Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Several stimuli induce programmed cell death by increasing Ca 2+transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca 2+ uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca 2+ uptake by mitochondria.FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM. FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca 2+ uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca 2+-dependent proapoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs.

BackgroundAdrenocortical carcinoma (ACC) is a rare endocrine malignancy. Tumor-related glucocorticoid excess is present in ~60% of patients and associated with particularly poor prognosis. Results of first clinical trials using immune checkpoint inhibitors were heterogeneous. Here we characterize tumor-infiltrating T lymphocytes (TILs) in ACC in association with glucocorticoids as potential explanation for resistance to immunotherapy.MethodsWe performed immunofluorescence analysis to visualize tumor-infiltrating T cells (CD3+), T helper cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+) and regulatory T cells (Tregs; CD3+CD4+FoxP3+) in 146 ACC tissue specimens (107 primary tumors, 16 local recurrences, 23 metastases). Quantitative data of immune cell infiltration were correlated with clinical data (including glucocorticoid excess).Results86.3% of ACC specimens showed tumor infiltrating T cells (7.7 cells/high power field (HPF)), including T helper (74.0%, 6.7 cells/HPF), cytotoxic T cells (84.3%, 5.7 cells/HPF) and Tregs (49.3%, 0.8 cells/HPF). The number of TILs was associated with better overall survival (HR for death: 0.47, 95% CI 0.25 to 0.87), which was true for CD4+− and CD8+subpopulations as well. In localized, non-metastatic ACC, the favorable impact of TILs on overall and recurrence-free survival was manifested even independently of ENSAT (European Network for the Study of Adrenal Tumors) stage, resection status and Ki67 index. T helper cells were negatively correlated with glucocorticoid excess (Phi=−0.290, p=0.009). Patients with glucocorticoid excess and low TILs had a particularly poor overall survival (27 vs. 121 months in patients with TILs without glucocorticoid excess).ConclusionGlucocorticoid excess is associated with T cell depletion and unfavorable prognosis. To reactivate the immune system in ACC by checkpoint inhibitors, an inhibition of adrenal steroidogenesis might be pivotal and should be tested in prospective studies.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.