]Ifng[superscript -/-] liver regenerates at a faster rate than the control one. Following HGF stimulation, hepatocytes from Socs1[superscrip -/-]Ifng[superscript -/-] mice display increased MET signaling, cell migration and proliferation. Forced expression of SOCS1 inhibits HGF-induced signaling pathways in different human or murine hepatoma cell lines. Furthermore, SOCS1 also decreases cell proliferation, anchorage-independent growth, and migration of HCC cell lines in cellulo, and results in significant inhibition of their growth as xenografts in immunodeficient mice. My findings show that activation of HGF-MET signaling results in transcriptional activation of SOCS1 gene, followed a physical interaction between SOCS1 and MET. Analysis of various SOCS1 mutants reveals that this interaction is mediated primarily via the SH2 and the kinase inhibitory region (KIR) domain of SOCS1. MET kinase activity is required for this interaction since SOCS1 binding to a kinase-dead MET mutant is dramatically reduced. MET promotes phosphorylation of SOCS1 on four tyrosine (Tyr) residues. Although these Tyr might represent potential binding sites for adaptors containing phospho-Tyr-binding domains, they do not appear to be involved in the interaction of SOCS1 with MET. I also show that SOCS1 induces polyubiquitination of MET via K48-ubiquitin chain elongation leading to its degradation by proteasomes. The SOCS1-mediated downmodulation of MET expression in HCC cells occurs independently of the Cbl-mediated lysosomal degradation pathway shared by many other RTKs. Taken together, my findings show that SOCS1 attenuates HGF-induced cellular functions by targeting the activated MET receptor for proteasomal degradation.

L'insuline, hormone clé du contrôle de l'homéostasie métabolique, exerce également des effets trophiques sur la croissance et la prolifération cellulaire. Des études épidémiologiques ont récemment montré que les individus obèses ou diabétiques de type 2 ont un risque plus élevé de développer des cancers et elles ont également suggéré que l’insuline jouerait un rôle dans ce développement tumoral. Ainsi, une signalisation adéquate en aval du récepteur de l’insuline est indispensable pour éviter des processus physiopathologiques. La signalisation de l’insuline est contrôlée par des mécanismes de rétrocontrôle, dont l’adaptateur moléculaire Grb14 qui agit comme un inhibiteur endogène de l’activité catalytique du RI. L’objectif de ma thèse a été d’étudier les conséquences métaboliques et mitogéniques de l’inhibition de Grb14 in#vivo spécifiquement dans le foie de souris. Dans une première étude, nous montrons que sept jours après l’invalidation de Grb14, les souris présentent une activation des voies de signalisation de l’insuline, qui s’accompagne d’une amélioration de la tolérance au glucose et de la production hépatique de glucose. Cependant, de façon paradoxale, la voie de la lipogenèse est très fortement diminuée. En décryptant le mécanisme moléculaire impliqué, nous montrons que l’inhibition de Grb14 permet la libération de la protéine p62/sqstm1 qui active le facteur de transcription Nrf2, ce qui entraine une inhibition du récepteur nucléaire pro-lipogénique LXR. De façon intéressante, l’invalidation de Grb14 chez des souris ob/ob permet de restaurer la glycémie et la stéatose hépatiques à des valeurs comparables aux témoins. Cette étude a ainsi permis de mettre en évidence une nouvelle voie de régulation de la lipogenèse hépatique. Dans une deuxième étude, nous nous sommes intéressés à l'action mitogénique de l'insuline. Nous montrons que 48 heures après l'inhibition de Grb14, les hépatocytes, qui sont des cellules quiescentes, entrent massivement dans le cycle cellulaire. Ce processus est dépendant de l’expression du RI et est médié par la signalisation PI3K/Akt/mTORC1 et la voie Rb/E2F1. Ces données révèlent ainsi que l'insuline est un puissant facteur mitogène dans le foie et que son action est étroitement contrôlée par l’adaptateur Grb14. D’un point de vue physiopathologique, nous avons pu mettre en évidence une diminution de significative de 58% de l’expression de Grb14 dans une collection de 70 CHC humains, apportant ainsi une explication moléculaire à une action pro-tumorigène de l’insuline dans le foie. L’ensemble de ces deux études permet de placer Grb14 au centre de la régulation des actions métaboliques et mitogéniques de l’insuline dans le foie. / Insulin is a key hormone controling metabolic homeostasis which also exerts having trophic effects on cell growth and proliferation. Epidemiological studies have recently shown that obese and type 2 diabetes patients are at higher risk of developing cancers, suggesting that insulin could be involved in tumor development. Proper signaling downstream the insulin receptor is thus essential to prevent pathophysiological processes. Insulin signaling is controlled by feedback mechanisms including the molecular adapter Grb14 which acts as an endogenous inhibitor of the IR catalytic activity. The aim of my PhD was to investigate the metabolic and mitogenic consequences of liver specific Grb14 inhibition in mouse. In the first study, we showed that after seven days of Grb14 invalidation, liver insulin signaling is enhanced, resulting in improved glucose tolerance and diminished hepatic glucose production. However, paradoxically, lipogenesis was greatly decreased. Deciphering the molecular mechanism, we show that Grb14 inhibition leads to the release of its partner p62/SQSTM1, inducing the activation of the Nrf2 transcription factor, which ultimatly inhibited the pro-lipogenic LXR nuclear receptor. Interestingly, Grb14 invalidation in ob/ob mice can restore blood glucose and hepatic steatosis comparable to control values. The study thus highlighted a new pathway controlling lipogenesis that could be targetted to improve metabolic diseases. In the second study, we were interested in insulin mitogenic action. We showed that 48 hours after Grb14 inhibition, hepatocytes that are quiescent cells, massively go through one cell cycle. This process depend on IR expression and is mediated by the PI3K/Akt/mTORC1 pathway and the Rb/E2F1 complex. Our data thus suggest that insulin is a potent mitogenic factor in the liver whose action is closely controlled by the Grb14 adapter in physiological conditions. Importantly, Grb14 expression is significantly decreased in a collection of human HCC, hence bringing out a molecular basis for a pro-tumorigenic action of hyperinsulinemia. Together these two studies reveal that Grb14 is a crucial gatekeeper of insulin metabolic and mitogenic actions in the liver.