Generation and characterization of a mitotane-resistant adrenocortical cell line

Abstract

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Liver X receptor inhibition potentiates mitotane-induced adrenotoxicity in ACC

“…This also suggests that, in contrast to H295R, MUC-1 favor alternative mechanism(s) of regulation and clearance of intracellular cholesterol, providing a possible escape mechanism from mitotane-induced lipotoxicity. Seidel et al have described similar findings in relation to FC and CE handling in a strain of HAC15 with induced resistance to mitotane (Seidel et al 2020). Detailed evaluation of lipid/cholesterol metabolism in ACC, using the full range of cell lines and banked tumour samples, is necessary to evaluate the significance for development of future therapeutic strategies.…”

Pharmacological profile and effects of mitotane in adrenocortical carcinoma

“…Symbols: à activation, ┤ inhibition further studies should be performed using in vivo models. 81 This might explain recurrence during and after ACC therapy.…”

“…The study revealed changes in lipoprotein and lipid homeostasis, which may collectively contribute to the characteristics of the resistant phenotype. Because this model evaluated mitotane resistance only in vitro , further studies should be performed using in vivo models 81 . This might explain recurrence during and after ACC therapy.…”

Pharmacological profile and effects of mitotane in adrenocortical carcinoma

“…The study pointed at the changes in lipoprotein and lipid homeostasis which collectively may contribute to the resistant phenotype. Although this model might help develop strategies to overcome mitotane resistancein vitro , further studies should be performed using in vivo models of mitotane resistance before conducting clinical trials [70].…”