Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-191).

Thesis--Ph. D., University of Hong Kong, 1978. / Typescript.

published_or_final_version / Diagnostic Radiology / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

Nasopharyngeal carcinoma (NPC) is squamous cell carcinoma derived from the epithelial layer of nasopharynx. The incidence is high in Southern China and South-east Asia. In Hong Kong, the prevalent of NPC subtype is undifferentiated NPC and is in close association with Epstein-Barr virus (EBV). MicroRNAs (miRNAs) are small non-coding RNAs. They play vital roles in regulating gene expression at post-transcriptional level. EBV also expresses viral miRNAs but the function remains unclear. In NPC diagnosis and monitoring, circulating EBV DNA level has been commonly used. However, in some cases, EBV DNA is below the detection threshold in the plasma of NPC patients making it impossible to be used in continuous monitoring of the patients. This study aimed to evaluate whether miRNAs (both NPC-derived and EBV-derived miRNAs) could be used as candidate circulating markers for disease monitoring. Candidate gene approach was used to select suitable circulating miRNA markers for NPC patients. Four candidate miRNAs including miR-21, miR-1301, miRBART7 and miR-BART22 were examined. The expression levels were first validated in paired NPC tissues and normal counterparts. Furthermore, circulating miRNA levels were evaluated in the plasma of NPC and normal individuals. To examine the changes of miRNA in response to radiotherapy, changes of circulating miRNA were monitored in 13 NPC patients before and after radiotherapy. In addition, functional assay in cell proliferation was performed to validate the potential role of the candidate miRNA in the pathogenesis of undifferentiated NPC. Of the 4 candidate miRNAs, miR-BART7 was consistently over-expressed in both tumor tissues and plasma samples of NPC. In addition, circulating miRBART7 was also detected in NPC patients in case of the plasma EBV DNA levels below the detection threshold. In response to radiotherapy, 10 of 13 (76.92%) patients had decreasing circulating miR-BART7 in the plasma samples collected at 3 month after radiotherapy. Furthermore, introducing miR-BART7 mimics into the undifferentiated NPC cell line HONE1 and normal nasopharyngeal-derived epithelial cell cultures NP69 and NP460 resulted in significant increases in cell proliferation rates of all the 3 cell lines. To summarize, miR-BART7 expression was significantly higher in NPC patients as a potential oncogenic miRNA. Evaluating the miR-BART7 levels is a possible screening approach in NPC diagnosis and post-treatment monitoring. The oncogenic role of miR-BART7 in the development of undifferentiated NPC deserves further investigation. / published_or_final_version / Surgery / Master / Master of Philosophy

 In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in a tightly latent form (latency I, II or III) expressing a limited number of viral latent proteins. Induction of EBV lytic cycle, which triggers expression of a much larger number of viral proteins, may lead to therapeutic effects against EBV-associated cancers. We previously found that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, induced EBV lytic cycle and mediated enhanced cell death in EBV-positive gastric carcinoma cells (latency II). In this thesis, we sought to investigate SAHA’s induction of EBV lytic cycle and its cellular consequences in EBV-associated epithelial malignancies, with particular focus on nasopharyngeal carcinoma (NPC) due to its strong association with EBV and high prevalence in southern Chinese populations. SAHA effected strong induction of EBV lytic cycle in EBV-positive epithelial malignancies, including gastric carcinoma and NPC, as evidenced by strong expression of EBV lytic proteins, replication of viral DNA and production of infectious viral particles. Immunofluorescent staining revealed that up to 70% EBV-positive epithelial cancers expressed EBV lytic proteins following treatment with micromolar concentrations of SAHA. However, SAHA could not induce EBV lytic cycle in NK lymphoma cells (both NPC and NK lymphoma express EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. EBV lytic cycle induction in NPC by SAHA required activation of protein kinase C-delta (PKC-) and acetylation of non-histone protein but required neither phosphatidylinositol 3’-kinase (PI3K), MAPK/ERK kinase (MEK), c-Jun aminoterminal kinase (JNK) nor p38 stress mitogen-activated protein kinase (MAPK) signaling pathway. Conflicting observations regarding the effect of EBV lytic cycle induction on apoptosis were reported. Thus, we investigated the relationship between EBV lytic cycle induction and apoptosis in NPC following treatment with SAHA. EBV-positive NPC showed a higher percentage of apoptosis and proteolytic cleavage of PARP, caspases-3, -7 and -9 over EBV-negative NPC and greater than 85% of NPC cells co-expressed EBV immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins and cleaved caspase-3. Tracking of expression of these lytic proteins over time demonstrated that NPC proceeded to apoptosis following EBV lytic cycle induction, contrary to the previously reported anti-apoptotic effect of EBV lytic proteins in Burkitt lymphoma. Analyses of cleaved caspase-3 expression upon RNAi knockdown and exogenous expression of Zta further supported that EBV lytic cycle directly led to apoptosis of EBV-positive NPC cells. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA’s induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. Finally, in vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC tumors established in nude mice. In conclusion, activation of EBV lytic cycle from latent cycle in EBV-positive epithelial malignancies including NPC by SAHA effected apoptosis and tumor growth suppression of the cancer cells and provided experimental evidence for virus-targeted therapy against EBV-positive cancers. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Nasopharyngeal carcinoma (NPC) is different from other head and neck squamous cell carcinomas and is closely related with Epstein-Barr virus infection. It is endemic in southern China and Southeast Asia, affecting between 20 and 30 per 100,000 populations. According to the World Health Organization (WHO) histological classification, there are three subtypes of NPC: WHO type 1 NPC is keratinizing squamous cell carcinoma; type 2 NPC is differentiated non-keratinizing carcinoma; type 3 NPC is undifferentiated non-keratinizing carcinoma. In southern China including Hong Kong, type 3 NPC (undifferentiated NPC) is dominant and constitutes over 90% of the total NPC. MicroRNA-138 (miR-138) is a small non-coding RNA which has been reported to be highly expressed in undifferentiated NPC. We hereby evaluated whether the miR-138 level could be used to differentiate NPC patients from the normal individuals and examine the potential oncogenic role in undifferentiated NPC cell line. To validate the hypothesis that miR-138 was an oncogenic microRNA, which is overexpressed in undifferentiated NPC patients, we first examined its expression level in nasopharyngeal tissues and peripheral blood. In our cohort, cancer tissues samples were collected from 42 primary NPC and 29 recurrent NPC patients. To evaluate the expression level in the cancer tissues, the miR-138 level was quantified by real-time quantitative polymerase chain reaction. For primary NPC, the expression level was compared with 29 normal nasopharyngeal epithelia. For recurrent NPC, the microRNA level was compared with the paired normal mucosa counterparts obtained from the same patients. In addition, plasma samples were also collected from 22 primary NPC, 21 recurrent NPC and 17 normal individuals. Our data suggested that there was no difference in the miR-138 expression level in primary NPC tissue and normal nasopharyngeal tissue from control. There was no difference in the circulating miR-138 levels in the primary NPC, recurrent NPC and normal control groups. The circulating miR-138 could not be used to differentiate NPC patients from the normal individuals. Further functional analysis on the undifferentiated NPC cell line HONE1 suggested that miR-138 overexpression could enhance NPC cell proliferation, migration and invasion in comparison with the mock control. With the use of high-throughput gene expression arrays, we observed that multiple cancer-related pathways were affected in miR-138 overexpressed NPC cells. Staining with Acridine orange (AO) and phosphorylated H2AX (γH2AX) showed that miR-138 overexpression is associated with an enhanced response to radiation. Our results are concordant with other similar studies suggested that miR-138 is an oncogenic microRNA which play an important part in the undifferentiated NPC tumorigenesis. Further studies, based on larger sample size, are warranted to explore the clinical use of this small RNA in diagnosis, prognosis and management of undifferentiated NPC. / published_or_final_version / Surgery / Master / Master of Philosophy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, we aimed to sequence the complete EBV genomes harbored in NPC tumor biopsies and compare against the non-NPC EBV strains to identify NPC-specific EBV variations. In the first part of the study, EBV genome contained in one primary NPC tumor biopsy was PCR-amplified and sequenced using next-generation and dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Accession number JQ009376), was generated by reference mapping and it appears to be a uniform strain in general despite minor heterogeneity. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC strains. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels). We found 76 non-synonymous SNVs shared amongst the Chinese GD1, GD2 and HKNPC1 isolates, while another 88 nonsynonymous SNVs were shared only by the two NPC tumor-derived strains HKNPC1 and GD2. In the second part of the study, SureSelect target enrichment technology was used instead of PCR to capture EBV DNA from total DNA. The study was scaled-up to sequence EBV strains in cell lines, saliva and NPC tumor, using the MiSeq Personal Sequencer and the Genome Analyzer IIx platforms. The reads were de novo assembled to generate 17 complete EBV genomes, out of which 9 were NPC-EBV strains. Phylogenetic analysis of all available EBV strains has demonstrated that all NPC strains were type 1 EBV. Phylogeny predicted by LMP-1 gene showed clear geographical pattern of where the EBV strains were isolated. A total of 5,011 variations were identified by comparing every EBV strain against the reference. MicroRNAs and EBERs are generally well conserved across all genomes. Comparative analysis of variations between NPC and non-NPC EBV strains discovered 904 NPC-specific variations, out of which 112 appeared in more than one NPC strains. Among these recurrent variations, 39 non-synonymous substitutions and seven deletions in coding region were found. About half of these recurrent variations were located in EBNA-3A, -3B and -3C, while the rest was found in latent, tegument, capsid and packaging-related proteins and transcription factors. There were two NPC EBV strains isolated from the primary tumors which later diagnosed to have distant metastasis. Unique variations were shared in these two EBV strains in regions between IR2 and IR3, where genes such as BPLF1, BOLF1 and EBNA-3A, -3B and -3C were located, and leftward of IR3, where BBLF2/3 and BBRF1 were found. In conclusion, we have demonstrated the feasibility of target capture and next-generation sequencing in whole genome sequencing of EBV. Comparison of reference mapping and de novo assembly of EBV sequences illustrated that both are feasible approaches, though de novo assembly is preferred since the method is less dependent on the reference genome. Large-scale sequencing of NPC and non-NPC EBV strains may facilitate the discovery of previously unknown variations of biological significance and reveal the diverse role of EBV in NPC pathogenesis. words) / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Nasopharyngeal carcinoma (NPC), endemic in southern China and Southeast Asia, was ranked 7th as the most common new malignancy in Hong Kong. Metastatic and recurrent NPC have a poor prognosis despite recent advancement in medicine. Inactivation of tumor suppressor genes (TSGs) through the loss of chromosomal regions is frequently reported in NPC. With the recent discovery of cancer stem cells (CSCs), which are refractory to current therapies, a new paradigm shift in the perspective of cancer therapy development has emerged. For the first time, this study aims to unravel the complexity of NPC tumorigenicity for identifying more effective targets by studying the possible interplay between CSCs and TSGs. NPC cell lines had different expression profiles of CSC markers, confirming not all CSC markers are applicable to every tumor type. Although CD24/CD44 were expressed in NPC, however CD24+CD44+ NPC cells did not initiate tumor formation. By utilizing a cancer hybrid cell model with a transferred single copy of chromosome 3, physiological β-catenin up-regulated core stem cell markers through the activation of Wnt signaling pathway in NPC. Moreover, the down-regulation of β-catenin suppressed chemoresistance and inhibited cell proliferation, colony formation, angiogenesis, the epithelial-mesenchymal transition (EMT) process, and the tumor microenvironment factors. Amongst the tumor microenvironment factors, chemokine Rantes and matrix metalloproteinase were down-regulated when β-catenin was knocked down. Therefore, activation of Wnt signaling provide an alternative platform for identifying putative CSCs in NPC, leading to the identification of several prospective CSC markers in NPC. Down-regulation of IKBB, a NF-KB inhibitor, in the majority of NPC patients indicated that IKBB plays a prominent role as a TSG in NPC. In this study, IKBB was found to exert its tumor suppressive functions by abrogating tumor formation, cell migration, invasion and angiogenesis. Angiogenic factors, including Rantes, Upar, IL6 and IL8, were significantly down-regulated by IKBB. In addition, IKBB also suppressed the binding activity of NF-KB. The involvement of Akt/Gsk-3β pathways was also observed. Taken together, IKBB regulated NPC tumorigenesis through NF-KB/Akt/Gsk-3β and interaction with tumor microenvironment. Collectively, this study demonstrated that the progression of NPC is not simply initiated by a single signaling pathway, but a dynamic and complex interplay between multiple signaling networks and the tumor microenvironment. NPC tumorigenesis is hypothesized to be driven by orchestrated interaction between CSCs and TSGs through crosstalk with the tumor microenvironment. Amongst the major players in the tumor microenvironment, Rantes/CCL5, IL6, and the matrix metalloproteinase are envisaged to induce angiogenesis, EMT, and metastasis in NPC. This dynamic intercommunication between CSCs and tumor suppressor IKBB signaling networks may shed better insights on modulation of the major hallmarks of cancer in orchestrating NPC development. The modulation of the major hallmarks of cancer by CSCs and IKBB, a TSG, involves promotion of aberrant proliferation, enhancement of invasion and metastasis, induction of angiogenesis, circumvention of tumor suppressors, and prevention of cell death. Taken together, selective and synergistic co-targeting these signaling networks and the tumor microenvironment will provide a more effective new modality of treatments for NPC. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy