A novel FOXA1/ ESR1 interacting pathway: A study of Oncomine™ breast cancer microarrays

Abstract

Forkhead box protein A1 (FOXA1) is essential for the growth and differentiation of breast epithelium, and has a favorable outcome in breast cancer (BC). Elevated FOXA1 expression in BC also facilitates hormone responsiveness in estrogen receptor (ESR)-positive BC. However, the interaction between these two pathways is not fully understood. FOXA1 and GATA binding protein 3 (GATA3) along with ESR1 expression are responsible for maintaining a luminal phenotype, thus suggesting the existence of a strong association between them. The present study utilized the Oncomine™ microarray database to identify FOXA1:ESR1 and FOXA1:ESR1:GATA3 co-expression co-regulated genes. Oncomine™ analysis revealed 115 and 79 overlapping genes clusters in FOXA1:ESR1 and FOXA1:ESR1:GATA3 microarrays, respectively. Five ESR1 direct target genes [trefoil factor 1 (TFF1/PS2), B-cell lymphoma 2 (BCL2), seven in absentia homolog 2 (SIAH2), cellular myeloblastosis viral oncogene homolog (CMYB) and progesterone receptor (PGR)] were detected in the co-expression clusters. To further investigate the role of FOXA1 in ESR1-positive cells, MCF7 cells were transfected with a FOXA1 expression plasmid, and it was observed that the direct target genes of ESR1 (PS2, BCL2, SIAH2 and PGR) were significantly regulated upon transfection. Analysis of one of these target genes, PS2, revealed the presence of two FOXA1 binding sites in the vicinity of the estrogen response element (ERE), which was confirmed by binding assays. Under estrogen stimulation, FOXA1 protein was recruited to the FOXA1 site and could also bind to the ERE site (although in minimal amounts) in the PS2 promoter. Co-transfection of FOXA1/ESR1 expression plasmids demonstrated a significantly regulation of the target genes identified in the FOXA1/ESR1 multi-arrays compared with only FOXA1 transfection, which was suggestive of a synergistic effect of ESR1 and FOXA1 on the target genes. In summary, the present study identified novel FOXA1, ESR1 and GATA3 co-expressed genes that may be involved in breast tumorigenesis.

Keywords: ChIP; Oncomine™; breast cancer; co-expression; multi-array; tumorigenesis.

Figure 1.

Analysis of overlapping FOXA1 and ESR1 co-expression genes. Venn diagrams depicting genes overlapping with FOXA1 and ESR1 with a cut-off frequency of (A) 4 (~16%) and (B) 5 (~20%) by meta-analysis with Oncomine™. (C) Pie chart of functional categories for FOXA1:ESR1 overlapping genes with a cut-off frequency of ≥5 studies. The Oncomine™ data analyzed consisted of 4 normal and 20 breast cancer microarrays data sets. FOXA1, forkhead box protein A1; ESR1, estrogen receptor 1.

Figure 2.

Analysis of overlapping FOXA1, ESR1 and GATA3 co-expression genes. (A) Pie chart of FOXA1, ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. (B) Pathway pie chart of FOXA1, ESR1 and GATA3 overlapping genes with a cut-off frequency of 4. The FOXA1 and ESR1 Oncomine™ microarray analysis consisted of 4 normal and 20 breast cancer microarray data. The GATA3 microarray data was extracted from published data by Wilson and Giguère (31). ESR1, estrogen receptor 1; FOXA1, forkhead box protein A1; GATA3, GATA binding protein 3.

Figure 3.

Gene expression analysis using RT-PCR and RT-qPCR. Several identified genes from the FOXA1:ESR1 overlapping cluster were examined following ectopic FOXA1 expression in ESR-positive MCF7 and T47D cell lines at 24 h post-transfection. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. (A) Gene expression of FOXA1:ESR1 overlapping genes using RT-PCR. (B) Gene expression of FOXA1:ESR1 overlapping genes using RT-qPCR. (C) FOXA1 overexpression following FOXA1 ectopic expression, as determined by RT-qPCR. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, vs. the control. ns, not significant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCL2, B-cell lymphoma 2; PGR, progesterone receptor; GATA3, GATA binding protein 3; FOXA1, forkhead box protein A1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CTRL, control; OE, overexpression; PS2, trefoil factor 1; SIAH2, seven in absentia homolog 2; CMYB, cellular myeloblastosis viral oncogene homolog.

Figure 4.

Schematic representation of the PS2 promoter. (A) Schematic diagram showing the presence of a functional estrogen response element (−407 nucleotide position) and two putative FOXA1 binding sites at −384 and −539 nucleotide positions, respectively. (B) In vitro binding assay. A total of 30 bp oligonucleotides containing FOXA1 binding sites were labeled with γ³²P radioisotope and incubated with nuclear lysate extracted from MCF7 cells. An unlabeled FOXA1 (cold probe) consensus sequence was used for competition at 100 and 150-fold molar excess. The reactions were subjected to electrophoresis in a 6% polyacrylamide gel at 180 V in 0.5X Tris/borate/ethylenediaminetetraacetic acid for ~1 h, and subsequently, the gel was dried and autoradiographed. (C) In vivo ChIP assay was performed for FOXA1 binding sites using an anti-FOXA1 antibody. The DNA elute from ChIP was subjected to polymerase chain reaction analysis from −321 to −464 and from −443 to −606 nucleotide positions for site 1 and site 2, respectively. FOXA1, forkhead box protein A1; ERE, estrogen response element; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; IgG, immunoglobulin G; Ntd, nucleotide; PS2, trefoil factor 1.

Figure 5.

Effect of FOXA1 and ESR1 on the PS2 promoter. For ChIP assay, MCF7 cells in the absence or presence of estradiol stimulation were sonicated, lysed and pre-cleared. ChIP with specific antibodies against FOXA1 and ESR1 along with corresponding control Ig G was performed. The nucleotide positions −573 to −315 and −506 to −344 represent the ERE and FOXA1 binding sequences, respectively, in the PS2 promoter. The eluted ChIP DNA samples were subjected to PCR analysis using ERE or FOXA1 site-specific primers. The PCR samples were electrophoresed in a 2% agarose gel. E2, estradiol; Ntd, nucleotide; IP, immunoprecipitation; IgG, immunoglobulin G; FOXA1, forkhead box protein A1; ERE, estrogen response element; ESR1, estrogen receptor 1; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; PS2, trefoil factor 1.

Figure 6.

Quantification of target genes regulated by FOXA1 and FOXA/ESR1 extracted from multi-array analysis. RT-qPCR was performed from FOXA1 and FOXA1/ESR1-transfected samples in the T47D cell line. (A) Overexpression of FOXA1 and ESR1 was confirmed by RT-qPCR in FOXA1 and FOXA1/ESR1-co-transfected cells. (B) Effect of FOXA1 and FOXA1/ESR1 transfection on the target genes (CMYB, B-cell lymphoma 2, SIAH2 and PS2) at 24 h post-transient transfection in T47D cells. The bar diagram represents data derived from triplicate experiments. *P≤0.05, **P≤0.001, ***P≤0.001, ****P≤0.0001. CTRL, control; FOXA1, forkhead box protein A1; ESR1, estrogen receptor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OE, overexpression; PS2, trefoil factor 1; SIAH2, seven in absentia homolog 2; CMYB, cellular myeloblastosis viral oncogene homolog.