Medicine (Austin & Northern Health) - Theses
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ItemInvestigating the mechanisms by which histone deacetylase inhibitors induce apoptosis in cancer cells( 2017)Cancer develops as a multi-step process through the accumulation of abnormal genetic alterations in tumour suppressor genes and oncogenes. Superimposed upon these genetic changes are changes in the epigenome which work together to induce the hallmarks of cancer. Histone deacetylase inhibitors (HDACi) are a class of epigenetic therapeutics approved for the treatment of cutaneous T-cell lymphoma (CTCL). These agents induce anti-tumour activity in a variety of ways, including inhibition of cell proliferation and induction of autophagy, differentiation and apoptosis. The mechanism by which HDACi induce apoptosis has been extensively investigated and shown to involve induction of a pro-apoptotic gene signature encompassing the up-regulation of pro-apoptotic genes such as BIM, BAX and BAK and the repression of anti-apoptotic genes such as BCL-2 and BCL-XL. However, the specific molecular mechanisms by which this pro-apoptotic signature is induced have yet to be clearly identified. Recent reports have demonstrated that HDACi-induced apoptosis is associated with upregulation of the AP-1 complex genes c-FOS, c-JUN and ATF3. The objective of this thesis was to determine whether induction of c-FOS, c-JUN or ATF3 is directly required for HDACi-induced apoptosis, and elucidate whether these changes in turn drive altered expression of the pro-apoptotic gene signature. The expression of cFOS, c-JUN and ATF3 expression was found to be robustly and selectively induced upon HDACi treatment in HDACi-sensitive tumour cell lines. These effects transcended tumour type and included melanoma, colorectal, breast, lung, gastric and haematological cell lines. Through systematic knockdown experiments, induction of ATF3 but not c-FOS or cJUN was found to be a functional driver of HDACi-induced apoptosis. This was demonstrated in HDACi-sensitive lung, colorectal and gastric cancer cell lines. These results were further confirmed using ATF3-/- MEFs which were significantly less sensitive to HDACi-induced apoptosis compared to wild-type MEFs. As HDACi treatment alters the expression of pro- and anti-apoptotic genes, we also determined the role of ATF3-induction in mediating these changes. Correlation of HDACi-induced ATF3 expression with the altered expression of intrinsic apoptotic members across 15 different cancer cell lines, revealed an inverse correlation between the magnitude of ATF3 induction and the repression of expression of the pro-survival gene BCL-XL. Furthermore, we demonstrated that ATF3 induction is directly required for HDACi-mediated repression of BCL-XL. A central role for repression of BCL-XL in HDACi-induced apoptosis was demonstrated in knockdown studies whereby siRNA-mediated silencing of BCL-XL was able to re-sensitise refractory cell lines to HDACi-induced apoptosis. Similarly, BH3 mimetics and BCL-XL-specific inhibitors could also re-sensitise refractory cell lines to HDACi-induced apoptosis both in vitro and in xenograft models in vivo. In addition to our finding that ATF3 induction is required for HDACI-induced apoptosis, it has previously been reported that proteasome inhibitor treatment can also induce ATF3 expression. Furthermore, the combination of HDACi and proteasome inhibitors has recently been approved for the treatment of multiple myeloma, although the mechanistic basis for this effect is unclear. We therefore postulated that additive or synergistic induction of ATF3 may underpin this effect. This thesis demonstrates that proteasome inhibitors robustly induce ATF3 in both colorectal cancer and multiple myeloma cell lines, and ATF3 induction is further enhanced by combination treatment with HDACi. We also demonstrate that these agents induce ATF3 through independent mechanisms, and that the combination treatment synergistically enhances apoptosis in these cell lines. Notably, knockdown of ATF3 attenuated the apoptotic response induced by the combination establishing ATF3 as a central component of the apoptotic response. Collectively, these findings demonstrate that HDACi-induced apoptosis is driven by ATF3 induction and subsequent repression of BCL-XL. We also demonstrate that combination treatment with a BCL-XL inhibitor can overcome inherent resistance to HDACi. Additionally, we demonstrate combination treatment with HDACi and proteasome inhibitors synergistically enhances apoptosis through additive induction of ATF3. These studies provide novel insight into the basis for differential response of cell lines to single agent HDACi therapy, and identify avenues for enhancing the activity of HDACi through rationally developed drug combinations.
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ItemImpact of a pregnancy-associated protein (PAPPA) on melanoma: a link between pregnancy and progression of cancer( 2016)Melanoma is the most common cancer diagnosed in pregnant women, and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. This thesis reports that migration, invasiveness and progression of melanoma are promoted by pregnancy-associated plasma protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of insulin-like growth factor (IGF)-1 by cleaving it from a circulating complex formed with IGF binding protein-4. The results presented in this thesis confirm that PAPPA is widely expressed by metastatic melanoma tumours and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in the avian embryonic neural crest model in vivo. Treatment with PAPPA-enriched pregnancy serum enhanced the motility of melanoma cells in vitro. Furthermore, this thesis reports that IGF-1 can induce the phenotypic and functional effects of epithelial-mesenchymal transition (EMT) in melanoma cells. This study establishes a clear relationship between the pregnancy-associated protein PAPPA, melanoma, and functional effects mediated through IGF-1, providing a plausible mechanism for accelerated progression of melanoma during pregnancy. This opens the possibility of therapeutic targeting of the PAPPA/IGF-1 axis.
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ItemOrigins of heterogeneity in melanoma( 2012)The cancer stem cell hypothesis has dominated discussions of cancer cellheterogeneity in recent years; however its relevance to melanoma is controversial. This thesis presents projects outlining sources of cellular heterogeneity in melanoma in relation to three models of cancer development: the cancer stem cell model, clonal evolution, and phenotypic plasticity. A common cancer stem cell culture method, which involves supplementing serum-free media with specific growth factors, yielded a model of melanoma that was not as representative of the original tumour as standard methods. Gene expression profiling revealed a biased towards expression of neural lineage genes, which could seriously confound therapeutic target selection. In contrast metastatic melanoma cell lines and a metastatic melanoma tissue sample were found to be heterogeneous for DNA copy number abnormalities, supporting the involvement of clonal evolution in the development of melanoma heterogeneity. Finally by segregating melanoma cells based on functional heterogeneity evidence for dynamic and reversible transitions between different phenotypes was observed. Gene expression profiling of these functionally distinct subpopulations identified genes relating to epithelial-to-mesenchymal transition and inflammation as relevant to melanoma cell invasion and drug resistance, including both cytotoxic and targeted therapies. Together these results suggest that heterogeneity between the cells of a melanoma results from ongoing genetic changes as well as cell-cell and environmental interactions, and that targeting molecules representing multiple phenotypes simultaneously might be necessary to successfully treat the disease.
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ItemTargeting therapy in colon cancer( 2011)Colon cancer is a common disease affecting Australians and is rarely curable once metastatic disease develops. Angiogenesis plays an important role in the development of metastases. In metastatic colon cancer, anti-angiogenesis agents such as bevacizumab have been integrated in to standard clinical practice. Despite only a modest benefit in delaying disease progression, these agents are expensive and associated with rare but catastrophic side effects, including cerebrovascular accident, pulmonary embolus and intestinal perforation. Standard treatments for metastatic colon cancer still consist of chemotherapy that is not targeted to cancer cells and affects all dividing cells in the body, including those in bone marrow, gastrointestinal tract and hair follicles, causing significant toxicity. In order to improve the toxicity of drugs such as doxorubicin, they have been encapsulated in nanoparticles, which are small structures usually smaller than 1µm in size. The aim of the first study was to investigate differences in the expression of angiogenic factors in the epithelium and stroma of normal colon, primary colon cancer and metastatic colon cancer. The hypothesis was that angiogenic factors would be upregulated in metastases compared with primary tumours and normal colon. Archival samples of primary colon cancer, adjacent normal colon and an associated metastasis were obtained from 32 patients. Immunohistochemistry was used to examine the tumours for vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)-2 expression. In the primary tumour, VEGF-A levels were significantly higher in tumour cells compared with the stroma but there was not a difference in expression between normal colon and cancer. The aim of the second study was to measure the changes in circulating biomarkers and circulating endothelial precursor cells (CEPC) in patients commencing treatment with chemotherapy and an angiogenesis inhibitor for metastatic colon cancer. The hypothesis was that biomarkers of angiogenesis would decrease as a result of treatment and that there would be a difference in biomarker levels between patients who responded and those who did not. Plasma biomarkers were measured in patients prior to treatment and then every two weeks for six weeks. There was a non-significant correlation between placental growth factor (PlGF) and basic fibroblast growth factor (bFGF) and tumour bulk as well as a significant correlation between CEPC and VEGF-A. In addition, CEPC fell in patients who responded poorly to treatment whereas those who did respond had low CEPC at baseline that did not change. The aim of the third study was to determine the biodistribution of a variety of hollow polymeric nanoparticles and measure the changes in biodistribution and tumour uptake that occurred as a result of coating with polyethylene glycol (pegylation) and antibody functionalisation. The hypothesis was that alteration in size and surface chemistry would affect biodistribution and that pegylation and antibody functionalisation would increase tumour uptake and reduce uptake by other organs. Biodistribution in mice of nanoparticles made using a layer-by-layer technique from poly(vinylpyrrolidone) (PVP), poly(methacrylic acid) (PMA) or DNA showed high liver and spleen uptake. This occurred despite alterations in size, polymer type and pegylation. Positron emission tomography (PET) and Gamma Camera imaging demonstrated that liver uptake of nanoparticles is nearly instantaneous and fluorescent microscopy showed that Kupffer cells were the cell-type responsible for uptake. Short circulation time appeared to be responsible for poor tumour uptake. Attaching the human A33 antibody, that specifically binds colon cancer cells, to PVP nanoparticles, appeared to reduce liver and spleen uptake but did not result in significant tumour uptake.
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ItemA prospective assessment of the total palliative care needs of patients with advanced malignancy( 1993)From Preface: This thesis is based on original work by the author who was the principal investigator of the prospective study. A proportion of patient interviews were conducted by two members of the Palliative Care Service and a State Registered Nurse. The majority of patient accrual and follow-up interviews, all data analysis and compilation of the results were completed by the principal investigator.
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ItemHypoxia and angiogenesis in renal cell carcinoma( 2009)Hypoxia is one of the hallmarks of cancer. It was first postulated to occur in solid tumours by Thomlinson and Gray in 1955.1 The presence of hypoxia has been demonstrated in different types of solid tumours.2 Intratumoral hypoxia is caused by the lack of functional blood vessels in proliferating tumour tissue, resulting in low intratumoral oxygen concentrations. If hypoxia is severe or prolonged, cell death occurs.3 Malignant cells can undergo genetic and adaptive changes that allow them to escape from dying of oxygen deprivation. These changes are associated with a more aggressive malignant phenotype 4,5 conferring resistance to radiation 6,7 and chemotherapeutic agents.3,8,9 Hence hypoxia is known to be a key factor responsible for tumour resistance in humans. Invasive polarographic oxygen sensor measurements have demonstrated hypoxia in solid tumours and it is generally defined to occur at an oxygen tension less than ten mmHg.10 Perhaps of more importance is that hypoxia has been demonstrated to be a prognostic indicator for local control after treatment with radiotherapy in glioma, head and neck and cervical cancers.11-13 It has also been able to predict for survival and the presence of distant metastases in soft tissue sarcomas.14 Finally, the significance of hypoxia in the activation and induction of functional molecules such as hypoxia inducible factors (HIFs) and VEGF, the modulation of gene expression (e.g. carbonic anhydrase IX), increased proto-oncogene levels, activation of nuclear factors and accumulation of other proteins (e.g. TP53) although progressing, is yet to be defined.15,16 Thus, it is of clinical interest to understand the levels of hypoxia and numbers of hypoxic cell populations in tumours, particularly those resistant to radiation and chemotherapy. In doing so clinicians and researchers may formulate more accurate prognostic information and develop treatments targeting hypoxic cells. Renal cell carcinoma (RCC) is a tumour resistant to radiation and chemotherapy that is yet to have its oxygen status investigated. Although the “gold standard” of oxygen tension measurement is the Polarographic Oxygen Sensor (POS or Eppendorf pO2 histograph), non-invasive means of measuring oxygen status via imaging, immunohistochemistry or serum tumour markers are more practical. As highlighted by Menon and Fraker, it is imperative that reliable, globally usable, and technically simplistic methods be developed to yield a consistent, comprehensive, and reliable profile of tumour oxygenation. Until newer more reliable techniques are developed, existing independent techniques or appropriate combinations of techniques should be optimized and validated using known endpoints in tumour oxygenation status and/or treatment outcomes.17 Hanahan and Weinberg 18 surmised that the field of cancer research has largely been guided by a reductionist focus on cancer cells and the genes within them- a focus that has produced an extraordinary body of knowledge. Looking forward in time, they believe that progress in cancer research would come from regarding tumours as complex tissues in which mutant cancer cells have conscripted and subverted normal cell types (endothelial cells, immune cells, fibroblasts) to serve as active collaborators in their neoplastic agenda. The interactions between the genetically altered malignant cells and these supporting coconspirators will prove critical to understanding cancer pathogenesis and to the development of novel, effective therapies.18 Essentially, the background outlined here not only highlights the core aim of this thesis: to better understand the oxygen status of renal cell carcinoma and the relationship of this to angiogenesis so that better targeted therapies may be pursued in the future; but it also places this research in the context of the future proposed by Hanahan and Weinberg,18 by clearly focusing on collaborators in the neoplastic agenda, rather than just tumour cells themselves, to better understand RCC.