Sir Peter MacCallum Department of Oncology - Theses
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ItemThe role of Pim-1 in breast cancer metastasis( 2017)Breast cancer is the most common cancer in women. Despite advances in treatment options, the spread of breast cancer to distant organs (metastasis) remains the major cause of morbidity and mortality in breast cancer patients. This is attributed primarily to the impairment of function in affected organs. Thus, there remains a vital need for better-targeted treatments that more effectively inhibit the development or progression of metastases. Pim-1 is a serine/threonine survival kinase that has been implicated in the development of metastasis in several haematological and solid cancers. However, little is known about its role in breast cancer. In our laboratory, we previously identified Pim-1 as upregulated in brain metastatic 4T1Br4 syngeneic mouse cells and tumours compared to parental 4T1 cells. This led us to propose that Pim-1 may play a role in mediating breast cancer brain metastasis. Therefore, the overall objective of this project was to examine the expression and functional role of Pim-1 in breast cancer metastasis, with a focus on organ-specific metastasis. We interrogated public databases to show that Pim-1 expression is low to absent in normal breast tissue and increased in breast tumour tissue. Furthermore we show that the murine (4T1Br4) and human (MDA-MB-231Br) brain metastatic breast cancer cell lines and tumours demonstrate the highest expression of Pim-1 mRNA and protein. To investigate the function of Pim-1 in breast cancer metastasis we tested the impact of inhibiting Pim-1, either by gene knock down using short hairpin RNAs or the pharmacological inhibitor SGI-1776, on the ability of 4T1Br4 and MDA-MB-231Br cells to migrate and invade in vitro. 4T1Br4 cells displayed increased migration and invasion propensity after Pim-1 knock down and this was coupled with a decrease in β4 integrin expression. Conversely, MDA-MB-231Br cells showed a decreased ability to migrate and invade after Pim-1 KD, as well as decreased cell surface expression of β1 and β3 integrins. Treatment with SGI-1776 dose-dependently decreased the ability of both 4T1Br4 and MDA-MB-231Br cells to migrate and invade, decreased cell surface expression of β3 integrin in 4T1Br4 cells, and both β1 and β3 integrins in MDA-MB-231Br cells. To examine the effect of Pim-1 inhibition in vivo, we assessed the metastatic spread of Pim-1 knock down MDA-MB-231Br cells in an experimental metastasis assay. After intracardiac injection of Pim-1 knock down cells, we observed a reduction in the number of circulating tumour cells and decreased bone metastasis, indicating a functional role for Pim-1 in breast cancer metastasis to the bone. Data from brain metastasis in this model were inconclusive. In summary, results from this project highlight the importance of Pim-1 in breast cancer metastasis and provide evidence that Pim-1 contributes to the migration and invasion of breast cancer cells both in vitro and in vivo, possibly via regulation of integrin expression, and indicate that Pim-1 is a relevant therapeutic target for the treatment of metastatic breast cancer.
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ItemCooperative tumourigenesis : analysis of novel tumour suppressors in ras oncogene driven epithelial tumours( 2015)Cancer is a cooperative process, involving mutations in multiple genes. Activation of a cancer-driving gene, the Ras small GTPase, via a mutation that locks Ras in the GTP-bound active form (RasV12), occurs in ~30% of human cancers. However, alone it is not sufficient for tumour formation. A loss of function screen previously performed in the vinegar fly, Drosophila melanogaster, identified 947 genes that potentiate RasV12-mediated tumourigenesis and metastasis (Zoranovic, et al. in prep.). This list has been narrowed down to 234 genes that 1) show increased tumourigenicity with RasV12 in vivo, 2) are in the top 100 genes down-regulated in human cancer, and 3) are known to regulate the cytoskeleton, polarity, adhesion or cell motility. This study has successfully confirmed involvement of autophagy-related genes Atg8a, Atg7 in regulating RasV12-mediated proliferation in the Drosophila eye epithelial tissue using the UAS/GAL4 system. The study identified the autophagy-related genes Atg1, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg12 and Atg101 that when knocked down cooperate with RasV12 and lead to increased tissue overgrowth in the Drosophila eye epithelium. Atg8a was chosen as the representative target gene to investigate this cooperation. It was observed that Atg8a cooperates with RasV12 through the Raf pathway. The role of p62 in this Ras-mediated cooperation with Atg8a was also examined and it was found that p62 levels increase in RasV12+ Atg8aRNAi expressing tissue in comparison with control. Investigations were also carried out to ascertain if knockdown of Atg genes cooperate with Ras through the JNK pathway. It was discovered that in the presence of oncogenic Ras, knock down of Atg8a increases the expression of the JNK target MMP1. The finding of this work could lead to use of this autophagy related genes as prognostic markers in Ras-driven oncogenesis and might reveal effective therapeutic targets to combat this deadly disease.