Background: Adrenocortical carcinoma (ACC) is a rare but devastating malignancy (5-year survival 18-57%), with limited treatment options. Currently mitotane is the first-line medical treatment for metastatic ACC and is used in the adjuvant setting after surgery to prevent recurrence. Mitotane is an adrenolytic drug that is thought to act by disruption of mitochondria with subsequent apoptosis activation. However, mitotane treatment comes with many drawbacks, such as severe side-effects and contralateral adrenal gland destruction, whilst both clinical and in vitro studies have shown that it is only effective in 25-30% of ACC patients. We performed a proteomics-based analysis of ACC tissues previously characterized in vitro as responders, partial responders and non-responders to mitotane, with the aim of identifying tissue biomarkers to improve future patient selection for mitotane therapy.

Methods: Protein lysates were obtained from fresh-frozen ACC samples (n=13 responders, n=10 partial responders, n=7 non-responders). Label-free liquid chromatography–mass spectrometry was performed on these samples. Differential protein abundance was assessed using DESeq2, followed by pathway analysis using STRING.

Results: On average, 2628 proteins were identified per tumor sample. We found over 50 proteins (80% being mitochondrial or mitochondrial-related) with lower expression in responders compared to non-responders. Pathway analysis revealed involvement of these proteins in the tricarboxylic acid cycle and associations with the mitochondrial cristae junction and mitochondrial contact site and cristae organizing system (MICOS) complex. In contrast, only four proteins were higher expressed in responders compared to non-responders.

Conclusion: Mitochondria-associated proteins are differentially expressed in patients who respond well to mitotane therapy compared to non-responders, fitting with the presumed action mechanism of mitotane. Immunohistochemical validation is necessary to further explore the potential of specific proteins to serve as biomarker for treatment response.