It was recently discovered that AR alternative splicing can give rise to COOH-terminally truncated AR protein isoforms that lack the AR LBD. These AR isoforms are missing the region of the receptor that would be predicted to mediate responses to traditional ADT, as well as new AR-targeted therapies such as abiraterone and MDV3100. Key observations leading to this discovery were initially reported in 2002, when it was demonstrated that 22Rv1 cells expressed two separate AR protein species of approximately 112 kDa and 75–80 kDa (Tepper et al. 2002
). By antibody mapping, it was demonstrated that the smaller 75–80kDa AR species was composed of the AR NTD and DBD, but lacked the AR LBD. Further biochemical characterization revealed that the AR ΔLBD isoform was constitutively nuclear and could bind DNA independent of androgens. Co-immunoprecipitation experiments indicated that the AR ΔLBD isoform did not interact with full-length AR, suggesting that this species functioned as an independent factor. In another study, Western blots with benign and cancerous prostate tissue demonstrated that AR immunoreactive species with a similar mobility to the AR ΔLBD isoform were frequently expressed in clinical PCa (Libertini et al. 2007
). Together, these studies provided the first evidence that AR protein species lacking the LBD could play an important role in modulating PCa therapy resistance.
The mechanism proposed for synthesis of the AR ΔLBD protein species in PCa cells was via proteolytic degradation of full-length AR by calpain-2 (Libertini et al. 2007
). This was based on the presence of a consensus calpain cleavage site in the AR hinge region and the observation that calpain-2 was able to cleave full-length AR protein in cell extracts derived from LNCaP and 22Rv1 cells, as well as the CRPCa CWR-R1 cell line which was also derived from the CWR22 xenograft model (Chen et al. 2010
; Libertini et al. 2007
). In addition, the full-length AR mRNA in 22Rv1 cells had been shown to be larger due to tandem duplication of AR exon 3, and this larger form of the AR appeared to have enhanced susceptibility to this mechanism of cleavage (Libertini et al. 2007
). However, several recent studies have suggested that alternative splicing could also be an important contributor to the synthesis of truncated AR species lacking the AR LBD in these and other cells. This new concept was initially based on the observation of differential siRNA targeting of the various AR species in 22Rv1 cells (Dehm et al. 2008
). For example, siRNA targeted to AR exon 7 abolished expression of full-length AR in 22Rv1 cells, but had no effect on the smaller 75–80kDa species. Conversely, siRNA targeted to AR exon 1 knocked down expression of all AR immunoreactive species in 22Rv1 cells. Functionally, androgen-dependent expression of AR target genes and androgen-dependent cell growth was shown to be attributable to full-length AR, whereas androgen-independent expression of AR target genes and constitutive, androgen-independent growth was shown to be supported by the smaller 75–80kDa species. These observations strongly suggested that different mRNA species encoded the different AR protein species observed in these cells, and provided the foundation for 3’-RACE and other approaches to identify their origin (Dehm et al. 2008
; Guo et al. 2009
; Hu et al. 2009
; Hu et al. 2011
; Marcias et al. 2010
). These efforts led to the identification of a series of alternatively spliced AR mRNAs expressed in 22Rv1 cells that resulted from cryptic exons located within AR introns 2 and 3. The AR isoforms or variants encoded by these alternatively spliced mRNAs have been shown to function as constitutively active, ligand-independent transcription factors that can support the CRPCa phenotype in various model systems. Specific details of the individual AR isoforms that have been identified in models derived from the CWR22 xenograft are discussed in the following section. A major barrier to clarity regarding this emerging topic is the nomenclature that has been employed by different investigators, leading to instances where different names have been given to the same isoform (e.g. AR 1/2/3/2b, AR-V4, AR5, and ARV6 are all encoded by contiguously-spliced AR exons 1/2/3/2b; AR-V7 and AR3 are both encoded by contiguously-spliced AR exons 1/2/3/CE3; and ARv567es
and AR-V12 are both encoded by skipping of exons 5–7). Also, there are instances where different isoforms have been given the same name (e.g. AR-V7 encoded by contiguously spliced AR exons 1/2/3/CE3 and ARV7 encoded by contiguously spliced AR exons 1/2/3/3/3’/4/5/6/7/8). Therefore, we will discuss these isoforms in the context of their exon constituency, and acknowledge all of the various names that have been given to these isoforms to date ().
Figure 4 Alternatively spliced AR isoforms identified in prostate cancer (PCa). (A) Schematic of the AR gene locus with locations of alternatively spliced, cryptic exons illustrated in red. The location of an intragenic tandem duplication identified in 22Rv1 cells (more ...)
Splicing of AR exons 1/2/2b and 1/2/3/2b
AR exon 2b was discovered following 5’-RACE experiments with RNA derived from 22Rv1 cells (Dehm et al. 2008
). AR exon 2b was shown to splice downstream of either AR exon 2 or 3, giving rise to truncated AR proteins containing the AR NTD, one or both zinc fingers of the AR DBD, and a short 11 amino acid COOH-terminal extension encoded by AR exon 2b. Expression of the 75–80kDa immunoreactive species in 22Rv1 cells was reduced upon transfection with exon 2b-specific siRNA, which corresponded with an inhibition of androgen-independent cell proliferation. These data confirmed that transcripts containing AR exon 2b were translated to functional proteins. Importantly, there was no effect of exon 2b siRNA on androgen-stimulated proliferation, further supporting the concept that truncated AR isoforms could support features of the CRPCa phenotype independent of full-length AR. Functionally, AR 1/2/2b and AR 1/2/3/2b were shown to function as constitutively active transcription factors in promoter-reporter assays. The AR 1/2/2b AR mRNA was also shown to be expressed in VCaP cells as well as the LuCaP 23.1 and 35 models of PCa progression.
Additional studies by other groups have confirmed the existence of mRNA species containing exon 2b in the 22Rv1 model system, but with variability in the precise 3’ composition of the transcript. For example, one transcript was reported with exon 2b (referred to as exon CE4 in the study) spliced between exons 2 and 3, with exon CE1 at the 3’ terminus (Hu et al. 2009
). This isoform was termed AR-V3 in this study, and was proposed to encode a 53 aa COOH-terminal extension due to a TAA trinucleotide deletion in the sequence of CE4 compared with the sequence of exon 2b. The reason for this discrepancy is not clear, but could be due to procedural differences or the fact that 22Rv1 cells harbor 2 genomic copies of AR exon 2b (Li et al. 2011
). Another transcript was also identified with exon 2b spliced between the duplicated copies of exon 3, with exon CE1 at the 3’ terminus (Hu et al. 2009
). This configuration, termed AR-V4, matched the splicing pattern for the AR5 mRNA reported in a separate study (Guo et al. 2009
). Importantly, AR5 mRNA expression was detected by RT-PCR at a low level in benign prostate tissue as well as PCa tissue. In yet another study, AR exon 2b (referred to as exon 2’ in that study) was shown to be spliced between the duplicated copies of exon 3 in the context of an mRNA species, termed V6, with contiguous splicing of AR exons 1–8 (Marcias et al. 2010
). Together, these data indicate that many AR mRNAs in the 22Rv1 cell line contain exon 2b spliced after exon 2 or 3, with possible variation in the composition of the 3’ untranslated region of these transcripts.
Splicing of AR exons 1/2/3/CE1 and 1/2/3/3/CE1
Two AR mRNA species composed of contiguously spliced AR exons 1/2/3/CE1 (termed AR-V1) and AR exons 1/2/3/3/CE1 (termed AR-V2) were demonstrated to be expressed in 22Rv1 cells following the bioinformatic identification of AR exon CE1 as an expressed sequence tag mapping to AR intron 3 (Hu et al. 2009
). The AR-V1 transcript composed of AR exons 1/2/3/CE1 was detected by quantitative RT-PCR at a higher level in RNA isolated from CRPCa tissue specimens vs. hormone naïve PCa. In another study, the AR 1/2/3/CE1 isoform was also detected in 22Rv1 cells, and termed AR4 (Guo et al. 2009
). No studies have been performed to demonstrate translation of either of these mRNAs into endogenous proteins.
Splicing of AR exons 1/2/3/CE2
Two separate AR mRNA species composed of contiguously spliced AR exons 1/2/3/CE2 have been identified, with different locations for the splice acceptor site used for incorporation of exon CE2 into these mRNAs (Hu et al. 2009
). As with the identification of AR exon CE1, exon CE2 was originally discovered by bioinformatic identification of an expressed sequence tag mapping to AR intron 3. The isoform generated from utilization of the most 5’ splice acceptor site in exon CE2, termed AR-V5, results in a protein predicted to encode a single aspartate residue following exon 3 coding sequence. The isoform generated from utilization of the most 3’ splice acceptor site in exon CE2, termed AR-V6, results in a protein predicted to encode a 6 amino acid COOH-terminal extension following exon 3 coding sequence. No studies have been performed to demonstrate translation of this mRNAs into an endogenous protein.
Splicing of AR exons 1/2/3/CE3
The best-characterized alternatively spliced AR isoform identified to date is encoded by contiguously splicing of AR exons 1/2/3/CE3. In one study, this isoform was named AR-V7 (Hu et al. 2009
), and in another study this same isoform was named AR3 (Guo et al. 2009
). As with AR exons CE1 and CE2, exon CE3 was originally discovered based on bioinformatic identification of an expressed sequence tag mapping to AR intron 3 (Hu et al. 2009
). Importantly, AR 1/2/3/CE3 mRNA was detected in various clinical PCa specimens as well as normal prostate tissue (Hu et al. 2009
). Quantitative RT-PCR demonstrated that the RNA levels of this isoform could predict biochemical recurrence following surgery (Hu et al. 2009
). The 16aa COOH-terminal extension encoded by AR exon CE3 has been amenable to antibody development (Guo et al. 2009
; Hu et al. 2009
), and the utilization of these isoform-specific antibodies has revealed widespread protein expression of this isoform in cell lines, xenografts, and clinical specimens. Importantly, protein expression of this isoform was shown to be increased in CRPCa vs. hormone naïve PCa (Guo et al. 2009
). Also, cytoplasmic levels of this isoform could predict biochemical recurrence following surgery (Guo et al. 2009
). It is interesting to note that the AR 1/2/3/CE3 isoform is also expressed at the mRNA and protein level in benign prostate tissue, indicating that there may also be a role for this splicing event in normal cells (Guo et al. 2009
). Functionally, AR 1/2/3/CE3 was demonstrated to function as a constitutively active, ligand-independent transcription factor that could induce a CRPCa growth phenotype in LNCaP cells grown in vitro
and as xenografts in vivo
(Guo et al. 2009
). Interestingly, in one study, gene expression profiling of LNCaP cells transfected with an expression vector encoding AR 1/2/3/CE3 demonstrated that the ligand-independent transcriptional activity of this isoform could largely recapitulate the gene expression program activated by ligand-induced full-length AR, including the PSA gene (Hu et al. 2009
). However, another study that used shRNAs to selectively knock-down the AR 1/2/3/CE3 or full-length AR isoforms in 22Rv1 and CWR-R1 cells demonstrated that AR 1/2/3/CE3 activated a significantly different gene expression program than ligand-activated AR (Guo et al. 2009
). One differentially-regulated gene that was identified was AKT1 (Guo et al. 2009
). The AR 1/2/3/CE3 isoform was shown to activate AKT1 expression at the mRNA and protein level, whereas full-length AR had no effect on AKT1 expression. Mechanistically, this could be attributed to the finding that AR 1/2/3/CE3 but not full-length AR could bind to two AREs located in the AKT1 promoter region. Overall, this body of work demonstrates that an AR variant derived from contiguous splicing of AR exons 1/2/3/CE3, termed AR-V7 or AR3, is a clinically validated AR isoform that could play an important role in supporting the CRPCa phenotype.
Insertion of an extra copy of exon 3 and a novel exon 3’ between AR exons 3 and 4
Additional alternatively-spliced AR isoforms have recently been cloned from 22Rv1 cells using a yeast functional assay to identify AR cDNAs with constitutive or ligand-induced transcriptional activity (Marcias et al. 2010
). This approach led to the identification of an additional cryptic exon in AR intron 3 termed 3’. Sequencing of the isolated cDNA, which was named ARV7 in this study, demonstrated that exon 3’ spliced between an extra copy of AR exon 3 and exon 4, giving rise to a truncated AR isoform containing the entire AR NTD, the entire DBD, an extra zinc finger encoded by the extra copy of exon 3, and a novel 23 aa COOH-terminal extension encoded by exon 3’.