FOXP1, an estrogen-inducible transcription factor, modulates cell proliferation in breast cancer cells and 5-year recurrence-free survival of patients with tamoxifen-treated breast cancer

Abstract

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of steroid hormones, including estrogen and progesterone. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. In the present study, we investigated the expression of FOXP1 in 133 human invasive breast cancers, obtained by core biopsy, by immunohistochemical analysis. Nuclear immunoreactivity of FOXP1 was detected in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including estrogen receptor alpha (ERα) and progesterone receptor, and negatively with pathological tumor size. In ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and increased ERα recruitment to ER binding sites identified by ChIP-on-chip analysis within the FOXP1 gene region. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Furthermore, FOXP1 enhanced estrogen response element-driven transcription in MCF-7 cells. Finally, FOXP1 immunoreactivity was significantly elevated in relapse-free breast cancer patients treated with tamoxifen. These results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity could be associated with the estrogen dependency of clinical breast cancers, which may predict favorable prognosis in the patients treated with tamoxifen.

Fig. 1

Immunohistochemistry of FOXP1 in breast cancer. Representative immunohistochemical staining of breast cancer tissues (a–d) and kidney (e, f) with anti-FOXP1 antibody (a, c, and e) or rabbit IgG (b, d, and f). Positive staining for FOXP1 was observed in the nuclei of breast cancer cells and kidney tubule cells. Immunoreactivity for FOXP1 tended to be strong in grade I breast cancer (a; total score, 8) and weaker in grade III breast cancer (c; total score, 3). Bar, 100 μm

Fig. 2

Transcriptional regulation of FOXP1 by estrogen in MCF-7 cells. a, b MCF-7 cells were treated with 100 nM 17β-estradiol (E2) for 48 h. Expression levels of FOXP1 mRNA (a) and protein (b) were examined at indicated time points by qRT-PCR and western blotting, respectively. The mRNA expression levels are shown as 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 the FOXP1 gene region in UCSC genome browser. Three ER binding sites (ERBSs) within the FOXP1 gene region were found by genome-wide ChIP-on-chip analysis [3]. d Estrogen-dependent recruitment of ERα to the FOXP1 ERBSs. MCF-7 cells were treated with 100 nM of E2 or vehicle for 45 min and then subjected to ChIP analysis with ERα-specific antibody. Immunoprecipitated DNA was examined by qPCR, and fold enrichments relative to vehicle control were plotted. The estrogen response element of the TFF1 gene (TFF1 ERE) was used as a positive control [19]

Fig. 3

FOXP1 promotes estrogen-dependent proliferation of MCF-7 cells. a Total cell lysates from MCF-7 cells transfected with pcDNA3 or pcDNA3-FOXP1-Myc (Myc-FOXP1) for 1, 2, and 4 days were immunoblotted with anti-FOXP1 or anti-β-actin antibodies. b MCF-7 cells were transfected with pcDNA3-FOXP1-Myc for 24 h and then treated with 100 nM 17β-estradiol (E2) for 4 days. Relative cell proliferation at indicated time points was examined using a WST-8 assay kit. **P < 0.01 compared with control vector; ***P < 0.001 compared with control vector (Student’s t test). c Total RNA from MCF-7 cells transfected with siRNA specific for FOXP1 (siFOXP1) or luciferase (siLuc) for 48 h was examined by qRT-PCR (upper). *P < 0.05 compared with control siRNA (siLuc; Student’s t test). Total cell lysates from MCF-7 cells transfected with siFOXP1 or siLuc for 1, 2, and 4 days were immunoblotted with anti-FOXP1 or anti-β-actin antibodies (lower). d MCF-7 cells were transfected with siRNA specific for FOXP1 or luciferase and then treated with 100 nM 17β-estradiol (E2) for 4 days. Relative cell proliferation at indicated time points was examined as b. *P < 0.05 compared with control siRNA (siLuc; Student’s t test)

Fig. 4

Effect of FOXP1 on ERE-mediated transcription and ER-target gene expressions in MCF-7 cells. a MCF-7 cells were transfected with a DNA mixture of 0.1 μg of estrogen response element (ERE)-tk-Luc, 0.02 μg of pRL-CMV, and increasing amounts of pcDNA3-FOXP1-Myc (Myc-FOXP1) or pcDNA3-Flag-FOXA1 (FOXA1). After a 12-h incubation, cells were treated with 17β-estradiol (E2; 100 nM) or vehicle (EtOH) for 24 h and then cell lysates were examined by luciferase assay. *P < 0.05; **P < 0.01; and ***P < 0.001 compared with control vector (pcDNA3) transfection (Student’s t test). b, c MCF-7 cells were transfected with pcDNA3 or pcDNA3-FOXP1-Myc (Myc-FOXP1) for 12 h and treated with E2 (100 nM) or EtOH for 24 h and then expression levels of SHP (b) and LRH-1 (c) were examined by qRT-PCR. *P < 0.05; ***P < 0.001 compared with control pcDNA3 (Student’s t test)