Sir Peter MacCallum Department of Oncology - Theses
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ItemThe control of melanoma by the Hippo pathway( 2018)Melanoma is an aggressive cancer with extremely unfavourable prognosis. Two main types of melanoma include cutaneous melanoma (CM) accounting for around 95% and uveal melanoma (UM) around 5%. In Australia, melanoma is in the top five most commonly diagnosed cancers, estimated to contribute to over 10% of all new cancer diagnoses in 2017 (Cancer Australia, 2018.). While the overall death rate caused by all cancer is decreasing, the mortality of melanoma has increased in recent years (Howlader et al., 2012; AIHW, 2017). Patients diagnosed only with primary melanoma have relatively high survival rates, whereas when patients are diagnosed with metastatic melanoma, the survival rate is very low (Gershenwald et al., 2017). Currently, the mechanisms that drive melanoma progression and metastasis remain poorly understood; but better therapies are definitely required. BRAF mutations are most common in melanoma, occurring in around 50% of this disease (Akbani et al., 2015), which provides a possibility for targeted therapy. Indeed, the United States Food and Drug Administration (USFDA) has approved BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) as the standard treatment for metastatic melanoma patients harbouring BRAF mutations. However, drug resistance occurs in the majority of these patients within two years of treatment (Long et al., 2016). Therefore there is an urgent need to understand the mechanism of BRAFi and MEKi resistance, and find new therapeutic strategies for melanoma. One gene that has been linked to BRAFi resistance is the YAP, which is the key downstream effector of a pathway called the Hippo pathway. The Hippo pathway is an important regulator of organ growth in development. Deregulation of the Hippo pathway stimulates the activity of the YAP oncoprotein, which can cause several human cancers (Zanconato, Cordenonsi and Piccolo, 2016). However, the impacts of YAP deregulation in melanoma are not thoroughly understood. In this project, the roles of YAP in melanoma were examined. Firstly, the impacts of knockdown, overexpression, and activation of YAP on anchorage-independent growth of melanoma cells were assessed using soft agar assays. The results showed that either YAP activation or overexpression promotes colony formation, whilst YAP knockdown reduces this, suggesting potential influences of YAP on melanoma tumorigenesis. Secondly, the effects of YAP in melanoma invasion and metastasis were investigated. Melanoma cells stably expressing an active YAP mutant (YAP-5SA) have a greater invasive ability, as determined with transwell invasion assays. A spontaneous murine metastasis model was used to investigate the impact of YAP on metastasis. The results demonstrated that YAP-5SA promotes metastasis to multiple organs such as the lung and the liver; YAP-5SA enhances vascularity and necrosis of primary melanoma. Thirdly, mechanisms responsible for YAP-induced invasion were explored. Four potential target genes of YAP, derived from RNA-sequencing data, were found crucial, as well as the key YAP transcription factor partners, TEAD1-4. Finally, a lipid-lowering drug called simvastatin was found to kill melanoma cells and inhibits YAP activity in vitro. A post-translational modification, geranylgeranylation, was found to be essential in the statin-induced melanoma cell death and YAP inactivation; RhoA and other geranylgeranylated proteins might be important in these phenotypes. To conclude, this study explored the role of YAP in melanoma metastatic progression, and identified crucial transcription factors and target genes that mediate YAP-induced impacts on melanoma invasion. Additionally, inhibition of YAP and its mechanism in melanoma cells was preliminarily assessed using simvastatin. Understanding the molecular mechanism of melanoma metastasis and inhibition may help us establish more effective therapies for this disease.
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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.