Identification of Genes Targeted by the Androgen and PKA Signaling

Oncogene (2006) 25, 7311–7323 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ONCOGENOMICS Identiﬁcation of genes targeted by the androgen and PKA signaling pathways in prostate cancer cells

G Wang, SJM Jones, MA Marra and MD Sadar

1Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada

Progression of prostate cancer to androgen independence receptors that function as ligand-dependent transcrip- is suspected to involve the androgen and protein kinase A tion factors. Ligand-activated AR, complexed with (PKA) signaling pathways. Here for the first time, the coactivator proteins and general transcription factors, transcriptomes associated with each pathway and common bind to cis-acting androgen response elements (AREs) transcriptional targets in response to stimulation of both located in the promoter and enhancer regions to activate pathways were identified in human prostate cancer cells or repress the transcription of specific target genes. using Affymetrix GeneChip technology (Human Genome Androgen-regulated genes are involved in morphogen- U133 plus2). Statistically significant changes in the levels esis and differentiation of the prostate gland as well as of 858 genes in response to androgen and 303 genes in the development and progression of prostate diseases response to activation of the PKA pathway were such as adenocarcinoma of the prostate (Lee et al., 1995; determined using GeneSpring software. Expression of a Prins, 2000; Nelson et al., 2002). The recognition that subset of these genes (22) that were transcriptional targets normal and neoplasticprostate epithelial cellsdepend for the androgen and/or PKA pathways were validated by on circulating androgens for their continued survival reverse transcriptase–polymerase chain reaction and and growth led to the development of effective endo- Western blot analyses. Application of small interfering crine-based therapy for prostate carcinoma (Huggins RNAs to the androgen receptor (AR) revealed that in and Hodges, 2002). Most prostate cancers initially addition to KLK3, levels of expression of KLK2 and respond to androgen-ablation therapy. However, these SESN1 were regulated by AR activated by both the tumors eventually become androgen independent (hor- androgen and PKA signaling pathways. SESN1 was mone refractory) and grow despite androgen ablation. identified as a gene repressed by activated AR. These The possible mechanisms of the development of results provide a broad view of the effects of the androgen hormone refractory prostate cancer can be divided into and PKA signaling pathways on the transcriptional two pathways – those involving AR and those that program of prostate cancer cells and indicate that only a bypass the receptor. These pathways are not mutually limited number of genes are targeted by cross-talk exclusive and frequently coexist in hormone refractory between AR and PKA pathways. prostate cancer (Debes and Tindall, 2004). Some Oncogene (2006) 25, 7311–7323. doi:10.1038/sj.onc.1209715; possible pathways involving AR-mediated survival of published online 5 June 2006 prostate cancer include amplification or mutations of the receptor, alteration of coactivators, deregulation Keywords: prostate cancer; androgen receptor; Affyme- of growth factors or cytokines and cross-talk with other trix; LNCaP hollow fiber model; cross-talk; PKA pathways such as mitogen-activated protein kinase and protein kinase A (PKA) pathways (Wang and Sadar, 2006). The cyclic adenosine monophosphate (cAMP)-PKA Introduction pathway is one of the most common and versatile signal transduction pathway in eukaryotic cells and is involved ONCOGENOMICS Prostate cancer is a major health problem in Western in regulation of cellular functions in almost all tissues in countries. It is the most frequently diagnosed cancer mammals. Various extracellular signals converge on this among men and the second leading cause of male cancer signaling pathway through ligand binding to G protein- death (Jemal et al., 2005). The androgenichormones, coupled receptors. The cAMP-PKA pathway is tightly testosterone and dihydrotestosterone exert their cellular regulated at several levels and itself is involved in effects by binding with the androgen receptor (AR), a the regulation of diverse cellular processes such as member of the family of intracellular steroid hormone cell cycle, proliferation and differentiation and regulation of microtubule dynamics, chromatin condensation and decondensation, nuclear envelope disassembly and Correspondence: Dr MD Sadar, Genome Sciences Centre, BC Cancer reassembly, as well as regulation of intracellular trans- Agency, 675 West 10th Avenue, Vancouver, BC, Canada V5Z1L3. E-mail: [email protected] port mechanisms and ion fluxes (Tasken and Aandahl, Received 30 August 2005; revised 9 March 2006; accepted 25 April 2006; 2004). Neuroendocrine differentiation of prostate cancer published online 5 June 2006 cells is implicated in androgen-independent prostate Genes regulated by the androgen and PKA signaling pathways G Wang et al 7312 cancer and can be induced in LNCaP cells by activation of the PKA pathway (Bang et al., 1994; Cox et al., 2000). Cross-talk between the AR and PKA signal transduction pathways occurs in androgen-depleted human prostate cancer cells maintained in culture (Sadar et al., 1999). These studies have shown that antiandrogens can block PKA induction of prostate- specific antigen (PSA) mRNA (Sadar, 1999) and androgen-responsive reporters (Nazareth and Weigel, 1996; Culig et al., 1997; Sadar, 1999). Activation of the PKA pathway inhibits the binding of the co-repressor SMRT (silencing mediator for retinoic acid and thyroid hormone receptor) to AR (Dotzlaw et al., 2002). These data suggest activation of AR via the PKA pathway. In the present study, we applied Affymetrix GeneChip (Human Genome U133 plus2) analysis to characterize the global program of transcription that reflects the cellular response to R1881, a synthetic androgen, and forskolin (FSK), an activator of adenylyl cyclase to synthesize cAMP which in turn stimulates PKA activity. Through the analysis of gene expression profiles in response to both of these two treatments we were able to identify common targets of cross-talk between the androgen and PKA pathways.

Results

A genome-wide view of androgen and FSK effects In the absence of androgen, PSA gene expression is induced by activation of the PKA pathway with FSK by a mechanism dependent on a functional AR (Sadar, 1999). To identify other genes targeted by cross-talk between the androgen and PKA pathways, experiments were undertaken to characterize the genome-wide expression profiles for androgen and FSK. LNCaP human prostate cancer cells were employed because these cells express endogenous functional AR and PSA (Horoszewicz et al., 1980; Young et al., 1991). LNCaP cells were exposed for 16 h to FSK (1 mM) or R1881 (10 nM) since this time and these concentrations have been previously shown to be optimal for the maximum induction of PSA mRNA (Sadar, 1999). Total RNA was extracted from the biological triplicate samples and analysed using Affymetrix Genechips (Human Genome U133plus2 GeneChip) to determine the global gene expression patterns. The Human Genome U133plus2 GeneChip contains greater than 54 000 probe sets and Figure 1 Cluster analysis of genes differentially expressed across can be used to analyse the expression level of more than all treatments. Unsupervised clustering analysis of all the 12 47 000 transcripts and variants, including approximately samples according to the 1435 genes designated significantly 38 500 well-characterized human genes. changed across all conditions by standard correlation using the Significance analysis of microarrays (Tusher et al., GeneSpring microarray analysis software. The genes are represented by each row and the experimental samples are represented 2001) was applied to identify genes differentially on each column (in triplicates). expressed across the different conditions. Using a one- way analysis of variance (Po0.01), 1435 probe sets were determined to be differentially expressed across the various conditions. A hierarchical two-dimensional (represented on each column). As expected each clustering algorithm based on similarity of expression triplicate was clustered together within each of the patterns was applied to these 1435 probe sets (Figure 1, experimental groups indicating a high level of reprodu- represented on each row) and experimental samples cibility between the biological replicates.

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7313 Changes in gene expression in response to androgen CDC6, EIF2B5, ITGB5, NCOA6IP, and decreased Genes differentially expressed in LNCaP cells upon levels of nuclear mitotic apparatus protein (NUMA)1 exposure to R1881 and FSK were identified as those transcripts were confirmed by RT–PCR using RNA cases where the level of transcript changed significantly isolated from cells treated with FSK (Figure 2d). from levels in the relevant controls. A P-value of p0.01 was used to identify genes that were differentially Changes in gene expression in response to both expressed by at least 1.3-fold with androgen stimulation. androgen and FSK These criteria revealed a total of 858 genes as Forty-six probe sets representing 32 genes were sig- significantly different between the mean expression nificantly changed at least 1.3-fold in response to both values for R1881-treated versus control samples. Many androgen and FSK. Applying the Venn diagram, of these genes have been previously reported to be expression of 22 genes was increased by both androgen regulated by androgen in normal prostate or prostate and FSK (Figure 3a). Table 1 provides a list of the cancer. Examples of these include the well-characterized common genes that increased in expression by both androgen-regulated genes such as PSA (KLK3), KLK2 treatments which include PSA (KLK3) and KLK2. and FKBP5 which were induced 6.4-fold (P ¼ 0.0017), Eight genes were differentially expressed by R1881 and 21.6-fold (P ¼ 4.08E-05), and 86.5-fold (P ¼ 0.0007), FSK in opposite directions (Table 2). Expression of respectively. A number of genes not previously reported SESN1 and NAV1 were the only genes to be decreased to be androgen regulated were also detected and include in response to both R1881 and FSK (Table 3). PKIB (À6.7-fold, P ¼ 0.002), SESN1 (À2-fold, Semi-Q-RT–PCR was employed to validate changes P ¼ 0.006), dynein light chain 2 (Dlc2) (2.1-fold, in expression of some of these genes including: those P ¼ 0.006) and RHOU (21.34-fold, P ¼ 1.63E-05). While increased in response to both androgen and FSK such as expression of the majority (784) of these genes increased PSA (KLK3) (4.5-fold induction in response to R1881, with androgen stimulation, 74 genes decreased in P ¼ 0.0065; 2.1-fold induction in response to FSK, expression and include SESN1 (À2-fold, P ¼ 0.006), P ¼ 0.024), 3-hydroxy-3-methylglutaryl coenzyme A OSR2 (À3.36-fold, P ¼ 0.002), MMP16 (À4.67-fold, reductase (HMGCR), insulin-induced gene 1 (INSIG1), P ¼ 0.003) and PKIB (À6.7-fold, P ¼ 0.002) (Figure 2a). KLK2, Dlc2 and FLJ22649; those decreased in response to both androgen and FSK such as SESN1; and Changes in gene expression in response to FSK genes differentially expressed in response to androgen Generally, there was a relatively lower fold change in and FSK in opposite directions such as MMP16 gene expression achieved by FSK as compared to and MGC61716. In LNCaP cells, these genes R1881. A total of 303 genes were detected as signifi- showed changes in gene expression by semi-Q-RT– cantly different with a P-value p0.05 and the fold PCR that were consistent with the Affymetrix data change of at least 1.3 with FSK stimulation as compared (Figure 3b). to control. Expression of the majority of these genes, To explore whether differential expression of these 204, was increased with FSK stimulation while expres- genes in response to androgen and FSK was consistent sion of 99 genes was decreased (Figure 2b). PSA in another androgen-responsive human prostate cancer (KLK3) was induced by FSK treatment by two-fold cell line, semi-Q-RT–PCR was employed using RNA (P ¼ 0.012), consistent with previous studies in this isolated from 22Rv1 cells that express the AR (Sram- cell line (Sadar, 1999). This time point of 16 h was koski et al., 1999; Tepper et al., 2002). 22Rv1 cells optimal to achieve maximum levels of PSA transcript showed similar trends in changes in expression of the induced by both FSK and androgen (R1881). Other PSA (6.2-fold induction in response to R1881, P ¼ 3.8E- genes may have differing kinetics which would have to 05; 1.8-fold induction in response to FSK, P ¼ 0.005), be examined empirically plus FSK effects may be as well as HMGCR, INSIG1, KLK2 and Dlc2 genes transient as opposed to R1881 which has a relatively as compared to LNCaP cells (Figure 3c). However, long half-life. there were some exceptions. Expression of SESN1 and MMP16 did not change in response to androgen or FSK (Figure 3c). FLJ22649 and MGC61716 transcripts could Validation of Affymetrix data by semi-quantitative not be detected in 22Rv1 cells after 40 cycles of PCR reverse transcriptase–polymerase chain reaction (data not shown). To confirm that gene expression changes observed were truly representative of the original samples, we selected several genes that showed distinctly different expression AR is required for changes in gene expression in response in response to either or both of R1881 and FSK for to FSK semi-Q-reverse transcriptase–polymerase chain reaction To clarify whether changes in gene expression in (RT–PCR) analysis. Consistent with the GeneChip data, response to both R1881 and FSK were dependent upon semi-Q-RT–PCR confirmed increased expression of the presence of a functional AR, AR-small interfering FKBP5, RHOU, ELL2, PTPRM, ORM and SOCS2 RNA (siRNA) was used to inhibit the expression of AR. and decreased expression of PKIB and OSR2 in LNCaP cells were transfected for 48 h with AR-siRNA response to R1881 when normalized to the house- (AR1) or nonspecific control siRNA (ARc) before keeping gene glyceraldehyde-3-phophate dehydrogenase R1881 or FSK treatment. After 16 h of treatment, (GAPDH) (Figure 2c). Similarly increased levels of whole cell lysate (WCL) and total RNA were harvested

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7314

Figure 2 Genes differentially expressed in response to androgen and FSK. (a) Changes in gene expression in response to androgen (Pp0.01 and fold change X1.3) and (b) FSK (Pp0.05 and fold change X1.3). The left panel showed the hierarchical two-dimensional clustering algorithm with the genes represented by each row and the experimental samples represented on each column. The right panel indicates genes that were validated by RT–PCR for R1881 (c), and FSK (d). Cells were treated with R1881 (10 nM) or ethanol carrier alone, FSK (1 mM) or DMSO carrier alone for 16 h.

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7315 consistent with ligand stabilizing the AR protein to increase levels as previously reported (Kemppainen et al., 1992; Zhou et al., 1995). FSK did not signiﬁcantly alter the levels of AR protein (P ¼ 0.091). Sixty-four hours after transfection of the AR-siRNA, the level of AR protein was reduced signiﬁcantly in cells treated with vehicle, R1881 and FSK (P ¼ 7.7 Â 10À7, 0.0089 and 0.0028, respectively). Similarly, the induction of PSA protein by R1881 (4.5-fold, P ¼ 0.013) and FSK (2.1-fold, P ¼ 0.048) was completely blocked by transfection of cells with AR-siRNA (P ¼ 0.014 and 0.003, respectively) (Figure 4b). Application of semi-Q RT–PCR detected increased expression of KLK2, PSA, HMGCR and INSIG1 in the presence of scramble siRNA (ARc). AR-siRNA blocked the baseline expression of KLK2 (Figure 4c), PSA (Figure 4d) and HMGCR (Figure 4e) (P ¼ 0.014, 0.007 and 0.0045, respectively), but not INSIG1 (P ¼ 0.176) (Figure 4f). In cells treated with R1881, AR-siRNA blocked the induced expression of KLK2, PSA, HMGCR as well as INSIG1 (P ¼ 0.002, 0.026, 0.006 and 0.01, respectively). In cells treated with FSK, AR-siRNA blocked the induced expression of KLK2 and PSA (P ¼ 0.007 and 0.035), but not HMGCR and INSIG1 (P ¼ 0.821 and 0.97). Thus, AR-siRNA blocks the FSK-induced expression of KLK2 and PSA, consistent with a role for the AR in this mechanism. However, induction of expression of INSIG1 and HMGCR by FSK was independent of the AR. Expression of SESN1 was repressed by both treatments (Figure 4g). In the presence of scramble siRNA (ARc), R1881 and FSK both inhibit the expression of SESN1 (P ¼ 0.002 and 4.86 Â 10À5, respectively), consistent with the results from the microarray data and RT–PCR validation. Application of AR-siRNA (AR1) attenuated the reduced expression of SESN1 by R1881 or FSK (P ¼ 0.01 and 0.02). This implies that SESN1 is a gene that is repressed by activated AR. Although, AR-siRNA also blocked basal levels of expression of SESN1 (P ¼ 0.00013).

In vivo expression of genes differential expressed in response to androgen and FSK To investigate the role of the genes differential expressed in response to androgen and FSK in the hormonal Figure 3 Common genes differentially expressed in response to progression prostate cancer, we applied the LNCaP androgen and FSK. (a) Venn diagrams showing the number of genes significantly upregulated, downregulated, or overlapping by hollow fiber model. This in vivo model uses hollow fibers R1881 and FSK treatments in LNCaP cells. Validation of common to provide a reproducible means of obtaining ‘pure’ changes in gene expression in response to R1881 and FSK in populations of LNCaP cells free of host cell contamina- LNCaP cells (b) and 22Rv1 cells (c) by semi-Q-RT–PCR. Numbers tion during different stages of progression to androgen beside the gel represent the fold change measured by Genechip independence for molecular analysis requiring RNA and technology in LNCaP cells. Total RNA (0.5 mg) was applied in a 10 ml reaction with the primers of each gene and GAPDH. All protein extracts (Sadar et al., 2002). Total RNA was experiments were performed independently at least three times. isolated from samples harvested from LNCaP cells grown in the implanted hollow fibers in nude mice at the time points of pre-castration (intact), 10 days after castration and 52 days after castration when the to investigate the levels of protein and mRNA of AR prostate cancer progressed to the stage of androgen and the commonly regulated genes. In the control independence which was indicated by increased expres- transfection with scramble siRNA (ARc), R1881 in- sion of PSA. Semi-Q-PCR was performed to detect creased levels of AR protein (Figure 4a). This was changes in expression of the genes identified in vitro in

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7316 Table 1 Expression of genes that signiﬁcantly increased in LNCaP cells in response to R1881 and FSK Symbol Affymetrix Fold change in P-value in Fold change in P-value in Description ID R1881 R1881 FSK FSK

ACSL3 201661_s_at 6.34 0.00321 1.32 0.025 Acyl-coenzyme A synthetase long-chain family member 3 223978_s_at 2.93 0.000387 C20orf155 225324_at 2.60 0.000778 Chromosome 20 open reading frame 155 232118_at 1.77 0.0401 CAMKK2 210787_s_at 6.65 0.00112 1.50 0.0171 Calcium/calmodulin-dependent protein kinase kinase 2, beta Dlc2 229106_at 2.10 0.00607 1.52 0.0255 Dynein light chain 2 EG1 225159_s_at 4.44 0.00328 Endothelial-derived gene 1 218438_s_at 1.32 0.00731 EVI5 209717_at 2.04 0.00479 1.32 0.0426 Ecotropic viral integration site 5 FADS1 208962_s_at 2.67 0.00156 1.34 0.0452 601066683F1 NIH_MGC_10 Homo sapiens cDNA clone IMAGE:3452925 51, mRNA sequence. FLJ22649 222753_s_at 3.77 0.00872 1.80 0.0111 Hypothetical protein FLJ22649 similar to signal peptidase SPC22/23 GNAI3 201179_s_at 2.11 0.0044 Guanine nucleotide binding protein (G protein), alpha 201181_at 1.64 0.0452 Inhibiting activity polypeptide 3 HMGCR 202540_s_at 3.59 0.00306 1.56 0.0256 3-Hydroxy-3-methylglutaryl-Coenzyme A reductase INSIG1 201625_s_at 3.83 0.00134 1.71 0.00353 Insulin-induced gene 1 201626_at 4.58 0.0023 1.68 0.0157 KIAA0690 216913_s_at 2.04 0.00851 1.53 0.00579 KIAA0690 KLK2 209854_s_at 31.36 0.00022 1.92 0.00744 Kallikrein 2, prostatic 210339_s_at 21.60 4.08 Â 10À5 1.95 0.0085 KLK3 204582_s_at 6.42 0.00172 2.06 0.0118 Kallikrein 3, (prostate-speciﬁc antigen) 204583_x_at 5.38 0.00405 2.03 0.0194 MAP7 215471_s_at 1.93 0.00846 1.35 0.00947 Microtubule-associated protein 7 NGLY1 220742_s_at 1.96 0.00498 1.31 0.0274 N-Glycanase 1 SMP1 217766_s_at 1.62 0.00744 1.46 0.0228 Small membrane protein 1 211144_x_at 11.44 0.0057 T-cell receptor (V-J-C) precursor; Human T-cell receptor TRGV9 209813_x_at 3.18 0.0046 Gamma chain VJCI-CII–CIII region mRNA, complete cds. UBE2J1 217823_s_at 6.16 0.00074 1.46 0.0439 Ubiquitin-conjugating enzyme E2, J1 (UBC6 homolog, yeast) 221844_x_at 2.87 0.00946 1.54 0.00339 Transcribed sequence with moderate similarity to protein sp:P39195 (H. sapiens) 228559_at 23.68 0.00125 5.90 0.0138 cDNA clone IMAGE:6043059, partial cds 229975_at 5.23 0.00227 1.92 0.0111 Transcribed sequences

Table 2 Genes signiﬁcantly differentially expressed in LNCaP cells in opposite directions in response to R1881 and FSK Symbol Affymetrix Fold change in P-value in Fold change in P-value in Description ID R1881 R1881 FSK FSK

APPBP2 202630_at 4.58 0.00218 À1.43 0.013 Amyloid beta precursor protein (cytoplasmic tail)-binding protein 2 MAF 209348_s_at 21.29 0.000168 À1.35 0.0149 V-maf musculoaponeurotic ﬁbrosarcoma oncogene homolog (avian) MGC61716 226486_at 2.30 0.000992 À1.64 0.0254 Hypothetical protein MGC61716 MJD 205415_s_at 1.67 0.00499 Machado–Joseph disease 233182_x_at À1.79 0.0246 (spinocerebellar ataxia 3, Olivopontocerebellar ataxia 3, autosomal dominant, ataxin 3) MMP16 207012_at À4.67 0.00283 1.35 0.0223 Matrix metalloproteinase 16 (membrane- inserted) PIAS1 217864_s_at 2.26 0.00907 Protein inhibitor of activated STAT, 1 217862_at À1.58 0.0492 SLC30A7 226601_at 2.83 0.00665 À1.39 0.0477 Solute carrier family 30 (zinc transporter), member 7 ZBTB10 219312_s_at 4.84 0.000413 228562_at 3.68 0.00396 Zincﬁnger and BTB domain containing 233899_x_at 4.15 0.00916 10 222863_at À1.36 0.0321

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7317 Table 3 Expression of genes that signiﬁcantly decreased in LNCaP cells in response to R1881 and FSK Symbol Affymetrix ID Fold change in R1881 P-value in R1881 Fold change in FSK P-value in FSK Description

NAV1 224772_at À2.40 0.00579 Neuron navigator 1 227584_at À2.07 0.0116 SESN1 218346_s_at À1.99 0.00589 À1.32 0.023 Sestrin 1 response to androgen and/or FSK. After castration, Genes differentially expressed in response to androgen expression of androgen-stimulated genes such as PSA Androgen-mediated gene expression involves binding of (KLK3), KLK2, Dlc2, ELL2, HMGCR, INSIG1, hormone with the AR protein, resulting in its nuclear RHOU and SOCS2 was decreased compared to intact translocation and interaction with AREs in regulatory levels, while the expression of androgen-suppressed regions of specific genes (Roche et al., 1992). Binding to genes such as MMP16 and SESN1 was increased AREs in the promoter and enhancer of genes facilitates (Figure 5). Interestingly, expression of genes that interactions with the general transcriptional machinery responded in vitro to FSK, such as CDC6, ITGB5 and leading to gene transcription (Roche et al., 1992). NUMA1 also changed in response to castration while Androgens play an important role in secretion, growth NCOA6IP, another gene that responded in vitro to and maintenance of function of the prostate. In this FSK, showed no change during hormonal progression. study, expression of 858 genes was significantly altered As prostate cancer progressed to the androgen-indepen- in LNCaP cells by exposure to androgen including dent stage, the expression of PSA (KLK3), KLK2, known androgen-regulated genes such as PSA (KLK3), ELL2, RHOU, SOCS2, MMP16, CDC6 and NUMA1 KLK2, FKBP5, ELL2, SOCS2, PTPRM, ORM and returned to the levels detected before castration. The OSR2 (Amler et al., 2000; Waghray et al., 2001; Xu expression of Dlc2, HMGCR, INSIG1 and ITGB5 et al., 2001; DePrimo et al., 2002; Nelson et al., 2002; remained decreased while SESN1 remained elevated in Segawa et al., 2002; Holzbeierlein et al., 2004). Previous androgen-independent cells at 52 days after castration as studies using cDNA microarrays revealed changes in compared to intact levels. PKIB, which decreased in expression of 146–517 genes in LNCaP cells in response vitro in response to androgen, showed no significant to androgen (DePrimo et al., 2002; Nelson et al., 2002). change during hormonal progression. Genes not previously reported to change in expression in response to androgen include RHOU and PKIB. RHOU (WRCH1) encodes a CDC42-related GTPase belonging to the RHO family and is a WNT target genes Discussion (Tao et al., 2001). Overexpression of RHOU induces the same effects as overexpression of Wnt1 in the regulation The androgen-signaling pathway plays an important of cell morphology, cytoskeletal organization and cell role in the prostate. Activation of the PKA pathway has proliferation (Tao et al., 2001). Thus, upregulation of also been shown to play a role in prostate biology and RHOU by androgen and in androgen independence may pathology. These two pathways cross-talk through the contribute to increased proliferation of prostate cancer AR in androgen-deprived prostate cancer cells (Sadar cells. PKIB, a member of the cAMP-dependent protein et al., 1999). The present studies investigated genes kinase inhibitor family, is an example of a gene whose commonly regulated by androgen and the PKA path- expression was decreased with androgen. The gene ways in prostate cancer cells and revealed the following. product of PKIB may interact with the catalytic subunit (1) Androgen stimulated changes in expression of 858 of cAMP-dependent protein kinase to act as a compe- genes. The majority (784) showed increased expression titive inhibitor (Van Patten et al., 1991). Decreased with only 74 genes showing decreased expression relative expression of PKIB by androgen may indirectly activate to controls. Genes not previously reported to be PKA. However, in vivo there was no significant change differentially expressed in response to androgen were in gene expression of PKIB in response to castration or also identified. (2) Activation of the PKA pathway by hormonal progression to androgen independence at the FSK altered the expression of 303 genes of which 204 time points examined. showed increased expression while 99 showed decreased expression relative to controls. This provides the first expression profile of genes altered by activation of the Genes differentially expressed in response to FSK PKA pathway in prostate cancer cells. (3) Only 32 genes FSK activates adenylyl cyclase to synthesize cAMP were commonly differentially expressed by stimulation which binds to the R subunits of the inactive holo- of both pathways. (4) Application of AR-siRNA enzyme (PKA). The catalytic subunits then dissociate revealed that the induction of members of the kallikrien and become active. The free catalytic subunit can family and repression of SESN1 by androgen and FSK phosphorylate serine and threonine residues; its required functional AR. (5) Genes differentially ex- entrance into the cell nucleus and subsequent phos- pressed in response to androgen and stimulation of the phorylation of transcription factors – for example, PKA pathway in vitro were also differentially expressed CREB, CREM, NF-kB and nuclear receptors – forms during hormonal progression in vivo. the basis of PKA regulation of transcriptional activation

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7318

Figure 4 AR is required for changes in expression of some genes in response to FSK. Western blot analyses of AR protein (a) and PSA protein (b) in LNCaP cells transfected with scrambled (ARc) or AR-siRNA (AR1) and then treated with R1881 or FSK for 16 h. b-Actin serves as a loading control. Semi-Q-RT–PCR analyses of transcript levels of KLK2 (c), PSA (d), HMGCR (e), INSIG1 (f) and SESN1 (g) in LNCaP cells transfected with scrambled (ARc) or AR-siRNA (AR1) and then treated with R1881 or FSK for 16 h. GAPDH serves as a loading control. The ratio of genes of interest to GAPDH was used to evaluate the difference between the two groups. The mean and s.d. of three independent experiments are shown. Signiﬁcant differences between each condition (P-value) were determined by a Student’s t-test: *Pp0.05; **Pp0.01.

(Daniel et al., 1998). However, FSK can also interact transcriptome of prostate cancer cells. A total of 303 with other proteins such as glucose transporters which genes were detected as differentially expressed in may inﬂuence gene expression (Morris et al., 1991). response to FSK. Some of these genes are involved in This is the ﬁrst report of the impact of FSK on the cell division, migration and transcriptional regulation.

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7319

Figure 5 In vivo expression of genes identified in vitro to be differentially expressed in response to androgen and FSK during hormonal progression in the LNCaP hollow fiber model. Total RNA from LNCaP cells harvested from implanted hollow fibers in nude mice at the time points of pre-castration (d0), 10 days (d10) and 52 days (d52) after castration. Semi-Q-RT–PCR was performed in a 10 ml reaction with the primers of each gene and normalized to GAPDH. Data are calculated as the mean expression normalized to GAPDH from three different animals (n ¼ 3). Insets show a representative gel from each animal for each of the time points. Significant differences between each condition (P-value) were determined by a Student’s t-test: *Pp0.05; **Pp0.01.

Activation of the PKA pathway by FSK or other Consistent with these observations, activation of the compounds can increase proliferation, alter cell mor- PKA pathway resulted in altered expression of genes phology, and induce neuroendocrine differentiation of involved in cell division such as increased CDC6 and prostate cancer cells (Bang et al., 1994; Shah et al., 1994; decreased NUMA. Transcription of CDC6 is regulated Chen et al., 1999; Cox et al., 2000; Farini et al., 2003). in response to mitogenicsignals through transcriptional

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7320 control mechanism involving E2F proteins (Vilaboa metastasis of prostate cancer. Here indeed expression of et al., 2004). Cdc6 is expressed in proliferating but not MMP16 increased in response to castration as predicted. quiescent mammalian cells and thought to play a MGC61716 (MTERFD2) plays a central role in the fundamental role in controlling initiation of DNA control of mitochondrial rRNA and mRNA synthesis replication (Yan et al., 1998). Consistent with this role, in mammalian mitochondria (Daga et al., 1993). here Cdc6 transcript was reduced in vivo with castration MGC61716 represents a gene that increases in expres- when proliferation decreases and then levels increased in sion in response to androgen but decreases in response androgen independence when proliferation resumes. to FSK. Interestingly, when compared to benign tissue the levels To date the only gene that has been reported to be of CDC6 are decreased in PC3 prostate cancer cells that induced by both androgen and activation of the PKA are devoid of both AR and p53 (Robles et al., 2002). pathway in prostate cancer cells by an AR-dependent NUMA is a mitotic centrosomal component that is mechanism is PSA (KLK3) (Sadar, 1999). Here, we essential for the organization and stabilization of spindle show for the ﬁrst time that KLK2 was also induced by poles (Zeng, 2000). NUMA responds to external signals both androgen and FSK through a mechanism depen- such as hormones that induce cell division (Gobert et al., dent on the AR. Considering that there is over 75% 2001). Here, NUMA1 transcript increased in vivo in homology between the 6.5 kb upstream regulatory response to castration as compared to intact and sequences of KLK2 and PSA, it is perhaps not androgen-independent levels. Trends in expression of surprising that these two genes have much the same these two genes were consistent with suggestion of expression patterns. KLK3 and KLK2 have both been decreased activation of the PKA pathway with castra- used for the detection of prostate cancer (Young et al., tion, followed by increased PKA activity with androgen 1992) and hormonal progression. independence. While most genes show an increase in expression in response to androgen through a mechanism involving the AR, only a few genes have been shown to be Genes differentially expressed in response to androgen repressed. These include Maspin (Zhang et al., 1997; and FSK Zou et al., 2002), a gene regulated by p53 (Zou et al., The Human Genome U133plus2 GeneChip contains 2000). Here, we identify another p53-regulated gene, oligonucleotides for 38 500 genes. Here, we detected SESN1, to be repressed in response to both androgen changes in expression of 858 and 303 genes in response and FSK by a mechanism that at least in part requires a to androgen and FSK, respectively, with 32 overlapping functional AR. Interestingly, in 22Rv1 cells that contain genes. This number is much greater than the expected reduced and mutated p53 (van Bokhoven et al., 2003), overlap of genes by random chance calculated to be 6.75 SESN1 was not repressed by androgen or FSK. The (858*303/38 500). relationship between p53 and the androgen axis is Genes differentially expressed in response to both complicated since p53 interacts with the AR to dis- treatments but not involving AR as a common rupt its N-terminal to C-terminal interaction thereby mechanism include those involved in cholesterol meta- inhibiting DNA-binding activity (Shenk et al., 2001). bolism such as HMGCR (Goldstein and Brown, 1990) Androgen also inhibits the expression (Rokhlin et al., and INSIG1 which both decreased in response to 2005) and nuclear accumulation of p53 (Nantermet castration; Dlc2 which may play a role in apoptosis et al., 2004). Withdrawal of androgen or application of (Day et al., 2004) and decreased in response to an antiandrogen can increase p53 expression and castration; FLJ22649, also known as SPCS3 (signal activity (Agus et al., 1999; Bouchal et al., 2005). While peptidase complex subunit 3 homolog) which cleaves the this may explain the high basal expression of SESN1 as signal sequence of the precursor proteins to release the shown here in the absence of androgen, a mechanism as mature form to the extracellular space (Stroud and to why AR-siRNA reduced these levels remains elusive Walter, 1999). and the subject of further investigation ongoing in our MMP16 and MGC61716 are examples of genes whose lab. Induction of SESN1 follows similar trends to other expression was signiﬁcantly altered by both treatments known p53-regulated genes with increases occurring but in opposite directions (i.e., MMP16 was repressed with cell cycle arrest (Velasco-Miguel et al., 1999). by androgen and increased by FSK). MMP16 belongs to Thus, decreased proliferation of prostate cancer cells the matrix metalloproteinase (MMP) family which is in response to castration would be expected to be involved in the breakdown of extracellular matrix in associated with increased levels of SESN1 as observed normal physiological processes, such as embryonic here. development, reproduction, tissue remodeling, as well as in disease processes, such as arthritis and metastasis (Jung et al., 2003). Decreased MMP16 expression by In vivo expression of genes differentially expressed in androgen can promote cell–cell aggregation and clone response to androgen and FSK during hormonal formation of prostate cancer cells. On the contrary, in progression the absence of androgens such as would be encountered PSA is an example of an androgen-regulated gene with with androgen ablation therapy, increased expression of several well-characterized AREs to which the AR binds MMP16 by stimulation of PKA may result in the to initiate transcription (Riegman et al., 1991; Cleutjens breakdown of extracellular matrix and facilitate the et al., 1996; Schuur et al., 1996). The re-expression of

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7321 PSA suggests that the AR plays an important role in regulation of cell cycle and apoptosis might yield results androgen-independent disease. Evidence supporting the relevant to understanding of hormone refractory pros- role of AR in hormonal progression of prostate cancer tate cancer. was recently summarized (Wang and Sadar, 2006). Consistent with these findings, in the present study, expression of androgen-stimulated genes such as PSA, Materials and methods KLK2, ELL2, RHOU and SOCS2 was reduced initially after castration with subsequent increased Cell culture and general methods levels in the androgen-independent stage. Expression LNCaP cells and 22Rv1 cells were obtained from the of androgen-suppressed genes such as MMP16 increased American Type Culture Collection (Bethesda, MD, USA) initially with castration with subsequent decreased and maintained in Roswell Park Memorial Institute (RPMI) levels in the androgen-independent stage while the media (StemCell Technologies, Vancouver, BC, Canada) with 10% fetal bovine serum (FBS) (StemCell Technologies, expression of PKIB did not change. The expression of Vancouver, BC, Canada). Before treatments, 1.2 Â 106 cells some other androgen-stimulated genes such as Dlc2, with passage numbers from 35 to 45 were seeded in Falcon HMGCR and INSIG1 was reduced initially after (Becton Dickinson Laboratories, Franklin Lakes, NJ, USA) castration but did not increase to the original levels in 10 cm culture dishes and 24 h afterwards, starved by 48 h the androgen-independent stage. These results indicate culture in serum-free media. After serum deprivation, cells the reactivation of the androgen-response pathway in were treated with either syntheticandrogen 10 n M R1881 the absence of androgens, but this reactivation may be (NEN Life Science Products Inc., Boston, MA, USA) or incomplete. ethanol carrier alone (0.000385% v/v), 1 mM FSK (Sigma- Expression of genes responsive to FSK also changed Aldrich Canada Ltd. Oakville, ON, Canada) or dimethyl with castration. These genes include CDC6, ITGB5 and sulfoxide (DMSO) carrier alone (0.01% v/v). NUMA1. Regulation of the PKA pathway by castration may involve both non-genomicand genomiceffectsof Whole-cell lysate and immunoblotting assays Whole-cell lysate (WCL) was isolated using ReadyPrep protein androgen. Testosterone is capable of elevating cAMP extraction kits (BioRad, CA, USA). Samples were stored at levels by binding to sex hormone-binding globulin À801C until use. Protein quantitation was performed using a associated with its receptor (Nakhla et al., 1999) which modified Bradford assay kit (BioRad, CA, USA). WCL from would result in increased PKA activity. In addition, or each sample were electrophoresed on an 8.5 or 12% sodium possibly alternatively, here we identify that androgen dodecyl sulfate–polyacrylamide gel (SDS–PAGE) gel. Briefly, decreased expression of PKIB, which is a competitive 20 mg of protein was mixed with appropriate volumes of 2 Â inhibitor of PKA. This would provide a novel mechan- SDS–PAGE loading buffer (0.5 M Tris pH 6.8, 2% SDS, 10% ism for androgen to indirectly activate the PKA glycerol, 0.05% bromophenol blue, 0.05% dithiothreitol), pathway. However, the expression of PKIB responded boiled for 5 min and electrophoresed at 150 V. Proteins were to androgen in 16 h in vitro (Figure 2a and c) while there transferred to Immobilon-P (Millipore, Billerica, MA, USA) for 60 min at 100 V. Membranes were washed in phosphate- was no change 10 days after castration in vivo (Figure 5). buffered saline. Western blot analyses were performed using One interpretation of these data could suggest transient Odyssey infrared imaging system (LI-COR, Lincoln, NE, regulation of PKIB by androgen. In the absence of USA) according to the manufacturer’s protocol. The antiandrogen, such as in patients receiving hormone bodies used in these studies were obtained from various ablation therapy for prostate cancer, the PKA pathway suppliers: AR-441 (Santa Cruz, Santa Cruz, CA, USA), PSA may contribute to androgen-independent activation of (clone ER-PR8 from DAKO, Glostrup, Denmark) and b-actin AR. With androgen independence there was re-expres- (Abcam, Cambridge, MA, USA). sion of the FSK-stimulated gene, CDC6, and decreased expression of FSK-suppressed gene, NUMA1 (Figure 5). siRNA assay These findings suggest the activation of PKA pathway in Double-stranded siRNAs were purchased from Dharmacon the progression of prostate cancer to the androgen- Research (Lafayette, CO, USA). A 21-nucleotide double- independent stage. stranded siRNA duplex generated against the amino-terminus of the AR at nucleotides 293–312 (50-aagcccatcgtagaggcccca- 30) as reported before (Wright et al., 2003) will be referred to in the following as the AR-siRNA (AR1). A single control Summary nucleotide double-stranded siRNA duplex, referred to as the In summary, a global view at the level of transcription control siRNA (ARc), was generated to the inverted sequence reveals the cellular response of androgen-responsive of AR at nucleotides 293–313 (50-accccggagatgctacccgaa-30) prostate cancer cells to stimulation of the androgen and and functioned as a nonspecific control siRNA for the RNAi PKA pathways and cross-talk between these pathways. experiments where indicated. As previously described, on day Aberrant activation of the AR through the PKA 0, LNCaP cells were seeded at an initial density of 1.5 Â 106 signaling pathway may play a role in the progression cells into Falcon 10 cm culture dishes and incubated for 24 h in of prostate cancer to the androgen-independent state. medium A (phenol red-free RPMI 1640 medium supplemented Further investigation of the mechanism involved in with 10% FBS lacking antibiotics). On day 1, the cells were serum-starved with phenol red-free RPMI 1640 medium, and activation of the AR by cross-talk with the PKA transfected with control or AR-siRNAs at a final concentra- pathway may lead to viable therapies for androgen- tion of 100 nM using the Oligofectamine reagent (Invitrogen independent prostate cancer. The development of new Life Technologies, Carlsbad, CA, USA) according to the tumor models to investigate activation of the AR and manufacturer’s instructions. The cells were incubated with

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7322 siRNAs for 48 h. The guidelines for siRNA silencing were Semi-Q-RT–PCR followed as detailed in the instructions provided at the RT–PCR was performed applying SuperScriptt one-step RT– Dharmacon Research website (http://www.dharmacon.com/). PCR with Platinum Taq kits (Invitrogen Life Technologies, The cells were then treated with various agents for 16 h and Carlsbad, CA, USA) according to the manufacturer’s instruc- harvested for immunoblot or RT–PCR. tions. The primers for each of the genes refer to Supplementary Table. Briefly, 0.5 mg of total RNA was applied in a 10 ml reaction with both the primers of each genes and GAPDH. RNA isolation and microarray analysis The reaction mixture was incubated at 501C for 30 min, Total RNA from three independent experiments was extracted denatured at 941C for 2 min prior to 28–30 cycles of: 941C for from cells using Trizol (Invitrogen Life Technologies, Carls- 15 s, 561C for 30 s and 721C for 1 min and 5 min at 721C for bad, CA, USA) according to the manufacturer’s protocol. final extension. The PCR products were electrophoresed on a RNA samples from cells were analysed by Affymetrix 1.3% agarose gel in the presence of ethidium bromide (0.001% Genechip microarray. The syntheses of cDNA and biotiny- v/v). The images were scanned using EagleEye II camera lated cRNA were performed according to the protocols (Stratagene, La Jolla, CA, USA). The density of the bands was provided by the manufacturer (Affymetrix, Santa Clara, CA, measured using ImageQuant Version 5.2 software (Amersham USA). Biotinylated fragmented cRNA probes were hybridized Biosciences, UK). The ratio of genes of interest to GAPDH to the HGU133 plus2 Genechips (Affymetrix), which con- was used to evaluate the fold change in levels of the difference tained probe sets for over 50 000 known transcripts and between the two groups. expressed sequence tags. Hybridization was performed at 451C for 16 h in a hybridization oven (Affymetrix). The Genechips Hollow fiber model of prostate cancer were then automatically washed and stained with streptavidin– Male athymic nude mice, 6–8 weeks of age, were purchased phycoerythrin conjugate in an Affymetrix Genechip Fluidics from Taconic Farms (Germantown, NY, USA). Hollow fiber Station. Fluorescence intensities were scanned with a Gene- model of prostate cancer was performed as described Array Scanner (Affymetrix). Hybridizations were carried out previously (Sadar et al., 2002). Briefly, LNCaP cells were independently for each condition using three biological suspended in RPMI plus 20% FBS, and loaded into the replicates. polyvinylidene difluoride hollow fibers (Spectrum Labora- tories, Rancho Dominguez, CA, USA) with the aid of an 18- 7 Expression profile analysis gauge needle at a seeding density of 3 Â 10 cells/ml. The fibers Comparative analysis between expression profiles for Affyme- were sectioned into about 2 cm pieces, heat sealed, and trix experiments was carried out using GeneSpringt software implanted subcutaneously in nude mice. A total of 24 fibers version 7 (Silicon Genetics, CA, USA). The ‘Cross gene error were implanted in each animal, in bundles of eight fibers at model for deviation from 1.0’ was active. Gene expression data three different regions in the animal. Castration of mice was was normalized in two ways: ‘per chip normalization’ and ‘per performed by making a small incision in the scrotum to excise gene normalization.’ For ‘per chip normalization,’ all expres- each testicle after ligation of the cord. Surgical suture was used sion data on a chip is normalized to the 50th percentile of all to close the incision. At each time point, eight fibers from the values on that chip. For ‘per gene normalization,’ the data for same mouse were harvested and total RNA was isolated from a given gene is normalized to the median expression level of LNCaP cells grown inside the fibers using Trizol (Invitrogen that gene across all samples. The data sets were then assigned Life Technologies, Carlsbad, CA, USA). to two groups for either experiment (R1881 and ethanol, FSK and DMSO). The expression profiles from three independent Acknowledgements experiments were compared using Student’s two-sample t-test (parametrictest, assume variancesequal) to identify genes that We thank Nasrin Mawji, Heidi Hare and Jean Wang for their were differentially expressed between the two groups. For technical assistance. This work is supported by funding from sample clustering, standard correlation was applied to measure NIH, Grant CA105403 (MDS). MAM and SJMJ are scholars the similarity of the expression pattern between different of the Michael Smith Foundation for Health Research. MAM samples. is a Terry Fox Young Investigator.

References

Agus DB, Cordon-Cardo C, Fox W, Drobnjak M, Koff A, Culig Z, Hobisch A, Hittmair A, Cronauer MV, Radmayr C, Golde DW et al. (1999). J Natl Cancer Inst 91: 1869–1876. Zhang J et al. (1997). Prostate 32: 106–114. Amler LC, Agus DB, LeDucC, Sapinoso ML, Fox WD, Kern Daga A, Micol V, Hess D, Aebersold R, Attardi G. (1993). S et al. (2000). Cancer Res 60: 6134–6141. J Biol Chem 268: 8123–8130. Bang YJ, Pirnia F, Fang WG, Kang WK, Sartor O, Whitesell Daniel PB, Walker WH, Habener JF. (1998). Annu Rev Nutr L et al. (1994). Proc Natl Acad Sci USA 91: 5330–5334. 18: 353–383. Bouchal J, Baumforth KR, Svachova M, Murray PG, von Day CL, Puthalakath H, Skea G, Strasser A, Barsukov I, Lian Angerer E, Kolar Z. (2005). J Pharm Pharmacol 57: 83–92. LY et al. (2004). Biochem J 377: 597–605. Chen T, Cho RW, Stork PJ, Weber MJ. (1999). Cancer Res 59: Debes JD, Tindall DJ. (2004). N Engl J Med 351: 1488–1490. 213–218. DePrimo SE, Diehn M, Nelson JB, Reiter RE, Matese J, Fero Cleutjens KB, van Eekelen CC, van der Korput HA, M et al. (2002). Genome Biol 3: RESEARCH0032. Brinkmann AO, Trapman J. (1996). J Biol Chem 271: Dotzlaw H, Moehren U, Mink S, Cato AC, Iniguez Lluhi JA, 6379–6388. Baniahmad A. (2002). Mol Endocrinol 16: 661–673. Cox ME, Deeble PD, Bissonette EA, Parsons SJ. (2000). J Biol Farini D, Puglianiello A, Mammi C, Siracusa G, Moretti C. Chem 275: 13812–13818. (2003). Endocrinology 144: 1631–1643.

Oncogene Genes regulated by the androgen and PKA signaling pathways G Wang et al 7323 Gobert GN, Hueser CN, Curran EM, Sun QY, Glinsky VV, Shah GV, Rayford W, Noble MJ, Austenfeld M, Weigel J, Welshons WV et al. (2001). Histochem Cell Biol 115: Vamos S et al. (1994). Endocrinology 134: 596–602. 381–395. Shenk JL, Fisher CJ, Chen SY, Zhou XF, Tillman K, Goldstein JL, Brown MS. (1990). Nature 343: 425–430. Shemshedini L. (2001). J Biol Chem 276: 38472–38479. Holzbeierlein J, Lal P, LaTulippe E, Smith A, Satagopan J, Sramkoski RM, Pretlow II TG, Giaconia JM, Pretlow TP, Zhang L et al. (2004). Am J Pathol 164: 217–227. Schwartz S, Sy MS et al. (1999). In vitro Cell Dev Biol Anim Horoszewicz JS, Leong SS, Chu TM, Wajsman ZL, Friedman 35: 403–409. M, Papsidero L et al. (1980). Prog Clin Biol Res 37: 115–132. Stroud RM, Walter P. (1999). Curr Opin Struct Biol 9: Huggins C, Hodges CV. (2002). J Urol 168: 9–12. 754–759. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor Tao W, Pennica D, Xu L, Kalejta RF, Levine AJ. (2001). A et al. (2005). CA Cancer J Clin 55: 10–30. Genes Dev 15: 1796–1807. Jung M, Romer A, Keyszer G, Lein M, Kristiansen G, Tasken K, Aandahl EM. (2004). Physiol Rev 84: 137–167. Schnorr D et al. (2003). Prostate 55: 89–98. Tepper CG, Boucher DL, Ryan PE, Ma AH, Xia L, Lee LF Kemppainen JA, Lane MV, Sar M, Wilson EM. (1992). J Biol et al. (2002). Cancer Res 62: 6606–6614. Chem 267: 968–974. Tusher VG, Tibshirani R, Chu G. (2001). Proc Natl Acad Sci Lee C, Sutkowski DM, Sensibar JA, Zelner D, Kim I, Amsel I USA 98: 5116–5121. et al. (1995). Endocrinology 136: 796–803. van Bokhoven A, Varella-Garcia M, Korch C, Johannes WU, Morris DI, Robbins JD, Ruoho AE, Sutkowski EM, Seamon Smith EE, Miller HL et al. (2003). Prostate 57: 205–225. KB. (1991). J Biol Chem 266: 13377–13384. Van Patten SM, Ng DC, Th’ng JP, Angelos KL, Smith AJ, Nakhla AM, Leonard J, Hryb DJ, Rosner W. (1999). Steroids Walsh DA. (1991). Proc Natl Acad Sci USA 88: 5383–5387. 64: 213–216. Velasco-Miguel S, Buckbinder L, Jean P, Gelbert L, Talbott Nantermet PV, Xu J, Yu Y, Hodor P, Holder D, Adamski S R, Laidlaw J et al. (1999). Oncogene 18: 127–137. et al. (2004). J Biol Chem 279: 1310–1322. Vilaboa N, Bermejo R, Martinez P, Bornstein R, Cales C. Nazareth LV, Weigel NL. (1996). JBiolChem271: 19900–19907. (2004). Nucl Acids Res 32: 6454–6467. Nelson PS, Clegg N, Arnold H, Ferguson C, Bonham M, Waghray A, Feroze F, Schober MS, Yao F, Wood C, Puravs E White J et al. (2002). Proc Natl Acad Sci USA 99: 11890– et al. (2001). Proteomics 1: 1327–1338. 11895. Wang G, Sadar MD. (2006). J Cell Biochem 98: 36–53. Prins GS. (2000). Mayo Clin Proc 75(Suppl): S32–S35. Wright ME, Tsai MJ, Aebersold R. (2003). Mol Endocrinol 17: Riegman PH, Vlietstra RJ, van der Korput JA, Brinkmann 1726–1737. AO, Trapman J. (1991). Mol Endocrinol 5: 1921–1930. Xu LL, Su YP, Labiche R, Segawa T, Shanmugam N, Robles LD, Frost AR, Davila M, Hutson AD, Grizzle WE, McLeod DG et al. (2001). Int J Cancer 92: 322–328. Chakrabarti R. (2002). J Biol Chem 277: 25431–25438. Yan Z, DeGregori J, Shohet R, Leone G, Stillman B, Nevins Roche PJ, Hoare SA, Parker MG. (1992). Mol Endocrinol 6: JR et al. (1998). Proc Natl Acad Sci USA 95: 3603–3608. 2229–2235. Young CY, Andrews PE, Montgomery BT, Tindall DJ. Rokhlin OW, Taghiyev AF, Guseva NV, Glover RA, (1992). Biochemistry 31: 818–824. Chumakov PM, Kravchenko JE et al. (2005). Oncogene 24: Young CY, Montgomery BT, Andrews PE, Qui SD, Bilhartz 6773–6784. DL, Tindall DJ. (1991). Cancer Res 51: 3748–3752. Sadar MD, Akopian VA, Beraldi E. (2002). Mol Cancer Ther Zeng C. (2000). Microsc Res Tech 49: 467–477. 1: 629–637. Zhang M, Magit D, Sager R. (1997). Proc Natl Acad Sci USA Sadar MD, Hussain M, Bruchovsky N. (1999). Endocr Relat 94: 5673–5678. Cancer 6: 487–502. Zhou ZX, Lane MV, Kemppainen JA, French FS, Wilson Sadar MD. (1999). J Biol Chem 274: 7777–7783. EM. (1995). Mol Endocrinol 9: 208–218. Schuur ER, Henderson GA, Kmetec LA, Miller JD, Lamparski Zou Z, Gao C, Nagaich AK, Connell T, Saito S, Moul JW HG, Henderson DR. (1996). JBiolChem271: 7043–7051. et al. (2000). J Biol Chem 275: 6051–6054. Segawa T, Nau ME, Xu LL, Chilukuri RN, Makarem M, Zou Z, Zhang W, Young D, Gleave MG, Rennie P, Connell T Zhang W et al. (2002). Oncogene 21: 8749–8758. et al. (2002). Clin Cancer Res 8: 1172–1177.

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