Surgery (RMH) - Theses
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ItemHormonally induced defects of DNA damage repair genes: an oncogenic driver of prostate cancer( 2018)Patients with defects in the mismatch repair pathway, driven by either MSH2 or MSH6 loss, experience a significant increase in the incidence of prostate cancer, while germline mismatch repair defects in either MLH1 or PMS2, exhibit no such increase. This PhD project demonstrates that androgen-receptor activation, a known driver of prostate cancer, can disrupt the MSH2 gene in prostate cancer model systems through the induction of structural variations. Prostate tumours from two contrasting risk cohorts were screened to confirm loss of MSH2 protein expression in a small number of patients. Surprisingly, it was also found that a small but significant fraction of high-risk cases exhibited reduced expression of MSH2 without complete loss. Stratifying a large independent TCGA prostate cancer cohort for MSH2 expression levels revealed that patients whose tumours exhibited either complete loss or aberrant levels of MSH2 had significantly worse survival outcomes and accelerated clinical progression. In contrast, aberrant MSH2 levels had no impact on clinical survival in colorectal cancer. This PhD project also demonstrates that reduced expression of MSH2 can be explained by androgen-induced microRNA regulatory mechanisms. Here, it is demonstrated that miR-21 and miR-141 may both target the MSH2 gene leading to reduced MSH2 protein staining and both microRNAs were seen to be upregulated in prostate cancer patients with reduced MSH2 levels. Interestingly, this thesis also shows that miR-21 and miR-141 are both regulated by androgens, implicating this mechanism as a second androgen driven method of MSH2 downregulation. Importantly this PhD project also found that aberrant MSH2 expression in prostate tumours does not induce the same enhanced immune cell mobilisation seen in colorectal tumours suggesting that the prostate is an immune privileged site. This is contrary to the findings of other studies and may warrant a re-evaluation of whether MSH2 deficient prostate cancers are likely to benefit from immunotherapies. To further investigate if the prostate tumour microenvironment is indeed in an immunosuppressive state, a detailed investigation of the transcriptomic profile of the cells of the tumour microenvironment was also conducted. This resulted in not only the discovery of immunosuppressive signatures in tumour infiltrating T-cells but also significant transcriptomic alterations in other cancer associated pathways such as osteogenesis, cell migration, epithelial mesenchymal transition (EMT), hormone signalling and angiogenesis throughout the tumour microenvironment compared to the cellular make-up of benign prostatic tissue. The data presented in this thesis constitute a significant contribution to the current understanding of how defects in the mismatch repair gene MSH2 may affect prostate cancer severity. Additionally, these studies demonstrate multiple mechanisms through which the prostate tumour microenvironment may enhance prostate cancer progression.
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ItemInvestigating the evolution of structural variation in cancer( 2017)Cancers arise from single progenitor cells that acquire mutations, eventually dividing into mixed populations with distinct genotypes. These populations can be estimated by identifying common mutational profiles, using computational techniques applied to sequencing data from tumour tissue samples. Existing methods have largely focused on single nucleotide variants (SNVs), despite growing evidence of the importance of structural variation (SV) as drivers in certain subtypes of cancer. While some approaches use copy-number aberrant SVs, no method has incorporated balanced rearrangements. To address this, I developed a Bayesian inference approach for estimating SV cancer cell fraction called SVclone. I validated SVclone using in silico mixtures of real samples in known proportions and found that clonal deconvolution using SV breakpoints can yield comparable results to SNV-based clustering. I then applied the method to 2,778 whole-genomes across 39 distinct tumour types, uncovering a subclonal copy-number neutral rearrangement phenotype with decreased overall survival. This clinically relevant finding could not have been found using existing methods. To further expand the methodology, and demonstrate its application to low data quality contexts, I developed a novel statistical approach to test for clonal differences in high-variance, formalin-fixed, paraffin-embedded (FFPE) samples. Together with variant curation strategies to minimise FFPE artefact, I applied the approach to longitudinal samples from a cohort of neo-adjuvant treated prostate cancer patients to investigate whether clonal differences can be inferred in highly noisy data. This thesis demonstrates that characterising the evolution of structural variation, particularly balanced rearrangements, results in clinically relevant insights. Identifying the patterns and dynamics of structural variation in the context of tumour evolution will ultimately help improve understanding of common pathways of tumour progression. Through this knowledge, cancers driven by SVs will have clearer prognoses and clinical treatment decisions will ultimately be improved, leading to better patient outcomes.
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ItemThe immune suppressive function of Transforming Growth Factor-β (TGF-β) in human diseases( 2016)he complex immune system has evolved to defend against pathogens while maintaining self-tolerance. Transforming growth factor-β (TGF-β) is a cytokine employed by many immune cells to suppress autoimmunity and inflammation. Therefore, dysregulation of TGF-β signalling leads to loss of autoimmunity inhibition, resulting in autoimmune diseases. Altered TGF-β levels have been observed in patients with autoimmune diseases and other inflammatory diseases. However, TGF-β level has never been approved as a diagnostic or prognostic marker due to a lack of sensitive and accurate quantitation methods. In cancer, TGF-β acts as a double-edged sword: a tumour-suppressor in early tumour development but a tumour-promoter in cancer progression and metastasis. Acting on the tumour environment, TGF-β induces immune tolerance, resulting in immune evasion. As such, TGF-β-mediated immune suppression is an obstacle to successful cancer immunotherapies and TGF-β blockade presents a viable strategic direction for developing cancer immunotherapies. However, as a pleiotropic cytokine that plays essential roles in a wide range of physiological processes, systemic blockade of TGF-β may result in a multitude of side-effects including severe autoimmune diseases. It is desirable for any TGF-β targeted treatment to be local and specific in order to minimise side-effects. In this thesis, a highly sensitive and reproducible TGF-β detection assay has been developed for measuring active TGF-β levels in serum samples from mouse and human. Using this method, we have demonstrated that patients with systemic lupus erythematosus have significantly reduced serum active TGF-β levels in comparison to healthy control subjects. Consistently, SLE-prone SHIP-deficient (SHIP-/-) and Lyn- deficient (Lyn-/-) mice also displayed lower serum active TGF-β levels than the wild- type (WT). This novel TGF-β quantitation method allows detection of active TGF-β to become a potential diagnostic or prognostic biomarker for SLE. We further explored whether TGF-β treatment ameliorates or delays autoimmune disease development in the Lyn-/- mice. Disappointingly, osmotic pump delivery of TGF-β1 didn’t prevent from or reduce autoimmune disease development in Lyn-/- mice. We examined three commonly used drug delivery methods of intraperitoneal (IP) and intravenous (IV) injections and osmotic pump implantation. Both IP and IV methods yielded a small and transient increase of in vivo TGF-β levels. All failed to achieve a lasting increase of active TGF-β in mice. New understanding and methods are urgently required to restore the TGF-β levels in mice effectively. To develop a targeted TGF-β blockade strategy, we generated an adenovirus expressing the soluble human TGF-β type II receptor fused with human immunoglobulin Fc chain (sTβRII-Fc) to trap the ligand. We have successfully infected B16 mouse melanoma cells with the adenovirus, expressing high levels of sTβRII-Fc that effectively neutralised TGF-β ligand. These adenovirus infected B16 cells were irradiated and applied as the vaccine to C57BL/6 mice. The vaccination of naïve immunocompatible C57BL/6 mice with these B16 cells resulted in the increase of CD8+ T cells in the nearby draining-lymph nodes and a higher percentage of antigen-specific CD8+ CTLs in the spleen. In mice with pre-implanted B16 tumours, the vaccination treatments resulted in a significant reduction of CD4+CD25+Foxp3+ Treg cells infiltrating the tumour site and an increase of antigen-specific CD8+ CTLs in the spleen. Consistently, mice receiving the TGF-β blockade B16 cell vaccine displayed slower tumour growth and prolonged survival than those without or with control vaccination treatments. This result demonstrated the validity of TGF-β targeted, tumour cell-based vaccine for cancer treatments. Significantly, mice pre-vaccinated with B16 cells carrying sTβRII-Fc expressing adenovirus were totally protected from small numbers of B16 tumour cells implanting and were largely protected from an even very large number of tumour cells. However, the control adenovirus achieved similar protection in mice against rumour implantation as the sTβRII-Fc virus, suggesting an additional adenovirus-mediated mechanism for enhancing anti-tumour immunity in cancer prevention. Taken together, this study demonstrates multiple mechanisms that may account for the enhancement of anti-tumour immunity by the TGF-β targeted vaccine and provides pre-clinical evidence for this novel vaccine to be developed into cancer therapeutics in the future.
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemIdentification of genes that regulate lymphatic endothelial cell migration through siRNA screening( 2013)The lymphatic vasculature is vital for tissue fluid homeostasis. It also enables effective immune surveillance, and aids the transport and absorption of fatty acids. Damaged or faulty lymphatic vessels can lead to an accumulation of lymph fluid, a condition known as lymphoedema. Metastatic cancer can also spread via the lymphatic vessels; expression of the lymphangiogenic growth factors VEGFC and VEGFD is correlated with tumour progression and negatively affects overall patient survival. Given the importance of lymphangiogenesis and vessel remodelling in development, and in pathological conditions such as cancer, it would be of interest to understand the molecules required for new lymphatic vessel growth to occur. One aspect of lymphangiogenesis that can be modelled in vitro is cell migration. The primary approach of this thesis was to utilise RNA interference technology to screen the entire human genome for molecules that regulate lymphatic endothelial cell (LEC) migration. An experimental procedure was established that was compatible with transfection of small interfering RNA (siRNA) in primary LECs, and enabled accurate quantification of LEC migration in a scratch-wound assay. Using this approach, a smallscale pilot screen of genes for 133 protein tyrosine kinases and tyrosine kinase-like molecules was performed. Several promising candidate genes were identified, whose roles in LEC migration were confirmed using siRNA and pharmacological inhibitors. The success of this approach enabled the screening of a genome-wide SMARTpool siRNA library, which was performed in a high-throughput manner with the aid of automation and robotics. A total of 154 genes were validated with medium or high confidence as regulators of LEC migration in vitro. This dataset enabled the collation of maps detailing signalling cascades and processes that may control the migration of this specialised cell type. While a proportion of the genes identified have been previously linked to cell migration or development of the vasculature, many were not known to play a role in cell migration or lymphatic biology. A parallel microarray study investigated the transcriptional response that occurred during LEC migration. This demonstrated that the majority of genes identified by the functional screen as being important for this process are not up-regulated during migration. However, gene ontology enrichment analysis found that genes associated with mitosis and the cell cycle are down-regulated during the early stages of cell migration. This prompted an investigation of the connection between cell cycle regulation and the ability of a cell to migrate effectively. Utilising high-content image analysis, the genome-wide LEC migration screen dataset was reanalysed to identify genes that, upon siRNA-mediated knockdown, led to cell cycle arrest. Importantly, this study showed that LECs are unable to migrate when arrested in S or G2/M phase, and identified a potential role for the Hippo/Warts pathway in ensuring that cells do not migrate during these phases. This thesis has revealed molecular regulators of lymphatic endothelial cell migration through a genome-wide siRNA screen. Identification of novel genes important for the lymphatics will increase the understanding of LEC signalling networks, and has implications for the development of therapeutic agents that could be used in preventing lymphogenous and distant organ tumour metastasis, or treating conditions such as lymphoedema or lymphangioma. Future investigations will define the biochemical signalling events that occur in LEC migration, and explore the significance of these pathways in vivo.
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ItemBiological function of the proteolytic processing of vascular endothelial growth factor-D in cancer( 2012)The Vascular Endothelial Growth Factors are a family of secreted homodimeric glycoproteins and primary regulators of angiogenesis, the growth of new blood vessels, and lymphangiogenesis, the growth of new lymphatic vessels. The mammalian family consists of VEGF, PlGF, VEGF-B, VEGF-C and VEGF-D and they exert biological effects in tissues by binding and activating their cognate receptors, VEGFR-1, VEGFR-2 and VEGFR-3, expressed on endothelial cells. VEGF-C and VEGF-D comprise a subset of the VEGF family as they are initially synthesised as precursor proteins composed of an N- and C-terminal propeptide flanking the central VEGF homology domain, which contains receptor binding sites. Proteolytic processing of these propeptides liberates the mature form consisting of homodimers of the VEGF homology domain, with enhanced affinity for receptors. VEGF-D binds VEGFR-2 and VEGFR-3, and when expressed in tissues promotes blood and lymphatic vessel growth. In animal models of cancer VEGF-D expression induces lymphangiogenesis, increasing lymph node metastasis, and stimulates tumour angiogenesis enhancing tumour growth. VEGF-D can be expressed in a range of prevalent human cancers and this expression correlates with lymph node metastasis and shorter overall patient survival in studies of breast, colorectal and ovarian cancer. Previous analysis of full-length VEGF-D in vivo demonstrated variability in the angiogenesis and lymphangiogenesis induced in tissues, however, processing was not blocked in these model systems so partially and fully processed VEGF-D may have contributed to the effects observed. The activity of full-length VEGF-D, which has not been processed, is not known. In addition, the bioactivity of partially processed VEGF-D and the effect that cleavage of the N- or C-terminal propeptides has on the biology of the molecule has not been defined. Further, it is not known if proteolytic processing is absolutely required for VEGF-D to promote tumour growth and spread. With the aim of determining the effects of the proteolytic processing of VEGF-D on its biological function and action in cancer, a form of VEGF-D was created with mutations at known cleavage sites, effectively blocking processing of both propeptides. Further, to explore the effects of partial processing and the role of the propeptides, additional mutants were generated in which only the N- or only the C-terminal propeptide were deleted and the cleavage site of the remaining propeptide was mutated to block processing. The research described in this thesis demonstrates that each processing mutant displays unique receptor binding and activation profiles, and activity in in vitro assays. In addition, processing of the N- and C-terminal propeptides is a critical determinant of the interactions of VEGF-D with neuropilins and heparin-containing proteoglycans. Tumour xenograft experiments showed that completely blocking VEGF-D processing significantly reduced tumoural angiogenesis and lymphangiogenesis, and abolishes the contribution of VEGF-D to tumour growth and spread. Further, VEGF-D processing mutants lacking the N- or the C-terminal propeptide displayed distinct effects on tumoural angiogenesis, lymphangiogenesis and tumour growth, however, did not promote metastasis to lymph nodes. This data demonstrates that proteolytic processing of VEGF-D is essential for this protein to enhance angiogenesis and lymphangiogenesis in cancer, and provides a possible strategy by which therapeutics can be targeted to block processing in tumours expressing VEGF-D, in order to restrict the growth and spread of cancer.
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ItemThe U12-dependent spliceosome is essential for regulating gene expression during zebrafish development( 2010)Removal of introns from pre-mRNA is an essential step in generating mature mRNA. The majority of introns are removed by the major, or U2-type, spliceosome, while the minor, or U12-type, spliceosome catalyses the removal of a small set of introns with characteristic features that are highly conserved in metazoans and plants. A novel zebrafish mutant, caliban (cal), with specific U12-type splicing defects, was used to study the biochemistry of the U12-type spliceosome and the role of U12-type introns in the regulation of gene expression. Greater than 99% of vertebrate introns are U2-type introns. However, a second class of U12-type introns exists that is defined by highly conserved consensus splice sites. U12-type introns represent about 0.5% of all introns in vertebrate genomes and usually occur alone alongside U2-type introns in pre-mRNA. U12-type introns are removed by a separate spliceosome that shares many components with the U2-type spliceosome, but contains several unique small nuclear RNAs (snRNAs) and spliceosomal proteins. Several lines of evidence suggest a function for U12-type splicing in the regulation of gene expression. These include the high evolutionary conservation of U12-type introns, their enrichment in certain functional gene groups and the demonstration that their excision can be rate-limiting in the generation of mature mRNAs. Despite these observations, little is known about the role and possible regulatory function of U12-type splicing in vivo. cal is a zebrafish development mutant with abnormalities in the intestinal epithelium, which is poorly polarised and unfolded compared to wildtype (wt). Mutant embryos also show a reduction in size of the liver and the pancreas and display a morphologically abnormal lens in the context of a smaller eye. The genetic lesion in cal was mapped to rnpc3, encoding the zebrafish orthologue of the human U11/U12 snRNP 65KDa protein, a specific component of the U12-type spliceosome. It was shown that cal embryos specifically retain U12-type introns compared to wildtype. Biochemical analyses of U12-type spliceosomal small nuclear ribonucleoproteins (snRNPs) demonstrate abnormal formation of the U11/U12 di-snRNP, which represents the first step in U12-type spliceosome assembly. However, it was also demonstrated that larger spliceosomal particles form and accumulate in cal embryos in the absence of Rnpc3/65K, suggesting a potential novel role for Rnpc3/65K in U12-type spliceosome disassembly and recycling. Whole transcriptome analysis of cal and wt embryos by microarrays and RNA sequencing demonstrated retention of U12-type introns on a global scale as well as a set of about 700 differentially expressed genes between cal and wt at two different developmental time points. Further analysis of gene expression data has led to the emergence of a model in which cal embryos are sustained through the first 48-72hpf by maternally deposited rnpc3 mRNA and Rnpc3/65K protein. After this time, defective U12-type splicing induces a cell cycle arrest in the endoderm-derived tissues of the liver, pancreas and intestine, which are highly proliferative between 72 and 108hpf. These results are leading to further studies with a focus on the role of U12-type splicing in human cancer. It was found that several prominent human tumour suppressor genes such as PTEN, LKB1 and PROX1, contain U12-type introns, and we propose a model by which an intermediate reduction of U12-type splicing efficiency can be tumourigenic by reducing the activity of particular tumour suppressor genes in a dose-dependent fashion. To test this hypothesis, conditional Rnpc3 knockout mouse models are currently being generated on a range of different colorectal cancer-susceptible backgrounds.