Sir Peter MacCallum Department of Oncology - Theses
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ItemRestoration of tumour suppression in prostate cancer( 2018)Aberration in signalling pathways due to the overexpression of oncogenes, and/or loss or mutations in tumour suppressors are key events during carcinogenesis. Hence, novel approaches to target oncogenes in order to restore tumour suppressors are upcoming and promising treatment strategies for cancer therapy. Prostate cancer (PC) is the most common malignancy among men in Australia and other western countries. While 5-year survival for primary PC is a remarkable 94% in Australia, 6% of patients develop resistance to conventional treatments, hence new effective therapies are requited for lethal PC. Recent efforts to molecularly stratify PC have not led to major changes in clinical management. Detailed understanding of the molecular events underlying PC progression is required for the discovery of innovative, targeted therapies for PC. This thesis is based on the following established key findings: (1) elevated levels of the E3 ligase E6AP in PC patients, (2) increased mRNA levels of MDM4, a negative regulator of tumour suppressor wild-type (wt) p53, in metastatic PC, and (3) mutation in the tumour suppressor TP53 is associated with metastatic PC. Therefore, we hypothesized that the oncogenic drivers E6AP, MDM4 and mutant p53 are involved in PC pathogenesis, opening the possibility of targeting these PC drivers to restore tumour suppression and inhibit PC progression. Based on this hypothesis, my thesis explores the implication of both E6AP and the MDM4-p53 axis in PC pathogenesis. Involvement of E6AP in PC: previous studies from our lab demonstrated that E6AP is an E3 ligase of the tumour suppressor PML and that high E6AP/low PML correlates with poor prognosis in PC patients. We, therefore hypothesized that targeting E6AP would restore PML-mediated tumour suppression in PC. Chapter 3 of this thesis describes the effect of E6AP knockdown in PC. In this study, we have shown that (A) E6AP knockdown inhibits PC cell growth in vitro and in vivo, and (B) E6AP knockdownmediated PC growth inhibition is compensated by restoration of PML, at least in part. High E6AP-low PML is observed only in a subset of PC; we speculated that E6AP effects in PC pathogenesis are also due to targeting other tumour suppressors. P27 is a key negative regulator of the cell cycle that is commonly expressed at low levels in cancers, including PC. Further, p27 was found to be negatively regulated by E6AP in neuronal cells. We, therefore, hypothesized that p27 is downregulated by E6AP in PC. Chapter 4 describes the significance of the E6AP-p27 axis in PC. In this study, we identified (A) E6AP knockdown elevates the expression of p27 and increases its nuclear accumulation in vitro and in vivo, (B) E6AP negatively regulates p27 expression by inhibiting its E2F1-dependent transcriptional activation, and (C) we describe an inverse correlation between the expression levels of E6AP and p27 in samples from PC patients. A role for the MDM4-p53 axis in PC pathogenesis: loss of p53 tumour suppressor activity is common in cancers, including PC. P53 activity is frequently curbed either by excessive negative regulation by members of the MDM family or through p53 mutation. TP53 mutation and high mRNA levels of MDM4 are associated with metastatic PC. We have shown oncogenic MDM4 function both in breast cancer with wt-p53 and mutant p53, albeit at least in part through distinct mechanisms. However, its role in PC with compromised p53 functions is unknown. Chapter 5 explores the significance of MDM4 and its p53-dependent and -independent functions in PC. In this study, we have shown (A) high levels of MDM4 in PC, (B) MDM4 downregulation using a genetic approach or small molecule inhibitors inhibit the growth of PC cells, and (C) p53- independent function of MDM4 appears to be exerted by inhibiting E2F1 transactivation of growth suppressive genes in PC. Interestingly, ablating MDM4 inhibited the growth of mutant p53 expressing PC in vitro and in vivo. This prompted us to test the combined effect of MDM4 downregulation and reactivating mutant p53 in PC. Chapter 6 determines the efficiency of reactivating mutant p53 using first-in-class mutant p53 reactivator, APR-246, either alone or in combination with targeting MDM4. In this study, we have demonstrated that: (A) p53 expression is elevated in PC, indicative of mutant p53 tumours, (B) mutant p53 expressing PC cells are sensitive to APR-246 treatment, (C) APR-246 treatment increases the expression of wt-p53 target genes in mutant p53 expressing PC cells, and (D) reactivating tumour suppressive function in mutant p53 using APR-246 in combination with MDM4 targeting compound efficiently inhibit the growth of PC in vitro. Overall, in this thesis we demonstrated that E6AP, MDM4 and mutant p53 are key oncogenic drivers of PC pathogenesis. Accordingly, targeting these oncogenes has shown promising anti-tumour effects in cell lines and in vivo xenograft models. Importantly, the findings described in this thesis have opened a novel avenue for rational drug development and personalized therapeutic approaches to combat PC and improve prognosis.
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ItemExploring the involvement of E6AP in prostate cancer: a combined transcriptomic and proteomic approach( 2017)In Australia, one in seven men are at a risk of being diagnosed with and one in 30 men at the risk of dying from prostate cancer (PC) by the age of 85 [1]. Limited treatment possibilities for castration resistant prostate cancer (CRPC) highlight the need for novel therapeutics. Restoring tumour suppressors in PC by proteasomal inhibition has demonstrated promising results in clinical trials, albeit with significant side effects [2]. Developing more specific and effective treatments for PC is therefore warranted. E6-Associated Protein (E6AP) is an E3 ubiquitin ligase and a transcription cofactor. Recent work from our laboratory and colleagues has demonstrated elevated expression of E6AP in a subset of PC patients [3]. Moreover, genetic manipulations of E6AP in PC cells exposed a role of E6AP in promoting growth and survival of PC cells in vitro and in vivo experimental systems [4]. Previous work from our laboratory unravelled that E6AP mediates this impact on PC cells via tumour suppressors such as promyelocytic leukaemia protein (PML; [4]) and p27 [5]. However, the effect of E6AP on PC cells is broad and it cannot be explained fully by these two tumour suppressors. To identify additional players that mediate E6AP phenotype, we utilised a combined transcriptomic (next generation RNA sequencing) and proteomic (SILAC) approaches. We assessed changes in the total transcriptome and proteome upon E6AP knockdown in CRPC cell line, DU145. We identified 16,130 transcripts using RNA-seq and 7,209 proteins using SILAC. A total of 2,763 transcripts and 308 proteins were considered significantly altered (± 1.5-fold, p-value < 0.05). A comparison of the omics data revealed candidates were either regulated transcriptionally alone, post-transcriptionally alone, or altered at both mRNA and protein level. Pathway analyses supported the known phenotypic effect of E6AP knockdown in PC cells as well as exposed novel links of E6AP with cancer metabolism, DNA damage repair and immune response. In parallel, we explored proteins regulated by E6AP in different PC cell lines, PC3 and LNCaP in addition to DU145 cells. We identified 4,814, 4,827 and 4,819 proteins inDU145, PC3 and LNCaP cells, respectively, upon E6AP knockdown using LC/MS-MS and data-independent acquisition in combination with label-free quantitation. A total of 225, 107 and 139 proteins were considered significantly altered (± 1.5-fold, p-value < 0.05) in DU145, PC3 and LNCaP cells, respectively, following knockdown of E6AP. Metabolism was the most deregulated biological pathway in all three PC cell lines upon E6AP knockdown, substantiating the novel link between E6AP and cancer metabolism. The significantly altered proteins and biological processes in various cell lines warrant further validation and investigation. Of the top ranked candidates, clusterin was pursued further. Clusterin is a stress-induced chaperone protein that modulates apoptosis, lipid transport, DNA repair and cell migration [6]. Clusterin is commonly deregulated in cancer, including prostate cancer. Our results demonstrate that E6AP negatively regulates clusterin at mRNA and protein level. Concomitant knockdown of E6AP and clusterin demonstrates that growth inhibition induced following knockdown of E6AP is in part restored by clusterin. Therefore, restoration of clusterin in the presence of low levels of E6AP can lead to tumour suppression. Overall, the results from this thesis identify novel candidates regulated by E6AP and this sheds new light on potential mechanisms by which E6AP promotes PC.