4-Hydroxyestradiol induces mammary epithelial cell transformation through Nrf2-mediated heme oxygenase-1 overexpression

Estrogen (17β-estradiol, E(2)) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE(2)), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE(2)-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE(2), but not E(2), increased the expression of heme oxygenase-1 (HO-1), a sensor and regulator of oxidative stress, in MCF-10A cells. Silencing the HO-1 gene in MCF-10A cells suppressed 4-OHE(2)-induced cell proliferation and transformation. In addition, subcutaneous administration of 4-OHE(2) markedly enhanced the growth of the MDA-MB-231 human breast cancer xenografts, which was retarded by zinc protoporphyrin, a pharmacological inhibitor of HO-1. 4-OHE(2)-induced HO-1 expression was mediated by NF-E2-related factor 2 (Nrf2). We speculate that an electrophilic quinone formed as a consequence of oxidation of 4-OHE(2) binds directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish association between Nrf2 and Keap1. 4-OHE(2) failed to interrupt the interaction between Keap1 and Nrf2 and to induce HO-1 expression in Keap1-C273S or C288S mutant cells. Lano-LC-ESI-MS/MS analysis in MCF-10A-Keap1-WT cells which were treated with 4-OHE(2) revealed that the peptide fragment containing Cys288 gained a molecular mass of 287.15 Da, equivalent to the addition of a single molecule of 4-OHE(2)-derived ortho-quinones.