Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (AR^v567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, AR^v567es functioned as a constitutively active receptor, increased expression of full-length AR (AR^fl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with AR^v567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of AR^v567es to AR^fl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected AR^v567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.

The androgen receptor (AR) is a principal driver of prostate cancer progression (1). This concept was first established by Huggins et al., with the demonstration that castration slowed, albeit temporarily, the progression of prostate cancer (2). Subsequent castration-resistant growth of prostate cancer has been attributed to a variety of mechanisms that include activation by receptor tyrosine kinases from growth factors, loss of cell cycle regulators, and rarely, genomic mutations in the AR allowing response to nonspecific AR ligands, such as progesterone or glucocorticoids (3–6). More recently, it was demonstrated that increased expression of the AR was the most common event associated with castration-resistant growth (7). Other studies support a process of metabolic adaptation, involving intracrine androgen synthesis (8–10). However, even when agents are used that decrease the tumoral androgen concentrations to very low levels, increased AR expression and signaling persists (11). In a percentage of tumors, the progression of prostate cancer is associated with activating AR mutations, but these events are infrequent (12). These observations suggest the possibility of alternative mechanisms, independent of androgenic ligands that maintain AR program activity in castration-resistant prostate cancers (CRPCs).

Recently, studies of cell lines and prostate cancers have identified several alternative splice forms of the AR (12–14). These AR variants have somewhat different structures, although each variant lacks portions of the ligand-binding domain (LBD), a feature predicted to produce a constitutively active receptor. Interestingly, the elevated expression of AR splice variants was found to be associated with more rapid disease recurrence following radical prostatectomy for localized disease, when compared with patients with lower expression of the variant (13, 15). Of additional interest, the splice forms were not expressed in the nucleus of normal prostate epithelium and rarely at substantial levels in primary prostate cancer. These data suggest that the presence of constitutively active splice variants of the AR arises following castration and plays a role in the progression of prostate cancer.

In this study, we report the identification and characterization of what we believe to be a previously unrecognized AR splice variant that comprises the full sequences of exons 1–4 and the full sequence of exon 8, skipping exons 5, 6, and 7 (hereafter referred to as AR^v567es, in which “v” denotes variant, and “es” denotes exons skipped). Because of the alternative splicing of exon 4 to the beginning of exon 8, a frame shift occurs that generates a new stop codon after the first 29 nucleotides of exon 8. Thus, the AR^v567es protein is not only smaller than the wild-type AR, but it terminates in a 10–amino acid sequence that we believe to be unique. We determined that AR^v567es is not only constitutively active but also increases expression of full-length AR (AR^fl) in the absence of ligand.

Identification of AR variants in human prostate cancer xenografts. To identify alterations in the AR that could contribute to the growth of CRPC, we used RT-PCR to measure AR transcript size in a panel of 25 different prostate cancer xenografts, termed the LuCaP series. Most of the LuCaP xenografts were derived from metastases obtained from men with CRPC, after prolonged exposure to androgen-deprivation therapy (ADT); however, their responses to castration, when grown in SCID mouse hosts, vary (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI41824DS1). We used 2 sets of primers to amplify exons 1–3 and exons 2–8 of the AR^fl cDNA (NCBI accession number NG_009014).

As shown in Figure 1A, 2 of the LuCaP xenografts, 86.2 and 136, express shorter AR transcripts in the region spanning exons 2–8, compared with AR^fl amplified in the remaining xenografts. We sequenced the short AR transcripts from LuCaP 86.2 and 136 and found identical cDNA sequences. In comparison to AR^fl, the variant AR mRNA from the LuCaP xenografts 86.2 and 136 lacks exons 5, 6, and 7, which encode the LBD of AR (AR^v567es; GenBank GU208210) (Figure 1A). While the full nucleotide sequence of exon 8 is present, due to the splicing of exon 4 to exon 8, a frame shift occurs in the ORF of AR^v567es. This frame shift results in a stop codon after the first 30 nucleotides, and thus, the amino acid sequence of exon 8 in AR^v567es is shortened to a 10–amino acid sequence, when compared with the amino acid sequence of exon 8 in the AR^fl (Supplemental Figure 1). The AR^v567es protein, therefore, is predicted to be 739 amino acids, compared with the 920–amino acid protein for AR^fl. The 2 xenografts, 86.2 and 136, were derived from 2 different patients who had undergone ADT during their clinical course. To address whether the AR^v567es variant was derived from a genomic mutation or alternative mRNA splicing, we extracted genomic DNA from the LuCaP 86.2 and 136 xenografts and determined the sequence of each AR exon and the intron flanking regions and found no differences when compared with the reference AR entry in GenBank (NM_000044). These results suggest that the AR^v567es variant transcript is a result of alternative mRNA splicing. We then designed primers to specifically detect the exon 4–8 junction present in AR^v567es, reexamined all the xenografts for the presence of the variant, and detected some level of variant AR in almost all xenograft samples (Figure 1, A and B). Interestingly, xenograft pairs comprising an androgen-sensitive derivative and a castration-resistant derivative expressed higher mRNA levels of AR^v567es in the castration-resistant sample (denoted by AI) compared with the androgen-sensitive sample (35 vs. 35AI and 96 vs. 96AI; P < 0.05).

Figure 1

Identification of a novel AR splice variant. (A) Agarose gels showing PCR amplification of AR from human prostate LuCaP xenografts. Three sets of primers were used for PCR amplification of AR. One set is specific for exons 1–3 (amplicon 1), and another is specific for exons 2–8 (amplicon 2). The final set is specific for the 4–8 junction present in AR^v567es (amplicon 3). Note that xenografts 86.2 and 136 have a smaller PCR product with the amplicon 2 primers, while several xenografts are positive for the deletion using the amplicon 3 primers. Inverted agarose images are shown. NTC, no template control. (B) A graph of relative amounts of AR^v567es (amplicon 3) using qt-RT-PCR (mean ± 1 SD). Note that when xenografts occur as castrate-sensitive and castrate-resistant pairs (e.g., 35 and 35AI), the castrate-resistant sample (labeled AI) shows increased levels of AR^v567es. The differences were significant at P < 0.05 for 35 versus 35AI and 96 versus 96AI.

The AR splice variant AR^v567es is constitutively active. To investigate the function of AR^v567es in both benign and cancer prostate cells, we cotransfected the P69 benign immortalized prostate epithelial cell line and the AR-null M12 human prostate cancer cell line, with the AR activity reporter construct pGL3-probasin ARE-ARR3-luciferase (ARR3-Luc) and a construct expressing AR^v567es (referred to as P69 AR^v567es cells and M12 AR^v567es cells, respectively). We also transfected the AR-null M12 cells with a wild-type AR^fl (referred to as M12 AR^fl cells). The pcDNA-AR^v567es expression vector expressed a protein of appropriate size for the AR^v567es cDNA in both benign and cancer cells (Figure 2A and Figure 3A). Interestingly, expression of AR^v567es in the benign P69 cells also resulted in expression of AR^fl (Figure 2A). Normally, AR^fl is only seen in these cells following dihydrotestosterone (DHT) treatment. Expression of the variant AR did not have this effect in the M12 cells (Figure 3A), which remained AR negative even after DHT treatment. As shown in Figure 2B and Figure 3B, expression of AR^v567es resulted in marked activity of the ARR3 reporter in the absence of the AR ligand DHT. The P69 AR^v567es cells, unlike the M12 AR^v567es cells, demonstrated a further enhancement in reporter activity following DHT treatment (Figure 2B and Figure 3B). The AR antagonist flutamide had no affect on the ARR3 reporter activity in M12 AR^v567es cells, whereas flutamide completely blocked DHT transactivation in the M12 AR^fl cells (Figure 3B). These data are consistent with a constitutively active AR^v567es without a functional LBD. In contrast, cells expressing an AR^fl required DHT to transactivate the ARR3 reporter construct.

Figure 2

AR^v567es enhances AR^fl activity in benign prostate epithelial cells. (A) P69 SV40T immortalized, nontransformed human prostate epithelial cells transfected with AR^v567es demonstrated a marked increase in AR^fl protein compared with P69 pcDNA empty vector control cells (P69 pc). (B) ARR3-Luc reporter assay of P69 pcDNA control cells versus P69 AR^v567es cells, showing increased transcriptional activity basally and in response to DHT, consistent with increased AR^fl expression and AR^v567es expression (mean ± SEM). ^#P < 0.05, P69 AR^v567es compared with P69 pc control cells in the absence of androgen; *P < 0.01, P69 AR^v567es compared with P69 pc cells with and without DHT added.

Figure 3

Constitutive activation of the AR^v567es in M12 prostate cancer cells. (A) The AR-null M12 human prostate cancer cells were transiently transfected with AR^fl or the splice variant AR^v567es. The Western immunoblot of transfected cells shows expression of either AR^fl or AR^v567es. AR was detected with AR sc441 antibody, which detects both full-length and variant AR. GAPDH was used as a loading control. (B) ARE luciferase assay with the ARR3-Luc reporter. The M12 pcDNA empty vector control cells show that no AR activity is detected for any of the treatments. The M12 AR^fl cells had very low reporter activity in the absence of androgen as well as in the presence of the AR antagonist, flutamide, but had a clear increase in luciferase activity when 10^–9 M DHT was added. In contrast, M12 AR^v567es cells showed maximal reporter activity regardless of treatment. Values are mean ± SEM. *P < 0.01, DHT vs. no added DHT or DHT plus flutamide compared with DHT alone for AR^fl. There were no differences among treatments for pcDNA or AR^v567es cells. (C) Immunofluorescence staining of AR^fl and AR^v567es. In the absence of ligand, AR^fl is in the cytoplasm and translocates to the nucleus after addition of DHT. However, the constitutively active AR variant is primarily intranuclear in the absence of DHT and no change is seen when DHT is added. Nuclei are shown with DAPI staining. Arrows indicate examples of cells that are positive for nuclear translocation of AR. Scale bars: 10 μm.

To further evaluate the mechanism involved in constitutive AR^v567es activation, we stained the M12 cells transiently transfected with AR^fl or AR^v567es with an antibody specific to the N terminus of AR (sc441). In the absence of DHT, AR^fl was localized predominantly in the cytoplasm but translocated into the nucleus in the presence of DHT. In contrast, cells expressing AR^v567es consistently showed AR localized in the nucleus, regardless of the presence of DHT (Figure 3C).

Expression of AR^v567es in LNCaP cells increases the sensitivity of the endogenous AR to ligand. When AR^v567es was stably transfected into the AR^fl-positive LNCaP cells (LNCaP AR^v567es cells), we observed not only expression of the variant AR but also an increase in the amount of endogenous AR^fl protein compared with mock-transfected LNCaP cells (LNCaP pc cells) (Figure 4A and Figure 5A). This finding was consistent with observations in P69 cells engineered to express AR^v567es (Figure 2A). LNCaP AR^v567es cells grown in medium containing charcoal-stripped (CS) serum had the cuboidal appearance of control LNCaP pc cells grown in the presence of DHT (Figure 4B), whereas the control LNCaP pc cells grown in CS medium exhibited the expected elongated, stressed appearance of LNCaP cells grown without DHT. This alteration in morphology in the absence of DHT suggested that AR^v567es might function dominantly as a constitutively active AR in prostate cells that also express a AR^fl.

Figure 4

Increased AR^fl activity in LNCaP cells transfected with AR^v567es. The AR-positive LNCaP cells were transfected with AR^v567es or empty vector (pcDNA). (A) Western blot using AR sc441 antibody, which detects both AR^fl and AR^v567es. In cells transfected with AR^v567es, the AR^fl protein is markedly increased over that of control cells. Lanes were run on the same gel but were noncontiguous. (B) LNCaP cells were grown in vitro in CS serum. Cells containing the constituently active AR^v567es had the cuboidal appearance of the androgen-treated LNCaP cells. Original magnification, ×100. (C) Luciferase ARR3-Luc reporter assays performed on LNCaP cells showed a significant increase in AR signaling in the absence of DHT for cells expressing the AR^v567es protein. When DHT was added, there was a further increase in AR transactivation in the AR^v567es cells (133.2 RLU) compared with LNCaP pc control cells (54.6 RLU). *P < 0.01, **P < 0.001. (D) mRNA from several AR-regulated genes was examined by qt-RT-PCR (RQ). Interestingly, IGF-IR, which is regulated by nongenomic activities of the AR, was suppressed by the presence of AR^v567es, suggesting a loss of nongenomic AR activity by the variant. *P < 0.01, **P < 0.001, ***P < 0.0001, compared with LNCaP pc cells with same treatment. (E) MTS assay of LNCaP pc and LNCaP AR^v567es cells treated with decreasing concentrations of DHT. ^#P < 0.05, compared with LNCaP control cells. Values are mean ± SEM.

Figure 5

AR^v567es increases endogenous AR^fl expression and activity to similar levels as overexpression of AR^fl in LNCaP cells. (A) Western blot of LNCaP cells transfected with empty vector, AR^fl, or AR^v567es. Immunoblot with AR C-19 antibody, which only detects AR^fl. Note the increase in AR^fl following transfection with either AR^fl or AR^v567es compared with empty vector control. The panel on the right shows immunoblot using AR sc441 antibody to demonstrate presence of AR^v567es in LNCaP AR^v567es cells. The graph depicts relative amounts of AR^fl present, using LNCaP pc plus DHT as the baseline. Values are mean ± SEM. (B) ARR3-Luc reporter assay on cell lines from A, grown with and without DHT (10^–9 M). Note that for the LNCaP pc cells, DHT resulted in more than a 10-fold increase (P < 0.01) in reporter activity compared with baseline. This increase in activity is difficult to discern in this figure due to the scale used to show changes with the AR^v567es construct. Values are mean ± SEM. *P < 0.01, LNCaP AR^fl cells compared with LNCaP control cells with same treatment; **P < 0.001, LNCaP AR^v567es cells compared with LNCaP AR^fl cells with same treatment.

Following the introduction of the AR^v567es construct into LNCaP cells (referred to as LNCaP AR^v567es cells), an increase in basal ARR3 luciferase reporter activity was observed as well as a marked increase in reporter activity in response to DHT (Figure 4C), a finding that is concordant with the responses measured in P69 cells expressing AR^v567es. We then evaluated the DHT-induced changes in the expression of several androgen-induced genes (kallikrein-related peptidase 3 [PSA, also known as KLK3], transmembrane protease, serine 2 [TMPRSS2], FK506 binding protein 5 [FKBP5], NK3 homeobox 1 [NKX3.1], insulin-like growth factor 1 receptor [IGF1R]) using RT-PCR in the LNCaP pc cells versus the LNCaP AR^v567es cells (Figure 4D). The LNCaP AR^v567es cells had significantly higher mRNA levels for PSA, TMPRSS2, and FKBP5 compared with those of LNCaP pc cells with and without DHT (P < 0.001 and P < 0.0001 compared with LNCaP pc cells with same treatment.). NKX3.1 mRNA levels in the LNCaP AR^v567es cells were equally high, regardless of DHT exposure. Interestingly, IGF1R mRNA levels decreased in cells expressing AR^v567es compared LNCaP pc cells (Figure 4D), suggesting there is a functional difference between activation of AR^fl by ligand compared with the constitutively active AR^v567es. We expect that the difference in IGF1R mRNA levels is due to the fact that a nongenomic AR pathway regulates IGF-IR transcription; since AR^v567es primarily resides in the nucleus, the nongenomic pathway is not activated in these cells. Proliferation assays demonstrated that the LNCaP AR^v567es cells proliferated in response to lower concentrations of DHT than control LNCaP pc cells (Figure 4E). Together, these data suggest that the presence of AR^v567es enhances the transcriptional response of the endogenous AR^fl to androgens.

Because AR can autoregulate its own transcription and we saw an increase in AR^fl protein in the LNCaP AR^v567es cells, we wanted to determine whether the increased effect of the variant on androgen-regulated gene expression was simply through an increase in AR^fl protein levels. We overexpressed AR^fl in LNCaP cells and then performed ARR3-Luc reporter assays. Overexpression of AR^fl did result in increased AR^fl protein expression and increased reporter activity compared with that of LNCaP pc cells (Figure 5, A and B). But more importantly, even though LNCaP AR^v567es cells had a similar increase in AR^fl protein expression, they had significantly increased reporter activity compared with LNCaP AR^fl cells, indicating that the variant AR is affecting transcriptional activity to a greater degree than when AR^fl alone is amplified (Figure 5, A and B).

AR^v567es binds to AR^fl. To further explore the mechanisms by which AR^v567es increases the activity of AR^fl, we expressed both AR^fl and a HA-tagged AR^v567es in the AR-null M12 cells and immunoprecipitated AR^v567es from cell lysates with an anti-HA antibody. Western blots were done on the immunoprecipitates using an N terminus–directed AR antibody (sc441), which recognizes both AR^fl and AR^v567es, and a C terminus AR–specific antibody (C-19), which recognizes only AR^fl, to detect any immunocomplex of the 2 ARs. As shown in Figure 6A, AR^fl coprecipitated with AR^v567es in the presence or absence of DHT, indicating a physical association of AR^v567es with AR^fl. M12 cells transfected with the empty pcDNA vector were used as a negative control, since these cells lack an endogenous AR. As a positive control, we cotransfected both an untagged and a Flag-tagged AR^fl into M12 cells, immunoprecipitated AR using a Flag antibody, and then immunoblotted with AR C-19 antibody (Figure 6A). In these cells, dimerization of AR^fl required ligand as opposed to the association of AR^fl and AR^v567es observed in the M12 AR^fl and AR^v567es cells in the absence of DHT. We then transfected M12 cells with either the AR^fl construct alone or both the AR^fl and AR^v567es constructs and performed immunofluorescence staining using the AR C-19 antibody, which detects only AR^fl protein. When both AR^fl and AR^v567es were present, AR^fl translocated to the nucleus in the absence of ligand, whereas it remained in the cytoplasm in cells expressing only AR^fl (Figure 6, B and C). To determine whether an interaction between AR^fl and AR^v567es occurs in tumors expressing both AR types, we immunoprecipitated AR^fl from lysates taken from castrate-resistant xenografts using the AR C-19 antibody and then immunoblotted them with AR sc441. We detected both full-length and variant AR in LuCaP 136 (strong AR^v567es band) and LuCaP 35 (weak AR^v567es band) xenografts (Figure 6D). These results suggest that in cells that endogenously express AR splice forms, the AR^v567es can functionally interact with AR^fl.

Figure 6

The splice variant AR^v567es forms a complex with AR^fl. (A) Immunoprecipitate with an HA antibody in M12 cells double transfected with AR^fl and HA-AR^v567es, followed by immunoblotting with AR C-19, which only recognizes AR^fl, and AR sc441, which recognizes both AR^v567es and AR^fl. As a positive control, Flag-tagged AR^fl was transfected into M12 cells and brought down with a Flag antibody. Lanes were run on same gel but were noncontiguous. (B) M12 cells transfected with the AR^fl construct alone or in combination with the HA-AR^v567es construct. Cells were grown in serum-free media and then treated with DHT 10^-9 M or vehicle (EtOH). AR^fl was immunolabeled with the AR C-19 antibody (red) and nuclei were immunolabeled with DAPI (blue). In cells containing both AR^fl and AR^v567es, AR^fl translocates to the nucleus in the absence of ligand. Scale bar: 10 μm. (C) Relative quantitative nuclear translocation of AR^fl. A minimum of 100 AR-positive cells were included for each construct. For comparisons, the population with the lowest percentage of translocation was considered 0 (AR^fl with no DHT), and the population with the highest percentage of translocation was considered 1 (AR^v567es with DHT). Values are mean ± SEM. **P < 0.001, ***P < 0.0001, compared with AR^fl with same treatment. (D) Tumor lysates were made from LuCaP 35 and 136 xenografts taken from castrated SCID mice, immunoprecipitated with AR C-19, and immunoblotted with AR sc441. AR^v567es was brought down with AR^fl in the LuCaP 136 xenograft.

AR^v567es increases the stability of AR^fl protein. Because we observed an increase in AR^fl protein levels without a long-term increase in AR^fl mRNA levels (Figure 7A) and there appeared to be an interaction between AR^v567es and AR^fl, we sought to determine whether the interaction between the 2 ARs had an effect on mRNA stability or AR protein degradation. We examined AR mRNA stability following treatment with actinomycin D and found no differences between LNCaP pc and LNCaP AR^v567es cells (Figure 7B). We then determined whether the interaction of AR^v567es with AR^fl affected AR protein degradation. When translation was halted with cycloheximide, there was a slowing in degradation of the AR^fl in cells containing both receptors, particularly in the presence of DHT, when compared with cells with only the endogenous AR receptor (Figure 7, C and D). These data are also consistent with studies showing that AR activation increases AR protein through increased translation efficiency and a decrease in the rate of AR degradation (16).

Figure 7

Stability of AR^fl in presence of AR^v567es. (A) LNCaP cells were transfected with either the empty vector (pcDNA) or the AR^v567es construct. After transfection, RNA was extracted from cells at the times noted, and qt-RT-PCR was performed for AR^fl. The relative levels of AR^fl at each time point were compared with levels at time 0 hours. Following transfection with the AR^v567es construct, there was an increase in AR^fl mRNA at 3 hours that rapidly returned to control levels by 6 hours after transfection. ^#P < 0.05 AR^v567es vs. pcDNA. Values are mean ± SEM. (B) PCR for AR^fl in actinomycin D–treated LNCaP pc and LNCaP AR^v567es cells. Note that AR^v567es did not significantly affect AR^fl mRNA stability. (C) The Western blot of cycloheximide-treated LNCaP pc and LNCaP AR^v567es cells demonstrates that AR^v567es increases AR^fl protein stability in the presence of DHT. (D) Graph depicting relative protein levels of AR^fl following treatment with cycloheximide in LNCaP pc versus LNCaP AR^v567es cells. *P < 0.01 versus 2 hour time point. Values are mean ± SEM.

Targeting full-length and variant AR expression with the histone deacetylase inhibitor SAHA. Since the AR^v567es does not contain a LBD, androgen ablation has no effect on signaling. Thus, in tumor cells expressing AR^v567es, alternative approaches for abrogating AR signaling will be required. The histone deacetylase inhibitor SAHA has been shown to alter levels of AR protein by inhibiting AR transcription (15). Therefore, we grew LNCaP pc and LNCaP AR^v567es cells in vitro to 80% confluence with either CS serum or with CS serum plus DHT. SAHA was then added to the medium at the concentrations indicated in Figure 8A. In the absence of DHT, SAHA markedly decreased AR^fl expression in LNCaP pc cells and AR^v567es and endogenous AR^fl in the LNCaP AR^v567es cells. However, in the presence of DHT, SAHA only effectively decreased AR^fl expression and had a minimal effect on AR^v567es expression. In order to determine whether this same effect of androgens would occur on tumors that were resistant to castration and in which the primary AR consisted of AR^v567es, we cultured LuCaP 86.2 tumors in vitro and treated the cells with SAHA or SAHA plus DHT. SAHA was effective at decreasing AR^v567es in the presence and absence of DHT (Figure 8, B and C). Although SAHA decreased AR^v567es protein levels in the LuCaP 86.2 cells, the resulting decrease in growth (Figure 8D) cannot be conclusively attributed to loss of AR^v567es, since histone deacetylases have multiple effects on cells that result in suppression of cell proliferation (17, 18).

Figure 8

The effect of the histone deacetylase inhibitor, SAHA, on AR^v567es-expressing cells. (A) LNCaP pc or LNCaP AR^v567es cells were grown to 80 percent confluence in CS medium, with or without DHT (10^–9 M). SAHA was then added at the concentrations noted, and after 16 hours, cell lysates were collected, and Western blots were run with AR sc441 primary antibody, which detects both full-length and variant AR. (B) Two LuCaP 86.2 xenografts were removed and digested with collagenase to single cell suspensions. Cells were then plated in RPMI 5% CS medium, with or without DHT (one xenograft was grown with DHT, another without). Six hours later, cells were treated with either vehicle control (VC) or SAHA (5 mM). Six and twenty-four hours following treatment, cells were trypsinized and counted and cell lysates were collected for Western blots (blotted with AR sc441 antibody or GAPDH as a loading control). (C) Density of AR^v567es bands corrected for GAPDH. ^#P < 0.05, *P < 0.01, compared with vehicle control 6 or 24 hours. Values are mean ± SEM. (D) Cell counts corresponding to treatments in B. ^#P < 0.05, compared with 6 hour control. Values are mean ± SEM.

AR^v567es-regulated gene expression. In order to compare and contrast the gene expression program regulated by AR^v567es and AR^fl, we first measured transcript abundance changes in LNCaP cells expressing AR^fl following exposure to androgen. Cells were grown in CS serum, with or without 10^–9 M DHT, for 24 hours in triplicate. Following RNA isolation, transcript abundance levels were measured using whole-genome microarrays. Because the magnitude of changes was quite high between the AR^fl CS and AR^fl DHT-treated groups, t test significance was set at a stringent q value of less than 0.01%. A separate experiment compared transcript abundance in LNCaP cells expressing AR^fl or AR^v567es grown in CS medium. Gene expression differences between AR^fl CS and AR^v567es CS groups were more subtle, and genes exhibiting q values of less than 10% were included in order to cross-compare similar numbers of gene expression changes between the experiments (Figure 9). We performed an analysis of Gene Ontology (GO) using the EASE software tool, which calculates overrepresentation statistics for GO terms in the significant list, with respect to all genes represented in the data set (Supplemental Tables 2 and 3, GO analysis statistics).

Figure 9

Expression profiles of LNCaP pc and LNCaP AR^v567es cells. (A) Venn diagrams showing the gene number per comparison for expression profiles of androgen-regulated and AR^v567es-regulated genes in LNCaP cell lines. Cells were grown in CS medium with or without DHT (10^–9 M) for 24 hours, in triplicate experiments. Microarray analysis using Agilent 44K whole human genome expression oligonucleotide microarray slides was performed. Statistical analysis was conducted using 2-sample, unpaired t test with the SAM software, with a q value of less than 0.01% considered statistically significant for AR^fl cells and a q value of less than 10% considered statistically significant for AR^v567es cells. I and IV indicate genes upregulated or downregulated by DHT in LNCaP cells; II and V indicate genes upregulated or downregulated by DHT in LNCaP cells or LNCaP AR^v567es cells compared with LNCaP cells with no DHT; and III and VI indicate genes regulated by AR^v567es in LNCaP cells compared with LNCaP cells without DHT. (B) The top AR-regulated genes in the LNCaP AR^v567es cells compared with LNCaP controls grown in CS medium. Note that the right hand column shows changes in genes previously described to be regulated by DHT in LNCaP cells. Results of GO analysis of genes upregulated or downregulated by DHT in AR cells or genes uniquely upregulated or downregulated in AR^v567es cells are listed in Supplemental Tables 2 and 3.

We determined that well-known androgen-regulated genes, such as PSA, TMPRSS2, and FKBP5, altered by DHT treatment in cells expressing AR^fl, were also altered in the context of AR^v567es expression in the absence of exogenous androgens, confirming the hypothesis that AR^v567es is capable of activating the AR-regulated gene expression program in the setting of castration. We further found that AR^v567es regulates a subset of genes that we believe to be unique that are not influenced by androgens in the context of the AR^fl. Analysis of GO terms enriched specifically in AR^v567es cells revealed that GO molecular function “transcription factor activity” is significantly increased in the absence of androgen, potentially signifying activation of other growth and survival pathways. Among the transcription factors upregulated by AR^v567es that are known to induce a proliferative program of gene expression were STAT3, which has antiapoptotic as well as proliferative effects, and JUN, which behaves as a positive regulator of cell growth by protecting cells from p53-dependent senescence and apoptosis (19–21). AR^v567es also activated genes involved in the metabolism of androgens. Interestingly, even in the absence of exogenous AR ligands, cells expressing AR^v567es demonstrated a signature of androgen metabolism, with enriched GO terms of biological processes “steroid biosynthesis” and “sterol metabolism.” Such ligand-independent stimulation of steroidogenic pathways in AR^v567es cells may provide a survival advantage in a low-androgen environment.

In addition to differences in the genomic signaling programs between AR^v567es and AR^fl, our data also indicate that these variant receptors may differentially regulate other AR activities. In this context, AR^v567es altered components of IGF pathway signaling in a manner distinct from that of the AR^fl. IGF-IR expression was enhanced by ligand stimulation of AR^fl, whereas AR^v567es suppressed IGF-1R expression (Figure 4D and Figure 9B). Pandini et al. have shown that the AR stimulates IGF-IR expression through a nongenomic pathway, involving binding of AR^fl to Src and downstream activation of the transcription factor MAPK (22). Our cell-based localization studies indicated that AR^v567es was rapidly targeted to the nucleus and, during this process, facilitated the movement of AR^fl as well. Thus, AR^v567es may have a primary effect through enhanced genomic AR activity and the concomitant suppression of nongenomic AR activity exerted by AR^fl.

AR^v567es enhances the growth of prostate cancer following ADT in vivo. Since AR^v567es is constitutively activated and also transactivates AR^fl in the absence of androgen, we next sought to determine whether AR^v567es influences prostate cancer responses to ADT in vivo. We implanted LNCaP AR^v567es cells or control LNCaP pc cells into immunocompromised nu/nu mice in subcutaneous locations. In the initial androgen-sensitive growth phase in eugonadal animals, there was no difference in tumor growth rate among the cell lines inoculated (data not shown). After tumors attained a size of approximately 0.2 cc, the mice were castrated, and tumor volumes were measured over a 13-week time period. We did not observe significant growth of the grafts comprised of LNCaP pc cells. However, grafts comprised of LNCaP AR^v567es cells were measurably larger than those of control cells by the 10-week time point (tumor volume of LNCaP pc vs. LNCaP AR^v567es cells; P < 0.01), and subsequent rapid growth required animal sacrifice by 13 weeks due to tumor size (Figure 10A). We examined the resulting tumors, resected at the study endpoint for AR transcript levels and splice variants. In the small LNCaP pc tumors surviving castration, transcript levels encoding AR^fl were not significantly different relative to levels in cells prior to castration, and the AR^v567es remained undetectable. In contrast, in LNCaP AR^v567es tumors the ratio of AR^v567es transcripts to those encoding AR^fl increased significantly from 0.5 to 4.3 (P < 0.05). These results support evolution of a tumor cell population favoring the selection of cells expressing higher levels of the AR^v567es variant.

Figure 10

Tumor growth of LNCaP AR^v567es cells and xenografts expressing various amounts of AR^v567es. (A) 1 × 10⁶ LNCaP pc or LNCaP AR^v567es cells were mixed 1:1 with Matrigel and injected s.c. into athymic nude mice (n = 10 per line). When tumors reached a volume of 100–200 mm³, mice were castrated and animals were followed until tumors regrew and reached a volume of 1,000 mm³ or met IACUC criteria for euthanasia. There was no difference in growth rate in intact mice. Following castration, the LNCaP AR^v567es tumors, which did not decrease in volume following castration, grew to a significantly larger volume, more quickly than those of controls. *P < 0.01, LNCaP AR^v567es tumor volume versus LNCaP pc tumor volume. Values are mean ± SEM. (B–D) The response of tumor volume to castration in 3 different xenografts (intact, n = 12 per xenograft; castrate, n = 12 per xenograft). Note that LuCaP 86.2, which has the majority of its AR in the AR^v567es form, had no castration response; LuCaP 136, which has both full-length and variant AR, had a modest response to castration; and LuCaP 35, which has the majority of its AR as AR^fl, had a marked decrease in tumor volume in response to castration. Values are mean ± SEM. (E) Western blots of representative xenografts before and 6 weeks after castration using AR sc441, which recognizes AR^v567es and AR^fl. Lanes were run on different gels.

We next examined the response of prostate cancers expressing endogenous AR^v567es to ADT. We chose 3 representative LuCaP xenografts, which have distinct differences in the expression of AR^fl and AR^v567es (Figure 10E). The LuCaP 35 xenograft expressed AR^fl, with little detectable AR^v567es. In addition, we have previously published that LuCaP 35 xenografts maintain high levels of intratumoral DHT following castration (1.7 ± 0.27 ng/g of tumor tissue precastration versus 1.5 ± 0.48 ng/g of tumor tissue after castration; ± SD; P = NS) (8, 9). The LuCaP 136 xenograft expressed AR^fl and had high intratumoral DHT levels (2.4 ± 0.9 ng/g) in eugonadal animals, but following castration there was a significant decrease in intratumoral DHT (0.15 ± 0.03 ng/g; P < 0.001) and increased expression of AR^v567es (Figure 10E). Finally, the LuCaP 86.2 xenograft, which had low levels of intratumoral DHT regardless of castration status (0.4 ± 0.04 ng/g precastration vs. 0.1 ± 0.04 ng/g after castration; P < 0.05), expressed primarily AR^v567es (Figure 10E). We implanted replicate xenografts subcutaneously into SCID mouse hosts, 24 animals per group, and after achieving a tumor volume of 0.2 cc, half of the animals in each group were subjected to ADT by surgical castration. Tumor responses correlated with the level of AR^v567es expression. No response to ADT was seen for the LuCaP 86.2 xenograft, which predominately expressed AR^v567es (Figure 10, B and E). The growth of LuCaP 136 tumors, which express a mix of AR^fl and AR^v567es following castration, was significantly suppressed following ADT, relative to eugonadal animals (14-week time point; P < 0.01), although there was a slow progressive increase in tumor volumes over time (Figure 10, C and E). LuCaP 35 tumors, which predominately express AR^fl, exhibited a sustained response to ADT, with minimal tumor growth measured over 14 weeks (Figure 10D).

Evolution of AR^v567es in prostate xenografts. Since AR^v567es is a splice variant and generation of the variant is posttranscriptional, acquisition of AR^v567es by the prostate could be considered an adaptive mechanism rather than a selected event, such as those encoded by a mutation in AR DNA. This would appear to be the case for the LuCaP 136 and 86.2 xenografts. When we evaluated the original tissue specimens from which these xenografts were derived, ascites cells and a bladder metastasis, respectively, only AR^fl mRNA was present in the original LuCaP specimen, whereas both AR^fl and AR^v567es mRNA were present in the LuCaP 86.2 original specimen (Supplemental Figure 2). This finding was consistent with the LuCaP 136 xenograft initially responding to castration in the mouse, but the 86.2 xenograft having no response. However, after castration and continued passage in SCID mice, AR^v567es mRNA became the dominant AR mRNA in the LuCaP 86.2 xenograft, and AR^v567es mRNA was generated in the LuCaP 136 xenograft (Supplemental Figure 2 and Figure 10E). Further, as mentioned earlier, in those LuCaP xenografts with pairs of castrate-sensitive and castrate-resistant tumors, there was a significant increase in AR^v567es expression in the castrate-resistant tumors compared with castrate-sensitive tumors (Figure 1). These data indicate that the generation of the splice variants is a dynamic process that occurs in response to castration pressures exerted on the AR signaling program.

AR splice variants are rarely expressed in benign prostate epithelium, and levels are increased in men with prostate cancer. To further evaluate the contribution of the AR^v567es variant to prostate cancer, we investigated the distribution of the AR splice variants directly in normal and neoplastic human prostate epithelium. We first acquired benign prostate epithelium by laser-capture microdissection (LCM) from prostate biopsies of 36 normal men, without evidence of prostate cancer, enrolled in a study of male contraception (acycline or DHT gel). Using quantitative RT-PCR (qt-RT-PCR) to quantitate transcripts isolated from benign epithelium, we were able to detect mRNAs encoding AR^fl in 35 subjects, and 6 out of 36 (17%) men expressed AR^v567es, 2 out of 36 (6%) men expressed the recently described AR splice variant termed AR-V7, and 1 out of 36 (3%) men expressed the AR splice variant termed AR3 (13, 14) (Table 1 and Figure 11A). The men who expressed AR variants, while they did not have prostate cancer, had either been castrated using the LHRH receptor antagonist acycline or had tissue androgens suppressed by administration of DHT gel.

Figure 11

Expression of the splice variant AR^v567es in human prostates. (A) AR variant PCR products from laser-captured samples of benign (B) and malignant tissue (T) from prostate tissue obtained at the time of prostatectomy from non-castrate men. Note that tumor or benign tissue may be positive in these samples. Samples 105–115 are PCR products from men, aged 35–55 years, with no evidence of prostate cancer, who were enrolled in a male contraception study. (B) Results of variant AR PCR products from metastases in a man who died from his prostate cancer. Table 1 shows results for all metastases samples. Variants include the AR^v567es described in the current report and 2 variants previously described, AR3 and AR-V7. GAPDH was used as a control for adequacy of RNA in the sample. Primers are described in the Methods section. If GAPDH could not be amplified, the sample was not included in the study. Inverted agarose images are shown for A and B. (C) Diagram of AR^v567es variant compared with previously published AR variants (AR^1/2/2b in ref. 30; AR-V7 in ref. 14; and AR3 in ref. 13). RRP, radical retropubic prostatectomy.

Table 1

Number of metastases positive for AR^fl or AR splice variants

Since we found AR^v567es in our group of normal men, we next evaluated whether in primary (untreated) prostate cancer specimens the variant was in the malignant epithelium, benign epithelium, or both; if primarily in the malignant epithelium, this might suggest that the presence of a variant was etiologic in cancer development. Therefore, we examined cDNA from laser-captured, matched benign and malignant epithelium of primary prostatectomy tissue. We found that the variants were expressed in both benign and malignant prostate epithelial tissue (Figure 11A). These data suggest that the presence of AR variants is not necessarily etiologic in the initiation of prostate cancer.

Variant AR expression in prostate cancer metastases. To determine the frequency of AR splice variant expression in advanced prostate cancers, we successfully amplified cDNA from 69 metastases, derived from 13 patients undergoing a rapid necropsy for tumor acquisition. All of these patients were documented to have either surgical or chemical castration, with tumor progression to a clinical state of CRPC. Tumor tissue was isolated by laser-assisted microdissection, and RNA integrity was verified. Despite amplification of GAPDH, no full-length or variant AR expression was detected using RT-PCR in 23 samples. These 23 metastases were from patients whose primary tumors had a neuroendocrine phenotype and would not be expected to depend on functional AR. Of the remaining 46 metastases, 37 out of 46 (80%) expressed AR^fl, 20 out of 46 (43%) expressed AR^v567es, 11 out of 46 (24%) expressed AR-V7, and 3 out of 46 (6%) expressed AR3 (Table 1 and Figure 11B). Interestingly, 9 out of 46 (20%) metastases expressed only the AR^v567es variant, but expression of AR-V7 or AR3 occurred concomitantly with AR^fl expression. Overall, 27 out of 46 (59%) AR-positive metastases expressed one or more AR splice variants. When evaluating all of the metastases from each individual patient collectively, 12 out of 13 patients had at a minimum one metastasis that was positive for at least one AR splice variant, and 10 out of 13 patients had at a minimum one metastasis that was positive for AR^v567es. Further, the presence of an AR variant was clustered among patients. For example, the majority of metastases for one patient may have been positive for a variant, whereas a different patient may have very few metastases that were positive for that variant. No patient had all samples positive for the same AR variant.

The progression of prostate cancer to an AR-independent state has recently become regarded as a misnomer (8–10, 23). Transcriptional activation of the AR has been suggested to occur through several nonsteroidal factors, such as IL-6, EGF, integrins, and insulin-like growth factors as well as by increased expression of AR cofactors (3, 24–29). Another mechanism for AR-regulated prostate cancer progression following castration is an alteration in AR protein. This alteration has been reported to occur by genomic mutations: either ligand-dependent activating mutations or ligand-independent activating mutations (12). Genomic mutations in the AR are not common, but those that result in an AR that functionally increases AR activity and lead to CRPC are even less common. Recently, constitutively active posttranslational splice variants of the AR have been reported in human prostate cell lines, xenografts, and human tissues (13, 14, 30).

In this paper, we describe a splice variant of the AR that we believe to be new, in which exon 4 has been spliced to exon 8, and thus, exons 5, 6, and 7 have been deleted. This alternative splicing event leads to a frame shift, so that 10 amino acids that we believe to be unique are encoded for and followed by a stop codon, resulting in an AR variant protein that is 180 amino acids shorter than the AR^fl protein. Thus, this variant, unlike the previously reported AR splice variants, retains the hinge region, which is necessary for complete nuclear translocation of the AR (31). Similar to previously reported AR splice variants, we show that the AR splice variant produced by this event is constitutively active (13, 14, 30). Unlike other reports, though, in which variants were cloned using a 3′ RACE method in a prostate cancer cell line or in which the authors created potential deletion mutants based on the different splicing sites in the AR, we identified AR^v567es through the analysis of 25 distinct human prostate xenografts. We originally sequenced the ORF of the AR in the xenografts using 2 sets of PCR primers, as described. Since these primers detect the predominant AR mRNA species, 2 of the xenografts, 86.2 and 136, had the splice variant as the majority of their AR. When specific primers were designed to detect the 4–8 junction present in AR^v567es, the variant was found in several additional xenografts. We also noted that in the same xenograft, the level of the AR^v567es was not constant and varied depending on passage number. When we examined the primary human tumors from which the LuCaP 86.2 and 136 xenografts were developed, we noted that the variant AR had always been present in LuCaP 86.2, while in LuCaP 136 the AR^fl was the predominate receptor. However, the AR variant became more predominant in the LuCaP 136 xenograft following long-term growth in mice as well as after it was grown in castrated mice. These data suggest that the level of splice variant AR is not fixed but is dynamic and likely regulated by environmental factors, including decreasing androgen levels (12, 13).

We expressed this new AR variant in both benign and prostate cancer cell lines and found that AR^v567es functions like a constitutively active AR in both types of cells and is resistant to the effects of flutamide on AR activity. Further, we report that in cells expressing endogenous AR^fl, AR^v567es increases the level of endogenous receptor by slowing the rate of protein degradation.

Since a constitutively active AR could lead to the progression of the tumor after castration, we examined expression of the AR^v567es variant in specimens of prostate cancer from men who had died from their cancer. All of these men were castrated at the time specimens were obtained; while a third of the samples did not have any detectable AR, over 50% of the specimens that contained detectable AR transcripts were positive for one or more of the constitutively active variants. These included biopsies from the prostate as well as lymph node, lung, liver, and bone. Interestingly, the presence of AR^v567es was not distributed randomly among the patients but rather the variant clustered within a patient’s specimens. Further, not all specimens within a patient were positive for an AR variant, consistent with a hypothesis of ongoing splicing activity that generates AR variants. We also examined specimens for the presence of 2 other AR variants recently reported to affect survival in prostate cancer, AR3 and AR-V7 (13, 14). AR^v567es was the most common of the 3 variants examined and several specimens contained more than one AR variant. Additionally, nearly all of the specimens that contained one or more of the variants also contained a AR^fl. These findings are not surprising, since there are multiple mechanisms by which the AR continues to be activated following castration.

Since AR splicing is determined in part by differences in androgen levels, we asked whether the splice variants might be present in men without cancer and in benign epithelium in men with cancer. In young men, ages 35–55, with normal PSA levels of less than 2.0 ng/ml and no clinical evidence of prostate cancer, who had a prostate biopsy as part of their participation in a male contraceptive study, we found that the AR^v567es variant was occasionally expressed in prostate epithelium of men receiving treatment but not in men receiving placebo. In a separate set of non-castrate older men, ages 59–70 years, who had a radical prostatectomy for prostate cancer, analysis of paired laser-captured benign and malignant prostate epithelial samples showed that AR splice variants could be found in benign, malignant, or both specimens from the men. These data suggest that expression of AR variants is not necessarily etiologic in cancer development, but if AR variants are present, they might serve to facilitate progression when hormone suppression is applied. This phenomenon has been demonstrated in a mouse model (32) as well in our current data, using various human xenografts grown in castrated versus intact SCID mice.

In this paper, we have shown that some prostate tumors may express only the variant AR receptor. However, while expression of AR splice variants is of interest and their functions have been examined as specific entities, the vast majority of splice variants are expressed in cells, along with a full-length receptor. Dimerization of full-length receptors occurs through both AF1 and AF2 sites as well as other N-terminal/C-terminal (N/C) interactions (33). Whereas most steroid receptors require the N/C interaction for binding of cofactors and subsequent receptor activation, the AR is unique in that the AR AF2 domain has evolved such that it preferentially interacts with FxxLF-like motifs contained in the AR N-terminal domain (23FQNLF27 and 433WHTLF437) over LxxLL-like motifs in the coregulatory proteins’ amino termini. As such, an AR lacking the LBD is still active, but now ligand cannot induce dimerization (33).

Although we and others have shown the constitutive function of the AR variants independent of AR^fl, the majority of tumors express both ligand-dependent and constitutively active splice receptors. In this study, we believe that we have shown for the first time that a constitutively active AR splice variant, AR^v567es, interacts with AR^fl. We demonstrated that we could pull down variant AR with AR^fl, not only in prostate cancer cells lines in which we overexpressed the variant, but also in human xenografts that endogenously express both full-length and variant AR protein. Further, we show that the variant causes translocation of the AR^fl in the absence of ligand, which results in enhanced AR transcriptional activity and increased cell proliferation in response to very low concentrations of ligand. However, the characterization of where the splice variant binds and interacts with AR^fl has yet to be determined.

Recent studies and data we presented here have shown that after standard androgen ablation, such as castration, levels of androgen in the prostate are still measurable. In our studies, using a panel of human xenografts that we believe to be unique, we further show a strong correlation between increased expression of the different AR variants in tumors and low intratumoral levels of androgens. In particular, the AR^v567es is expressed most abundantly in tumors with the lowest levels of androgens. Thus, the expression of the AR variants, including the AR^v567es variant that we believe to be unique, provides a mechanism to enable cancer progression in the face of decreased androgens.

AR^v567es contains the entire DNA-binding domain and nuclear localization sequence (Supplemental Figure 1). It is clear that AR^v567es is constitutively active and can function without ligand or AR^fl. On the other hand, we have also shown that AR^v567es can form a heterodimer with AR^fl and enhance its activity (Figure 4). Thus, for the first time to our knowledge, we show that a constitutively active AR variant generated by castration in human tumors may function in a ligand-independent manner or enhance the ligand-dependent function of the AR^fl. Either one or both of these mechanisms may be active in a tumor cell when both variant and AR^fl coexist.

Since AR^v567es does not bind ligand and has no N/C interaction, it might be expected that its interaction with AR coregulators and its binding to androgen response elements (AREs) may be altered. We have shown the latter in this study, since we saw enhanced activation of ARE-containing reporter constructs in cells overexpressing AR^v567es compared with cells simply overexpressing AR^fl. Further, in cells expressing AR^v567es, we observed a decrease in genes that are upregulated via the nongenomic pathway, including IGF-1R. This would suggest that the repertoire of genes activated by the splice variant receptors will have similarities and differences with that of the full-length ligand-dependent AR. In this study, we have seen that there are significant differences in profiles of androgen-regulated genes in LNCaP cells that only express the endogenous AR and those that express the splice variant, even when androgens are added to the culture medium of the control LNCaP cells. These differences in gene expression by cells that express the AR splice variants may be significant, because the AR has tumor-promoting and -suppressing properties. Since the AR splice variants are associated with cancer progression, the differences in gene expression may help to delineate those AR-regulated genes involved in tumor progression.

Using data from the gene arrays comparing xenografts with high and low levels of expression of the AR^v567es variant, we were able to identify a panel of splicing factors that were differentially expressed. Specific regulation of these factors by androgens has not been determined, although several of the factors have been implicated in generating variability of prostate tumors (34–37). If specific splicing factors can be identified that generate these AR splice variants, inhibition of these factors at the time of androgen ablation could significantly enhance the effects of androgen ablation and prevent or delay development of castration-resistant disease.

In summary, we demonstrate in this report that AR splice variants that contribute to castration resistance are common in prostate cancer. There appears to be a family of AR variants, generated after androgen ablation, that results in a constitutively active AR. Identification of these variant receptors may serve as a biomarker for men destined for early recurrence and who are candidates for therapy directly targeting the AR rather than ligand.

View Supplemental data

We would like to thank Jennifer Wu and Marco Marcelli for critical review of the manuscript and Martín Escandón for assistance with manuscript preparation. Grants from the NIH-NCI (PO1 CA 85859 to S.R. Plymate, R. Vessella, and P.S. Nelson), the Pacific Northwest Prostate Cancer SPORE (P50 CA 097186 to S.R. Plymate, R. Vessella, and P.S. Nelson), and the Veterans Affairs Research Program (to S.R. Plymate) supported this work.

Address correspondence to: Stephen R. Plymate, University of Washington School of Medicine, Harborview Medical Center, Box 359625, 325 9th Avenue, Seattle, Washington 98104, USA. Phone: 206.897.5336; Fax: 206.897.5396; E-mail: splymate@u.washington.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(8):2715–2730. doi:10.1172/JCI41824.

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