Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for over 600,000 deaths per year. The most common treatment strategy for intermediate and advanced stage unresectable HCC is transarterial chemoembolization (TACE), which involves the local administration of a chemotherapeutic drug combined with arterial occlusion resulting in ischemic tumor necrosis. However, TACE suffers from inadvertent exposure of noncancerous liver parenchyma to embolic agents resulting in liver injury. In some cases, over-embolization has lead to infection, necrosis of unaffected liver tissue, and even liver failure which suggests the need for a biocompatible, multifunctional embolic material which can deliver anticancer drugs with high target specificity. Our laboratory has recently developed a method to fabricate porous silicon (pSi) microparticles with defined physicochemical properties based on photolithography and anodic etching. These microparticles function as multistage drug delivery systems that can circumvent the biobarriers present in the systemic circulation enabling site-specific localization and release of chemotherapy and imaging agents. The versatility of the fabrication process enables the realization of microparticles ranging in size from 600nm to 116[mu]m in diameter with varying shapes, including discoidal, cylindrical and hemispherical, and varying porosity with pore sizes ranging from 6nm to greater than 50nm in diameter. Nanoparticles, such as quantum dots, siRNA-loaded nanoliposomes, gadolinium-based contrast agents, gold and iron oxide nanoparticles, are loaded in pSi microparticles by tailoring their pore sizes and surface chemistries. This thesis presents preliminary results on the applicability of biocompatible, engineered pSi microparticles as an embolic agent for HCC chemoembolization therapy. Hemispherical microparticles with 116[mu]m diameter were successfully fabricated and suspended in phosphate buffered saline (PBS). A microvascular construct was rapid prototyped in polydimethylsiloxane (PDMS) as an in vitro experimental platform to study the embolization behavior of pSi microparticles. Oxidized pSi microparticles were introduced into the microfluidic device at an appropriate flow rate and time-lapse images were taken showing the formation of occlusions at the bifurcation within minutes of administration. Furthermore, penetration through the bifurcation was completely hindered suggesting that pSi microparticles can potentially be used as a biocompatible, multifunctional chemoembolization agent. Although these results are promising, further investigations are warranted.

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Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23415号 / 医博第4760号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 浅野 雅秀, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Bc. Magdaléna Krhutová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: The changes in expression of long non-coding RNAs in hepatocellular carcinoma Hepatocellular carcinoma (HCC) is one of the highly prevalent cancers globally. A number of new cases of HCC and deaths rises every year. Molecular mechanisms of HCC are being intensively studied, yet they are not still fully understood. In addition to genetic alterations, epigenetics also plays an important role in HCC pathogenesis. Long non- coding RNAs (lncRNAs) are RNA molecules that are not capable of coding proteins, and their length is 200 nucleotides or more. Various studies have already revealed lncRNAs involved in tumorigenesis through binding to DNA, RNA and proteins. New studies also demonstrate significant changes in the expression of biotransformation enzymes in HCC, and interactions with microRNAs (miRNAs) and lncRNAs. This diploma thesis deals with the issue of long non-coding RNAs in relation to HCC. It summarizes the epidemiological situation, risk factors, and current possibilities of diagnosis and therapy of this disease. It also summarizes recently described genetic and epigenetic mechanisms contributing...

Could NAMPT inhibition become a potential treatment option in hepatocellular carcinoma?

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with a poor prognosis. Currently, prognosis for HCC patients remains poor as few therapies are available. The clinical need for more effective HCC treatments remains unmet partially because HCC is genetically heterogeneous and HCC driver genes amenable to targeted therapy are largely unknown. Mutations in the TP53 gene are found in ~30% of HCC patients and confer poor prognosis to patients. Identifying genes whose depletion can inhibit HCC growth, and determining the mechanisms involved, will aid the development of targeted therapies for HCC patients. Therefore, the first half of this thesis focuses on identifying genes that are required for cell growth in HCC independent of p53 status. We performed a kinome-wide CRISPR screen to identify genes required for cell growth in three HCC cell lines: HepG2 (p53 wild-type), Huh7 (p53-mutant) and Hep3B (p53-null) cells. The kinome screen identified 31 genes that were required for cell growth in 3 HCC cell lines independent of TP53 status. Among the 31 genes, 8 genes were highly expressed in HCC compared to normal tissue and increased expression was associated with poor survival in HCC patients. We focused on TRRAP, a co-factor for histone acetyltransferases. TRRAP function has not been previously characterized in HCC. CRISPR/Cas9 mediated depletion of TRRAP reduced cell growth and colony formation in all three cell lines. Moreover, depletion of TRRAP reduced its histone acetyltransferase co-factors KAT2A and KAT5 at the protein level with no change at the mRNA level. I found that depletion of KAT5, but not KAT2A, reduced cell growth. Notably, inhibition of proteasome- and lysosome-mediated degradation failed to rescue protein levels of KAT2A and KAT5 in the absence of TRRAP. Moreover, tumor initiation in an HCC mouse model failed after CRISPR/Cas9 depletion of TRRAP due to clearance via macrophages and HCC cells depleted of TRRAP and KAT5 failed to grow as subcutaneous xenografts in vivo. RNA-seq and bioinformatic analysis of HCC patient samples revealed that TRRAP positively regulates expression of genes that are involved in mitotic progression. In HCC, this subset of genes is clinically relevant as they are overexpressed compared to normal tissue and high expression confers poor survival to patients. I identified TOP2A as one of the mitotic gene targets of the TRRAP/KAT5 complex whose inhibition greatly reduces proliferation of HCC cells. Given that this was the first time the TRRAP/KAT5 complex has been identified as a therapeutic target in HCC, the second half of this thesis focuses on identifying the mechanism via which depletion of this complex inhibits proliferation of HCC cells. I discovered that depletion of TRRAP, KAT5 and TOP2A reduced proliferation of HCC cells by inducing senescence. Typically, senescence is an irreversible state of cell cycle arrest at G1 that is due to activation of p53/p21 expression, phosphorylation of RB, and DNA damage. Surprisingly, induction of senescence after loss of TRRAP, KAT5 and TOP2A arrested cells during G2/M and senescence was independent of p53, p21, RB and DNA damage. In summary, this thesis identifies TRRAP as a potential oncogene in HCC. I identified a network of genes regulated by TRRAP and its-cofactor KAT5 that promote mitotic progression. Moreover, I demonstrated that disruption of TRRAP/KAT5 and its downstream target gene TOP2A result in senescence of HCC cells independent of p53 status. Taken together, this work suggests that targeting the TRRAP/KAT5 complex and its network of target genes is a potential therapeutic strategy for HCC patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18147号 / 医博第3867号 / 新制||医||1002(附属図書館) / 31005 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 千葉 勉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20809号 / 医博第4309号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM