Barrett's oesophagus (BO) is the major precursor of oesophageal adenocarcinoma (OA) and we do not understand the dynamics of the evolution of BO in order to identify patients at high risk of cancer. Studies have proposed that BO is a monoclonal lesion, however recent work has shown that there are multiple independent clones present. Project 1: Determines the evolution of polyclonal dysplasia through sequencing and mapping clones onto tissue sections. I show that several cases are polyclonal but in each case only one clone progresses to cancer, suggesting oesophageal cancers are monoclonal outgrows from polyclonal Barrett's dysplasia. Project 2: Aims to understand the clonal relationship between cells in glands displaying basal crypt dysplasia-like atypia (BCDA), as it is unclear whether those cells in the upper part of the gland arise from the same stem cell that generates the gland bases. Glands displaying BCDA show a common mutation between the dysplastic base and non-dysplastic surface suggesting a common cell of origin. Project 3: 50% of patients who undergo oesophagectomy for OA develop post-oesophagectomy Barrett's (neo- BO) within 3-5 years possibly due to a field effect, wherein pre-neoplastic cells remain post-resection in histologically normal areas of epithelium predisposing the patient to cancer recurrence. Here I show that no genetic link between the neo-BO and the cancer is present. Immunohistochemical analysis shows that neo- Barrett's glands are gastric in nature. Project 4: The stem cell dynamics and clonal expansion rates of BO are unknown. Here I employed diversity analysis of methylation patterns of CpG islands in the promoter regions of non-expressed genes as a molecular clock. My data suggests that 3-4 stem cells are found in each Barrett's gland. Methylation patterns within a gland were less diverse compared to adjacent and distant glands, suggesting BO is characterized by long periods of stasis followed by bursts of clonal expansions.

Endogenous retroviruses (ERVs) are fossils of ancient retroviral infection in the germline. In primates they represent around 5% of the genome sequence. During time spent in the genome, being transmitted in a Mendelian fashion, copies of ERVs have accumulated mutations, which rendered them inactive. However, some of them (the most recently integrated ones) are still able to transcribe and produce viral proteins, although few are capable of re-infection. In the past often considered as unharmful 'junk DNA', recent evidence link ERVs with cancer and several inflammatory diseases. For example, a few reports demonstrate that ERVs are involved in tumour development using shRNA knock-down and over-expression systems, and their overexpression tends to correlate with inflammation status, generating the hypothesis that they can act as pathogen-associated molecular patterns (PAMPs) and bind to innate sensors. Focusing on the Human (Homo sapiens) and the rhesus macaque (Macaca mulatta), the main aims of this thesis are to look for further evidence linking ERVs to tumour development, with possible implications for therapies, and test the hypothesis that ERVs are PAMPs by seeing if individuals with higher levels of ERV expression exhibit a higher innate immune response. The work on ERVs in cancer involved the human ERV type-K HML2 lineage (HERV-K (HML2)), an ERV lineage found in humans, in Merlin-deficient tumours. These are schwannomas that arise from Schwann cells and for which effective drug therapy is urgently needed. The work on ERVs in inflammation involved the Papio cynocephalus ERV (PcEV), in rhesus macaques infected with simian immunodeficiency virus (SIV) infection. The main outcomes are as follows: regarding HERV-K (HML2) in human schwannomas, (i) HERV-K (HML2) proteins are overexpressed in schwannoma compared to Schwann cells; (ii) these proteins are released from the tumour; (iii) regulation of HERV-K (HML2) expression in the tumour appears to involve the transcription factor TEAD; (iv) schwannomas are potentially treatable using anti-HERV-K (HML2) monoclonal antibodies and antiretroviral drugs since both decreased proliferation in vitro. Regarding PcEV in SIV-infected macaques: (i) PcEV is transcriptionally active; (ii) PcEV can be retrieved at low levels in the blood of some macaque animals; (iii) the levels of PcEV in cells correlates strongly with the strength of the innate response as measured by cellular levels of STAT1 transcripts - an interferon-stimulated gene (ISG). Other recent research has shown that human ERV lineages, namely HERV-W and HERV-H, have been co-opted and are involved in placentation and pluripotency during development, respectively. The present work suggests that ERVs are involved in a wide range of biological process and supports the need for further research into the biological significance of ERVs for their hosts.

The majority of women with ovarian cancer (OC) have advanced disease at diagnosis and 5-year survival rates of less than 25%. Women with stage I disease have significantly better 5-year survival rates of over 90%. Recent large studies using CA 125 and transvaginal ultrasound have failed to improve mortality in a screened population. There is therefore a pressing need for new diagnostic biomarkers in OC. The primary aim of my project, as a first step in developing a diagnostic circulating tumour DNA (ctDNA) biomarker for high grade serous ovarian cancer (HGSOC), was to investigate low-cost high-throughput next generation sequencing assays in plasma samples collected from women with newly diagnosed OC. The secondary aim was to apply these methods to other non-invasive samples including cervical liquid based cytology samples that might contribute to earlier diagnosis or screening for women with OC. ctDNA was detected in 30-49% of women with newly diagnosed OC from the UKOPS (n=54) and CTCR-OV04 (n=156) cohorts using targeted sequencing. Using the trimmed median absolute deviation (t-MAD) score, a quantitative measure of genome wide copy number aberration generated from shallow whole genome sequencing (sWGS) data, ctDNA was detected in 39-41% of the women with newly diagnosed disease. To improve sensitivity of ctDNA detection I developed an optimised method for targeted sequencing that has the potential to lower the limit of detection of ctDNA in HGSOC by 100 fold. I have also shown that the size profile of HGSOC ctDNA fragments is different to that of wildtype DNA fragments and shown that selecting for DNA fragments between 90-150 bp can increase rates of ctDNA detection in HGSOC. ctDNA detection increased to 53-67% of women with newly diagnosed OC using the size selected t-MAD score. I have evaluated the utility of cervical sampling for earlier diagnosis of OC by testing and optimising DNA extraction, library preparation and sequencing methods. I have detected tumour DNA in routine cervical cytology samples collected from women subsequently diagnosed with cervical and endometrial cancers. In summary I have developed methods for ctDNA detection in women with newly diagnosed HGSOC that can be applied and refined in larger prospective studies of women undergoing follow-up for treated HGSOC, women with symptoms suggestive of OC and women at high risk of OC.

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer. Our laboratory has recently identified both ANKRD11 and FBXO31 as candidate tumour suppressor genes in the chromosome band 16q24.3, which is the smallest region of overlap for breast cancer LOH. This thesis focuses on the functional analysis of these two novel genes and implicates a role for them as breast cancer tumour suppressors. ANKRD11: a novel p53 coactivator involved in the rescue of mutant p53. The ability of p53 to act as a transcription factor is critical for its function as a tumour suppressor. Ankyrin repeat domain 11 (ANKRD11) was found to be a novel p53-interacting protein which enhanced the transcriptional activity of p53. ANKRD11 expression in breast cancer cell lines was shown to be down-regulated when compared to ANKRD11 expression in finite life-span HMECs and non-malignant immortalized breast epithelial cells. Restoration of ANKRD11 expression in MCF-7 (p53 wild-type) and MDA-MB-468 (p53[superscript R273H] mutant) cells suppressed the oncogenic properties of these breast cancer cell lines through enhancement of p21[superscript waf1] expression. ShRNA-mediated silencing of ANKRD11 reduced the ability of p53 to activate p21[superscript waf1] expression in response to DNA damage. ANKRD11 was shown to associate with the p53 acetyltransferase, P/CAF, and exogenous ANKRD11 expression increased the levels of acetylated p53. Exogenous ANKRD11 expression enhanced the DNA-binding properties of the p53[superscript R273H] mutant to the CDKN1A promoter, implicating a role for ANKRD11 in the restoration of mutant p53[superscript R273H] function. These findings demonstrate a role for ANKRD11 as a p53 coactivator and illustrate the potential of ANKRD11 in the restoration of mutant p53[superscript R273H] function. ANKRD11 has roles beyond that of p53 coactivation. This thesis also presents preliminary findings to suggest that ANKRD11 may be involved in the regulation of eukaryotic cell division. Furthermore, ANKRD11 was shown to function as an estrogen receptor coactivator. Taken together, these finding suggest that ANKRD11 is a multi-functional cancer-related protein. FBXO31: the 16q24.3 senescence gene. A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumour cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. Exogenous FBXO31 expression inhibited the oncogenic properties of the MCF-7 breast cancer cell line. In addition, compared to the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumour cell lines and primary tumours. FBXO31 protein levels were cell cycle regulated, with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G1 phase of the cell cycle. FBXO31 was also shown to be a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumour suppressor by generating SCF[superscript FBXO31] complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325445 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, Discipline of Medicine, 2008

Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer. Our laboratory has recently identified both ANKRD11 and FBXO31 as candidate tumour suppressor genes in the chromosome band 16q24.3, which is the smallest region of overlap for breast cancer LOH. This thesis focuses on the functional analysis of these two novel genes and implicates a role for them as breast cancer tumour suppressors. ANKRD11: a novel p53 coactivator involved in the rescue of mutant p53. The ability of p53 to act as a transcription factor is critical for its function as a tumour suppressor. Ankyrin repeat domain 11 (ANKRD11) was found to be a novel p53-interacting protein which enhanced the transcriptional activity of p53. ANKRD11 expression in breast cancer cell lines was shown to be down-regulated when compared to ANKRD11 expression in finite life-span HMECs and non-malignant immortalized breast epithelial cells. Restoration of ANKRD11 expression in MCF-7 (p53 wild-type) and MDA-MB-468 (p53[superscript R273H] mutant) cells suppressed the oncogenic properties of these breast cancer cell lines through enhancement of p21[superscript waf1] expression. ShRNA-mediated silencing of ANKRD11 reduced the ability of p53 to activate p21[superscript waf1] expression in response to DNA damage. ANKRD11 was shown to associate with the p53 acetyltransferase, P/CAF, and exogenous ANKRD11 expression increased the levels of acetylated p53. Exogenous ANKRD11 expression enhanced the DNA-binding properties of the p53[superscript R273H] mutant to the CDKN1A promoter, implicating a role for ANKRD11 in the restoration of mutant p53[superscript R273H] function. These findings demonstrate a role for ANKRD11 as a p53 coactivator and illustrate the potential of ANKRD11 in the restoration of mutant p53[superscript R273H] function. ANKRD11 has roles beyond that of p53 coactivation. This thesis also presents preliminary findings to suggest that ANKRD11 may be involved in the regulation of eukaryotic cell division. Furthermore, ANKRD11 was shown to function as an estrogen receptor coactivator. Taken together, these finding suggest that ANKRD11 is a multi-functional cancer-related protein. FBXO31: the 16q24.3 senescence gene. A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumour cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. Exogenous FBXO31 expression inhibited the oncogenic properties of the MCF-7 breast cancer cell line. In addition, compared to the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumour cell lines and primary tumours. FBXO31 protein levels were cell cycle regulated, with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G1 phase of the cell cycle. FBXO31 was also shown to be a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumour suppressor by generating SCF[superscript FBXO31] complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325445 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, Discipline of Medicine, 2008

A 9.4 T magnetic resonance imaging (MRI) system was used to evaluate the response of a human tumour xenograft model to radiation therapy. The apparent diffusion coefficient (ADC) and the transverse relaxation time (T2) of human glioblastoma multiforme (GBM) tumour xenografts in NIH-iii nude mice were measured before, and at multiple points after, treatment of the tumours with 200 kVp x-rays. The response was characterized as a function of a number of variables of interest in the clinical treatment of cancer with external beam radiation therapy. Mean tumour ADC and T2 responses after single fractions of radiation were investigated, with measurements being made until 14 days after treatment. Single fraction doses ranged from 50 cGy to 800 cGy. Fractionated treatments were used to deliver 800 cGy in two or three fractions with fraction spacings of 24 or 72 hours. The role of hypoxia on ADC and T2 response was investigated by using an externally-applied, suture-based ligature to induce a state of reduced oxygenation in tumours during treatment, after which ADC and T2 were measured using serial MRI. Finally, tumours were dissected in order to provide insight into possible pathophysiological mechanisms explaining the observed responses. Tissue sections were prepared and reviewed by a pathologist. This work adds to the body of literature describing tumour ADC and T2 response to anticancer therapy, and adds to the understanding of ADC and T2 response to radiation therapy in particular. This works supports that of others suggesting the use of ADC and T2 as potential biomarkers for tumour response to treatment. / Medical Physics

No description available.

Non-Hodgkin’s lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases. The control of lymphomagenesis still remains a challenge. Early growth response gene (Egr) 2 and 3 are zinc finger transcription factors. Egr2 plays an important role in the development of both central nervous system and lymphocytes. However the mechanism of action in lymphocytes is still unknown. In order to fully understand the function of Egr2, in lymphocytes, we developed Egr2 and 3 double knockout mice (Egr2-/-Egr3-/-) by crossbreeding lymphocyte specific Egr2 knockout mice (CD2-Egr2-/-) with Egr3 knockout mice (Egr3-/-), as previous reports suggested that Egr3 compensates for the role of Egr2. In the absence of Egr2 and 3, the homeostasis of T cells is dysregulated with hyper-homeostatic proliferation of effector like phenotype cells. More importantly the development of spontaneous B and T cell lymphoma was found in more than 70% of Egr2-/-Egr3-/- mice. The lymphoma cells from Egr2-/-Egr3-/- mice were highly proliferative and metastatically spread into other non-lymphoid organs, such as lung, liver and kidney. In additional to this lymphoma development the Egr2-/-Egr3-/- mice showed signs of chronic inflammatory disorder. This inflammatory disorder was characterised by glomerulonephritis and an increase in serum cytokines, which may provide the microenvironment for the lymphoma development. To explore the molecular mechanism of tumour development in Egr2-/-Egr3-/- mice, the transcriptional profile of Egr2 was studied by microarray and ChIP-on-chip. We found firstly that Egr2 directly binds to the promoter regions of Ikaros and FOXO3. The deletion of Egr2 and 3 in lymphocytes led to the downregulation of Ikaros, Aiolos and FOXO3 expression. The impaired expression was found to be associated with proliferative disorder and the development of T and B cell lymphoma. Secondly Egr2 strongly inhibits STAT3 transcriptional activity by regulating SOCS3, which is a known inhibitor of STAT3. The breakdown of this regulation could be an important mechanism in lymphomagenesis. A model is proposed which defines Egr2 and Egr3 as the backbone of important tumour suppressor genes that control cell fate decision and regulates homeostasis in the lymphoid system. Thus, our results suggest that Egr2 and 3 are important regulators of lymphocyte function by their involvement in multiple cell signalling pathways, which could potentially be key genes for future cancer therapy.

The Large Tumor Suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in a broad spectrum of human cancers. It is a central player of the emerging Hippo-LATS tumour suppressor pathway, which plays important roles in cell proliferation, apoptosis, and stem cell differentiation. Despite the ample data supporting a role of LATS1 in tumour suppression, how LATS1 is regulated at the molecular level remains largely unknown. In this study, we have identified Itch, a HECT class E3 ubiquitin ligase, as a novel binding partner of LATS1. Itch can complex with LATS1 both in vitro and in vivo through the PPxY motifs of LATS1 and the WW domains of Itch. Significantly, we found that over-expression of Itch promoted LATS1 degradation by polyubiquitination through the 26S proteasome pathway. On the other hand, knockdown of endogenous Itch by shRNAs provoked stabilization of endogenous LATS1 proteins. Finally, through several functional assays, we also revealed that change of Itch abundance alone is sufficient for altering LATS1-mediated downstream signaling, negative regulation of cell proliferation, and induction of apoptosis. Together, our study identifies E3 ubiquitin ligase Itch as the first negative regulator of LATS1 and presents for the first time a possibility of targeting LATS1/Itch interaction as a therapeutic strategy in cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2011-04-26 22:25:46.008