Rapid induction of androgen receptor splice variants by androgen deprivation in prostate cancer

Abstract

Purpose: Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.

Experimental design: Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone.

Results: AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (<1%) relative to AR-FL in resistant VCaP xenografts and CRPC clinical samples. AR-V7 protein expression was similarly low relative to AR-FL in castration-resistant VCaP xenografts and androgen-deprived VCaP cells, but the weak basal AR activity in these latter cells was further repressed by AR-V7 siRNA.

Conclusions: AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain.

Figure 1

Expression of AR-stimulated genes and androgen synthesis in abiraterone-resistant VCaP xenografts. A, mice bearing recurrent VCaP xenografts were treated with abiraterone until relapse (0.5 mg/ml in drinking water for 4–6 weeks). RNA extracted from 4 sets of tumor samples pre- (pre-abi) or post-treatment (Abi-resistant) was analyzed by microarray (Affymetrix HuGene 1.0 ST). Expression of 12 androgen-stimulated genes is shown as heat map (red, high expression; green, low expression). B, the Log₂ ratio for expression of androgen stimulated genes (>2-fold) in abiraterone-relapsed versus pretreatment xenograft is plotted versus their fold androgen induction. R² is presented as an indication of the correlation between androgen-induction and change induced by abiraterone treatment, and showed no trend towards lower expression in the relapsed tumors. C, expression of 9 genes involved in androgen synthesis is shown as heat map. D, DHT, testosterone, and androstenedione levels in 6 sets of abiraterone-relapsed VCaP xenograft tumor samples versus pre-treatment levels in tumor biopsies were measured using mass spectrometry. Each sample was measured in duplicate.

Figure 2

Genes and pathways altered in abiraterone-relapsed xenografts. A and B, heat map presentations of expression of top 30 most consistently (A) upregulated genes or (B) downregulated genes in abiraterone resistant tumors. Genes shown in bold and red in (A) were shown previously to be repressed by DHT in VCaP cells. C and D, gene ontology analysis on (C) 181 upregulated genes or (D) 132 downregulated genes (p<0.05). E, expression of 13 androgen-suppressed genes shown as heat map.

Figure 3

Structural alterations in AR in abiraterone-resistant xenografts. A and B, expression of AR-V7 transcripts versus AR-FL in a series of VCaP xenografts (1, 2, 3, 4, 7, 8) were examined using (A) semi-quantitative RT-PCR (-1, pre-abiraterone; -2, post-abiraterone) or (B) real-time RT-PCR. C, VCaP xenografts were established and biopsied at three stages: androgen-dependent tumor (AD), 4d post-castration (CS), and castration-resistant relapsed tumor (CR). Expression of AR-V7 and AR-FL were examined in 4 sets of these tumor samples. D, fold change in expression of AR-V7 and AR-FL in different stages of xenograft tumors are summarized.

Figure 4

Androgen preferentially suppresses expression of AR-V7 versus AR-FL. A, VCaP or VCS2 cells were treated with (A) 10 nM DHT or vehicle (ethanol) for 24 h and mRNA for AR-V7 or AR-FL were measured using qRT-PCR (GAPDH as internal normalization control). B, C4-2 and high-passage LNCaP (LN-HP) cells were androgen deprived for 3 or 10 days, respectively, before being treated with 10 nM DHT for overnight. RNA samples were subjected to qRT-PCR for AR-V7 and AR-FL. C, VCaP cells were treated with increasing doses of DHT (0–10 nM) for 24 h. RNA samples were subjected to qRT-PCR for AR-V7 and AR-FL expression, and the levels shown are normalized to the levels in the absence of added DHT. D, VCaP cells were transfected with siRNA against exon 7 of AR-FL (siEX7) for 48 h and then treated with 0–10 nM DHT for 24 h. RNA samples were subjected to qRT-PCR. E and F, VCaP cells were pretreated with/out DHT for 2 h followed by addition of (E) actinomycin D (10 μM) for 0–6 h or (F) cycloheximide (CHX, 10 mg/μl) for 0–24 h. Note: all cells were androgen starved by culturing in steroid-depleted medium prior to treatments.

Figure 5

AR-V7 contributes for AR activity in hormone depleted conditions. A, AR protein in androgen dependent (AD) or castration resistant (CR) VCaP xenograft tumors was immunoblotted, with β-tubulin as loading control. The AR-V7 and AR-FL bands were quantified using NIH Image J software in comparison to band intensity on blots with serial dilutions of AR, and values were further normalized to β-tubulin. Ratios of AR-V7 versus AR-FL expression are presented. B, VCaP cells were transfected with siRNA against non-target control (siCtrl), cryptic exon 3 (siCE3), or exon 1 (siEX1) followed with treatment of DHT or vehicle for 24 h, and lysates were then immunoblotted with AR N- or C- terminal antibodies or an AR-V7 specific antibody. β-actin and β-tubulin were used as loading control. The AR-V7 and AR-FL bands were quantified as in A and presented as ratios of AR-V7 versus AR-FL. C, effects of siCE3, siEX7 (siRNA against exon 7) and siEX1 (siRNA targeting exon 1 of both AR-FL and AR-V7) on a panel of androgen stimulated genes in androgen starved VCaP cells. D, Androgen-deprived VCaP cells were transfected with siCtrl or siCE3 for 24 h and then treated with enzalutamide (enza, 10 μM) or vehicle (DMSO) for overnight. SiCtrl-transfected VCaP cells treated with DHT were included as a positive control for AR activation. RNA samples were subjected to qRT-PCR for PSA, PLZF, KLK2 and GAPDH (as internal control).

Figure 6

AR-V7 expression in CRPC patient samples. Expression of AR-V7 and AR-FL were assessed by qRT-PCR in 20 CRPC bone marrow biopsy tumor samples, with GAPDH co-amplified as an internal control. The levels for each are normalized to those in androgen starved VCaP cells (VCaP cells treated with ethanol vehicle control). The bottom panel shows the ratios of AR-V7 versus AR-FL, which are similarly normalized to the ratio in androgen starved VCaP cells. *AR-FL in Sample 28 was 17.0-fold relative to androgen-deprived VCaP cells; **AR-V7 and AR-FL in Sample 49 were 14.0-fold and 860-fold, respectively, relative to those in the androgen-deprived VCaP cells.