title: Novel approaches in the management of bladder cancer
Creators: name: Greensmith, RMD
Publisher: name: University of Liverpool Repository
Year: 2017

description: Background: In recent decades, despite survival improvements in the majority of high incidence cancers, survival rates for patients with bladder cancers have remained static. Key challenges in the treatment of bladder cancer are the acquisition of chemotherapy resistance, and progression to lethal muscle- invasive bladder cancer. Aims: This thesis aims to explore methods of pharmacological inhibition of chemotherapy resistance, mechanisms of bladder tumour invasion, and gene expression in bladder cancer. Rationale: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) overexpression has been associated with chemotherapy resistance in a number of cancers including bladder. Therefore, the utility of the purported specific inhibitor of NRF2, brusatol in the attenuation of cisplatin chemotherapy resistance was investigated. In bladder cancer, cluster of differentiation-40 (CD40)-ligation with membrane bound CD40-Ligand (CD40L) has been shown to induce marked cell death. However, little is known about the role of CD40-ligation in tumour cell invasion. With CD40 currently under investigation as a putative target for anti-cancer therapy, it was a timely goal of this thesis to understand more about its role in tumour cell invasion. Until recently, knowledge of gene expression profiles in bladder cancer has been limited. Understanding of gene expression patterns in cancer may allow for enhanced prognostication, identification of potential drug targets and move the management of bladder cancer towards the ideal of personalised medicine. Methods: In vitro cytotoxicity assays were employed to determine the effect of brusatol on cisplatin sensitivity. Western blotting was employed to determine the effects of brusatol on proteins downstream of NRF2. An organotypic model of bladder cancer was used to investigate the effect of CD40-ligation on bladder tumour cell invasion. Non-cleavable membrane bound CD40-ligand delivered by adenoviral vector was used in CD40 expressing cell lines of invasive and non-invasive origin. The effect of CD40-ligation was investigated downstream by western analysis and RNA microarray. Whole-transcript based microarray analysis was performed on bladder biopsies obtained from 19 patients, and 10 controls. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks with statistically significant enrichment of differentially expressed genes. Samples from a further 6 patients (muscle invasive bladder cancer (MIBC) n = 3, normal tissue n = 3) were tested for osteopontin expression by immunohistochemistry. Results: Brusatol increased the sensitivity of bladder cancer cell lines to cisplatin in a dosage and cell line dependent manner. Furthermore, brusatol exhibited marked toxicity as a standalone treatment. However, evidence suggests that brusatol does not act as a specific inhibitor of NRF2. CD40-ligation with non-cleavable membrane-bound CD40-ligand led to marked cell death in CD40-positive non-muscle invasive cell lines. However, in muscle- invasive CD40-positive cell lines marked inhibition of tumour invasion was also observed. Expression analysis of family with sequence similarity member 83 d (FAM83D), fibronectin (FN1), matrix metalloproteinase 1 (MMP1) and anillin (ANLN) revealed downregulation of FN1, FAM83D, and ANLN, and upregulation of MMP1, with a concurrent upregulation of MMP1 and a paradoxical increase of FN1 in EJ cells, with no change in FN1 and MMP1 expression in RT112 cells. xiii Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was significantly overexpressed in invasive cancer compared to healthy tissue. Conclusions: It is unlikely that brusatol is a specific inhibitor of NRF2 however its marked cytotoxicity as a stand-alone agent and potentiating effects in combination with cisplatin are rationale for further investigation. CD40-ligation attenuates the invasion of MIBC cells in an organotypic model of bladder cancer. However, it is unclear whether the attenuation of invasion is due to the inhibition of invasive pathways, or direct induction of apoptosis. Nevertheless, with the usage of CD40-agnoistic antibodies currently in clinical trials, attenuation of tumour invasion is a promising effect. Gene expression analysis identifies divergent pathways that may be useful in the stratification of bladder cancers, and the identification of drug targets. Furthermore, this study supports a key role for osteopontin in bladder cancer, which warrants further investigation as a marker prognostic of muscle invasive disease.