Repression of androgen receptor mediated transcription by ... - Nature

Oncogene (2002) 21, 5609 – 5618 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Repression of androgen receptor mediated transcription by the ErbB-3 binding protein, Ebp1 Yuexing Zhang1,2, Joseph D Fondell3, Qianben Wang3, Xianmin Xia1, Aiwu Cheng3, Michael L Lu4 and Anne W Hamburger*,1,2 1

Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland, MD 21201, USA; 2Department of Pathology, University of Maryland, Baltimore, Maryland, MD 21201, USA; 3Department of Physiology, University of Maryland, Baltimore, Maryland, MD 21201, USA; 4Harvard Medical School, Department of Surgery, Boston, Massachusetts, MA 02115, USA

Members of the ErbB family of receptors have been implicated in regulation of androgen receptor (AR) activity. Ebp1, an ErbB-3 binding protein recently cloned in our laboratory, possesses an LXXLL motif important in mediating interactions with nuclear hormone receptors. Therefore, we sought to determine if Ebp1 could bind AR and influence AR transcriptional activation potential. We demonstrate in this study that Ebp1 bound to AR in vitro and in vivo, and that this binding was increased by androgen treatment. The C terminal 79 amino acids of Ebp1 were sufficient to bind AR. The N terminal domain of AR was responsible for binding Ebp1. Ligand-mediated transcriptional activation of both artificial and natural AR regulated promoters was inhibited by ectopic expression of ebp1 in transient transfection systems. Ebp1 deletion mutants that either lacked the C terminal AR binding region or had a mutated LXXLL motif failed to inhibit AR activated transcription. PSA expression from its endogenous promoter was also decreased in LNCaP prostate cancer cells overexpressing Ebp1. The growth of AR positive LNCaP cells was inhibited by ectopic expression of ebp1, but mutants that failed to repress transcription did not inhibit cell growth. These studies suggest that Ebp1 may play a role in the function of the AR and provide a link between ErbB receptors and the AR. Oncogene (2002) 21, 5609 – 5618. doi:10.1038/sj.onc. 1205638 Keywords: prostate cancer; androgen receptor; ErbB receptors; Ebp1 Introduction The molecular basis for hormone independent progression in prostate cancer is poorly understood. One current model suggests that non-steroid receptor signal transduction pathways may activate the androgen *Correspondence: AW Hamburger, Greenebaum Cancer Center, University of Maryland at Baltimore, 655 W Baltimore St., Baltimore, Maryland, MD 21201, USA; E-mail: [email protected] Received 31 January 2002; revised 24 April 2002; accepted 29 April 2002

receptor (AR) in the androgen deprived patient (Jenster, 2000). Increasing data demonstrate that members of the ErbB family of receptors are involved in regulation of AR activity. In vitro, AR is activated in a ligand independent manner by EGF, although the mechanisms of activation are unclear (Culig et al., 1994). Most recently, the role of ErbB2 in rendering cells androgen independent or more sensitive to extremely low levels of androgen has been demonstrated. Craft et al. (1999) showed that androgen independent sublines of human prostate cancer xenografts expressed higher levels of ErbB2 than androgen dependent sublines. Furthermore, overexpression of ErbB2 in androgen-dependent LNCaP prostate cancer cells resulted in androgen independent cell growth. Overexpression of ErbB2 also activated AR-mediated transcription from the prostate specific antigen (PSA) gene in an androgen-independent fashion. In other studies by Yeh et al. (1999), ectopic expression of ErbB2 sensitized prostate cancer cells to extremely low levels of androgen. This activation was dependent on both the presence of an intact AR and the kinase activity of ErbB2. Induction of PSA transcription via overexpression of ErbB2 has recently been demonstrated to depend on activation of AKT (Protein kinase B) (Wen et al., 2000). Taken together, these studies demonstrate cross talk between ErbB and AR signal transduction pathways during prostate cancer progression. The functional role of the ErbB receptor family in clinical prostate cancer is currently being examined (Barton et al., 2001). The EGF receptor is expressed in both benign prostatic hyperplasia and prostate cancer (Mellon et al., 1992). Although ErbB2 is expressed in normal prostate epithelial cells (Ware et al., 1991), many studies have determined that ErbB2 is overexpressed and/or amplified at the DNA level in a subset of prostate cancer patients. Overexpression of ErbB2 has been associated with shortened patient survival (Morote et al., 1999). Elevated serum levels of the ErbB2 extracellular domain have also been correlated with hormone refractory disease after endocrine therapy. A study (Signoretti et al., 2000) using primary prostatic tissue demonstrates an increase in ErbB2 expression with progression to androgen independence. Furthermore, immunohistochemical

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studies have shown EGFR, ErbB2, and ErbB3 to be frequently coexpressed in both the normal prostate and prostate cancer (Myers et al., 1994; Prigent et al., 1992). The role of the ErbB3/4 ligand Heregulin (HRG) in prostate cancer progression is also poorly understood. HRG was found to be more highly expressed in prostate stromal cells than in prostate epithelial cells (Lyne et al., 1997). In addition, higher levels of HRG were observed in normal tissue, as compared with prostate cancer tissue. The effects of HRG on proliferation of prostate cancer cells has not been extensively studied. In breast cancer, HRG can either inhibit cell growth and induce differentiation or stimulate cell proliferation depending on cellular context and growth conditions (Bacus et al., 1993). HRG inhibits growth and induces differentiation of AR positive, ErbB 1 – 3 positive LNCaP cells (Lyne et al., 1997; Grasso et al., 1997) but has little effect on proliferation of ErbB 1 – 3+, AR negative DU145 and PC-3 cells. HRG also induces the expression of the tumor suppressor protein p53 and the cyclin kinase inhibitor p21 in LNCaP cells (Bacus et al., 1996). To better understand HRG-mediated signal transduction pathways initiated by the kinase-inactive ErbB3 receptor, we performed a yeast two-hybrid screen using the non-phosphorylated cytoplasmic domain of ErbB3 to identify ErbB3 interacting proteins. We isolated an ErbB3 binding protein, Ebp1, that interacts with the juxtamembrane domain of ErbB3 (Yoo et al., 2000). Ebp1 is the human homologue of a previously identified cell cycle regulated mouse protein p38-2G4 (Radomski and Jost, 1995). Treatment of serum-starved AU565 breast cancer cells with the ErbB3 ligand HRG resulted in dissociation of Ebp1 from ErbB-3 and translocation of Ebp1 from the cytoplasm into the nucleus (Yoo et al., 2000). These findings suggest that Ebp1 may be a downstream member of an ErbB3 signal transduction pathway. Ebp1 has several important structural motifs, including potential PKC phosphorylation sites, an amphipathic helical domain predicted to mediate protein/protein or protein/DNA interactions (Radomski and Jost, 1995), and an LXXLL motif present in many nuclear hormone receptor coregulatory proteins (Heery et al., 1997). In addition, recent work in our laboratory demonstrated that Ebp1 protein is expressed in human prostate cancer cell lines and normal human prostate epithelial cells (Xia et al., 2001a). Because of its observed role in ErbB signaling and the fact that it contains an LXXLL motif shown to be important in binding nuclear receptors, we explored whether Ebp1 might functionally interact with AR. We demonstrate here that Ebp1 directly interacts with AR and that ectopic expression of ebp1 inhibits AR signaling in human prostate cancer cell lines. This is the first time that a protein that interacts with the ErbB3 receptor has been demonstrated to interact directly with AR. Oncogene

Results Ebp1 interacts with the androgen receptor Due to the potential importance of ErbB family members in both the progression and genesis of prostate cancer and the fact that Ebp1 is expressed in prostate cancer cell lines and normal prostate epithelial cells and contains an LXXLL motif, we postulated that Ebp1 may interact with AR. Therefore, COS-7 cells were transfected with pSG5-hAR, and incubated in the presence or absence of 25 nM testosterone (Aarnisalo et al., 1998) in phenol red free RPMI 1640-2% CSS media for 48 h. Lysates of control and treated cells were incubated with equal amounts of GST-Ebp1 or GST. Immunoblot analysis of precipitated proteins revealed that AR bound to GST-Ebp1 in nonstimulated cells and that this binding was increased after testosterone treatment (Figure 1a, lanes 2 and 3). Examination of cell lysates by Western blotting revealed that testosterone treatment did not increase the levels of AR proteins (Figure 1a, lanes 5 and 6). The specificity of the Ebp1AR interaction was suggested by the fact that GST alone did not bind AR (lane 1). Untransfected COS-7 cells were also incubated with GST-Ebp1. No bands at MW 110 were observed (lane 4) further suggesting the interaction of Ebp1 and AR was specific. To determine if Ebp1 could interact with endogenously expressed AR, AR positive MCF-7 cells were treated with the synthetic androgen R1881 for 48 h in RPMI 1640-2% CSS. Cells were harvested and lysates were incubated with GST-Ebp1 as described. GSTEbp1 was able to associate with AR in the absence of R1881, but this binding was increased by R1881 treatment (Figure 1b). We next used immunoprecipitation analysis to determine if endogenous Ebp1 could interact with AR in vivo. COS-7 cells were transfected with pSG5hAR, and incubated in the presence of R1881 for 48 h. Cell lysates were then immunoprecipitated with antibody to His-tagged Ebp1. Western blot analysis indicated that AR was present in Ebp1 immunoprecipitates supporting the interaction of Ebp1 and AR (Figure 1c). To determine the region of Ebp1 necessary for binding AR, a series of GST-Ebp1 truncated fusion proteins was prepared (Xia et al., 2001b). COS-7 cells were transfected with an AR expression plasmid and treated with R1881 for 48 h. Cell lysates were incubated with equal amounts of GST fusion proteins encoding different regions of Ebp1 (Figure 2a). We found that the last 79 amino acids of Ebp1, containing the LXXLL motif, were sufficient to bind AR (Figure 2b). GST-Ebp1 aa 1 – 136 and 133 – 306 failed to bind AR. We also tested whether an ebp1 mutant plasmid (ebp1 LXXAA), in which leucine residues of the core LXXLL motif (L335 and L336) were mutated to alanines, could still bind AR. This GST-Ebp1 fusion protein failed to bind AR. We next mapped the region of AR responsible for binding Ebp1. For this purpose, we transfected COS-7


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Figure 1 Physical interaction between Ebp1 and the androgen receptor. (a) COS-7 cells were transfected with the pSG5-hAR expression vector (lanes 1 – 3) or left untransfected (lane 4) and grown in the presence (+) or absence (7) of 25 nM testosterone. Cells were harvested 48 h after transfection and lysates incubated with equal amounts of either GST (lane 1) or GST-Ebp1 bound to glutathione-Sepharose (lanes 2 – 4). The GST-Ebp1 or GST-associated proteins were resolved by SDS – PAGE and analysed by Western blotting using an anti AR antibody as described in Materials and methods. The input represents 5% of the protein used in the pull down assay. 5=untreated lysate; 6=testosterone treated lysate; 7=untransfected COS-7 cells. (b) MCF-7 cells were grown in the presence (+) or absence (7) of 100 nM R1881 for 48 h. Cell lysates were incubated with either GST (lane 1) or GST-Ebp1 (lanes 2 and 3). Associated proteins were analysed as described in (a). (c) COS-7 cells were transfected with an AR expression vector as in (a). Cells were incubated in the presence (+) of 100 nM R1881. Cell lysates were prepared and incubated with Protein G/A (PG/A), control rabbit IgG or an antibody to His-Ebp1 as described in Materials and methods. Immunocomplexes were separated by SDS – PAGE and proteins transferred to PVDF membranes. Immunoblots were probed with an antibody to AR

Figure 2 Binding of AR by Ebp1 mutant proteins. Equal amounts of GST-Ebp1 wild type and mutant fusion proteins (a) or GST alone were prepared and incubated with lysates of R1881 treated COS-7 cells transfected with pSG5-AR. (b) Ebp1 associated proteins were analysed by Western blotting using a monoclonal anti AR antibody. The arrow indicates the position of AR. An aliquot of the cell lysates (input) was also directly loaded onto the gel and analysed by Western blotting

cells with mammalian expression vectors expressing HA tagged fusion proteins of VP-16 full-length AR (FL), N terminal AR (AR-N, 1 – 500), DNA binding domain of AR (DBD, 501 – 660), ligand binding domain of AR (LBD, 661 – 919) and N/DBD of AR

Figure 3 The AR N-terminal domain (NTD) interacts with Ebp1. COS-7 cells were transfected with the indicated expression vectors for HA tagged VP-16 AR fusions proteins : AR-FL (full length), AR-N (1 – 500), N/DBD (1 – 660), DBD (501 – 660), or LBD (661 – 919). Forty-eight hours later, extracts were prepared and incubated with GST-Ebp1 or GST as indicated. Associated proteins were analysed by Western blotting using an antibody to the HA tag

(1 – 660). The equivalent expression of these constructs was confirmed by Western blot analysis using anti-HA antibodies (data not shown). Lysates of COS-7 cells transfected with these expression plasmids were incubated with GST or GST-Ebp1. As shown in Figure 3, full length AR and N terminal AR were specifically precipitated by GST-Ebp1. A weaker interaction was observed using the N/DBD fusion Oncogene


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protein. We were unable to detect a physical interaction between DBD or LBD under these conditions. A

Ebp1 represses AR mediated transactivation As we demonstrated that Ebp1 was able to physically associate with AR, we questioned whether Ebp1 could influence androgen mediated activation of the AR. We transfected COS-7 cells with the pARE2DS-Luc reporter plasmid, the AR expression vector pSG5hAR, and an expression vector encoding full length ebp1. Cells were treated with or without 25 nM testosterone as indicated for 24 h. We found that ectopic expression of ebp1 resulted in a dose-dependent decrease in the androgen induced transcriptional activation of the AR using both reporters (Figure 4a). In contrast, ebp1 had no effect on the estrogen induced responsiveness of an ERE-luciferase reporter plasmid (Figure 4b) or the thyroid hormone mediated activity of a TRE-luciferase reporter plasmid (Figure 4c). To further delineate the region of Ebp1 that might be important for transcriptional repression, we created a deletion mutant (ebp1D45)(aa 327 – 372) that lacks part of the AR binding domain (aa 293 – 372) and an LXXLL mutant plasmid (ebp1 LXXAA), in which leucine residues of the core LXXLL motif (L335 and L336) were mutated to alanines. We demonstrated that wild type and mutant proteins were expressed at comparable levels as confirmed by Western blot analysis (Figure 5a,b). We found that deletion of the last 45 amino acids of Ebp1 decreased the ability of Ebp1 to inhibit the activity of the ARE promoter. We also found that mutation of the LXXLL motif resulted in an abrogation of the ability of Ebp1 to repress AR activated transcription (Figure 5c). To determine if ebp1 could affect AR-mediated transcription from a natural promoter, we examined the ability of ebp1 to repress transcription from a PSA promoter linked reporter gene. LNCaP cells were transfected with wild type AR, ebp1 and a PSAluciferase reporter plasmid. The addition of R1881 resulted in a 2.5-fold increase of PSA promoter activity (Figure 5d). Significant (P40.05) repression of AR mediated transactivation of this reporter in the presence of ebp1 was observed. We also analysed the ability of the two ebp1 mutants to affect PSA promoter activity. As with the pARE2DS-luciferase plasmid, we found that either deletion of the Ebp1 AR binding domain or mutation of the LXXLL motif abrogated the ability of Ebp1 to inhibit AR mediated transactivation (Figure 5d). To rule out potential artifacts due to the use of a transfected reporter system, we examined the effect of Ebp1 on the expression of the endogenous PSA gene, as serum levels of PSA are an important marker in the diagnosis and progression of prostate cancer (Kallioniemi and Visakorpi, 1996). We first attempted to examine the effect of transiently transfected ebp1 on the expression of endogenous PSA in LNCaP cells using Northern blot analysis. However, due to low transfection efficiencies, we were unable to observe any Oncogene

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Figure 4 Ebp1 inhibits the transcriptional activity of an androgen regulated promoter. (a) COS-7 cells were transiently transfected with either pcDNA3 (1 mg) or pcDNA3-ebp1 at the indicated concentrations, the pSG5 AR expression plasmid, and the ARE2DS-luciferase reporter construct as described. Cells were stimulated overnight with 25 nM testosterone in 2% CSS and lysates assayed for luciferase activity. Each point represents the mean+s.e. of three wells (representative of three experiments). (b) MCF-7 cells were transfected with an ERE luciferase reporter plasmid and 1 mg of Ebp1. Twenty-four hours later, cells were stimulated overnight with 1076 M b-estradiol and luciferase activity assessed. (c) COS-7 cells were transfected with a human thyroid hormone receptor b expression plasmid and a luciferase reporter plasmid controlled by 2 TREs and 1 mg of an ebp1 expression plasmid. Twenty-four hours later, cells were stimulated overnight with 1078 M T3 and luciferase activity assessed


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Figure 5 Ebp1 repression of AR transcriptional activity. COS-1 cells (a – c) were transfected with pARE2DS-Luc (0.25 mg/well), pSG5-hAR (0.5 mg/well) and 0.5 mg of pcDNA3 (C) or wild type (WT), deletion (D45), or LXXAA (LX) mutant ebp1 expression plasmids. Twenty-four hours after transfection, cells were treated with 100 nM R1881 for 24 h. Forty-eight hours after transfection, lysates (30 mg) of cells transfected with each of the ebp1 expression plasmids or control vector were analysed by Western blotting using a polyclonal antibody against full-length Ebp1 (a,b). Relative luciferase activity of the cell lysates was also measured as described in Materials and methods (c). Each point represents the mean+s.e. of three wells (representative of three experiments). (d) LNCaP cells were transfected with a PSA reporter luciferase construct (0.5 mg/well) and 0.5 mg of pcDNA3 or wild type, deletion (D45), or LXXAA mutant ebp1 expression plasmids. Twentyfour hours after transfection, cells were treated with 100 nM R1881 for 24 h

significant changes in PSA mRNA expression levels. Therefore, we generated stable cell lines transfected with ebp1. Ebp1 protein expression was increased about 2 – 3-fold (Figure 6a) in the stable transfectants consistent with the model that high levels of Ebp1

Figure 6 Effects of stable expression of ebp1 on endogenous PSA gene expression in LNCaP cells. (a) Lysates (30 mg) of pcDNA control or ebp1 transfected cells were analysed by Western blotting using a polyclonal antibody to Ebp1 as described in Materials and methods. (b) Twenty mg of total RNA from LNCaP cells stably transfected with pcDNA (lanes 1 and 2) or ebp1 (lanes 3 and 4) were subjected to Northern blot analysis using an a-32P-dCTP labeled PSA probe. Where indicated, cells were treated with 100 nM R1881 for 24 h before harvest (+). (c) Ethidium bromide stained gel. A representative experiment of three is shown. (d) Graphical analysis of PSA mRNA levels as determined by densitometry is shown with the relative levels of PSA normalized to 28 S RNA. The data represent the mean+s.e. of three experiments

protein expression are incompatible with cell growth (Lessor et al., 2000). In cells transfected with pcDNA3 alone, the expression of the PSA transcript was increased sixfold after 48 h of R1881 treatment. The basal level of PSA expression was unchanged in ebp1 transfected cells as compared to vector controls. Although an increase in PSA levels was still observed in ebp1 transfectants in response to R1881, the PSA mRNA level reached only half that observed in the control transfectants (Figure 6b – d). Oncogene


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Ebp1 inhibits colony growth of LNCaP cells We have previously shown that ectopic expression of ebp1 inhibits growth of human breast cancer cell lines (Lessor et al., 2000). To determine whether the ability of Ebp1 to inhibit growth of prostate cancer cells correlates with its ability to repress AR-mediated transcription, we transfected AR positive LNCaP cells with wild type ebp1 or the deletion and LXXAA mutants. We found that ectopic expression of wild type ebp1 inhibited cell growth (Figure 7). The ebp1 deletion mutant (ebp1D45) was unable to significantly (P40.05) inhibit colony growth of LNCaP cells. The LXXAA mutant was also significantly (P40.05) less effective than wild type ebp1 in inhibiting growth of the AR positive LNCaP cell line. Discussion Cross-talk between the ErbB and AR signal transduction pathways has been suggested to be an important component of prostate cancer progression. While overexpression of ErbB1 and 2 and EGF has been associated with increased malignancy, the ErbB3/4 ligand HRG appears to be associated with slower cell growth (Grasso et al., 1997). However, a mechanism to link activation of ErbB3 receptors by HRG with inhibition of prostate cancer cell growth has not yet been reported. In this study, we present evidence that an ErbB-3 binding protein, Ebp1, can interact with AR and inhibit AR-mediated transcription. Previous data from our laboratory have indicated that Ebp1 is released from ErbB3 in serum-starved breast cancer cells after treatment with HRG (Yoo et al., 2000). We have also demonstrated a similar release of Ebp1 from ErbB3 after HRG treatment in prostate cancer cells (Zhang, manuscript in preparation). Thus, it is possible

Figure 7 Effect of wild type and Ebp1 mutants on colony growth. AR positive LNCaP cells were transfected with 2 mg of pcDNA3, wild type ebp1, ebp1 D45 or the LXXAA mutant as indicated. The numbers of colonies that survived G418 selection were compared to the number of colonies of cells transfected with vector alone. Each point represents mean+s.e. of three wells. Representative of two experiments Oncogene

that release of Ebp1 from ErbB3 and its subsequent interaction with AR may play a role in the ability of HRG to inhibit cell growth. This is the first time that a protein that interacts with ErbB3 has been demonstrated to directly interact with the AR. We demonstrated that Ebp1 could interact with AR in the absence of ligand, but that ligand treatment enhanced binding. The fact that the interaction of Ebp1 with AR was increased by hormone treatment contrasts with the behavior of other nuclear hormone corepressors as their interaction is disrupted by ligand treatment (Nagy et al., 1999; Perissi et al., 1999; Fondell et al., 1996). The factors governing the association of Ebp1 with AR are not known. It is possible that androgen-induced phosphorylation of AR (Kemppainen et al., 1992) may affect its binding to Ebp1. Similarly, Ebp1 is a phosphoprotein in vivo and heregulin treatment increases its phosphorylation (Lessor and Hamburger, 2001). Androgen treatment may similarly induce changes in Ebp1 phosphorylation that result in increased affinity for AR. Studies examining changes in Ebp1 phosphorylation after androgen treatment are currently underway in our laboratory. We next tested the ability of Ebp1 to affect hormone dependent AR transactivation of target genes. We found the ectopic expression of ebp1 inhibited AR mediated transcription from both artificial and natural promoters. Experiments using ebp1 deletion mutants demonstrated that the AR binding region was necessary for the inhibition of AR mediated transcription. In addition, there was a good correlation between the ability of Ebp1 mutants to bind AR, repress AR mediated transcription, and inhibit proliferation of AR+ LNCaP cells. However, mutation of the LXXLL motif appeared to have abrogated transcriptional repression more effectively than deletion of the C terminal 45 amino acids of Ebp1. Conversely, deletion of the C terminal 45 amino acids was more effective at preventing Ebp1’s inhibition of cell growth than mutation of the LXXLL motif. The reasons for this discrepancy are unclear. However, it is known that Ebp1 can also inhibit transcription of cell cycle regulated genes such as Cyclin E (Xia et al., 2001b). This activity maps to the C-terminal 72 amino acids. It is likely that the growth inhibition exerted by Ebp1 is multifactorial and may depend on both the ability of Ebp1 to modulate AR mediated pathways and to affect cell cycle regulated genes. Ebp1-mediated suppression of the PSA endogenous promoter also supports the role of Ebp1 as a repressor of transcription of ARregulated genes. The fact that the inhibition observed using the endogenous PSA promoter was not as great as that observed using transiently expressed constructs may be due to the differences in the degree of Ebp1 overexpression in transfected cells. Stable transfectants express lower levels of Ebp1 than transiently transfected cells due to the negative effect of Ebp1 on cell growth (Lessor et al., 2000). Nonetheless, these findings suggest that Ebp1 may function to negatively regulate AR target gene expression in vivo.


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We also determined that the isolated N terminal, but not DBD or LBD domains of AR, bound Ebp1. The AR has an overall domain structure common to nuclear hormone receptors, comprised of an N terminal activation domain (activation function 1(AF1)), a central DNA-binding domain (DBD), and a C-terminal ligand binding domain (LBD) (Wilson et al., 1991). AR is unique among the nuclear hormone receptors in that coactivators primarily bind to the AF1 activation domain in the N terminal region of the AR receptor (Alen et al., 1999; He et al., 2000). Thus, Ebp1 may compete with coactivators for binding sites in the N-terminal domain, resulting in overall transcriptional repression. Similarly, Hayes et al. (2001) recently demonstrated that Smad 3 represses androgen receptor-mediated transcription and binds the N terminal activation domain of AR. Cyclin D has also been demonstrated to repress the transcriptional activation activity of AR and bind its N terminal domain (Reutens et al., 2001; Knudsen et al., 1999). Cyclin D has recently been demonstrated to inhibit AR in part by competing with coactivators (Petre et al., 2002). In our study, LXXLL mutants were unable to either bind AR in vitro or inhibit AR mediated transcriptional activation. This finding is somewhat puzzling as coactivator proteins with mutated LXXLL motifs can still bind AR and potentiate androgen receptor activity (Alen et al., 1999). However, our data indicate that the LXXLL mediated interaction of AR with Ebp1 is not typical of interactions of AR with coactivator proteins. For example, Ebp1 does not affect the transcriptional activity of other nuclear receptors such as ER or TR that typically can be activated by LXXLL containing proteins. In addition, Ebp1 cannot physically bind ER (data not shown). Thus, Ebp1 appears to be unable to interact with AF2 in the C terminal domain of either ER or AR. These data suggest that unique properties of the Ebp1 LXXLL motif such as its flanking sequences may contribute to the differences in its interactions with AR as compared to coactivators. For example, glutamines in the LXXLL flanking sequence of SRC-1 are critical for interaction of SRC1 with AR (Bevan et al., 1999). Similarly, there are several glutamines in the sequences that flank the LXXLL motif of Ebp1. It is possible that disruption of the LXXLL motif changes the ability of these flanking sequences to mediate AR binding and transcriptional repression. Current studies are underway in our laboratory to mutate these flanking sequences and determine effects on the transcriptional repressor activity of Ebp1. The molecular mechanism of the Ebp1 inhibition of AR transactivation is not yet known. We have found, for example, that recombinant Ebp1 does not inhibit the binding of AR to its target DNA as measured by EMSA assays (data not shown). Similarly, the PIASy protein has been demonstrated to inhibit the transcriptional activity of the AR, but does not inhibit the DNA binding activity of the AR (Gross et al., 2001). One alternative mechanism, based on the action of

other nuclear hormone receptor repressors, is that Ebp1 may represses AR-mediated transcription by recruiting molecules with histone deacetylase activity to promoters. Corepressors such as NCoR-1 and SMRT bind to the unliganded nuclear hormone receptors TR, RAR, ER, or GR, and recruit histone deacetyltransferases, leading to condensation of nucleosomal structures for repression of transcription (Whitfield et al., 1999). Similarly, the ability of cyclin D1 to inhibit AR activation was demonstrated to partially require deacetylase activity (Petre et al., 2002). The interaction of Ebp1 with both an ErbB receptor and AR is unique to the best of our knowledge. However, interactions of transcriptional regulators that bind both transmembrane growth factor receptors and nuclear receptors are not unprecedented. For example, STAT5B can mediate the inhibitory effects of growth hormone on PPARa transcriptional activity via its interaction with both receptors (Zhou and Waxman, 1999). Functional interactions involving the binding of STAT factors and glucocorticoid receptors have also been described. STAT5A and the glucocorticoid receptor form a molecular complex that enhances prolactin-induced transcription from the b-casein promoter, but inhibits glucocorticoid stimulated transcription from a glucocorticoid response element (Stocklin et al., 1996). These studies establish a model in which Stat proteins cross talk with nuclear receptors by direct protein – protein interactions that modulate gene expression. Increasing data in the literature suggest that the ErbB receptor system may play a role in androgen receptor mediated signal transduction and the progression of prostate cancer. Ebp1 may be involved in such a process by providing a link between signals received by ErbB receptors and the AR receptor. An understanding of Ebp1 function could provide a better understanding of cross-talk between the ErbB family receptors and AR in prostate cancer progression.

Materials and methods Cell culture All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained at 378C in a humidified atmosphere of 5% CO2 in air. Cell lines were routinely cultured in either DMEM-F12 media supplemented with 10% Fetal Bovine Serum (FBS) (COS-1, COS-7, MCF7) or RPMI 1640 media supplemented with 10% FBS (LNCaP). Plasmids pARE2DS-LUC containing two copies of a natural ARE cloned into the murine ornithine decarboxylase promoter and pSG5-hAR were gifts from Dr Olli Janne (Aarnisalo et al., 1999). The PSA reporter luciferase construct was a gift from Dr Martin Gleave and contains 7630/+12 of the 5’ PSA flanking region. The ERE reporter plasmid was previously described (Yoo et al., 1998) as was a human thyroid hormone receptor b plasmid and a luciferase reporter controlled by a Oncogene


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thyroid hormone responsive elements (Sharma and Fondell, 2000). A series of mammalian expression vectors encoding fusion proteins of VP16-AR (full length), AR-N (1 – 500), DBD (501 – 660), LBD (661 – 919) and N/DBD (aa 1 – 660) were constructed by cloning the corresponding AR fragments in frame with residues 411 – 456 of the VP 16 transactivation domain in the pACT vector (Promega, Madison, WI, USA) as previously reported (Lu et al., 2001). Preparation of deletion and mutant constructs of ebp1 The bacterial expression vectors encoding full-length and truncated glutathione S-transferase (GST)-Ebp1 fusion proteins and a mammalian expression vector (pcDNA3) encoding full length ebp1 were previously described (Xia et al., 2001b). A mammalian expression vector encoding a truncated ebp1 (ebp1 D45) was constructed by inserting a BamHI-EcoRI PCR fragment encoding nucleotides 262 – 1240 (GenBank U87954) (aa 1 – 327) into pCDNA3. We also mutated L4 and L5 (L335A, L336A) of the LXXLL motif on the basis of the critical roles of these leucine residues in binding to nuclear receptors. The ebp1 LXXLL motif mutant (LXXAA) was generated by mutating nucleotides 1260 – 1269 CTC CTC to GCC GCC using the QuickChange Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The orientation and integrity of the cDNA inserts in all constructs were confirmed by automated DNA sequencing in the core laboratory of University of Maryland School of Medicine. Affinity purification of GST fusion proteins In vitro expression and purification of recombinant GSTEbp1 fusion proteins were performed essentially as described (Xia et al., 2001b). Briefly, the recombinant vector was introduced into BL21 cells. An overnight culture of a colony of the transformants was diluted 1 : 10 and grown for 1 h at 378C before induction for 4 h at 258C with IPTG (0.1 mM). The cultures were centrifuged at 10 000 g for 15 min at 48C and the cells resuspended in ice cold phosphate-buffered saline (PBS). Bacterial cells were lysed by sonication on ice for 15 consecutive 15 s intervals. Following addition of Triton X-100 to a final concentration of 1% (v/v) and centrifugation, the supernatants were incubated with glutathione-agarose beads (50% slurry) for 30 min at room temperature. Beads were used directly in GST-pull down assays after extensive washing with buffer. GST-pull down assays, immunoprecipitation and Western blot analysis For studies demonstrating interactions of AR with various fragments of Ebp1, COS-7 cells were transfected with the pSG5-hAR expression plasmid encoding wild type AR and treated with 25 nM testosterone (Sigma) or 100 nM of the synthetic androgen R1881(methyltrienolone) (NEN, Boston, MA, USA) where indicated. Cells were rinsed with PBS and lysed in buffer consisting of 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mm EDTA, 1% Triton X-100, 1 mg/ml leupeptin and 1 mM PMSF. Cell lysates (1 mg of protein) were mixed with equal amounts of GST or GST-Ebp1 loaded onto glutathione Sepharose beads and incubated overnight at 48C with gentle rotation. An aliquot of bound GST or GST-Ebp1 fusion constructs was also analysed by Coomassie blue staining of SDS – PAGE gels to confirm equal loading of fusion proteins. The pelleted beads were then washed in lysis buffer, mixed with SDS sample buffer, boiled, and proteins separated on SDS gels. After electrophoresis, the proteins were transferred Oncogene

to Immobilin-P membranes, and immunoblotted as described (Xia et al., 2001a). The blots were probed with a monoclonal anti AR antibody (Santa Cruz) diluted in PBS supplemented with 2% milk for 2 h. Blots were washed three times with PBST and proteins detected using an ECL kit (Amersham). For studies determining interaction of Ebp1 with different AR domains, constructs encoding the different AR fusion proteins described above were transfected into COS-7 cells. The equal expression of the different AR fusion proteins was confirmed by Western blot analysis using anti-HA antibodies as previously described (Lu et al., 2001). Cell lysates were incubated with full-length GST-Ebp1 as described above. Blots were probed with a monoclonal antibody to HA (12CA5) (Roche, Indianapolis, IN, USA). Cell lysates were also immunoprecipitated where indicated as described previously (Xia et al., 2001b). Briefly, total cell extracts were prepared by direct lysis of cells with buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM NaCl, 1% Triton X-100, 0.5 mM DTT and 1 mM PMSF. Proteins concentrations were measured using a detergent compatible kit (BioRad, Hercules, CA, USA). Cell lysates were precleared with Protein A/Protein G agarose and immunoprecipitated for 4 h at 48C with 2 mg of an antibody directed against a His-tagged Ebp1 recombinant protein and 20 ml packed Protein A/G agarose beads. The immunoprecipitates were washed and resuspended in Laemmli sample buffer. Proteins were resolved by SDS – PAGE and analysed by Western blotting as described. The Ebp1 antibody was prepared as follows. The cDNA encoding a recombinant His-tagged Ebp1 fusion protein was cloned into pQE31 (Qiagen, Valencia, CA, USA) and expressed in the E. Coli host strain M15. The His-6 tagged fusion protein was purified from bacterial lysates using Ni+/nitrilotriacetic acid agarose according to the instructions provided by the manufacturer (Qiagen). The antiserum was generated in New Zealand White rabbits by Lampire Biologicals (Pipersville, PA, USA). The antiserum was purified by affinity chromatography using His-Ebp1 linked CNBr-activated Sepharose as previously described (Xia and Serrero, 1999). Luciferase reporter assays COS-1, COS-7 (16105) or LNCaP (2.56105) cells were plated in 6-well plates in complete media. When cells reached 50 – 60% confluence, they were transfected with 0.25 mg of pARE2DS-LUC, or the PSA reporter plasmid, 0.5 mg of pSG5-hAR, and 0.5 mg of pcDNA3 or wild type, truncated (D45), or LXXAA mutant ebp1 expression plasmids using the Lipofectamine reagent (Life Technologies, Rockville, MD, USA). Equivalent expression of all Ebp1 proteins was determined by Western blot analysis using a polyclonal antibody to full-length Ebp1. Complete medium was replaced after 24 h with phenol red and serum free DMEM-F12 or RPMI 1640 with or without 100 nM R1881 (Slagsvold et al., 2001) (NEN, Boston, MA, USA). Luciferase activity was determined as previously described (Xia et al., 2001b). A bgalactosidase reporter vector was also included to adjust for transfection efficiency. All transfection experiments were carried out in triplicate wells and repeated three times. Creation of stably transfected cell lines To establish stable transfectants, subconfluent LNCaP cells in 100 mm tissue culture dishes were transfected with 10 mg of pcDNA3, or pcDNA3-ebp1 expression plasmids using Lipofectamine according to the manufacturer’s protocol.


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Cells were selected in G418 (1000 mg/ml) for 5 weeks and resistant colonies were cylinder cloned and expanded. Northern blot analysis Total RNA was extracted using STAT RNA-60 and fractionated by electrophoresis prior to blotting on Hybond-N+ filters (Amersham Pharmacia Biotech). A 760 bp fragment of the PSA cDNA (kindly provided by Dr Natasha Kyprianou, University of Maryland) was labeled with a-32P-dCTP using the Prime-a-gene labeling system (Promega, Madison, WI, USA). Hybridization was performed as previously described (Yoo et al., 1998). Colony inhibition assays LNCaP cells (46104) were seeded into individual wells of 6well plates in complete media. Cells were transfected with

2 mg of ebp1 wild type or mutant expression plasmids or pcDNA3. The number of colonies surviving after 4 weeks of G418 (500 mg/ml) selection was determined microscopically after staining with Crystal violet. Statistical analysis Results were analysed using a two-tailed Student’s t-test. Significance was established at P40.05. Acknowledgments This work was supported in part by the Susan Komen Foundation and NIH R01 CA76047 and R21 088882-01 (to AW Hamburger). We thank Dr Olli Janne for the androgen responsive reporter gene and AR expression plasmids, and Dr Martin Gleave for the PSA reporter construct.

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