published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

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

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

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

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy