Project Funding Details

Adrenocortical carcinoma (ACC) is a rare disease with a low response rate to conventional therapy (mitotane alone or combined with cytotoxic drugs). ACC cells increase fatty acid (FA), cholesterol and 27-hydroxycholesterol (27HC) content in response to mitotane. Lipid metabolism increases in several models of drug-resistant tumors. However, a clear understanding of the involvement of lipid metabolism in mediating resistance to conventional treatments in ACC patients is still lacking. We found that upon treatment with mitotane and cytotoxic drugs, H295R cells increase expression of key enzymes involved in FA synthesis, uptake, mitochondrial and peroxisomal FA oxidation. These enzymes are increased in a mitotane- and chemo-resistant ACC cell model (MUC-1 cells), and in H295R cells treated with 27HC or over-expressing SF-1, whose transcriptional activity further increases in cells enriched with 27HC (by silencing its catabolic enzyme). Selective inhibitors for FA metabolism decrease ACC cell growth. Additionally, we show that mitotane activates antioxidant responses that require 27HC-SF1 axis. This mechanism can explain 27HC-mediated protection from ferroptosis seen in other cell models. We hypothesize that mitotane, by favoring 27HC accumulation in ACC cells, would increase SF-1 activity to selectively enhance transcription of genes for FA metabolism and antioxidant responses, favoring protection from ferroptosis and survival. We will characterize lipidomic, transcriptomic, proteomic and bioenergetic profile of mitotane-treated cells in a long-term setting and define changes in SF-1 cofactors. The derived information will be studied in response to 27HC to demonstrate overlapping effects (TASK1). We will evaluate effects on FA synthesis and uptake (TASK2), peroxisomal and mitochondrial FA catabolism and the interconnection between them (TASK3), antioxidant responses (TASK4) initiated as a survival mechanism. The experimental design will define the role for 27HC in orchestrating all the observed responses and the convergence of mitotane/27HC action on SF-1. TASK5 will evaluate repurposing of drugs recently demonstrated to act as CYP27A1 inhibitors, to prevent both metabolic and antioxidant responses initiated by ACC cells upon mitotane treatment. H295R and MUC1 cells will be manipulated (TASK1) in order to establish 27HC and SF-1 role in mitotanedependent responses to overcome cell death. Mitotane will be combined with inhibitors of FA synthesis (TASK2), and FA oxidation (TASK3) and ferroptosis (TASK4) to evaluate changes in lipids content, growth, migration and metabolism. Particularly relevant will be the data obtained on mitotane-resistant MUC1 cells. In vivo experiments will be performed with the most effective drugs. TASK5 will use CYP27A1 inhibitors to summarize the effects of single agents used in previous tasks. Results will uncover the role of 27HC/SF-1 on lipid metabolic reprogramming induced by mitotane. Establishment of novel key targets for the therapy, to converge on CYP27A1 inhibitors, for their ability in shutting-down all pro-survival mechanisms in response to mitotane. ACC cells shape their metabolism and activate antioxidant responses selecting highly aggressive phenotypes resistant to standard therapeutics. Key enzymes will be identified to improve patients' response to therapy and survival. Above all, identification of 27HC as a central player in mitotane resistance will allow clinical translation thanks to old drug repurposing.