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

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Clinical Oncology / Master / Master of Philosophy

The majority of sporadic colorectal cancer (CRC) cases affects individuals over the age of 50, but about 10% of cases occur in young adults under 50 in Hong Kong. Apart from germline mutation of the DNA mismatch repair genes that predisposes to early-onset CRC with a high-level of microsatellite instability (MSI-H), it is unknown if the mechanisms that give rise to CRC in other young adults differ from those in older individuals. In an effort to understand the genetic and epigenetic basis of early and late-onset CRC outside the Lynch Syndrome setting, we performed a detailed characterization of 36 MSI-H and 198 non-MSI-H tumours from patients of varying ages. This characterization was based primarily on the presence of the CpG island methylator phenotype (CIMP), as measured by the level of DNA methylation; and presence of genetic instability, as measured by DNA copy number aberrations, as well as mutations in BRAF, KRAS, or TP53. Our findings revealed that early (≤50) and late-onset (>50) CRCs have different genetic and epigenetic features. In non-MSI-H cancers, CIMP-H was associated with early-onset, while CIMP-L and KRAS mutation was associated with late-onset. However, in MSI-H tumours, late-onset disease was associated with CIMP-H and BRAF mutation. In addition, promoter methylation of MLH1 in early-onset MSI-H patients had a higher frequency of occurring in the germline that was locus specific, whereas nearly all late-onset MSI-H patients showed somatic regional methylation at the MLH1 locus, as well as regional methylation on other chromosomes. This is the first study to show regional methylation at chromosome 9p21 and 7p14, which encompass the CIMP markers P16 and ELMO1, respectively. We also observed an association between regional methylation and CIMP-H, but in MSI-H cases it was linked with late-onset, whereas in non-MSI-H cases it was irrespective of age. This suggests that mechanisms of methylation seeding and spreading may be different in early and late-onset disease. Moreover, CIMP-H non-MSI-H cases had significantly worse prognosis (p=0.021 for overall survival, p=0.004 for disease-free survival) and poor response to chemotherapy compared to CIMP-L or CIMP-Neg cases. Lastly, a methylation score assigned to non-MSI-H patients based on the degree of methylation of known CIMP markers was a significant prognostic factor of disease-free survival (p=0.004), and patients with a high methylation score showed a poor response to chemotherapy. Thus, our results suggest that different genetic and epigenetic mechanisms may drive tumourigenesis in early and late-onset disease. Although further research will be needed to elucidate the exact nature of these mechanisms, our findings should help to improve current classification of CRC patients with a goal towards personalized treatment. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine