Metformin Inhibits Nuclear Localization Of Estrogen Receptor Alpha In Breast Cancer Cells, In Part, By Inhibition Of Cyp-Mediated Epoxyeicosatrienoic Acid (Eet) Biosynthesis

Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+HER2-, breast cancer cells, CYP3A4 promotes tumor growth, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The biguanide diabetes drug metformin is currently being studied in breast cancer clinical trials. We have discovered that metformin binds to the active site heme of CYP3A4, thereby inhibiting EET biosynthesis (1). Structure-based design led to the discovery of the 100-fold more potent metformin analog N1-hexyl-N5-benzyl-biguanide (HBB). HBB specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity in breast cancer cells, suppresses the growth of breast cancer cell lines (IC50=3-30 uM), and inhibits growth of the MCF-7 ER+ mammary tumor model, similar to CYP3A4 gene silencing. Although CYP3A4 synthesizes EETs, how EETs may promote tumor growth is unknown. Recently, we found that HBB inhibits nuclear transit of estrogen receptor alpha (ERα) and 70kD FITC-dextran, but the mechanism was unknown (2). Metformin was discovered to inhibit nuclear pore complex (NPC) function, but the mechanism was also unknown (3). We therefore hypothesized that CYP-derived EETs function as second messengers that regulate nuclear translocation of ERα in breast cancer by promoting permeability of the NPC. This hypothesis implies that biguanide drugs suppress nuclear translocation of the ERα through inhibition of CYP-derived EETs that serve as second messengers to open the NPC. Methods: Breast cancer cells were serum-starved for 16 hours and then treated with metformin, HBB, the (±)-14,15-EET regioisomer, or EET agonists, for varying times (30 min to 6 hours). Cells were fixed, permeabilized, and incubated with antibodies specific for ERα. After incubation with fluorescent secondary antibodies and Hoechst 33342 dye, cell images were acquired with a confocal microscope and the nuclear ERα fluorescence signal was quantified relative to the Hoechst signal. Quantitative PCR was used to measure the expression of ERα regulated genes. Results: (±) 14,15-EET (5 uM) treatment for 1 hour increased nuclear ERα by 66 ± 26% (n=9, p=0.031) in MCF-7 cells and 77 ± 24% (n=7, p=0.015) in ZR-75 cells. Treatment with EET agonists C22 (5 uM) and EET-A (5 uM) for 1 hour increased nuclear ERα by 32 ± 13% (n=7, p=0.047) and 39 ± 14% (n=7, p=0.02) in MCF-7 cells. Metformin (5 mM) treatment for 6 hours reduced nuclear ERα in MCF-7 by 35 ± 4.9 % (mean ± SEM, n=8, p=0.003). HBB (20 uM) treatment for 2 hours reduced the expression of three ERα regulated genes in MCF-7 cells: estrogen receptor alpha (ESR1) was reduced by 15 ± 1.7% (n=3, p=0.001), progesterone receptor (PGR) by 21 ± 0.6% (n=3, p=0.02) and L-type amino acid transporter 1 (SLC7A5) by 14 ± 0.2% (n=3, p=0.002). Conclusion: CYP epoxygenase activity in ER+HER2- breast cancer cells produces EETs, which function as second messengers that promote nuclear transit of ERα. Metformin and HBB inhibit nuclear ERα translocation, in part, through inhibition of CYP3A4 AA epoxygenase activity and this inhibition reduces ERα mediated gene expression, providing a novel mechanism for ERα regulation. 1. Cell Chem Biol. 2017 Oct 19; 24(10) 1259 - 1275. 2. Abstract of 109th AACR meeting (#LB-023), April 2019 3. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Jianxun Lei, Shaoping Wu, Julissa Molina-Vega, Paloma Cervantes, Aline R. Rom9Mand, Dylan C. Castillejo Mijangos, John R. Falck, Carol Lange, Jinhua Wang, David A. Potter. Metformin inhibits nuclear localization of estrogen receptor alpha in breast cancer cells, in part, by inhibition of CYP-mediated epoxyeicosatrienoic acid (EET) biosynthesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4372.