ARv7 Represses Tumor-Suppressor Genes in Castration-Resistant Prostate Cancer

Abstract

Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.

Keywords: AR variant v7 (ARv7); androgen receptor (AR); castration-resistant prostate cancer (CRPC); transcription.

Figure 1.. LNCaP95 Cell Growth Is Dependent on both ARfl and ARv7

(A) Top: schematic of the full-length AR (ARfl) and the AR variant 7 (ARv7), with the N-terminal domain (NTD), DNA-binding domain (DBD), hinge region (H), ligand-binding domain (LBD), and cryptic exon (CE3). Bottom: western blot of shGFP, shARv7, or shARfl LNCaP cells using an N-terminal, pan-AR antibody. Actin signals serve as a loading control. (B) Proliferation assay of indicated cells grown in 2D culture. Data are the mean of three independent experiments ±SEM. (C) Representative scanning electron microscopy images of LNCaP95 cells after 7 days of growth in 3D/PEGda cryogels. Scale bar, 100 mm. (D) Quantification of 3D cell growth data in (C). Data are the mean of four independent experiments relative to day 0 ±SEM. **p % 0.01; ***p % 0.001; ****p % 0.0001 by Student’s t test. See also Figure S1.

Figure 2.. ARv7, Unlike ARfl, Functions as a Transcriptional Repressor in CRPC Cells

(A and B) Volcano plots of differentially expressed genes in shARv7 (A) or shARfl (B) cells, compared with shGFP control. Significantly altered genes (fold change >±1.5; adjusted p value <0.05) are highlighted in red (activated) or blue (repressed). Select AR targets and significant outliers are labeled. (C) Top: violin plots of log₂ fold changes of ARv7-regulated (blue) and ARfl-regulated (red) genes (relative to the shGFP control) in response to DHT stimulation. Only genes with an adjusted p value of <0.05 are shown. Bottom: bar plots of the mean log₂ fold changes of the ARv7-regulated (blue) and ARfl-regulated (red) target genes as above. ****p % 0.0001 by ANOVA and Tukey’s honest significant difference test (HSD). (D) Comparison of log₂ fold changes of significantly dysregulated genes (adjusted p value <0.05) in response to shARv7 or shARfl (as defined in A and B). Colors indicate genes primarily dysregulated by shARfl (red), shARv7 (blue), or both (purple). Select classical AR targets are labeled. See also Figure S2; Tables S1 and S2.

Figure 3.. ARfl and ARv7 Binding to Chromatin Is Interdependent

(A) Euler diagram of the overlap (R1 bp) of ARv7 (blue) and ARfl (red) cistromes, profiled in the absence of hormone (ETOH) or after 4 h of DHT (10 nM) treatment. The numbers of unique and overlapping binding sites are indicated. (B) Top: Euler diagram of the overlap of ARv7 (blue), ARfl (red), and AR N-terminal cistromes (ARN; purple) in the absence of hormone. Bottom: signal profiles (500-bp interval around the center of ARv7 peaks) of ARv7, ARfl, and ARN cistromes at different ARv7-binding sites. Left panel: 2,629 ARv7, ARfl, and ARN shared peaks. Middle panel: 199 ARv7 and ARN shared peaks. Right panel: 595 “ARv7-unique” peaks. (C) Signal profiles of ARv7 (blue) and ARfl (red) cistromes in response to ARv7 (shARv7; dashed line) or GFP KD (shGFP; solid lines) at “ARv7-unique” (left) or “high confidence” AR sites (union of ARN, Arv7, and ARfl peaks) (right). (D) Apparent FRET efficiencies representative of the level of AR isoform interactions, as shown. Values are the mean of 34–48 cells ±SEM. (E) Signal profiles of ARfl (left) and ARv7 (right) cistromes centered on AR isoform peaks. Cells were induced for 3 days and treated for 4 h with vehicle (ETOH) or 10 nM DHT (DHT). See also Figure S3 and Table S3.

Figure 4.. ARv7 Binds to Transcriptional Co-repressors NCOR1, NCOR2, and NRIP1

(A) MARCoNI assay (using a pan-AR antibody) of select corepressor peptides and cell lysates from indicated LNCaP95 cells. Results are the mean of three experiments ±SD. *p % 0.05, ***p % 0.001, Student’s t test. (B) Quantification of ARv7:NCOR1,2 coIP signals from nuclear lysates of shGFP, shARv7, and shARfl cells. Values are the means ±SEM of 5–6 replicates (n). n.s., not significant; *p % 0.05,**p % 0.01, ***p % 0.001, paired one-tailed t test. (C) Heatmap of the union of dysregulated genes (adjusted p value <0.05) in response to NCOR1 or NCOR2 KD (siNCOR1 or siNCOR2) relative to control (siCtrl) in the background of shGFP, shARv7, and shARfl cells. See also Figure S4.

Figure 5.. ARv7 Negatively Regulates H3K27ac and FOXA1 Chromatin Binding

(A–C) Top: heatmaps of H3K27ac (A), ARv7 (B), and ARfl ChIP-seq (C) in indicated cells, depicting only signals for AR “high-confidence” binding sites (union of ARN, Arv7, and ARfl peaks). Clusters (I and II) are based on the H3K27ac data. Bottom: bar graphs of the average normalized ChIP-seq counts, as indicated. Data are the mean of each cell line data within each cluster ±SEM (cluster I, n = 3,284 peaks; cluster II, n = 4,268 peaks). ****p < 0.0001 by ANOVA and Tukey’s HSD. (D) Violin plots of the differentially expressed (adjusted p value <0.05) ARv7-target (blue) and ARfl-target (red) genes (within 10 kb of an AR site). The number of up- and downregulated genes and median values (black dot) are shown. The Fisher’s exact test between clusters has a p = 0.074 for shARv7 and p = 1 for shARfl. (E) Histogram of the fraction of ChIP-seq peaks in clusters I and II from (A) that contain an AR-binding (left) or FOXA1-binding motif (right). (F) Heatmaps (left) and bar graphs (right) for FOXA1 ChIP-seq in indicated cell lines and clusters, as in (A) to (C). See also Figure S5 and Table S4.

Figure 6.. ARv7 Represses Genes with a Tumor-Suppressive Function

(A) GSEA of ARfl- and ARv7-specific gene signatures (Table S5), compared with genes ranked by transcriptome data from CRPC tumors with IHC-defined ARv7 expression. The number of genes in each signature is indicated in parentheses. The normalized enrichment score (NES), the nominal p value (NOM p), and the FDR q value (FDR q) are shown. (B) GSEA-determined enrichment profile of the ARv7-repressed gene signature (ARv7 rep) as shown in (A). Genes are ranked by their expression in IHC-defined ARv7 high (red, left, ARv7 pos. corr.) versus low patient tumors (blue, right, ARv7 neg. corr.). (C) Heatmap of relative expression of 57 target genes defined by the leading edge in (B), in shGFP and shARv7 LNCaP95 cells. Genes in red were also identified in (D). (D) Hockey-stick plot of positively selected genes identified in a genome-wide CRISPR KO screen. Genes were ranked according to their r score and log₁₀ p value. Target genes from the ARv7-repressed gene signature in (A) are indicated, and genes highlighted in red overlap with the leading-edge analysis in (C). (E) Hierarchical clustering of the 4 ARv7-target genes from (D) on gene expression data from patient samples (Decipher-GRID). Three clusters, based on the average expression of the four genes, are shown: low (blue), mixed (orange), and high (red). (F) Kaplan-Meier graphs of prostate-specific antigen recurrence-free survival for the three patient clusters defined in (E). Log-rank test: n.s, not significant; ****p < 0.0001. (G and H) Expression of the 4 ARv7-regulated genes in (D) in patient subgroups (Decipher-GRID). Samples are grouped by patients that did (n = 492) or did not (n = 1,134) develop metastasis (G), or did (n = 236) or did not (n = 1,211) succumb to the tumor (PCa-specific mortality; H). Standard t test: ***p % 0.001, ****p % 0.0001. Box plots show the median, and the first and third quartile. Whiskers extend to 1.5 the interquartile range and data beyond that are shown as individual points. See also Figure S6; Tables S5 and S6.