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 ARfl 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 ARfl 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 ARfl, the variant AR mRNA from the LuCaP xenografts 86.2 and 136 lacks exons 5, 6, and 7, which encode the LBD of AR (ARv567es; 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 ARv567es. This frame shift results in a stop codon after the first 30 nucleotides, and thus, the amino acid sequence of exon 8 in ARv567es is shortened to a 10–amino acid sequence, when compared with the amino acid sequence of exon 8 in the ARfl (Supplemental Figure 1). The ARv567es protein, therefore, is predicted to be 739 amino acids, compared with the 920–amino acid protein for ARfl. 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 ARv567es 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 ARv567es variant transcript is a result of alternative mRNA splicing. We then designed primers to specifically detect the exon 4–8 junction present in ARv567es, 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 ARv567es 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 1Identification 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 ARv567es (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 ARv567es (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 ARv567es. The differences were significant at P < 0.05 for 35 versus 35AI and 96 versus 96AI.
The AR splice variant ARv567es is constitutively active. To investigate the function of ARv567es 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 ARv567es (referred to as P69 ARv567es cells and M12 ARv567es cells, respectively). We also transfected the AR-null M12 cells with a wild-type ARfl (referred to as M12 ARfl cells). The pcDNA-ARv567es expression vector expressed a protein of appropriate size for the ARv567es cDNA in both benign and cancer cells (Figure 2A and Figure 3A). Interestingly, expression of ARv567es in the benign P69 cells also resulted in expression of ARfl (Figure 2A). Normally, ARfl 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 ARv567es resulted in marked activity of the ARR3 reporter in the absence of the AR ligand DHT. The P69 ARv567es cells, unlike the M12 ARv567es 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 ARv567es cells, whereas flutamide completely blocked DHT transactivation in the M12 ARfl cells (Figure 3B). These data are consistent with a constitutively active ARv567es without a functional LBD. In contrast, cells expressing an ARfl required DHT to transactivate the ARR3 reporter construct.
Figure 2ARv567es enhances ARfl activity in benign prostate epithelial cells. (A) P69 SV40T immortalized, nontransformed human prostate epithelial cells transfected with ARv567es demonstrated a marked increase in ARfl protein compared with P69 pcDNA empty vector control cells (P69 pc). (B) ARR3-Luc reporter assay of P69 pcDNA control cells versus P69 ARv567es cells, showing increased transcriptional activity basally and in response to DHT, consistent with increased ARfl expression and ARv567es expression (mean ± SEM). #P < 0.05, P69 ARv567es compared with P69 pc control cells in the absence of androgen; *P < 0.01, P69 ARv567es compared with P69 pc cells with and without DHT added.
Figure 3Constitutive activation of the ARv567es in M12 prostate cancer cells. (A) The AR-null M12 human prostate cancer cells were transiently transfected with ARfl or the splice variant ARv567es. The Western immunoblot of transfected cells shows expression of either ARfl or ARv567es. 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 ARfl 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 ARv567es 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 ARfl. There were no differences among treatments for pcDNA or ARv567es cells. (C) Immunofluorescence staining of ARfl and ARv567es. In the absence of ligand, ARfl 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 ARv567es activation, we stained the M12 cells transiently transfected with ARfl or ARv567es with an antibody specific to the N terminus of AR (sc441). In the absence of DHT, ARfl was localized predominantly in the cytoplasm but translocated into the nucleus in the presence of DHT. In contrast, cells expressing ARv567es consistently showed AR localized in the nucleus, regardless of the presence of DHT (Figure 3C).
Expression of ARv567es in LNCaP cells increases the sensitivity of the endogenous AR to ligand. When ARv567es was stably transfected into the ARfl-positive LNCaP cells (LNCaP ARv567es cells), we observed not only expression of the variant AR but also an increase in the amount of endogenous ARfl 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 ARv567es (Figure 2A). LNCaP ARv567es 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 ARv567es might function dominantly as a constitutively active AR in prostate cells that also express a ARfl.
Figure 4Increased ARfl activity in LNCaP cells transfected with ARv567es. The AR-positive LNCaP cells were transfected with ARv567es or empty vector (pcDNA). (A) Western blot using AR sc441 antibody, which detects both ARfl and ARv567es. In cells transfected with ARv567es, the ARfl 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 ARv567es 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 ARv567es protein. When DHT was added, there was a further increase in AR transactivation in the ARv567es 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 ARv567es, 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 ARv567es cells treated with decreasing concentrations of DHT. #P < 0.05, compared with LNCaP control cells. Values are mean ± SEM.
Figure 5ARv567es increases endogenous ARfl expression and activity to similar levels as overexpression of ARfl in LNCaP cells. (A) Western blot of LNCaP cells transfected with empty vector, ARfl, or ARv567es. Immunoblot with AR C-19 antibody, which only detects ARfl. Note the increase in ARfl following transfection with either ARfl or ARv567es compared with empty vector control. The panel on the right shows immunoblot using AR sc441 antibody to demonstrate presence of ARv567es in LNCaP ARv567es cells. The graph depicts relative amounts of ARfl 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 ARv567es construct. Values are mean ± SEM. *P < 0.01, LNCaP ARfl cells compared with LNCaP control cells with same treatment; **P < 0.001, LNCaP ARv567es cells compared with LNCaP ARfl cells with same treatment.
Following the introduction of the ARv567es construct into LNCaP cells (referred to as LNCaP ARv567es 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 ARv567es. 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 ARv567es cells (Figure 4D). The LNCaP ARv567es 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 ARv567es cells were equally high, regardless of DHT exposure. Interestingly, IGF1R mRNA levels decreased in cells expressing ARv567es compared LNCaP pc cells (Figure 4D), suggesting there is a functional difference between activation of ARfl by ligand compared with the constitutively active ARv567es. We expect that the difference in IGF1R mRNA levels is due to the fact that a nongenomic AR pathway regulates IGF-IR transcription; since ARv567es primarily resides in the nucleus, the nongenomic pathway is not activated in these cells. Proliferation assays demonstrated that the LNCaP ARv567es cells proliferated in response to lower concentrations of DHT than control LNCaP pc cells (Figure 4E). Together, these data suggest that the presence of ARv567es enhances the transcriptional response of the endogenous ARfl to androgens.
Because AR can autoregulate its own transcription and we saw an increase in ARfl protein in the LNCaP ARv567es cells, we wanted to determine whether the increased effect of the variant on androgen-regulated gene expression was simply through an increase in ARfl protein levels. We overexpressed ARfl in LNCaP cells and then performed ARR3-Luc reporter assays. Overexpression of ARfl did result in increased ARfl protein expression and increased reporter activity compared with that of LNCaP pc cells (Figure 5, A and B). But more importantly, even though LNCaP ARv567es cells had a similar increase in ARfl protein expression, they had significantly increased reporter activity compared with LNCaP ARfl cells, indicating that the variant AR is affecting transcriptional activity to a greater degree than when ARfl alone is amplified (Figure 5, A and B).
ARv567es binds to ARfl. To further explore the mechanisms by which ARv567es increases the activity of ARfl, we expressed both ARfl and a HA-tagged ARv567es in the AR-null M12 cells and immunoprecipitated ARv567es 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 ARfl and ARv567es, and a C terminus AR–specific antibody (C-19), which recognizes only ARfl, to detect any immunocomplex of the 2 ARs. As shown in Figure 6A, ARfl coprecipitated with ARv567es in the presence or absence of DHT, indicating a physical association of ARv567es with ARfl. 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 ARfl into M12 cells, immunoprecipitated AR using a Flag antibody, and then immunoblotted with AR C-19 antibody (Figure 6A). In these cells, dimerization of ARfl required ligand as opposed to the association of ARfl and ARv567es observed in the M12 ARfl and ARv567es cells in the absence of DHT. We then transfected M12 cells with either the ARfl construct alone or both the ARfl and ARv567es constructs and performed immunofluorescence staining using the AR C-19 antibody, which detects only ARfl protein. When both ARfl and ARv567es were present, ARfl translocated to the nucleus in the absence of ligand, whereas it remained in the cytoplasm in cells expressing only ARfl (Figure 6, B and C). To determine whether an interaction between ARfl and ARv567es occurs in tumors expressing both AR types, we immunoprecipitated ARfl 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 ARv567es band) and LuCaP 35 (weak ARv567es band) xenografts (Figure 6D). These results suggest that in cells that endogenously express AR splice forms, the ARv567es can functionally interact with ARfl.
Figure 6The splice variant ARv567es forms a complex with ARfl. (A) Immunoprecipitate with an HA antibody in M12 cells double transfected with ARfl and HA-ARv567es, followed by immunoblotting with AR C-19, which only recognizes ARfl, and AR sc441, which recognizes both ARv567es and ARfl. As a positive control, Flag-tagged ARfl 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 ARfl construct alone or in combination with the HA-ARv567es construct. Cells were grown in serum-free media and then treated with DHT 10-9 M or vehicle (EtOH). ARfl was immunolabeled with the AR C-19 antibody (red) and nuclei were immunolabeled with DAPI (blue). In cells containing both ARfl and ARv567es, ARfl translocates to the nucleus in the absence of ligand. Scale bar: 10 μm. (C) Relative quantitative nuclear translocation of ARfl. 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 (ARfl with no DHT), and the population with the highest percentage of translocation was considered 1 (ARv567es with DHT). Values are mean ± SEM. **P < 0.001, ***P < 0.0001, compared with ARfl 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. ARv567es was brought down with ARfl in the LuCaP 136 xenograft.
ARv567es increases the stability of ARfl protein. Because we observed an increase in ARfl protein levels without a long-term increase in ARfl mRNA levels (Figure 7A) and there appeared to be an interaction between ARv567es and ARfl, 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 ARv567es cells (Figure 7B). We then determined whether the interaction of ARv567es with ARfl affected AR protein degradation. When translation was halted with cycloheximide, there was a slowing in degradation of the ARfl 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 7Stability of ARfl in presence of ARv567es. (A) LNCaP cells were transfected with either the empty vector (pcDNA) or the ARv567es construct. After transfection, RNA was extracted from cells at the times noted, and qt-RT-PCR was performed for ARfl. The relative levels of ARfl at each time point were compared with levels at time 0 hours. Following transfection with the ARv567es construct, there was an increase in ARfl mRNA at 3 hours that rapidly returned to control levels by 6 hours after transfection. #P < 0.05 ARv567es vs. pcDNA. Values are mean ± SEM. (B) PCR for ARfl in actinomycin D–treated LNCaP pc and LNCaP ARv567es cells. Note that ARv567es did not significantly affect ARfl mRNA stability. (C) The Western blot of cycloheximide-treated LNCaP pc and LNCaP ARv567es cells demonstrates that ARv567es increases ARfl protein stability in the presence of DHT. (D) Graph depicting relative protein levels of ARfl following treatment with cycloheximide in LNCaP pc versus LNCaP ARv567es 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 ARv567es does not contain a LBD, androgen ablation has no effect on signaling. Thus, in tumor cells expressing ARv567es, 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 ARv567es 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 ARfl expression in LNCaP pc cells and ARv567es and endogenous ARfl in the LNCaP ARv567es cells. However, in the presence of DHT, SAHA only effectively decreased ARfl expression and had a minimal effect on ARv567es 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 ARv567es, we cultured LuCaP 86.2 tumors in vitro and treated the cells with SAHA or SAHA plus DHT. SAHA was effective at decreasing ARv567es in the presence and absence of DHT (Figure 8, B and C). Although SAHA decreased ARv567es protein levels in the LuCaP 86.2 cells, the resulting decrease in growth (Figure 8D) cannot be conclusively attributed to loss of ARv567es, since histone deacetylases have multiple effects on cells that result in suppression of cell proliferation (17, 18).
Figure 8The effect of the histone deacetylase inhibitor, SAHA, on ARv567es-expressing cells. (A) LNCaP pc or LNCaP ARv567es 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 ARv567es 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.
ARv567es-regulated gene expression. In order to compare and contrast the gene expression program regulated by ARv567es and ARfl, we first measured transcript abundance changes in LNCaP cells expressing ARfl 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 ARfl CS and ARfl 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 ARfl or ARv567es grown in CS medium. Gene expression differences between ARfl CS and ARv567es 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 9Expression profiles of LNCaP pc and LNCaP ARv567es cells. (A) Venn diagrams showing the gene number per comparison for expression profiles of androgen-regulated and ARv567es-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 ARfl cells and a q value of less than 10% considered statistically significant for ARv567es 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 ARv567es cells compared with LNCaP cells with no DHT; and III and VI indicate genes regulated by ARv567es in LNCaP cells compared with LNCaP cells without DHT. (B) The top AR-regulated genes in the LNCaP ARv567es 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 ARv567es 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 ARfl, were also altered in the context of ARv567es expression in the absence of exogenous androgens, confirming the hypothesis that ARv567es is capable of activating the AR-regulated gene expression program in the setting of castration. We further found that ARv567es regulates a subset of genes that we believe to be unique that are not influenced by androgens in the context of the ARfl. Analysis of GO terms enriched specifically in ARv567es 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 ARv567es 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). ARv567es also activated genes involved in the metabolism of androgens. Interestingly, even in the absence of exogenous AR ligands, cells expressing ARv567es 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 ARv567es cells may provide a survival advantage in a low-androgen environment.
In addition to differences in the genomic signaling programs between ARv567es and ARfl, our data also indicate that these variant receptors may differentially regulate other AR activities. In this context, ARv567es altered components of IGF pathway signaling in a manner distinct from that of the ARfl. IGF-IR expression was enhanced by ligand stimulation of ARfl, whereas ARv567es 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 ARfl to Src and downstream activation of the transcription factor MAPK (22). Our cell-based localization studies indicated that ARv567es was rapidly targeted to the nucleus and, during this process, facilitated the movement of ARfl as well. Thus, ARv567es may have a primary effect through enhanced genomic AR activity and the concomitant suppression of nongenomic AR activity exerted by ARfl.
ARv567es enhances the growth of prostate cancer following ADT in vivo. Since ARv567es is constitutively activated and also transactivates ARfl in the absence of androgen, we next sought to determine whether ARv567es influences prostate cancer responses to ADT in vivo. We implanted LNCaP ARv567es 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 ARv567es cells were measurably larger than those of control cells by the 10-week time point (tumor volume of LNCaP pc vs. LNCaP ARv567es 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 ARfl were not significantly different relative to levels in cells prior to castration, and the ARv567es remained undetectable. In contrast, in LNCaP ARv567es tumors the ratio of ARv567es transcripts to those encoding ARfl 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 ARv567es variant.
Figure 10Tumor growth of LNCaP ARv567es cells and xenografts expressing various amounts of ARv567es. (A) 1 × 106 LNCaP pc or LNCaP ARv567es 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 ARv567es 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 ARv567es 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 ARv567es 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 ARfl, 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 ARv567es and ARfl. Lanes were run on different gels.
We next examined the response of prostate cancers expressing endogenous ARv567es to ADT. We chose 3 representative LuCaP xenografts, which have distinct differences in the expression of ARfl and ARv567es (Figure 10E). The LuCaP 35 xenograft expressed ARfl, with little detectable ARv567es. 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 ARfl 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 ARv567es (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 ARv567es (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 ARv567es expression. No response to ADT was seen for the LuCaP 86.2 xenograft, which predominately expressed ARv567es (Figure 10, B and E). The growth of LuCaP 136 tumors, which express a mix of ARfl and ARv567es 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 ARfl, exhibited a sustained response to ADT, with minimal tumor growth measured over 14 weeks (Figure 10D).
Evolution of ARv567es in prostate xenografts. Since ARv567es is a splice variant and generation of the variant is posttranscriptional, acquisition of ARv567es 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 ARfl mRNA was present in the original LuCaP specimen, whereas both ARfl and ARv567es 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, ARv567es mRNA became the dominant AR mRNA in the LuCaP 86.2 xenograft, and ARv567es 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 ARv567es 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 ARv567es 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 ARfl in 35 subjects, and 6 out of 36 (17%) men expressed ARv567es, 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 11Expression of the splice variant ARv567es 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 ARv567es 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 ARv567es variant compared with previously published AR variants (AR1/2/2b in ref. 30; AR-V7 in ref. 14; and AR3 in ref. 13). RRP, radical retropubic prostatectomy.
Table 1Number of metastases positive for ARfl or AR splice variants
Since we found ARv567es 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 ARfl, 20 out of 46 (43%) expressed ARv567es, 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 ARv567es variant, but expression of AR-V7 or AR3 occurred concomitantly with ARfl 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 ARv567es. 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.
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