Constitutively active androgen receptor splice variants expressed in castration-resistant prostate cancer require full-length androgen receptor

Abstract

Androgen receptor (AR) splice variants lacking the ligand binding domain (ARVs), originally isolated from prostate cancer cell lines derived from a single patient, are detected in normal and malignant human prostate tissue, with the highest levels observed in late stage, castration-resistant prostate cancer. The most studied variant (called AR-V7 or AR3) activates AR reporter genes in the absence of ligand and therefore, could play a role in castration resistance. To explore the range of potential ARVs, we screened additional human and murine prostate cancer models using conventional and next generation sequencing technologies and detected several structurally diverse AR isoforms. Some, like AR-V7/AR3, display gain of function, whereas others have dominant interfering activity. We also find that ARV expression increases acutely in response to androgen withdrawal, is suppressed by testosterone, and in some models, is coupled to full-length AR (AR-FL) mRNA production. As expected, constitutively active, ligand-independent ARVs such as AR-V7/AR3 are sufficient to confer anchorage-independent (in vitro) and castration-resistant (in vivo) growth. Surprisingly, this growth is blocked by ligand binding domain-targeted antiandrogens, such as MDV3100, or by selective siRNA silencing of AR-FL, indicating that the growth-promoting effects of ARVs are mediated through AR-FL. These data indicate that the increase in ARV expression in castrate-resistant prostate cancer is an acute response to castration rather than clonal expansion of castration or antiandrogen-resistant cells expressing gain of function ARVs, and furthermore, they provide a strategy to overcome ARV function in the clinic.

Fig. 1.

Discovery of ARVs in additional prostate cancer models. (A and B) Next generation AR mRNA sequencing in VCaP. (A) Exon 3 truncation ARVs described here (blue) or previously (black) were initially identified by Sanger sequencing; 454 junctions supporting Sanger sequences were determined by TopHat in supervised mode (i.e., input of predetermined junctions). TopHat does not detect noncanonical splice sites (AR-V10) or exon runon (AR-V11). SOLiD coverage is represented on a log scale. The greatest number of SOLiD reads mapped to the native AR exons, consistent with the relative abundance of AR-FL in these cells. SOLiD specifically detected the unique AR-V11 sequence that was not identifiable using TopHat. (B) Unsupervised TopHat analysis of 454 junctions identifies putative ARVs distinct from exon 3 truncations involving exon skipping. A putative cryptic intron 5 exon is shown in red, with a large number of SOLiD reads relative to adjacent intron sequence. (C) Sequences of Myc-CaP ARVs were mapped to intergenic regions (dashed lines) of chromosome X (not drawn to scale) using the University of California, Santa Cruz (UCSC) Genome Browser (NCBI37/mm9 assembly). The genetic origins of mAR-V2– and mAR-V4–specific sequences are represented by blue or red boxes, respectively. Adjacent genes are shown with their physical position. The ARV and AR-FL proteins are depicted with the native AR exons numbered (not drawn to scale). (D) Myc-CaP were transfected with siRNA against total AR (exons 1 and 3), mAR-V2, or mAR-V4 (four individual siRNA per ARV shown as a–d). A nontarget siRNA (N) was used as a negative control. Growth media was standard 10% FBS. Western blots were done at 24 h posttransfection.

Fig. 2.

Androgen represses AR-FL and ARV transcription. (A) Western blot of prostate cancer xenograft tumors from intact mice on various days postcastration. Testosterone was implanted after the indicated number of days postcastration. Duration of testosterone replacement was 8 or 4 d for VCaP and LuCaP35, respectively. AR-FL blots underwent extensive additional washing to avoid ECL signal saturation; therefore, the actual large AR-FL to ARV protein ratio is not reflected here. (B and C) qRT-PCR of AR isoforms with normalization to β-actin. (B) Mean expression in xenografts (n = 4; error bars = SEM); 14 d postcastration tumors were used for VCaP. (C) Expression in individual clinical prostate cancer metastases.

Fig. 3.

LBD truncation is insufficient for nuclear translocation and androgen-independent transcriptional activity. (A) Peptide sequences of the C-terminal extension for each ARV used in comparative studies. (B and C) Transient transfected Cos-7. (B) Western blot. (C) AR immunofluorescence ± 1 nM R1881. (Magnification: 200×.) (D and E) Parental or stably infected DU145. (D) Representative transfected 4× ARE-Luc activity normalized to Renilla-Luc. (E) Western blot ± 1 nM R1881.

Fig. 4.

Effect of ARVs on prostate cancer tumor growth in castrated mice. (A and B) Western blot of parental or stably infected prostate cancer cell lines. (C) Parental or stable LNCaP cells were bilaterally grafted into precastrated mice (n = 6–10). Mean tumor volumes are graphed; error bars = SEM. The human and mouse ARV LNCaP lines were grafted on separate days. Tumor volumes varied between experiments because of the inherent in vivo variability of LNCaP. (D) Parental or stable Myc-CaP cells were bilaterally grafted into intact mice (n = 4–8). Castrate-resistant growth was assessed after castration-induced tumor regression. Mean tumor volume is depicted as the percentage of the tumor volume at the time of castration (error bars = SEM).

Fig. 5.

Gain of function ARVs are not resistant to the antiandrogen MDV3100. (A) Precastrated mice were grafted with LNCaP/GFP or AR-V7 sublines (n = 6–7). When castrate-resistant tumors arose, the mice were treated with MDV3100. Fold-change tumor volume after treatment is plotted relative to the volume on day 0 of treatment (error bars = SEM). (B) Western blot of LNCaP/AR-V7 cell line (in vitro culture) or tumors from castrated mice treated for 25 d with MDV3100 or vehicle. (C and D) Anchorage-independent soft-agar growth in 10 μM MDV3100 for parental DU145 and stable LNCaP lines. (C) Images of representative plate quartile. (D) Mean total colony number (n = 3; error bars = SEM).

Fig. 6.

Gain of function ARVs remain dependent on AR-FL. (A) AR-V7 immunofluorescence in LNCaP/AR-V7 treated with 10 μM MDV3100. (Magnification: ×200.) (B) Proliferation of stable LNCaP cells after transfection with 10 nM nontarget (NT) or AR-FL siRNA. (C) qRT-PCR for AR-FL 2 d after siRNA transfection (n = 3; error bars = SEM). Western blot 6 d after transfection with nontarget (N) or AR-FL (A) siRNA. (D and E) qRT-PCR for the indicated AR-regulated genes 2 d after siRNA transfection (n = 3; error bars = SEM). (D) Within each LNCaP line, gene expression after AR-FL siRNA is expressed relative to the mean signal of the NT siRNA control, which was set at 1. (E) In the NT siRNA controls, the mean expression level of each gene within LNCaP/GFP was used as the reference sample for comparison with the corresponding expression in the LNCaP/ARV lines. Gene expression was normalized to AR-FL.