AR overexpression potentiates Wnt/β-Catenin transcriptional activities
While the role of activated Wnt/β-Catenin signaling in prostate cancer is not entirely clear, recent evidence indicates that β-Catenin contributes to prostate cancer progression through its enhancement of the AR transcriptional activity in the presence of androgens [13]. Reciprocally, using transient transfections of prostate cancer cells lines, we explored whether the AR overexpression had any effect on the Wnt/β-Catenin signaling pathway. As expected, expression of Wnt1 in the prostate cancer cells PC3 (Figure 1A), CWR22Rv1 and LNCaP (Figure 1B) resulted in the activation of the luciferase reporter gene driven by a LEF-dependent promoter [24]. This activation ranged from 2 to 20 fold compared with control cells transfected with GFP, depending on the cell type and assay conditions. The expression of the AR alone had little effect on LEF-dependent transcription in these cells under these assay conditions, although the transfection of relatively high amounts of the AR did lead to a 2–5 fold activation of the reporter gene (data not shown). Interestingly, the co-expression of Wnt1 and AR resulted in increased transactivation, compared to the effect of Wnt1 alone (7-fold in PC3, 4-fold in CWR22Rv1, and 2.5-fold in LNCaP cells) (Figure 1A &1B). This effect is more dramatic in PC3 cells than in CWR22Rv1 and LNCaP cells, probably due to the fact that PC3 cells have no detectable levels of the AR expression, while both CWR22Rv1 and LNCaP cells express endogenous mutant forms of the AR. We also tested the ability of the AR to potentiate other signaling components of the Wnt signaling pathway, such as the constitutively activated receptor LRP6 with a truncated N terminus (LRP6ΔN) [24], or the stabilized β-Catenin mutant (β-S37ACatenin) [25]. While the activation of the Wnt/β-Catenin signaling pathway in PC3 cells transfected with LRP6ΔN led to a 12-fold increase in the luciferase activity compared with the GFP control, the co-expression of the AR increased the activity 46-fold compared with LRP6ΔN signaling alone (Figure 1C). Cotransfection of β-S37ACatenin together with the AR resulted in a 6-fold activity increase in LEF mediated transcription compared with β-S37ACatenin activity alone (Figure 1C). Similar effects were also observed when the Wnt signaling pathway was activated by co-expression of Dishevelled or Casein Kinase I with the AR in PC3 cells. In this case, the AR enhanced the signaling activity of Dishevelled and Casein Kinase I by 6- and 10-fold respectively (data not shown). Based on these results, we conclude that the AR can potentiate Wnt transcriptional activity in prostate cancer cells.
Since CWR22Rv1 cells express mutant forms of ARH874Y [26], and the LNCaP cells express ART877A [27], we explored whether those mutant forms of the AR could also stimulate Wnt signaling. Wnt1 together with either wild-type or mutant AR expression constructs were cotransfected into PC3 cells. Indeed, the mutant ARH874Y also enhanced Wnt1 signaling with at least 5-fold increases when compared to Wnt1 activity alone, although this activity was reduced compared with that of the wild-type AR (Figure 1D). To prove that the difference in activity was not due to difference in expression, the levels of AR and ARH874Y were examined. As shown in Figure 1D, the expressions of both forms of AR were equivalent. Similar results were obtained for the ART877A mutation as well (data not shown). These results indicate that the mutated AR expression in CWR22Rv1 and LNCaP cells can potentiate Wnt signaling.
To confirm our observation that the AR potentiates Wnt signaling, bicalutamide was used to determine whether an AR antagonist could inhibit this potentiation. At concentrations as high as 10 μM, bicalutamide did not have any effect on the transcriptional activities of the LEF-dependent luciferase reporter in PC3 cells when cells were transfected with Wnt1 or AR alone. However, it inhibited approximately 60% of the transcriptional activity promoted by the co-expression of the AR and Wnt1 (Figure 1E), suggesting that when bound to an antiandrogen, the AR is less capable of potentiating Wnt signaling. Noticeably, this inhibition was observed in cells cultured with 5% charcoal-stripped serum without the addition of an AR agonist such as 5α-dihydrotestosterone (DHT). This indicates the mechanism by which bicalutamide inhibits the synergy between Wnt and AR is not likely to be the displacement of an agonist, but rather through the binding of bicalutamide to the AR. To characterize this inhibition further, bicalutamide was tested in a dose-dependent manner with concentrations ranging from 0.1 nM to 25 μM in the LEF-dependent luciferase reporter assay, using PC3 cells co-transfected with both Wnt1 and AR. Bicalutamide showed an IC50 of 1.38 μM in this study (Figure 1F).
AR interacts with Wnt signaling to promote prostate cancer cell proliferation
To investigate whether the synergy between the AR and Wnt transcriptional activities would lead to accelerated tumor cell growth, we set out to measure the growth of PC3 cells under the conditions of overexpressing the AR alone, Wnt1 alone, or the combination of both. The transfection efficiency was monitored by the expression of a control GFP. Around 90% of the PC3 cells had green fluorescence under a fluorescent microscope, indicating a high level of transfection in those cells. Cell numbers were then obtained after seven days post-transfections and incubation in the absence or presence of 0.1 nM DHT using Guava technology. As shown in Figure 2A, cells transfected with either Wnt1 or AR alone do not show significant differences in cell numbers compared with control cells transfected with GFP (p < 0.2) in the absence or presence of 0.1 nM DHT. However, cotransfection of Wnt1 and AR increased the number of PC3 cells significantly compared with either one of them alone: with AR (p < 0.005), or with Wnt1 (p < 0.007) in the absence of DHT; and with AR alone (p < 0.027), or with Wnt alone (p < 0.003) in the presence of 0.1 nM DHT. These results suggest that the interaction between these two signaling pathways at castration levels of androgens promotes prostate cancer cell growth more significantly compared with the activation of either pathway alone.
We further confirmed this growth stimulation effect in the parental LNCaP cell line, which expresses an endogenous ART877A, as well as in an engineered LNCaP-Flag-AR cell line that also stably expresses a Flag-tagged wild-type AR (Figure 2B). Since the efficiency of transfection is usually less than 50% in these cells compared with around 90% in PC3 cells, we used commercially-available recombinant Wnt3A in this set of experiments to ensure the exposure of Wnt ligand to all cells. In addition, since these cells tend to cluster, the assessment of proliferation was carried out by measuring the incorporation of [3H]-thymidine rather than counting cells directly. As expected, LNCaP-Flag-AR cells proliferated faster than LNCaP cells in both the presence and the absence of Wnt3A treatment because of the wild-type AR overexpression. As shown in Figure 2C, the treatment of LNCaP cells with Wnt3A increased cell proliferation up to 50%, possibly due to the interaction between the endogenous mutant AR and the Wnt3A signaling. Furthermore, in the LNCaP-Flag-AR cells, Wnt3A dramatically increased the cell proliferation by nearly 100% due to the overexpression of the wild-type AR. The cells shown in 2C were cultured in charcoal-stripped serum with no addition of DHT. Similar results were also obtained in cells exposed to low levels of androgen (such as 0.01 nM and 0.1 nM DHT, data not shown). Taken together, these results suggest that at castration levels of androgens, the amplified wild-type or mutant AR can synergistically interact with the Wnt signaling pathway, resulting in the stimulation of prostate cancer cell growth.
AR agonists inhibit the AR potentiation of the Wnt signaling pathway
We next examined the effect of androgens on the transcriptional synergy between Wnt1 and the AR. As expected, 10 nM DHT stimulated the AR signaling pathway, inducing the expression of the luciferase reporter gene driven by the PSA promoter in PC3 cells transfected with either the AR, or with both the AR and Wnt (Figure 3A). Interestingly, 10 nM DHT stimulated PSA-luciferase activity similarly in cells transfected with the AR alone or with both the AR and Wnt1 (Figure 3A), implying that additional β-Catenin did not necessarily translate into further activation of the AR signaling pathway. On the other hand, 10 nM DHT inhibited approximately 70% of the expression of the LEF-driven luciferase reporter in PC3 cells co-transfected with Wnt and AR (Figure 3B). However, it had no inhibitory effect when cells were transfected with Wnt1 alone (Figure 3B), indicating that similar to bicalutamide, its mechanism of action is through binding to the AR. We also noticed that DHT not only abrogated the AR-Wnt synergy, but also reduced the LEF-dependent transcription to levels lower than what was observed in cells transfected only with Wnt 1 (Figure 3B). This may be due to the fact that DHT can recruit β-Catenin into the AR signaling pathway, an observation that has been confirmed by other groups [17–20].
In order to confirm that the inhibition of the AR-Wnt potentiation by any AR ligand is independent of its pharmacological activity, we explored the effect of nilutamide, a small non-steroidal molecule that is an agonist of the mutant ART877A expressed in LNCaP cells and possibly also an agonist of the mutant ARH874Y found in the CWR22 xenograft model [27–30]. As shown in Figure 3C, nilutamide stimulated the PSA promoter-driven transcription of the luciferase reporter when cotransfected with either the ART877A or the ARH874Y mutants into PC3 cells. However, it had less of an effect on the wild-type AR-mediated transcription. This result is consistent with previous reports [27–30]. In addition, we observed that nilutamide impeded at least 60% of the LEF-dependent transcription activity in PC3 cells cotransfected with Wnt1 and either wild-type AR, ART877A, or ARH874Y (Figure 3D). Recently, bicalutamide was identified as an agonist for the ARW741L and ARW741C mutations [31]. This effect was confirmed in our transactivation assays performed in PC3 cells cotransfected with the mutant AR expression plasmids and the PSA promoter-driven luciferase reporter construct. Bicalutamide indeed stimulated the transcriptional activity through these two particular AR mutants, but not through the wild-type AR or the LNCaP or CWR22 AR variants (Figure 3E). Bicalutamide also repressed the synergy between Wnt1 and either the wild type AR or any of the mutant AR isoforms in PC3 cells (Figure 3F). This set of experiments demonstrates that AR ligands, agonists, or antagonists can prevent the AR synergy with the Wnt signaling pathway.
To further examine the effects of androgens on prostate cancer growth mediated by the Wnt and AR signaling, we took advantage of the stable LNCaP-Flag-AR cell line that overexpresses the wild-type AR (Figure 2B) mimicking the hormone -refractory state [3]. We examined if this cell line behaves differently from its parental LNCaP cell line in the presence or absence of Wnt3A treatment. Cell proliferation was assessed by measuring [3H]-thymidine incorporation. Similar to LNCaP cells [32], LNCaP-Flag-AR cells showed a biphasic response to DHT treatments (Figure 4A). Wnt3A stimulated LNCaP-Flag-AR cell growth noticeably by 2 fold with 0.01 nM DHT or no DHT treatment. The maximum increase of LNCaP-Flag-AR cell growth by DHT at 0.5 nM or 1 nM concentration was 4-fold. Higher concentrations of DHT at 5 nM and 10 nM reduced cell proliferation by approximately 50% either with or without Wnt3A treatment (Figure 4A). These results indicate that normal physiological levels of serum DHT concentration may inhibit cell proliferation driven by the AR overexpression and Wnt signaling.
We further confirmed our observation by performing soft agar growth assays to measure anchorage-independent growth of LNCaP and LNCaP-Flag-AR cells treated either with Wnt3A or with DHT. As seen in Figure 4B, LNCaP-Flag-AR showed an increased soft agar growth compared with the parental LNCaP cell line. This agrees with past reports that the AR overexpression increased the oncogenic malignancy and transformation of prostate cancer cells. Wnt3A treatment appeared to stimulate colony numbers on soft agar, but not the colony size. Similarly, as observed with the cell proliferation assay by [3H]-thymidine incorporation in LNCaP-Flag-AR cells, 10 nM DHT treatment did not stimulate, but rather reduced the soft agar growth of the LNCaP-Flag-AR cells. When these cells were treated both with Wnt3A and 10 nM DHT, the results were similar to that of 10 nM DHT alone (data not shown). These results suggest that the restoration to physiological levels of androgens could decrease the malignant tendency of castration-refractory prostate cancer cells, possibly by inhibiting the input of the AR into the Wnt/β-Catenin signaling pathway.
AR is promoting Wnt signaling at the chromatin level
Since both the AR and β-Catenin translocate into the nucleus upon androgen and Wnt signaling respectively, we performed immunohistochemistry staining using a tissue microarray with the AR and β-Catenin specific antibodies to explore the possible co-localization of the AR and β-Catenin in human prostate cancer samples. Four normal prostate tissue samples and nine prostate tumors were included in this tissue microarray. In the tissue array tested, the AR expressed at a much higher level in the tumor samples compared with the normal prostate tissues. In order to observe clear nuclear staining in the tumor samples, we calibrated the staining to conditions that gave a very low or negative signal in normal prostate tissues (Figure 5A). Those conditions revealed a strong nuclear AR staining in prostate cancer cells with Gleason Score (GS) 6 (Figure 5C) and above. These studies demonstrated a considerable AR staining in the nucleus of the nine tumors. In the meantime, β-Catenin was localized to the cell membrane in all four normal prostate tissue samples (Figure 5B), whereas we observed nuclear β-Catenin staining in three out of nine prostate tumors from patients with Gleason Score 6 (Figure 5D) and above. Since the sections of these stainings are consecutive, it is possible to identify the same cells with both AR and β-Catenin nuclear staining (see arrows). These results are not only consistent with the reports that associate high levels of Wnt-1 and β-Catenin expression with advanced metastatic, hormone-refractory prostate carcinoma [33, 34], but they also agree with the suggestion of a functional interaction between the AR and β-Catenin upon co-localization in the nucleus of prostate cancer cells at late stages.
To investigate further the interaction between the AR and β-Catenin in the nucleus, we examined through chromatin immunoprecipitation assays (ChIP), whether the AR can be recruited to the promoter region of Wnt target genes in LNCaP cells. Since it has been shown that Wnt signaling can activate both Myc and Cyclin D1, which contain LEF/TCF binding sites in their promoter regions [35, 36], we asked if Wnt signaling could bring the AR to these regions as well. Indeed, Wnt3A stabilized β-Catenin and increased its binding to the myc and cyclin D1 promoter regions (Figure 6A, 6B). In addition, the treatment of LNCaP cells with Wnt3A resulted in the recruitment of the AR to the promoter regions of both myc and cyclin D1 (Figure 6C, 6D), implying a direct transcriptional involvement of the AR at the promoters of these Wnt target genes upon activation of Wnt signaling. Interestingly, LNCaP cells treated with a physiological level of DHT (10 nM) might have reduced amounts of β-Catenin at the myc and cyclin D1 promoters compared with untreated samples (Figure 6A, 6B), possibly due to the fact that cell stimulation with DHT causes recruitment of β-Catenin to the promoter and enhancer regions of PSA (Figure 6E, 6F). We also found that DHT recruited the AR to the myc promoter and possibly the cyclin D1 promoter in the absence of Wnt signaling (Figure 6B, 6C), indicating that direct AR binding sites may be present in these promoter regions. This finding is in agreement with the report that endogenous AR was bound to a TCF-4 responsive element in the c-Myc promoter [37].
We also examined the promoter and enhancer regions of PSA, a well characterized AR regulated target gene. As reported [38], DHT treatment promoted the binding of the AR at both of these regions (Figure 6E, 6F). Interestingly, in the absence of androgens the AR was also recruited to the PSA promoter and enhancer regions as a result of Wnt3A signaling (Figure 6E, 6F). This suggests that upon Wnt stimulation, the stabilized β-Catenin could bind to the AR, leading to its signaling activation. This was further confirmed when β-Catenin was found at the promoter and enhancer regions of PSA upon treatment with Wnt3A (Figure 6G, 6H). These ChIP results demonstrate a novel mechanism by which Wnt stimulates the transcriptional activity of the AR in the absence of androgens.