Mitotane inhibits sterol-o-acyltransferase leading to lipid-mediated endoplasmic reticulum stress and apoptosis of adrenocortical carcinoma cells

Context: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC) and in clinical use for more than 50 years. Mitotane counteracts both tumor growth and tumoral steroid hormone production but treatment is associated with severe side effects. The molecular mechanism of mitotane is still unknown, which hampers progress in treatment of ACC. Objective: To identify the mechanism of action and molecular target of mitotane. Methods: We combined expression genomics, proteomics and lipidomics in an in vitro study using the NCI-H295 ACC reference cell line and four non-steroidogenic cell lines. Results: Pathway analysis of gene expression data demonstrated activation of endoplasmic reticulum (ER) stress and altered lipid metabolism. Strongly increased expression of ER-stress marker C/EBP-homologous protein (CHOP, 26.7 ± 0.1 fold, p < 0.001) and activated XBP1-mRNA splicing (23.2 ± 3.2 fold, p < 0.001) was in sharp contrast to weak ER-stress activation in non-steroidogenic cell lines (HepG2: 2.6/1.4-fold, HEK293: 2.5/3.7, HeLa: 3.5/6.4, IMR-32: 1.5/2.1). ER-stress experimentally triggered by thapsigargin mimicked mitotane effects on steroidogenesis and apoptosis. Mass spectrometry revealed specific mitotane-induced lipid alterations with elevated free cholesterol (2.53-fold, p < 0.05), oxysterols like 7-dehydrocholesterol (5.4-fold, p < 0.001) and fatty acids, e.g. C20:4 (arachidonic acid; 3.2-fold, p < 0.01) in NCI-H295 cells but not in other cell lines. Inhibition of Acyl-CoA-Cholesterol-Acyl-transferase (SOAT1) by mitotane (IC50 = 22µM) was identified as the mechanism underlying these events. Conclusions: Mitotane confers adrenal specific cytotoxicity and down-regulation of steroidogenesis by lipid-induced ER-stress through inhibition of SOAT-activity. This finding opens new avenues for improved ACC treatment. Cancer specific lipid metabolism may be a treatment target in other types of cancer.