At the interface between endoplasmic reticulum and mitochondria: inhibition of SOAT1 as new treatment strategy against adrenocortical carcinoma

米托坦是肾上腺皮质癌（ACC）的标准治疗。这种罕见的肿瘤预后非常差（中位生存期<3.5年），只有约30%的患者受益于米托坦治疗，这与严重的不良反应有关。因此，有必要提高治疗的准确性，并开发更有效的药物。在第一个资助期内，我们能够首次确定米托坦的分子靶点，即甾醇-O-酰基转移酶1（SOAT 1）。使用基因表达分析，脂质质谱和功能的方法，我们已经表明，米托坦的影响，特别是在肾上腺皮质激素产生细胞的脂质组成，从而强烈激活内质网（ER）应激信号通路。这是由于米托坦对SOAT 1的直接抑制。SOAT 1位于ER和线粒体的界面，位于所谓的线粒体相关膜（MAM）内。我们还表征了米托坦与膜和脂蛋白的相互作用，并鉴定了潜在的协同药物。在五个子项目中，我们将继续努力：1）为了研究建立SOAT 1表达作为米托坦个性化治疗的标志物，我们将在300多个临床上非常精确表征的肿瘤样本中进行SOAT 1特异性免疫组织化学。我们的假设是SOAT 1表达能够预测对米托坦的反应（无论是在辅助治疗还是姑息治疗中）。2)根据其他SOAT 1抑制剂更有效和耐受的假设，我们将描述它们在ACC细胞中的作用。值得注意的是，SOAT 1抑制剂在20世纪90年代被开发为降脂药物，但部分由于肾上腺毒性而停止开发。现在，我们将通过质谱法评估细胞内脂质和代谢物的变化，通过代谢通量分析评估能量代谢，通过流式细胞术评估细胞凋亡，并通过不同细胞系统中的基因表达分析评估由此产生的适应性过程。3)由于我们在初步研究中注意到MAMs对肾上腺类固醇合成和米托坦作用非常重要，因此在第三个子项目中，我们将使用SOAT抑制剂作为工具，以更好地了解MAMs在脂质诱导的ER应激中的作用。4 + 5）在最后两个子项目中，我们将研究米托坦也可能抑制其他膜结合O-酰基转移酶的假设，以及米托坦诱导的ER应激通过诱导某些信号模式（危险相关膜模式）导致免疫原性细胞死亡的假设。我们相信，这些研究将在未来显着改善ACC患者的治疗，也可能为SOAT 1作为治疗其他肿瘤的靶分子提供基础。

Mitotane is the standard of care for adrenocortical carcinoma (ACC). This rare tumour comes with a very poor prognosis (median survival <3.5 years) and only about 30% of patients benefit from mitotane therapy, which is associated with severe adverse effects. Therefore, it is necessary to increase the accuracy of the therapy and to develop more effective drugs. In the first funding period, we were able to identify for the first time a molecular target of mitotane, namely sterol-O-acyltransferase 1 (SOAT1). Using gene expression analysis, lipid mass spectrometry and functional methods, we have shown that mitotane influences the lipid composition particularly in hormone producing cells of the adrenal cortex, thereby strongly activating the endoplasmic reticulum (ER) stress signalling pathway. This is due to direct inhibition of SOAT1 by mitotane. SOAT1 is located at the interface of the ER and the mitochondria within the so-called mitochondria-associated membranes (MAM). We also characterized the interaction of mitotane with membranes and lipoproteins and identified potentially synergistic drugs. In five sub-projects, we will now continue our efforts: 1) To investigate establish SOAT1 expression as a marker for a personalized therapy with mitotane we will perform SOAT1 specific immunohistochemistry in over 300 clinically very precisely characterized tumour samples. Our hypothesis is that SOAT1 expression is able to predict response to mitotane (both in adjuvant and palliative setting). 2) Following the hypothesis that other SOAT1 inhibitors are more effective and tolerable, we will characterize their effect in ACC cells. Of note, SOAT1 inhibitors were developed in the 1990ies as lipid lowering drugs but development was stopped partly due to adrenal toxicity. Now, we will assess changes in intracellular lipids and metabolites by mass spectrometry, energy metabolism by metabolic flux analyses, apoptosis by flow cytometry and the resulting adaptive processes by gene expression analyses in different cell systems. 3) Since we have noted in preliminary studies that MAMs are of great importance for adrenal steroid synthesis and mitotane action, in the third subproject we will use SOAT inhibitors as a tool to better understand the role of MAMs in lipid-induced ER stress. 4 + 5) In the last two subprojects, we will investigate the hypotheses that mitotane might also inhibit other membrane-bound O-acyltransferases, and that the mitotane-induced ER stress leads to immunogenic cell death by inducing certain signal patterns (danger associated membrane patterns). We are confident that these studies will significantly improve the therapy of ACC patients in the future and might also provide the basis for SOAT1 as a target molecule for the treatment of other tumours.