Although mutations of the breast cancer susceptibility gene 1 (BRCA1) may play important roles in breast and prostate cancers, the detailed mechanism linking the functions of BRCA1 to these two hormone-related tumors remains to be elucidated. Here, we report that BRCA1 interacts with androgen receptor (AR) and enhances AR target genes, such as p21 (WAF1/CIP1) , that may result in the increase of androgen-induced cell death in prostate cancer cells. The BRCA1-enhanced AR transactivation can be further induced synergistically with AR coregulators, such as CBP, ARA55, and ARA70. Together, these data suggest that the BRCA1 may function as an AR coregulator and play positive roles in androgen-induced cell death in prostate cancer cells and other androgen/AR target organs.
// Hsueh-Chou Lai 1, 2 , Chun-Chieh Yeh 1, 2 , Long-Bin Jeng 1 , Shang-Fen Huang 2 , Pei-Ying Liao 2 , Fu-Ju Lei 1, 2 , Wei-Chun Cheng 1, 2 , Cheng-Lung Hsu 3 , Xiujun Cai 4 , Chawnshang Chang 2, 4, 5 , Wen-Lung Ma 1, 2 1 Graduate Institution of Clinical Medical Science, and Graduate Institution of Cancer Biology, China Medical University, Taichung 40403, Taiwan 2 Sex Hormone Research Center, Organ Transplantation Center, Research Center for Tumor Medical Science, and Department of Gastroenterology, China Medical University/Hospital, Taichung 40403, Taiwan 3 Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung University/Memorial Hospital, Taoyuan 333, Taiwan 4 Chawnshang Chang Liver Cancer Center, Department of General Surgery, Sir Run-Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China 5 George Whipple Laboratory for Cancer Research, Department of Pathology, The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY 14623, USA Correspondence to: Wen-Lung Ma, email: maverick@mail.cmu.edu.tw Chawnshang Chang, email: chang@urmc.rochester.edu Keywords: AR, HCC recurrence, CTC, CD90, anoikis Received: December 15, 2015 Accepted: May 28, 2016 Published: June 20, 2016 ABSTRACT Purpose: Although hepatectomy and liver transplantation surgery for hepatocellular carcinoma (HCC) are effective treatment modalities, the risk of recurrence remains high, particularly in patients with a high number of circulating tumor cells (CTCs) expressing cancer stem/progenitor cell markers. Androgen receptor (AR) signaling has been shown to suppress HCC metastasis in rodent models of HCC. In this study, we investigated whether AR is associated with postoperative HCC recurrence. Experimental Design: CTCs were obtained from patients with HCC who had undergone hepatectomy to investigate whether they are associated with disease outcome. AR knockout was introduced in two mouse models of spontaneous HCC (carcinogen- and hepatitis B virus-related HCC) to delineate the role that AR plays in HCC recurrence. Biological systems analysis was used to investigate the cellular and molecular mechanisms. Results: We found that the expression of AR in CTCs was negatively associated with HCC recurrence/progression after hepatectomy. Our results suggest that AR-mediated suppression of HCC recurrence/progression is governed by a three-pronged mechanism. First, AR suppresses the expression of CD90 in CTCs by upregulating Histone 3H2A. Second, AR suppresses cell migration at the transcriptome level. Third, AR promotes anoikis of CTCs via dysregulation of cytoskeletal adsorption. Conclusions: The results indicate that AR expression may be the gatekeeper of postoperative HCC recurrence. Therefore, targeting AR in presurgical down-staging procedures may serve as a secondary prevention measure against HCC recurrence in the future.
Hormone therapy for prostate cancer eventually fails leading to a stage called hormone-resistant (HR) disease. To investigate the issue about the characteristics and the radiation response in HR prostate cancer, we established HR cell sub-lines, 22RV1-F and 22RV1-DF, from 22RV1 cells with androgen deprivation for 16 weeks, and obtained LNCaP-HR from LNCaP with long-term bicalutamide treatment. We examined their sensitivities to radiation therapy and the underlying mechanisms. In vitro and in vivo faster tumor growth rate was noted in the HR prostate cancer cells when compared with control. Moreover, HR prostate cancer cells had greater capacity to scavenge reactive oxygen species, and suffered less apoptosis and senescence, and subsequently were more likely to survive from irradiation as measured by clonogenic assay in vitro and growth delay in vivo. The decreased p53 and increased mouse double minute 2 oncogene (MDM2) might be the potential underlying mechanisms for the more aggressive growth and more radioresistance in HR prostate cancer cells. In conclusion, HR prostate cancer cells appeared to be more aggressive in tumor growth and in resistance to radiation treatment. Regulation of the expressions of p53 and MDM2 should be the promising treatment strategies for relative radioresistant prostate cancer.
Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC).Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5'azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator's binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay.A tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region - 62 to - 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration.CLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression.
Early studies suggested that the signature motif, LXXLL, within steroid hormone receptor p160 coregulators may play important roles for the mediation of receptor-coregulator interaction. Interestingly, several androgen receptor (AR) coregulators, such as ARA70 and ARA55, may not use such a unique motif to mediate their coregulator activity. Here we apply the phage display technique to identify some new signature motifs, (F/W)XXL(F/W) and FXXLY (where F is phenylalanine, W is tryptophan, L is leucine, Y is tyrosine, and X is any amino acid) that can influence the interaction between AR and AR coregulators. Sequence analyses found that several AR coregulators, such as ARA70, ARA55, ARA54, and FHL2, contain FXXL(F/Y) motifs. Both glutathione S-transferase pull-down assays and transient transfection reporter assays demonstrate that these AR coregulators may use the FXXL(F/Y) motif to interact with AR and exert their AR coregulator activity. Exchanging the amino acid of Phe, Trp, or Tyr in this newly identified signature motif cluster may influence these peptides to interact with AR. The motif-containing peptides, as well as ARA70 or ARA54, may require selective flanking sequences for the better interaction with AR. In addition to influencing the AR transactivation, these motifs in AR-interacting peptides/proteins were also able to influence the AR N-/C-terminal interaction. Together, our data suggest that AR interacting peptides and/or AR coregulators may utilize the (F/W)XXL(F/W) and FXXLY motifs to mediate their interaction with AR and exert their influences on the AR transactivation.
Using bicalutamide-androgen receptor (AR) DNA binding domain-ligand binding domain as bait, we observed enrichment of FxxFY motif-containing peptides. Protein database searches revealed the presence of receptor-interacting protein kinase 1 (RIPK1) harboring one FxxFY motif. RIPK1 interacted directly with AR and suppressed AR transactivation in a dose-dependent manner. Domain mapping experiments showed that the FxxFY motif in RIPK1 is critical for interactions with AR and the death domain of RIPK1 plays a crucial role in its inhibitory effect on transactivation. In terms of tissue expression, RIPK1 levels were markedly higher in benign prostate hyperplasia and non-cancerous tissue regions relative to the tumor area. With the aid of computer modeling for screening of chemicals targeting activation function 2 (AF-2) of AR, we identified oxadiazole derivatives as good candidates and subsequently generated a small library of these compounds. A number of candidates could effectively suppress AR transactivation and AR-related functions in vitro and in vivo with tolerable toxicity via inhibiting AR-peptide, AR-coregulator and AR N-C interactions. Combination of these chemicals with antiandrogen had an additive suppressive effect on AR transcriptional activity. Our collective findings may pave the way in creating new strategies for the development and design of anti-AR drugs.
The proline-rich tyrosine kinase 2 (Pyk2) was first identified as a key kinase linked to the MAP kinase and JNK signaling pathways that play important roles in cell growth and adhesion. The linkage between Pyk2 and the androgen receptor (AR), an important transcription factor in prostate cancer progression, however, remains unclear. Here we report that using the full-length androgen receptor-associated protein, ARA55, coregulator as bait, we were able to isolate an ARA55-interacting protein, Pyk2, and demonstrated that Pyk2 could repress AR transactivation via inactivation of ARA55. This inactivation may result from the direct phosphorylation of ARA55 by Pyk2 at tyrosine 43, impairing the coactivator activity of ARA55 and/or sequestering ARA55 to reduce its interaction with AR. Our finding that Pyk2 can indirectly modulate AR function via interaction and/or phosphorylation of ARA55 not only expands the role of Pyk2 in AR-mediated prostate cancer growth but also strengthens the role of ARA55 as an AR coregulator. The proline-rich tyrosine kinase 2 (Pyk2) was first identified as a key kinase linked to the MAP kinase and JNK signaling pathways that play important roles in cell growth and adhesion. The linkage between Pyk2 and the androgen receptor (AR), an important transcription factor in prostate cancer progression, however, remains unclear. Here we report that using the full-length androgen receptor-associated protein, ARA55, coregulator as bait, we were able to isolate an ARA55-interacting protein, Pyk2, and demonstrated that Pyk2 could repress AR transactivation via inactivation of ARA55. This inactivation may result from the direct phosphorylation of ARA55 by Pyk2 at tyrosine 43, impairing the coactivator activity of ARA55 and/or sequestering ARA55 to reduce its interaction with AR. Our finding that Pyk2 can indirectly modulate AR function via interaction and/or phosphorylation of ARA55 not only expands the role of Pyk2 in AR-mediated prostate cancer growth but also strengthens the role of ARA55 as an AR coregulator. androgen receptor c-Jun NH2-terminal kinase nuclear receptors 5α-dihydrotestosterone proline-rich tyrosine kinase 2 kinase-negative Pyk2 androgen receptor-associated protein DNA-binding domain luciferase mouse mammary tumor virus Renilla luciferase prostate-specific antigen platelet-derived growth factor phosphatidylinositol 3-kinase mitogen-activated protein fetal calf serum minus uronolactone. The androgen receptor (AR),1 a transcription factor, requires coregulators to exert its optimal or proper function in the control of cell growth and death (1Chang C. Kokontis J. Liao S.T. Science. 1988; 240: 324-326Crossref PubMed Scopus (729) Google Scholar, 2Chang C. Saltzman A. Yeh S. Young W. Keller E. Lee H.J. Wang C. Mizokami A. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 97-125Crossref PubMed Scopus (245) Google Scholar, 3Sampson E.R. Yeh S.Y. Chang H.C. Tsai M.Y. Wang X. Ting H.J. Chang C. J. Biol. Regul. Homeost. Agents. 2001; 15: 123-129PubMed Google Scholar, 4McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar). Several AR coregulators including ARA24, ARA55, ARA70, ARA160, and ARA267 were isolated in our previous studies (5Hsiao P.W Lin D.L Nakao R. Chang C. J. Biol. Chem. 1999; 274: 20229-20234Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 6Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. 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Invest. 2001; 81: 51-59Crossref PubMed Scopus (41) Google Scholar). The significance of Pyk2 interaction/phosphorylation of ARA55 in prostate cancer progression is currently unclear. Here we demonstrate that Pyk2 is an ARA55-interacting protein that represses AR transactivation via phosphorylation of ARA55. This new signal pathway from Pyk2-ARA55-AR may represent a novel mechanism to modulate AR function in prostate cancer. 5α-dihydrotestosterone (DHT) and doxycycline were obtained from Sigma. PDGF-B-chain homodimer (PDGF-BB) and hygromycin B were purchased from Invitrogen. The anti-AR polyclonal antibody, NH27, was produced as described (31Hu S. Yeh Y.C. Rahman M. Lin H.K. Hsu C.L. Ting H.J. Kang H.Y. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11256-11261Crossref PubMed Scopus (130) Google Scholar). A monoclonal antibody for ARA55 (anti-Hic-5) was purchased from Transduction Laboratories. Antibodies for Pyk2 (anti-Pyk2) and phosphotyrosine (anti-Tyr(P)) were purchased from Upstate Biotechnology. His probe (H-3) was purchased from Santa Cruz Biotechnology. The pKH3Pyk2 expression plasmid was kindly provided by Dr. Jun-Lin Guan, Cancer Biology Laboratory, Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell University. The pEF-PKM expression plasmid (kinase-negative Pyk2) was kindly provided by Dr. Aknori Takaoka, Dept. of Immunology, University of Tokyo, Japan and Dr. Joseph Schlessinger, Dept. of Pharmacology, New York University Medical Center. The PKM cDNA was subcloned into theEcoRI site of the pcDNA4A expression vector. The PC-3(AR)2 cell line was provided by T. J. Brown, University of Toronto, Ontario, Canada. We also constructed ARA55 into the pcDNA4-His, GAL4, and VP16 vectors using the BamHI enzyme. The Pyk2 C terminus (675–1009) that was isolated from the yeast library was constructed into the GAL4 and VP16 vectors. DU145, PC-3, H1299, and MCF-7 human cancer cells, as well as COS-1 monkey kidney cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (25 units/ml), streptomycin (25 μg/ml), and 10% fetal calf serum (FCS). PC-3(AR)2 human cancer cells were maintained in RPMI enzyme 1640 containing penicillin (25 units/ml), streptomycin (25 μg/ml), and 10% charcoal-stripped FCS. LNCaP human prostate cells were maintained in RPMI 1640 containing penicillin (25 units/ml), streptomycin (25 μg/ml), and 10% FCS. A CytoTrap yeast two-hybrid system (Stratagene) and pMyr plasmid library (Stratagene) consisting of a DNA sequence encoding a myristylation membrane localization signal fused with human prostate and testis cDNAs were used for yeast two-hybrid screening. The pSos vector containing the hSos gene fused with the full-length ARA55 cDNA using the BamHI cloning site served as bait. Expression of the myristylation sequence-tagged target protein is induced by galactose but not glucose and is anchored to the cell membrane. The library was screened by co-transformation of the pSos-ARA55 bait construct into a temperature-sensitive cdc25H yeast strain that cannot grow at 37 °C. Once the bait protein physically interacts with the target protein, the hSos protein is recruited to the membrane activating the Ras signaling pathway and allowing the temperature-sensitive mutant yeast strain to grow at 37 °C. The potential tyrosine kinase target site on wild type ARA55 was mutated by site-directed mutagenesis using two primers 5′-GGACCACCTGTTCAGCACGGTATG-3′ and 5′-CATACCGTGCTGAACAGGTGGTCC-3′, substituting phenylalanine for tyrosine at amino acid 43 (Y43F). The mutant ARA55 PCR product was constructed into the pcDNA4A expression vector for the AR transactivation assay and constructed into the GAL4 and VP16 expression vectors for mammalian two-hybrid assay. DU145 cells, an ARA55-negative human prostate cancer cell line, was transfected with pBig or pBig-ARA55 for 24 h using SuperFect (Qiagen, Chatsworth, CA). The cells were then selected using 100 μg/ml hygromycin B (32Strathdee C.A. McLeod M.R. Hall J.R. Gene. 1999; 229: 21-29Crossref PubMed Scopus (117) Google Scholar). A single colony was chosen, amplified, and confirmed by reporter gene and Western blotting assay. DU145, H1299, MCF-7, LNCaP, PC-3(AR2), and COS-1 cells were grown in appropriate medium. Transfection was performed by modified calcium phosphate precipitation as previously described (33Mizokami A. Yeh S.Y. Chang C. Mol. Endocrinol. 1994; 8: 77-88Crossref PubMed Scopus (78) Google Scholar) or by using SuperFect according to the manufacturer's procedure. After incubation for 24 h with charcoal-stripped medium, the medium was changed, and cells were treated with ethanol or DHT for another 24 h and then harvested for the luciferase assay. The MMTV-LUC or PSA-LUC plasmids were used as the reporter genes, and SV40-pRL (Promega) was used as an internal control. The Dual-luciferase reporter 1000 assay system (Promega) was employed to measure LUC activity. Transfections in DU145, H1299, or COS-1 cells were performed using the calcium phosphate precipitation method. The cells were transiently co-transfected with the pG5-LUC reporter plasmid and 3.5 μg of both the GAL4 and pVP16-hybrid expression plasmids. The SV40-pRL plasmid was used as an internal control. For the interaction between ARA55 and AR the cells were treated with 10 nm DHT for another 24 h and then harvested for luciferase assays as previously described. H1299 cells were co-transfected with Pyk2 and His-ARA55 expression plasmids by SuperFect. Cells were lysed using radioimmune precipitation buffer (RIPA) following the protocol from Santa Cruz Biotechnology and supplemented with protease inhibitor mixture tablet (Roche Molecular Biochemicals) and 1 mm phenylmethlsulfonyl fluoride. Anti-His probe antibody (Santa Cruz) was used to immunoprecipitate the complex from the whole cell lysate. The complex was then resolved on a 10% SDS-polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane and then blotted with anti-Pyk2. The Pyk2 bands were resolved by an alkaline phosphatase detection kit (Bio-Rad). In the PC-3 whole cell lysate, an anti-Hic-5 antibody was used to immunoprecipitate the endogenous ARA55 and Pyk2 complex followed by the previously described procedure. After transfection with various combinations of pcDNA4A-ARA55, pSG5AR, Pyk2, and PKM by SuperFect, H1299 cells were treated with l0 nm DHT for another 24 h and then starved in DMEM with 0.1% FCS and 10 nm DHT for 16 h. The cells were treated with 1.5 nm PDGF-BB and then harvested. His probe was used to precipitate the His-ARA55 and AR complex that was resolved by Western blotting using anti-AR NH27 and anti-Hic-5 antibodies, respectively. For the ARA55 phosphorylation experiment, RIPA was supplemented with 1 mm pyrophosphate, 50 mmsodium fluoride, and 2 mm sodium vanadate. The anti-Hic-5 antibody was used to immunoprecipitate ARA55, and then the anti-phosphotyrosine antibody was applied for Western blotting. Full-length ARA55 was used as bait to screen prostate and testis libraries using the CytoTrap yeast two-hybrid system. The Pyk2 C-terminal sequence (amino acids 675–1009) was isolated, and its interaction with ARA55 was reconfirmed in a yeast growth assay. When we co-transfected the pSos-ARA55 and pMyr-Pyk2 C terminus plasmids into the temperature-sensitive mutant yeast, cell colonies appeared on both SD/glucose (-UL) agar and SD/galactose (-UL) agar plates at 25 °C and also on SD/(-UL)/galactose agar plates at 37 °C (Fig.1A). Glucose represses the expression of target proteins, preventing yeast growth at 37 °C. Then we constructed ARA55 and the Pyk2 C terminus into both the GAL4 and VP16 vectors and tested their interaction via the mammalian two-hybrid system in various human cell lines. As shown in Fig.1B, GAL4-ARA55 and VP16-Pyk2 C terminus (left panel) and GAL4-Pyk2 C terminus and VP16-ARA55 (right panel) can interact strongly in H1299, DU145, and COS-1 cells. ARA55 also interacts with Pyk2 in vivo by co-immunoprecipitation. As shown in Fig.2A, exogenous Pyk2 can be co-immunoprecipitated with His-ARA55 from H1299 whole cell extracts using an anti-His antibody. In addition, endogenous ARA55 and Pyk2 can be co-immunoprecipitated in PC-3 cells using an anti-Hic-5 antibody, confirming the in vivo interaction of the two proteins (Fig.2B). Together, results from Figs. 1 and 2 demonstrate that ARA55 can interact with Pyk2 using various in vitro andin vivo systems in several yeast and mammalian cells. Interestingly, whereas Pyk2 can interact with ARA55, an AR-interacting protein, Pyk2 cannot interact with AR in the same mammalian two-hybrid assay (Fig. 3) or co-immunoprecipitation assays (data not shown) suggesting that any effect of Pyk2 on AR activity may require interaction with ARA55.Figure 3Pyk2 interacts with ARA55 but not AR in the mammalian two-hybrid assay. COS-1 and H1299 cells were transiently co-transfected with 3 μg of pG5-LUC reporter plasmid, 10 ng of SV40-pRL internal control plasmid, as well as 3.5 μg of GAL4DBD, VPl6, GAL4-ARA55, GAL4-Pyk2C terminus, or VP16-AR as indicated. 24 h after transfection the cells were treated with 10 nm DHT for another 24 h. The LUC activity of the sample transfected with GAL4DBD and VPI6 was set as 1-fold. All values represent the mean ± S.D. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 50 μg of whole cell lysate of each cell line were separated on a SDS-PAGE gel and blotted by anti-Pyk2 and anti-ARA55 antibodies. Pyk2 is almost ubiquitously expressed among these cell lines with the exception of H1299 (Fig. 4). In contrast, ARA55 was only expressed in PC-3, PC-3(AR2), and COS-1 cells (Fig. 4). H1299 cells were then used for further study because of relatively lower expression of both Pyk2 and ARA55. Results from Fig. 4 also offered us the opportunity to study Pyk2 function in ARA55-positive versus -negative cells. To study the potential influence of Pyk2 on AR function via interaction with ARA55, we compared the effect of Pyk2 on AR transactivation in ARA55-negative versus ARA55-positive cells. As shown in Fig.5, whereas Pyk2 can significantly repress AR transactivation in ARA55-positive PC-3(AR)2 cells, Pyk2 has only a marginal effect in LNCaP, MCF-7, and DU145 cells that lack endogenous ARA55. These results further suggest that Pyk2 may suppress AR transactivation through the interaction with ARA55. We then used PC-3(AR2), an ARA55-positive cell line stably transfected with AR to further characterize the effect of Pyk2 on AR transactivation. As shown in Fig. 6, Pyk2 can suppress AR transactivation in a dose-dependent manner using the PSA or MMTV promoters linked to the LUC (Fig. 6A) or chloramphenicol acetyltransferase (CAT) reporter systems (data not shown). In contrast, PKM with a lysine to alanine substitution at amino acid 475 of Pyk2 (20Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar) only slightly affected AR transactivation (Fig.6A). To verify that ARA55 is required for Pyk2 to suppress AR activity, we then stably transfected DU145 with ARA55 using a doxycycline-inducible system. As with PC-3(AR2), transfection of Pyk2, ARA55, and AR into parent ARA55-negative DU145 cells resulted in suppression of AR transactivation in a dose-dependent manner (data not shown). Treatment of the stably transfected pBIG-ARA55 DU145 cells with doxycycline to induce ARA55 expression resulted in enhanced AR transactivation that could be suppressed by the exogenous Pyk2. In contrast, DU145 cells stably transfected with the pBIG vector demonstrated no increased AR activity upon doxycycline treatment and were unaffected by exogenous Pyk2. (Fig. 6B). These results support the essential role of ARA55 in suppression of AR transactivation by Pyk2. Since PKM, a kinase-negative Pyk2 mutant (20Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar), failed to repress AR transactivation, we suspected that Pyk2 may need to phosphorylate ARA55 to suppress AR function. Sequence analysis revealed a potential Pyk2 tyrosine kinase phosphorylation site, HLYST, on ARA55 at amino acids 41–45. We therefore mutated this potential Pyk2 target site to HLFST and tested its effect on the AR transactivation. Results in Fig.7A demonstrate that PDGF-induced Pyk2 can enhance the tyrosine phosphorylation of ARA55(lane 3 versus lane 2) in vivo. In contrast, PDGF-induced Pyk2 only marginally increased the phosphorylation of mutant ARA55 (lane 4 versus lane 3). As shown in Fig. 7B wild type ARA55 alone, but not Pyk2, enhances AR transactivation (lanes 4 versus 2 and lanes 3 versus 2, respectively), but addition of Pyk2 suppresses the ARA55-enhanced AR transactivation (lane 5 versus lane 4). Interestingly, the mutant ARA55, like wild type ARA55, enhances AR transactivation (lane 6 versus lane 2), but addition of Pyk2 only marginally suppresses mutant ARA55-induced AR transactivation (lane 6 versus lane 7). The data from Fig. 7,A and B demonstrate that Pyk2 enhances the phosphorylation of ARA55 at residue 43 to suppress AR transactivation. Pyk2 may also inhibit ARA55-induced AR transactivation by blocking the interaction between AR and ARA55 or by sequestering ARA55 away from AR. Results from the mammalian two-hybrid assay in H1299 cells show that wild type Pyk2, but not kinase-negative PKM, can block the interaction between AR and ARA55 (Fig.8A). Interestingly, the interaction between AR and the Y43F ARA55 mutant can only be partially blocked by either Pyk2 or PKM. We then used co-immunoprecipitation to verify the ability of Pyk2 to block the interaction between AR and ARA55 in H1299 cells. As shown in Fig. 8B, anti-His antibody precipitates the complex containing AR and wild type or mutated ARA55. Addition of Pyk2 in the presence of PDGF, a growth factor that activates Pyk2, can then block the interaction between AR and wild type ARA55 but not the interaction between AR and mutated ARA55 (lanes 3 and 4 versus lanes 7 and 8). PKM only slightly blocks the interaction between AR and ARA55 (lanes 5 and6). Results from both the mammalian two-hybrid and co-immunoprecipitation assays demonstrate that Pyk2, but not kinase-negative PKM, can block the interaction between AR and wild type ARA55. Cross-talk between various kinase signals and the androgen/AR pathway has been well documented. The phosphorylation of AR co-regulators to modulate AR transactivation, however, is currently unclear. Pyk2 is an important tyrosine kinase that can be induced by various extracellular stimuli (24Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 25Hiregowdara D. Fu H. Avraham Y. London R. Avraham S. J. Biol. Chem. 1997; 272: 10804-10810Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Brinson A.E. Harding T. He P.A. Li Y. Diliberto X. Hunter D. Herman B. Earp H.S. Graves L.M. J. Biol. Chem. 1998; 273: 1711-1718Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 27Zheng C. Xing Z. Bian Z.C. Guo C. Akbay A. Warner L. Guan J.L. J. Biol. Chem. 1998; 273: 2384-2389Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28Miyazaki T. Takaoka A. Nogueira L. Dikic I. Fujii H. Tsujino S. Mitani Y. Maeda M. Schlessinger J. Taniguchi T. Genes Dev. 1998; 12: 770-775Crossref PubMed Scopus (67) Google Scholar) resulting in the activation of the MAPK and JNK kinase pathways (20Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 23Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). The linkage from the Pyk2 pathway to NR transactivation, however, remains largely unknown. Here we provide evidence demonstrating that Pyk2 can interact with and phosphorylate ARA55 to suppress AR transactivation. This finding may represent a new mechanism to modulate AR function and supports the role of ARA55 as an AR co-regulator (6Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. Mizokami A. Chang C. J. Biol. Chem. 1999; 274: 8316-8321Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Tissue distribution analysis indicates that ARA55 is differentially expressed in various stages of prostate cancer (6Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. Mizokami A. Chang C. J. Biol. Chem. 1999; 274: 8316-8321Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 12Fujimoto N. Mizokami A. Harada S. Matsumoto T. Urology. 2001; 58: 289-294Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Stanzioneet al. (30Stanzione R. Picascia A. Chieffi P. Imbimbo C. Palmieri A. Mirone V. Staibano S. De Franco R. Rosa G. Schlessinger J. Tramontano D. Lab. Invest. 2001; 81: 51-59Crossref PubMed Scopus (41) Google Scholar) also reported that Pyk2 expression declines with increasing prostate cancer grade. These findings, in addition to our data showing that ARA55 and Pyk2 modulate AR function, suggest that the regulation of AR function may be altered in prostate cancer by changes in Pyk2 and ARA55 expression. The significance of these alterations in the progression of prostate cancer from androgen dependence to androgen independence, however, has yet to be determined. Results from Figs. 5 and 6 clearly demonstrate that Pyk2 needs ARA55 to suppress AR function. Pyk2, a tyrosine kinase linked to the MAPK and JNK signaling pathways and thereby the regulation of cell growth and adhesion, may also utilize non-ARA55-mediated pathways to exert its physiological functions. The discovery of new pathways that cross-talk with Pyk2→ARA55→AR signaling may therefore expand the importance of Pyk2 in the control of prostate cancer growth. The inability of PKM to suppress AR function via wild type ARA55 and the inability of Pyk2 to suppress AR function via mutated ARA55 suggests that the Pyk2 phosphorylation of ARA55 at residue 43 is critical in the suppression of AR function. Since this phosphorylation site is not located in the AR interaction domain of ARA55, amino acids (251–444) (6Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. Mizokami A. Chang C. J. Biol. Chem. 1999; 274: 8316-8321Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), it is possible that other mechanisms may also be involved in the suppression of AR function by Pyk2-ARA55. Nevertheless, our mammalian two-hybrid and co-immunoprecipitation assays indicate that Pyk2 blocks the interaction between AR and ARA55. Therefore, it is possible that Pyk2 may have multiple ways to communicate with ARA55 to suppress AR transactivation. In summary, Pyk2 can dramatically repress AR transactivation by inhibiting the coregulatory activity of ARA55. This interruption may entail both the direct phosphorylation of ARA55 to impair its coregulatory activity and/or sequestering ARA55 to reduce its interaction with AR. Our findings not only expand the role of Pyk2 in AR-mediated prostate cancer growth but also support the importance of ARA55 in the control of AR function. We thank Drs. Jun-Lin Guan, Aknori Takaoka, and Joseph Schlessinger for valuable plasmids and Dr. T. J. Brown for the PC-3(AR2) cell line. We also thank Karen Wolf for manuscript preparation.
Early reports showed that androgen receptor (AR) NH2- and COOH-terminal (N-C) interaction was important for full AR function. However, the influence of these interactions on the AR in vivo effects remains unclear. Here we tested some AR-associated peptides and coregulators to determine their influences on AR N-C interaction, AR transactivation, and AR coregulator function. The results showed that AR coactivators such as ARA70N, gelsolin, ARA54, and SRC-1 can enhance AR transactivation but showed differential influences on the N-C interaction. In contrast, AR corepressors ARA67 and Rad9 can suppress AR transactivation, with ARA67 enhancing and Rad9 suppressing AR N-C interaction. Furthermore, liganded AR C terminus-associated peptides can block AR N-C interaction, but only selective peptides can block AR transactivation and coregulator function. We found all the tested peptides can suppress prostate cancer LNCaP cell growth at different levels in the presence of 5alpha-dihydrotestosterone, but only the tested FXXLF-containing peptides, not FXXMF-containing peptides, can suppress prostate cancer CWR22R cell growth. Together, these results suggest that the effects of AR N-C interactions may not always correlate with similar effects on AR-mediated transactivation and/or AR-mediated cell growth. Therefore, drugs designed by targeting AR N-C interaction as a therapeutic intervention for prostate cancer treatment may face unpredictable in vivo effects.
The androgen receptor (AR) is a member of the steroid receptor superfamily that binds to the androgen response element to regulate target gene transcription. AR may need to interact with some selected coregulators for maximal or proper androgen function. Here we report the isolation of a new AR coregulator with a calculated molecular mass of 267 kDa named the androgen receptor-associated protein 267-α (ARA267-α). ARA267-α contains 2427 amino acids, including one Su(var)3-9,Enhancer-of-zeste, and Trithorax (SET) domain, two LXXLL motifs, three nuclear translocation signal (NLS) sequences, and four plant homodomain (PHD) finger domains. Northern blot analyses reveal that ARA267-α is expressed predominantly in the lymph node as 13- and 10-kilobase transcripts. HepG2 is the only cell line tested that does not express ARA267-α. Yeast two-hybrid and glutathione S-transferase pull-down assays show that both the N and C terminus of ARA267-α interact with the AR DNA- and ligand-binding domains. Unlike other coregulators, such as CBP, which enhance the interaction between the N and C terminus of AR, we found that ARA267-α had little influence on the interaction between the N and C terminus of AR. Luciferase and chloramphenicol acetyltransferase assays show that ARA267-α can enhance AR transactivation in a dihydrotestosterone-dependent manner in PC-3 and H1299 cells. ARA267-α can also enhance AR transactivation with other coregulators, such as ARA24 or PCAF, a histone acetylase, in an additive manner. Together, our data demonstrate that ARA267-α is a new AR coregulator containing the SET domain with an exceptionally large molecular mass that can enhance AR transactivation in prostate cancer cells.
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