Abstract
256 Background: Nrf2 controls expression of multiple target genes, each containing an antioxidant response element (ARE). These targets mediate cellular protection through antioxidant response, cellular detoxification (including glutathione conjugation), and altered drug uptake/efflux. Some targets (e.g., metallothionein, glutathione reductase) are associated with UC characteristics, outcomes, and chemotherapy resistance. Nrf2 regulation is partially understood and includes negative regulation by Keap1 and phosphorylation. De-novo or acquired cisplatin resistance is a problem in UC. We hypothesised that it might be mediated by Nrf2. Methods: Nrf2 expression was assessed in UC clinical samples by immunohistochemistry. We then utilised a panel of UC cell lines, including a cisplatin resistant RT112 sub-line (RT112CP), to test Nrf2 axis expression and activation correlated to cisplatin sensitivity/resistance. Effects of AKT or HER2 kinase activity on Nrf2 activation were tested by chemical inhibition. Results: In UC clinical samples we detected Nrf2 over-expression in 64% of cancers compared to surrounding normal tissue. Varied Nrf2 expression levels were also detected in different UC cell lines whereas Keap1 demonstrated an inverse profile of both RNA and protein expression. Nrf2 expression was 4 fold greater in RT112CP cells which exhibited comparative cisplatin resistance (10 fold greater IC50 in proliferation assays) compared to parental cells. Nrf2 over expression was functional in this model system as indicated by increased activity in ARE luciferase reporter assays, raised expression of Nrf2 transcriptional targets (metallothionein, glutathione reductase) and glutathione levels. Furthermore, depletion of Nrf2 in RT112CP cells by siRNA or transfection of a dominant negative Nrf2 construct partially restored cisplatin sensitivity. Finally chemical inhibition of AKT or HER2 reduced Nrf2 activation in ARE reporter assays. Conclusions: Nrf2 is overexpressed in a subset of UC. In cell line models it is functionally activated and contributes to cisplatin resistance, which is reversible. This might be amenable to detection and therapeutic reversal in the clinic. No significant financial relationships to disclose.
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