Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer

Abstract

Suppression of androgen production and function provides palliation but not cure in men with prostate cancer (PCa). Therapeutic failure and progression to hormone-refractory PCa (HRPC) are often accompanied by molecular alterations involving the androgen receptor (AR). In this study, we report novel forms of AR alteration that are prevalent in HRPC. Through in silico sequence analysis and subsequent experimental validation studies, we uncovered seven AR variant transcripts lacking the reading frames for the ligand-binding domain due to splicing of "intronic" cryptic exons to the upstream exons encoding the AR DNA-binding domain. We focused on the two most abundantly expressed variants, AR-V1 and AR-V7, for more detailed analysis. AR-V1 and AR-V7 mRNA showed an average 20-fold higher expression in HRPC (n = 25) when compared with hormone-naive PCa (n = 82; P < 0.0001). Among the hormone-naive PCa, higher expression of AR-V7 predicted biochemical recurrence following surgical treatment (P = 0.012). Polyclonal antibodies specific to AR-V7 detected the AR-V7 protein frequently in HRPC specimens but rarely in hormone-naive PCa specimens. AR-V7 was localized in the nuclei of cultured PCa cells under androgen-depleted conditions, and constitutively active in driving the expression of canonical androgen-responsive genes, as revealed by both AR reporter assays and expression microarray analysis. These results suggest a novel mechanism for the development of HRPC that warrants further investigation. In addition, as expression markers for lethal PCa, these novel AR variants may be explored as potential biomarkers and therapeutic targets for advanced PCa.

Figure 1. Cloning of novel AR variants

A. Novel AR variants lacking LBD generated by splicing of 4 cryptic exons. The 8 canonical exons of the AR gene were represented by numbered open boxes and shown (not to scale) in relation to the genomic positions of the 4 cryptic exons (CE1 to CE4) in shaded boxes. The identical forward primer, P1/P2/P3(F), in exon 2 was paired with 3 reverse primers (P1R, P2R, P3R) (Supplemental Table II) designed based on GenBank entries for the three transcribed genomic fragments in intron 3 (supplemental Table III). Sequencing of the amplicons (from CWR22Rv1 cells) defined the 5′ junctions of CE1, CE2, and CE3, and 5′ and 3′ junctions of CE4, as marked by vertical lines with the corresponding genomic coordinates (Human Genome Assembly March 2006, HG18). Note there were 4 CE1 containing variants (AR-V1, V2, V3, V4), and that the two CE2-containing variants (AR-V5, V6) differed by an 80bp contiguous 5′ extension in CE2. Stop codons were marked with the arrowheads in the schematically illustrated transcripts. The 7 translated protein sequences corresponding to the 7 transcripts were shown, starting from the last 4 amino acids encoded by exon 3 (AR-V1, V2, V4, V5, V6, V7) or exon 2 (AR-V3), and followed by variable lengths of variant specific sequences in light gray that matched the cryptic exons B. Detection of the AR variant transcripts by semi-quantitative RT-PCR in clinical prostate specimens using the same sets of P1, P2 and P3 primers. Note that AR-V3, with expected size of 1126 bp by P1 primer set (Supplemental Table II), was not detected in these clinical specimens. HRPC (autopsy): metastatic HRPC samples from autopsies; HRPC (TURP): HRPC samples from transurethral resection of prostate (TURP); PCa (RRP): Hormone Naïve PCa from radical retropubic prostatectomy (RRP) specimens C. Amplification of full-length coding region for AR-V1 and AR-V7, using primer sets P4 and P5 (Supplemental Table II), from one HRPC autopsy sample, one TURP sample, and the CWR22RV1 cell line. An identical forward primer, P4(F) and P5(F) located upstream of the translation start codon in exon 1, were paired with reverse primers, P4(R) and P5(R), located downstream of the stop codon in cryptic exon 1 and cryptic exon 3.

Figure 2. Quantification of AR variant transcripts in clinical specimens

A. Representative gel images of amplified AR variant transcripts detected using primer sets designed for real-time RT-PCR assays. An identical forward primer, P6/P7/P8(F) in exon 3 was paired with different reverse primers P6(R), P7(R), and P8(R) (Supplemental Table II), to amplify the AR-V1, AR-V7 and prototype AR transcripts, respectively. SF3A3 was used as a reference gene transcript (Methods and Materials). Normal (RRP): normal prostate tissues from radical retropubic prostatectomy (RRP) specimens; PCa (RRP): Hormone Naïve PCa from RRP specimens; HRPC (TURP): HRPC samples from transurethral resection of prostate (TURP); HRPC (autopsy): metastatic HRPC samples from autopsies (Supplemental Table I) B. Quantitative results of AR-V7 in 124 clinical prostate specimens by real time PCR. Normalized expression values (in log₂ scale) for AR-V7 derived from comparative threshold analysis were shown in 4 groups of clinical specimens. Normal (n=17): normal prostate tissues from radical retropubic prostatectomy (RRP) specimens; Hormone Naïve PCa (n=82): PCa samples from RRP specimens; HRPC (TURP) (n=4): HRPC samples from transurethral resection of prostate (TURP); HRPC (autopsy) (n=21): metastatic HRPC samples from autopsies (Supplemental Table I) C. Kaplan-Meier plot comparing progression free survival in patients with less than median AR-V7 expression (n = 38) to those with greater than median AR-V7 expression (n = 28). The survival curves were compared using the Log-rank test. Follow-up years were marked on the X axis. Censored subjects were marked with vertical ticks in blue. Note that the PSA recurrence status was annotated in years, not months.

Figure 3. AR-V7 protein detection and analysis using a variant specific antibody

A. Detection of AR-V7 protein product in cell lines expressing high levels of AR-V7 transcript (Supplemental Figure 1). Following immunoblot analysis for AR-V7 (upper panel), the same membrane was stripped and subjected to immunoblot analysis with anti-AR(N20) antibody (middle panel) to detect the prototype AR. Loading of total protein was monitored by Ponceau S staining of the PVDF membrane (Lower panel) B. Detection of AR-V7 protein following enrichment of all NTD-containing AR proteins by immunoprecipitation (IP) using the anti-AR(441) antibody. Note that following enrichment, AR-V7 was detected in cell lines expressing highest levels of AR-V7 mRNA, VCaP and CWR22Rv1 cells, but not in LNCaP cells, which expressed low levels of AR-V7 (Supplemental Figure 1). Control: mouse IgG. anti-AR: anti-AR(441) monoclonal antibody C. Detection of AR-V7 protein in HRPC. Western blot analysis were performed to detect AR-V7 in whole tissue lysates and enriched AR protein extracts derived from 4 hormone naïve human PCa tissue (RRP5, 6, 7 and 8) and two hormone refractory human PCa tissues (TURP1 and 2). Protein loading was monitored by Ponceau S staining of the PVDF membrane (Middle panel). IP with the anti-AR(441) antibody was performed to enrich the AR proteins and immunoblotted (IB) with anti-AR(N20) to detect the prototype AR, and AR-V7 by the antiAR-V7 antibody (Lower panel) D. Biochemical analysis of cellular localization of AR-V7 protein. VCaP and CWR22Rv1 cells were grown in phenol red-free RPMI 1640 containing charcoal stripped serum (CSS) with or without 10nM R1881. The cytosolic fraction (C) and nuclear fraction (N) of lysates with equivalent number of cells were isolated and subjected to immunoblot analysis of AR-V7, prototype AR by anti-AR(N20) antibody, and β-actin.

Figure 4. Constitutive function of AR-V7

A. Constitutive nuclear localization of transfected AR-V7 in the absence of androgen. PC-3 cells were transfected with pEGFP-AR and pEGFP-AR-V7 to express the prototype AR or AR-V7, and examined for the localization of GFP-tagged AR proteins in the absence or presence of 5 nM R1881 B. AR-V7 constitutively activates an AR luciferase reporter. PC-3 cells were transfected with vector control (EGFP), a LBD truncated AR mutant (EGFP-Q640X), AR-V7 (EGFP-AR-V7), and prototype AR (EGFP-AR), and subjected to luciferase assays and western blot analysis following culturing in the absence or presence of R1881 C. Androgen-independent induction of AR responsive genes by AR-V7 in LNCaP cells. LNCaP cells were transfected with pcDNA-AR-V7 to express the untagged AR-V7 protein or the control pcDNA vector, and cultured with or without 10nM R1881 before being harvested for western blot analysis or RNA extraction for expression microarray analysis. The genes shown were the top 20 ranked genes by fold induction following R1881 treatment in pcDNA empty vector transfected LNCaP cells. Expression ratios of the test sample versus the common reference (pcDNA empty vector transfected LNCaP without R1881) were represented by red (Greater than 1) and green colors (less than 1).