GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex

Abstract

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.

Keywords: AR signaling; GATA2; prostate cancer; small molecule inhibitor; steroid receptor coactivator.

Fig. 1.

Prognostic significance of GATA2 in PC and its role in AR signaling. Examples of strong (index 9) staining in PC (A) and weak (index 3) staining in benign prostate (B). (Magnification: 200×.) (C) Immunostaining for GATA2 in tumor compared with benign prostate epithelium. (D) PCs with more intense GATA2 immunostaining (upper vs. lower half of the cohort) had shorter BCR-free survival. (E and F) In two publicly available PC gene-expression datasets, Taylor et al. (17) (E) and TCGA (F), patients with high GATA2 transcriptional activity had shorter BCR-free survival (upper 25% vs. lower 75% of each cohort). (G and H) The GATA2 gene signature strongly enriches for AR-regulated genes. GSEA reveals that the transcriptomic footprint of GATA2 significantly enriches for genes regulated by AR. (G) Transcripts induced after treatment with si-GATA2 were enriched for transcripts up-regulated by si-AR. (H) Transcripts repressed after treatment with si-GATA2 were enriched for transcripts down-regulated by si-AR. (I–K) GATA2 activity scores in human PC specimens correlate strongly with AR activity. We computed GATA2 and AR activity scores for all specimens in several publicly available gene-expression datasets from previously reported human PC cohorts: GSE21034 (17) (I), GSE32269 primary tumors (29) (J), and GSE32269 metastatic tumors (29) (K).

Fig. 2.

GATA2 is critical for the expression and transcriptional function of both AR-FL and ARVs in PC. (A). RT qPCR of GATA2 and AR mRNA expression in LNCaP cells following 72 h of silencing GATA2. (B) Immunoblot analyses of GATA2 and AR or AR3/v7 ARV following 72 h of silencing GATA2. (C) RT qPCR of KLK3 following 72 h of silencing GATA2 in LNCaP cells. Cells were androgen-starved for 48 h in 10% CSS medium and then were treated with 1 nM of R1881 or vehicle for 24 h. (D) Cell viability (MTT absorbance) of PC cell lines 96 h posttransfection with si-nontarget (si-NT) or si-GATA2. Absorbance values are normalized to the corresponding NT siRNA for each cell line. (E and F) Parental LNCaP cells and LNCaP cells stably transfected with tetracycline-inducible AR-FL (LNCaP-TetR-AR-FL) were transfected with si-GATA2 or si-NT for 48 h in the presence or absence of doxycycline (DOX), as indicated. (E) Immunoblot analyses were conducted for the expression levels of GATA2, AR, and β-actin. (F) RT qPCR for KLK3 mRNA following 24 h of treatment with vehicle or 1 nM R1881. (G and H) LNCaP cells stably transfected with tetracycline-inducible AR3/v7 (LNCaP-TetR-ARV7 cells) were transfected with si-NT or si-GATA2 for 48 h in the presence or absence of doxycycline, as indicated. (G) Immunoblot analysis of AR3/v7 and GATA2 expression. (H) RT qPCR was performed for AR3/v7 and FKBP5 (a known AR3/v7 target gene).

Fig. 3.

GATA2 colocalizes with AR and FOXA1 on the chromatin and cooperates with them to recruit master transcriptional regulators. (A) ChIP-Seq demonstrates that, in LNCaP cells, GATA2 strongly colocalizes on chromatin with AR and FOXA1. (B) Integrative analysis with other ChIP-Seq datasets shows that the steroid receptor coactivators SRC-1, SRC-2, and SRC-3, key transcriptional regulators CBP and p300, and transcription factors such as c-Myc preferentially bind to genomic regions where GATA2, AR, and FOXA1 are colocalized (triple-positive peaks). (C) Distribution of normalized sequence tag density for SRC-1, SRC-2, SRC-3, CBP, p300, cMYC, and RNA pol II confirm the higher recruitment of these key transcriptional regulators at triple-positive peaks. In addition, acetylated histone H3 (Ac. H3) shows higher recruitment at 500 bp upstream and downstream of the triple-positive peaks. Color labeling in B and C follows the pattern shown in A. (D) Peak overlap with gene features (promoter, 5′ UTR, 3′ UTR, exons, introns), distal regulatory elements (enhancers), and intergenic regions shows enrichment of triple-positive peaks at distal enhancers: 25% of triple-positive peaks overlap with enhancers, compared with 10–17% of single-positive peaks (1.82-fold enrichment, P < 0.0001, χ² test) and 17–20% of double-positive peaks (1.41-fold enrichment, P < 0.0001, χ² test). (E) Recombinant GATA2 protein increases the recruitment of recombinant AR, SRC-2, and p300 to DNA harboring a classical ARE (from the KLK3 promoter). Immunoblot analyses were conducted following a DNA pull-down assay performed in the presence of R1881.

Fig. 4.

The GATA2 SMI K7174 suppresses expression and transcriptional activity of both AR-FL and AR3/ARv7 and suppresses growth of AR⁺/GATA2⁺ PC cell lines in vitro and in vivo. (A) Immunoblot analysis for AR and GATA2 expression levels in PC cell lines following treatment with K7174 for 72 h. (B) Relative mRNA expression of AR target genes KLK3, TMPRSS2, and FKBP5 following treatment with K7174, as indicated, for 72 h. Columns show the mean of biologic triplicates ± SD. (C) Cell viability (MTT assay) of PC cell lines following treatment with K7174 (20 μM) for 96 h. Results are reported as percent of corresponding vehicle control. Columns show the mean of biologic quadruplicates ± SD. (D) In vivo activity of K7174 against PC cells. Treatment of mice with vehicle or K7174 (25 mg⁻¹⋅kg⁻¹⋅d, 5 d/wk, i.p. injection, n = 4 per cohort) was initiated when the mean tumor volume was ∼200 mm³. The x axis depicts days after initiation of treatment.