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ItemThe contribution of genetic variations in the region of the parathyroid hormone-like hormone, PTHLH, gene to breast cancer susceptibilityFreeman, Adam Noel ( 2016)Aims: • Analyse the evidence for PTHLH and PTHrP, its protein product, playing a role in breast cancer and update the empirical definition of the gene. • Describe the 3-Dimensional structure of the PTHLH region and determine its system of regulatory interactions, including remote regulatory elements affecting PTHLH. • Integrate existing tools in addition to the novel perspectives generated above to enable a comprehensive annotation and analysis of genetic variants identified through molecular epidemiological techniques including Genome-Wide Association Studies (GWAS) and somatic DNA sequencing of tumour tissue to derive putative molecular mechanisms for the region’s involvement in breast cancer susceptibility. Methodology: • Review published studies and databases, integrating findings from diverse sources. • Acquire and analyse DNA and RNA sequencing, regulatory, expression, algorithm-inferred, and proximity-ligation data from multiple public data sources including ENCODE, ROADMAP, dbGAP, COSMIC and other to update the definition of PTHLH, and advance concepts of structure and regulatory function in the region. • Acquire and analyse primary GWAS data, performing imputation with multiple algorithms and references, and annotating associated variants with a suite of tools. • Use genome browsers including UCSC, WUSTL, and Golden Helix SVS, and their associated databases and tools, to analyse and visually integrate findings. Results: • PTHrP has multiple discretely functional segments active throughout the cell. It likely plays a bivalent and context-dependent role in cancer biology. Analysis of somatic variation in cancer suggests PTHrP may have a tumourigenic role within the nucleus. • PTHLH sits within a 1.3Mb TAD featuring multiple sub-structures that are integrated with the region’s regulatory function. There are activated chromatin hubs (ACHs) at protein-coding genes with evidence of extensive interaction between them. This is facilitated by the TAD’s structure, collocating them at the neck of the TAD. • The ACHs at MRPS35, KLHL42, and CCDC91 each monopolise a subordinate regulatory sub-net with a hierarchical structure. They each appear to act as important remote regulatory elements that integrate regulatory signals generated within their respective sub-nets, transferring them to PTHLH, and other genes, via ACH-ACH interactions. • There appear to be multiple discrete GWAS breast cancer association signals in the PTHLH region. Annotation of the associated variants suggests three particular regulatory elements may be its key drivers. In the context of the regulatory concepts developed in this thesis, the variants may affect a particular regulatory signal at multiple points in its assembly. PTHLH is the likely downstream target of this signal. • There are multiple poorly-describe coding, and non-coding, genes in the region that are also potential actors in breast cancer and should be investigated. Conclusion: • The PTHLH region is likely involved in the pathogenesis of breast cancer through the modification of PTHLH expression. There are likely to be other mechanisms in parallel that are yet to be fully described.
ItemRoles of cyclin-dependent kinase substrates: cell cycle and beyondbinte Roesley, Siti Nur Ain ( 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.
ItemEpithelial - mesenchymal plasticity in circulating and disseminated tumour cellsTACHTSIDIS, ANTHONY ( 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.
ItemEpithelial to mesenchymal transition in human breast cancer cellsGunasinghe, N. P. A. Devika ( 2011)Breast cancer is the most frequently diagnosed malignancy in females and accounts for the highest cancer related mortality worldwide. According to the available statistics, more than 90% of breast cancer related deaths occur not because of the primary tumour but as a result of the secondary metastases. Therefore, understanding the precise mechanisms involved in the progression of secondary metastases from the primary tumour is of utmost importance as a prognostic indicator for estimating metastatic risk, deciding the treatment plan, and monitoring of treatment response. The process of Epithelial to Mesenchymal Transition (EMT) has been widely accepted as a major mechanism that is involved in the metastatic cascade. The aim of the current thesis was to explore the effects of epithelial versus mesenchymal manipulation of breast cancer cells on the sequential events of metastatic progression. Since it is widely accepted that one of the cardinal features during EMT is loss or down-regulation E-cadherin, we modulated this molecule in our experimental model, the MDA-MB-468 (MDA-468) breast cancer cell line, to generate cells with a stable forced epithelial phenotype and epithelial cells with stable mesenchymal traits. The E-cadherin overexpressing cells were generated by transfecting MDA-468 cells with a plasmid vector encoding full length E-cadherin (468-CDH1), while dominant negative E-cadherin expressing cells were produced by transfecting the cells with a plasmid vector encoding a chimera of the E-cadherin cytoplasmic tail connected to the interleukin 2 receptor extracellular domain (468-dnCDH1). In addition, E-cadherin knocked down cells were generated using short hairpin technology (468-shCDH1-B and 468-shCDH1-D). The EMT-inducing effects of epidermal growth factor (EGF) and hypoxia were characterised in terms of EMT marker expression, morphology, proliferation, and migration. These assessments were also performed on the E-cadherin manipulated cells and also to examine the effects of these manipulations on EGF response. The effective introduction of each manipulation was confirmed, however, very little effect on EMT marker expression was seen. Morphology was affected by the shCDH1-mediated knock down, but not other treatments. Cell migration was inhibited by CDH1 transfection and stimulated in the most complete shCDH1 knock down, as was invasive colony outgrowth. Opposite trends on proliferation were noted, with tendencies for higher proliferation with more functional E-cadherin, and reduced proliferation in shCDH1 cells. Importantly, the published effects of EGF on apoptosis of MDA-468 cells were not seen, and instead an intense but reversible EMT was seen in the cells that became detached. Despite the lack of constitutive effects on EMT marker status, sufficient behavioural responses were seen to warrant in vivo analysis. When these E-cadherin manipulated cells were inoculated into the mammary fat pad of SCID mice, the cells in which E-cadherin was optimally knocked down (468-shCDH1-B) formed significantly slower growing, smaller tumours compared to the vector control while the growth rate was also diminished, albeit less dramatically, in the moderately E-cadherin knocked down (468-shCDH1-D) cells. The 468-dnCDH1 cells did not develop tumours efficiently and those that formed displayed a significantly slower growth than the vector control. This contradicts the accepted knowledge that loss of E-cadherin is indicative of aggressive tumour behaviour. However, there is a possibility that these tumours are associated with late diagnosis due to their smaller size, escaping chemotherapeutics due to their slower proliferation rate, yet being capable of forming secondary metastases, thus indeed being more aggressive. In contrast, the 468-CDH1 tumours grew marginally faster than the vector control. Overall, the results are suggestive of E-cadherin facilitating the establishment of MDA-468 primary tumours thus indicating the necessity of re-evaluation of the role of E-cadherin other than its function as a tumour suppressor. All tumour groups, except 468-shCDH1-B, displayed typical histological features of MDA-468 tumours, including widespread central necrosis surrounded by a thin rim of viable tumour tissue while the degree of necrosis was significantly lower in 468-shCDH1-B group. Vimentin positive cells, depicting EMT was seen at the invasion front and the tumour-necrosis border, whereas in 468-shCDH1-B tumours almost all cells were vimentin positive, in contrast to the lack of constitutive effects in vitro. Thus, E-cadherin knockdown promoted an intense EMT in vivo, while hypoxia and stromal influences also induced EMT in the MDA-468 cells. The local lymphovascular invasion (LVI) was a common feature associated with all tumours, and the majority of invaded tumour cells were E-cadherin positive, including the tumour emboli seen in 468shCDH1. A gradual transition from a vimentin positive mesenchymal phenotype, to concurrent vimentin and E-cadherin positive metastable status, to an E-cadherin-expressing epithelial phenotype was observed in some LVI, highlighting the occurrence of EMT reversal through mesenchymal to epithelial transition (MET). These observations provide evidence of an extremely early MET in metastatic progression. Observation of E-cadherin positive tumour cells in both local and distant lymph nodes also further emphasise the mechanism MET during tumour progression. Lung metastases were common across all tumour groups and although no significant difference was observed among the groups despite their different E-cadherin status, an obvious trend was seen towards primary tumours with E-cadherin expression having an advantage for forming secondary deposits in the lungs. The overall findings of the current thesis suggest that EMT is a transient process that rather swiftly reverts through MET during the establishment of secondary tumours even at the primary site. These studies highlight the requirement for a re-appraisal of the precise role of E-cadherin in tumour progression.