ECE2020 Audio ePoster Presentations Adrenal and Cardiovascular Endocrinology (121 abstracts)
Cytochrome P450 (CYP) 2W1 affect steroid secretion in adrenocortical cell line and tumor tissues
Barbara Altieri 1 , Silviu Sbiera 1 , Isabel Weigand 1 , Marco Volante 2 , Sonja Steinhauer 1 , Antonia Lorey 1 , Sabine Kendl 1 , Max Kurlbaum 1 , Sabine Herterich 3 , Silvia Della Casa 4 , Annamaria Colao 5 , Massimo Terzolo 6 , Matthias Kroiss 1 , Martin Fassnacht 1 & Cristina Ronchi 1,7
1University Hospital of Würzburg, Division of Endocrinology and Diabetes, Department of Internal Medicine I, Würzburg, Germany; 2University of Turin, San Luigi Hospital, Department of Oncology, Turin, Italy; 3University Hospital of Würzburg, Central Laboratory, Würzburg, Germany; 4Catholic University of the Sacred Heart, Division of Endocrinology and Metabolism, Rome, Italy; 5Federico II University, Department of Clinical Medicine and Surgery, Naples, Italy; 6University of Turin, San Luigi Gonzaga Hospital, Division of Internal Medicine I, Turin, Italy; 7Institute of Metabolism and System Research, University of Birmingham, Birmingham, United Kingdom
Background: The human cytochrome P450 (CYP) 2W1 is involved in the metabolism of several endogenous substrates, including lysophospholipids and fatty acids. Using a polyclonal antibody, we previously demonstrated a high CYP2W1 immunoreactivity in adrenocortical tumors, particularly in those secreting steroids. The aim of the study was to better elucidate the relationship between CYP2W1 and steroid secretion in adrenocortical carcinoma (ACC) H295R cell line and in a larger series of ACC tissues associated with different steroid secretion patterns.
Methods: H295R cells were stably transfected in order to achieve CYP2W1 overexpression, which was confirmed both at mRNA and protein levels. Mock- transfected H295R cells were used as controls. Steroid secretion was evaluated by liquid chromatography tandem-mass spectrometry (LC-MS/MS) using a panel of 13 steroids both at baseline (t0) and after 48h of 1 µM forskolin treatment (t1). For the immunohistochemistry (IHC) analysis 203 ACC tissue samples were collected, most of which pertained tumors that secreted cortisol and/or aldosterone alone (n = 64, 31.5%), followed by endocrine inactive tumors (n = 49, 24.1%), multiple secreting tumors, including cortisol in combination with other steroids (n = 46, 22.7%), and androgen-secreting ACC (n = 44, 21.7%). IHC was performed using a new highly specific monoclonal CYP2W1 antibody (sc374426, Santa Cruz). The relationship between cytoplasmic CYP2W1 immunoreactivity, measured by semi-quantitative H-score, and the steroid secretion pattern at the time of diagnosis was evaluated.
Results: In vitro, aldosterone levels were significantly higher in CYP2W1-transfected cells than in controls both at t0 (21.5 ± 9.2 fold change (FC), P = 0.02) and t1 (18.3 ± 6.8 FC, P = 0.04). Cortisol was significantly increased at t0 (3.9 ± 1.1 FC, P = 0.01), whereas cortisone was higher at t1 (2.4 ± 0.6 FC, P = 0.01). On the contrary, androgen levels were significantly lower in CYP2W1-cells, including DHEA (–2.1 ± 0.9 FC, P = 0.01 at t0), DHEAS (−4.1 ± 0.1 FC, P < 0.001 at t0; −2.00 ± 0.0 FC, P < 0.001 at t1), androstenedione (−3.3 ± 0.1 FC, P = 0.04 at t0; −3.7 ± 0.1 FC, P = 0.03 at t1), and DHT (−2.0 ± 0.2 FC, P = 0.02 at t1). At IHC analysis, the majority of ACC tissues (56.3%) had CYP2W1 immunoreactivity of H-score = 1. Particularly, CYP2W1 staining was lower in androgen-secreting tumors than in multiple-secreting cases (P = 0.003), whereas a trend to lower immunoreactivity was observed compared to only cortisol- or aldosterone-secreting ones (P = 0.11). No significant difference was found between inactive and secreting-ACC.
Conclusion: CYP2W1 may induce cortisol and aldosterone secretion and inhibit androgens secretion both in vitro and in ACC tissues. Further experiments investigating the steroidogenic pathway are needed to confirm our results.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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