published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Ovarian cancer is a highly metastatic disease having a poor prognosis (<25%). The factors and underlying mechanisms that regulate ovarian cancer metastasis, however, are still incompletely understood. p70 S6 kinase (p70S6K), a serine/threonine kinase, is frequently activated in high-grade malignant human ovarian cancer. The aim of this study is to investigate the molecular mechanisms by which p70S6K may promote ovarian cancer metastasis. The results show that p70S6K is a critical regulator of the actin cytoskeleton, peritoneal adhesion and dissemination, and multicellular aggregates/spheroids formation in the acquisition of the metastatic phenotype. The regulation of p70S6K on the actin cytoskeleton is through two important functions: as an actin cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70S6K induced a marked reorganization of the actin cytoskeleton and directional migration of ovarian cancer cells. Actin binding and immunofluorescence studies showed that p70S6K had a direct interaction with the actin filaments with no other proteins involved. This interaction did not affect actin polymerization kinetics but cross-linked the actin filaments to inhibit cofilin-induced actin depolymerization. In addition, p70S6K mediated the activation of Rac1 and Cdc42 GTPases and their downstream effector p21-activated kinase 1, but not RhoA. Peritoneal adhesion and dissemination is regulated by p70S6K through integrin expression. Expression of p70S6K siRNA efficiently inhibited ovarian cancer cell adhesion to fibronectin and laminin among different peritoneal extracellular matrix components, as well as to human primary peritoneal mesothelial cells. These effects were associated with the expression of alpha5 and beta1 integrin. Studies into the mechanisms suggest that p70S6K may upregulate alpha5 integrin by a transcriptional mechanism whereas beta1 integrin is regulated at a post-transcriptional level. Enhanced expression of alpha5 and beta1 integrin by active p70S6K mediated the subsequent peritoneal adhesion. In ovarian cancer xenografts, p70S6K and beta1 integrin interference significantly inhibited peritoneal dissemination through reduction in the number and weight of tumors. Multicellular spheroids are present in the malignant ascites of ovarian cancer patients. Using a 3-dimensional culture system, expression of p70S6K siRNA resulted in inhibition of multicellular spheroid formation, which was mediated by N-cadherin but not E- or P-cadherin. In addition to spheroid formation, inhibition of p70S6K was associated with reduced growth of spheroids and disaggregation capabilities on different extracellular matrix components. Taken together, these findings indicate that p70S6K plays an important role in the biology of ovarian cancer metastasis through regulation of several critical steps in dissemination and migration, suggesting that p70S6K could be explored as a potential therapeutic target in ovarian cancer. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Hypoxia, a state of low oxygen, is a feature of most solid tumours as a consequence of poor tumour vascularisation. The mechanisms, which allow cancer cells to survive and continue to grow in hypoxia, are coordinated by the transcription factor HIF. The tumour suppressor gene van Hippel-Lindau (vHL) that targets HIF for degradation is mutated in the vast majority of renal cell carcinomas (RCCs), highlighting the importance of hypoxia to tumour biology. There is, therefore, an important need to understand the adaptive changes mediated by hypoxia and to target this clinically. One class of genes regulated by HIF are microRNAs (miRNAs). MiRNAs are short, single stranded RNA that primarily inhibit protein expression from target m RNA. The first aim of this project was to identify novel hypoxia regulated miRNAs in bladder cancer and assess their functional significance. It was found that a number of miRNAs were induced in hypoxic conditions. The hypoxic induction of miR-210 was conserved in all cell lines tested. MiR-145 was found to be highly induced by hypoxia in RT4, a cell line derived from a low- grade, non-muscle invasive tumour. We showed that miR-145 was a novel, HIF target gene with two hypoxia response elements identified within the promoter. Functionally we found that miR-145 induces apoptosis in RT4 cells. MiR-100 was downregulated in hypoxia, but this downregulation did not involve HIF. Regulation of miR-100 was of interest, as it is known to target FGFR3, a gene commonly overexpressed or mutated in bladder cancer. Concomitant with a decrease in miR-100, both the mRNA and protein level of FGFR3 were found to increase in hypoxia in RT4 and RT112 cells. Increased FGFR3 expression in hypoxia was involved in sustaining activation of the downstream signaling targets phospho-PKB and phospho-ERK. In addition, we demonstrate a role for FGFR3 in regulating both 2D and 3D growth and of miR-100 in regulating 3D growth of RT4 cells. We also showed that miR-100 decreased the protein levels of mammalian target of rapamycin (mTOR). However, transfection of miR-l00 into RT4 cells did not affect the sensitivity of this cell line to rapamycin. The genetic and biochemical changes that occur in (hypoxic) tumours may alter their responsiveness to chemotherapeutic agents such as rapamycin. The second aim of this project was to investigate the responsiveness of RCCs to clinically approved chemotherapeutic agents, with the goal of correlating any differences in response to alterations in expression of specific genes. Although hypoxia regulated miR-100 did not affect sensitivity to rapamycin, we extended these studies and investigated the role of vHL status on response of renal cancer cell lines to sorafenib, sunitinib, rapamycin and metformin. We found that the presence of vHL increased resistance to rapamycin. Sensitivity to these drugs was also tested in 10 primary cell lines. There was varying sensitivity to these drugs across the cell lines representing the heterogeneity of renal cancer. We analysed the expression of a number of genes in the m TOR and hypoxic pathways in these tumours, we found the expression of a known hypoxic gene REDDl correlated with sensitivity to rapamycin. REDDl expression levels were also higher in tumour tissue when compared to normal renal parenchymal tissue and was associated with other prognostic markers such as CA9, miR-210 and vascular invasion suggesting a role as a diagnostic or prognostic marker and in patient selection for treatment with rapamycin.

Sze, Siu Ching. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 109-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).