Investigating the Role of Cholesterol and Lipid Trafficking in Mitotane Resistance in Adrenocortical Carcinoma

Introduction: Adrenocortical Carcinoma (ACC) is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by poor response rates and resistance following tumour recurrence. Mitotane’s efficacy has been attributed to intracellular accumulation of toxic free cholesterol (FC) predominantly through inhibition of cholesterol storage through SOAT1. Yet SOAT1 specific inhibitors demonstrate inferior efficacy to mitotane in inducing ACC cell death. We hypothesize that mitotane’s efficacy to induce toxic FC accumulation in ACC cells is also mediated through enhanced breakdown of stored cholesterol within intracellular lipid droplets (LDs). Methodology: ATCC-H295R (mitotane sensitive) and MUC-1 (mitotane resistant) ACC cells were evaluated for neutral lipid content using BODIPY493/503 under baseline and cholesterol loaded conditions using Amnis ImageStream, additionally cells were treated with mitotane (H295R - 20, 40, 50µM; MUC1 - 50, 100, 200µM) for 6hr. Analysis of LDs using CE-BODIPY and FA-BODIPY identified cholesterol ester (CE) and triacylglycerol (TAG)-containing LDs, respectively. Lipid droplet-associated proteins (LDAPs) Perilipin (PLIN) 1–4 and hormone sensitive lipase (HSL) were evaluated using western blotting and PCR. Lipid uptake receptors; SRB1, LDLR, LRP1 and CD36 were measured by flow cytometry. Results: Mitotane treatment, within therapeutic range, decreased staining for LDs significantly in H295R. This was also reflected by decreased expression of LDAPs, PLIN1 and PLIN3. The decrease in H295R LDs was associated with increased activation of HSL (pHSL and LIPE). However, this effect was only evident in MUC-1 at supratherapeutic mitotane (200µM). H295R and MUC-1 demonstrated similar overall LD numbers at baseline and under cholesterol supplementation. Expression of PLIN3 was high in both cell lines, while PLIN1, PLIN2 and PLIN4 demonstrated distinct LD profiles in each. Investigation of LD content showed that H295R preferentially store CEs while MUC-1 store only TAG, irrespective of cholesterol-loading. Mitotane treatment significantly reduces both CE and TAG LDs in H295R and MUC-1. Expression of lipid uptake receptors also demonstrated significant variability between cell lines including SRB1 and LRP1. Conclusion: We highlight that lipolysis through LD breakdown and activation of HSL represents a putative additional mechanism for mitotane induced FC cytotoxicity in ACC. We also demonstrate significant differences in cholesterol handling and LDAPs between mitotane sensitive and mitotane resistant models, in particular, the absence of CE LDs in MUC-1. We therefore propose a mechanism of resistance to mitotane through absent CE storage. Further understanding of cholesterol and lipid handling in ACC offers novel therapeutic exploitation, especially in the setting of mitotane resistant disease.