Epithelial ovarian cancer is a leading fatal malignancy in women. The high mortality rate of this cancer is due to the poor prognosis and that the majority of patients are diagnosed at late stage. Therefore, a combination of cytoreduction and adjuvant chemotherapy is the only choice for this disease at late stage. Platinum-based chemotherapy regimens are the first line treatment for ovarian cancer. However, the repetitive challenges of platinum-based agents and the frequent relapse cause acquired chemoresistance which is the major obstacle for clinical management of this disease. The underlying molecular mechanism for acquired chemoresistance remains largely unclear. Therefore, understanding the molecular mechanism in acquired chemoresistance of ovarian cancer is urgently needed. Emerging evidence has suggested that dysregulation of miRNAs is significantly involved in acquired chemoresistance in human cancers. In this study, the expression status and functional roles of miR-199b-5p were characterized in the chemoresistance of ovarian cancer. By miRCURY LNA™ miRNA array, miR-199b-5p was one of the downregulated miRNAs identified in acquired chemoresistant ovarian cancer cells. Further Q-PCR analysis found that miR-199b-5p exhibited a pattern of progressive reduction from early to advanced stage and from low to high grade ovarian cancer tissue samples (N=79). Interestingly, the expression of miR-199b-5p could be restored upon 5-Aza-2’-deoxycytidine (5-Aza-dC) treatment in ovarian cancer cells. Methylation-specific PCR and bisulfite genomic sequencing revealed that the promoter region of miR-199b-5p (Chromosome9q34) was frequently hypermethylated in chemoresistant ovarian cancer cells. Functionally, enforced expression of miR-199b-5p remarkably diminished the cisplatin-resistance in miR-199b-5p low expressing ovarian cancer cells, whereas depletion of miR-199b-5p by siRNA approach augmented cisplatin-resistance in miR-199b-5p high expressing ovarian cancer cells. A tumor xenograft mouse model further confirmed that miR-199b-5p could sensitize ovarian cancer cells to cisplatin-mediated tumor growth inhibition in vivo. On the other aspect, computational analysis plus luciferase reporter assay and western blotting identified JAG1, a key ligand of Notch1 signaling frequently associated with chemoresistance of human cancers, as a direct target of miR-199b-5p in ovarian cancer cells. Intriguingly, an inverse relationship between the downregulation of miR-199b-5p and the upregulation of JAG1 was found by in situ hybridization (ISH) and immunohistochemical (IHC) analyses in a commercial ovarian cancer tissue array (OVC1021). Functionally, enforced expression of JAG1 increased cisplatin resistance of ovarian cancer cells. In contrast, depletion of JAG1 by siRNA approach abrogated the cisplatin-resistance of ovarian cancer cells. This finding was in consistent with the effect of enforced expression of miR199b-5p in cisplatin-resistant ovarian cancer cells. Mechanistically, the Notch1 luciferase reporter assay and western blotting analysis demonstrated that the Notch1 signaling activity could be activated by JAG1 or conversely be inhibited by enforced expression of miR-199b-5p. Further investigation using the Notch specific inhibitor γ-secretase showed that JAG1-Notch1 signaling is a crucial pathway associated with chemoresistance of ovarian cancer in vitro and in vivo. Taken together, this is the first study showing that the epigenetic silencing of miR-199b-5p leads to aberrant activation of JAG1-Notch1 signaling and such signaling cascade plays a critical role in tumor progression and chemoresistance of ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Breast cancer is one of the most prevalent cancers affecting women worldwide. In the breast, estrogen receptor alpha (ERα), upon binding with ligands, activates gene transcription and promotes cell growth and proliferation. Tamoxifen, a selective antagonist of ERα in breast, has been proved to be effective therapeutically. In spite of this, resistance remains a prominent issue and underlying mechanisms are not yet fully understood. Aberrant regulation of ER expression at genetic and transcriptional levels has been implicated as the mechanisms accounting for tamoxifen resistance. However, regulation of ERα expression at translational level including protein synthesis and degradation has not yet been characterized and its relevance to tamoxifen resistance has not been described. At level of protein synthesis, eukaryotic translation initiation factor 4E (eIF4E) selectively enhances the translation of 4E-sensitive mRNAs which contain long and complex 5’-untraslated regions (5’-UTR). eIF4E is often over-expressed in cancers. In silico analysis revealed that ERα contained a highly structured 5’-UTR similar to reported eIF4E-sensitive mRNAs, suggesting that ERα mRNA might be eIF4Esensitive. We showed by polysome fractionation and subsequent Q-PCR quantification that the ERα mRNAs were more actively translated in the cell line expressing higher levels of eIF4E. Consistently, transient transfection of eIF4E into an ERα-positive cell line resulted in enhanced protein expression of ERα. Moreover, subcelluar fractionation showed that eIF4E was bound with ERα mRNAs in the nucleus thus participating in transportation of mRNAs from the nucleus into the cytoplasm. Therefore, eIF4E could positively modulate protein synthesis of ERα by enhancing mRNA export in the nucleus as well as translation in the cytoplasm. Their positive correlation was validated in vivo using 106 Chinese breast cancer samples (Chi-square test, p=0.004). It was also found that elevated expression of eIF4E could mediate resistance to tamoxifen treatment and enhance cell survival. This could be due to enhanced expression of ERα or activation of PI3K/Akt pathway upon eIF4E over-expression. At the level of degradation, ERα is conjugated to poly-ubiquitin chains catalyzed by multiple enzymes and degraded by 26S polysomes. Carboxyl-terminus of Hsc70- interacting protein (CHIP) is an E3 enzyme specific for ERα degradation through interaction with ERα’s ligand-binding domain (LBD). Various splicing variants of ERα have been reported and implicated in tamoxifen resistance by interfering with functions of ERα wild type. Variants ERαΔ4, ERαΔ5, ERαΔ6/7 and ERαΔ7 with different degrees of truncation in their LBDs and differential expression were detected or reported in human breast cancers. Their interactions with CHIP may be different, resulting in variations in degradation. We found that the degradation of ERαΔ6/7 through ubiquitin-proteasome pathway was impaired whilst the degradation of other variants were less affected. This finding suggests that the binding site of CHIP to ERαmight be located within the peptide sequences encoded by exon6. Furthermore, as ERαΔ6/7 plays a dominant negative role in regulating functions of ERα wild type, aborted degradation of this variant may result in accumulation of this variant in the cell, inhibiting and inactivating ERα, making the cells refractile to tamoxifen treatment. / published_or_final_version / Pathology / Master / Master of Philosophy

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Epstein-Barr virus (EBV) is one of the most successful human pathogens in the world and establishes a lifelong persistent infection in 95% of adult population worldwide. It is associated with a number of malignancies including Burkett’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma(NPC) and gastric carcinoma. EBV was the first virus reported to produce microRNAs (miRNAs) and it encodes 44 mature miRNAs from 2 viral transcripts, BART and BHRF1. The BART transcript is abundantly expressed in all latently infected cells, particularly in epithelial cells. The BART miRNAs (miR-BARTs) were shown to be involved in apoptosis inhibition, immune evasion, metastasis, viral and cellular transcripts regulation. The high expression profile and the diverse functions of miR-BARTs suggest that they may play a critical role in the development of EBV-associated NPC. In order to understand the importance of miR-BARTs in NPC development, in this thesis, I conducted a study on the miR-BARTs function in nasopharyngeal carcinogenesis. In the first part, I characterized the cellular target and function of an abundantly expressed miR-BART in NPC. In the second part, I established a novel recombinant EBV construction system for genetic studies of miR-BARTs in nasopharyngeal epithelial (NP) cells. In the first part of my study, I characterized the cellular target and function of miR-BART3* in NPC. As predicted by bioinformatics, tumor suppressor protein DICE1 was a cellular target of miR-BART3*. The specific targeting between miR-BART3* and DICE1 3’UTR was validated by luciferase assays and the downregulation of both endogenous DICE1 protein and mRNA was observed in EBV+epithelial cells and miR-BART3* expressing cells. In addition, restoration of DICE1 protein expression by inhibition of miR-BART3* was also demonstrated in EBV+epithelial cells. Moreover, miR-BART3* was shown to promote cell proliferation via suppression of DICE1. Analysis of22 human nasopharyngeal(NP)biopsy samples demonstrated the inverse correlation between miR-BART3* and DICE1 expression. Taken together, miR-BART3* downregulates the tumor suppressor DICE1 protein to promote cell proliferation and transformation in NPC. Besides the candidate approach, genetic studies can provide a systematic view of the functions of all miR-BARTsand shed light on the importance of miR-BARTs in NPC under a more physiological condition. At present, bacterial artificial chromosome (BAC) technology is commonly used for recombinant EBV construction. However, the intrinsic disadvantages of BAC prevent its use in NP epithelial cells. Therefore in the second part of my study, I established a novel CRISPR/Cas9-mediated recombinant EBV construction system and constructed a miR-BART deleted recombinant EBV. The CRISPR/Cas9 system was demonstrated to be effective in EBV genome editing and Akata cells were infected by the recovered recombinant mutant virus. Infected Akata cells served as the source for NP cell infection through co-culture. The new CRISPR/Cas9 system have many advantages over the conventional EBV BAC method. My work reported in this thesis not only illustrated the importance of miR-BARTs in NPC, but also provide a new technology platform for further study of miR-BARTs in NP epithelial cells. (An / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

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

published_or_final_version / Microbiology / Master / Master of Philosophy