Medicine (St Vincent's) - Theses
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ItemRoles of cyclin-dependent kinase substrates: cell cycle and beyond( 2015)The cyclin-dependent kinases (CDKs) are serine/threonine specific kinases that are key regulators of the cell cycle. However, several reports indicated their roles in other pathways. Therefore, it is important to identify novel CDK substrates in order to gain a better understanding of the pathways they regulate and in turn, the diseases where they are deregulated. In this thesis, I describe two substrates; Brahma (Brm), an ATPase subunit of the SWI/SNF chromatin remodeling complex and Breast Cancer Metastasis Suppressor 1 (BRMS1), a metastasis suppressor in human cancers. Brm has long been modeled to be part of the pRb/E2F complex, regulating entry into S phase of the cell cycle. In addition, several studies have indicated that Brm interacts with Cyclin E and CDKs. Here, I demonstrate that Drosophila Brm is phosphorylated in vitro by both Cyclin A and Cyclin E/CDK2. Furthermore, using Drosophila as an animal model, a phospho-mimic of Brm was able to bypass the developmental G1 arrest in the wing discs’ zone of non-proliferating cells (ZNC), indicating its role in inhibiting S phase entry. In addition, based on the phenotypes obtained from the expression of Brm phospho-mutants and the current literature, I postulate that CDK-mediated phosphorylation of Brm also has roles in maintaining genomic stability, cell signaling and expression of cell adhesion systems. Our laboratory had previously identified the regulation of Drosophila EGFR/Ras signaling by Brm-DN and its antagonism by Gem. In this thesis, I have further characterised the roles of Brm-DN and Gem in Drosophila development and have identified Rhomboid (a positive regulator of EGFR/Ras signaling) to be the target gene that is transcriptionally regulated by Brm and Gem. Finally, BRMS1 is a metastasis suppressor, better known to exert its functions by being a part of the SAP30/mSin3/HDAC complex to modulate transcription. Furthermore, it was previously reported to be in complex with RBP1, a CDK substrate, which inhibits S phase entry. In this thesis, we have found that BRMS1 is phosphorylated by CDKs both in vivo and in vitro. Using Mass Spectrometry, we have further identified the phosphorylated site to be Serine 237. Interestingly, mutation of this phosphorylation site had no impact on cell cycle progression and BRMS1’s role as a transcriptional regulator, however, it modulated BRMS1’s role in inhibiting cell migration. Taken together, this thesis confirms the CDK-mediated phosphorylation of two proteins and has expanded our understanding of the various roles that CDK substrates have beyond the cell cycle. Overall, this work provides further insights into the role of CDK substrates in cellular behaviour, tissue growth and differentiation, and the development of cancer and metastasis.
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ItemEpithelial - mesenchymal plasticity in circulating and disseminated tumour cells( 2013)Metastasis, or the spread of cancer from the site of origin to a distant site, is the leading cause of cancer-associated death. The work presented in this thesis revolves around the process of metastasis in order to understand some of the underlying mechanisms. Specifically, the focus of this project was on cancer cells moving through the blood circulation, known as circulating tumour cells (CTC), as well as cancer cells that have escaped into a distant site but are undetectable by conventional screening methodologies, known as disseminated tumour cells (DTC). Collectively, CTC and DTC are often referred to as minimal residual disease. Whilst there is an exponentially growing amount of attention on minimal residual disease research, in addition to a large push towards the use of these cells (especially CTC) clinically, their clinical utility currently remains in limbo. The detection and enumeration of CTC/DTC in cancer patients has been demonstrated to associate with worse disease-free survival and overall survival, yet a relatively small proportion of cancer patients with detectable CTC/DTC do not fit the predicted relapse and/or survival trends. Thus, there is growing emphasis in published literature to delve beyond the simple detection of CTC/DTC, and into their characterization. The belief is that this will then enable one to identify a patient who bears CTC/DTC that are of actual biological concern. Particularly, the ability of cancer cells to switch between epithelial and mesenchymal states, or even display both epithelial and mesenchymal properties, has been proposed by our lab to be important in the generation and function of CTC/DTC. This ‘flexibility’ in cellular status has been termed in our lab as epithelial – mesenchymal plasticity (EMP). This project is divided into three main sections pertaining to; (i) establishment and optimisation of CTC/DTC capture and detection/analysis protocols, (ii) the use of xenograft mouse models for studying EMP in minimal residual disease, and (iii) the study of EMP in minimal residual disease of metastatic breast cancer patients. A number of other laboratories around the world also propose a role for EMP in CTC/DTC, and several publications have come forth in very recent years attempting to look into this phenomenon. However, to the best of our knowledge, (i) the work presented in this thesis is the first characterising the EMP status of CTC/DTC in Australia, and (ii) the only of its kind (globally) utilising the MDA-MB-468 and ED03 xenograft models to study minimal residual disease using our specific experimental design and ‘in-house’ developed assays.