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. 2017 Jan 3;8(1):164-178.
doi: 10.18632/oncotarget.10516.

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

Sin-Aye Park  1 , Mee-Hyun Lee  1 , Hye-Kyung Na  2 , Young-Joon Surh  1   3   4
Affiliations

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

Sin-Aye Park et al. Oncotarget. .

Erratum in

Abstract

Estrogen (17β-estradiol, E2) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE2), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE2-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE2, but not E2, 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-OHE2-induced cell proliferation and transformation. In addition, subcutaneous administration of 4-OHE2 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-OHE2-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-OHE2 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-OHE2 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-OHE2 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-OHE2-derived ortho-quinones.

Keywords: 4-hydroxyestradiol; Nrf2; breast cancer; catechol estrogen; heme oxygenase-1.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. 4-OHE2 induces both expression and/or activity of HO-1 in human breast cells
(A) Each cell line was treated with E2 or 4-OHE2 (20 μM) for the indicated time periods, and the expression of HO-1 was assessed by Western blot analysis. (B) MCF-10A cells were treated with various concentrations of 4-OHE2 (0.2, 2, 5, 10, and 20 μM) for 6 h and the whole-cell lysates were subjected to Western blot analysis. (C) MCF-10A cells were treated with 4-OHE2 (20 μM) for 12 h and the HO activity was measured as described in Materials and Methods. n = 3; *P < 0.001.
Figure 2
Figure 2. 4-OHE2-induced HO-1 expression is associated with cell proliferation
(A) MCF-10A cells stably expressing mock, negative control (NC) shRNA or HO-1 shRNA were selected with 0.5 μg/ml puromycin. Cells were exposed to 4-OHE2 (20 μM) for 6 h and analyzed for expression of HO-1 mRNA and protein by RT-PCR and Western blot analysis, respectively. HMOX1: HO-1 mRNA; ACTB: β-actin mRNA. (B) MCF-10A-mock or MCF-10A-shHO-1 cells were treated with DMSO or 4-OHE2 (5 μM) for 72 h, and the cell proliferation was measured as described in Materials and Methods. (C-E), Cell migration was measured by using Culture-Inserts, and the wound closure was monitored by photography at indicated time points as described in Materials and Methods. (C) The representative images of migration assay are from MCF-10A-mock or MCF-10A-shHO-1 cells treated with DMSO or 4-OHE2 (20 μM) for 48 h. (D) MCF-10A cells were treated with DMSO, 4-OHE2 (20 μM), or ZnPP (5 and 10 μM), separately or in combination for 24 h. n = 3; *P < 0.001. (E) The representative images of migration assay are from MDA-MB-231 cells transfected with control vector (pcDNA) or HO-1 plasmid in the absence or presence of ZnPP (10 μM) for 12 h. F-G, The anchorage-independent cell transformation assay was performed in MCF-10A or MDA-MB-231 cells as described in Material and Methods. Colonies were counted by using an inverted microscope (Nikon Diaphot 300). (F) MCF-10A-mock, MCF-10A-shNC, or MCF-10A-shHO-1 cells were treated with DMSO or 4-OHE2 (20 μM) once every 3 days for 3 weeks. Scale bars: 200 μm. n = 4; *P < 0.001. (G) MDA-MB-231 cells were treated with DMSO, 4-OHE2 (20 μM), or ZnPP (10 μM), separately or in combination. Scale bars: 200 μm. n = 4; *P < 0.001.
Figure 3
Figure 3. 4-OHE2 induces the nuclear translocation of Nrf2 and transcriptional activity of ARE
(A) MCF-10A cells treated with or without 4-OHE2 (20 μM) were harvested at the indicated intervals, and total RNA was analyzed by RT-PCR to check the level of Nrf2 mRNA. NFE2L2: Nrf2 mRNA; ACTB: β-actin mRNA. (B) MCF-10A cells were treated with E2 or 4-OHE2 (20 μM, each) for 3 h. The nuclear extracts were separated and subjected to Western blot analysis. (C) Nuclear, cytosol or whole extracts from MCF-10A cells were prepared at the indicated intervals after treatment with 4-OHE2 (20 μM), followed by Western blot analysis. (D) After the cells were treated with DMSO or 4-OHE2 (20 μM) for 3 h, immunofluorescence staining of Nrf2 was conducted as described in Materials and Methods. Scale bars: 10 μm. (E) MCF-10A cells were co-transfected with pCMV-β-galactosidase and either the luciferase reporter gene fusion construct (pTi-luciferase) or WT ARE and for 18 h, followed by treatment with DMSO or 4-OHE2 for 6 h. The cells were analyzed for the ARE transcriptional activity as described in Materials and Methods. n = 3; *P < 0.001.
Figure 4
Figure 4. 4-OHE2-induced HO-1 expression is mediated through Nrf2 activation
(A) MCF-10A cells were co-transfected with luciferase reporter plasmid construct harboring the HO-1 binding site (pGL2-HO-1) and pCMV-β-galactosidase with control siRNA or Nrf2 siRNA (20 nM) for 18 h, followed by treatment with 4-OHE2 for additional 6 h. 4-OHE2-mediated transcriptional activation of HO-1 was measured by the luciferase reporter assay as described in Materials and Methods. n = 3; *P < 0.001. (B) Cells were transfected with control siRNA or Nrf2 siRNA (20 nM) for 18 h and exposed to 4-OHE2 (20 μM) for another 6 h. The cell lysates were subjected to Western blot analysis. (C) The effect of Nrf2 on the expression of HO-1 was assessed by using embryonic fibroblasts from Nrf2 WT (Nrf2+/+) and Nrf2-null (Nrf2−/−) mice. The cells were exposed to 4-OHE2 (20 μM) for 6 h and subjected to Western blot analysis. (D) MCF-10A cells were treated with DMSO or 4-OHE2 (20 μM) for 6 h and harvested for the ChIP assay. Chromatin immunoprecipitated DNA was analyzed by PCR with primers for distal E2 (−9.0 kb region) ARE, E1 (−4.0 kb region) ARE, and two non-specific regions (NS-1 and NS-2) of the HO-1 promoter.
Figure 5
Figure 5. The thiol modification of Keap1 is responsible for 4-OHE2-induced Nrf2 activation and HO-1 expression
(A) Recombinant human Keap1 protein was reacted with DMSO or 4-OHE2 (20 μM) for 30 min, and then incubated with BPM (50 μM) for additional 30 min. The resulting protein was subjected to Western blot analysis with an anti-biotin antibody. (B) MCF-10A-Keap1 WT cells were incubated with DMSO, 4-OHE2 (20 μM), or DTT (100 μM) for 1 h and then lysed with RIPA buffer. The cell lysates were subjected to the BPM-labeling assay as described in Materials and Methods. n = 3; *P < 0.001. (C) MCF-10A cells were pre-treated with DTT (100 μM) or NEM (25 μM) for 1 h, followed by 6 h incubation with 20 μM 4-OHE2. Whole cell lysates were subjected to Western blot analysis. (D) MCF-10A cells stably expressing HA-Keap1-WT, HA-Keap1-C151S, HA-Keap1-C273S, or HA-Keap1-C288S were pre-treated with MG-132 (10 μM) for 1 h and then treated with DMSO or 4-OHE2 (20 μM). The proteins were lysed with RIPA buffer and immunoprecipitated with anti-HA antibody. The immunoprecipitated proteins were subjected to Western blot analysis with an anti-Nrf2 or anti-HA antibody. (E) MCF-10A-Keap1 WT or each mutant cells were incubated with DMSO or 4-OHE2 (20 μM) for 6 h, and the whole cell lysates were assessed by Western blot analysis. (F) MCF-10A cells were co-transfected with an expression vector for Myc-Nrf2 and an expression vector for either WT Keap1 or mutant Keap1 for 18 h. The transfected cells were treated with DMSO or 4-OHE2 (20 μM) for additional 6 h, and then cell lysates were subjected to Western blot analysis. (G) MCF-10A cells stably expressing WT Keap1 were incubated with DMSO or 4-OHE2 (20 μM) for 1 h, and the proteins were subjected to in-gel digestion. Digested peptide fragments were subjected to mass spectrometry as described in Materials and Methods.
Figure 6
Figure 6. Inhibition of HO-1 activity by ZnPP impairs 4-OHE2-induced tumorigenesis in mice
(A) The representative images of human mammary tumor (MDA-MB-231) xenografts in mice treated with vehicle, 4-OHE2, 4-OHE2 plus ZnPP, or ZnPP for 3 weeks (n = 12/group). (B) The effect of each treatment on the tumor volume of MDA-MB-231 cell xenograft was measured with digital calipers and calculated by the formula 0.52 × length × width2. Vehicle vs 4-OHE2 or 4-OHE2 vs 4-OHE2 + ZnPP; *P < 0.001 (Two-sided t-test). (C) The effects of each treatment on the tumor mass were measured. *P < 0.001 (Two-sided t-test). (D) The effect of each treatment on the expression of the indicated genes was examined by immunohistochemical analysis as described in Materials and Methods (Magnification, × 400). NC: negative control, Scale bars: 50 μm.
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