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.