OBJECTIVES 1. To explore the potential role of Nicotinamide Nucleotide transhydrogenase as a molecular target in the treatment of adrenocortical carcinoma (ACC). 2.To determine the prognostic significance of NNT expression in the clinical course of ACC. 3.To determine how NNT inactivation affects adrenal steroidogenesis. METHODOLOGY In vitro Using siRNA-mediated knockdown, I will study the impact of silencing NNT on different ACC cell lines assessing the following outputs: - ACC pro liferation rates, apoptosis, and cell cycle stage - ROS production, super oxide levels and mitochondrial bioenergetics - Steroidogenic enzyme activity Ex vivo I will determine NNT expression levels in a tissue microarray of human normal adrenal and ACC samples. I will relate these findings to clinical outcomes. In vivo I will study the effects of NNT inactivation on ACC proliferation in vivo using a mouse xenograft model. To this end I will generate a stable knockdown of human ACC cel ls using doxycycline-inducible NNT shRNA. Subsuquently, I will inoculate the cells subcutaneously to athymic mice and monitor tumour proliferation in comparison to mice transplanted with wild-type ACC cells for 2 months. Tumour growth will be monitored using Bioluminescence. Effects on steroidogenesis will be assessed with urine steroid analysis by gas chromatography/mass spectrometry.