Surgery (St Vincent's) - Theses
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ItemIdentifying functional drivers of epithelial-mesenchymal transition (EMT) in human breast cancer: the integrin/ILK axis( 2018)Breast cancer is the leading cause of cancer in women worldwide, and over 90% of deaths caused by breast cancer are due to metastases, many of which are not responsive to current therapies. The ability for cells to acquire a metastatic phenotype includes epithelial mesenchymal transition (EMT), invoked as a critical component of the metastatic cascade. During the process of EMT, epithelial cells undergo a temporary conversion acquiring molecular and phenotypic changes that facilitate the loss of epithelial features, and the gain of mesenchymal phenotype. Such transformation promotes cancer cell migration and invasion. EMT is typically characterized as a loss of the epithelial cell adhesion proteins E-cadherin and cytokeratins, coupled with the gain of mesenchymal-associated molecules N-cadherin and vimentin. However, these proteins may not always be present in cancer systems. For example, one of the limitations in the use of vimentin as a prognostic marker in breast carcinomas is the likelihood that vimentin-positive cells may have migrated away from the primary mass, and become buried in the surrounding stroma, which is also vimentin-positive. Therefore, the identification of new markers which better represent EMT in breast carcinomas, and allow for a more specific detection of EMT-derived or EMT-prone breast cancer cells in the tumour vicinity, could have a dramatic impact on breast cancer prognosis. The work presented in this thesis describes a comprehensive characterization of two human breast cancer EMT model systems: the in vitro PMC42 cell system and the in vivo EDW-01 patient derived xenograft system. Specifically, the focus of this project was to perform a sequence of studies to assess the regulation of α2 and β1 integrin (ITGα2, ITGβ1), and ILK. The functional role of the integrin/ILK axis in the mesenchymal state, and in the epithelial-to-mesenchymal transition is explored and assessed.
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ItemUsing a murine bioengineering model to study cancer cell biology: the effects of mammographic density on breast cancer progression( 2017)Breast cancer (BC) remains a leading cause of cancer-related morbidity and mortality for women worldwide. Mammographic density (MD) has been well recognized as a strong risk factor for BC, independent of its masking effect for small tumours on a mammogram. Due to the common presentation of high MD (HMD) in the community, especially in pre-menopausal women, MD is arguably the most important risk factor for BC when taking into account of its high population-attributable risk. However, much remains to be learned about the biological mechanism underlying MD-associated BC risk. My team previously utilised a murine engineering biochamber model to show that human breast tissues of high and low MD not only remained viable, but also maintained their radiological and histological features in relation to their MD status for at least 6 weeks. Extending from the prior findings, this PhD study aimed to further explore the biological mechanisms behind MD-associated BC risk, and is divided into four main sections: (i) I first attempted to develop the murine biochamber model further by implanting collagenase digested and flow cytometry sorted single cells to pave the way for manipulations of specific cell types that might be responsible for HMD. I found that collagenase digested and flow cytometry sorted single cells of high or low MD breast tissue reconstituted glandular organoids in murine chambers, albeit in limited numbers; (ii) second, based on a collection of human high and low MD breast tissues from prophylactic mastectomy procedures over a period of 5 years, I evaluated the histological differences between within-individual high and low MD mammary specimens of all participants, and found that the HMD tissue microenvironment was significantly altered compared with that of LMD -- HMD was characterised by increased levels of collagen organisation and quantity, aromatase immunoreactivity and immune cell infiltration of various subtypes; (iii) The serendipitous finding of increased immune cell presence in HMD led to my subsequent examination of the potential differences in immune cell representation between patient-matched high and low MD tissues, the immune infiltrates of both innate and adaptive system, and cytokines such as IL-6 and IL-4, and I found that immune cells of various subtypes were significantly raised in HMD tissue compared with LMD; and (iv) parallel to the human breast tissue studies, I also tested whether high and low MD human tissue had any direct effect on cancer cell growth and dissemination; using our murine biochamber model, I showed that compared to co-inoculation with LMD tissue, HMD tissue stimulated the progression of MCF10DCIS.com cells that represented cells of ductal carcinoma in situ (DCIS), to lesions resembling invasive ductal carcinomas, as well as their metastases in the mice hosting the murine biochambers. Over the past twenty years, the body of evidence on various aspects of MD and its associated BC risk has been expanding, however, to the best of my knowledge, (i) my study contained the largest cohort of high-risk women in Australia to characterise immunohistochemical and immune cell differences between high and low MD, and (ii) the work presented in this thesis is the first to utilise the murine biochamber xenograft model to test human breast organoids formation from single cells and to evaluate the direct effect of MD on human breast cancer cell progression in vivo. Collectively my work has defined that HMD is characterised by an increase in stromal cells, extracellular matrix and inflammation. I have shown that HMD stimulated the progression of early stage BC cells, which highlights the importance of MD being considered for BC diagnoses, treatments and surveillances.
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ItemInvestigating epithelial-mesenchymal plasticity in breast cancer using circulating tumour cells and circulating tumour DNA( 2017)Breast cancer is the most frequent invasive cancer among women worldwide with mortality primarily caused by metastasis. One of the key proposed processes underlying metastasis, including the escape to the bloodstream, is epithelial- mesenchymal plasticity (EMP). This refers to the dynamic transition between epithelial and mesenchymal phenotypes of cells within the primary tumour mass. Within the bloodstream, tumour cells and tumour DNA, which are referred to as circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), respectively, have showcased their potential use as liquid biopsy in cancer management. The presence of CTCs has been shown to associate with poor prognosis in metastatic cancers, which becomes worse with higher CTC numbers. By virtue of carrying genetic and epigenetic features of primary tumours, ctDNA has demonstrated its utility in detecting and monitoring cancer progression. Various studies conducted on the molecular characterisation of CTCs have generated data supporting the role of EMP in generating CTCs. However, the dynamic changes in expression, especially of genes associated with EMP, between primary tumours, CTCs and metastases remain far from conclusive. In keeping with this paradigm, as cancer cells in the primary tumours shift from the epithelial to the mesenchymal phenotype, any released ctDNA may have epithelial and/or mesenchymal features depending on its cellular origin. To date, research on the utility of EMP-associated methylation markers to detect ctDNA is lacking. In light of the suggested role of EMP in different key steps of cancer progression and metastasis, this thesis has aimed to study EMP reflected in CTCs and ctDNA to provide further insights into the CTC molecular characteristics and assist in the detection of ctDNA. This thesis is comprised of two principal areas of study: (1) the gene expression profiling of CTCs in two human breast cancer xenograft models, and (2) the locus- specific methylation profiling of breast cancer cell lines and breast tumours. Firstly, the expression profiles of EMP-associated genes were characterised in CTCs at the population level and the single cell level, and were compared with the expression profiles of primary tumours and (where possible) metastases, for two xenograft models, the MDA-MB-468 and ED-03. In pooled CTCs relative to primary tumours from both models, a significant increase in expression of mesenchymal markers (SNAI1, VIM, SERPINE1 and NOTCH1), and surprisingly, of a typical epithelial marker CDH1, were observed. A decrease/loss of EPCAM was reproducibly observed in CTCs of both models, while decreased CD24 and EGFR in CTCs were only seen in the MDA-MB-468 model. Genes associating with hypoxia (HIF1A, BNIP3 and APLN) and cellular metabolism (PPARGC1A) were also significantly elevated in CTCs of both models. In additional studies, a direct lysis method was successfully optimised to assist the gene expression study in single cells. The subsequent analysis of single CTCs revealed heterogeneity of CTCs, with co-expression of epithelial and mesenchymal markers, and high expression levels of epithelial markers in individual CTCs. The results reinforced the complex gene expression profiles and alterations seen in pooled CTCs. Secondly, a panel of DNA methylation markers, including those associated with EMP, was developed and tested in breast cancer cell lines, primary tumours and whole blood of normal controls to identify suitable markers for ctDNA detection. In a panel of breast cancer cell lines spanning the epithelial-mesenchymal spectrum, the majority of epithelial cell lines were methylated for cancer-associated markers (i.e., RASSF1A, RARß) and epithelial methylation-based markers (i.e. VIM, DKK3 and CRABP1). Mesenchymal cell lines were exclusively methylated for mesenchymal methylation-based markers (GRHL2, MIR200C and CDH1); however, the level of methylation was quite low. The methylation profiles of the studied genes classified primary tumours into intermediate phenotypes while few tumours were mesenchymal. In addition, MIR200C, RASSF1A, AKR1B1 and TWIST1 were methylated at high frequency in our cohort. Among these, RASSF1A and AKR1B1 showed no methylation in whole blood of normal controls, suggesting their potential use as markers for ctDNA detection from plasma of the breast cancer patients in our cohort. Preliminary experiments established ddPCR assays for these two markers, allowing further testing on patient cell-free DNA samples for the detection of ctDNA. The results of this thesis challenge the conventional model of EMT, where cells in epithelial tumours become mesenchymal, with associated migratory properties, and later re-epithelialise (MET) at a distant metastasis. Firstly, the complex and consistent alterations in the epithelial and mesenchymal markers in CTCs across the two models is suggestive of a ‘hybrid’ phenotype. The overall findings from the CTC work that CTCs were not as mesenchymal as expected also suggest that other processes than EMP directly influence the generation and survival of CTCs. Secondly, nearly all the examined breast tumours exhibited an intermediate rather than a strong epithelial phenotype based on the methylation profiles. This suggested that a plasticity is already present at the solid tumour state. These findings provide an alternative view of EMP in both primary breast cancer and the disseminated forms, and provide an important platform for further research in this field.
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ItemBiocellular aspects of high mammographic density as a risk factor for breast cancer( 2016)High mammographic density (MD) is one of the strongest risk factors for breast cancer after high-risk mutations, with a 4-6 fold increased risk comparing the highest to lowest MD quartiles. MD is of great clinical relevance, given that the attributable risk of breast cancer (BC) due to high MD in the population may be as high as 30%, and the already widespread use of mammography for breast assessment. However, the biological basis for high MD and its associated cancer risk is poorly understood. A validated xenograft model where the dynamic effects of drug interventions and gene perturbations on human MD tissue can be investigated in the preclinical setting will be valuable. Prior to commencement of the PhD thesis, there were no animal models of human MD that could maintain the MD differential of tumour-free breast tissues. In the first published study, we developed a xenograft model of human MD, where matched high and low MD human tissues were precisely-sampled under stereotactic-guidance from fresh mastectomy tissues and maintained in separate vascularized biochambers in SCID mice. This study demonstrated that the high and low MD biochamber tissues retained their differential radiographic density and histologic features of the original human tissue. The high compared to low MD biochamber tissues were composed of increased stromal and decreased adipose percentage areas, reflecting the MD phenotype of the original human breast samples. The MD xenograft model was then extended to examine the changes in radiographic density and histology that occurred during murine pregnancy, lactation and postpartum involution states, and after exposure to exogenous endocrine treatment. These studies demonstrated the dynamic nature of the MD xenograft model, with decreased stromal and increased adipose tissue percentage areas observed in high MD biochamber tissues during murine lactation and postpartum involution, and also in Tamoxifen-treated compared to placebo-treated mice. High and low MD biochamber tissues had increased radiographic density with postpartum involution, and increased duration of implant. The radiographic density decreased in high MD biochamber tissues of Tamoxifen-treated compared to oestrogen-treated mice. As increased Cox-2 levels have been observed in breast tumours and stromal regions of high MD tissues, we investigated the expression of Cox-2 in the epithelial and stromal cells of matched high and low MD breast tissues. We demonstrated increased staining in both epithelial and stromal cells of high MD breast tissues. We then showed that the differential Cox-2 expression in high and low MD human breast tissues was maintained in murine biochambers, and was sensitive to hormonal supplementation. Collectively, this thesis indicates that the MD model will be valuable for investigating the mechanisms of how modifying factors, such as lifestyle behaviours and endocrine treatments like Tamoxifen, can reduce MD in cancer-free human breast tissues, and would be useful in research aiming to develop preventative therapies to reduce MD-related risk, such as Cox-2 inhibition.
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ItemThe contribution of genetic variations in the region of the parathyroid hormone-like hormone, PTHLH, gene to breast cancer susceptibility( 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.
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ItemEpithelial-to-Mesenchymal Transition (EMT) in human breast cancer: investigating microRNAs in breast cancer EMT( 2015)Breast cancer is the most common malignancy among women worldwide, with mortality primarily associated with metastasis. In recent years, microRNAs (miRNAs) have emerged as a new class of master regulatory molecules with the potential to influence carcinoma progression. They are a class of small RNA molecules that regulate gene transcript stability and processing by binding to discreet motifs in the 3’ and 5’ untranslated regions of mRNAs. They regulate important mechanisms in development, including epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), which have also been associated with cancer metastasis. miRNA profiling of control and EMT-induced PMC42-ET, PMC42-LA and MDA-MB-468 cultured human cell lines using microarray and Next Generation Sequencing (NGS) was undertaken. Several miRNAs were reproducibly up- or downregulated between the untreated cells, and in response to epidermal growth factor. Variations in miRNA expression were also assessed bioinformatically using publically available data from >50 human breast cancer cell lines. Whilst a number of these have already been implicated in cancer, other novel miRNAs consistently associated with EMT/MET were also identified. The expression levels of >20 miRNAs were experimentally validated and stable miRNA manipulations (overexpression and knockdown) in cell lines with low endogenous expression were achieved by lentiviral transduction. Recent evidence has demonstrated a crucial role for the miR-200 family in carcinoma progression, tumourigenesis and metastasis in various cancers including breast cancer. miR-200 regulation of EMT and MET was identified in our microarray and NGS datasets and was characterised in the two EMT models commonly used in our laboratory – the PMC42 and MDA-MB-468 human breast cancer cell lines. In vitro functional changes upon miR-200 manipulation showed a strong correlation between high miR-200 levels and the epithelial phenotype. In vivo changes mirrored the in vitro changes, where miR-200c knockdown resulted in reduced primary tumour growth and increased axillary lymph node metastasis in the MDA-MB-468 xenograft model. Transcriptionally, it was observed that the MDA-MB-468 miR-200c knockdown tumours showed upregulation of epithelial genes (such as E-cadherin, Grhl2, EpCAM) and a corresponding downregulation of mesenchymal genes (such as BNIP3, FN1), suggesting that the MDA-MB-468 miR-200c knockdown cells that were able to survive and eventually form a tumour had to transcriptionally activate epithelial-associated genes to achieve tumour development. Despite this, circulating tumour cells in the peripheral blood of MDA-MB-468 miR-200c knockdown tumour-bearing mice showed indications of a hybrid state, trending towards a more mesenchymal profile. Apart from the miR-200 family, eight other miRNAs—namely miR-744, miR-153, miR-708, miR-483, miR-100, miR-34b/c, miR-146a and miR-29a, were short-listed as candidate EMT-associated miRNAs identified from microarray, NGS and bioinformatic analyses. Collectively our findings suggest that no one particular miRNA alone was capable of functionally altering MDA-MB-468 breast cancer cells. Interestingly, when the cells were driven to a more mesenchymal state, there was a trend towards increased lymph node metastasis, suggesting that a mesenchymal profile is advantageous in that process. The overall findings of this thesis suggest that miRNAs do play a key role in the area of breast cancer EMT and each step in the metastasis cascade can be regulated by different miRNAs. A cooperative network of miRNA changes results in downstream changes in tumourigenesis and metastasis. These studies highlight the need to further investigate context-dependent miRNA manipulations and validate miRNAs that play critical regulatory roles to properly understand the complex role of various important miRNAs in breast cancer EMT.