Endocrine Abstracts

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 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 20/40/50 µM; MUC1 – 50/100/200 µM) for 6 hrs. Analysis of LDs using CE-BODIPY and FA-BODIPY identified cholesteryl 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.

Results

Mitotane treatment, within its therapeutic range, decreased staining for neutral lipid droplets significantly in H295R. This was also reflected by decreased expression of LDAPs PLIN1 and PLIN3. Decreased LDs was associated with increased activation of HSL (pHSL and LIPE). However, this effect was only evident in MUC-1 at supratherapeutic 200 µM mitotane. H295R and MUC-1 demonstrated similar overall neutral 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 H295R preferentially store CEs, while MUC-1 store only TAG, irrespective of cholesterol-loading. Mitotane treatment significantly reduces CE and TAG LD stores in H295R and MUC-1. Pharmacological inhibition of HSL decreases mitotane-induced toxicity and CE-LD reduction, however, does not affect TAG-LDs. Additionally, toxic mitotane increases glycerol production in H295R and MUC-1.

Conclusion

We highlight that LD breakdown and activation of HSL represents a putative additional mechanism for mitotane induced FC cytotoxicity in ACC. We also demonstrate significant overall differences in cholesterol handling and LDAPs between mitotane-sensitive and mitotane-resistant models, in particular, the absence of CE-LDs in MUC-1. However, inhibition of HSL and CE-LD breakdown does not fully attenuate mitotane toxicity. Increased glycerol production and decreased TAG-LDs following mitotane treatment highlights an additional toxic mechanism through TAG lipolysis. Further understanding of cholesterol and lipid handling in ACC offers potential for novel therapeutic exploitation, especially in mitotane-resistant disease.