The Importance of Death Receptor Pathway in Hepatocellular Carcinoma and It's Potential in the Development of Oligonucleotide Nanomedicine

Hepatocellular carcinoma (HCC; primary liver cancer) is one of the most common cancers with high mortality rate worldwide. Hepatocarcinogenesis is triggered by multiple factors, such as cancer etiologies, treatment options, host factors, and lifestyle. It is a complex progressive disease involving sustained inflammatory damage, fibrosis, and cirrhosis. Numerous numbers of intrinsic and extrinsic factors leading to the alteration of various molecular and cellular events, compromising the balance between survival and apoptotic signals in the cells. Fas (CD95, Apo-1, TNFRSF6) is a member of the cell death receptor in tumor necrosis factor receptor superfamily. It has a major function in inducing apoptosis upon engagement by its natural ligand FasL (CD95L, CD178, TNFSF6). In cancer, however, the role of the Fas/FasL axis is extensive, including in non-apoptotic and cancer-promoting functions. In this study, we showed the importance of Fas/FasL axis in HCC. By using different experimental settings, we demonstrated that Fas and FasL expressions were associated with p53/PUMA intrinsic apoptosis pathway, tumor growth, inflammation, and cancer stemness. In in vivo study by utilizing human HCC clinical samples and normal liver, the presence of Fas and FasL were analyzed. The expression of Fas was found significantly higher in HCC nodules compared cirrhosis and normal donor livers, while FasL was high in both HCC and cirrhosis compared to normal. In contrast, PUMA expression was dysregulated from normal to cirrhosis and to be lowest in HCC. In in vitro study by using hepatic normal and cancer cell lines, the modulation of Fas were analyzed by two methods: 1. extrinsic apoptotic pathway activation by anti-Fas in both acute and chronic exposure, and 2. gene silencing of Fas. By two methods, Fas modulation was found to be diverse for cancer cells compared to normal cells. In normal cells, Fas was shown to be correlated mainly in the pro-apoptotic pathway and cells growth, while in cancer cells, in addition to apoptosis and cells growth, Fas is also related to cancer stemness. In particular, for acute anti-Fas exposure, modulation of transcription factors, apoptotic and inflammation genes was strongly noticed. Due to the significance of Fas in HCC, the last part of the thesis described the development of potential oligonucleotide aptamers against Fas as a molecular probe for HCC. Aptamers are single-stranded RNA or DNA oligonucleotides with a low molecular weight that specifically and efficiently bind to a target molecule. The screening and evaluation of DNA aptamers recognizing Fas were developed through a cell-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in Fas+ and Fas- HCC cell lines. The aptamers candidate pools by SELEX were cloned and will be subjected to sequencing for subsequent bioinformatics screening and validation assay. Future Fas aptamers can be potentially used for theranostics (therapy and diagnostic) approach, biosensors, and drug nano-delivery system. In summary, our data indicated the importance of extrinsic cell death receptor pathway Fas/FasL axis in HCC. This study contributes to the understanding and the development of potential future therapy against HCC.