published_or_final_version / Pathology / Master / Master of Medical Sciences

Nasopharyngeal carcinoma (NPC) has distinctive ethnic and geographic distributions, with the highest incidence in Southern China. Epstein-Barr virus (EBV) infection, non-viral environmental risk factors, and host genetics contribute to the development of NPC. In our previous studies, Fibulin-2 (FBLN2), located at chromosome 3p25.1, has been identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by using a chromosome 3 NotI genomic microarray screen, followed by functional assays. FBLN2 belongs to the fibulin family of extracellular matrix glycoproteins. It encodes a large protein consisting of cysteinerich and cysteine-free segments, three anaphylatoxin (AT) modules, a series of cbEGFlike repeated, and a fibulin module. Although FBLN2 was also identified as a candidate TSG in other cancers, its molecular characterization is still largely unknown. In the present study, lentiviral constitutive and inducible transgene expression systems, fluorescent protein labelling and reporter systems, and shRNA-mediated knockdown system were optimized and established for studies in NPC. With the use of lentiviral systems, the FBLN2-mediated signaling pathways and the functions of FBLN2-related p65 signaling pathway were revealed. Lentiviral pWPI-FBLN2 infected HONE1, HK1, and C666 cell lines consistently reduced p65 phosphorylation at serine S536. Also, FBLN2 was shown to inactivate RhoA and Cdc42, resulting in decreased stress fiber and filopodia formation. Full-length and truncated FBLN2 fragments, with the exception of anaphylatoxin module, reduced phosphorylation of p65 as well as suppressed HUVEC tube formation. The p65 pathway was then chosen for in-depth studies. Inactivation of p65 by p65 stable knockdown and IκBα super repressor overexpression showed reduced cell migration, invasion, angiogenesis, in vitro cell growth, and in vivo tumor growth. In contrast, overexpression of wild type p65 and phospho-mimic S536E p65 promotes cell migration, invasion, angiogenesis, in vitro cell growth, and cell cycle progression. Molecular studies suggested that tumorassociated angiogenesis is regulated by p65 through expression of pro-angiogenic factors and the p65 activity controls epithelial-to-mesenchymal transition (EMT)-like properties in NPC. Western blotting and qPCR analyses showed that inactivation of p65 reduced expression of pro-angiogenic factors and mesenchymal markers. Overexpression of p65 induced expression of pro-angiogenic factors and mesenchymal markers as well as enhanced EMT-like properties. The elimination of the p65 feedback mechanism by IκBα knockdown largely induced expression of pro-angiogenic factors and mesenchymal markers, as well as changes in cell morphology. In conclusion, these results suggest that FBLN2 suppresses tumor growth, tumor-associated angiogenesis, migration, and invasion through the regulation of Erk1/2, p65, and Rho GTPase pathways. The important roles of the p65 pathway in angiogenesis and EMT were also revealed. These findings provide a strategic new insight into the understanding of mechanistic role of FBLN2 in NPC and provide a better understanding for the molecular genetic basis of NPC tumorigenesis. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

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

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

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy