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

Ovarian cancer is the most lethal gynecological cancer. This is mainly due to widespread peritoneal dissemination and malignant ascites, in which angiogenesis, the formation of new blood vessels, is critical to both ascites development and its metastasis. Loss of E-cadherin is a well-established marker that characterizes the progression of metastatic tumors, including ovarian cancer. The release of a soluble form of E-cadherin (sE-cad) has been frequently associated with a rapid reduction of functional E-cadherin at the cell surface. Importantly, sE-cad is significantly present in ascites from women with stage III/IV ovarian cancer when compared to women with benign ovarian cysts. However, despite the clinical significance, most studies have focused on its role in weakening cell-cell adhesion, whether sE-cad itself has any biological function is not fully understood. Here it is shown for the first time a potent angiogenic role for sE-cad released from ovarian carcinoma. Soluble form of E-cadherin promoted the migration, permeability, and tubulogenesis of endothelial cells. These activities were also observed with a sE-cad/Fc chimera, and targeted inhibition using E-cadherin blocking antibodies completely prevented the sE-cad mediated effects. In addition, it was further revealed that sE-cad could be released from ovarian cancer cells in form of exosomes, a form of extracellular vesicles that play an important role in distant intercellular communication. sE-cad-positive exosomes were able to stimulate the angiogenic phenotype in vitro and functional neovascularization in a Matrigel implant model in vivo. The use of E-cadherin blocking antibodies resulted in diminished angiogenesis, confirming that the effect was sE-cad-positive exosomes specific. In search of the underlying mechanism by which sE-cad-positive exosomes promoted angiogenesis in endothelial cells which lacked E-cadherin, sE-cad was found to heterodimerize with VE-cadherin. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector β-catenin, but not p120 catenin. Similarly, the angiogenic phenotype could be reversed by inhibition of VE-cadherin, PI3K/Akt and β-catenin. A mass spectrometric proteomic analysis of the isolated exosomes revealed distinct membrane-bound proteases, especially disintegrin and metalloproteinase 10 (ADAM10) and matrix metalloproteinase 25 (MMP25) commonly associated with ovarian cancer progression, are implicated in sE-cad production. Small interfering RNA-mediated down-regulation of ADAM10 and MMP25 significantly inhibited sE-cad production. Moreover, hepatocyte growth factor, a multifaceted cytokine which is frequently elevated in ovarian cancer ascites, was shown to increase the expression of ADAM10 and MMP25 concomitant with an elevated level of sE-cad. Together, these results uncover a novel angiogenic role of sE-cad and a new mechanism of the action of sE-cad in tumor progression. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Oncogenic mutations in lung cancer further our knowledge about cancer initiation and progression, and may guide personalized treatment. The fact that targeted therapy is most effective in subsets of patients with defined molecular targets indicates the need for classification of clinically-related molecular tumor phenotypes based on the presence of oncogenic mutations, including EGFR mutations and EML4-ALK rearrangements. The identification of EGFR mutations, in up to half of lung adenocarcinomas in Asians, could predict clinical sensitivity to tyrosine kinase inhibitor (TKI). However, testing for mutations is not always possible due to tumor tissue availability. The therapeutic decision sometimes remains a clinical one especially for elderly lung cancer patients but no known mutation status. We studied the survival outcomes of targeted therapy versus conventional chemotherapy in elderly patients with lung cancer when we did not yet have routine EGFR mutation testing and demonstrated comparable survival outcomes in targeted therapy compared to chemotherapy, implying that survival with targeted therapy could be better if the treatment population could be selected with EGFR mutations. Though testing for EGFR mutation in tumor biopsy have later become routine practice and remains the accepted reference for therapeutic decision, the detection of EGFR mutations in plasma DNA with high diagnostic performance will be useful adjunct for diagnostic and therapeutic monitoring. Among patients with EGFR mutations in tumor biopsy, the concurrent detection of EGFR mutation in plasma DNA was found to confer a less favorable prognosis in terms of overall survival than those patients with EGFR mutations in tumor biopsy but the corresponding mutation was not detected in plasma. Other oncogenic mutations with therapeutic implications in lung tumors are yet to be fully explored, like ALK, KRAS, ROS1 or NTRK1 mutations. It is not exactly the tumor – but the mutations in the tumor that need to be explored with reference to clinical behavior. Even with EGFR mutation with well-established clinical implications, further exploration into its mechanistic functions will help in understanding of drug resistance. Lung cancer cell lines established from patients with known mutation profiles could be useful tools for studying the biology of known molecular targets as well as for therapeutic testing. Four new lung adenocarcinoma and one mesothelioma cell lines were established from patients with different clinical characteristics and oncogenic mutation profiles. These cell lines with defined mutation profiles will provide tools for exploration of lung cancer and mesothelioma biology with respect to molecular therapeutic targets. The Large Tumor Suppressor 2 (LATS2) gene was a differentially expressed gene between EGFR mutant and wildtype lung adenocarcinomas. The differential LATS2 expression levels were predictive of survival in patients with resected lung AD and may modulate tumor growth via different signaling pathways in EGFR mutant and wild-type tumors. The identification of oncogenic mutations has led to a new paradigm of targeted therapy in lung cancer. Further improvements in outcome of lung cancer management will stem from research into the biology of oncogenic mutations and their clinico-pathological correlations, which would fuel parallel development of clinically efficacious targeted therapies. / published_or_final_version / Medicine / Master / Doctor of Medicine

Epithelial ovarian cancer is a common female malignancy with a relatively high mortality rate worldwide. This may due to a lack of efficient diagnostic methods at early stage and worsen by complications caused by metastasis at advanced stage. For successful metastasis, cancer cells detached from the original growing sites have to survive in the body circulation before conquering a distant location within the body. Resistance to anoikis (apoptosis induced without appropriate extracellular matrix) is therefore utmost important during metastatic spreading. In addition, a pre-metastatic niche remodeled by cancer cells is also a pre-requisite for metastatic colonization. Emerging evidence has suggested the dysregulation of miRNAs is associated with different aspects of tumorigenesis. However, the specific roles of miRNAs in anoikis resistance and in remodeling of distant niche remain unknown thus far. This study attempted to investigate the functional roles of miR-141, in particularly anoikis resistance of ovarian cancer cells and the reprogramming of stromal cells. The miR-200 family is frequently upregulated and associated with human cancer metastasis. In this study, by cDNA array profiling together with biochemical and functional studies, miR-141, a member of miR-200 family, was identified as an oncomiR enhancing cell viability in low serum medium and anoikis resistance. Moreover, enforced expression of miR-141 led to bigger tumor sizes and promoted metastatic colonization in mouse models. Further studies demonstrated miR-141 directly targets tumor suppressive KLF12 in ovarian cancer cells, depletion of KLF12 could mimic function of miR-141. Clinical study revealed the upregulated miR-141 was significantly correlated with the downregulated KLF12, serous subtype, advanced and distant metastatic ovarian cancer. Furthermore, Genechip profiling, Human Apoptosis Array and Luciferase reporter assay revealed the upregulated miR-141 and downregulated KLF12 enhanced anoikis resistance via elevation of survivin which protect cells against intrinsic apoptotic activity. On the other aspect, miR-141 was found to be a secretary miRNA and commonly detected in the serum of ovarian cancer patients. The upregulated miR-141 expression was also correlated to levels of common cancer biomarker CA125. Importantly, the serum miR-141 level was significantly correlated with the tumor burden of patients during treatments, indicating it could be used as a non-invasive biomarker for ovarian cancers. Finally, based on miR-141 as tumor-secreted and circulated miRNA, a series of functional studies demonstrated that miR-141 could be transferred to hFF-1 fibroblast cells. Intriguingly, ovarian cancer cells cultured in miR-141-fibroblast culturing medium showed a remarkable increase of cell migration, suggesting that the remodeled-miR-141 fibroblast cells can secrete stimulating factors and promote ovarian cancer cells aggressiveness. This is the first study showing miR-141 could reprogram fibroblast cells to be a niche for ovarian cancer cell dissemination and metastatic progression. However, further investigations for verifying such functions are warranted. In conclusion, this study provides strong evidence that miR-141 is oncoMir enhancing ovarian cancer cell plasticity in metastasis e.g. anoikis resistance. Moreover, the finding of secretary form miR-141 not only gives the feasibility to be a potential biomarker for detecting ovarian cancer but also shows a possible mechanism of how miRNAs reprogram the distant niche for metastatic colonization. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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

published_or_final_version / abstract / toc / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy