biomedicines Article Estrogen-Like Effect of Mitotane Explained by Its Agonist Activity on Estrogen Receptor-α Elisa Rossini 1,†, Edoardo Giacopuzzi 2,3,† , Fabrizio Gangemi 4,† , Mariangela Tamburello 1, Deborah Cosentini 5, Andrea Abate 1, Marta Laganà 5, Alfredo Berruti 5 , Salvatore Grisanti 5,‡ and Sandra Sigala 1,*,‡ 1 Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; [email protected] (E.R.); [email protected] (M.T.); [email protected] (A.A.) 2 Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK; [email protected] 3 National Institute for Health Research Oxford Biomedical Research Centre, Oxford OX4 2PG, UK 4 Unit of Physics, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; [email protected] 5 Medical Oncology Unit, Department of Medical and Surgical Specialties, Radiological Sciences, and Public Health, University of Brescia at A.S.S.T. Spedali Civili di Brescia, 25123 Brescia, Italy; [email protected] (D.C.); [email protected] (M.L.); [email protected] (A.B.); [email protected] (S.G.) * Correspondence: [email protected] † These Authors equally contributed to this paper. ‡ These authors are co-senior authors of this paper. Citation: Rossini, E.; Giacopuzzi, E.; Abstract: Mitotane is the cornerstone of medical treatment of adrenocortical carcinoma. Estrogenic- Gangemi, F.; Tamburello, M.; like side effects frequently occur in patients, and previous studies explored the chemical nature Cosentini, D.; Abate, A.; Laganà, M.; of the interaction between estrogen receptor-α (ER-α) and toxic compounds, including the DDD Berruti, A.; Grisanti, S.; Sigala, S. derivatives. We used molecular docking and molecular dynamics (MD) simulations to explore the Estrogen-Like Effect of Mitotane possible interaction between mitotane and the ER-α receptor and the induced conformational changes. Explained by Its Agonist Activity on The ER-α expressing MCF-7 cells were exposed to mitotane with/without tamoxifen, and the cell Estrogen Receptor-α. Biomedicines α 2021, 9, 681. https://doi.org/ viability/proliferation was evaluated by MTT assay and direct count. The transient ER- silencing 10.3390/biomedicines9060681 was performed using two ER-α siRNA (50 nM) and verified by Western blot. MDA-MB-231 cells were used as a negative control. Mitotane showed a similar docking configuration to 17β-estradiol and Academic Editors: Anna Angelousi bisphenol A (BPA) and a significant binding affinity to ER-α. MD simulations showed that mitotane and Danae Delivanis preserves the active conformation of ER-α more than both BPA and Bisphenol C, classifying it as an agonist. Exposure of MCF-7 cells to mitotane led to the concentration-dependent increase Received: 29 May 2021 of cell viability and proliferation, which was reduced in the presence of tamoxifen and nullified Accepted: 14 June 2021 by the transient ER-α knock-down. Integrating bioinformatics approaches with cell biology and Published: 16 June 2021 pharmacological methods, we demonstrated that mitotane directly binds and activates ER-α. Publisher’s Note: MDPI stays neutral Keywords: estrogen receptor α; mitotane; adrenocortical carcinoma; bioinformatics analysis with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Adrenocortical carcinoma (ACC) is a rare malignancy with a reported incidence of 0.7–2 cases per million population per year [1,2]. It is more frequent in females and can Copyright: © 2021 by the authors. occur at any age, with a peak in the fifth and sixth decades of life. As a result of cancer- Licensee MDPI, Basel, Switzerland. specific altered transcriptional modules [3], about 50–60% of ACCs are hormone functioning This article is an open access article at presentation. Hypercortisolism (Cushing’s syndrome) or mixed Cushing’s and virilizing distributed under the terms and conditions of the Creative Commons syndromes are observed in 50–80% of hormone-secreting ACCs [1,2]. In patients with Attribution (CC BY) license (https:// resected ACC, the presence of clinical signs of cortisol excess is an independent poor creativecommons.org/licenses/by/ prognostic factor [4]. 4.0/). Biomedicines 2021, 9, 681. https://doi.org/10.3390/biomedicines9060681 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 681 2 of 13 Mitotane (o,p’-DDD) is the cornerstone of medical treatment of ACC, both in adju- vant [5,6] and metastatic settings [7]. The drug has selective cytotoxic activity against cells of the zona reticularis and zona fasciculata of the adrenal cortex, which leads to a marked inhibition of adrenocortical steroid synthesis, resulting in the control of hormone excess-associated syndromes [8]. Mitotane therapy invariably leads to adrenal insufficiency because of the functional suppression of the remaining normal adrenal gland, requiring chronic exogenous cortisol replacement therapy [1,2]. Therefore, patients with ACC re- ceiving long-term mitotane face both direct (mitotane inhibition of steroidogenesis) and indirect (exogenous cortisol replacement therapy) endocrine effects. One common clinical observation in patients undergoing mitotane administration is the high incidence of estrogenic-like side effects. Indeed, approximately one-third of adult male patients develop gynecomastia eventually associated with other signs of secondary hypogonadism such as hair loss, libido loss, muscle mass loss, and psychological alterations [9,10]. In premenopausal fertile women, mitotane induces the formation of benign ovarian cysts, amenorrhea, or abnormal uterine bleeding such as menorrhagia and metrorrhagia [11,12], and it has been associated with increased frequency of peripheral precocious puberty in female children [13]. Early studies demonstrated that most ACC patients treated with mitotane have increased levels of sex hormone-binding globulin (SHBG), a glycoprotein involved in binding and transport of circulating plasma testosterone and 17β-estradiol (E2) to target tissues [9,14]. In an in vitro study, Nader et al. demonstrated that mitotane increases gene expression of SHBG in an estrogen-receptor-α (ER-α)-dependent manner [15]. Thus, it has been postulated that at least some of the estrogen-like effects of mitotane therapy could be explained in terms of ER-α-dependent increase of SHBG and subsequent perturbation of bioavailability of plasma testosterone and E2 for target tissues. ER-α and ER-β have been described in the normal adrenal cortex during fetal de- velopment and in postnatal life [16,17]. ERs are expressed at different intensities in both normal and neoplastic adrenal cortices [17]; however, the pathophysiological relevance of their expression in adrenal cell proliferation is not completely elucidated. The ER-α subtype appears to be generally poorly expressed, while ER-β is expressed mainly in the glomerular and fasciculata areas [18,19]. Data concerning their expression in ACC are controversial, although results obtained with immunohistochemical analyses indicated that ER-α expression is weak/undetectable [17,20]. Contrasting results suggest indeed low ER-β and/or high ER-α expression in ACC, leading to an increase in the ER-α/ER-β ratio, compared to that observed in healthy tissue [19]. Previous studies suggested that mitotane could bind the human ER-α with a relative binding affinity of about 1000-fold weaker than the natural ligand, E2 [21–23]. However, these studies mainly explored the chemical nature of the interaction between ER-α and potential environmental toxic compounds. At physiological conditions, ER-α exists as a dimer and can present itself in different conformations stabilized by the binding of either agonists or antagonists [24]. Five distinct functional domains can be identified in the ER-α protein moving from the N-terminus to the C-terminus, namely, the N-terminal domain (NTD), the DNA-binding domain (DBD), the D domain, the ligand-binding domain (LBD), and the F domain. The crystal structure of DBD and LBD have been determined by crystallographic studies [25–27]. Besides the natural ligand E2, several molecules have been identified that can interact with ER-α, providing either agonist or antagonist effect, with tamoxifen and fulvestrant representing the prototype for selective estrogen receptor modulators (SERM) and selective estrogen receptor degraders (SERD), respectively [28]. Several of these receptor–ligand complexes have been also studied at the structural level, leading to the identification of the main conformational changes involved in the activation and inactivation of the receptor [24] and providing a strong background to predict the effect of new ligands based on molecular docking and dynamic simulations. Biomedicines 2021, 9, 681 3 of 13 In this study, integrating bioinformatics approach with cell biology and pharmaco- logical methods, we demonstrated that mitotane directly bounds ER-α as a direct agonist, inducing cell proliferation of the ER-α positive breast cancer cell line MCF-7 cells. These results provide the biological and pharmacological basis of the clinically observed estrogen- like effect of mitotane. 2. Material and Methods 2.1. Ligand Docking Experiments To investigate the interaction between mitotane and ER-α and whether this ligand would act as an agonist or antagonist, we compared the behavior of this simulated ligand- protein complex with those containing bisphenol