Estrogen receptor α (ERα) mediates 17β-estradiol (E2)-activated expression of HBO1

Dulbecco's modified Eagle's medium (DMEM), phenol red-free DMEM (PR-free DMEM), U0126 and 17β-estradiol (E2) were purchased from Sigma. Lipofectamine 2000, Trizol Reagent and fetal bovine serum (FBS) were purchased from Invitrogen. Charcoal-stripped fetal bovine serum (CS-FBS) was purchased from Biowest. PVDF membrane, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF) and X-tremeGENE siRNA Transfection Reagent were purchased from Roche. RNA PCR Kit (AMV) Ver.3.0 was purchased from TaKaRa. Polinl-2-plus kit was a product of GBI. Anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology. ERα siRNA, mouse monoclonal anti-ERα, anti-GAPDH, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse IgG secondary antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal anti-HBO1 antibody (Catalog No: 13751-1-AP) was purchased from Protein Tech Group, Inc. The enhanced chemiluminescence (ECL) assay kit was purchased from Tiangen. Dual-luciferase reporter assay system was bought from Promega. ICI 182,780 was purchased from Tocris Bioscience.

Human MCF-7, T47 D, MDA-MB-453 and MDA-MB-435 breast cancer cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. MCF-7 cells were cultured in DMEM supplemented with 10% fetal calf serum (FBS), 10 ug/ml of insulin, 100 U/ml of penicillin and 50 ug/ml of streptomycin. T47 D cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml of penicillin and 50 ug/ml streptomycin. SiRNA was transfected with X-tremeGENE siRNA Transfection Reagent according to the manufacture's instructions.

Proteins were detected by western blot analysis as described previously [10]. The cells were lysed by lysis buffer, separated in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was incubated with primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody. Then the membrane was developed using the ECL detection system.

112 primary breast cancer specimens were consecutively obtained from pathological archives of Huashan Hospital, Fudan University from 2005 to 2008. There was no any bias for selection. Tissues were formalin-fixed and paraffin-embedded for histopathologic diagnosis and immunohistochemical study. The malignancy degree of tumor was scored according to the Scarff-Bloom-Richardson system.

Serial sections (5 μm thick) were mounted on glass slides coated with 10% polylysine. Sections were dewaxed in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by immersion in 0.3% methanolic peroxide for 30 minutes. Next, sections were microwaved in citrate buffer for antigen retrieval. Rabbit polyclonal anti-HBO1 (1:100 dilutions) was used as primary antibodies. Staining was completed with Polink-2-plus kit, in accordance with the manufacturer's instructions. Color reaction product was developed with diaminobenzidine. All sections were counterstained with hematoxylin. Two pathologists separately blinded to the clinical outcome of the patients evaluated all samples. The immunoreactivity intensity was evaluated as 0 (absent); 1 (weak nuclear staining); 2 (moderate nuclear staining of intermediate level); 3 (more coarse nuclear staining). Positive cells were quantified as a percentage of the total number of tumor cells with observation of 1000 cells in 5 high power field (×400), and assigned to one of five categories: 0: < 5%, 1: 5-25%, 2: 26-50%, 3: 51-75% and 4: > 75%. HBO1 expression was scored semi-quantitatively using the Remmele-score (immunoreactive score (IS) = score of percentage of positive tumor cells × score of staining intensity). Then the slide of IS > 3 was classified as a positive case [11]. Reproducibility of the scoring method used by both observers was greater than 95%.

Total mRNA samples of T47 D and MCF-7 breast cancer cells were extracted using trizol reagent according to the manufacturer's instructions. One microgram of total RNA extracted from the cells was subjected to reverse transcription (RT). The RT and real-time PCR were performed by using TaKaRa RNA PCR Kit (AMV) Ver.3.0 and SYBR Premix Ex Taq II according to the manual respectively. Primers used for real-time PCR were as follows: HBO1-F 5'-GATGCCCACTGTATCATAACC-3' and HBO1-R 5'-TTCTTCCTGAGTTCAGCCACT-3'; GAPDH-F 5'-GGCTGAGAACGGGAAGCTTGTCAT-3' and GAPDH-R 5'-CAGCCTTCTCCATGGTGGTGAAGA-3'. Real-time PCR was performed using 7500 multicolor real-time PCR detection system (ABI) with the following cycling conditions: (i) 30s at 95°C and (ii) 40 cycles, with 1 cycle consisting of 5s at 95°C, 34s at 60°C. GAPDH was employed as an internal control under the same experimental conditions. Data were analyzed by using 7500 software (ABI). The values were obtained through normalizing to GAPDH copies.

Statistical data analyses were performed using SPSS 11.5 statistical software package. The relationship between protein levels and different clinical and pathological features were explored using cross tabulation and Pearson's x². P values less than 0.05 were selected.

In order to explore the biological role of HBO1 in human breast cancer in vivo, we examined the expression of HBO1 in tumor samples of primary breast cancer (n = 112) by IHC analysis. We found that HBO1 which was predominantly detected in nucleus (Figure 1B-C) was highly expressed in breast cancer tissues (Table 1) and significantly correlated with ERα (p < 0.001) and PR (p = 0.002) (Table 2). Further statistical analysis revealed HBO1 protein level correlated positively with histology grade in ERα positive tumors (p = 0.016) rather than ERα negative tumors (Table 2). For benign breast tissues, low HBO1 immunoreactivity was observed and epithelial cells displayed a minimal granular staining (Figure 1A). Moderately and poorly differentiated breast cancer tissues showed intense HBO1 staining (Figure 1B-C). To further study the relationship between HBO1 and ERα, we examined HBO1 expression level in several breast cancer cell lines by western blot, which showed that ERα positive breast cancer cell lines exhibited higher HBO1 protein than ERα negative breast cancer cell lines (Figure 1D).

In order to further investigate the relationship between ERα and HBO1, we treated breast cancer cells with 17β-estradiol (E2). Quantitative real-time PCR was used to determine the effect of E2 on the mRNA level of HBO1. With the dose of E2 increasing from 10^-9 to 10^-7 M, the mRNA level of HBO1 gradually increased and reached a plateau at 10^-8 M in T47 D cells (Figure 2A). Hereby, 10^-8 M of E2 was applied for all subsequent experiments. Western blot was also performed to confirm that E2 could upregulate HBO1 protein expression in T47 D (Figure 2B) and MCF-7 cells (Figure 2C). Taken together, these results demonstrated that E2 increased expression of HBO1 at transcriptional level.

To further study the role of ERα in HBO1 expression, ICI 182,780 was further applied in T47 D cells (Figure 3A) and MCF-7 cells (Figure 3B) to block the effect of E2. As shown in Figure 3A, E2 increased HBO1 protein expression (lane 2), which was significantly reduced by ICI 182,780 (lane 4). Similar results were obtained in MCF-7 cells (Figure 3B). ICI 182,780 was reported to act by binding ERα, causing disassociation of ERα associated proteins and resulting in impaired receptor dimerisation and increased receptor degradation [12, 13]. As expected, ERα expression was decreased by ICI 182,780 (Figure 3A and 3B). These results indicated that E2-induced HBO1 upregulation could be inhibited by ICI 182,780. In order to figure out the role of ERα in E2-induced HBO1 upregulation, ERα siRNA was transfected into T47 D and MCF7 cells to knockdown ERα. We observed that E2-induced HBO1 upregulation was inhibited by ERα siRNA (lane 4), indicating that HBO1 might be a downstream signaling molecule for ERα.

Using TRSEARCH software based on the sequence database, the promoter region of the HBO1 gene has no putative binding sites for ERα (data not shown). And previous studies have shown that steroidal estrogens are able to rapidly and transiently activate the ERK1/2 pathway [13], so we examined whether the ERK1/2 pathway was involved in the effect of E2 on HBO1 expression in breast cancer cells. As shown in Figure 4, E2-increased HBO1 protein expression was significantly suppressed by treatment with inhibitor of MEK1/2 (U0126) in T47 D (Figure 4A) and MCF-7 (Figure 4B) cells as analyzed by western blot. These results indicated that ERK1/2 signaling pathway was involved in the E2-induced HBO1 upregulation in breast cancer cells.