The role of the liver X receptor in mitotane therapy in adrenocortical carcinoma
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Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with limited treatment options. The Liver X Receptor α (LXRα) is highly expressed in the adrenal cortex, and functions to regulate cholesterol and lipid homeostasis. Proposed herein, is a novel therapeutic approach, through modulating intracellular free cholesterol (FC), via LXRα inhibition, in combination with current first line chemotherapeutic mitotane. High expression of LXRα was demonstrated in both H295R (primary, treatment-sensitive) and MUC-1 (metastatic, treatment-resistant) ACC cell lines. In both models, LXRα inhibitors; GSK2033 and SR9243, successfully decreased efflux pump mRNA expression. Combination treatment using mitotane and LXRα inhibition demonstrated more effective induction of apoptotic cell death, at usually sub-therapeutic concentrations of mitotane in H295R. Whilst LXRα inhibition potentiated mitotane-induced cell death in MUC-1, this was only observed at supra-therapeutic concentrations. However, in both models, toxicity was enhanced through cholesterol-methyl-β-cyclodextrin loading. These findings were confirmed using a dominant-negative LXRα construct and pharmacological synergism analysis using CompuSyn. Intracellular FC levels were assayed using filipin staining, whereby LXRα inhibition potentiated mitotane-induced FC lipotoxicity in H295R and MUC-1 cell lines. Investigation of neutral lipid content using BODIPY™493/503 revealed an abundance of lipid droplets in H295R which decreased following mitotane and LXRα inhibition. Additionally, in H295R, mitotane induced a dose-dependent decrease in the expression of lipid droplet-associated proteins, PLIN1 and PLIN3, alongside increased activity of hormone sensitive lipase. Further investigation of intracellular lipid stores revealed H295R as extremely cholesteryl ester rich. However, MUC-1 were found to be cholesteryl ester poor and preferentially store triacylglycerol, under baseline and cholesterol-loaded conditions. These data demonstrate a complementary pathway to enhance mitotane-induced FC toxicity, through LXRα mediated efflux pump inhibition, whereby cytotoxicity is enhanced by cholesterol loading. Additionally, the importance of cholesterol and lipid storage is highlighted in the context of cell survival and mitotane-resistance in adrenocortical cells. Ultimately, these data identify a novel strategy to broaden the therapeutic window of mitotane, and highlight the importance of understanding cholesterol and lipid metabolism in the setting of primary and metastatic ACC.
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