SAT-354 Characterization of Cell Death Induced by SOAT1 Inhibition in Adrenocortical Carcinoma Cells

Abstract

Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). We have found that mitotane inhibits Sterol-O-Acyl transferase 1 (SOAT1) which leads to depletion of cholesterol esters and increase of free cholesterol in the ACC cell line NCI-H295. We demonstrated activation of endoplasmic reticulum response pathway that resulted in decreased cell viability. Aim: To characterize the mechanisms underlying cell death resulting from SOAT1 inhibition with inhibitors mitotane, ATR101, AZD3988 and Sandoz58-035. Methods: SOAT1 inhibition was quantified in vitro, ER-stress marker expression by qPCR, cell viability by cell titer glo assay, lipid composition by GC-MS, and lipid peroxidation with BODIPY 581/591 C11. Cell death in NCIH295R cells induced by mitotane was determined by mitotane treatment alone and in combination with either the pan caspase inhibitor zvad-fmk, necroptose inhibitor necrostatin-1, ferroptosis inhibitor liproxstatin or ferroptosis activators erastin and RSL-3. Results: Mitotane and ATR101 but not AZD3988 and Sandoz58-035 induced lipid peroxidation in NCI-H295R cells in a concentration dependent manner. ATR101, AZD3988 and Sandoz58-035 were inhibitors of SOAT1 with IC50 of 3 nM, 13 nM and 0.9 nM. Correspondingly, expression of ER-stress marker CHOP and splicing of X-box protein 1 mRNA was activated by mitotane, ATR101 and poorly by AZD3988. Mitotane and ATR101 but not AZD3988 and Sandoz58-035 treatment induced the accumulation of free cholesterol in NCI-H295 cells after 6h. Cell viability was impaired with mitotane, ATR101 and AZD3988 but not Sandoz58-035. None of the inhibitors could reverse the effects of mitotane while synergistic effects were observed for RSL3. Erastin was shown to reverse the effects of mitotane treatment. Conclusion: Although SOAT1 inhibition was confirmed for all compounds, downstream effects on ER-stress markers and cell viability exhibit marked differences. Hence it is likely that targets different from SOAT1 are relevant for in vitro cytotoxic activity of mitotane and ATR101. Caspases do not seem to be predominantly involved in mitotane-induced cell death. Although we observed a synergistic effect of mitotane with RSL3, ferroptosis inhibitors were not able to block mitotane-induced cell death. The precise mechanism of cell death and immunogenic cell death pathways require further investigation.