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. 2011 Sep 13;30(19):3962-76.
doi: 10.1038/emboj.2011.328.

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

Biswajyoti Sahu  1 , Marko Laakso, Kristian Ovaska, Tuomas Mirtti, Johan Lundin, Antti Rannikko, Anna Sankila, Juha-Pekka Turunen, Mikael Lundin, Juho Konsti, Tiina Vesterinen, Stig Nordling, Olli Kallioniemi, Sampsa Hautaniemi, Olli A Jänne
Affiliations

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

Biswajyoti Sahu et al. EMBO J. .

Abstract

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
AR and FoxA1 cistromes in LNCaP-1F5 cells and redistribution of the AR cistrome by FoxA1 depletion. (A) Consensus cis-element for FoxA1 was the most enriched DNA motif in the parental AR cistrome of LNCaP-1F5 cells. (B) Overlap between AR- and FoxA1-binding sites (FDR <2%) in LNCaP-1F5 cells. (C) Depletion of FoxA1 in LNCaP-1F5 cells treated for 72 h with siRNA specific for FoxA1 mRNA (siFoxA1) or control siRNA (parental). (D) Overlap of ARBs (FDR <2%) in parental (AR) and FoxA1-depleted (AR-siFoxA1) LNCaP-1F5 cells. (E) Class I ARBs are independent of FoxA1, despite potential colocalization of AR- and FoxA1-binding sites. The binding sites are shown for NKX3-1 and TMPRSS2 in parental and siFoxA1 cells. (F) Class II ARBs require FoxA1 as a pioneer factor. For LPAR3 and DNM1L, ARBs are present only in parental cells. (G) Class III ARBs involve FoxA1 as a repressor; AR binding occurs only in siFoxA1 cells. There are two ARB subtypes: (i) FoxA1 prevents AR recruitment to the same locus where FoxA1 is bound (EVL) and (ii) new ARBs appear at sites not previously occupied by FoxA1 (EDN2).
Figure 2
Figure 2
FoxA1 depletion in VCaP cells. (A) FoxA1 mRNA and protein levels. VCaP cells were treated for 72 h with siRNA specific for FoxA1 mRNA (siFoxA1) or control siRNA (parental). (B) Overlap of ARBs (FDR <2%) in parental and FoxA1-depleted VCaP cells. (C) Directed ChIP validation of new ARBs in parental (white bars) and FoxA1-depleted (solid bars) VCaP cells. The cells were exposed to 100 nM DHT (+) or vehicle (−) for 2 h prior to ChIP assays. Mean+s.e.m. values for duplicate samples are shown.
Figure 3
Figure 3
Dual role of FoxA1 in androgen-dependent transcription programme. (A) Venn diagram showing the numbers of androgen-regulated genes in parental and siFoxA1 cells. The cells were exposed to 100 nM DHT for 24 h, after which RNA was isolated for expression profiling by microarray using the cut-off of ⩾1.7-fold change. (B, C) Correlation between androgen-regulated (up- and down-regulated) and androgen-independent (stably expressed) genes and the incidence of binding sites unique to AR, shared by AR and FoxA1, and unique to FoxA1 within a window of ±100 kb of TSSs of the genes in parental cells (B) and in siFoxA1 cells (C). ***Significantly different (P<0.001) from stably expressed genes. (D) Unsupervised hierarchical clustering of androgen-regulated transcripts in parental and siFoxA1 cells before (Control) and after exposure to DHT. Heatmaps of biological duplicate samples are shown. (E) Violin plots (Hintze and Nelson, 1998) summarizing the changes in androgen-dependent transcripts in the three classes of genes defined by FoxA1.
Figure 4
Figure 4
Top-scoring cis-elements by de novo motif search. (A) Top-scoring cis-element for the ARBs in parental cell AR cistrome. (B) Top-scoring cis-element for the FoxA1-binding sites in parental cell FoxA1 cistrome. (C) Top-scoring cis-element for the FoxA1-independent ARBs. (D) Top-scoring cis-element for ARBs that required FoxA1 pioneering. (E) Top-scoring cis-element for the new ARBs that appeared upon FoxA1 depletion. (F) CTCF as the over-represented cis-element in the unique FoxA1-binding sites in parental cells and the new ARBs in siFoxA1 cells. (G) Top-scoring cis-element for the FoxA1-pioneered ARBs identified by an extended de novo motif search, in which the number of nucleotides in the search was 35 instead of 15 as in (A–F).
Figure 5
Figure 5
H3K4me2 marks in parental and FoxA1-depleted cells. (A) AR-/FoxA1-binding sites and H3K4me2 marks in parental and siFoxA1 cells at CLDN8 and DNM1L loci. H3K4me2 marks are shown by raw tag counts and the solid black bar shows the binding sites after peak calling on the UCSC genome browser. (B) AR-/FoxA1-binding sites and H3K4me2 marks in parental and siFoxA1 cells at FECH and HIVEP1 loci. (C) AR-/FoxA1-binding sites and H3K4me2 marks in parental and siFoxA1 cells at the ETS2 locus. AR recruitment upon FoxA1 depletion occurs concomitantly with the appearance of new H3K4me2 marks. (D) Distribution of AR-/FoxA1-binding sites and H3K4me2 marks in parental cells (left panel), siFoxA1 cells (middle panel), and in the three classes of ARBs defined by FoxA1 (right panel). H3K4me2 marks for FoxA1-independent ARBs are shown separately for parental (P) and FoxA1-depleted cells (F).
Figure 6
Figure 6
Comparison of DHS sites in parental and FoxA1-depleted LNCaP-1F5 cells. (A) DHS sites together with AR- and FoxA1-binding sites in parental and siFoxA1 cells at the NKX3-1 and DBI loci. The constitutively open sites are shown marked by red arrows. (B) DHS sites together with AR- and FoxA1-binding sites in parental and siFoxA1 cells at the EDN2 and PTGES loci. The de novo DHS sites are marked by green arrows. EDN2 and PTGES genes are androgen-regulated only in siFoxA1 cells. (C) Proportion of the DHS sites in the three ARB classes defined by FoxA1. DHS sites for FoxA1-independent ARBs are shown separately for parental (P) and FoxA1-depleted cells (F).
Figure 7
Figure 7
FoxA1 depletion in LNCaP-1F5 cells brings about redistribution of GR binding and commensurate changes in GR-dependent transcription programme. (A) Overlap analysis of GRBs (FDR <2%) in parental (GR) and FoxA1-depleted cells (GR–siFoxA1). (B) Top-scoring cis-elements, as identified by de novo motif search in different subclasses of GRBs. (C) The number of genes up- and down-regulated by Dex (Dex–up and Dex–down, respectively) exposure in parental and FoxA1-depleted (siFoxA1) LNCaP-1F5 cells.
Figure 8
Figure 8
FoxA1 protein expression in prostate cancer tissue specimens and prostate cancer-specific survival. (A) Representative FoxA1 and AR staining patterns in prostate cancer and adjacent non-cancerous tissues of two patients. (B) Correlation between FoxA1 and AR staining intensity in the prostate cancer patient cohort (n=350). (C) Disease-specific survival of 350 patients with prostate cancer according to the intensity of FoxA1 staining in the primary tumour. χ2=4.36. P=0.04 (log-rank test). (D) Disease-specific survival of patients with high AR staining classified according to FoxA1 staining in the same specimen; either negative–moderate (n=18) or high (n=222). χ2=2.57. P=0.10 (log-rank test).
Figure 9
Figure 9
Examples of prostate cancer-associated genes with altered ARBs and expression profiles upon FoxA1 depletion. Distribution of ARBs in parental (blue) and siFoxA1 (red) cells in eight loci known to encode genes that are involved in prostate cancer development and/or progression. The shaded numbers with arrows (↑, up-regulation; ↓, down-regulation; ↕, no effect) refer to fold induction of the encoded mRNA after a 24-h exposure to 100 nM DHT in LNCaP-1F5 cells, as determined by the microarray experiments.
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