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. 2016 Oct 25;113(43):E6600-E6609.
doi: 10.1073/pnas.1612835113. Epub 2016 Oct 6.

FOXA1 Overexpression Mediates Endocrine Resistance by Altering the ER Transcriptome and IL-8 Expression in ER-positive Breast Cancer

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Free PMC article

FOXA1 Overexpression Mediates Endocrine Resistance by Altering the ER Transcriptome and IL-8 Expression in ER-positive Breast Cancer

Xiaoyong Fu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)-chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Keywords: FOXA1; breast cancer; endocrine resistance; estrogen receptor; transcriptional reprogramming.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
FOXA1 gene amplification in preclinical ER+ Endo-R cell models. (A) Overall CN across the genome for MCF7L-TamR related to P cells. Log2(CN ratio) is shown on the vertical axis. Each point represents the log-transformed CN ratio for each targeted exon, ordered by genomic coordinates and colored by chromosome using red and blue for subsequent chromosomes. Black lines show inferred segments. The arrows point to the segment with the highest focal amplification exons. (B) Zoom into the 60-Mb region containing FOXA1 in A. CN ratios for the four FOXA1-targeted exons are shown as purple squares, and the remaining targeted exons are red circles. The segment containing FOXA1 contains just the four targeted exons corresponding to FOXA1. (C) Representative images of bright-field and the FOXA1-FISH of MCF7L-P and TamR cells show the enrichment of gene amplification in MCF7L-TamR vs. P cells. Green and red signals indicate the locations where FOXA1 and chromosome 14 centromere reference (REF) probes were hybridized, respectively. [Scale bar, 100 μm (bright field) and 20 μm (FISH).] (D) Stacked bar chart summarizes the percentage of cells (n = 65) with FOXA1/REF foci ratio within indicated ranges. (E) Normalized FOXA1-CN values from multiple Endo-R cells were calculated from the results of a real-time gPCR assay. The normal diploid MCF10A cell line was used as the normalization control (CN = 2, marked by a dashed line). (F) Box-whisker plots show the FOXA1-CN across the five molecular subtypes of breast cancer (n = 831) in the TCGA dataset (24).
Fig. S1.
Fig. S1.
Cell growth of multiple Endo-R cell models. (AE) Prestarved cells cultured with PRF medium and 5% CS-FBS were treated with E2 (control), ED, or Tam for indicated days. Cell numbers were either counted by an in situ cytometer (Celigo) or quantified with a colorimetric assay. **P < 0.01, ***P < 0.001, unpaired two-sided t test for all of the comparisons between E2-treated and antiestrogen-treated cells.
Fig. S2.
Fig. S2.
FOXA1 amplification and CN in breast cancer cell lines. (A) Overall CN across the genome for MCF7RN-TamR related to P cells. Log2(CN ratio) is shown on the vertical axis. Each point represents the log-transformed CN ratio for each targeted exon, ordered by genomic coordinates and colored by chromosome using red and blue for subsequent chromosomes. Black lines show inferred segments. The arrows point to the segment with the highest focal amplification exons. (B) Zoom into the 60-Mb region containing FOXA1 in A. CN ratios for the four FOXA1-targeted exons are shown as purple squares, and the remaining targeted exons are red circles. The segment containing FOXA1 contains just the four targeted exons corresponding to FOXA1. (C) Representative images of bright field and FOXA1-FISH in MCF7RN-P and TamR cells. [Scale bar, 100 μm (bright field) and 20 μm (FISH)]. (D) Stacked bar chart summarizes the percentage of cells (n = 65) with FOXA1/REF foci ratio within indicated ranges. (E) Normalized FOXA1-CN [log2(CN/2)] across a panel of breast cancer cell lines (n = 59) from the Cancer Cell Line Encyclopedia (20). Base line (y axis at 0) indicates the CN of 2. ER+ cell lines are in red.
Fig. S3.
Fig. S3.
FOXA1 mRNA and CN values in breast tumors. (A) Box-whisker plots show the FOXA1 mRNA levels (log2) across the five molecular subtypes of breast cancer (n = 845) in the TCGA dataset (24). ****P < 0.0001, Bonferroni post hoc test (multiple testing corrected). (B) FOXA1-CN was derived from a study of whole-genome array-based comparative genomic hybridization (GEO accession no. GSE56765) (26). (C) FOXA1-CN was compared between primary breast luminal tumors (n = 22) and their matched lymph-node metastases (from B), using the two-tailed paired t test.
Fig. 2.
Fig. 2.
Increased FOXA1 expression in multiple Endo-R cell models. (A) FOXA1 mRNA levels determined by qRT-PCR across various Endo-R cell models. The amount of FOXA1 mRNA from MCF7L-P cells was set as normalization control (= 1). (B) FOXA1, ER, and PR protein expression using selected antibodies in Western blot analysis across various Endo-R cell models. GAPDH was used as a loading control. (C) Scatter dot plots of FOXA1 Allred score in MCF7L Endo-R xenograft tumors measured by IHC. Xenograft tumors in ovariectomized nude mice with estrogen pellets (E2), or without E2 but treated with Tam or without Tam (ED), were harvested when the tumor volume reached 1,000 mm3. Data are represented as mean ± SEM (n > 5 in each group). ***P < 0.001, Bonferroni post hoc test (multiple testing-corrected). (D) Western blots showing the protein levels of FOXA1 and ER in MCF7L-TamR cells with gene knockdown. (EH) Cell growth within a 6-d period after siRNA knockdown of nonspecific (N.S.) target, ER, or FOXA1 in MCF7L, MCF7RN, ZR75-1, and 600MPE Endo-R cell models. Cell growth in N.S. knockdown was used as normalization control (100%). Data represent means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two-sided t test for all comparisons between N.S. and ER/FOXA1 knockdown.
Fig. S4.
Fig. S4.
Protein expression of ER and its classical regulated genes. (AC) Bar charts show the protein levels measured by RPPA assays using cell lysates from three Endo-R cell models. (D) Bar charts show the protein levels in MCF7L/FOXA1 cells ± Dox for 2 or 5 d. Protein levels were presented as means ± SEM from biologically triplicated samples with triplicated loading dots in the array (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-sided t test to compare the expression between P and Endo-R cells, and between +Dox and −Dox cells.
Fig. 3.
Fig. 3.
FOXA1 overexpression is associated with endocrine resistance and poor clinical outcome. (A) FOXA1 IHC of MCF7L-P and TamR, and MCF7L/FOXA1 ± Dox cells. (Scale bar, 100 µm.) (B) Heat map of differentially expressed genes (|Gfold| >1.5) after FOXA1 overexpression in MCF7L/FOXA1 cells. The levels of ER protein in ± Dox cells are shown at the bottom panel. DAVID functional annotation shows the enriched GO terms for the genes up- or down-regulated upon FOXA1 overexpression. (C) GSEA shows the correlation of the gene expression profile of MCF7L/FOXA1 cells with a gene set derived from the Endo-R xenograft tumors (3). (D) Spearman correlation of FOXA1 mRNA levels and the signature scores of the Endo-R gene set in ER+ breast tumors (n = 752). (E and F) Cell growth was measured in MCF7L/FOXA1 and ZR75-1/FOXA1 cells with 0, 0.25, or 1 µg/mL Dox in presence of E2 or various endocrine therapies. E2-treated cells were used as normalization controls for anti-estrogen groups (ED, Tam, and Ful). The induced FOXA1 proteins are shown by Western blots. Data represent means ± SEM, *P < 0.05, **P < 0.01, two-sided t test for all indicated comparisons. (G) Kaplan–Meier plots show RFS in ER+ patients receiving Tam but without chemotherapy (n = 615), who were stratified by FOXA1 mRNA levels at the top quartile (25%) vs. the rest (75%). (H) Same analysis in ER+ patients without endocrine treatment (n = 500). P value was calculated by using the log-rank test. Analysis was performed using an online tool and resource at kmplot.com/analysis (33).
Fig. 4.
Fig. 4.
FOXA1 overexpression in ER+ breast cancer cell lines induces proteomic perturbations in multiple oncogenic signaling pathways. Heat maps of RPPA data representing differentially expressed proteins (one-way ANOVA, P < 0.05) in MCF7L/FOXA1 (A), ZR75-1/FOXA1 (B), and 600MPE/FOXA1 (C) cells upon Dox addition for 2 or 5 d. (DF) Signaling perturbations by FOXA1 overexpression in 14 cancer-related KEGG pathways were evaluated in these cell models of A, B, and C, by averaging the expression levels of proteins available from RPPA (numbers in parentheses) within the same pathway, followed by subtraction of basal levels in −Dox cells. A paired one-sided t test was applied and the P value was plotted as minus log10-transformed. The perturbations in pathways with P < 0.05 (P = 0.05 is marked by a dashed line) were statistically significant. Venn diagrams showing the overlapping proteins up- (G) or down-regulated (H) across all three cell models upon FOXA1 overexpression (day 5 of +Dox vs. −Dox, P < 0.05). (I) Enriched KEGG pathways represented by the commonly up-regulated proteins in all three cell models. Significance in enrichment was calculated by Fisher’s exact test (P = 0.05 is marked by a dashed line).
Fig. S5.
Fig. S5.
Inverse correlation of proteomic profiles by FOXA1 alteration in MCF7L-P and TamR cells. (A) MCF7L cells with inducible YFP control or FOXA1 overexpression were harvested for protein lysates after 3-d incubation under different doses of Dox. Western blotting was performed using the indicated antibodies. (B) Heat map of the 89 clustered proteins measured by RPPA that were differentially expressed (one-way ANOVA, P < 0.05) in the MCF7L-TamR cells with FOXA1 vs. N.S. (control) knockdown. Arrows indicate the proteins PR and GATA3. (C) Overall comparison of proteomic changes in signaling pathways between the MCF7L/FOXA1 ± Dox cells and the MCF7L-TamR/si-FOXA1 cells. Proteins were assigned to 13 cancer-related pathways defined by KEGG (34). The protein signals in each pathway (protein number in parentheses) were averaged and subtracted by those from the controls, representing the pathway perturbations scaled by the left y axis. A paired one-sided t test was applied to assess pathway perturbations in MCF7L/FOXA1 cells with FOXA1 overexpression and in MCF7L-TamR cells with FOXA1 knockdown. The minus log10-transformed P value was scaled by the right y axis. The gray dashed line marks the P value at 0.05. (D) Pearson correlation was used to assess the concordance of pathway proteomic changes (represented by t statistics) between MCF7L-P and TamR cells with the corresponding FOXA1 manipulations (overexpression in P and knockdown in TamR cells).
Fig. 5.
Fig. 5.
Integrative analysis revealed IL8 as a target of increased FOXA1 in ER transcriptional reprogramming. (A) Integrated RNA-seq and FOXA1 ChIP-seq data in MCF7L-P and TamR cells. Genes aligned in RNA-seq were calculated for their expression log2 ratio of fragments per kilobase of transcript per million mapped reads (FPKM) in TamR vs. P cells, by which the genes were sorted in a descending order. FOXA1 binding events (tags) within ± 20 kb of each gene’s TSS were counted and represented by average normalized RPM for every 300 consecutive genes along the order of sorted genes from RNA-seq. These FOXA1 tags were plotted separately for P (in blue) and TamR (in red) cells. (B) Heat maps of genes with high expression [log2(FPKM)] and with enriched FOXA1 binding (log2 ratio of RPM) in TamR vs. P cells. Heat maps of the expression in these genes (log2 ratio of FPKM) in MCF7L/FOXA1 +Dox vs. −Dox cells and in TamR cells with si-ER vs. si-N.S. knockdown are also shown. (C) Venn diagram showing the overlap genes, including IL8, CTGF, and LOX, between the FOXA1-overexpression (O.E.) up-regulated genes and the MCF7L-TamR signature genes. P value was calculated by Fisher’s exact test. (D) IL-8 gene expression measured by qRT-PCR in four Endo-R cell models. Data represent means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two-sided t test for all comparisons between Endo-R and P cells. (E) Venn diagram showing the overlap among the predicted genes with the binding of FOXA1 and EGF/E2-stimulated ER within ± 20 kb of TSS in MCF7 cells. The gene sets with highlighted numbers (in red) were used for the following analysis. (F) The genes induced (UP) or not altered (NA) by FOXA1 overexpression were intersected with the FOXA1 and ER cistromes. Gene enrichment within the FOXA1-UP gene set for the genes associated with FOXA1 binding and ER binding induced only by EGF, E2, or both was compared with the enrichment within the FOXA1-NA gene set. ***P < 0.001, Fisher’s exact test.
Fig. S6.
Fig. S6.
Cistromic profiling of FOXA1 in MCF7L-P and TamR cells. (A) Venn diagram showing the overlap of FOXA1 binding events between MCF7L-P and TamR cells. (B) Enriched motifs in the differential FOXA1 binding events in MCF7L-P and TamR cells.
Fig. 6.
Fig. 6.
Increased FOXA1 and ER regulate IL-8 expression in ER+ breast cancer. (A) Schematic diagram of ER and FOXA1 binding within the IL8 gene locus as defined by EGF-stimulated ER ChIP-on-chip (37) and FOXA1 ChIP-sEq. (7) in MCF7 cells. (B) Snapshot of FOXA1 continuous peaks from ChIP-seq data showing the binding pattern upstream of the IL8 gene TSS in MCF7L-P and MCF7L-TamR cells treated with Tam or E2. (C) FOXA1-ChIP (Left) and ER-ChIP (Right) followed by qPCR of binding regions in MCF7L-P and TamR cells. Quantification of amplified binding regions was calculated as fold enrichment by normalizing to an intergenic sequence as a negative control. (D and E) Measurement of IL-8 mRNA by qRT-PCR in MCF7L-TamR and 600MPE-TamR cells with either ER or FOXA1 knockdown. N.S., nonspecific; #1 and #2, two different siRNA sequences. (F and G) ELISA of IL-8 protein in culture media of MCF7L/FOXA1 and 600MPE/FOXA1 cells −/+Dox in the absence/presence of ER knockdown. (H) Representative H&E staining and IL-8 IHC images from E2-treated and Endo-R MCF7L xenograft tumors. (Scale bars, 100 μm and 50 μm, respectively.) (I) Scatter dot plots of IL-8 Allred score in H. Data represent means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two-sided t test for indicated comparisons. (J) Representative IHC images from two ER+ tumors showing low (#1) vs. high (#2) FOXA1 and the negative vs. positive IL-8 staining, respectively. (Scale bar, 50 μm.) (K) Proportions of positive vs. negative IL-8 tumors within the groups of tumors showing the same FOXA1 Allred score (AS). Correlation of IL-8 positivity and FOXA1-AS was evaluated by Fisher’s exact test.
Fig. S7.
Fig. S7.
Altered ER, PR, and IL-8 protein levels in Endo-R models. (A) Representative ER and PR IHC images from E2-treated and Endo-R MCF7L xenograft tumors. (Scale bar, 200 μm.) (B) Scatter dot plots of ER and PR Allred score in A. Data represent means ± SEM **P < 0.01, ***P < 0.001, two-sided t test for indicated comparisons. (C) MCF7L-P and TamR cells were processed for IL-8 immunofluorescence staining. DAPI was used for nucleus counterstaining. (Scale bar, 100 μm.)
Fig. 7.
Fig. 7.
IL-8 mediates the effect of FOXA1 on cell growth and invasion in endocrine resistance. (A) Cell growth within 5 d in MCF7L-P and TamR cells with IL-8 knockdown by two different sequences. N.S. knockdown was used as normalization control. (B) A stable MCF7L-TamR/IL-8 cell line was established to express Dox-inducible IL-8, encoded by an IL8 cDNA without 3′-UTR sequence. Two different IL8 siRNA sequences, targeting either the IL8 coding DNA sequence (#1) or the 3′-UTR region (#2), were transiently transfected into MCF7L-TamR/IL-8 cells ± Dox at two different doses. A 6-d cell growth measurement was performed using methylene blue staining. Cell growth under N.S. knockdown was used as the normalization control. (C) Western blots of GFR downstream signaling mediators in MCF7L-TamR cells with siRNA knockdown of N.S. or IL-8. (D and E) Measurement of IL-8 and FOXA1 mRNA by qRT-PCR in MCF7L cell lines with inducible FOXA1 overexpression, or concomitant IL-8 knockdown under ± Dox. (F) Cell growth within 7 d in four MCF7L lines with Dox induction (0.5 μg/mL), treated with E2 (as control) or antiestrogen (ED, Tam, or Ful). Cell growth in the E2 group was set as 100%. (G) Cell invasion measurement in MCF7L-P and TamR cells transfected with N.S. or IL-8–targeting siRNAs. Cells were seeded onto Matrigel-coated, 24-well Transwell plates and cultured for 48 h. The invading cells were counted under a microscope for a total of nine random fields. Data are presented as mean number of cells per field. (H) Cell invasion measurement for MCF7L/FOXA1 cells ± Dox with siRNA knockdown of N.S. or IL-8. Cell invasiveness was evaluated as above. Data represent means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two-sided t test for indicated comparisons.
Fig. S8.
Fig. S8.
Altered transcriptome and proteome upon FOXA1 or IL-8 perturbation. (A) Heat map of the expression of differentially expressed genes (|Gfold| >0.5) in the MCF7L-TamR cells with FOXA1 knockdown, aligned with the expression of the same genes in the MCF7L-TamR cells with IL-8 knockdown. Pearson correlation analysis was performed for the altered expression levels of genes upon FOXA1 knockdown vs. the altered levels of the same genes upon IL-8 knockdown in MCF7L-TamR cells. (B) Heat map of partial RPPA data (centered log2) of the MCF7L/FOXA1 +Dox cells with siRNA knockdown of N.S. or IL-8. Proteins were chosen based on the previous RPPA data showing the high expression levels in MCF7L/FOXA1 +Dox vs. −Dox cells (log2 ratio >0, Right).
Fig. S9.
Fig. S9.
Cell invasion upon FOXA1 or IL-8 knockdown. (A) Cell invasion measurement in MCF7L-TamR cells with inducible FOXA1 knockdown. Cells at the same number were seeded onto Matrigel-coated 24-well Transwell plates for 48 h. Invading cells were stained and counted under a microscope. (B) Cell invasion in 600MPE-P and TamR cells transfected with N.S. or IL-8-targeting siRNAs. (C) Cell invasion in 600MPE/FOXA1 cells ± Dox with siRNA knockdown of N.S. or IL-8. Data are presented as means ± SEM of invading cells per field from a total of nine random observed fields. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided t test for indicated comparisons.

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