Sir Peter MacCallum Department of Oncology - Theses

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Author: Raghu, Dinesh × End date: 2019 × Subject: prostate cancer ×

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    Restoration of tumour suppression in prostate cancer
    Raghu, Dinesh ( 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.