Novel Androgen Receptor Coregulator GRHL2 Exerts Both Oncogenic and Antimetastatic Functions in Prostate Cancer

Abstract

Alteration to the expression and activity of androgen receptor (AR) coregulators in prostate cancer is an important mechanism driving disease progression and therapy resistance. Using a novel proteomic technique, we identified a new AR coregulator, the transcription factor Grainyhead-like 2 (GRHL2), and demonstrated its essential role in the oncogenic AR signaling axis. GRHL2 colocalized with AR in prostate tumors and was frequently amplified and upregulated in prostate cancer. Importantly, GRHL2 maintained AR expression in multiple prostate cancer model systems, was required for cell proliferation, enhanced AR's transcriptional activity, and colocated with AR at specific sites on chromatin to regulate genes relevant to disease progression. GRHL2 is itself an AR-regulated gene, creating a positive feedback loop between the two factors. The link between GRHL2 and AR also applied to constitutively active truncated AR variants (ARV), as GRHL2 interacted with and regulated ARVs and vice versa. These oncogenic functions of GRHL2 were counterbalanced by its ability to suppress epithelial-mesenchymal transition and cell invasion. Mechanistic evidence suggested that AR assisted GRHL2 in maintaining the epithelial phenotype. In summary, this study has identified a new AR coregulator with a multifaceted role in prostate cancer, functioning as an enhancer of the oncogenic AR signaling pathway but also as a suppressor of metastasis-related phenotypes. Cancer Res; 77(13); 3417-30. ©2017 AACR.

Figure 1. GRHL2 is a novel AR-interacting protein

(A) Graphical plots (generated by MS-ARC) showing AR- and ARv567es-associated proteins, clustered according to molecular function. The length of the line represents the Mascot score. (B) Peptide coverage of GRHL2 from RIME experiments. (C-D) Validation of AR:GRHL2 interaction by co-immunoprecipitation. AR protein in R1-AD1 and R1-D567 cells (left) and LNCaP cells (right) was immunoprecipitated and GRHL2 was detected by immunoblotting (C). GRHL2 protein in LNCaP cells was immunoprecipitated and AR was detected by immunoblotting (D). For all co-immunoprecipitations, IgG served as a negative control.

Figure 2. GRHL2 is commonly amplified and over-expressed in prostate cancer

(A) Oncoprints representing GRHL2 copy-number alterations from the Grasso and Taylor datasets were generated using cBioPortal. mCRPC, metastatic castration-resistant prostate cancer (mCRPC); Mets, metastases. (B) GRHL2 gene amplification in the Grasso cohort is associated with increased mRNA expression. Boxes show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes) values. P value was determined an unpaired t test. (C) GRHL2 expression is elevated in primary prostate cancer. In the Taylor graph, lines represent the mean ± standard deviation (SD). P values were determined using unpaired (Taylor) or paired (TCGA) t tests. (D) GRHL2 expression is associated with Gleason grade. Lines in the graph represent the mean ± SD. P values were determined using unpaired t tests. (E) Representative staining of non-malignant and cancer tissues are shown on the left (bar = 100 microns). A graph of staining scores is shown on the right. P value was determined using an paired t test. (F) Cellular co-expression of GRHL2 and AR in prostate tissues, as demonstrated by dual immunofluorescence. Shown are two representative prostate tumors. Nuclei were stained with DAPI. Spearman r values represent the correlation between AR and GRHL2 staining (see Materials and Methods). Scale bars are 50 microns.

Figure 3. Regulation of GRHL2 expression by AR in prostate cancer

(A) GRHL2 protein levels in a panel of prostate cancer cell lines (tubulin is the loading control). The graph on the right shows the correlation between tubulin-normalized AR and GRHL2 in all cell lines. (B) Cell lines were treated with 1 nM DHT for 4 hours and GRHL2 levels were assessed by RT-qPCR. Error bars represent ± SD. (C) LNCaP (left) or R1-D567 (right) cells were transfected with siRNA targeting AR (siAR) or control siRNA (siNC) for 48 hours. LNCaP cells were additional treated with 1 nM DHT (or ethanol as a control) for 24 hours. Tubulin or GAPDH are loading controls. (D) GRHL2 mRNA expression in two clinical cohorts. Boxes in the right graph show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes) values. P values were determined using a Wilcoxon matched-pairs signed rank test (GSE48403) or unpaired t tests (GSE28680). FPKM, fragments per kilobase of exon per million mapped reads; NS, not significant. (E) AR binding sites (from ChIP-seq) proximal to the GRHL2 gene in non-malignant and prostate tumor samples (35). Each track depicts ChIP-seq AR binding intensity for a given sample. (F) Validation of 3 putative AR binding sites (A-C; shown below the ChIP-seq tracks in E) by ChIP-qPCR. Error bars represent ± standard error of the mean (SEM).

Figure 4. Regulation of prostate cancer cell growth and AR expression and signaling by GRHL2

(A) Prostate cancer cell lines were transfected with 3 distinct siRNAs and Trypan blue growth assays were performed. Error bars represent ± SD. (B) LNCaP cells were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and gene expression was measured by RT-qPCR after 48 hours. Error bars represent ± SEM of 3 independent experiments. (C) Prostate cancer cell lines were transfected with GRHL2 siRNA (siGRHL2) or a control (siNC) and, 2 days later, protein expression was assessed by Western blotting. Tubulin or GAPDH are shown as loading controls. (D) LNCaP cells were transfected with a GRHL2 over-expression vector (GRHL2-OE) or a control (Empty) and protein expression was assessed by Western blotting after 48 hours. GAPDH is shown as a loading control. (E) AR expression is higher in metastatic CRPC (mCRPC) tumors with GRHL2 copy number gain or amplification compared to tumors with no change in GRHL2 copy number (diploid). Data is from the Grasso cohort and was obtained via cBioPortal. P value was determined using an unpaired t test.

Figure 5. GRHL2 enhances AR's transcriptional activity and associates with AR on chromatin

(A) PC3 cells were transfected with plasmids expressing AR, GRHL2 and a probasin-derived AR-responsive reporter and subsequently treated with 1 nM DHT or vehicle (Veh) control. Transcriptional activity values as assessed by luciferase assays represent the mean (±SEM) of 6 biological replicates; results are a representative of three independent experiments. An unpaired t test was used to assess the affect of GRHL2 on AR activity. (B) Examples of AR-specific, GRHL2-specific and shared binding sites (left). (C) Select motifs enriched in AR-specific, GRHL2-specific and shared peaksets. Motifs were identified using a de novo Gibbs motif sampling approach. P values for enrichment over genomic background are shown. (D) Genomic location summary of AR-specific, GRHL2-specific and shared binding events. (E) Distribution of normalized sequence tag density for H3K27ac, H3K4me2, RNAPII, FoxA1, P300 and Med12 in AR-specific, GRHL2-specific and shared binding events. (F) A gene signature based on shared GRHL2/AR binding events is upregulated in metastatic CRPC (two left panels) and associated with recurrence following radical prostatectomy (right panel).

Figure 6. GRHL2 regulates AR, ERBB and PI3K/Akt signaling pathways

(A) GRHL2 knockdown is associated with decreased expression of an AR-regulated gene set (“Hallmark: Androgen reponse”), as demonstrated by GSEA. (B) Correlation between GRHL2- and AR-regulated gene signatures in primary prostate cancer (left, TCGA cohort) and metastatic CRPC (right, SU2C cohort). (C) GRHL2 knockdown is associated with decreased expression of the PI3K/Akt pathway (“Hallmark: PI3K/Akt/MTOR signaling”), as demonstrated by GSEA. (D) GRHL2 knockdown results in decreased expression of the ERBB2-regulated gene set (“Reactome: Signaling by ERBB2”), as demonstrated by GSEA. (E) GRHL2 knockdown (siGRHL2) leads to reduced expression of ERBB2, ERBB3 and EGFR. Values for the negative control (siNC) were set to 1, and error bars are ± SEM. P values were determined using one-sample t tests (*, P < 0.05; **, P < 0.01). (F) GRHL2 binding events in LNCaP cells, identified by ChIP-seq, proximal to ERBB2, ERBB3 and EGFR. (G) GRHL2 knockdown results in decreased phosphorylation of Akt (pAkt) and ERK (pERK) in LNCaP and 22Rv1 cells, as assessed by Western blotting.

Figure 7. GRHL2 suppresses EMT and invasion of prostate cancer

(A) The growth of two distinct clones with stable knockdown of GRHL2 (shGRHL2.1 and shGRHL2.2) were compared to a control (shNC) in Trypan blue growth assays. Error bars represent ± SD. (B) Stable knockdown of GRHL2 decreases epithelial marker (E-cad, ZO-1) and increases mesenchymal marker (N-cad, ZEB1, Vim) expression at the protein level in LNCaP cells. E-cad, E-cadherin; N-cad, N-cadherin; Vim, Vimentin. (C) Stable knockdown of GRHL2 increases migration of LNCaP cells in a scratch-wound assay. (D) Stable knockdown of GRHL2 increases invasion of LNCaP cells. Values for the negative control (NC) were set to 1, and error bars are ± SEM. P values were determined using unpaired t tests (**, P < 0.01). (E) Stable knockdown of GRHL2 promotes cancer cell invasion in CAM invasion assays. Data represents the mean percentage of images with invasion into the mesoderm ± SEM. P value was determined using an unpaired two-sided t test (***, P < 0.001; ****, P < 0.0001). (F) Representative images from the CAM assays. Cancer cell:matrigel grafts (CM) were placed on top of the ectoderm (ET) layer and cancer cell invasion into the CAM mesoderm (MD) was assessed in day 14 chick embryos. Endoderm, EN. Shown are cytokeratin (CK) IHC (left) and haematoxylin and eosin (right) images. Scale bars = 100 microns.