Association of double-positive FOXA1 and FOXP1 immunoreactivities with favorable prognosis of tamoxifen-treated breast cancer patients

Abstract

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of the steroid hormones estrogen and progesterone. Forkhead box A1 (FOXA1) is a member of the forkhead box transcription factor family and functions as a pioneer factor of the estrogen receptor (ER) in breast cancer. In the present study, we demonstrate that FOXA1 mRNA was upregulated by estrogen and that estrogen receptor-α (ERα) recruitment to ER-binding sites in the vicinity of the FOXA1 gene was increased by estrogen in ERα-positive MCF-7 breast cancer cells. The estrogen-induced FOXA1 upregulation was repressed by 4-hydroxytamoxifen treatment. We also demonstrated that the proliferation and the migration of MCF-7 cells were decreased by FOXA1-specific small interfering RNA (siRNA; siFOXA1). Furthermore, siFOXA1 decreased the estrogen response element-driven transcription and the estrogen-dependent upregulation of ERα target genes in MCF-7 cells. Next, the immunohistochemical analyses of FOXA1 were performed using two groups of breast cancer specimens. The nuclear immunoreactivity of FOXA1 was detected in 80 (74%) of 108 human invasive breast cancers and was negatively correlated with tumor grade and positively correlated with hormone receptor status, including ERα and progesterone receptor, pathological tumor size, and immunoreactivity of FOXP1, another FOX family transcription factor. FOXA1 immunoreactivity was significantly elevated in the relapse-free breast cancer patients treated with tamoxifen. Notably, the double-positive immunoreactivities of FOXA1 and FOXP1 were significantly associated with a favorable prognosis for the relapse-free and overall survival of patients with tamoxifen-treated breast cancer, with lower P values compared with FOXA1 or FOXP1 immunoreactivity alone. These results suggest that FOXA1 plays an important role in the proliferation and migration of breast cancer cells by modulating estrogen signaling and that the double-positive immunoreactivities of FOXA1 and FOXP1 are associated with a favorable prognosis of tamoxifen-treated breast cancer.

Fig. 1

Estrogen-induced expression of FOXA1 in the MCF-7 cells. a, b MCF-7 cells were treated with 100 nM 17β-estradiol (E₂) (a) or with 100 nM E₂ and 1 nM 4-OHT (b) for 48 h. The FOXA1 mRNA level was examined at indicated time points by qRT-PCR; the results are shown as a fold change over the expression level at 0 h. ***P < 0.001 compared with 0 h (by Student’s t test). c Schematic representation of estrogen receptor-binding sites (ERBSs) in the vicinity of FOXA1 gene in chromosome 14. Cytogenetic location of the FOXA1 gene in chromosome 14 and UCSC Genome Browser view customized with estrogen-dependent ERα binding sites (ERBSs) determined by ChIP-on-chip at the 14q region [26] are shown. Two significant ERBSs (ER_8127 and 8128) at a cutoff value P < 1e−3 are identified in the vicinity of FOXA1 gene region. d Estrogen-dependent recruitment of ERα to the FOXA1 ERBSs. The MCF-7 cells were treated with 100 nM of E₂ or vehicle for 45 min and then subjected to ChIP analysis with the ERα-specific antibody. Immunoprecipitated DNA was quantified by qPCR; the results were represented as the fold enrichment of ERα occupancy in the immunoprecipitated DNAs vs. input DNAs. The estrogen response element of the Trefoil factor 1 (TFF1) gene (TFF1 ERE) and non-ERBS within the myoglobin (MB) gene region were used as the positive and negative controls, respectively

Fig. 2

Knockdown of FOXA1 suppresses cell proliferation and migration of the MCF-7 cells. a Total RNA was prepared from the MCF-7 cells transfected with 20 nM of siRNA specific for FOXA1 (siFOXA1) or control siRNA (siControl) for 48 h; the FOXA1 mRNA level was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ***P < 0.001 compared with the siControl (Student’s t test). b MCF-7 cells were transfected with 20 nM of siFOXA1 or siControl for 4 days in Dulbecco’s modified Eagle medium (DMEM). The relative cell proliferation at indicated time points was examined using a WST-8 assay kit and determined photometrically as the absorbance at OD = 450 nm. ***P < 0.001 compared with the siControl (Student’s t test). c, d The MCF-7 cells were transfected with 20 nM of siFOXA1 or siControl for 48 h in DMEM, and then the cells were transferred into transwells for an additional 48 h. The representative photographs (c) and numbers of migrated cells (d) are shown. e Effect of FOXA1 on estrogen response element (ERE)-mediated transcription in the MCF-7 cells. The MCF-7 cells were transfected with a DNA mixture of 0.1 μg of ERE-TK-LUC, 0.02 μg of pRL-CMV, and 20 nM of siFOXA1 or siControl. After 12 h, the cells were treated with 17β-estradiol (100 nM) or vehicle (EtOH) for 24 h, and then the cell lysates were prepared for and used in the luciferase assay. f, g Effect of siFOXA1 on the expression of ERα target genes. MCF-7 cells were transfected with 20 nM of siFOXA1 or siControl. After 12 h, cells were treated with E₂, 4-OHT, or EtOH for 24 h. mRNA levels of progesterone receptor (PgR) (f) and growth regulation by estrogen in breast cancer 1 (GREB1) (g) were examined by qRT-PCR; the results are shown as a fold change over EtOH treatment

Fig. 3

Immunohistochemistry of FOXA1 in breast cancer. The representative images of the immunohistochemical staining of breast cancer tissues with the anti-FOXA1 antibody are shown. Positive staining for FOXA1 was observed in the nuclei of the breast cancer cells. The FOXA1 immunoreactivities in grade III breast cancer were significantly lower than those in grade I breast cancer (5.83 vs. 2.38, P = 0.002). Bar, 100 μm

Fig. 4

Relapse-free survival and overall survival according to the FOXA1 and/or FOXP1 immunoreactivities in the patients with tamoxifen-treated breast cancer with or without distant metastasis within 5 years after surgery or entire follow-up period (n = 113). The Kaplan–Meier survival curves were plotted using JMP 9 software, and the statistical significances were determined using the log-rank test. a, b Patients with the positive FOXA1 immunoreactivity showed good relapse-free survival and overall survival (a and b, respectively). c, d Relapse-free survival and overall survival according to the FOXP1 immunoreactivity in the patients with tamoxifen-treated breast cancer with or without distant metastasis within 5 years after surgery or entire follow-up period. Patients with positive FOXP1 immunoreactivity showed good relapse-free survival and overall survival (c and d, respectively). e, f Patients with double-positive FOXA1 and FOXP1 immunoreactivities showed better relapse-free survival and overall survival (e and f, respectively)