Dissertations / Theses on the topic 'Hepatocellular Carcinoma HSC'

Liver cancer is the 6th most common cancer worldwide and Australia’s fastest growing malignancy both in incidence and mortality. Although great improvement in early diagnosis and treatment approach have been achieved during last two decades, patients with HCC still have a dismal prognosis with a mortality-to-incidence ratio close to 1, meaning that the disease is almost uniformly fatal. One of explanations for such a dismal prognosis for HCC is the existence of heterogeneity in HCC, in which HCC is composed of liver cancer stem cells and mature HCC cells. Previous data suggested that liver cancer stem cells are responsible for HCC initiation, early metastasis and treatment resistance. However, accurate characterization of liver cancer stem cells is still required as the reliability of those reported approaches in identify liver cancer stem cells is variable. To better characterization of liver cancer stem cells, we found by qPCR and western blotting that Oct4, a stem cell gene associated with a range of functions in HCC cells with stem cell feature, is ubiquitously expressed in all HCC tumors in our cohort, whereas other liver cancer stem cell markers had high variability in their expression. By utilizing a human Oct4 promoter driving an enhanced green fluorescent protein reporter, we successfully established OCT4+EGFP reporter HCC cells. More importantly, Oct4+ cells had all the classic features of liver cancer stem cells including increased sphere formation in vitro, tumor forming potential in vivo, along with up-regulated expression of stemness genes and increased resistance towards Sorafenib, the only drug approved for advanced HCC. Upon success in characterization for liver cancer stem cell, we, for the first, demonstrated by western blotting that JAG2, one of the five ligands in Notch signaling, is significantly increased in liver cancer stem cells. Moreover, JAG2 is the only Notch ligands aberrantly expressed in liver cancer stem cells characterized by qPCR and western blotting. In addition, Sorafenib resistant HCC cells, which are enriched for liver cancer stem cells, also have profound increase in the expression of JAG2 at both RNA and protein level. Since the reported functions of Notch signaling in HCCs are conflicting and an effective approach targeting Notch signaling with tolerable side effect is missing, we then studied the expression profile of Notch signaling components in HCC by qPCR and western blotting. We found that JAG2 is significantly increased in 65% of HCC tissues in our cohort, which is in consistence with microarray data extracted from NCBI and RNA sequence results provided by TCGA. More importantly, we showed that patients with hyper-Notch activity had worse prognosis than that with hypo-Notch activity demonstrated by immunohistochemical staining and Kaplan-Meier survival assay. These results indicate an important biological role of JAG2 mediated Notch signaling in regulation of liver cancer stem cells and HCC. Using Compound E, a selective γ-secretase inhibitor, we demonstrated that inhibition of Notch signaling leads to slowed cell proliferation and decreased liver cancer stem frequency, which were testified by Brdu assay and flow cytometry respectively. Moreover, selective inhibition of JAG2 alone by shRNA in HCC cells results in a similar phenotype along with impaired function in liver cancer stem cells. We also demonstrated a great translational potential of selective targeting JAG2 in HCC as verified by colony-forming assay that JAG2 knockdown HCC cells became significantly sensitive to the killing effect of Sorafenib. In term of mechanism, we identified that Notch2, one of the four Notch signaling receptors, is solo responsive to JAG2 mediated signaling testified by western blotting and immunofluorescence analysis. Inhibition of Notch2-dependent JAG2 signaling leads to a mature HCC phenotype including less liver cancer stem cells with impaired biological function. Targeting JAG2-initiated Notch2 signaling in HCC would greatly improve prognosis of patients with HCC.

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.

Für Lebertransplantationen bei Patienten mit hepatozellulärem Karzinom stellt sich angesichts der defizitären Organspendesituation die berechtigte Frage, unter welchen Bedingungen diese Form der Therapie ein gutes Outcome für die Patienten verspricht und somit keine Verschwendung der ohnehin knappen Ressourcen darstellt. Ziel dieser Arbeit war es, ein Kollektiv aus 98 Patienten, die an einem hepatozellulären Karzinom erkrankten und im Zeitraum von 1994 bis einschließlich 2010 am Universitätsklinikum Leipzig eine Lebertransplantation erhielten, retrospektiv zu charakterisieren und den Einfluss mehrerer Faktoren auf das Outcome der Patienten zu untersuchen. Bei den Faktoren handelte es sich um die Wartezeit, den präoperativen Einsatz der TACE, den präoperativen AFP-Serumspiegel, sowie die Tumorzahl und -größe. Der Nachbeobachtungszeitraum lag bei 3 Jahren. Die Charakterisierung des Kollektivs erbrachte folgende Ergebnisse: Das Kollektiv bestand zu rund 80% aus Männern. Das mediane Alter zum Zeitpunkt der Transplantation lag bei 59 Jahren. Die Transplantationszahlen bei HCC-Patienten sind am UKL seit Einführung des MELD-Scores 2006 deutlich angestiegen. Die mediane Wartezeit hat sich seit Einführung des MELD-Scores nicht wesentlich verändert. Sie betrug 7,3 Monate in der Prä-MELD-Ära und 6,9 Monate in der MELD-Ära. Mit über 60% war der Alkoholabusus die häufigste Ursache für die Entstehung des hepatozellulären Karzinoms. An zweiter Stelle stand die Hepatitis-C-Infektion. In der Diagnostik des HCC spielte die Computertomographie die größte Rolle. Die Sensitivität des AFP zur Erfassung des HCC (>400 ng/ml) war mit Werten unter 30% sehr niedrig. Die TACE war die mit Abstand am häufigsten durchgeführte, neoadjuvante Maßnahme. Zum Zeitpunkt der Transplantation befanden sich rund 75% der Patienten in einem Stadium bis maximal T2. Das Auftreten von solitären und multifokalen HCCs war in etwa gleich häufig (46,9% vs. 53,1%). Die Milan-Kriterien waren bei knapp 39% der Patienten im postoperativen Explantat-Befund überschritten. Nach Transplantation traten bei 26 Patienten Abstoßungsreaktionen auf. 8 Patienten mussten aufgrund eines Transplantatversagens retransplantiert werden. Das postoperative Überleben (intention-to-treat) betrug 75,5% (6 Monate), 71,4% (1 Jahr) und 63,3% (3 Jahre). Die entsprechenden Rezidivraten lagen bei 11,2%, 14,3% und 22,4%. Rezidiven traten am häufigsten in der Spenderleber auf, gefolgt von einem Befall der Lymphknoten und Knochen. Ein signifikanter Einfluss auf das Outcome der Patienten konnte für das AFP, die Tumorzahl und die Milan-Kriterien nachgewiesen werden: Präoperative AFP-Spiegel unter 100 ng/ml zeigten eine signifikant niedrigere Rezidivrate. Multifokale Tumoren waren mit einem signifikant schlechteren 3-Jahres-Überleben verknüpft. Bei Erfüllung der Milan-Kriterien (im postoperativen Explantat-Befund) war die Rezidivrate signifikant und die Überlebensrate deutlich besser. Für die Wartezeit konnte seit Einführung des MELD-Scores eine positive Entwicklung festgestellt werden. Das 3-Jahresüberleben hat sich bei Wartezeiten unter 12 Monaten um 22,5% verbessert. Die Rezidivrate ist bei Wartezeiten über 12 Monate um 15,3% gesunken. Für den Einfluss der TACE auf das Outcome der Patienten konnten keine signifikanten Unterschiede festgestellt werden. Auch andere Studien belegten bisher lediglich einen Vorteil für das erfolgreiche Downstaging gegenüber Patienten, bei denen die TACE erfolglos blieb. Für die Untersuchung des tatsächlichen Nutzens einer TACE vor Transplantation werden daher Studien mit höherem Evidenzgrad benötigt.

Carcinome hépatocellulaire (CHC) est le sixième cancer et la deuxième cause de mortalité par cancer dans le monde. Dans la majorité des cas, le CHC se développe sur une maladie chronique associée à des étiologies variées telles que l'infection par le virus de l'hépatite B ou l’hépatite C, la consommation excessive d'alcool et des maladies métaboliques. Le développement des maladies chroniques du foie qui conduisent à la cirrhose puis au cancer induisent des modifications de la composition chimique des cellules et des tissus. En effet, la carcinogenèse hépatique est un processus en plusieurs étapes caractérisé par la progression de nodules de régénération, de nodules dysplasiques de bas grade puis de grade et enfin du CHC. Le traitement du CHC reste difficile et la transplantation du foie est la seule option thérapeutique curative à long terme. Le problème est qu'il n'y a pas de marqueur objectifs et quantifiables pour contrôler la qualité d’un greffon. Des biomarqueurs spécifiques marquant la progression du CHC font également défauts.Dans ce travail de thèse, nous avons évalué l’intérêt de la microspectroscopie infrarouge (IR) pour le diagnostic de la stéatose, qui est le facteur le plus important affectant la reprise de la fonction hépatique après greffe de foie. La microspectroscopie infrarouge permet de détecter de façon qualitative et quantitative les caractéristiques biochimiques liées aux différents constituants moléculaires présents dans l'échantillon biologique. Nos travaux ont montré que la progression de la stéatose hépatique correspond non seulement à l'accumulation de lipides, mais également à des changements spectaculaires dans la composition qualitative du tissu. En effet, le bas grade de stéatose présente une diminution de la teneur en glycogène et une augmentation concomitante de lipides par rapport au foie normal. La stéatose intermédiaire montre une augmentation de glycogène et des changements majeurs sont observés en ce qui concerne les lipides, avec une contribution significative des acides gras estérifiés, des chaînes de carbone allongées et des lipides insaturés. Ces caractéristiques sont encore plus prononcées dans les hauts degrés de stéatose. De plus, nous avons mis en évidence que des changements biochimiques majeurs se produisent dans la partie non-stéatosique du tissu malgré son aspect normal sur le plan histologique, ce qui suggère que l’organe dans son ensemble reflète le degré de la stéatose.La deuxième partie de la thèse est focalisée la carcinogenèse hépatique. Il s’agit d’un processus en plusieurs étapes qui se caractérise dans la plupart des foies cirrhotiques par la progression de nodules hyperplasiques de régénération vers des lésions précancéreuses telles que les nodules dysplasiques de bas grade puis de haut grade et enfin le CHC. Le diagnostic différentiel entre nodules dysplasiques en particulier de haut garde et CHC reste extrêmement difficile. Nous avons abordé le potentiel de la microspectroscopie IR pour le diagnostic des nodules cirrhotiques. Nous avons observé de profondes modifications de la composition biochimique du foie pathologique. En effet, des changements importants ont été détectés dans la composition des lipides, des protéines et des sucres mettant en évidence la reprogrammation métabolique dans la carcinogenèse. Les principaux changements ont été observés dans le domaine de fréquence 950-1480 cm-1 dans lequel plusieurs bandes permettaient la discrimination des nodules cirrhotiques, dysplasiques et tumoraux. Enfin, nous avons montré que le diagnostic peut être réalisé à l’aide d’un microscope de laboratoire qui peut être facilement mis en œuvre en milieu hospitalier<br>Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the second most common cause of death in the world. Hepatocarcinogenesis is a multistep process characterized in patients with chronic liver diseases by a spectrum of hepatic nodules that mark the progression from regenerative nodules to dysplastic lesions followed by HCC. Liver transplantation remains the curative therapeutic option able to treat both the HCC and the underlying liver disease. The issue is that there is no objective and quantifiable marker for quality control of liver graft. Specific biomarkers of early stages of HCC are also an unmet need.In this study, we have evaluated the potential of infrared (IR) microspectroscopy for the diagnosis of steatosis, one of the most important factors affecting the liver allograft function. Vibrational microspectroscopy, such as Fourier transform infrared microspectroscopy (FTIR), allows detecting spectral characteristics associated with different molecular components present in the biological sample, both qualitatively and quantitatively. Our first working hypothesis was that the progression of liver steatosis corresponds not only to the accumulation of lipids but also to dramatic changes in the qualitative composition of tissue. Indeed, a lower grade of steatosis showed a decrease in glycogen content and concomitant increase in lipids in comparison with normal liver. Intermediate steatosis exhibited an increase in glycogen and major changes in lipids, with a significant contribution of esterified fatty acids with elongated carbon chains and unsaturated lipids, and these features were more pronounced in a high grade of steatosis. Furthermore, we have shown, that FTIR approach allows a systemic discrimination of morphological features, leading to a separate investigation of steatotic vesicles and the non-steatotic counterpart of the tissue. This highlighted the fact that dramatic biochemical changes occur in the non-steatotic part of the tissue also despite its normal histological aspect, suggesting that the whole tissue reflects the grade of steatosis. The second part of the thesis focused on hepatocarcinogenesis; a multistep process that is characterized in most cirrhotic livers by the progression from hyperplastic regenerative nodules to low grade dysplastic nodules (LGDN), high grade dysplastic nodules (HGDN) and finally small HCC which corresponds either to vaguely nodular well differentiated HCC so called early HCC or to distinctly nodular moderately differentiated hepatocellular carcinomas. Since the differential diagnosis between precancerous dysplastic nodules and early HCC remains extremely difficult, we addressed the potential of FTIR microspectroscopy for grading cirrhotic nodules. The study was focused on 39 surgical specimens including normal livers as controls, dysplastic nodules, early HCC and the progressed HCC. Profound alterations of the biochemical composition of the pathological liver were demonstrated by FTIR microspectroscopy. Indeed, dramatic changes were observed in lipids, proteins and sugars highlighting the metabolic reprogramming in carcinogenesis. The major changes were observed in the frequency domain 950-1480 cm-1 in which several bands allowed significant discrimination of cirrhotic nodules, dysplastic lesions and HCC. Finally, a significant discrimination between benign, dysplastic nodules and early HCC remained possible using a FTIR microscope equipped with a laboratory-based infrared source that can be easily implemented in hospital environment. In conclusion, our study positions FTIR microspectroscopy as a versatile and powerful approach for investigating liver diseases, such as steatosis, dysplastic lesions and cancer. Further studies on larger series of patients as well as on biopsies will allow confirming the clinical reliability of such spectral signatures. Therefore, we anticipate that FTIR microspectroscopy will open new avenue in clinical diagnosis

Intra-arterial or percutaneous locoregional therapies (LRT) are often employed to maintain potential liver transplant (LT) recipients with hepatocellular carcinoma (HCC) within T2/Milan criteria. Predictors of survival when LRT is used as destination therapy in those who are either ineligible or unwilling for LT remain poorly defined. We evaluated predictors of 3-year survival with destination LRT in a population of cirrhotic patients diagnosed with HCC, presenting within T2 criteria, and either ineligible or unwilling for LT. The cohort surviving 3 years had a significantly lower model for end-stage liver disease (MELD) score at HCC diagnosis (9.7 vs. 11.4, P= 0.037) and MELD following initial locoregional therapy (10.7 vs. 13.3, P= 0.008) compared to those not surviving three years despite similar demographic, tumor, and treatment variables. LRT as destination therapy results in modest intermediate term survival, with liver function at presentation and immediately following initiation of LRT predicting intermediate survival with this approach.

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l’aide d’une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d’étudier leur force d’activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d’une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l’expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L’expression de la protéine Us3 est placée sous contrôle d’un promoteur cellulaire spécifique d’HCC. L’hypothèse est qu’en l’absence d’activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l’apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d’HCC et cellules hepatocyte-like<br>Our long-term purpose is to develop transcriptionally targeted oncolytic vectors, derived from herpes simplex virus type 1 (HSV-1), designed to eradicate hepatocellular carcinomas (HCC). We have identified several HCC-specific promoters, as well as other cancer-specific promoters, that maintain their specificity when expressed from the virus genome. More precisely, we have demonstrated that these promoters are able to drive reporter gene (luciferase) expression from the virus genome in HCC-derived cells, both in cultured cells and in nude mice, but not in fresh human hepatocytes or in the WRL38 hepatocyte-like cells. HSV-1 infection induces, but then inhibits, a cellular antiviral apoptotic response, and the early virus protein US3 is a key actor in inhibiting apoptosis. We have hypothesized that inhibition of US3 expression in hepatocytes should led to early apoptotic death of these cells, therefore precluding virus multiplication and spread. In contrast, expression of US3 in cancer cells is expected to block apoptosis, leading to the achievement of the virus life cycle, cell lysis, and virus spread within the tumours. We report in this communication the construction and properties of two different potentially oncolytic HSV-1 vectors. One of them expresses US3 protein under the control of the HCC-specific promoter ANGPTL3, while the second promoter contains 9 repeats of the hypoxia responsive elements of vascular-endothelial growth factor (VEGF) (9xHRE promoter). Growth curves of these viruses were performed on different HCC cell lines to show their oncolytic properties

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

Proposal and initial experience with new surgical techniques and technologies for extending hepatocellular carcinoma treatment beyond actual indications. Laparoscopic hepatic resections in cirrhotic patients with HCC. Extreme hepatic resections in cirrhotic patients with advanced HCC: a new surgical approach.

Functional characterization of a novel genetic variant predisposing to advanced fibrosis and hepatocellular carcinoma development in nonalcoholic fatty liver disease Introduzione La steatosi epatica non alcoolica (NAFLD) rappresenta la più comune tra le malattie epatiche croniche nei paesi occidentali e rappresenta una causa emergente di cirrosi epatica e carcinoma epatocellulare (HCC). Numerosi studi hanno sottolineato l'importanza di vari tratti ereditari nel modificare la suscettibilità e il decorso di questa malattia. Tuttavia, la componente ereditabile di NAFLD resta in larga misura sconosciuta. Scopo dello studio Nell’ipotesi che una componente dell’ereditarietà della NFLD sia rappresentata da varianti genetiche rare con un forte impatto sull’attività proteica, il primo obiettivo di questo studio è stato quello di identificare, mediante Whole Exome Sequencing (WES), nuove varianti genetiche rare che determinassero un'alterazione dell'attività proteica e si associassero agli stadi avanzati della NAFLD. Abbiamo rilevato un'associazione tra una variante nel gene interferon regulatory factor 3 (IRF3) e lo sviluppo di carcinoma epatocellulare (HCC) correlato alla NAFLD. Abbiamo successivamente esaminato la regolazione specifica delle isoforme di IRF3 nei pazienti a rischio di NAFLD in relazione al danno epatico, abbiamo cercato di comprendere il ruolo di IRF3 nella biologia dell’epatocita e, infine, di mimare l'impatto della perdita di funzione dei trascritti alternativi di IRF3 sfruttando l'approccio di CRISPR/Cas9 genome editing in modelli in vitro. Infine, abbiamo testato l'impatto dell'inibizione della via di IRF3 sul tasso di proliferazione di cellule epatiche (HepG2) da parte dell'amlexanox. Quest’ultimo è un inibitore dell'asse TBK1-IRF3 in studio per il trattamento delle complicanze legate all'obesità.   Metodi e Pazienti • Abbiamo effettuato il sequenziamento dell'intero esoma (WES) di 72 pazienti NAFLD-HCC italiani e 50 individui sani. I pazienti HCC sono stati confrontati con la popolazione europea (Europei non finlandesi inclusi nell’ Exome Aggregation Consortium, N = 33,370). • Le associazioni sono state validate in un altro gruppo di pazienti con HCC correlato a NAFLD (N = 105), in 211 pazienti con fibrosi avanzata (N = 211) e altri 270 individui Italiani sani. Inoltre, è stato utilizzato un database più ampio per valutare la popolazione europea (genome aggreagation consortium, N = 64.603). • Sono state eseguite analisi trascrittomiche su 125 soggetti gravemente obesi (coorte trascrittomica) sottoposti a biopsia epatica percutanea eseguita durante un intervento chirurgico bariatrico. L'RNA-Seq è stato eseguito su RNA estratto da biopsie epatiche congelate rapidamente. • Per valutare l'effetto di IRF3-CL è stata introdotta una variante somatica nell'accettore di splicing dell’esone 7 specifico di IRF3-CL. In breve, abbiamo generato una linea di cellule HepG2 esprimente Cas9 sotto il controllo di un promotore inducibile da dxiciclina usando vettori lentivirali (Dharmacon, Lafayette, U.S.A.). L’RNA guida specifico è stato disegnato utilizzando il CRISPR design tool (http://crispr.mit.edu/) e clonato in un vettore di espressione (Addgene # 51133). Infine, le cellule trattate con doxiciclina sono state trasfettate con il vettore di espressione. Popolazioni clonali derivate da singole cellule sono state ottenute con il metodo della diluizione limite. In seguito, la presenza di mutazioni nel locus specificato è stata studiata mediante test nucleasi T7 (New England Biolabs, Ipswich, USA) e ulteriormente confermata con sequenziamento diretto (metodo di Sanger).   Risultati La variante IRF3 rs141490768 è risultata più frequente nel gruppo di pazienti con HCC rispetto alla popolazione europea (OR = 37,1, p = 4,5 * 10-7) e, allo stesso modo, era più frequente nei pazienti con fibrosi avanzata (N = 211, p = 0,049; OR = 5,8; IC al 95% = 0,7-21). Considerando tutti i pazienti con epatopatia avanzata (N = 388), l'associazione è rimasta fortemente significativa (OR = 11; IC 95% = 4-24; p = 6,16 * 10-6). rs141490768 codifica una variante loss-of-function (A418T) nell'isoforma regolatoria IRF3-CL, suggerendo che una maggiore attività dell'IRF3 può predisporre al NAFLD-HCC. Inoltre, anche due trascritti non codificanti a funzione sconosciuta (rispettivamente IRF3-NC1 e IRF3-NC2) si sovrappongono al locus rs141490768. Mediante Burden test abbiamo riscontrato che vi era un arricchimento in varianti rare che alterano specificamente IRF3-CL sia nei pazienti con HCC (p = 5,69 * 10-7; OR = 35,5; IC al 95% = 11-90) che in quelli con fibrosi avanzata (OR = 6,4; IC al 95% = 0,8-24; p = 0,04). Le analisi di trascrittomica hanno rivelato livelli elevati di espressione di IRF3, IRF3-CL e IRF3-NC1. Inoltre, i livelli di mRNA di IRF3-CL erano direttamente correlati con lo stadio della malattia, mentre quelli di IRF3-NC1 erano inversamente correlati (β = 1,3 e -0,77, rispettivamente; p &lt;0,05, entrambi). Mediante analisi immunoistochimica abbiamo evidenziato un’overespressione e iperattivazione di IRF3 in base alla severità del danno epatico. Inoltre, IRF3 è risultato overespresso nel tessuto tumorale rispetto al tessuto sano in una coorte di 20 pazienti del database TCGA e in campioni istologici. Sfruttando modelli in vitro abbiamo visto come l'esposizione ad acidi grassi liberi riesca attivare IRF3 in cellule HepG2, supportando il suo coinvolgimento nella patogenesi NAFLD. Allo stesso modo, anche stimoli proliferativi erano in grado di attivare questo fattore trascrizionale. Per mimare l'impatto della perdita di funzione di IRF3-CL e valutarne l’effetto sulla biologia degli epatociti, abbiamo sviluppato una linea di HepG2 parzialmente difettiva per IRF3-CL. Come previsto, le cellule IRF3-CL +/- presentavano un’attivazione di IRF3 più sostenuta in risposta al siero. Inoltre l'esposizione delle HepG2 all'amlexanox, da solo in combinazione con sorafenib, risultava in grado di ridurre il tasso di crescita cellulare. Questo effetto risultava più evidente nelle cellule IRF3-CL+/-.   Conclusioni In conclusione, la variante IRF3 rs141490768 è associata ad un aumentato rischio di sviluppare stadi avanzati di NAFLD. Inoltre, l'overespressione di IRF3 è stata associata alla gravità della malattia epatica, in parallelo con l'induzione della risposta infiammatoria, l’alterazione del metabolismo lipidico e la proliferazione cellulare. Anche se saranno necessari ulteriori studi meccanicistici per chiarire la complessa rete di interazioni tra le varie isoforme di IRF3, i nostri esperimenti in vitro confermano che IRF3-CL esercita un effetto inibitorio sull'attivazione dell'isoforma principale, che induce la proliferazione cellulare. Complessivamente, i dati suggeriscono che il meccanismo alla base dell'associazione della variante rs141490768 con la progressione NAFLD a fibrosi grave e HCC è correlato a un’attivazione aberrante della via di IRF3. Infine, i risultati supportano la necessità di ulteriori studi per esaminare il possibile ruolo dell'amlexanox (o altri inibitori Ikk-epsilon) nel trattamento della NAFLD e indicano questa classe di farmaci come buoni candidati da valutare ulteriormente per il trattamento di NAFLD HCC in combinazione con sorafenib.<br>Functional characterization of a novel genetic variant predisposing to advanced fibrosis and hepatocellular carcinoma development in nonalcoholic fatty liver disease Introduction Nonalcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in the Western countries and represent an emerging cause of liver cirrhosis and hepatocellular carcinoma (HCC). Several studies underlined the importance of heritability in modifying the susceptibility and progression of NAFLD. However, the specific determinants of NAFLD heritability remain largely unkonwn. Aims In the hypothesis that rare genetic variants with a strong impact on protein activity account for a fraction of NAFLD missing heritability, the first aim of the current study was to identify by Whole Exome Sequencing (WES) novel rare genetic variants determining an alteration of protein activity associated with advanced stage NAFLD. As we detected an association with a variant in Interferon regulatory protein 3 (IRF3) and hepatocellular carcinoma (HCC) related to NAFLD, we next examined the specific regulation of IRF3 isoforms in patients at risk of NAFLD in relation to liver damage, and tried to understand IRF3 regulation and to model the impact of IRF3 alternative transcripts downregulation by exploiting CRISPR/Cas9 gene editing approach in in vitro models. Finally, we tested the impact of IRF3 pathway inhibition by amlexanox, a TBK1-IRF3 axis inhibitor under study for the treatment of obesity related complications, on the proliferation of hepatoma cells (HepG2).   Patients and methods • Variants discovery was performed by WES in 72 Italian NAFLD-HCC patients and 50 healthy individuals. HCC patients were compared to those in the European population (Exome Aggregation Consortium Non-Finnish Europeans, N=33,370). • Validation was performed in NAFLD HCC patients (N=105) and in 211 patients with Advanced fibrosis (N=211), further 270 Italian healthy individuals. Furthermore, a larger database was used to evaluate the European population (genome aggregation consortium, N=64,603). • Transcriptomic analyses were performed on 125 severely obese individuals (Transcriptomic cohort) who underwent to percutaneous liver biopsy performed during bariatric surgery. Bulk RNA-Seq was performed on RNA extracted from flash-frozen liver biopsies. • To evaluate the effect of IRF3-CL a somatic variant in the IRF3-CL specific exon 7 splicing acceptor was introduced. Briefly, a Doxycycline (Therm-Fisher, Waltham, US) inducible Cas9 expressing HepG2 cell line was produced by lentiviral infection exploiting was produced exploiting Edit-R Inducible Lentiviral Cas9 Nuclease vectors (Dharmacon, Lafayette, U.S.A.). Specific guide RNA was designed using CRISPR design tool (http://crispr.mit.edu/) and cloned into an sgRNA expression vector (Addgene #51133) and doxycycline treated cells were transfected with the expression vector. Single cell derived populations were obtained by limiting diluition method and presence of mutations in the specified locus was investigated by T7 nuclease assay (New England Biolabs, Ipswich, US) and further confirmed by Sanger sequencing.   Results The rs141490768 SNP in IRF3 resulted enriched in HCCs compared with European population (OR=37.1, p=4.5*10-7). We validated this association in the replication cohort (N=211, p=0.049; OR=5.8; 95% CI = 0.7-21). In the overall series of patients with advanced NAFLD (N=388), the association remained significant (OR=11; 95% CI= 4-24; p=6.16*10-6). The rs141490768 encodes a loss-of-function variant (A418T) in the IRF3 inhibitory isoform IRF3-CL, suggesting that increased IRF3 activity may predispose to NAFLD-HCC. Furthermore, two non-coding transcripts of unknown function (referred as IRF3-NC1 and IRF3-NC2, respectively) also overlap with the rs141490768 locus. Burden test analysis revealed enrichment in rare variants altering specifically IRF3-CL in both our HCC discovery (p=5.69*10-7; OR= 35.5; 95% CI = 11-90) and the patients of our advanced fibrosis cohort with available WES data (OR=6.4; 95% CI= 0.8-24; p=0.04). Transcriptomic analyses revealed high expression levels of IRF3, IRF3-CL, and IRF3-NC1. Moreover, the IRF3-CL mRNA levels were directly correlated with the disease stage, whereas those of IRF3-NC1 were inversely correlated (β=1.3 and -0,77, respectively; p&lt;0.05, both). Immunohistochemical analysis revealed overexpression and hyperactivation of IRF3 according to NAFLD transition to fibrogenic steatohepatitis and HCC. Furthermore, IRF3 was overexpressed in tumor tissue compared with healthy tissue in a cohort of 20 patients of the TCGA database, and in histological samples. In HepG2 hepatoma cells, exposure to free fatty acids triggered IRF3 activation, supporting its involvement in NAFLD pathogenesis. Furthermore, IRF3 was also responsive to proliferation stimuli. To model the impact of IRF3-CL loss of function effect on hepatocyte biology, we developed IRF3-CL+/- HepG2 by CRISPR-Cas9 genome editing. As expected, IRF3-CL+/- HepG2 more sustained IRF3 activation in response to serum. Exposure of HepG2 to amlexanox, alone in combination with sorafenib, was able to impair cell growth rate, but more so in IRF3-CL+/- cells.   Conclusion In conclusion, the rs141490768 IRF3 variant was associated with an increased risk to develop advanced NAFLD. Moreover, IRF3 upregulation was associated liver disease severity, in parallel with induction of the inflammatory response, altered lipid metabolism and cell proliferation. Even if further mechanistic studies are required to clarify the complex network of interactions among alternative IRF3 transcripts, our in vitro experiments confirm that IRF3-CL exerts an inhibitory effect on the activation of the main IRF3 isoform, which promotes cell proliferation. Altogether, data suggest that the mechanism underpinning the association of the rs141490768 variant with NAFLD progression to severe fibrosis and HCC is related to facilitation of IRF3 pathway activation. Finally, results support the necessity of further studies to examine the possible role of amlexanox (or other Ikk-epsilon inhibitors) in NAFLD treatment and points out this class of chemicals as good candidates to be further evaluated for the treatment of NAFLD HCC in combination with Sorafenib.

CHARACTERIZATION OF STAPHYLOCOCCAL NUCLEASE AND TUDOR DOMAIN CONTAINING PROTEIN 1 (SND1) AS A MOLECULAR TARGET IN HEPATOCELLULAR CARCINOMA AND NON-ALCOHOLIC STEATOHEPATITIS Nidhi Jariwala, PhD A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Life Sciences Virginia Commonwealth University, 2017 Devanand Sarkar, M.B.B.S., PhD. Associate Professor, Department of Human and Molecular Genetics Virginia Commonwealth University Richmond, Virginia SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). In the present study, we characterize oncogenic functions of SND1 in HCC employing a novel transgenic mouse model (Alb/SND1) and present SND1 as a potential molecular target in HCC management. We show that Alb/SND1 mice develop spontaneous HCC with partial penetrance and exhibit more highly aggressive HCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibit a relative increase in inflammatory markers and spheroid-generating tumor initiating cells (TiC). Mechanistic investigations defined roles for Akt and NF-κB signaling pathways in promoting TiC formation in Alb/SND1 mice. Intravenous administration of the selective SND1 inhibitor 3', 5'-deoxythymidine bisphosphate (pdTp) inhibited tumor formation without effects on body weight or liver function. We conclude that SND1 drives pro-oncogenic transcriptomic and proteomic changes in hepatocyte resulting in aggressive HCC. SND1 specific RNA interactome is identified with RNA immunoprecipitation sequencing (RIPSeq) approach. With an adjusted p value of2-fold enrichment over control, 282 mRNAs were identified to significantly associate with SND1 protein. We focused on the tumor suppressor Protein Tyrosine Phosphatase non-receptor type 23 (PTPN23) because its regulation by SND1 and its role in HCC are not known. In current study, we confirm that SND1 post-transcriptionally downregulates PTPN23. Pursuing functional studies with tetracycline inducible overexpression system, we validate that PTPN23 inhibits tyrosine kinase signaling, proliferation, epithelial to mesenchymal transition, migration, invasion and in vivo tumorigenesis. Alb/SND1 mice also manifest steatosis and fibrosis at one year of age. Coupled with a pro-inflammatory hepatic phenotype, we conclude that Alb/SND1 livers present NASH. High fat diet causes severe NASH and aggressive NASH induced HCC in Alb/SND1 mice. Serum and hepatic lipid profiling shows that hepatocyte specific SND1 overexpression associate with elevated triglyceride and cholesterol LDL levels. Contrarily, hepatocyte specific deletion of SND1 (SND1ΔHEP) in vivo, significantly protects against age dependent steatosis. Association of SND1 in NASH pathology is novel discovery and we present preliminary evidence confirming role of SND1 in promoting NASH.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world and accounts for the vast majority of all liver cancers. HCC develops in response to various factors including viral infections, aflatoxin, alcohol and metabolic diseases. Recent studies have highlighted substantial differences in the acquired genomic alterations depending on the causative agent. Despite such a mutagen-dependent genetic heterogeneity, HCC is almost invariably associated with an underlying inflammatory state, whose direct contribution to the acquisition of critical genomic changes is not yet clear. The aim of my PhD project has been to understand how chronic inflammation and fibrosis affect the cancer genome. We mapped the acquired genomic alterations in human and mouse HCCs induced by defects in hepatocyte biliary transporters. These HCCs arise as a result of chronic exposure to non-neutralized bile acids that cause the onset of chronic inflammation and develop into cancer in the absence of exogenous direct (viruses) or indirect (alcohol) mutagens. We first studied the mutational landscapes of human and mouse cancer genomes and found a surprisingly low number of somatic point mutations with no impairment of cancer genes. We next studied the acquisition of somatic copy number variations (CNVs) and used well-established approaches for detecting CNVs from SNP arrays and whole genome sequencing data. We also developed a novel method, GeneCNV, for the identification of CNVs from targeted re-sequencing screenings. Overall, we observed the acquisition of massive gene copy number gains and rearrangements in both human and mouse HCCs. Amplifications preferentially occurred at late stages of cancer development and frequently targeted the mitogen-activated protein kinase (MAPK) signalling pathway, in particular, direct regulators of c-Jun NH2-terminal kinases (JNKs). We showed that that pharmacological inhibition of JNK impairs the adenoma-to- carcinoma progression in mouse. This suggests that JNK inhibition may be a useful therapeutic approach to block HCC onset in bile salt export pump (BSEP) deficiency patients waiting for liver transplantation. Altogether, this study showed that human BSEP-HCCs and mouse Mdr2-KO HCCs acquire a similar genomic signature, thus highlighting the remarkable analogy between human and mouse tumours with similar etiopathogenesis. This genomic signature differs from that of other HCCs profiled so far, which were for the most part virus induced. This demonstrates that HCC in the absence of external agents develops through genomic alterations that can be clearly distinguished from those determined by other etiological factors.

First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.

Background and aims: Sorafenib is the reference therapy for advanced Hepatocellular Carcinoma (HCC). No method exists to predict in the very early period subsequent individual response. Starting from the clinical experience in humans that subcutaneous metastases may rapidly change consistency under sorafenib and that elastosonography a new ultrasound based technique allows assessment of tissue stiffness, we investigated the role of elastonography in the very early prediction of tumor response to sorafenib in a HCC animal model. Methods: HCC (Huh7 cells) subcutaneous xenografting in mice was utilized. Mice were randomized to vehicle or treatment with sorafenib when tumor size was 5-10 mm. Elastosonography (Mylab 70XVG, Esaote, Genova, Italy) of the whole tumor mass on a sagittal plane with a 10 MHz linear transducer was performed at different time points from treatment start (day 0, +2, +4, +7 and +14) until mice were sacrified (day +14), with the operator blind to treatment. In order to overcome variability in absolute elasticity measurement when assessing changes over time, values were expressed in arbitrary units as relative stiffness of the tumor tissue in comparison to the stiffness of a standard reference stand-off pad lying on the skin over the tumor. Results: Sor-treated mice showed a smaller tumor size increase at day +14 in comparison to vehicle-treated (tumor volume increase +192.76% vs +747.56%, p=0.06). Among Sor-treated tumors, 6 mice showed a better response to treatment than the other 4 (increase in volume +177% vs +553%, p=0.011). At day +2, median tumor elasticity increased in Sor-treated group (+6.69%, range –30.17-+58.51%), while decreased in the vehicle group (-3.19%, range –53.32-+37.94%) leading to a significant difference in absolute values (p=0.034). From this time point onward, elasticity decreased in both groups, with similar speed over time, not being statistically different anymore. In Sor-treated mice all 6 best responders at day 14 showed an increase in elasticity at day +2 (ranging from +3.30% to +58.51%) in comparison to baseline, whereas 3 of the 4 poorer responders showed a decrease. Interestingly, these 3 tumours showed elasticity values higher than responder tumours at day 0. Conclusions: Elastosonography appears a promising non-invasive new technique for the early prediction of HCC tumor response to sorafenib. Indeed, we proved that responder tumours are characterized by an early increase in elasticity. The possibility to distinguish a priori between responders and non responders based on the higher elasticity of the latter needs to be validated in ad-hoc experiments as well as a confirmation of our results in humans is warranted.

Background and aims: Sorafenib is the reference therapy for advanced Hepatocellular Carcinoma (HCC). No method exists to predict in the very early period subsequent individual response. Starting from the clinical experience in humans that subcutaneous metastases may rapidly change consistency under sorafenib and that elastosonography a new ultrasound based technique allows assessment of tissue stiffness, we investigated the role of elastonography in the very early prediction of tumor response to sorafenib in a HCC animal model. Methods: HCC (Huh7 cells) subcutaneous xenografting in mice was utilized. Mice were randomized to vehicle or treatment with sorafenib when tumor size was 5-10 mm. Elastosonography (Mylab 70XVG, Esaote, Genova, Italy) of the whole tumor mass on a sagittal plane with a 10 MHz linear transducer was performed at different time points from treatment start (day 0, +2, +4, +7 and +14) until mice were sacrified (day +14), with the operator blind to treatment. In order to overcome variability in absolute elasticity measurement when assessing changes over time, values were expressed in arbitrary units as relative stiffness of the tumor tissue in comparison to the stiffness of a standard reference stand-off pad lying on the skin over the tumor. Results: Sor-treated mice showed a smaller tumor size increase at day +14 in comparison to vehicle-treated (tumor volume increase +192.76% vs +747.56%, p=0.06). Among Sor-treated tumors, 6 mice showed a better response to treatment than the other 4 (increase in volume +177% vs +553%, p=0.011). At day +2, median tumor elasticity increased in Sor-treated group (+6.69%, range –30.17-+58.51%), while decreased in the vehicle group (-3.19%, range –53.32-+37.94%) leading to a significant difference in absolute values (p=0.034). From this time point onward, elasticity decreased in both groups, with similar speed over time, not being statistically different anymore. In Sor-treated mice all 6 best responders at day 14 showed an increase in elasticity at day +2 (ranging from +3.30% to +58.51%) in comparison to baseline, whereas 3 of the 4 poorer responders showed a decrease. Interestingly, these 3 tumours showed elasticity values higher than responder tumours at day 0. Conclusions: Elastosonography appears a promising non-invasive new technique for the early prediction of HCC tumor response to sorafenib. Indeed, we proved that responder tumours are characterized by an early increase in elasticity. The possibility to distinguish a priori between responders and non responders based on the higher elasticity of the latter needs to be validated in ad-hoc experiments as well as a confirmation of our results in humans is warranted.

Die vorliegende Arbeit beschäftigt sich mit einem neuartigen proteinbasierten, suizidgentherapeutischen Ansatz zur sicheren und effektiven Behandlung von soliden Tumoren. Verwendet wurden zellpermeable Fusionsproteine auf der Grundlage des bakteriellen Enzyms Cytosin Desaminase, welches spezifisch die Umsetzung der inaktive, nichttoxische Substanz (Prodroge) 5-Fluorcytosin in den hochwirksamen, stark toxischen Wirkstoff 5-Fluoruracil katalysiert. Dieser bewirkt die selektive Zerstörung von Tumorzellen. Durch die Fusion der bakteriellen Cytosin Desaminase (bCD) mit der Sequenz des Zellpermeabilität vermittelnden Peptides HBV-Translokationsmotiv (TLM) des Hepatits B-Virus (HBV) wurden zunächst zellpermeable E.coli Cytosin Desaminase Suizidfusionskonstrukte generiert. Für die bakteriell synthetisierten HBV-TLM-Fusionsproteine konnten eine Hexamerisierung sowie eine spezifische enzymatische Aktivität bei der Umsetzung von Cytosin zu Uracil als strukturelle und funktionelle Voraussetzungen für einen Einsatz in der Suizidgentherapie nachgewiesen werden, die vergleichbar mit dem wt-Protein waren. Bei Versuchen zur Internalisierung der zellpermeablen Fusionsproteine wurde für die Fusionsproteine mit C-terminal fusioniertem HBV-TLM (bCD-HBV-TLM) eine Aufnahme in das Zytoplasma von Hepatomzellen mittels konfokaler Laserscanmikroskopie und differentieller Zellfraktionierung nachgewiesen, nicht jedoch für Fusionsproteine mit N-terminalem HBV-TLM (HBV-TLM-bCD). Die gezeigte Internalisierung des Proteins HBV-TLM-bCD erfolgte effizient und schnell und war unabhängig vom endosomalen Aufnahmeweg. Bei der nachgewiesenen Translokalisation blieb die enzymatische, suizidgentherapeutische Aktivität des zellpermeablen Suizidproteins (HBV-TLM-bCD), d.h. die katalytische Wirkung bei der Umsetzung der Prodroge 5-Fluorcytosin vollständig erhalten, so dass sich dieses Fusionsprotein für einen therapeutischen Einsatz in der Suizidgentherapie eignet. Zusätzlich zur antitumoralen Wirkung können durch einen gezielten, lokal begrenzten therapeutischen Einsatz der vorgestellten zellpermeablen bCD-HBV-TLM-Fusionsproteine starke Nebenwirkungen, wie sie bei einer konventionellen Chemotherapie zu beobachten sind, weitgehend vermieden werden.<br>This work investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors. The concept of the used suicide gene therapy system is based mainly on the transfer of the cell permeable bacterial suicide enzyme cytosine deaminase which specifically convert the inactive, non-toxic prodrug 5-fluorocytosine into the toxic metabolite 5-fluorouracil finally executing the efficient destruction of tumor cells. Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus (HBV), E.coli cytosine deaminase (bCD) suicide fusion proteins were generated. HBV-TLM fusion proteins formed hexamers (as do parental wt bCD) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtbCD protein. However, only bCD-HBV-TLM fusion proteins, but not HBV-TLM-bCD fusion proteins were found to be taken up to the cytoplasm of target hepatoma cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of bCD-HBV-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since bCD-HBV-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-fluorocytosine strongly is suggested. Future therapeutic local application of cell permeable bCD-HBV-TLM fusion proteins together with a systemic 5-fluorocytosine prodrug application could result in profound antitumor activities without apparent side effects.

BACKGROUND AND AIMS: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia and centromers. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The αvβ6 integrin mediates local activation of TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in Pkhd1del4/del4 mice. METHODS: In Pkhd1del4/del4 mice we studied, at different ages (1-12 months): a) portal fibrosis (Sirius Red) and portal hypertension (spleen weight/body weight); b) αvβ6 mRNA and protein expression (RT-PCR, IHC); c) α-SMA and TGFβ1 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45 and FACS analysis of whole liver infiltrate); f) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); g) cytokine effects on monocyte/macrophage proliferation (MTS assay) and migration (Boyden chamber); h) TGFβ1 and TNFα effects on β6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFβ receptor type II (TGFβRII); i) TGFβ1 effects on collagen type I (COLL1) mRNA expression by cultured cholangiocytes. RESULTS: Pkhd1del4/del4 mice showed a progressive increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1 showed a similar age-dependent increase. FACS analysis showed that 50-75% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted from the basolateral side significantly increased amounts of CXCL1 and CXCL10 (p<0.05). Both cytokines were able to stimulate macrophage migration (p<0.05). Basal expression of β6 mRNA by cultured Pkhd1del4/del4 cholangiocytes (0.015±0.002 2^-dCt) was potently stimulated by the macrophage-derived cytokines TGFβ1 (0.017±0.002 2^-dCt, p<0.05) and TNFα (0.018±0.003 2^-dCt, p<0.05). Inhibition of TGFβRII completely blunted TGFβ1 (0.014±0.003 2^-dCt, p<0.05) but not TNFα effects (0.017±0.001 2^-dCt, p=ns) on β6 mRNA. COLL1 mRNA expression by cultured Pkhd1del4/del4 cholangiocytes (0.0009±0.0003 2^-dCt) was further and significantly increased after TGFβ1 stimulation (0.002±0.0005 2^-dCt, p<0.05). CONCLUSIONS: Pkhd1del4/del4 cholangiocytes possess increased basolateral secretory functions of chemokines (CXCL1, CXCL10) able to orchestrate macrophage homing to the peribiliary microenvironment. In turn, by releasing TGFβ1 and TNFα, macrophages up-regulate αvβ6 integrin in Pkhd1del4/del4 cholangiocytes. αvβ6 integrin activates latent TGFβ1, further increasing the fibrogenic properties of cholangiocytes.

Le Carcinome Hépatocellulaire a une incidence mondiale élevée et est associé à un mauvais pronostic. Les traitements curatifs existants ne sont applicables qu’à une minorité de patients. La radiothérapie sélective interne (SIRT) est un traitement palliatif de plus en plus utilisé. Elle consiste à l’injection sélective intra-tumorale de microsphères d’yttrium-90 par infusion intra-artérielle, et repose sur deux étapes : une étape pré-thérapeutique de simulation du traitement avec l’injection de macroagrégats d’albumines marqués au 99mTc et le traitement en lui-même. Cependant les caractéristiques de ces deux vecteurs diffèrent et peuvent conduire à des variations de biodistribution et à une dosimétrie approximative. Ce travail a pour but de développer un vecteur radiothéranostique unique pour la SIRT : les microparticules à base d’amidon (SBMP), afin de pallier aux différents problèmes rencontrés en clinique. L’optimisation du radiomarquage par le 68Ga et le 188Re sous forme de kits lyophilisés prêts-à-l’emploi, a permis d’obtenir une pureté radiochimique &gt; 98 % et &gt; 95 % respectivement. Une étude préliminaire par imagerie TEP/TDM in vivo chez le rat, suite à l’injection intraartérielle des 68Ga-SBMP a montré une biodistribution spécifique des microparticules avec plus de 95 % de l’activité retrouvée dans le foie et plus particulièrement dans les tumeurs. Les SBMP offrent plusieurs avantages répondant à différents problèmes actuels et constituent un agent théranostique prometteur pour la SIRT. Une présentation de la SIRT, des différentes microparticules en développement pour la SIRT et des modèles animaux de tumeur hépatique existants seront également développées dans ce travail<br>The Hepatocellular Carcinoma has a high incidence worldwide and is associated with a bad prognostic. The existing curative treatments can only be apply in a minority of cases. The selective internal radiation therapy (SIRT) is a palliative treatment that is increasingly used. This technique is define by the selective intratumoral injection of yttrium-90microspheres via intra-arterial infusion. It involves two steps : a pre-therapeutic one for treatment simulation purpose with the injection of serum albumin macroaggregates radiolabeled with 99mTc and the treatment itself. However the characteristics of these two vectors are different and can lead to variations in biodistribution and approximate dosimetry. This works aims to develop a unique radiotheranostic vector for the SIRT: the starch-basedmicroparticles (SBMP), in order to overcome the different currents clinical problems. The optimization of the radiolabeling by the 68Ga and the 188Re in the form of ready-to-use radiolabeling kits allowed to obtain a radiochemical purity &gt; 98 % and &gt; 95 % respectively. A preliminary in vivo study by PET/CT imaging in rat, following the intra-arterial injection of 68Ga-SBMP displayed a specific biodistribution of the microparticles with more than 95 % of the activity found in the liver and mostly in the tumors. The SBMP offer several advantages that answer different current issues and area promising theranostic agent for the SIRT. A presentation of the SIRT, the different microparticles in development and the existing animal models of hepatic tumor will also be developed in this work

Fu Zhenming.<br>Thesis submitted in: December 2003.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (leaves 138-162).<br>Abstracts in English and Chinese.<br>List of abbreviations --- p.i<br>Abstract (in English) --- p.ii<br>摘要（中文） --- p.iii<br>Acknowledgement --- p.iv<br>Chapter Chapter 1. --- Introduction and Objectives of Study --- p.1<br>Chapter 1.1 --- Hepatocellular carcinoma in general --- p.2<br>Chapter 1.1.1 --- "Epidemiology, risk factors" --- p.2<br>Chapter 1.1.2 --- Pathology and staging --- p.4<br>Chapter 1.1.3 --- Treatment --- p.6<br>Chapter 1.1.4 --- Improvement of early detection and treatment of HCC --- p.7<br>Chapter 1.2 --- General aspects of mitochondria and mitochondrial DNA (mtDNA) --- p.10<br>Chapter 1.2.1 --- Structure and dynamics of mitochondria --- p.10<br>Chapter 1.2.1.1 --- General introduction of mitochondria --- p.10<br>Chapter 1.2.1.2 --- Respiration chain of mitochondria --- p.11<br>Chapter 1.2.2 --- The mitochondrial genome --- p.14<br>Chapter 1.2.2.1 --- Strucure --- p.14<br>Chapter 1.2.2.2 --- Genes for structure proteins --- p.16<br>Chapter 1.2.2.3 --- Genes for translation --- p.17<br>Chapter 1.2.2.4 --- Imported proteins and RNAs --- p.17<br>Chapter 1.2.3 --- Mitochondrial DNA maintenance --- p.19<br>Chapter 1.2.4 --- Mitochondrial DNA replication --- p.25<br>Chapter 1.2.5 --- Mitochondrial DNA transcription --- p.30<br>Chapter 1.2.6 --- Mitochondrial DNA translation --- p.32<br>Chapter 1.3 --- MtDNA diseases --- p.35<br>Chapter 1.4 --- MtDNA mutation and HCC --- p.35<br>Chapter 1.5 --- Aims of the study --- p.39<br>Chapter Chapter 2. --- Materials and Methods --- p.41<br>Chapter 2.1 --- Materials --- p.42<br>Chapter 2.1.1 --- Chemicals --- p.42<br>Chapter 2.1.2 --- Primers --- p.42<br>Chapter 2.1.3 --- Enzymes --- p.45<br>Chapter 2.1.4 --- Cell line --- p.45<br>Chapter 2.1.5 --- Collection of specimens --- p.46<br>Chapter 2.2 --- Methodology --- p.47<br>Chapter 2.2.1 --- "DNA extraction from hcc tissues, cell line Hep3B and PBMCs" --- p.47<br>Chapter 2.2.1.1 --- DNA extraction from HCC tissues --- p.47<br>Chapter 2.2.1.2 --- DNA extraction from cell line Hep3B --- p.49<br>Chapter 2.2.1.3 --- DNA extraction from and PBMCs --- p.50<br>Chapter 2.2.1.3.1 --- Preparation of PBMCs --- p.50<br>Chapter 2.2.1.3.2 --- DNA extraction from and PBMCs --- p.51<br>Chapter 2.2.2 --- Detection of mt whole genome mutation by direct sequencing --- p.51<br>Chapter 2.2.2.1 --- Design of mtDNA primers --- p.51<br>Chapter 2.2.2.2 --- PCR amplification of the whole mt genome --- p.51<br>Chapter 2.2.2.3 --- Direct sequencing of the whole mt genome --- p.52<br>Chapter 2.2.2.3.1 --- Primer used in sequencing --- p.52<br>Chapter 2.2.2.3.2 --- Purification of the PCR products of the whole mt genome --- p.53<br>Chapter 2.2.2.3.3 --- Dye terminator cycle sequencing reaction --- p.53<br>Chapter 2.2.2.3.4 --- Purification of extension products --- p.54<br>Chapter 2.2.3 --- Detection of mtDNA control region mutation --- p.55<br>Chapter 2.2.3.1 --- PCR amplification of D310 in the mtDNA control region --- p.55<br>Chapter 2.2.3.2 --- Screening of D310 mutation by PFLDA --- p.55<br>Chapter 2.2.3.2.1 --- Making 8% denatured gel mixture --- p.55<br>Chapter 2.2.3.2.2 --- Setting up and Pouring the denatured gel --- p.56<br>Chapter 2.2.3.2.4 --- Preparing and Loading the PCR products --- p.57<br>Chapter 2.2.3.2.5 --- Electrophoresis --- p.57<br>Chapter 2.2.3.2.6 --- "Gel fixing, silver staining and color development " --- p.58<br>Chapter 2.2.3.3 --- Direct sequencing of D310 in the mtDNA control region --- p.59<br>Chapter 2.2.4 --- Detection of mt DNA coding region mutation --- p.60<br>Chapter 2.2.4.1 --- PCR amplification of the 5 respiratory chain subunit genes --- p.60<br>Chapter 2.2.4.2 --- Restriction enzyme digestion of 5 genes in mtDNA coding region --- p.60<br>Chapter 2.2.4.3 --- Screening of mtDNA coding region mutation by SSCP --- p.61<br>Chapter 2.2.4.3.1 --- Making 6% 49:1 acrylamide/Bis SSCP gel mixture --- p.61<br>Chapter 2.2.4.3.2 --- "Setting up the SSCP gel, loading sample, fixing, staining and developing of the gel " --- p.62<br>Chapter 2.2.4.4 --- Sequencing conformation of the mtDNA coding region mutation --- p.62<br>Chapter 2.2.5 --- Statistics --- p.63<br>Chapter 2.2.5.1 --- The chi-square test --- p.63<br>Chapter 2.2.5.2 --- The Friedman test --- p.63<br>Chapter 2.2.5.3 --- Wilcoxon signed ranks test --- p.63<br>Chapter Chapter 3. --- Results --- p.64<br>Chapter 3.1 --- Detection mt DNA whole genome mutation --- p.65<br>Chapter 3.1.1 --- Identification of mtDNA whole genome by direct sequencing --- p.65<br>Chapter 3.2 --- Detection mt DNA D-loop mutation --- p.76<br>Chapter 3.2.1 --- Screening of C-tract alteration in HCC tissus by PCR fragments length detection assay (PFLDA) --- p.76<br>Chapter 3.2.2 --- Screening of coding region alteration in HCC tissues by SSCP --- p.77<br>Chapter 3.2.2.1 --- Identification of C-tract alterations in HCC and non-tumorous tissues by direct sequencing --- p.77<br>Chapter 3.2.3 --- Identification of C-tract alterations by direct sequencing --- p.82<br>Chapter 3.2.3.1 --- Identification of C-tract alterations in HCC tissues by direct sequencing --- p.82<br>Chapter 3.2.3.2 --- Identification of C-tract alteration in PBMC of normal subjects by direct sequencing --- p.82<br>Chapter 3.2.3.3 --- Identification of C-tract alteration in PBMC of HCC patients by direct sequencing --- p.82<br>Chapter 3.2.4 --- Statistics of the analysis of C-tract alterations --- p.82<br>Chapter 3.3 --- Detection mt DNA mutation in the coding region --- p.87<br>Chapter Chapter 4. --- Discussion --- p.98<br>Chapter 4.1 --- Detection mtDNA whole genome mutation --- p.99<br>Chapter 4.2 --- Detection mtDNA D-loop mutation --- p.107<br>Chapter 4.3 --- Detection mtDNA mutation in the coding region --- p.119<br>Chapter 4.4 --- Possible mechanisms of mtDNA mutation in HCC carcinogenesis --- p.125<br>Chapter 4.5 --- Proposals for prospective studies --- p.126<br>Chapter 4.5.1 --- Function of C7 in D310 --- p.128<br>Chapter 4.5.2 --- Function changes of mtDNA coding region mutation --- p.130<br>Chapter 4.5.3 --- Detection of D310 C-tract mutation in patients' plasma --- p.131<br>Chapter 4.5.4 --- Relationship between nMSl and mtMSI --- p.132<br>Chapter 4.6 --- Summary --- p.134<br>References --- p.137

博士<br>國立臺灣大學<br>生物化學暨分子生物學研究所<br>103<br>Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer and the third most common cause of cancer death worldwide. The progression of HCC relies on the formation of new blood vessels, and VEGF is critical in this process. Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and its expression level was positively correlated with that of VEGF-A. Additionally, L-FABP significantly promoted tumor growth and metastasis in a xenograft mouse model. We also studied the mechanisms of L-FABP activity in tumorigenesis: L-FABP was found to be associated with VEGFR2 on membrane rafts and subsequently activate the Akt/mTOR/P70S6K/4EBP1 and Src/FAK/cdc42 pathways. This resulted in up-regulation of VEGF-A expression accompanied by an increase in both angiogenic potential and migration activity. Taken together, our results suggest that L-FABP may be a potential target for HCC chemotherapy. Inhibition of VEGFR2 activity has been proposed as an important strategy for the clinical treatment of hepatocellular carcinoma (HCC). In this study, we identified corosolic acid (CA), which exists in the root of Actinidia chinensis (藤梨), as having a significant anti-cancer effect on HCC cells. We found that CA inhibits VEGFR2 kinase activity by directly interacting with the ATP binding pocket. CA down-regulates the VEGFR2/Src/FAK/cdc42 axis, subsequently decreasing F-actin formation and migratory activity of Huh7 cells in vitro. In an in vivo model, CA exhibites an effective dose (5 mg/kg/day) on tumor growth, and we further demonstrate that CA has a synergistic effect with sorafenib within a wide range of concentrations. In conclusion, we elucidate the effects and molecular mechanism for CA on HCC cells and suggest that CA could serve as a therapeutic or adjuvant target for patients with aggressive HCC.

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

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Current treatments are subpar, with late stage diagnosis and poor prognosis contributing to limited treatment options. The evolutionarily conserved, ubiquitously expressed transcription factor LSF is overexpressed in HCC, and its expression is positively correlated with disease severity. Certain small molecules, known as Factor Quinolinone Inhibitors (FQIs), specifically inhibit LSF DNA-binding activity, inhibit HCC cell proliferation in vitro and prevent tumor growth in an endogenous mouse liver cancer model without apparent toxicity. The targeting of transcription factors by small molecule inhibitors has been historically difficult (Dunker and Uversky, 2010), warranting further molecular investigation into the requirement for LSF in HCC to confirm that the anti-tumor effects of FQIs are the consequence of LSF inhibition. This body of work investigates a dual approach for inhibiting LSF function in order to determine the molecular consequences for HCC cells. To identify the specific point of the cell cycle where LSF is required for HCC proliferation, synchronous HCC cells were treated with FQI or with short interfering RNA to reduce levels of LSF. The results indicate that LSF is required for proper mitotic progression in HCC cells. Specifically, these data show a reduction of key mitotic regulators Aurora Kinase B and Cdc20, at the level of mRNA and protein expression. Time-lapse microscopy also demonstrated an increase in the time for progression through mitosis, with a prometaphase/metaphase delay. Immunofluorescence analysis revealed a prometaphase delay plus aberrant cell division and generation of multi-nucleated cells. These findings were consistent with both FQI1 treatment and RNA interference. Additionally, shorter incubation with FQI1 surprisingly revealed a distinct, non-transcriptional regulation of mitosis in HCC cells, suggesting that mitotic regulation by LSF is multi-faceted. As a targeted therapy for use in the clinic, the in vivo toxicity of FQIs is critical to investigate. Whole blood provides populations of rapidly dividing normal cells that can test susceptibility to anti-mitotic compounds. When mice were treated with FQI1, the blood analysis showed no toxicity. Taken together, these findings indicate that LSF is a mitotic regulator in HCC, further supporting the therapeutic promise of molecular therapies targeting LSF.<br>2019-03-04T00:00:00Z

碩士<br>國立臺灣大學<br>應用力學研究所<br>89<br>In this study，we measure the waveform of the blood velocity in common hepatic artery using Doppler ultrasound. Image processing and fast Fourier transform(FFT) were used to get peak velocity waveform of the normalized amplitude of individual harmonics. The harmonics of velocity waveform and RI etc. were investigated for the hemodynamics of hepatocellular carcinoma (HCC). The subjects studied were divided control group and HCC group. The Hcc group studied were further categorized into varies groups based on theirs age，sex，portal vein thrombosis(PVT)，liver cirrhosis，α-fetoprotein(αFP)，iodocyanide green(ICG) after 15 minutes and ICG after 20 minutes in turns. Results showed that the first harmonic values in control group were lower than those in HCC group(0.772±0.049 vs. 0.806±0.042，P<0.05). RI values in control group were higher than those in HCC group(0.781±0.026 vs. 0.688±0.040，P<0.01). The second harmonic values decreased with age. The first harmonic values in non-PVT were smaller than that in PVT group(0.790±0.084 vs. 0.842±0.043，p<0.01). The RI values in non-PVT were larger than that in PVT group(0.413±0.058 vs. 0.653±0.098，p<0.01). The first harmonic values in liver cirrhosis group were higher than those in liver non-cirrhosis (0.801±0.079 vs. 0.757±0.067，p<0.05). The first harmonic values in αFP<20 group were smaller than those inαFP>20 group(0.756±0.089 vs. 0.812±0.076，p<0.05). With single t-test statistics showed that velocity harmonics were better than RI，in the classifications of PVT，liver cirrhosis andαFP . However considering multi-variables statistics，RI was better in the classifications of PVT and tumor size.

碩士<br>大同大學<br>生物工程學系(所)<br>97<br>Hepatceullar carcinoma (HCC) is the leading and second cause of death in male and female of Taiwan; respectively, which indicated that HCC is one of the major threats to public health. Several evidences showed that viral factors, alcoholic assumption and drugs abuse led to malignant abnormalities of liver. Among these etiologies, viral factor, such as hepatitis virus B (HBV), caused most of malignant abnormalities of liver diseases in Taiwan, including acute and chronic hepatitis, cirrhosis, and HCC. Since HCC is still a main threat to public health until now, therefore we intended to global survey differential gene expression profile between HCC tumor tissues and its normal counterpart. To address this issue, patients with HCC tumor were enrolled (n=15, tumor size < 5cm) and analyzed transcriptome globally by Affymetrix U133A Chip. Our results showed that the most differential expressed (down-regulation) genes in HCC and its normal counterpart belonged to alcohol metabolism; such as: cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1), alcohol dehydrogenase 1A (class I), alpha polypeptide (ADH1A)、alcohol dehydrogenase 1B (class I), beta polypeptide (ADH1B). In addition, drug metabolism gene- nicotinamide N-methyltransferase (NNMT), iron binding genes-metallothionein (MT) were also found to be down-regulated in HCC tumor tissues. Using real-time PCR and immunohistochemistry, we found that expression of these genes was positively correlated with differentiation status of HCC. For example, over-expression of CYP2E1 was found in well-differentiated HCC tumor tissues; whereas in poor differentiated HCC, the expression of CYP2E1 is down-regulated in tumor tissues. Since the differentiation of tumor is significantly correlated with prognosis of HCC patients, therefore, the expression of these genes will be statistically analyzed with stage, survival rate, and clinical manifestations of HCC patients when the sample size of HCC patients is enlarged. These data will provide us a further insight about pathogenesis of HCC and its clinical relevance.

Lau Yee Lam.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.<br>Includes bibliographical references (leaves 147-157).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>Acknowledgements --- p.iv<br>Abbreviations --- p.v<br>List of Figures --- p.viii<br>List of Tables --- p.xi<br>Contents --- p.xii<br>Chapter Chapter 1 --- Introduction<br>Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1<br>Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1<br>Chapter 1.1.2 --- Etiology of HCC --- p.2<br>Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3<br>Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5<br>Chapter 1.1.5 --- Confirmation of candidate genes --- p.6<br>Chapter 1.2 --- Ras-Oncogene --- p.8<br>Chapter 1.2.1 --- Ras superfamily --- p.8<br>Chapter 1.2.1.1 --- Rho family --- p.9<br>Chapter 1.2.1.2 --- Rab family --- p.10<br>Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11<br>Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14<br>Chapter 1.3 --- RhoC --- p.16<br>Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16<br>Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19<br>Chapter 1.4 --- Rabl4 --- p.20<br>Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20<br>Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23<br>Chapter 1.5 --- Aims of study --- p.23<br>Chapter Chapter 2 --- Materials and Methods<br>Chapter 2.1 --- Materials --- p.25<br>Chapter 2.1.1 --- Cell lines --- p.25<br>Chapter 2.1.2 --- Cell culture reagents --- p.26<br>Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29<br>Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30<br>Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31<br>Chapter 2.1.6 --- Vectors for cloning --- p.39<br>Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39<br>Chapter 2.1.8 --- Restriction digestion reagents --- p.42<br>Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42<br>Chapter 2.1.10 --- Ligation reagents --- p.44<br>Chapter 2.1.11 --- Bacterial culture medium --- p.44<br>Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46<br>Chapter 2.1.13 --- Reagents for flow cytometry --- p.48<br>Chapter 2.1.14 --- Detection of apoptosis --- p.48<br>Chapter 2.2 --- Methods --- p.50<br>Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50<br>Chapter 2.2.1.1 --- cDNA preparation --- p.50<br>Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50<br>Chapter (2) --- Total RNA isolation --- p.50<br>Chapter (3) --- First-strand cDNA synthesis --- p.51<br>Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52<br>Chapter 2.2.1.3 --- Western blotting --- p.53<br>Chapter (1) --- Cell culture --- p.53<br>Chapter (2) --- Protein extraction --- p.53<br>Chapter (3) --- Quantification of proteins --- p.53<br>Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54<br>Chapter (5) --- Western blotting luminol detection --- p.56<br>Chapter 2.2.2 --- Cloning protocol --- p.57<br>Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57<br>Chapter 2.2.2.2 --- Purification of PCR product --- p.58<br>Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53<br>Chapter 2.2.2.4 --- Insert/vector ligation --- p.59<br>Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60<br>Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61<br>Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61<br>Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62<br>Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63<br>Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64<br>Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66<br>Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66<br>Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66<br>Chapter 2.2.3.3 --- DAPI staining --- p.67<br>Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68<br>Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68<br>Chapter 2.2.4 --- Analysis of cell cycle --- p.69<br>Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69<br>Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69<br>Chapter 2.2.5 --- Detection of apoptosis --- p.70<br>Chapter 2.2.5.1 --- Transfection --- p.70<br>Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70<br>Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71<br>Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71<br>Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71<br>Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72<br>Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72<br>Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72<br>Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73<br>Chapter 2.2.7.3 --- Cell invasion assay --- p.73<br>Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75<br>Chapter 2.2.8.1 --- RT-PCR --- p.75<br>Chapter 2.2.8.2 --- Western blotting --- p.75<br>Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76<br>Chapter 2.2.9.1 --- Cloning and transfection --- p.76<br>Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76<br>Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77<br>Chapter 2.2.10 --- Statistics --- p.77<br>Chapter Chapter 3 --- Results<br>Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78<br>Chapter 3.1.1 --- RT-PCR --- p.78<br>Chapter 3.1.2 --- Western blotting --- p.81<br>Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85<br>Chapter 3.3 --- Characterization of RhoC --- p.93<br>Chapter 3.3.1 --- Cell cycle analysis --- p.93<br>Chapter 3.3.2 --- Apoptosis --- p.95<br>Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97<br>Chapter 3.3.4 --- Cell invasion ability --- p.99<br>Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102<br>Chapter 3.4 --- Characterization of Rabl4 --- p.107<br>Chapter 3.4.1 --- Cell cycle analysis --- p.107<br>Chapter 3.4.2 --- Apoptosis --- p.109<br>Chapter 3.4.3 --- Roles in intracellular transportation --- p.111<br>Chapter Chapter 4 --- Discussion<br>Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117<br>Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119<br>Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122<br>Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122<br>Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123<br>Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124<br>Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125<br>Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126<br>Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132<br>Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132<br>Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133<br>Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134<br>Chapter 4.5 --- Conclusion --- p.138<br>Chapter 4.6 --- Future prospects --- p.140<br>Appendix --- p.143<br>References --- p.147

碩士<br>國立陽明大學<br>微生物及免疫學研究所<br>97<br>Hepatocellular carcinoma (HCC) is the leading cause of malignant tumors in Taiwan, and its death rate is also high because of its high probability of recurrence and metastasis. It is very important to find the mechanisms of HCC recurrence and metastasis, and such new knowledge may help us to design new drugs for curing this terribly malignant cancer. We first focused on the level of miR-34a in clinical HCC tissues. We found there is a negative correlation between the expression level of miR-34a and the occurrence of primary HCC recurrence and metastasis. Secondly, we proved that miR-34a has the ability to inhibit the migration of HCC cell lines in vitro and established Mahlavu-34a cell line for in vivo experiment. Finally, we used expression microarray to find the target genes of miR-34a which are also related to HCC recurrence and metastasis. This study will help to develop the effective drugs to cure hepatocellular carcinoma in the future. It will be a gift for those people who suffer from the pain of HCC.

碩士<br>長庚大學<br>基礎醫學研究所<br>96<br>Up to now, hepatocellular carcinoma (HCC) is still one of the major cancer types in Taiwan. For tumorigenesis, oncogene addiction is an important cause and alteration in the expression level of protein tyrosine kinases (PTKs) is related to many human diseases, including cancer. In human, there are about 90 PTKs and they are involved in many signaling pathways that lead to angiogenesis, cell proliferation, migration, survival and apoptosis. We established a sensitive platform, qRT-PCR, for profiling of PTKs in HCC. Profiling of PTKs in 8 normal and 15 tumor samples revealed that 15 PTKs were up-regulated in HCC and 8 PTKs down-regulated. We found the receptor tyrosine kinase, neurotrophic tyrosine kinase receptor type 3 (NTRK3), and its ligand, neurotrophin 3 (NT3), were significantly down-regulated in tumor samples compared with normal samples. Analysis of 40 HCC samples by immunohistochemistry confirmed that protein level of NTRK3 and NT3 were down-regulated. Epigenetic modification is one of the regulatory mechanisms of gene expression in cancer and bioinformatic analysis predicts that both NTRK3 and NT3 have CpG islands in their promoter region. Here we showed that epigenetic modification might be a regulatory mechanism of NTRK3 and NT3 in HCC.

Gho Wai-Man.<br>Thesis submitted in: August 2006.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (leaves 114-123).<br>Abstracts in English and Chinese.<br>ABSTRACT --- p.I<br>摘要 --- p.IV<br>ACKNOWLEDGEMENT --- p.VI<br>TABLE OF CONTENT --- p.VII<br>LIST OF TABLES --- p.XII<br>LIST OF FIGURES --- p.XIII<br>ABBREVIATIONS --- p.XVI<br>Chapter CHAPTER 1 --- INTRODUCTION --- p.1<br>Chapter 1.1 --- Introduction --- p.2<br>Chapter 1.2 --- Epidemiology --- p.2<br>Chapter 1.3 --- Etiological factors --- p.6<br>Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6<br>Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7<br>Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9<br>Chapter 1.3.2 --- Aflatoxin Exposure --- p.10<br>Chapter 1.3.3 --- Alcohol Abuse --- p.11<br>Chapter 1.3.4 --- Liver Cirrhosis --- p.12<br>Chapter 1.4 --- Genetic alterations in hcc --- p.16<br>Chapter 1.4.1 --- Chromosomal Gain --- p.16<br>Chapter 1.4.2 --- Chromosomal Loss --- p.17<br>Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19<br>Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19<br>Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19<br>Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20<br>Chapter 1.6 --- Aim of thesis --- p.28<br>Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29<br>Chapter 2.1 --- Materials --- p.30<br>Chapter 2.1.1 --- Chemicals --- p.30<br>Chapter 2.1.2 --- Buffers --- p.31<br>Chapter 2.1.3 --- Cell culture --- p.31<br>Chapter 2.1.4 --- Nucleic acids --- p.32<br>Chapter 2.1.5 --- Enzymes --- p.32<br>Chapter 2.1.6 --- Equipment --- p.32<br>Chapter 2.1.7 --- Kits --- p.33<br>Chapter 2.1.8 --- Software and Web Resource --- p.33<br>Chapter 2.2 --- Dna extraction --- p.34<br>Chapter 2.2.1 --- Cell Lines --- p.34<br>Chapter 2.2.2 --- Primary HCC --- p.34<br>Chapter 2.2.3 --- Lymphocytic DNA --- p.35<br>Chapter 2.3 --- Rna extraction --- p.36<br>Chapter 2.4 --- Dna sequencing --- p.38<br>Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38<br>Chapter 2.4.2 --- Cycle Sequencing --- p.39<br>Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41<br>Chapter 2.5.1 --- FISH Probe Preparation --- p.41<br>Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41<br>Chapter 2.5.1.2 --- Nick Translation --- p.41<br>Chapter 2.5.2 --- FISH --- p.42<br>Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43<br>Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43<br>Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44<br>Chapter 2.9 --- Bisulfite dna sequencing --- p.44<br>Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46<br>Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49<br>Chapter 2.11.1 --- ATF5 Transfection --- p.49<br>Chapter 2.11.2 --- Cell Growth Assay --- p.50<br>Chapter 2.11.3 --- Xenograft Development --- p.51<br>Chapter 2.12 --- codelink expression microarray --- p.51<br>Chapter 2.13 --- Statistical analysis --- p.53<br>Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54<br>Chapter 3.1 --- Introduction --- p.55<br>Chapter 3.2 --- Materials and methods --- p.58<br>Chapter 3.2.1 --- Cell Lines --- p.58<br>Chapter 3.2.2 --- Mutational Analysis --- p.58<br>Chapter 3.2.3 --- Copy Number Loss --- p.59<br>Chapter 3.2.4 --- Epigenetic Control --- p.59<br>Chapter 3.3 --- Results --- p.67<br>Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67<br>Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73<br>Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73<br>Chapter 3.4 --- Discussion --- p.82<br>Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85<br>Chapter 4.1 --- Introduction --- p.86<br>Chapter 4.2 --- Materials and methods --- p.88<br>Chapter 4.2.1 --- Cell Lines --- p.88<br>Chapter 4.2.2 --- Plasmids and Transfection --- p.88<br>Chapter 4.2.3 --- Cell Growth Assay --- p.88<br>Chapter 4.2.4 --- Xenograft Development --- p.88<br>Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89<br>Chapter 4.2.6 --- Quantitative RT-PCR --- p.90<br>Chapter 4.2.7 --- Statistical analysis --- p.90<br>Chapter 4.3 --- Results --- p.91<br>Chapter 4.3.1 --- Cell Proliferation --- p.91<br>Chapter 4.3.1.1 --- In-Vitro Examination --- p.91<br>Chapter 4.3.1.2 --- In-Vivo Examination --- p.91<br>Chapter 4.3.2 --- Microarray A nalysis --- p.91<br>Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103<br>Chapter 4.4 --- Discussion --- p.106<br>Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110<br>Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111<br>Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112<br>Chapter CHAPTER 6 --- REFERENCES --- p.114

碩士<br>長庚大學<br>生物醫學研究所<br>97<br>Using transcriptome data to profile the global gene expression has been widely applied to identify genes whose expression pattern is associated with clinical pathological features of tumors which may be used for the diagnosis and prognostic predictions of hepatocellular carcinoma (HCC). Recently, researchers have focused on the expression profiling of microRNAs (miRNAs) to provide more comprehensive results. A significant challenge in the post-genomic era is how to use large-scale and multiple dimensions data to extract and understand the underlying biology of hepatocarcinogenesis. Previous studies indicate that 12-O-Tetradecanoylphorbol-13-acetate (TPA) induce G1 growth arrest in HepG2 cells. Deregulation of growth control mechanism may play a key role in the cancer development. We hypothesize that the genes and miRNAs whose expression altered by TPA treatment may be related to growth control of human hepatoma cells. We proposed that expression level of critical regulatory components in particular networks may be moderately modulated at both transcriptional and translational level during tumorgenesis. In this study, we used a systems biology approach to identify regulatory networks by integrating mRNAs and miRNAs expressions and provide a new dimension of target identification which may be ignored by conventional microarray analysis for mRNAs and miRNAs alone.

Hepatocellular carcinoma (HCC) remains one of the most challenging health problems worldwide. Currently, HCC is the fifth most common cancer, and the second leading cause of cancer-related death globally. Although HCC is more prevalent in Asian and African nations, important evidence indicates that the incidence of HCC is rising in developed countries. Various risk factors for HCC development are well defined, such as hepatitis B virus (HBV) infection and hepatitis C virus (HCV) infection, cirrhosis (that is, chronic liver damage caused by inflammation and fibrosis) alcohol abuse and metabolic syndrome. Other cofactors such as tobacco use and intake of aflatoxin B1 (a fungal carcinogen present in food supplies associated with mutations in the tumor suppressor gene TP53) are wellcharacterized contributors to HCC. However, the exact molecular mechanisms underlying the development of HCC are still unclear. Over the past decade, there has been an improvement in the understanding of the molecular pathogenesis of HCC. Genomic analyses have provided a clear picture of the main drivers responsible for tumor initiation and progression. Each HCC has an average of 40 genomic aberrations, among which few are considered drivers. The most frequent mutations are able to affect WNT–β-catenin pathway activation (because of mutations in β-catenin, encoded by CTNNB1 gene), functions of the cellular tumour antigen p53 (encoded by TP53 gene), and telomere maintenance (because of mutations in telomere reverse transcriptase, encoded by TERT gene).A recent study has shown that occurrence of somatic mutations in CTNNB1 and TP53 genes is a rare event in HCC patients from Southern Italy. Indeed, no CTNNB1 exon 3 mutations or TP53 mutations were detected in any case. The aim of this study was to investigate genetic heterogeneity of CTNNB1, TP53, and hTERT promoter in paired tumorous and nontumorous liver specimens of a large cohort of patients with HCC from Southern Italy.

After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined.<br>Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A).<br>In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters.<br>Dong, Qingming.<br>Adviser: Ming-Liang He.<br>Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: .<br>Thesis submitted in: December 2008.<br>Thesis submitted in: December 2008.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.<br>Includes bibliographical references (leaves 132-154).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.