published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) is the most common tumors worldwide. In contrast to other cancers, the prognosis of HCC is extremely poor, with less that 5% of 5-year survival rate worldwide. From our previous studies, we isolated Chromodomain Helicases/ATPase DNA binding protein1-Like (CHD1L) gene from chromosome 1q21, and characterized it as a specific oncogene in HCC. By using 2D-PAGE and MALDI-TOF mass spectrometry approach, Translationally Controlled Tumor Protein (TCTP) was identified as a CHD1L target, which was preferentially expressed in CHD1L-transfected cells. TCTP is a highly conserved protein and expressed in almost all mammalian tissues. It has been reported that TCTP interacts with microtubules in a cell-cycle-dependent manner, and functions as a prosurvival factor and inhibiting apoptosis. To better understand the molecular mechanisms of HCC progression, the effect of TCTP overexpression in HCC and the mechanism by which TCTP regulated cell-cycle progression were elucidated in this study. CHD1L is a unique oncogene belongs to SNF2-like subfamily. Mechanistic studies found that CHD1L protein directly binds to the promoter region (nt -733 to -1,027) of TCTP and activated TCTP transcription. Investigation of clinical HCC specimens found that overexpression of TCTP was not only significantly associated with the advanced tumor stage (P = 0.037) and overall survival time of HCC patients (P = 0.034), but also an independent marker associated with poor prognostic outcomes. Functional studies demonstrated that TCTP has tumorigenic abilities and overexpression of TCTP contributed to the mitotic defects of tumor cells. Further mechanistic studies demonstrated that TCTP promoted the ubiquitin-proteasome degradation of Cdc25c during mitotic progression, which caused the failure in the dephosphorylation of Cdk1 on Tyr 15 and decreased Cdk1 activity. The consequence of chromosome missegregation and mitotic catastrophe results in aneuploidy, which is frequently observed in cancer. In addition, the correlation between TCTP overexpression and metastatic potential of HCC was elucidated by examined the expression levels of TCTP using a tissue microarray (TMA) containing 60 pairs of primary HCCs and their matched metastases. Further studies demonstrated that overexpression of TCTP shows high incidence of extrahepatic metastasis and positive correlation was found between TCTP and MMP-2 or MMP-9 (Spearmen correlation coefficient=0.466, and 0.352, respectively, P<0.001 for both). In vitro functional studies showed that TCTP protein associated with promoter regions of MMP-2 and MMP-9 and activates their transcriptions. Molecular analyses revealed that TCTP served as a JunD coactivator and formed complexes with JunD and bind with consensus AP-1 sites on MMP-2 and MMP-9 promoters to enhance their expression in HCC cells. More importantly, high co-expression of TCTP and MMP-2 or MMP-9 was significantly associated with poor disease-free survival (log rank= 8.146, and 11.677 respectively, P =0.017 and 0.003 respectively). In summary, two novel molecular mechanisms (CDH1L/TCTP/Cdc25C/Cdk1) and (TCTP/JunD/MMP-2, MMP-9) were revealed during HCC progression and metastasis. Also, the prognostic value of TCTP and MMP-2 or MMP-9 coexpression for HCC was highlight in this study. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies worldwide with very poor prognosis. It is generally believed that accumulation of irreversible alterations in critical oncogenes and tumor suppressor genes during the long-term inflammation finally leads to the hepatocellular pathogenesis. Although under intensive investigation, the molecular pathogenesis of HCC still remains to be further elucidated. In this study, we aimed to identify novel genetic alterations critical to the pathogenesis of HCC, especially in hot regions with recurrent chromosomal instability. Amplification of broad regions of 8q is one of the most frequent genetic alterations in HCC, suggesting the existence of oncogenes in addition to MYC at 8q24. By screening the publicly available microarray database and clinical samples, we found frequent amplification and overexpression of Serum and Glucocorticoid Kinase 3 (SGK3) in clinical HCC specimens, and SGK3 genomic activation was significantly associated with poor outcome of HCC patients. Functional assays revealed that SGK3 could increase G1/S cell cycle progression, cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. We provided evidences that SGK3 could promote HCC growth and survival through inactivating GSK3-β and BAD respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1, has more significance than overexpression of AKT in predicting poor outcome of HCC patients. Our findings suggested the existence of an AKT-independent SGK3 pathway, which may function in parallel with AKT pathway in the pathogenesis of HCC. In addition to large chromosomal alterations, small changes in nucleotides may also make substantial contributions to carcinogenesis. Recent advances in high-throughput deep sequencing technology have provided a powerful tool to understand the whole cancer transcriptome and identify novel genetic alterations related to cancer progression. In this study, we identified a high proportion of allele imbalance in genes related to cellular stress response by sequencing the whole transcriptome of 3 paired HCC tissues. A novel nucleotide variation which resulted in a R438H amino acid change was identified in the coding region of the gene Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), and the variant 438H form of OSGIN1 was found to be specifically retained in the tumor tissues in a cohort of HCC patients. OSGIN1 was found to be closely associated with chemotherapeutic reagents and exhibited strong tumor suppressive function in HCC by directly inducing cell apoptosis. The wild type OSGIN1 was found to have stronger tumor suppressive function than the variant allele, and this might be due to their different ability to localize to mitochondria. The significantly decreased basal apoptotic index in HCC patients carrying OSGIN1 variant allele and their poor prognosis further suggested that the specific retention of 438H OSGIN1 might be important in HCC progression. In summary, we found a frequently amplified oncogenic SGK3 signaling pathway, as well as the allele-specific imbalance of tumor suppressive OSGIN1 in the pathogenesis of HCC. Further characterization of their mechanisms in hepatocarcinogenesis may help provide novel prognostic biomarkers and therapeutic targets in HCC treatment. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and particularly common in China and Southeast Asia. It ranks the 2nd and 4th most common fatal cancer in males and females, respectively, in Hong Kong. Current treatments are not always effective, as recurrence and metastasis in HCC are difficult to tackle and the underlying mechanisms not fully understood. Aberration of Wnt signaling has been implicated in HCC; in this study, we investigated the underlying mechanisms of how aberrant Wnt signaling promoted HCC development. With Taqman Low Density Array (LDA) analysis on 38 pairs of HCC and the corresponding non-tumorous livers for 59 Wnt signaling related-genes, we found significant overexpression of the Wnt signaling intermediate, Dishevelled (Dvl)-3, in HCC (p = 0.014). This observation in LDA was confirmed in 36 additional HCC cases. Among a total of 74 cases studied, 28.38% showed more than 3-fold overexpression in the tumors as compared with the corresponding non-tumorous livers. Dvl3 overexpression positively correlated with the presence of venous invasion. We also observed significant correlation of Dvl3 expression with accumulation of β-catenin, a downstream effecter of Wnt/β-catenin signaling (p=0.028). We further characterized the functional roles of Dvl3 in contributing to the stem cell-like and metastatic properties of HCC. We found that Dvl3 knockdown in HCC cells suppressed cell proliferation, sphere formation, tumorigenicity in immunodeficient mice, chemo-resistance, and expression of stemness genes. We then examined whether Wnt/β-catenin was effectively modulated by Dvl3 and found that Dvl3 overexpression and knockdown, respectively, promoted and reduced the TOP/FOP luciferase reporter activity in HCC cells. This was accompanied by the expression of β-catenin target genes, EpCAM and LGR5, both of which are associated with HCC stemness. Furthermore, rescue with wild-type or constitutively active β-catenin partially restored the in vivo tumorigenicity suppressed by Dvl3 knockdown, indicating a partial role of β-catenin in mediating the effects of Dvl3 on HCC stemness. In addition, since cell migration is a critical determinant in metastasis, we assessed the HCC cell migratory ability in vitro using transwell migration assays and observed suppression of the cell migration ability upon Dvl3 knockdown. Also, the in vivo orthotopic model confirmed a role of Dvl3 in promoting metastasis, as stable Dvl3 knockdown in HCC cells resulted in a reduction in lung metastasis. Interestingly, the effect of Dvl3 on cell migration was independent of β-catenin, as knockdown of β-catenin had no effect on HCC cell migration in vitro. It was also not related to the phosphorylation of MYPT in Rho-ROCK signaling, which itself was previously implicated in HCC cells metastasis and reported as a downstream signaling of Dvl in development. In summary, our study has identified roles of Dvl3 in HCC stemness properties and cell migration and this may provide functional implication of Dvl3 overexpression, which significantly correlated with venous invasion in human HCCs. Also, β-catenin is partly responsible for the role of Dvl3 in HCC stemness but independent of that in cell migration. Functional characterization of Dvl3 in HCC may help future development of therapy targeting Dvl3 of Wnt signaling pathways. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Liver cancer is the fifth most commonly diagnosed and the second most lethal malignancies worldwide, in which hepatocellular carcinoma (HCC) represents the majority subtype. High mortality rate of HCC is due to lack of effective treatments and early detection methods. Activation of cadherin-17 (CDH17)/β-catenin axis is found by our team in HCC and targeting components of this axis associated with anti-tumorigenesis. With limited knowledge on this axis in HCC, I plan to study molecules related to this axis as a way to uncover the cellular mechanism of this axis in liver tumorigenesis. Gene profiling data was re-analyzed to search for CDH17-associated genes in HCC clinical samples. The patient cohort was segregated into CDH17-high and CDH17-low group according to tumor/adjacent non-tumor expression ratio of CDH17. Serine peptidase inhibitor, Kazal type 1 (SPINK1) was found highly expressed in CDH17-high cases and its over-expression accounted for 73 % of total studied cases. Gene manipulation and inhibitor study in HCC cell lines suggested SPINK1 as a downstream molecule of CDH17/β-catenin axis in HCC. Further in silico analysis predicted potential binding sites of two transcriptional factors downstream of CDH17/β-catenin axis, lymphoid enhancer-binding factor 1 (LEF1) and T-cell factor 7 (TCF7), on SPINK1 promoter. Deletion or mutation of their binding sites on SPINK1 promoter suppressed the transcription of SPINK1 gene, while transient suppression of these two transcriptional factors resulted in reduction of SPINK1 level. As the direct link between SPINK1 and CDH17/β-catenin axis was confirmed, SPINK1 was hypothesized to possess tumorigenic properties like its upstream molecule CDH17. Suppression of SPINK1 using RNA interference in PLC and MHCC97-H HCC cells hampered growth, migration and colony formation abilities of suppressed cells. These phenotypic alterations accompanied with an inactivation of tumorigenic c-Raf/MEK/ERK pathway. These findings demonstrate the tumorigenic properties of SPINK1 in HCC. Next, the therapeutic potential of targeting SPINK1 in HCC was tested by using purified monoclonal antibody raised against recombinant SPINK1 protein (C4). C4 was capable in suppressing SPINK1 level based on results of immunocytochemisty, enzyme-linked immunosorbent assay and immunoneutralization. Treatment of HCC cells using C4 suppressed growth, migration and colony formation ability of cells by inactivating MAPK pathway. In subcutaneous tumor xenograft study, treating tumor-bearing mice with C4 at 8 mg/kg three times weekly inhibited tumor growth by around 65 %. These findings demonstrate C4 is a potential therapeutic for counteracting liver tumorigenesis. In conclusion, I have demonstrated for the first time SPINK1 as a novel downstream molecule of CDH17/β-catenin axis involved in HCC progression via activating MAPK pathway. Targeting this molecule with its specific monoclonal antibody is a potential approach for cancer therapy. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the fifth most prevalent human malignancy and the third leading cause of cancer deaths in the world. MicroRNAs (miRNA) are conserved, small noncoding RNA molecules that regulate gene expression of protein-coding gene posttranscriptionally. Dysregulation of miRNA is implicated in many human malignancies including HCC, yet little is known regarding the regulatory mechanisms of these small noncoding RNAs. Hypoxia is a prevalent! tumor microenvironment in HCC because of its rapid growth often to large size and plays a key role in modulating tumor aggressiveness. In the present study, we investigated the effects of hypoxia on microRNA expression in human HCCs, identified and characterized hypoxia-inducible microRNAs that are important for the development of aggressive phenotypes. To initialize the study, we examined the miRNA expression profiles with TaqMan human microRNA Low-Density Array and identified a panel of microRNAs differentially expressed in HCC cells under hypoxic conditions. We observed that miR-210 was consistently upregulated by hypoxia in a total of 7 different HCC cell lines, via a HIF1α-dependent mechanism. In human HCCs, miRL210 overexpression significantly correlated with poorer overall and disease-free survival of patients, as well as aggressive pathologic features, including advanced tumor stages of HCC and the presence of venous invasion. These findings established miRL210 as a surrogate marker of aggressive HCC with high metastatic potential. In most human malignancies, cancer metastasis contributes to about 90% of cancer-related mortality. Given the correlation of miR-210 levels with poorer patient survival and aggressive clinical features of HCC, we then characterized the metastatic role of miRL210 by functional assays in the second part of the study. The findings from in vitro and in vivo experiments using both gain- and loss-of-function approaches led us to conclude that the hypoxic induction of miRL210 enhanced metastatic potential of HCC cells. The pro-metastatic effect of miRL210 was attributed, at least partly, to the downregulation of TIMP2 by hypoxia, through a feedback loop circuit consisting of HIF1α, miRL210, and HIF3α. The impact of miR-210 on HCC metastasis was not the only scope of this study since hypoxia has long been recognized as a major obstacle in chemotherapy. Given that activation of the HIF1α-miR-210 axis was frequently observed in hypoxic HCC cells, in the last part of the study we also investigated whether hypoxic induction of miRL210 promoted cell survival against cytotoxic treatments, including cisplatin and 5-flurouracil. Here, we demonstrated that induction of HIF1α-miR-210 axis conferred chemoresistance to HCC cells under hypoxic conditions, and inhibition of miR-210 re-sensitized HCC cells to these cytotoxic drugs. Mechanistically, we also revealed that RAD52 was a direct functional target of miRL210 that linked hypoxia to chemoresistance in HCC cells. The overall findings of this study have enriched our understanding of miR-210 as a mediator of hypoxic responses in HCC, in particular metastasis and chemoresistance. We have highlighted the clinical significance of this microRNA by showing that miR-210 can serve not only as a prognostic marker for HCC progression, but also as a mediator for the hypoxic tumor microenvironment to modulate tumor aggressiveness. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy