FOXA1 represses the molecular phenotype of basal breast cancer cells

Abstract

Breast cancer is a heterogeneous disease that comprises multiple subtypes. Luminal subtype tumors confer a more favorable patient prognosis, which is, in part, attributed to estrogen receptor (ER)-α positivity and antihormone responsiveness. Expression of the forkhead box transcription factor, FOXA1, similarly correlates with the luminal subtype and patient survival, but is also present in a subset of ER-negative tumors. FOXA1 is also consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, breast cancer cell lines representing the basal subtype do not express FOXA1. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed expression microarray analyses on FOXA1-positive and ER-positive (MCF7, T47D), or FOXA1-positive and ER-negative (MDA-MB-453, SKBR3) luminal cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Gene set enrichment analyses revealed FOXA1 silencing causes a partial transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to a subset of both luminal and basal genes within luminal breast cancer cells, and loss of FOXA1 increases enhancer RNA transcription for a representative basal gene (CD58). These data suggest FOXA1 directly represses a subset of basal signature genes. Functionally, FOXA1 silencing increases migration and invasion of luminal cancer cells, both of which are characteristics of basal subtype cells. We conclude FOXA1 controls plasticity between basal and luminal breast cancer cells, not only by inducing luminal genes but also by repressing the basal phenotype, and thus aggressiveness. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward more aggressive cancers.

Figure 1. FOXA1 is expressed in the absence of ER in breast tumors and luminal cell lines

(A) Representative FOXA1 IHC of ER-positive (n=32) and ER-negative (n=13) breast tumor sections. FOXA1 expression (brown) is counterstained with hematoxylin. Scale bars = 100 µm. (B) FOXA1 is expressed in all ER-positive and ~50% of ER-negative tumors. ER-negative tumors express significantly less FOXA1 than ER-positive tumors (*p < 1×10−6). Scores were computed by multiplying signal intensity (1=lowest; 3=highest) by the percentage of positive cells (1=10%, 2=20%, etc.) (10). (C) Quantitative real-time PCR of FOXA1 mRNA in a diverse group of breast cancer cell lines. Bars represent the mean of three experiments (cells harvested on separate occasions) ± s.e.m. relative to GAPDH. (D) Immunoblot analysis of a cohort of breast cancer cell lines for FOXA1 and ER (BaA=Basal A; BaB=Basal B).

Figure 2. Identification of a FOXA1-dependent luminal transcriptome

(A) Representative immunoblots confirming FOXA1 knockdown (~60%–87%) with siA1#4 at 36 (n=1) and 72 hours (n=3) post-transfection. NT = non-targeting siRNA. (B) Schematic of the experimental design for each cell line. Technical replicates from each experiment were performed in triplicate and were processed for microarray analysis. Biological replicates were combined via error-weighted ANOVA. (C) Venn diagrams of commonly changed gene probes at 72 hours post-transfection (p < 0.001).

Figure 3. Loss of FOXA1 decreases enrichment for luminal genes, while increasing enrichment for basal genes

(A) Heat maps depicting expression changes of the genes within the Neve et al. (RN) (7) luminal and basal A classifier lists upon knockdown of FOXA1 (siA1#4) at 72 hours post-transfection. Genes are ordered from highest to lowest classification power. A propensity of red or green is indicative of a directional shift in global expression of the gene classifier. (B) GSEA enrichment plots utilizing a subset of the luminal and basal discriminatory gene sets generated by Neve et al. (RN) (7) and Charafe-Jauffret et al. (ECJ) (8). Vertical lines represent individual genes of the respective classifier that contribute to the enrichment score. Genes are ranked by signal to noise ratio: left (most positive) to right (most negative). Values below 0 indicate reduced enrichment of a signature gene set, while values above 0 indicate a gain in enrichment.

Figure 4. Loss of FOXA1 induces basal mRNA and protein expression

(A) MCF7, (B) T47D, (C) MB-453, and (D) SKBR3 cells were transiently transfected with non-targeting (NT) or siRNA targeting FOXA1 (siA1#4). (Left) Quantitation of mRNA changes for a subset of differentially expressed luminal and basal classifying genes at 72 hours post-transfection using real time RT-PCR. Bars represent the mean of three experiments ± s.e.m. relative to GAPDH (*p < 0.05). (Right) Representative immunoblots at 72 hours post-transfection showing induction of the basal protein, Annexin 1, in response to FOXA1 silencing (n=3).

Figure 5. FOXA1 transcriptionally regulates luminal and basal gene expression in luminal breast cancer cells

(A–C) FOXA1 ChIP of a subset of basal and luminal genes in MCF7 and MB-453 cells. (A) Representative images and (B–C) quantitation of FOXA1 ChIP. Bars represent the fold change in binding relative to normal goat IgG ± s.e.m. (n=3) where the Y-axis is log2. Genes underlined represent sites predicted to bind FOXA1 in a previously published MCF7 ChIP-chip dataset. The asterisk represents a site predicted in a previously published MB-453 ChIP-seq dataset. The remaining sites were either predicted to bind in both MCF7 and MB-453 datasets, or are regions surrounding consensus elements within the gene promoter (see Table 3). Genes listed twice represent independent binding locations for that gene. (D) Quantitation of eRNA transcripts generated upstream and downstream of the common FOXA1 binding site in CD58 at 72 hours post-transfection with non-targeting (NT) or siRNA targeting FOXA1 (siA1#4). A region ~10 kilobases upstream of CD58 not bound by FOXA1 in previously published datasets was used as a negative control (NC). See Supplementary Figure 6 for a schematic of the associated binding sites and primer locations for CD58. Bars represent the mean of three experiments ± s.e.m. relative to GAPDH (*p < 0.05).

Figure 6. Loss of FOXA1 increases migration and invasion of luminal breast cancer cells

(A–D) MCF7 and MB-453 cells were transiently transfected with non-targeting (NT) or siRNA targeting FOXA1 (siA1#1). At 48 hours post-transfection, cells were plated in modified Boyden chambers to analyze (A) migration at 24 hours or (B) invasion at 48 hours. (C) Number of viable cells (trypan blue excluded) at 48 hours post-transfection. Bars in A–C represent the mean of three experiments ± s.e.m. relative to NT (*p < 0.05; **p < 0.01). (D) Representative immunoblots confirming knockdown of FOXA1 with siA1#1 at 72 hours post-transfection (MCF7, n=3; MB-453, n=2).