Esophageal cancer (EC), which is geographically diverse, has only a 10.7% five-year survival rate. One of the histologic forms, esophageal squamous cell carcinoma (ESCC), in Hong Kong accounts for 81.5% of the total EC cases and its five-year survival rate is only ~14%, due to its high frequency of metastasis. In our previous studies, functional complementation study of chromosome 9 defects led to the discovery of a novel tumor suppressor gene, Deleted in Esophageal Cancer 1 (DEC1), mapping to 9q32. DEC1 was shown to reduce tumorigenicity in a mouse model and its expression was shown to be associated with lymph node metastasis, early onset of ESCC, and familial ESCC development in a tissue microarray (TMA) study. Moreover, DNAJ (Hsp40) homologue subfamily B member 6 (DNAJB6), a molecular co-chaperone protein and the focus of the current study, was identified as a DEC1-interacting protein through a yeast two-hybrid screening. The interaction was further confirmed by the GST pull-down assay and co-localization studies. Using a TMA constructed with ESCC tissues from Hong Kong, the clinical relevance of DNAJB6 expression was demonstrated. In the present study, the role of DNAJB6 in ESCC was investigated using cell line-based in vivo and in vitro studies. DNAJB6 was shown to be down-regulated in ESCC cell lines. The two isoforms of DNAJB6 have distinct subcellular localizations, with DNAJB6a mainly localized to the nucleus and DNAJB6b diffused throughout the cell. Existence of a functional nuclear localization signal peptide and a functional nuclear export signal peptide was verified in DNAJB6a and DNAJB6b, respectively. In vitro evidence of possible DNAJB6a truncation was found. In vivo subcutaneous nude mice tumorigenicity assays showed that over-expression of DNAJB6a, but not DNAJB6b, suppresses tumor growth at the primary site, while DNAJB6a silencing enhances tumor growth. The suppressive effect of DNAJB6a depends on nuclear localization of the protein and the HPD tripeptide motif in the N-terminal J domain. In vitro function studies show that DNAJB6a over-expression impairs cell proliferation by suppressing G1/S transition. AKT1 phosphorylation is down-regulated in DNAJB6a over-expressed cells, leading to up-regulation of p27KIP1 protein expression and down-regulation of cyclin E1 protein expression, the G1/S transition promoter, in an AKT1-dependent manner. DNAJB6a silencing results in the opposite effect. Over-expression of DNAJB6b, but not DNAJB6a, instead suppresses lung colonization in an experimental metastasis assay, and prolongs survival of the mice. Silencing of DNAJB6a in immortalized normal esophageal epithelial cells initially induces a senescence-like phenotype with greatly reduced proliferation possibly due to oncogenic stress from up-regulation of AKT1 phosphorylation and cyclin E1 protein expression, but promotes EMT-like molecular alterations by up-regulating STAT3 phosphorylation and TWIST1 protein expression and resumes proliferation after prolonged culture. In summary, these results suggest that DNAJB6 plays a critical role in ESCC initiation, development, and metastasis and provides valuable insight into the understanding of ESCC tumorigenesis and metastasis. This suggests its usefulness as a biomarker candidate for detecting early ESCC tumor initiation. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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

Esophageal squamous cell carcinoma (ESCC) is a common cancer worldwide with a high mortality rate. High occurrence of ESCC is observed in Southeast Asia. Identification and characterization of ESCC important tumor suppressor genes will be highly beneficial to the understanding of the disease and for the early diagnosis and improvement of therapy for the cancer. In our previous microcell-mediated chromosome transfer (MMCT) studies, the transfer of an intact chromosome 13 into the recipient ESCC cell line revealed the tumor suppressive ability and putative tumor suppressive function of chromosome 13 in ESCC. One candidate gene, Plant-Homeodomain Finger Protein 11(PHF11), was identified from the study and selected for further functional studies in this current study. PHF11, located on chromosomal region 13q14, contains two plant homeodomain fingers and is a member of the PHD finger protein family. PHF11was reported to be associated with asthma and atopic diseases, yet no studies of PHF11havebeen reported in cancer to date. This study is the first to report the functional role of PHF11in tumor suppression. In this current study, two isoforms of PHF11, PHF11aand b, were reintroduced into ESCC cell lines by methods of transient tranfection and lentiviral-infection. In vitro studies showed both isoforms have cell proliferation and colony-formation inhibition abilities. In the nude mouse tumorigenicity assay, however, it was revealed that only thePHF11aisoform was tumor suppressive in vivo. No differences in angiogenesis-related factors and apoptosis-related factors were observed in PHF11a-and b-expressing cells. Further studies by Western blotting analysis and flow cytometry analysis showed that PHF11amay play a role in delaying cell cycle progression by the down-regulation of cyclin expression, while the PHF11bmay be functionally inactive, The results of this current study further confirm the tumor suppressive role of PHF11ain ESCC, whereas the PHF11b isoform was unable to suppress tumor formation in vivo. Further study of the PHF11 isoforms to identify their differential functions and interacting partners will provide a better understanding of the mechanism by which PHF11a suppressestumor growth. / published_or_final_version / Clinical Oncology / Master / Master of Philosophy

abstract / Anatomy / Master / Master of Philosophy

Background: Esophageal squamous cell carcinoma (ESCC) has been increasing in incidence, but knowledge of the genetic basis of this disease remains limited. In general, esophageal carcinoma can be divided into two main types: Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC). The pathogenesis of esophageal carcinoma still remains unclear, although some risk factors like chronic irritation, or chronic inflammation which may be caused by diseases such as gastroesophageal reflux disease (GERD) or unhealthy lifestyles like smoking have been proved to be related to the carcinogenic process. Diagnosis and treatment for this kind of cancer have continue to develop and evolve, but the 5-year overall survival rate is still relatively low. Therefore, it is clinically important to identify any potential genetic changes which may help us to discover some useful biomarker targets for the further development of more direct and harmless targeted therapy for our esophageal cancer patients. Objectives: In this study, I aimed to identify some potential oncogenic microRNA (miRNA) and to study their clinical meaning in ESCC patients. Methods: Microarray was applied to identify differentially expressed miRNAs in ESCC tumour tissue, compared with corresponding adjacent non-tumour esophageal tissue. One candidate oncogenic miRNA, miR-665, was investigated in the present study. After testing the expression level of miR-665 in ESCC cell lines and patients’ samples with RT-PCR, miR-665 stably expressing cells was established using two ESCC cell lines (KYSE30 and KYSE510). Functional characterization was then conducted using in vitro and in vivo assays to examine the effect of miR-665 towards the development of ESCC. Bioinformatic software such as Target Scan was used to generate a list of predicted target genes that may be modulated by miR-665. Results: The high expression of miR-665 has been confirmed in ESCC tissues and cell lines, showing the potential carcinogenic function of miR-665. Ectopic expression of miR-665 also demonstrates its ability to enhance tumour growth and invasion in vitro and in vivo. Bioinformatic analysis of miR-665 predicted targets showed putative binding sites for miR-665 within the 3’UTR region of NLK. Conclusions: This study has identified a novel miRNA and a related gene which might play an important role in the pathogenesis of ESCC, affecting the cancer process and tumour growth. This may help to find potential new biomarker for the future improvement and development of new treatment of ESCC patients. / published_or_final_version / Clinical Oncology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Clinical Oncology / Master / Master of Philosophy