Dissertations / Theses on the topic 'Hepatocellular carcinoma'

The efficacy of current hepatocellular carcinoma (HCC) surveillance has been questioned, with some guidelines removing alpha-fetoprotein (AFP) from screening recommendations and relying on liver ultrasound alone. A prospectively enrolled Australian HCC surveillance cohort showed a combined surveillance method of ultrasound with AFP is more efficacious than ultrasound alone in HCC detection. Survival analyses showed having Asian ethnicity conferred a protective effect on HCC survival. Antiviral therapy improved HCC survival in hepatitis C. Extracellular vesicles (EV) have been shown to be mediators and effectors of cancer gene pathways. In this thesis EV were phenotyped with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. TEM qualified the ultrastructure of EV. NTA and flow cytometry demonstrated EV concentration to be increased in hepatitis and HCC. Flow cytometry demonstrated hepatocyte-specific EV concentration to be increased in HCC. EV can carry short segments of RNA including miRNA. In our studies differential expression of plasma EV miRNA was found in HCC compared to cirrhosis and hepatitis. Gene set enrichment analyses of miRNA expression profiles were performed using nCounter and OpenArray, with the finding of more than 80% concordance in cancer gene pathways including p53, epithelial mesenchymal transition, TNF- signalling via NF-, TGF-, kRAS, PI3K/AKT/mTOR, IL-6 JAK/STAT, and Wnt/-catenin. In conclusion, traditional HCC surveillance methods are efficacious. EV and its miRNA profiling may provide a sensitive surveillance, diagnosis and prognostication tool for HCC. nCounter and OpenArray technologies showed differential miRNA expression profiles in HCC plasma EV with enriched gene pathways corresponding to genomic studies in HCC tissue. These findings support future investigations into the utility of plasma EV as a circulating biomarker of HCC.

Hepatocellular carcinoma (HCC) is an often fatal condition due to late diagnosis, resistance to existing anticancer agents, as well as underlying liver disease that can limit the use of hepatotoxic chemotherapy. Proprotein convertases (PCs) are serine proteases that convert a variety of growth factors, cell surface glycoproteins, receptors and metalloproteinases into their active forms, thus regulating the biological activity of these proteins. PCs have been found to be upregulated in various malignancies. Growth factors implicated in HCC, such as IGF-1, HGF, VEGF and PDGF, have all been shown to be converted into their active forms by PCs. In this study, I explored the hypothesis that expression of proprotein convertases, specifically PCSK9, furin and PC5, is elevated in HCC. This was evaluated through construction of a Tissue Microarray and staining for these proteins. We found that PCSK9 expression was significantly downregulated in HCC tumours associated with poorer survival. PCSK9 is upregulated in the context of liver regeneration and has been involved in cholesterol metabolism, with development of monoclonal antibodies against PCSK9 to treat hypercholesterolemia. Its altered expression in aggressive HCC tumours potentially indicates that HCC is able to modulate its local microenvironment to enable a constant energy supply. There has recently been a move in oncology research to study suppression of suppress tumour growth by modifying energy supply and metabolism (for eg, metformin in prostate and breast cancer). Further confirmation at the mRNA level is required to confirm the altered expression of PCSK9, however this appears to be a promising finding and potential chemotherapeutic target.
Contexte et hypothèses: Le carcinome hépatocellulaire (CHC) est le 5e cancer le plus courant dans le monde entier et la 3ème cause de décès par cancer dans le monde entier, avec une survie médiane à 5 ans de 8,9%. La reconnaissance tardive en raison du manque de biomarqueurs pour détecter la maladie résécable, une résistance aux agents anticancéreux, ainsi qu'une maladie du foie sous-jacente limitant l'utilisation de chimiothérapie hépatotoxique sont des facteurs qui diminuent le taux de survie. Les proprotéines convertases (PCs) sont des sérine-protéases qui convertissent une variété de facteurs de croissance, glycoprotéines de surface cellulaire, les récepteurs, et les métalloprotéinases à leurs formes actives, contrôlant ainsi l'activité biologique de ces protéines. On a démontré l'expression augmentée de PCs dans de diverses tumeurs malignes. On a prouvé que les facteurs de croissance impliqués dans le CHC, tels que l'IGF-1, HGF, VEGF et PDGF, sont convertis à leurs forme actives par les PC. Notre hypothèse est que l'expression de proprotéines convertases est élevée dans le CHC, permettant l'activation de différentes protéines essentielles dans le développement et la progression du CHC. L'objectif de recherche était d'évaluer l'expression des PCs PCSK9, furine et PC5 dans le CHC par rapport aux stroma environnant, zones péri-cirrhotiques, et foie normal afin de déterminer si un gradient d'expression existe. PCSK9 en particulier est connu comme étant plus exprimé chez le foie régénérateur post-hepatectomie. Les diapositives de pathologie de CHC stockés dans le département de pathologie du CUSM ont été examinés par une pathologiste, et les zones appropriées (tumeur de CHC, interface de tumeur et du foie, le foie cirrhotique, et d'autres échantillons d'hépatite et de foie normal) dans les blocs de tissu correspondants ont été creusés et ont été incorporées dans un microarray de tissu (TMA). Des lignes cellulaires de CHC etablies, dont le HepG2 et le Huh7, avec des profils d'expression de PC connus, ont été incorporées sous forme de pastilles de cellules dans la TMA, afin de servir de témoins positifs et négatifs. La TMA a été sectionnée en diapositives, qui ont été colorées avec des anticorps de la PCSK9, furine et PC5. On a découvert que le niveau d'expression de PCSK9 était diminuée dans les CHC avec un pire prognostique. L'expression augmentée de PCSK9 dans les CHC plus aggressifs pourrait indiquer un rôle du PCSK9 dans la tumorigenèse, directement ou indirectement. Il se peut que les CHCs plus aggressifs sont capables de modifier l'environnement local pour apprivoiser l'énergie métabolique, et que le PCSK9 permet que le cholestérol soit utilisé comme source d'énergie. La confirmation de son importance fonctionnelle avec mRNA pourrait potentiellement mener au développement de chimiothérapie ciblée avec des anticorps contre le PCSK9 (stratégie en étude pour l'hypercholestérolémie). Compte tenu des options chimiothérapeutiques actuellement limitées pour le CHC, une telle constatation pourrait améliorer la prise en charge clinique du CHC.

Hepatocellular carcinoma (HCQ is one of the commonest cancers worldwide. There is, however, a marked geographical variation in incidence and it has been suggested that the pathogenesis may vary in different parts of the world. A retrospective analysis of 110 HCC patients was initially undertaken which confirmed that only 29% of British patients had markers of hepatitis B infection, suggesting a possible role for other environmental agents in the pathogenesis, and that 80% of patients had underlying cirrhosis. The nature of the strong relationship between HCC and cirrhosis has not been established but it has been postulated that increased hepatocyte turnover in the cirrhotic liver may predispose to DNA damage by environmental mutagens. Cell proliferation is required to express the strongly promutagenic DNA base lesion 0'-methylguanine, produced by alkylating agents, as a mutation. &- methylguanine is repaired by the DNA repair enzyme 06-methylguanine-DNA methyltTansferase (06-MT). A microassay was developed which could reliably measure 06-MT levels in liver biopsy samples. Using this approach 06-MT levels were found to be significantly lower in cirrhotic liver when compared to non-cirrhotic and normal liver tissue. No correlation was found between lymphocyte and liver levels from individual patients with liver disease indicating that the deficiency in DNA repair is disease-a nd tissue-specific. Three polyclonal antibodies were subsequently raised to 06-MT peptides and characterised by immunoblotting in an attempt to establish the tissue distribution of the enzyme in liver. Although none of the antisera were able to detect &-MT in tissue sections they were used to analyse structural differences in the enzyme between cirrhotic and non-cirrhotic liver using SDS-PAGE followed by immunoblotting and fluorography. A band of M, 24,000r,e presentingn ative enzyme, was visualised by fluorography in all liver extracts. Densitometry of these bands correlated with the enzyme activity determined by the direct enzyme assay, validating the assay findings. Other small molecular weight bands were seen in all liver extracts and comparison with immunoblots suggested that these bands represent C-terminal truncated enzyme. The spectrum of smaller molecular weight enzyme forms was similar in cirrhotic and non-cirrhotic liver. It was, thus, concluded that although 06-MT levels were lower in'cirrhosis this was not accounted for by structural differences in the enzyme. DNA mutations (G to A) produced by the failure to repair 06-methylguanine are known to activate oncogenes and turnour suppressor genes such as p53. However only 5/55 (9%) of HCC expressed mutant p53. Other factors potentially involved in hepatocarcinogenesis include the growth factor TGF-a and a growth factor receptor encoded by the c-erb B-2 proto-oncogene. Expression of TGF-a and the C-erbB -2 oncoprotein were seen in 8/28 (28%) and 2/26 (8%) of HCC respectively, findings which differ from those observed in HCC from the Far East. Deficient DNA repair by &-MT provides one possible reason why cirrhosis is an important risk factor for the development of HCC. However, failure to repair 06-mothylguanine does not result in mutations within the p53 gene in British HCC. Furthermore, the finding of low expression of mutant p53, TGF-a and the c-erb B-2 oncoprotein in HCC from Britain compared to HCC from the Far East and Africa suggests geographical differences in the molecular mechanisms involved in hepatocarcinogenesis between areas of high and low HCC prevalence.

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.

Hepatocellular carcinoma (HCC), the commonest primary liver tumour, is curable if detected early. It is often diagnosed late, carrying a devastating prognosis. In part, this is due to lack of accurate diagnostic markers. Using proton nuclear magnetic resonance (1H NMR) spectroscopy and ultra performance liquid chromatography mass spectrometry (UPLC-MS), metabolic profiles of plasma, serum and urine were generated from an animal model of HCC and three cohorts of patients with HCC from Nigeria, Egypt and the UK. Plasma 1H NMR spectra from an animal model of HCC displayed decreases in lipoproteins, acetyl-glycoprotein, acetoacetate and glucose (p≤0.001). Blood 1H NMR studies of Nigerian and Egyptian patients with HCC compared to healthy controls and those with cirrhosis revealed alterations in levels of LDL (p=0.002, 0.12), VLDL (p=<0.001, 0.77), lactate (p=0.03, 0.12), N-acetylglycoproteins (p=<0.001, 0.001) and acetoacetate (p=0.52, 0.06). Using UPLC-MS blood profiling, lysophosphatidylcholine (24, 0, 0) (LPC), a major cell membrane component, was identified as altered between groups. Urine 1H NMR spectral profiles were distinguished in a Nigerian, mostly HBV-infected HCC cohort, with a sensitivity/specificity of 100%/93% and 89.5%/88.9% from healthy subjects and patients with cirrhosis. Urinary creatinine (p<0.001, 0.06), carnitine (p=0.04, <0.001) and creatine (p=0.05, 0.29) were contributory. In an Egyptian, mostly HCV-infected HCC cohort, sensitivity/specificity was 100%/94% and 81%/71%, displaying reduced creatinine (p=0.003, <0.001), glycine (p=<0.001, 0.88), trimethylamine-N-oxide (p=<0.001, 0.18), hippurate (p=<0.001, 0.66), citrate (p=<0.001, 0.12) and increased carnitine (p=0.29, 0.30) and creatine (p=0.12, 0.33) levels. In the UK cohort, sensitivity/specificity was 53.6%/96%. UPLC-MS of urine from the Nigerian HCC cohort identified elevated acetylcarnitine levels (p=0.07, <0.001). Both in blood and urine, HCC induces a metabolic profile change that can distinguish patients from healthy or cirrhotic controls. Furthermore, there exists some similarity between distinct populations. Underlying pathways may include heightened cellular proliferation, altered cellular, aberrant lipid metabolism and cancer cachexia.

The urgent unmet need to develop hepatocellular carcinoma (HCC) therapies is addressed here by characterising a novel mouse model of HCC in the context of ongoing liver damage and overnutrition. Male C57Bl/6J mice were treated with Diethylnitrosamine (DEN) and thioacetamide (TAA), and some were provided with an atherogenic high fat diet (HFD). Inflammation, steatosis, fibrosis, 87 genes, liver lesions and intratumoural leukocyte subsets were quantified up to 24 weeks of age. Mice treated on the DEN/TAA/HFD regime for 24-36 weeks were then treated with one of several compounds, ARI-4175, ARI-3996, ARI-6000 or vehicle controls. These compounds target the DPP4 enzyme family. The DPP4 enzyme family includes four atypical serine proteases: DPP4, Fibroblast activation protein (FAP), DPP8, and DPP9. These proteins mediate a diverse range of biological processes by releasing the N-terminal dipeptide of substrates that have a proline or alanine at the penultimate position. Livers were assessed for tumour and histopathological burdens. The expression and activity of DPP4, FAP, DPP8 and DPP9 were investigated to assess their biomarker utility and roles in liver carcinogenesis. Adding HFD to DEN/TAA increased fibrosis, steatosis and inflammation, and the incidence of both HCC and non-HCC dysplastic lesions. All lesions contained α-SMA positive fibroblasts. Macrophage marker F4/80 was not significantly different between treatment groups, but the macrophage-associated genes Arg-1 and Cd47 were differentially expressed. Fibrosis, cancer and cell death associated genes were upregulated in DEN/TAA/HFD livers. Fewer Kupffer cells and plasmacytoid dendritic cells were in tumours compared to control liver. FAP+ fibroblastic populations can be identified with a novel reagent 4613b FAP+ fibroblastic populations were acutely depleted from livers with FAP specific pro-drug ARI-3996. Depletion of FAP+ fibroblast populations with FAP-specific prodrugs ARI-3996 and ARI-6000 as a result of long term administration did not decrease liver fibrosis. ARI-6000 treatment showed a nonsignificant trend towards less inflammation and fibrosis. ARI-4175 administration increased liver fibrosis and decreased tumour burden in the liver. Liver DPP4, DPP8, and DPP9 enzymatic activity expression decreased with DEN/TAA/HFD treatment and DPP9 was enriched in tumours and dysplastic lesions. In conclusion, combining hepatotoxins with an atherogenic diet produced more intrahepatic tumours, dysplastic lesions and fibrosis compared to hepatotoxins alone. This new HCC model provides a relatively rapid means of examining primary HCC and potential therapies in the Preface xii context of multiple hepatotoxins including those derived from overnutrition. The DPP-family have shown potential as both HCC markers and potential therapeutic targets due to their tissue specific expression in the case of FAP and roles in liver carcinogenesis in the case of DPP4 and DPP9.

2010/2011
SUMMARY Introduction. Hepatocellular Carcinoma (HCC) ranks fifth in frequency of cancers in the world. Orthotopic Liver Transplantation (OLT) or liver resection represents the best treatments for HCC. However, most patients cannot be subjected to potential curative OLT or resection because of extensive tumor involvement of the liver, metastasis, invasion of the portal vein or advanced underlying hepatocellular disease at the time of diagnosis. Systemic chemotherapy or chemoembolization represent a good alternative for the treatment, however drug therapy of cancer in general is hampered by multidrug resistance (MDR) that is a phenomenon caused by the up-regulation of the ABC-transporters (ABC) leading to chemotherapy failure. To overcome these problems new therapeutic approaches, such gene therapy, are needed. Selective down-regulation of an essential and specific cancer gene such as telomerase (hTERT) could represent an emerging strategy that could prevent cancer progression and diminish numerous side effects derived from drug usage. The present study include two tasks whose aims are: Task 1: a) Assess if the extent of tumoral differentiation results in a different ABCB1, ABCC1 and ABCG2 expression. b) Assess whether the treatment with a chemotherapeutic drug(s) may affect the expression of the three ABC transporters under study. Task 2: To overcome the obstacle of MDR-induced chemoresistance using new therapeutic approaches such as gene therapy, silencing a cancer essential and specific gene. Results and discussion. Task 1: We assessed the ABCB1, ABCC1 and ABCG2 expression in three hepatic cell lines: IHH (non tumoral control), HuH7 (differentiated tumoral cells) and JHH6 (undifferentiated tumoral cells). Only ABCG2 expression correlates with the degree of tumoral differentiation. Through confocal microscopy analysis we observed that the Doxorubicin (Dox) is able to reach the cell’s nucleus within 10 min. After 24h and 48h Dox is completely concentrated into the nucleus where some nuclear damage occurs. The presence of damaged nuclei could explain the decreased mRNA in most of the ABCs under study. The treatment with Dox doses lower than the LC50 for 24h and 48h has different consequences for all the ABC considered in the three cell lines, with an mRNA expression pattern not in line with the protein one in most of the cases, suggesting that the possible mechanism that determines the ABCs protein upregulation in the tumoral cell lines (HuH7 and JHH6) is not the de-novo transcription but probably something related to the protein turnover. After the treatment ABCC1 protein expression increases in the tumoral cell lines but not in the non tumoral one (IHH). Regarding ABCB1 and ABCG2, these transporters seem to play a role only in HuH7 and JHH6 cells respectively. We were not able to correlate the tumorigenic potential of the two tumoral cell lines with the ABC expression since the different behaviour of ABCs and the different contribution to MDR. Thus in order to better clarify the contribution of each single ABC to MDR our future steps will consider the use specific inhibitors. Task 2: From our in vivo data, among four cancer related genes we selected hTERT as the best candidate for silencing experiments due to its exclusive expression in tumoral samples. A functional non-inflammatory siRNA targeting hTERT was designed: SirTel 1. Silencing experiments were conducted in JHH6 cell line. The hTERT silencing effect was dose dependent, at least at the three considered doses (25-50-100nM). For all the subsequent determinations the experimental concentration was 25nM. After 72h of silencing we observed a significant reduction in both hTERT mRNA expression and enzymatic activity (p<0.001). The effects observed in the cells after silencing are: - Morphological changes, from a fibroblast-like to an hepatocyte-like shape; - Increased albumin expression. The expression of this hepatic hallmark increases after silencing in JHH6 cells that, due to their poor degree of differentiation, at basal conditions do not express quantifiable levels of albumin. The peak of the higher albumin expression corresponds to the maximum hTERT silencing effect. - Decreased cell viability (p<0.01). Interestingly, the siRNA induced a reduction in cell viability higher than Dox. - Cell cycle arrest in G1 phase (p<0.01). All data were validated using a hTERT negative cell line (primary culture of human fibroblast). After 72h silencing, we observed that hTERT expression reaches its minimum, and the expression is recovered after 264h although it does not reach the initial expression levels. Re-exposing the cells to additional 25nM of siRNA induces a reduction of mRNA levels by 76% compared to the amount already present after the first treatment. Taken together all this results suggest the pivotal role of hTERT silencing in a HCC derived cell line. Therefore, hTERT represent a promising candidate for gene-therapy strategies in HCC.
RIASSUNTO Introduzione. Il carcinoma epatico occupa il quinto posto per incidenza tra i diversi tipi di tumore a livello mondiale. Il trapianto di fegato o la resezione epatica rappresentano le terapie d’elezione per questo tipo di tumore, tuttavia molti dei pazienti non possono essere sottoposti a questo tipo di interventi visti l’estensione del tumore nell’organo, la presenza di metastasi, l’invasione portale e la presenza di altre disfunzioni epatiche al momento della diagnosi. La chemioterapia sistemica o la chemioembolizzazione rappresentano una valida alternativa per il trattamento della patologia, tuttavia l’utilizzo di farmaci chemioterapici può portare all’insorgenza di chemioresistenza (multidrug resi stance, MDR) determinando l’inefficacia del trattamento. Parte attiva del fenomeno della chemioresistenza è rappresentato dai trasportatori ACB che sono coinvolti nell’espulsione del farmaco dalla cellula, di fatto impedendone l’azione. Lo sviluppo di nuove terapie geniche potrebbe sostituire l’utilizzo di farmaci per il trattamento del tumore. L’inibizione selettiva di componenti essenziali e specifici per le cellule tumorali come lo è la Telomerasi può rappresentare una strategia emergente che potrebbe prevenire la progressione tumorale ed evitare l’insorgenza dei numerosi effetti collaterali che derivano dalla correnti terapie farmacologiche. Il presente studio comprende due task i cui obbiettivi sono i seguenti: Task 1: a) valutare se il diverso grado di differenziamento tumorale corrisponde ad una diversa espressione dei trasportatori ABCB1, ABCC1 e ABCG2. b) valutare se il trattamento farmacologico influisce sull’espressione dei trasportatori ABC analizzati in questo studio. Task 2: sviluppo di nuovi approcci terapeutici basati sulla terapia genica. Risultati e Discussion. Task 1: è stata valutata l’espressione di mRNA dei trasportatori ABCB1, ABCC1 e ABCG2 in tre linee cellulari epatiche: IHH (cellule epatiche immortalizzate, il controllo non tumorale), HuH7 (cellule tumorali differenziate) e JHH6 (cellule tumorali scarsamente differenziate). ABCB1 è il principale ABC espresso nelle Huh7 e solamente l’espressione di ABCG2 correla col grado di differenziamento tumorale delle cellule. Tramite l’utilizzo della microscopia confocale è stata osservata la capacità della Doxorubicina (Dox), uno tra i più utilizzati farmaci antineoplastici, di raggiungere il nucleo delle cellule entro 10 min dalla somministrazione. Trascorse 24h la Dox è totalmente localizzata all’interno del nucleo causando la morte delle cellule più sensibili. A 48h sono evidenti i danni nucleari e cellulari nelle cellule sopravissute. Il trattamento con dosi di Dox inferiori alla LC50 per 24h e 48h ha diverse conseguenze sull’espressione dei tre trasportatori nelle diverse linee cellulari studiate. In particolare in molti casi l’espressione di mRNA non correla con l’espressione proteica suggerendo che, in questi casi, la regolazione sia dovuta a fenomeni di diminuito turnover proteico piuttosto che di nuova sintesi. A seguito del trattamento con il farmaco, l’espressione di ABCC1 aumenta nelle sole linee cellulari tumorali (Huh7 e JHH6). ABCG2 sembra avere un ruolo rilevante nelle sole cellule JHH6 mentre ABCB1 è espresso unicamente nelle HuH7 dove vi è un aumento dell’espressione proteica a seguito di trattamenti con la Dox. In questo studio non è stato possibile correlare il potenziale tumorigenico delle linee cellulari con l’espressione degli ABC visto il loro peculiare profilo di espressione ed il loro diverso contributo nella chemioresistenza. Per questi motivi in futuro ci si propone di trattare le cellule con inibitori specifici per ciascun ABC al fine di meglio comprendere i loro diversi ruoli nell’insorgenza della resistenza alla Dox. Task 2: Da studi condotti in vivo, il gene della subunità catalitica della Telomerasi (hTERT) è stato selezionato per gli esperimenti di silenziamento genico vista la sua esclusiva espressione nei tessuti tumorali. Tramite analisi bioinformatiche è stato disegnato un siRNA anti-hTERT (SirTel 1) capace di evitare l’induzione di infiammazione nelle cellule che lo internalizzano. Gli esperimenti di silenziamento sono stati effettuati nella linea cellulare JHH6. Dopo 72h di silenziamento con 25nM di SirTel 1 si è osservata una riduzione significativa nell’espressione di hTERT e nella sua attività enzimatica (p<0,001). Gli effetti del silenziamento di hTERT riscontrati sono i seguenti: - Cambiamenti morfologici delle cellule silenziate le quali passano dall’avere un forma fibroblasto-simile ad una forma epatocita-simile. - Aumento dell’espressione di albumina, un marker epatocitario, il cui picco di espressione corrisponde al massimo effetto di silenziamento. - Diminuzione della vitalità cellulare (p<0,01). SirTel 1 risulta più efficace nel ridurre la vitalità cellulare della Dox, il farmaco antineoplastico più utilizzato nelle terapie tumorali. - Arresto del ciclo cellulare nella fase G1 (p<0,01). Tutti i risultati sono stati validati utilizzando cellule hTERT negative come i fibroblasti primari. Il silenziamento raggiunge i suoi massimi livelli trascorse le 72h dalla trasfezione e l’espressione della Telomerasi sembra ritornare ai livelli iniziali attorno alle 264h dalla iniziale somministrazione. La riesposizione con una seconda dose di siRNA riduce ancor più l’espressione di hTERT portandola a circa il 20% dell’espressione iniziale (p<0,001). Questi risultati dimostrano il ruolo fondamentale della Telomerasi per la sopravvivenza e la proliferazione delle cellule tumorali epatiche. Per questi motivi hTERT costituisce un candidato promettente per le future terapie geniche applicate all’HCC.
XXIV Ciclo
1982

With the recent advances in the knowledge of hepato-carcinogenesis, there has been encouraging development in the molecular targeted therapy for patients with advanced hepatocellular carcinoma (HCC). Sorafenib, an anti-angiogenic multi-targeted receptor tyrosine kinase inhibitor, has become the standard of treatment in HCC patients with Child-Pugh A cirrhosis. Nevertheless, the benefits and safety profile of sorafenib in the majority of the unselected advanced HCC patients and other patient subgroups are still unclear. More importantly, the survival benefit associated with sorafenib use is generally modest in Asian population. Therefore, an unmet medical need remains for more effective therapeutic agents. This thesis studied the impact of molecular targeted therapy in the treatment of advanced HCC patients and it contains 10 original studies divided into six sections. The first section provides a concise overview of the epidemiology, risk factors, and current treatment options for HCC patients. Also, the molecular biology and opportunity for the use of targeted therapy in advanced HCC were discussed. The second section is about a new prognostic score system that we developed — Advanced Liver Cancer Prognostic System (ALCPS). Our study results showed that ALCPS was able to objectively estimate the 3-month survival probability of advanced HCC patients and thus could enhance patient selection for targeted therapy or clinical trials. The third section is about the use of sorafenib in the treatment of advanced HCC patients. The results of our single centre phase II study showed that sorafenib had good efficacy and acceptable tolerability in treating advanced HCC patients in hepatitis B endemic area. Furthermore, our retrospective study results confirmed that the overall survival benefits and overall treatment-related adverse events of sorafenib were comparable in elderly and young advanced HCC patients. More importantly, our other retrospective analysis showed that Child-Pugh (CP) A and CP B patients tolerated sorafenib similarly and derived similar clinical and progression-free survival benefit. Among CP B patients, most benefits were observed in patients with score 7. Nevertheless, CP B patients were more susceptible to developing cirrhotic complications. Last but not least, our study also demonstrated that drop in serum alpha-fetoprotein level > 20% in the first 6 weeks of sorafenib treatment was a useful early surrogate endpoint for evaluating antitumor response and survival benefits. All these results are instrumental in guiding future rational use of sorafenib in advanced HCC population. The fourth section is about the role of targeted therapies in treating sorafenib-refractory advanced HCC patients. In a single arm phase II study, we showed that bevacizumab and erlotinib combination was not effective in treating advanced HCC patients who had failed prior sorafenib treatment. The fifth section of the thesis comprises results of four early phase novel clinical trials that may potentially improve the therapeutic outcomes in advanced HCC patients. First, our phase I/II study demonstrated that another anti-angiogenic agent — PTK787 had encouraging and possible synergistic activity when combined with intravenous doxorubicin in treating advanced HCC patients. Second, our multi-center phase II study results demonstrated promising activity with good tolerability of a novel combination — sorafenib together with capecitabine and oxaliplatin (SECOX) in the treatment of advanced HCC patients. Third, in a phase I study, we showed that pazopanib, a novel anti-angiogenic agent, had a manageable safety profile and preliminary activity in advanced HCC patients. Moreover, pazopanib reduced tumor vessel leakage, as shown by contrast-enhanced magnetic resonance imaging indicating a direct effect on HCC vasculature that might be associated with its antitumor activity. Lastly, in another phase I study, we evaluated safety, pharmacological parameters, and potential antitumor activity of pegylated recombinant human arginase 1 (peg-rhArg1) in advanced HCC patients. Our results illustrated that arginine depletion in humans can be achieved safely with peg-rhAgr1 in a dose-response manner and peg-rhArg1 had manageable safety profile and preliminary evidence of activity in advanced HCC patients. In the last section, the future perspectives about the use of molecular targeted therapy in the treatment of advanced HCC patients were discussed.
published_or_final_version
Medicine
Master
Doctor of Medicine

TAX1 Binding Protein 2 (TAX1BP2) has been found to be a centrosome duplication regulating protein. Previous findings have demonstrated that over-expression of TAX1BP2 suppresses centrosome over-duplication. Recently, our lab has revealed that TAX1BP2 is a novel tumor suppressor in hepatocellular carcinoma (HCC) regulated by cyclin-dependent protein kinase 2 (CDK2), nevertheless, the molecular mechanism of how TAX1BP2 regulates centrosome duplication and the link between its centrosome duplication regulatory ability and the tumor suppressing property remain elusive. With the aim to understand the roles of TAX1BP2 in HCC, the present study intended to investigate the link between centrosome duplication regulating ability and tumor suppressing property. Polo-like kinase 4 (PLK4) is a special member of the Polo-like kinase family as its structure is diverged from other family members. Instead of having two Polo-boxes, it carries one Polo-box and one cryptic Polo-box. It has been shown that PLK4 is involved in the formation of centrioles, an important component of centrosome, and is a key regulator of centrosome duplication. Based on the functional similarity, it was hypothesized that PLK4 may function as a regulator of TAX1BP2. To define if PLK4 regulate TAX1BP2, the interaction between PLK4 and TAX1BP2, both in vivo and in vitro, was first confirmed using affinity pulldown and co-immunoprecipitation assays. To understand the significance of the physical interaction, in vitro and in vivo kinase assay were used to study the phosphorylation activity between PLK4 and TAX1BP2. It was demonstrated that TAX1BP2 is a potential substrate of PLK4. Centrosome duplication assay was also performed to investigate if over-expression of PLK4 abolished the centrosome over-duplication suppressing ability of TAX1BP2. In order to delineate the signaling pathway of TAX1BP2, the interaction between TAX1BP2 and its cellular interacting partners was investigated in this study. Ten proteins were isolated as potential interacting partners of TAX1BP2 using Tandem affinity purification (TAP) coupled with Mass Spectrometry protein fingerprinting. Two of the ten proteins, the Ezrin and Mortalin, were confirmed to be binding partners of TAX1BP2 using affinity pull-down assay and TAP, respectively. The identification of the interacting partners suggested that TAX1BP2 may modulate centrosome duplication via alteration of the subcellular localization of Mortalin. These findings helped to delineate the signaling pathway of TAX1BP2 and enabled the better understanding of the roles of TAX1BP2 in tumor suppressor function of HCC. In summary, we demonstrated that TAX1BP2 contains a centrosome duplication regulatory domain (CDRD) and its centrosome duplication regulating ability is critical for its tumor suppressing property. Moreover, three novel interacting partners of TAX1BP2, including Ezrin, PLK4 and Mortalin, are identified. Our findings provide a new insight into the roles of TAX1BP2 in centrosome duplication, hepatocarcinogenesis and metastasis.
published_or_final_version
Anatomy
Master
Master of Philosophy

Background and Aims: Hepatocellular carcinoma (HCC) often presents at a late stage which limits the use of curative therapy. Hence the pressing need for increasing research into newer therapies such as immunotherapy. Alpha-Fetoprotein (AFP) is an oncofetal antigen elaborated in most HCCs and is a tumour rejection antigen in animal models for HCC making it a target for the development of T cell based immunotherapy. The effect of AFP on antitumour immune responses in patients with HCC has not been explored in depth. We aimed to study the effects of AFP on the immune system cells including dendritic cells (DCs). In man, naturally occurring anti-AFP CD8 T cell responses have been detected in patients with HCC. One vital step for the design of epitope-based therapeutic vaccines is the identification and characterization of T-cell epitopes on AFP. Several AFPderived peptides have been identified and T cells recognizing these epitopes have been studied in patients with HCC. However, the role of anti-AFP CD4+ T cell responses (Th1 cells or regulatory T cells) in HCC patients has not been studied. Also, our aim was to study the role of Th1 cells in HCC patients and investigate any possible association between the expansion of these cells with clinical features of the disease such as stage of disease, serum AFP levels and tumour invasiveness. Results and Conclusions: In our first study, the treatment of monocyte-derived DCs with AFP led to DC dysfunction as detected by the down-regulation of surface molecules (CD40 and CD86) and inhibition of their T cell-stimulatory capacity. In addition, AFP treatment induced apoptosis of DCs and reduced their ability to produce TNF-alpha and IL-12. Our ex vivo results showed that the ability of monocytes, isolated from patients with elevated levels of serum AFP (>1,000 ng/ml), to produce TNF-alpha was impaired. In the second study we identified an AFP-derived T cell epitope that was recognized by circulating CD4+ T cells from patients with HCC and normal or mildly elevated AFP level in the early stage of the disease. This response was absent in healthy donors and in patients with chronic liver disease, which suggested that this response had been previously expanded in vivo in response to the tumour. The induction or activation of regulatory T cells (T-regs) by tumours or pathogens may suppress protective immunity. In the third study, we demonstrated that AFP contained an epitope which activated the expansion of inducible TGF-beta producing T-regs. In our fourth study we revealed that CD4 T-cell response expanded in the early stages of disease (Child–Pugh A score), which is usually associated with low concentrations of serum AFP. Furthermore, CD8 T cell response was largely detected in HCC patients with a Child–Pugh B or C score. Necrosis of tumour cells can activate both innate and adaptive antitumor immunity. In a further study by our group, development of higher frequencies of AFP-specific CD4+ T cells after embolisation therapy was noted. Necrosis produced by Transarterial Chemoembolization (TACE) or Chemoembolization (TAE) unmasks tumour rejection Antigen-specific T cell responses. This represented a method of in situ immune response induction that could be combined with immunotherapy to increase the frequency of AFP-specific T cells with the aim of controlling tumour growth and improving survival. Also, two further HLA-DR-restricted AFP-derived CD4+ T cell epitopes were detected. From our studies thus far, we concluded that predictive factors for detecting an AFP-specific Th1 response in patients with HCC included a serum AFP of <1000 ng/ml, Okuda stage 1 and treatment with TACE/TAE.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Introduction. Cancer is the most common given name to a series of molecular and physiological events which result in genetic instability and biochemical imbalance in cells. Among the seven events that drives cell cancer, the role of chronic inflammation and tumor microenvironment in cancer development were highlighted in this work . To experimentally evaluate some of these events was chosen as an experimental model, cirrhosis resulting from chronic hepatitis (viral and alcoholic) as a progressor of hepatocellular carcinoma (HCC), the most common liver cancer. Goals. To identify and correlate proteins/enzymes involved in chronic inflammatory process of cirrhosis and HCC establishment in cancer progress. Patients and Methods. The profile of soluble proteins in inflammatory tissue and in HCC were analyzed using Functional Proteomics techniques of , such as 2D DIGE, coupled to mass spectrometry. The interaction within the identified proteins signaling pathways was performed by using MetaCore software. Results. In this study, where identified proteins that never been described in the literature, using differential gel electrophoresis within the different biological scenarios analyzed here, such as macrophage metalloelastase - MMP12; Collagenase 3 - MMP13; endoplasmina - HSP90B1; heat shock protein HSP 90β - HSP90AB1; Protein S100 A6; Disintegrin and metalloproteinase with a thrombospondin motifs 9 - ADAMTS9, those playing an important role in carcinogenesis. Discussion Relevant observations due to signaling pathways within the proposed biological scenario were analysed, as well as specific pathways in each etiology. Conclusion. Proteins/enzymes involved in cirrhosis and chronic inflammatory progression and the development of HCC were identified and related by their functionality. The interaction between identified proteins within each biological scenario was evaluated from the perspective of its signaling pathways, and differences between those pathways were demonstrated. Tumor microenvironment plays and undergoes significant influence on the variation of gene expression.
IntroduÃÃo. O cÃncer à o nome mais comum dado a uma sÃrie de eventos moleculares e fisiolÃgicos que resultam na instabilidade genÃtica e no desequilÃbrio bioquÃmico nas cÃlulas. Dentro os sete eventos celulares que regem o cÃncer destaca-se neste trabalho o papel da inflamaÃÃo crÃnica e do microambiente tumoral no desenvolvimento dos tumores. Para avaliar experimentalmente alguns destes eventos foi escolhido como modelo experimental, a cirrose resultante da hepatite crÃnica (viral e alcoÃlica) como progressora da neoplasia mais comum no fÃgado, o carcinoma hepatocelular. Objetivo. Identificar e inter-relacionar as proteÃnas/enzimas envolvidas no processo inflamatÃrio crÃnico da cirrose e o estabelecimento dos processos neoplÃsicos no carcinoma hepatocelular. Pacientes e MÃtodos. Utilizando de tÃcnicas de ProteÃmica diferencial, 2D DIGE acoplada à espectrometria de massa, foram analisadas, entre vÃrios cenÃrios biolÃgicos, o perfil das proteÃnas solÃveis em tecidos inflamatÃrios e no prÃprio carcinoma hepatocelular. Uma abordagem por meio da interaÃÃo das proteÃnas anotadas em vias de sinalizaÃÃo foi realizada por meio do programa MetacoreÂ. Resultados. Neste estudo, proteÃnas que nÃo haviam sido separadas e purificadas por meio de eletroforese em gel diferencial foram anotadas, como MacrÃfago metaloelastase - MMP12; Colagenase 3 â MMP13; endoplasmina - HSP90B1; ProteÃna S100 A6; Desintegrina e metaloproteinase com repetiÃÃo de trombospondina 9 - ADAMTS9, estas desempenhando importante papel na carcinogÃneses. DiscussÃo Relevantes observaÃÃes das vias de sinalizaÃÃo que regem cada cenÃrio biolÃgico proposto, foram realizadas e vias especÃficas em cada etiologia foram analisadas. ConclusÃo. As proteÃnas/enzimas envolvidas no processo inflamatÃrio crÃnico da cirrose e o surgimento/progressÃo do carcinoma hepatocelular foram identificadas e caracterizadas quanto à sua funcionalidade e a interaÃÃo das mesmas, com outras proteÃnas diferenciais anotadas em cada cenÃrio biolÃgico foi avaliada dentro da perspectiva de suas vias de sinalizaÃÃo correspondentes. O microambiente tumoral exerce e sofre importante influÃncia na variaÃÃo da expressÃo gÃnica.

Astrocyte Elevated Gene 1 (AEG1) is an oncogene for hepatocellular carcinoma (HCC). Its role in HCC pathogenesis has been well studied. A pan cancer analysis of gene expression in multiple databases identified TATA-box binding protein associated factor 2 (TAF2) as the gene that is most frequently co-expressed with AEG1. TAF2 is a protein that is involved in transcription of genes by RNA polymerase II. It is a factor that is dispensable for basal transcription but, required for activated transcription. It has also been shown to be involved in regulating cyclin levels and hence cell cycle progression. Bioinformatic analysis on data from different cancer databases confirmed the positive correlation of TAF2 expression with AEG1 expression, the over expression of TAF2 in HCC patients and poor survival of HCC patients with increasing TAF2. We confirmed the over expression of TAF2 in HCC cell lines using western blotting and HCC liver using immunohistochemistry. We established cell lines with stable knockdown of TAF2 expression. These clones showed significant decrease in their ability to invade and migrate but not their proliferation ability. This is in contrast to what has been observed in previous studies. We hypothesize that the knockdowns do not show any decrease in cellular proliferation since the remaining TAF2 in the cells is sufficient to produce cyclins and keep cell cycle undisturbed. The knockdown of TAF2 causes an increase in E-cadherin level and decrease in Snail protein expression which is a known negative regulator of E-cadherin. Knockdown of TAF2 causes cells to become more epithelial leading to a decrease in their ability to migrate and invade. This study shows that TAF2 is a potential oncogene that needs to be further studied.

The World Health Organisation (WHO) estimates that 150 and 240 million people are chronically infected with hepatitis C and B viruses (HCV/HBV) worldwide, of whom approximately 500,000 develop hepatocellular carcinoma (HCC) annually. The 5-year survival for HCC is less than 10% and up to 80% of cases are inoperable at presentation with no hope of cure. The treatment of patients with advanced HCC is frequently complicated by immunosuppression, leading to the attenuation of cancer-directed immune responses and the reactivation/exacerbation of HCV/HBV infections. In contrast, immunotherapies such as cancer selective oncolytic viruses, could theoretically suppress rather than stimulate underlying oncogenic virus infections, and improve clinical outcomes. This project investigates the potential for oncolytic viruses to be employed as dual anti-cancer and anti-viral therapies in pre-clinical models of hepatitis virus-associated HCC. The results described herein show that oncolytic virus stimulation of normal primary and malignant liver cells induces interferon secretion, leading to the inhibition of HCV. Further work using oncolytic wild-type reovirus showed that it stimulates interferon responses in primary hepatocytes, liver-resident immune cells and HCC cell lines, potently inhibiting HCV and HBV replication. Reovirus also activated liver resident natural killer cells to kill HCC, whilst infection of primary hepatocytes was non-toxic. Finally, in preclinical murine models of HCV-positive HCC, reovirus exerted a specific antiviral effect on HCV replication, detectable at very early times post-treatment, and distinct to its direct and immune-mediated tumour killing. Application of oncolytic virus therapy in an HCV/HBV positive HCC setting represents a novel, and potentially safe means of simultaneously targeting both tumours and the underlying oncogenic virus, providing a much-needed opportunity to improve clinical outcomes in HCC patients.

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.

Thyroid hormones elicit many cellular and metabolic effects. Most of them are mediated by the thyroid hormone 3,5,3’-L-triiodothyronine (T3) nuclear receptors (THRs), which act as transcription factors. THRs have also been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors. Here, experiments were performed to determine the role of THRs in hepatocellular carcinoma development. Using the Resistant-Hepatocyte model (RH model) of rat hepatocarcinigenesis, we found downregulation of THRβ1 and THRα1 in most of early preneoplastic lesions and of HCCs. THRβ1 and, to a lesser extent, THRα1 downregulation was observed only in preneoplastic lesions positive for the progenitor cell marker cytokeratin-19 (KRT-19), which are characterized by a higher proliferative activity compared to KRT-19 negative ones. Accordingly, THRs target genes were strongly down-regulated in KRT-19+ lesions. In rat HCC THRβ1 decrease was not due to hypermethylation of the THRβ1 promoter but was associated with an increased expression of miR-27a, -181a and -204, known to target this receptor in other cell types. Accordingly, transfection of hepatoma cell lines with these miRNAs led to down-regulation of THRβ1. Downregulation of THRβ1 expression was observed also in the vast majority of the analyzed human HCCs compared to peritumorous liver of the same patients or to normal liver. These results show that downregulation of THRs, especially THRβ1, is an early and relevant event in liver cancer development, species- and etiology-independent. They also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC.

L'inflammation hépatique chronique peut entraîner des maladies chroniques du foie (MCF), notamment l'hépatite, la cirrhose et le carcinome hépatocellulaire (CHC), qui est la tumeur maligne primaire du foie la plus fréquente et le troisième cancer le plus fréquent en termes de mortalité dans le monde. Indépendamment des facteurs étiologiques, toutes les MCFs partagent de nombreux mécanismes physiopathologiques communs : réponse aux protéines non pliées (UPR), inflammation chronique et fibrose. A l'aide d'un modèle animal et d'échantillons cliniques nous avons étudié un autre dénominateur commun des MCFs et du CHC : la molécule neurotrope de guidage axonal netrin-1 et ses récepteurs à dépendance UNC5A, UNC5b et UNC5C. La nétrine-1, connue de ces propriétés pro-oncogéniques dans d'autres tumeurs solides, est induite pendant l'inflammation hépatique, alors que le signal pro-apoptotique des récepteurs UNC5 est atténué avec un équilibre ligand/récepteur globalement augmenté dans la cirrhose et le CHC. L'inflammation chronique est médiée par de multiples acteurs du système immunitaire. L'implication du système nerveux autonome (SNA) dans l'inflammation hépatique et la progression du CLD reste mal comprise. En étudiant l'implication des signaux neuronaux pré-synaptiques et post-synaptiques dans la cirrhose et le CHC, nous avons observé le remaniement de l'équilibre entre les systèmes nerveux sympathique (adrénergique) et parasympathique (cholinergique) intra-hépatiques. Le modèle animal de CHC cirrhotique a montré l'établissement progressif d'une orientation cholinergique à travers les différents stades de la fibrose. Le signal cholinergique global du CHC était associé à un microenvironnement anti-inflammatoire et à une survie plus faible chez les patients. La progression observée du CLD vers le CHC in vivo était accompagnée de la surexpression de marqueurs neuronaux immatures dans le CHC. Dans l'ensemble, nous avons montré que le bras parasympathique du SNA est impliqué dans la physiopathologie du CHC, suggérant l'utilisation de médicaments ciblant le SNA, dont beaucoup sont cliniquement sûrs et bien caractérisés, dans les études sur le CHC. En tentant d'établir le lien entre la netrine-1 ayant des propriétés anti-apoptotique et chimiotactique et SNA intrahépatique, nous avons identifié la corrélation positive entre le système netrine-1/UNC5s et le signal cholinergique dans le CHC. Le ciblage de la netrine-1 par l'anticorps monoclonal NP137, actuellement étudié dans les essais cliniques dans le traitement des tumeurs solides avancées, a montré un remaniement du SNA, confirmant la sensibilité du SNA à l'axe netrine-1/UNC5 dans le contexte hépatique pathologique. Les anomalies moléculaires les plus courantes dans le CHC au niveau du promoteur du gène TERT et au niveau du gène CTNNB1, ont montré une association avec le système netrin-1/UNC5, alors que les mutations dans T53, le régulateur bien connu de NTN1 et UNC5s n'ont pas montré d'implication dans le remodelage de l'axe netrin-1/UNC5 dans les échantillons cliniques de CHC, également insensibles au statut fonctionnel de la protéine p53. Globalement, les échantillons cliniques hébergeant une mutation dan le gène CTNNB1 sont corrélés à la polarité adrénergique du CHC, tandis que les mutations dans le gène TP53 apparaissent comme positivement associées à la polarité cholinergique du CHC. Dans l'ensemble, les résultats de ma thèse suggèrent le rôle pro-cancérogène de la nétrine-1 et l’implication de la neuroregulation associée via l’orientation de SNA dans le CLD et le CHC
Chronic liver inflammation can lead to chronic liver diseases (CLD), including hepatitis, cirrhosis and hepatocellular carcinoma (HCC), which is the most common malignant primary liver tumor and is the 3rd most common cancer in terms of mortality worldwide. Regardless of the etiological factor all CLDs share numerous common patho-physiogycal mechanisms: unfolded protein response (UPR), chronic inflammation and fibrosis. Using animal model and clinical samples we studied another common denominator of CLDs and HCC, which is the system of axon guidance cue netrin-1 and its dependence receptors UNC5s. Netrin-1, known as pro-oncogenic in other solid tumors, is induced during hepatic inflammation, and the pro-apoptotic signal of UNC5 receptors is attenuated with overall increased ligand/receptor balance in cirrhosis and HCC. Chronic inflammation is mediated by multiple actors of the immune system. The implication of autonomic nervous system (ANS) in hepatic inflammation and CLD progression remains poorly understood. Looking at the involvement of pre-synaptic and post-synaptic neuronal signals in the cirrhosis and HCC, we observed the reshaping of the balance between intrahepatic sympathetic (adrenergic) and parasympathetic (cholinergic) nervous systems. In vivo model of cirrhotic HCC showed the progressive establishment of cholinergic orientation throughout the different stages of fibrosis. The overall cholinergic signal of HCC was associated with anti-inflammatory microenvironment and poorer survival in patients. The observed CLD-to-HCC progression in vivo was accompanied by the overexpression of immature neuronal markers in HCC. Altogether, we showed that the parasympathetic arm of the ANS is implicated in the patho-physiology of HCC, and encourage for the use of ANS-targeting drugs in HCC studies, many of which are clinically safe and well characterized. Trying to establish the connection between pro-survival and chemotactic netrin-1 and intrahepatic ANS, we found the positive association between netrin-1/UNC5s and cholinergic signal in HCC. Netrin-1 targeting by the monoclonal antibody NP137, currently studied in the clinical trials in the treatment of advanced solid tumors, showed remodeling of ANS orientation, confirming the sensitivity of ANS to netrin-1/UNC5 axis in the hepatic pathological context. The most common molecular anomalies in HCC in the TERT promoter and CTNNB1 gene, showed an association with netrin-1/UNC5 system, whereas mutations in T53, the well-known regulator of NTN1 and UNC5s expression did not show any implication in netrin-1/UNC5 axis reshuffling in clinical HCC samples, also unsensitive to functionality status of p53. In average, CTNNB1-mutated clinical samples correlated with adrenergic polarity of HCC, whereas TP53 mutations appear to be positively associated with cholinergic polarity of HCC. Taken together, my thesis results suggest the putative pro-cancerogenic role of the netrin-1 and the implication of the neuroregulation via ANS in the CLD and HCC development

Le cancer primitif du foie est le sixième cancer le plus fréquent dans le monde et, en 2018, il était la quatrième cause de mortalité liée au cancer. Le carcinome hépatocellulaire (CHC), qui est souvent diagnostiqué à un stade avancé, représente 90 % des cas de cancer primitif du foie. La résection chirurgicale, la transplantation ou l'ablation locale ne sont possibles que chez des patients à un stade précoce de la maladie. Les agents de chimiothérapie et certains médicaments de thérapie ciblée sont peu efficaces en termes de survie, d'où l'urgence de développer de nouveaux traitements et stratégies. L'autophagie joue un rôle double dans le cancer selon le stade de développement du cancer, son type et son micro-environnement. Selon sa fonction (suppresseur de tumeur ou oncogène), l'autophagie est considérée comme une cible thérapeutique dans le traitement du cancer, bien que peu de composés manipulant ce processus aient été caractérisés. Notre étude a examiné les effets sur la viabilité et la mort des cellules, ainsi que les mécanismes sous-jacents, de médicaments qui ciblent la machinerie de l'autophagie. Nous avons découvert que ces médicaments exerçaient un effet cytotoxique prononcé dans différentes lignées cellulaires humaines dérivées de cancer du foie comparé à une quasi-absence d’effet délétère dans des hépatocytes primaires humains. Cette combinaison de composés induit un effet inhibiteur synergique par rapport à l’action de chaque composé pris séparément. Nous montrons également que les mécanismes d'induction et de blocage simultanés de l'autophagie entraînent une mort cellulaire apoptotique. Il en résulte une accumulation d'autolysosomes, une augmentation des niveaux d'espèces réactives de l'oxygène (ROS), une induction de la perméabilité de la membrane du lysosome (LMP) et une libération de cathepsines dans le cytosol. Dans l'ensemble, notre étude montre que l'induction et le blocage simultanés de l'autophagie est une stratégie prometteuse pour le traitement anticancéreux du carcinome hépatocellulaire
Worldwide, liver cancer is the sixth most common cancer and, in 2018, ranked as the fourth most common cause of cancer-associated death. Hepatocellular carcinoma (HCC), which is often diagnosed at an advanced stage accounts for 90% of primary liver cancer cases. Surgical resection, transplantation or local ablation are only possible in early-stages patients. Chemotherapeutic agents and some molecular targeted drugs are weakly effective in terms of survival, hence the urgent need to develop new treatments and strategies. Autophagy plays a dual role in cancer depending on the developmental stage of cancer, its type and its microenvironment. Depending on its function (tumor suppressor or oncogenic), autophagy is considered as a therapeutic target in cancer treatment, although few such compounds manipulating this process have been characterized. Our study investigated the effects on cell viability and cell death, and the underlying mechanisms, of drugs that target the autophagy machinery. We found that these drugs exerted a pronounced cytotoxic effect against all the different human liver cancer cell lines studied compared to nearly absent effect in human primary hepatocytes. The combination drug therapy exhibited synergistic inhibitory effect compared to each drug separately. We showed that the mechanisms of simultaneously inducing autophagy and blocking autophagy result in apoptotic cell death. It resulted in accumulation of autolysosomes, an increase in reactive oxygen species (ROS) levels, an induction of lysosome membrane permeabilization (LMP) and a release of cathepsins into the cytosol. Overall, our study showed that the simultaneous induction and blockade of autophagy is a promising strategy for anticancer treatment in hepatocellular carcinoma

Au cours de la dernière décennie, l’Amérique latine a connu la plus forte incidence de cancer du foie. Les derniers rapports ont montré l'apparition précoce et inhabituelle de CHC dans cette région. C'est particulièrement le cas de Pérou, où une distribution bimodale basée sur l'âge a été rapportée. Cette distribution délimitait deux sous-populations, une jeune et une autre plus vieux (≤ 44 et > 44 ans). Les principales caractéristiques sont le sex-ratio équilibré (M: F; 1,6), le nombre élevé de tumeurs mal différenciées et indifférenciées (86%), le grosse tumeurs (12,3 cm-diamètre), le valeur élevée de AFP dans le sérum (1,42E + 05 ng/ml), un nombre considérable de foies non cirrhotiques (89%) et taux d'infection élevé par le VHB (74%). Ces particularités du développement du CHC péruvien soulignent la nécessité de mieux comprendre les principes moléculaires. Pour ces raisons, une analyse génomique intégrative a été menée en utilisant des profils d'expression génique et de méthylation de l'ADN à partir d'échantillons paires tumeur/non-tumorale de patients péruviens. Globalement, le CHC péruvien a été caractérisé au niveau transcriptomique par un gain de signaux prolifératifs impliquant des modificateurs épigénomique, une perte massive de marqueurs hépatocytaires, une détection et signalisation aberrantes des hormones et des métabolites primaires, tels que les rétinoïdes. Cliniquement, le CHC péruvien présentait un phénotype hybride entre les classes proliférantes et non-proliférantes, présumé mutuellement exclusif. Que cette épidémiologie du génome humain soit liée à des facteurs ethniques ou à des indices étiologiques reste à explorer
N the last decade Latin American is the region with highest incidence rise for liver cancer. Latest reports have shown the occurrence of unusual early onset of HCC in this region. This is specially prevailing in Peru, where a bimodal age-based distribution has been reported. This distribution delineated two subpopulations, an early-onset and a late-onset (≤44 and > 44 years old). The main characteristics of this HCC presentation are equilibrated sex ratio (M:F, 1.6), high number of poorly and undifferentiated tumors (86%), big tumors (12.3 cm-diameter), high AFP values in serum (1.42E+05 ng/ml), a considerable number of non-cirrhotic livers (89%) and high HBV infection rate (74%).These peculiarities of HCC development in Peruvian patients emphasize the needs to provide further insight into the molecular principles that, with HBV infection or other factors, could lead to this HCC presentation. For these reasons an integrative genomic analysis was conducted using gene expression and DNA methylation profiles of Peruvian patients' surgical specimens (tumour/non-tumour pairs). Overall, Peruvian HCC was characterized at the transcriptomic level by a gain of proliferative signals involving epigenome modifiers, massive loss in hepatocyte markers, aberrant sensing and signalling of hormones and primary metabolites, such as the retinoids. From the clinical standpoint, Peruvian HCC featured a hybrid phenotype between proliferative and non-proliferative classes that are presumed mutually exclusive in the unifying classification. Whether this human genome epidemiology is linked to ethnic factors or to etiological cues remains to be explored

Hepatocellular carcinoma (HCC) is a devastating malignancy originating from hepatocytes. There is an urgent need for novel therapeutic approaches. Currently explored gene therapy systems have not yet achieved significant survival benefits. The aim of this thesis was the development and evaluation of novel genetic approaches to this malignancy.

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2009-2010
published_or_final_version
Clinical Oncology
Doctoral
Doctor of Philosophy

Hepatocellular carcinoma (HCC) accounts for 70-85% of liver cancer, which is the sixth most common cancer in the world. Prognosis of HCC is dismal with little chance of complete recovery after diagnosis. It is of essence to discover the key molecules involved in the tumor progression. This could help earlier detection of HCC and establish targeted molecular therapies. Cyclin E1 (CCNE1) is a cyclin molecule responsible for the transition from G1 to S phase of the cell cycle and is often dysregulated in human cancers. CCNE1 is reported with overexpression in about 30-70% of HCC cases. It expresses in tumor cells as a ladder of proteins and as low molecular weight CCNE1. The study is aimed to investigate the role of CCNE1 in HCC. From the local cohort of HCC patients, 6 out of 13 patients (46.2%) of HCC tumor tissues were found with CCNE1 overexpression compared with the non-tumor tissues by western blotting. The presence of three CCNE1 isoforms in HCC was detected. The expression of total CCNE1 and each isoform varied independently among the studied HCC cell lines, with HepG2 having the highest expression and 97L the lowest. To extend our study on the regulation of CCNE1 expression, the expression of selected four genes associating with the CCNE1 expression and functions was studied by quantitative PCR (qPCR). F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) and cullin 3 (CUL3), the two genes responsible for CCNE1 degradation, had increased expression in the HCC cell lines with higher CCNE1 expression. Cyclin A (CCNA2), the downstream cyclin molecule of CCNE1, also had higher expression in these cell lines. In contrast, the expression of cyclin dependent kinase 2 (CDK2), the catalytic partner of CCNE1, had the least difference among the six HCC cell lines compared to other three genes. To characterize the role of CCNE1 isoforms in HCC, CCNE1 isoform 1, 2, and 3 were overexpressed in PLC cells and such overexpression remained even after 8 passages in culture. In flow cytometric analysis, GFP signal in cell culture population was viewed to observe the transduction efficiency. The vector control showed the strongest GFP signal, followed by CCNE1 isoform 3 showing dim signal. CCNE1 isoform 1 and 2 almost showed no signal. In the functional studies, the overexpression of CCNE1 isoform 3 could increase proliferation and migration of HCC cells. In summary, CCNE1 could promote proliferation and migration of HCC cells through elevated expression of CCNE1 isoform 3.
published_or_final_version
Surgery
Master
Master of Philosophy