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.

Mutação 249Ser no TP53 no Carcinoma Hepatocelular (CHC), frequente em países da África e Ásia, é uma evidência molecular de exposição à aflatoxina. O objetivo deste estudo é analisar a freqüência de 249Ser em 74 amostras de CHC no Brasil. A mutação foi analisada por RFLP e sequenciamento. A presença de vírus da hepatite B (VHB) foi analisada por PCR em tempo real. 249Ser foi encontrada em 13/74 (28%) e VHB em 13/74 (16%). A mutação foi encontrada em maior freqüência em tumores indiferenciados (OR = 2,415, IC = 1,001 - 5,824). O tamanho médio de tumores com 249Ser foi de 9,4 cm contra 5,5 cm de amostras sem a mutação (p=0,012). Não foi encontrada relação entre VHB e 249Ser. Os resultados indicam que 249Ser é um fator importante na carcinogênese do CHC no Brasil sendo associada à uma forma maior e menos diferenciada de tumor. / TP53 249Ser mutation has been proved a molecular evidence for aflatoxin-related Hepatocellular Carcinoma (HCC) and is frequent in Africa and Asia. The aim of our study was to analyze the frequency of 249Ser mutation in HCC from Brazil. We studied 74 samples of paraffin embedded HCC. 249Ser mutation was analyzed by RFLP and sequencing. Presence of HBV DNA was determined by Real-Time PCR. 249Ser was found in 21/74 (28%) samples while HBV DNA was found in 13/74 (16%). Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, IC = 1.001 - 5.824). The mean tumor size of 249Ser HCC was 9.4 cm versus 5.5cm on wild type (p=0.012). HBV DNA was not related with 249Ser. Results indicate that 249Ser is an important factor of HCC carcinogenesis in Brazil and is associated with large and poorly differentiated tumors.

Staphylococcal nuclease and tudor-domain containing 1 (SND1) is an oncogene for a wide variety of cancers, including hepatocellular carcinoma (HCC). SND1 is a multifunctional protein regulating gene expression of proto-oncogenes and tumor suppressor genes, making SND1 a prime target for developing cancer therapeutics. This notion is especially attributed to HCC as most patients are diagnosed in advanced stages and the therapeutic options available for these patients are severely limited. In this study, we evaluated the therapeutic potential of a replication-defective adenovirus vector delivering SND1 shRNA (Ad.SND1sh) to human HCC cell lines, HepG3, HuH-7, and Hep3B. Adenovirus infection in HCC cells was confirmed by Western blotting and immunofluorescence. The efficacy of Ad.SND1sh to knockdown SND1 expression was confirmed via Western blot, qRT-PCR, and immunofluorescence. Ad.SND1sh did not significantly affect proliferation of the three human HCC cells but significantly inhibited their invasive and migratory capacities, as determined by wound healing and Matrigel invasion assays, respectively. As a corollary, Ad.SND1sh treatment resulted in a decrease in mesenchymal markers, such as N-cadherin, Twist, Snail, and Slug, without affecting levels of epithelial marker E-Cadherin, indicating that SND1 knockdown induces mesenchymal conversion in HCC cells. Additionally, reductions in liver cancer stem cell marker CD133 and HCC marker α-fetoprotein (AFP) were observed with SND1 knockdown. HCC cells with aberrant expression of these markers are associated with tumor initiation, recurrence, and multi-drug resistance. Our findings indicate that Ad.SND1sh may potentially be an effective therapy for advanced HCC and needs to be studied further for its clinical application.

Liver cancer is the fourth leading cause of cancer-associated deaths globally, and among primary liver cancers, hepatocellular carcinoma (HCC) encompasses 75-85% of all cases. HCC is a highly lethal disease due to limited treatment options – only a small subset of patients qualify for surgical resection or transplantation; the remaining patients often display resistance to radiation therapy or chemotherapy. Overexpression of the oncogene astrocyte elevated gene-1 (AEG-1) is associated with poorer survival and increased tumor recurrence in HCC, and numerous studies show its role in initiation of hepatocarcinogenesis. A prior study also demonstrated AEG-1 expression inhibits senescence by diminishing the ATM/Chk1/Chk2/p53/p21 DNA damage response (DDR) pathway. The aim of this study is to understand if AEG-1 expression promotes radioresistance in HCC. A CRISPR/Cas9 plasmid system was used to delete AEG-1 in the QGY-7703, HuH7 and DihXY cell lines, which model HCC. The cell lines were then treated with ionizing radiation (IR). We find that knockout of AEG-1 in these cell lines induces sensitivity to IR at 2.5 Gy. In response to radiation, AEG-1 wildtype cells more profoundly upregulate ATR, Chk1, and Chk2 signaling; and also more rapidly induce γH2AX, ATM, and BRCA1 signaling, which sense dsDNA breaks to initiate homologous recombination repair. We conclude that AEG-1 expression protects HCC cells from IR through two mechanisms: 1) rapidly initiating the DNA damage response; and 2) increasing replication fork stabilization. These findings indicate AEG-1 can be a therapeutic target in combination with radiation treatment to improve outcomes for HCC patients who demonstrate radioresistance.

acase@tulane.edu

MiR-17-92 is an oncogenic miRNA cluster implicated in the development of several human cancers; however, it remains unknown whether miR-17-92 cluster is able to regulate hepatobiliary carcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of miR-17-92 cluster in primary liver cancer.<br>In-situ hybridization and qRT-PCR analysis showed that miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared to the non-neoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation and invasiveness, in vitro. Overexpression of miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in SCID hairless outbred mice. The tumor suppressor PTEN was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells. Accordingly, overexpression of PTEN open reading frame protein (devoid of 3’UTR) prevented miR-92a- or miR-19a-induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings in this study disclose a novel IL-6/Stat3 miR-17-92 cluster PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.<br>We also found the miR-17-92 is highly expressed in tumor tissue compared to non-tumor adjacent tissue in hepatocellular carcinoma patient tissue. Forced overexpression of the miR-17-92 cluster in cultured human hepatocellular carcinoma cells enhanced tumor growth in vitro; on contrast, inhibition of miR-17-92 cluster inhibited cell growth. MiR-17-92 cluster promote diethylnitrosamine-induced hepatocarcinogenesis in liver-specific miR-17-92 cluster transgenic mice. Binding sequence and mice whole genome microarray analysis revealed about 300 possible targets. RNA-sequencing data analysis showed both individual miRNAs and the host gene of miR-17-92 cluster was highly expressed in hepatocellular carcinoma patients and had negative correlation with several genes (CREBL2, PRRG1, and NTN4), among which, CREBL2 may play an important role in the hepatocarcinogenesis. / acase@tulane.edu

Woodchucks are the preferred animal model to study chronic viral hepatitis and the development of hepatocellular carcinoma (HCC), which occurs as a result of infection with woodchuck hepatitis virus. Significant elevations in the phosphomonoester peak in 31P-MRS spectrum correlated to the presence of HCC. Ex vivo 31P-NMR determined that HCC tissue had significantly elevated concentrations of PC compared to uninfected control tissues, confirming that PME is specific to the tumour’s growth. Finally, a recombinant vaccinia virus was constructed to stimulate the immune systems of infected woodchucks against cells expressing core antigens. Despite reductions in surface antigen expression and viral load, elevations in serum GGT and the PME in 31P-MRS indicated that there was tumour growth in treated woodchucks. In conclusion, the PME peak represents a potential biomarker of cancerous growth when used in conjunction with serological tests to detect HCC in the liver due to chronic hepatitis virus infection. / May 2009

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of liver carcinogenesis. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of Huh-7 hepatoma cell line. HDGF has growth stimulating activity for various types of cells. Recent evidence indicates that HDGF upregulation is associated with poor survival outcome and tumor progression in HCC, non-small cell lung carcinoma and melanoma. However, the exact function and molecular mechanism of HDGF overexpression during HCC progression remain largely unknown. In the first project (Chapter 2) of this thesis study, we started with characterizing in HDGF release and response to exogenous HDGF between benign HepG2 and malignant SK-Hep-1 hepatoma cells. It was found that serum deprivation significantly stimulated the HDGF secretion in SK-Hep-1 cells but not HepG2 cells. Interestingly, SK-Hep-1 cells did not increase the secretion of vascular endothelial growth factor (VEGF), a potent angiogenic factor, during serum deprivation. Besides, SK-Hep-1 cells were more responsive to the growth- and migration-promoting effect of exogenous HDGF. We also validated the angiogenic functions of recombinant HDGF protein in vitro and in vivo. In the second project (Chapter 3), we investigated the influence of cellular HDGF level on the neoplastic potential of hepatoma cells. Adenovirus vectors encoding HDGF, Ad-HDGF, and antisense HDGF, Ad-HDGF (-), were generated to modulate the cellular HDGF levels in SK-Hep-1 cells. Adenovirus-mediated HDGF gene delivery increased the HDGF expression and release, and stimulated the proliferation, migration and anchorage-independent growth of SK-Hep-1 cells. In contrast, infection with Ad-HDGF (-) reduced the HDGF expression and secretion, and attenuated the oncogenic behaviors of SK-Hep-1 cells. Implanting HDGF-overexpressing SK-Hep-1 cells led to the accelerated growth of xenografted hepatoma in SCID mice while implantation of HDGF-downregulated SK-Hep-1 cells caused retarded tumor growth. Histological analysis revealed the increased proliferation and neovascularization in HDGF-overexpressing tumors. This could be attributed to elevated VEGF expression and activation of the nuclear factor kappa B (NF£eB) activities by HDGF upregulation in SK-Hep-1 cells. In the third project (Chapter 4), we delineated the mechanism underlying HDGF-induced VEGF secretion and activation of NFB pathway in SK-Hep-1 cells. Adding recombinant HDGF protein enhanced the VEGF release by SK-Hep-1 cells particularly during serum starvation. This was associated with a concomitant increment in VEGF protein and mRNA levels in SK-Hep-1 cells. Like many mitogens, HDGF increased the production of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide in a dose-dependent manner. Pretreatment with antioxidants abolished the HDGF-induced VEGF secretion. NF£eB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase¡V2 (COX-2). Application of HDGF stimulated NF£eB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NF£eB (p65) by HDGF. HDGF treatment also elevated the COX-2 protein levels and activities in SK-Hep-1 cells. In addition, blockade of COX-2 by NS-398 attenuated the HDGF-induced VEGF secretion, suggesting the involvement of COX-2. Finally, it was found that HDGF stimulated the phosphorylation of Akt, Erk1/2, and p38 MAPK. Inhibition of Akt by LY294002 also diminished the HDGF-induced VEGF secretion. These studies suggest that HDGF induces oxidative stress to activate NF£eB/COX-2/Akt pathway, thereby stimulating VEGF expression and release. In summary, this thesis study brings functional and mechanistic insights on how aberrant HDGF expression contributes to angiogenesis and tumorigenesis during liver carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most common and devastating malignant tumors in Taiwan. Due to an imbalanced between reactive oxygen species (ROS) production and detoxification, oxidative stress, has been implicated in liver carcinogenesis. Superoxide dismutases (SODS) play a key role in the detoxification of superoxide radicals and thus protect cells from damage induced by free radicals. Manganese superoxide dismutase (MnSOD or SOD2) is a member of the superoxide dismutase family located in mitochondria. SOD2 transforms toxic superoxide, a byproduct of the mitochondrial electron transport chain, into hydrogen peroxide and diatomic oxygen. Though reduced SOD2 protein level and activities have been reported in hepatoma tissues, it remains unclear how SOD2 expression affected the tumorigenic processes of hepatoma cells. Expression analysis of an array of human HCC cell lines revealed that SOD2 were down-regulated in poorly differentiated SK-Hep-1 hepatoma cells. Moreover, SOD2 is downregulated in 68.8% of resected HCC samples (97 out of 141 cases). Adenovirus-mediated SOD2 gene delivery increased the cellular SOD2 protein level and H2O2 production, but reduced the superoxide anion level in SK-Hep-1 cells. Furthermore, SOD2 restoration significantly reduced the proliferation, motility, and colony formation of SK-Hep-1 cells. In vivo animal model, the finding of SOD2 overexpression inhibited the proliferation of Sk-Hep-1 hepatoma cells while reduced the tumor growth in mice. Flow cytometry analysis showed that SOD2 gene transfer inhibited the growth of hepatoma cells through induction of cell cycle arrest at G2/M phase. This was associated with declined cdc2/cdk1 and cyclin B1 expression and upregulation p21Cip1 by SOD2 gene delivery. However, SOD2 overexpression had no effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9.In conclusion, SOD2 overexpression suppresses the tumorigenicity of hepatoma cells and may hold promise for HCC treatment.