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. 2019 Oct 29;116(44):22269-22274.
doi: 10.1073/pnas.1912700116. Epub 2019 Oct 14.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis

Alexia Belavgeni  1   2 , Stefan R Bornstein  3   4   5   6   7 , Anne von Mässenhausen  1   2 , Wulf Tonnus  1   2 , Julian Stumpf  1 , Claudia Meyer  1   2 , Evelyn Othmar  1   2 , Markus Latk  1   2 , Waldemar Kanczkowski  3 , Matthias Kroiss  8   9 , Constanze Hantel  10 , Christian Hugo  1 , Martin Fassnacht  8   9 , Christian G Ziegler  3 , Andrew V Schally  11   12   13   14   15 , Nils P Krone  3   16 , Andreas Linkermann  17   2
Affiliations

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis

Alexia Belavgeni et al. Proc Natl Acad Sci U S A. .

Abstract

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis VSports手机版. .

Keywords: adrenal; adrenocortical carcinoma; endocrine tumors; ferroptosis; regulated necrosis V体育安卓版. .

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Conflict of interest statement

Competing interest statement: S. R. B. and A. V V体育ios版. S. hold patents on MIA602. A. V. S. patents are assigned to the University of Miami and Veterans Affairs. Reviewer W. K. was a coauthor with A. L. on this manuscript on a 2018 paper that is a committee report. Apart from this, all authors declare no competing interest regarding any of the presented data in this manuscript.

Figures (VSports最新版本)

Fig. 1.
Fig. 1.
ACCs are sensitive to ferroptosis induction. (A) Structure of mitotane and time lapse video screen shots of H295R cells induced to undergo necrosis by mitotane. Membrane blebbing was sensitive to caspase inhibition by zVAD-fmk. (B) Caspase inhibition does not prevent mitotane-induced cellular necrosis after 6 h of incubation. Jurkat T cells serve as controls. (C) Expression of GPX4 in H295R cells in comparison with HT1080 cells, the standard cell line for ferroptosis research. (D) H295R cells are sensitive to the type 2 ferroptosis inducer RSL3 (HT1080 cells serve as controls). (E) RSL3-induced ferroptosis in H295R cells is prevented by addition of the small molecule ferrostatin-1 (Fer-1). All experiments were repeated at least 3 times; representative examples of primary data are shown.
Fig. 2.
Fig. 2.
ITS supplements prevent ferroptotic cell death in H295R cells. (A) H295R cells were treated with the type 2 ferroptosis inducer RSL3 for 6 h in the presence or absence of ITS (see also SI Appendix, Fig. S2, for details). Insulin, transferrin, selenium, and sodium selenite do not prevent mitotane-induced necrosis, either when applied (B) as single components or (C) in combination. (D) The absence of linoleic acid, however, resulted in failure of ITS to inhibit mitotane-induced necrosis. All experiments were repeated at least 3 times; representative examples of primary data are shown.
Fig. 3.
Fig. 3.
Mitotane induces a specific form of cell death. (A) H295R cells, Jurkat T cells, HT29 cells, and HT1080 cells were treated with different doses of mitotane for 24 h. At 50 µM, the number of annexin V/7AAD double negative cells significantly decreased in the H295R cells, but all other cell lines were resistant. (B) Western blot for human phosphorylated MLKL, the downstream mediator of necroptosis. Mitotane does not induce necroptosis, as controlled by HT29 cells that were treated with TNFα, smac mimetics, and a pan-caspase inhibitor zVAD (together indicated as TSZ). GAPDH serves as a loading control. (C) Mitotane does not induce ferroptosis, as indirectly demonstrated by the absence of a protective effect of Fer-1 or Nec-1. All experiments were repeated at least 3 times; representative examples of primary data are shown.
Fig. 4.
Fig. 4.
Analyses of public databases on human ACCs suggest a role for glutathione peroxidases and ferroptosis. (A) Gene expression and DNA methylation of epigenetically silenced genes in human ACCs, according to Zheng et al. in ref. . (B) Selected differentially expressed genes in normal adrenocortical tissue, adrenocortical adenomas and ACCs indicate a role for ferroptosis sensitivity in ACCs. (C) Cystathionase (CTH cystathionine gamma-lyase) catalyzed reaction and generation of cysteine, initially identified in ref. . (D) Five µM of MIA602 are sufficient to kill more than 50% of H295R cells in cell culture.
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