Le foie est un organe essentiel à la vie chez tous les mammifères. C’est un organe central du métabolisme énergétique et de la détoxification des substances xénobiotiques auxquelles l’individu est exposé. Le foie est la cible d’agressions diverses, telles que les virus, l’alcool, les substances chimiques présentes dans l’alimentation ou l’environnement. Il peut également subir destransformations pathologiques profondes, lors du diabète ou de l’obésité par exemple.La stéatose hépatique, caractérisée par une accumulation de triglycérides sous forme de vésiculesgénérant une réponse inflammatoire, est connue depuis de nombreuses années. Son étude a permisde définir un modèle en deux étapes (« two hits ») indispensables à la genèse d’une stéatohépatite ou NASH. La première est l’accumulation de lipides, la seconde consiste en la genèse d’un stress oxydant et la libération de cytokines. La NASH est une des conséquences pathologiques du syndrome métabolique au cours duquel une résistance des tissus à l’insuline se développe.Récemment, la composition des lipides accumulés dans la NASH a été décrite et montre la présence de cholestérol libre et de différents métabolites des acides gras dont la toxicité est grande mais variable. De façon surprenante, une nouvelle hypothèse tend à émerger quant aux rôles protecteurs de certaines catégories de lipides. En effet, le stockage des triglycérides sous forme de vésicules pourrait être un mécanisme de survie cellulaire (Neuschwander-Tetri, 2010). Il s’agirait principalement d’une tolérance à la mort cellulaire par nécrose ou apoptose. Dans ce contexte,l’activation de l’autophagie serait capitale et la nécrose ne serait plus un mécanisme non contrôlé,mais au contraire un système finement régulé.Des données expérimentales récentes suggèrent l’existence d’un réseau complexe d’interactions moléculaires qui lient, dans la NASH comme dans le cas de la cancérogenèse, le métabolisme énergétique, la réponse inflammatoire systémique et tissulaire et des altérations subcellulaires, telles que les lésions des mitochondries et du réticulum endoplasmique.Nous avons utilisé le cas particulier du préconditionnement ischémique, une technique chirurgicale qui consiste, grâce à de courtes périodes d’occlusion vasculaire avant l’ischémie, à conférer au tissu une protection contre les lésions d’ischémie/reperfusion (I/R), pour étudier les mécanismes de survie mis en place par les hépatocytes stéatosiques au cours d’un stress d’I/R. Dans deux contextes différents, celui d’une ischémie chaude au cours d’une hépatectomie partielle et celui d’une ischémie froide au cours de la transplantation hépatique, nous avons montré que l’autophagie peut jouer un rôle central dans la protection des hépatocytes stéatosiques. Cependant, il est envisageable qu’un dysfonctionnement de l’autophagie pourrait conduire à la genèse d’altérations cellulaires comme une instabilité génomique, caractéristique de la transformation cancéreuse. L’équilibre entre la survie et la mort cellulaire dépend donc de l’intégration de cette signalisation complexe, qui concerne l’état énergétique de la cellule, la réponse aux stress transitoires et l’adaptation aux stress chroniques. Dans ce contexte, l’autophagie semble jouer un rôle central dans l’intégration de la réponse aux stress (Kroemer et al 2010), ce qui pourrait favoriser directement ou indirectement la transformation cancéreuse d’une cellule.L’amélioration de la compréhension des mécanismes impliqués dans la tolérance et la survie des hépatocytes chargés de lipides en réponse à un stress inflammatoire, ischémique ou du réticulum endoplasmique semble donc essentielle. Elle permettrait en effet la mise en place de nouvelles stratégies thérapeutiques qui pourraient améliorer la prise en charge des patients, augmenter le nombre de greffons disponibles pour les greffes, et la prévention des risques cancérogènes pour le foie. / Liver is a an essential to life organ in all mammals. It plays a central role in energy and drug metabolism. Liver is constantly challenged by damaging compounds such as viruses, alcohol and chemicals from food intake or from the environment. It can also undergo some deep pathological transformations, e.g. in diabetes or obesity. Liver steatosis has been known for many years, it is defined as an accumulation of triglycerides vesicles generating an inflammatory response in hepatocytes. A « two step hypothesis » has been proposed for the genesis of Non Alcoholic Steatohepatitis (NASH). The first step is the fat accumulation, the second step involves the generation of an oxidative stress and the release ofcytokines. NASH is one of the pathological consequences of metabolic syndrome, when insulin resistance occurs in the tissues.The composition of accumulated fat in NASH has been recently described and reveals the presence of free cholesterol and different fatty acids metabolites with a high but variable toxicity. Surprisingly, a new hypothesis tends to emerge about the protective effects of some types of lipides.Triglyceride storage in vesicles could indeed be a survival mechanism for cells (Neuschwander-Tetri, 2010). It is assumed that it would mainly result in an tolerance to cell death by necrosis orapoptosis. In this context, (activation of) autophagy would play a key-role and necrosis, usually an uncontrolled mechanism, would become accurately regulated. Similarly to oncogenesis, recent experimental data in NASH suggest that energy metabolism,systemic and tissular inflammatory response and subcellular alterations such as impaired mitochondria and ER are connected in a complex network of molecular interactions. Ischemic preconditioning (IP) is a surgical technique consisting of brief periods of vascular occlusion which confer protection against subsequent ischemia/reperfusion via endogenous protective mechanisms. We investigated the survival mechanisms set up by steatotic hepatocytes during I/R, with or without IP. In the following two situations, warm ischemia during partial hepatectomy and cold ischemia during liver transplantation, we pointed out that autophagy can play a central role in steatotic hepatocytes protection. However, an autophagy dysfunction might result in the generation of cellular impairments such as genomic instabilities, typical features of oncogenic transformation. Therefore, the balance between cell survival or death depends on the integration of a complex signaling, taking into account the cellular energetic state, the cell response to transient stress and its adaptation to chronical stress. In that context, autophagy seems to play a central role in the integration of stress response (Kroemer et al Mol Cell 2010), which could promote, directly or indirectly the malignant cell transformation.Therefore, it seems essential to improve the understanding of mechanisms involved in tolerance and survival of lipid-full hepatocytes in response to an inflammatory, ischemic or ER stress. Indeed, this would help developing new therapeutical strategies to improve patients care, increase the number of available grafts for transplants, and prevent cancer risks in liver.

Magnetic resonance imaging is utilized as an important tool in radiation oncology for delineation of healthy and cancerous tissues, and evaluating the functionality of those tissues, structures, and organs. Currently, the clinical imaging protocol at Virginia Commonwealth University includes anatomical imaging for tissue and structure delineation, and to observe treatment induced changes. Diffusion weighted imaging (DWI) is also acquired for calculation of apparent diffusion coefficient (ADC) values to provide quantitative information on tissue diffusivity and microstructure. However, anatomical images and ADC values may not display the true extent of changes in tissue. This work seeks to further utilize the capabilities of MRI and expand its role in treatment response monitoring for liver cancer patients treated with stereotactic body radiotherapy (SBRT). To do so, an imaging protocol and image analysis methodology to evaluate treatment changes on pre- and post-treatment image sets was developed. An extension of DWI, termed intravoxel incoherent motion (IVIM) imaging, was utilized to quantitatively assess levels of perfusion and diffusion within the liver and tumor. Acquisition of high-quality diffusion weighted images of the liver necessitated the development of an MR safe respiratory motion management device, which was designed, constructed and evaluated in this work. An imaging protocol was developed providing anatomical and functional images of the liver, acquired under breath hold, utilizing the respiratory motion management device. An IVIM parameter calculation and texture analysis workflow was developed using MATLAB, and applied to acquired data sets from multiple studies, including past clinical cases, investigator, healthy volunteer, and liver cancer patient . Differences in IVIM and texture analysis parameters were investigated for healthy and diseased tissue, and for select dose regions from pre- and post-treatment imaging sessions. Significant differences, at a voxel level, were found between healthy and diseased tissue, and pre- and post-treatment volumes, for multiple parameters, including apparent diffusion coefficient, pure diffusion, and perfusion, as well as for various texture features. Overall, this study showed the potential of IVIM and texture analysis to be used for discriminating between healthy and diseased tissues in the liver, and for indication of treatment response.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, especially in Asia and Africa countries. The distribution pattern shows geographical variation and pathogesis in multifactors as environment, infection, nutrition, metabolism, and endocrine contribute to hepatocarcinogenesis. Alpha fetal protein (AFP), the major tumor marker used at present accounts only 50% HCC diagnostic efficiency. This study aims to identify potential tumor markers or suppressor genes for further application in early HCC diagnoses and treatments. Therefore we utilized available cDNA microarray databases in conjunction with other bioinformatic resources to identify our candidate genes related to HCCs. cDNA microarray technology and bioinformatic resources which enable investigators to obtain comprehensive data with respect to gene-expression profiles, is progressing rapidly. The Okabe¡¦s and Stanford¡¦s HCC database were our major data-mining material. A total of 85 potential tumor markers and 106 potential tumor suppressors were found via preliminary in silico datamining. We furthermore narrowed down to 14 candidate tumor markers and 7 candidate tumor suppressor genes by the way of quantitative RT-PCR technologies were applied in various HCC cancer cell lines and 21 patient¡¦s in pair, tumor/non-tumor tissues to confirm gene expression profile. The results revealed that 6 genes (PRO2000, PYGB, STMN1, AFM, C8FW, NNMT) conformed to our data-mining studies.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Hepatocarcinogenesis is considered a multifactorial and mulitstep process that involves the activation of oncogenes or the inactivation of tumor suppressor genes. Tumor suppressor gene PTEN (also known as MMAC or TEP1) is located on human chromosome 10q23. The 403¡Vamino acid PTEN protein encodes dual specificity protein phosphatases. Mutation of the PTEN is a common event in advanced stage of diverse human cancers. In our previous studies, immunohistochemical analysis indicated that reduced PTEN expression was found in nearly 40% of HCC specimens. Furthermore, restored PTEN expression by adenovirus gene delivery effectively inhibited the in vitro and in vivo tumorigenicity of Mahlavu cells, a human HCC cell line with PTEN inactivation. In the present study, we further characterize whether PTEN gene delivery still suppressed the oncogenic potential in HCC cell lines with functional PTEN. By expression and sequencing analysis, we identified human SK-Hep-1 cells as the hepatoma cell line with functional PTEN expression. The optimal condition for adenovirus vector to infect SK-Hep-1 cells was determined at the multiplicity of infection (MOI) of 50-100. Tough SK-Hep-1 cells were effectively transduced with exogenous PTEN gene, the enhanced PTEN expression by adenovirus gene delivery did not alter the phosphoryation extent of Akt in SK-Hep1 cells. Nevertheless, PTEN gene delivery reduced the proliferation of SK-Hep-1 cells by ~20%. In addition, the motility of PTEN-transduced SK-Hep-1 cells significantly decreased comparing to cells of control groups. Western blot analysis suggested the decreased cell motility might be attributed to the reduced phosphorylation of focal adhesion kinase (FAK) by PTEN gene delivery. Above all, PTEN gene delivery profoundly reduced the colony formation of SK-Hep-1 cells in soft-agar. However, PTEN gene delivery did not affect the secretion of matrix metallo-proteinases (MMPs) release. Animal studies will be carried out in the future to validate the present in vitro findings. In summary, PTEN gene delivery holds promise for treatment of HCC even when the hepatoma cells possess functional PTEN gene.

PURPOSE: HCC is a complicated disease with high mortality rates and limited treatment options. No universal clinical or molecular classification established to inform better treatment options. There has been very limited success in determining a molecular profile that represent valid drivers in HCC patients and thus no targeted agents have obtained marketing approval. However, emerging data suggest the FGF19-pathway as a HCC driver and a potential therapeutic target. This research study aims to investigate whether the HCC prognostic risk factor, serum AFP, is predictive of FGF19 protein expression as assessed by immunohistochemistry in advanced HCC patients. METHODS: A cross-sectional analysis was performed from baseline data collected in a Phase 1 study conducted at various centers across the US, EU, and Asia. Only advanced HCC patients with adequate liver function were eligible for enrollment. Demographic data, detailed history of HCC, and any prior treatments or surgeries were recorded. Baseline laboratory values and prognostic factors including performance status (ECOG), lab values (i.e. bilirubin, albumin), and the number, size and biomarker status of the tumor(s) were collected. Differences between groups were assessed by t test, or Chi-square test, as appropriate. Multivariate logistic stepwise regression analyses were performed including all parameters with highly significant correlations in the multivariate analysis. RESULTS: Only AFP, metastatic disease, and prior surgery met the criteria to be incorporated into the final model. Results indicated that high AFP had a statistically significant (p-value = .01) positive association (Wald chi-square statistic = 6.601) with positive FGF19 IHC status. The odds ratio for being FGF19 IHC+ was 12.216 among the high AFP subjects as compared to low AFP subjects, and also statistically significant but had a very wide 95% confidence interval (1.811, 82.79). CONCLUSIONS: The results indicated that HCC patients with high serum AFP levels have a twelve fold higher chance of having a positive FGF19 IHC status than those with low AFP levels. Further studies are warranted in order to replicate the data in a larger sample size to understand future clinical implications once treatment options become available for FGF19 IHC positive patients.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.

No description available.

Obesity is tightly linked to the development of liver steatosis which is an important risk factor for the development of metabolic dysfunction associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC). Specifically, obesity increases the risk of developing HCC by over 80% indicating the urgent need to understand and treat the obesity-MASLD-HCC spectrum of diseases. Environmental toxicants including some commonly used pesticides have been linked to the development of obesity, MASLD and HCC however, the mechanisms contributing to these effects are incompletely understood. Mechanistically, defects in fatty acid metabolism including increases in de-novo lipogenesis, mitochondrial dysfunction, and reductions in brown adipose tissue (BAT) metabolic activity are important contributing factors to MASLD and HCC. A key regulator of both liver and BAT metabolism is the AMP-activated protein kinase (AMPK). These data suggest that environmental factors may contribute to obesity, MASLD and HCC by inhibiting AMPK while therapeutic activation of AMPK may exert beneficial effects. In this thesis we find that two commonly used pesticides, chlorpyrifos and deltamethrin, suppressed the metabolic activity of cultured brown adipocytes. Subsequent studies in mice found that chlorpyrifos promoted obesity and liver steatosis and that this was linked to reductions in BAT metabolic activity and AMPK action. In contrast, when deltamethrin was delivered to mice it had no effect on obesity. Lastly, we evaluated the effects of the rheumatoid arthritis drug and AMPK activator Salsalate in mice and found that it reduced MASLD and HCC and enhanced the effectiveness of Lenvatinib, a tyrosine kinase inhibitor used in HCC treatment. This work furthers our understanding of the mechanisms by which commonly used pesticides may promote obesity and MASLD and provides evidence for the potential repurposing of Salsalate as a combinatorial treatment strategy for MASLD driven HCC. / Thesis / Doctor of Philosophy (Medical Science) / Environmental chemicals may affect the body’s metabolic functions and contribute to cancer. In this thesis we studied the effects of two common pesticides chlorpyrifos and deltamethrin, their contribution to obesity and liver disease and tested a new treatment strategy in liver cancer. We found that chlorpyrifos, but not deltamethrin, inhibited brown adipose tissue activity, the body’s metabolic sink which promoted diet induced obesity and fatty liver disease. Next, we studied whether salsalate, a drug with metabolic properties used to treat rheumatoid arthritis, could also be used to combat fat accumulation in the liver and block liver cancer. Salsalate in combination with Lenvatinib, an established cancer therapy for liver cancer, slowed down cancer growth and reduced factors that increase the risk and severity of liver cancer development. This work highlights the need to understand the effects of pesticides on our metabolism and capacity of metabolic strategies in helping combat liver cancer.

Le carcinome hépatocellulaire (CHC) est le principal cancer primitif du foie et est associé à un très mauvais pronostic. Notre équipe a mis en évidence que la Reptine et la Pontine, des AAA+ ATPases homologues, sont surexprimées dans le CHC par rapport au foie non tumoral. Au cours de ce travail de thèse, j’ai contribué à démontrer que l’extinction de la Reptine par l’induction de shRNA suffit à arrêter la croissance de tumeurs déjà établies, et même à induire leur régression dans des xénogreffes chez la souris. Ces résultats encourageants suggèrent que la Reptine pourrait être une cible thérapeutique dans le CHC. L’utilisation de siRNA en thérapeutique n’étant pas envisageable actuellement, il parait plus pertinent de tenter de cibler la Reptine via son activité ATPase. Le principal objectif de ma thèse était donc de déterminer l’implication de l’activité ATPase de la Reptine pour ses propriétés oncogéniques dans le CHC. Nos résultats ont montré que des mutants inactifs de la Reptine (D299N et E300G) ont un effet dominant négatif et ne sont pas capables de complémenter l’absence de la Reptine endogène, ce qui conduit à une diminution significative de la croissance des cellules HuH7 et Hep3B, et à une induction de l’apoptose. Ceci indique que l’activité ATPase de la Reptine est nécessaire pour la croissance et la survie des cellules de CHC. Enfin, grâce à une étude transcriptomique, nous avons identifié de nouveaux gènes dont l’expression est régulée par la Reptine et/ou la Pontine. Parmi ces gènes, certains pourraient être impliqués dans les fonctions oncogéniques de la Reptine et/ou de la Pontine dans le CHC. Finalement, ce travail a permis de mettre en évidence l’implication de l’activité ATPase de la Reptine, et d’apporter des éléments permettant de mieux comprendre le mécanisme d’action de la Reptine dans le CHC. / Hepatocellular carcinoma (HCC) is the main primary cancer of the liver and is often associated with poor prognosis. Our team has demonstrated that Reptin and Pontin, two AA+ ATPases, are overexpressed in HCC compared to normal liver. Moreover this overexpression is also associated with poor prognosis. In the course of my PhD, I demonstrated that shRNA-mediated silencing of Reptin is sufficient to inhibit tumor growth and even can promote their regression in xenografted mice. These encouraging results suggest that Reptin might represent a novel therapeutic target in HCC. As the use of siRNA as therapeutic tools is still debated, the targeting of Reptin enzymatic activity might represent a more relevant approach to impair its functions. To this end I first proposed to determine the involvement of Reptin ATPase activity in HCC oncogenesis. My results show that ATPase inactive Reptin mutants (D299N and E300G) play dominant negative roles toward Reptin functions and are unable to complement for the depletion of endogenous Reptin, thereby leading to a significant decrease of cell growth and to a significant increase of apoptosis in HuH7 and Hep3B cells. These results show that Reptin’s ATPase activity is necessary for HCC cell growth and survival. Moreover, using a transcriptomic approach that compared gene expression upon siRNA-mediated Reptin or Pontin silencing, we identified specific genes whose expression is under the control of those proteins and whose functions might provide mechanistic explanation to Reptin’s involvement in HCC. Collectively, the results obtained during my PhD thesis have characterized the contribution of Reptin ATPase activity to HCC growth and development and might represent a founding step in the understanding of Reptin’s biology in cancer development.

Introduction: Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work has demonstrated pIgR to be upregulated in hepatocellular carcinoma, even though it is not expressed in normal liver cells. High pIgR levels are associated with poor survival and distant metastases for a number of cancers such as nasopharyngeal cancers, lung and oesophageal cancers. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). My aim was to assess pIgR's role in PDAC by interrogating human PDAC tissue samples as well using cell biology experimental tools. Methods: pIgR expression was manipulated (siRNA and shRNA) in cell lines to evaluate its subsequent effect on cell behaviour in 2D assays as well as 3D organotypics models. Tissue Microarrays of patients with PDAC were analysed after pIgR, αSMA, E-Cadherin and Picrosirius Red staining to assess their role as a combined bio-marker panel. Results: Cytokines such as interleukin 4 (IL4) and Tumour Necrosis Factor (TNFα) could not modulate pIgR expression in PDAC cell lines despite this effect being seen in other studies using colorectal and nasopharyngeal cancer cell lines. Downregulation in pIgR expression in Capan1 cell line resulted in reduction of cellular proliferation (n= 3, P < 0.05, Friedman test), adhesion (n= 3, P < 0.05, Kruskal-Wallis) and migration (n= 3, P < 0.05, Kruskal-Wallis). In 3D organotypic models, pIgR downregulation resulted in reduced cancer cell invasion (n= 9, P < 0.05, Kruskal- Wallis) and diminished contraction of gels (n= 9, P < 0.05, Kruskal-Wallis). In human PDAC, decreased E-cadherin expression correlates with increased pIgR expression through pancreatic intra-epithelial neoplasia (PanIN) progression. There was no IgA expression in PDAC. pIgR expression had no clinical correlation with routine prognostic measures such as differentiation, lymph node metastasis (n= 88, P=0.5012, Kruskal-Wallis). Even in combination with stromal indices (α-smooth muscle action (SMA) and Picrosirius red), low pIgR scores had no statistically significant impact on prognosis but had a trend towards better survival (n= 88, P=0.2791, Mann-Whitney U test). Conclusion: pIgR may be involved in progression from pre-neoplastic lesions such as PanIN to PDAC. pIgR may have a biological impact on cellular motility and invasion due to yet to be deciphered signalling cascades with marked effect on cellular phenotype. Careful analysis is required to study the impact of pIgR on prognostic impact bearing in mind the histological sub-types of pancreatic cancer.