Mitotane Targets Lipid Droplets to Induce Lipolysis in Adrenocortical Carcinoma

Abstract

Introduction Adrenocortical Carcinoma (ACC) is a rare aggressive cancer with low overall survival. Adjuvant mitotane improves survival but is limited by poor response rates and resistance. Mitotane’s efficacy is attributed to the accumulation of toxic free-cholesterol, predominantly through cholesterol storage inhibition. However, targeting this pathway has proven unsuccessful. We hypothesize that mitotane-induced free-cholesterol accumulation is also mediated through enhanced breakdown of lipid droplets. Methodology ATCC-H295R (mitotane-sensitive) and MUC-1 (mitotane-resistant) ACC cells were evaluated for lipid content using specific BODIPY dyes. Protein expression was evaluated by immunoblotting and flow cytometry. Cell viability was measured by quantifying propidium iodide positive cells following mitotane treatment and pharmacological inhibitors of lipolysis. Results H295R and MUC-1 cells demonstrated similar neutral lipid droplet numbers at baseline. However, evaluation of lipid machinery demonstrated distinct profiles in each model. Analysis of intracellular lipid droplet content showed H295R preferentially store cholesteryl esters, while MUC-1 store triacylglycerol. Decreased lipid droplets were associated with increased lipolysis in H295R and in MUC-1 at toxic mitotane concentrations. Pharmacological inhibition of lipolysis attenuated mitotane-induced toxicity in both models. Conclusion We highlight that lipid droplet breakdown, and activation of lipolysis represents a putative additional mechanism for mitotane-induced cytotoxicity in ACC. Further understanding of cholesterol and lipids in ACC offers potential novel therapeutic exploitation, especially in mitotane-resistant disease.

Pharmacogenomic analysis in adrenocortical carcinoma reveals genetic features associated with mitotane sensitivity and potential therapeutics

“…A total of 1612 genes were differentially expressed (|logFoldChange| ≥ 1.5, P<0.05) between responder and non-responder tumors ( Figure 3B ). Evidence has accumulated that mitotane dysregulated lipid metabolism and raised the potential correlation between mitotane responsiveness and capacity of handling lipids ( 37 , 38 ). From our transcriptome data, to be noted, expression of genes involved in steroidogenesis ( CYP11B1 ) and lipid metabolism ( CYP27A1, ABCA1, PLIN2, PLIN4, NR1H3 , etc) were significantly upregulated in mitotane-sensitive tumors ( Figure 3C ), implying elevated capacity for handling intracellular lipids.…”

“…Oxysterol/LXR involves in adrenal steroidogenesis and serves as a safety valve to limit free cholesterol levels, thereby protecting adrenal cortex from lipotoxicity ( 48 ). Because mitotane could cause lipotoxicity in ACC cells through targeting lipolysis and cholesterol storage ( 19 , 38 ), we hypothesize that ACCs expressing higher level of CYP27A1 and ABCA1 might tightly depend on its capacity of handling cholesterol flux, thus vulnerable to disturbance of lipid homeostasis induced by mitotane.…”

Preclinical Models of Adrenocortical Cancer

“…Warde et al recently showed that mitotane sensitivity correlates with intracellular lipid content; while MUC-1 and H295R cells store similar amounts of intracellular lipid droplets, MUC-1 (mitotane resistant) cells are rich in triacylglycerols, whereas H295R (mitotane sensitive) cells are rich in cholesterol esters [77]. Lipid content as measured by Hounsfield units is used in some diagnostic algorithms for ACC; however, these results show that further distinguishing the particular lipids involved may provide more useful clinical information [78,79].…”