Regulated necrosis of adrenocortical carcinomas
PhD Student: Alexia Belavgeni
Supervisor at TUD: Andreas Linkermann, Stefan Bornstein
Supervisor at KCL: Rocio Sancho
Start Date: 01.10.2017
Adrenocortical carcinomas (ACCs) are rare, devastating tumors with low prognosis that are derived from the cortex of the adrenal glands. The majority of the ACCs are functional, producing hormones that results in respective clinical syndromes. The current effective therapeutic approach for ACCs is complete removal of the adrenal glands. More malignant or metastatic types of ACCs are treated with mitotane, a pesticide derivative of DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane). However, it’s mode of action remains to be elucidated. Apoptosis, necroptosis, pyroptosis and ferroptosis are regulated cell death pathways. The aforementioned three pathways are caspase-controlled systems, while ferroptosis is an oxidisation controlled-system, free of caspase control. Failure of the antioxidant/redox systems to maintain the redox balance of the cell leads to lipid peroxidation. Rupture of the cellular membrane, characteristic event of necroptosis, ferroptosis and pyroptosis, remains an unclear part so far. More specifically, glutathione peroxidase 4 (GPX4) constitutes as the main inhibitor of ferroptosis, through the conversion of potentially toxic lipid hydroperoxides (L-OOH) to non-toxic lipid alcohols (L-OH) while metabolising reduced glutathione (GSH) to glutathione disulphide (GSSG). Therefore, the abundance of GSH in the cell constitutes the limiting factor for the GPX4 function. Induction of ferroptosis can be achieved in many ways by different ferroptosis inducers (FINs). Molecules that inhibit the system Xc-, an antiporter of glutamate and cystine, e.g. erastin are referred to as type I FINs. Type II FINs, e.g. RSL3, directly target the enzymatically active pocket of GPX4. Type III FINs, e.g. FIN56, induce GPX4 degradation, while other molecules, such as FINO2, are referred to as type IV FINs that are able to target in unclear so far manners GPX43. Ferroptosis has been shown to be important in a several pathophysiological conditions, such as stroke, acute tubular injury, and cancer. Being interested in ACCs and given the side effects of mitotane, we aim to investigate the sensitivity of the human ACC cell line NCI-H295R cells (will be referred from now on as H295R cells for simplicity reasons), to different ferroptosis inducers. In parallel, we aim to elucidate the mechanism of action of mitotane, which could potentially lead to new drug targets. With these data, we will be able to understand in depth the basic molecular pathways that connect the physiological processes of the ACC cells and ferroptosis.
In order to test the sensitivity of H295R cells to mitotane, different concentrations were tested and the induced cell death was assessed with flow cytometry analysis, during which cells were stained with annexin V, marker for phosphatidylserine exposure, and 7AAD, a nucleus stain. Based on the obtained data 50 μΜ of mitotane was used as the standard concentration. Interestingly, live imaging experiments of H295R cells treated with 50 μΜ mitotane revealed cell death and blebbing of the cellular membrane. However, when zVAD-fmk, a pan-caspase inhibitor, was added this phenomenon was prevented without rescuing the cells from the mitotane-induced cell death. These findings were confirmed by FACS analysis. Use of emricasan, a pan-caspase inhibitor, also was not able to rescue the cells from the observed cell death. Therefore, apoptosis was ruled out as a possible mechanism of mitotane. Following similar experimental design, Nec-1s, a necroptosis inhibitor, was added in treatment of the ACC cells with mitotane, without being able to reverse the cell death. Similar results were obtained with Fer-1, a ferroptosis inhibitor. These data indicate that the mechanism of mitotane does not involve any of the classical regulated pathways of cell death. Aiming to find out whether H295R cells were sensitive to necroptosis or ferroptosis, western blot analysis of molecules involved in these pathways were performed. It is now known that RIPK1, RIPK3 protein receptor-interacting protein kinases) and MLKL (mixed lineage kinase domain-like protein) are important molecules for necroptosis. Even though RIPK1 was detected in low levels in the H295R cells, RIPK3 and MLKL were not detected. As a consequence, induction of the ACC cell line with necroptosis inducers did not result in cell death. These data proposed a resistance to the classical method used to induce necroptosis. On the other hand, when performing western blot for ferroptosis important molecules, such as GPX4, ACSL4, we discovered a higher expression of GPX4 compared to HT1080, human fibrosarcoma cells, which as used as a positive control to ferroptosis. In our knowledge, H295R show the highest expression of GPX4. Based on this finding, it was of our interest to investigate ferroptosis inducers. Our data showed that H295R cells were extremely sensitive to RSL3 induced cell death, which was successfully reversed with addition of Fer1. Interestingly, addition of ITS+1 supplementation, standing for human insulin, bovine transferrin, selenium and linoleic acid, was able to completely prevent this necrotic cell death. Further investigation on the matter pointed towards the importance of linoleic acid in the medium of the cultured cells.
The aforementioned findings pointed towards a high sensitivity of H295R cells to ferroptosis, proposing a new therapeutic approach which could be followed in the clinical practise. The mechanism of mitotane still remains a field to be investigated, due to the complexity that it seems to have in its function. It is of our interest to expand our knowledge in the investigation of the sensitivity of H295R cells to other FINs and ferroptosis inducing substances aiming to better comprehend the ferroptosis pathway. The successful preparation of cas9 expressing H295R cells will help us answer our questions genetically.
Publications:
The role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes in the [omim][BF4]-mediated toxic mode of action in mussel hemocytes. A. Belavgeni, S. Dailianis. Fish Shellfish Immunol. 2017;68:144-153.
Miniaturising acute toxicity and feeding rate measurements in Daphnia magna. K. Grintzalis, W. Dai, K. Panagiotidis, A. Belavgeni, M.R. Viant. Ecotoxicol Environ Saf. 2017;139:352-357.
The impact of expired commercial drugs on non-target marine species: A case study with the use of a battery of biomarkers in hemocytes of mussels. N. Politakis, A. Belavgeni, I. Efthimiou, N. Charalampous, C. Kourkouta, S. Dailianis. Ecotoxicol Environ Saf. 2018;148:160-168.
Prominin-2 Suppresses Ferroptosis Sensitivity. A. Belavgeni, S.R. Bornstein, A. Linkermann. Dev Cell. 2019;51:548-549.
Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis. A. Belavgeni, S.R. Bornstein, A. von Massenhausen, W. Tonnus, J. Stumpf, C. Meyer, E. Othmar, M. Latk, W. Kanczkowski, M. Kroiss, C. Hantel, C. Hugo, M. Fassnacht, C.G. Ziegler, A.V. Schally, N.P. Krone, A. Linkermann. Proc Natl Acad Sci U S A. 2019;116:22269-22274.
Don't trick me twice! W. Tonnus, A. Belavgeni, Y. Xu, A. Linkermann. Kidney Int. 2019;95:736-738.
The pathological features of regulated necrosis. W. Tonnus, C. Meyer, A. Paliege, A. Belavgeni, A. von Massenhausen, S.R. Bornstein, C. Hugo, J.U. Becker, A. Linkermann. J Pathol. 2019;247:697-707.
Stress will kill you anyway! A. Belavgeni, S.R. Bornstein, A. Linkermann. Cell Death Dis. 2020;11:218.
Ferroptosis and Necroptosis in the Kidney. A. Belavgeni, C. Meyer, J. Stumpf, C. Hugo, A. Linkermann. Cell Chem Biol. 2020;27:448-462.
Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury. W. Tonnus, C. Meyer, C. Steinebach, A. Belavgeni, A. von Mässenhausen, N.Z. Gonzalez, F. Maremonti, F. Gembardt, N. Himmerkus, M. Latk, S. Locke, J. Marschner, W. Li, S. Short, S. DollS, I. Ingold, B. Proneth, C. Daniel, N. Kabgani, R. Kramann, S. Motika, P.J. Hergenrother, S.R. Bornstein, C. Hugo, J.U. Becker, K. Amann, H.J. Anders, D. Kreisel, D. Pratt, M. Gütschow, M. Conrad, A. Linkermann. Nat Commun. 2021;12(1):4402.
The role of regulated necrosis in endocrine diseases. W. Tonnus, A. Belavgeni, F. Beuschlein, G. Eisenhofer, M. Fassnacht, M. Kroiss, N.P. Krone, M. Reincke, S.R. Bornstein, A. Linkermann. Nat Rev Endocrinol. 2021;17(8):497-510.
BEX1 Is Differentially Expressed in Aldosterone-Producing Adenomas and Protects Human Adrenocortical Cells From Ferroptosis. Y. Yang, M. Tetti, T. Vohra, C. Adolf, J. Seissler, M. Hristov, A. Belavgeni, M. Bidlingmaier, A. Linkermann, P. Mulatero, F. Beuschlein, M. Reincke, T.A. Williams. Hypertension. 2021;77:1647-1658.