Evolution of the Androgen Receptor Pathway During Progression of Prostate Cancer

Research Article

Evolution of the Androgen Receptor Pathway during Progression of Prostate Cancer

Peter J.M. Hendriksen,1,2 Natasja F.J. Dits,1 Koichi Kokame,3 Antoine Veldhoven,1 Wytske M. van Weerden,1 Chris H. Bangma,1 Jan Trapman,2 and Guido Jenster1

Departments of 1Urology and 2Pathology, Josephine Nefkens Institute, Erasmus Medical Center, Rotterdam, the Netherlands and 3National Cardiovascular Center Research Institute, Osaka, Japan

Abstract carcinoma is only temporary (1). The function of the androgen receptor, however, markedly differs between the normal prostate The present work focused on the potential involvement of and prostate carcinoma. Whereas the androgen receptor is a key selective adaptations of the androgen receptor pathway in regulator for prostatic function and survival in the normal prostate the initiation and progression of prostate cancer. We defined epithelium, the androgen receptor has been converted into an the androgen receptor pathway by selecting 200 genes inducer of uncontrolled cell growth in prostate carcinoma (1, 2). that were androgen responsive in prostate cancer cell lines The recent identification of frequent chromosomal rearrangement and/or xenografts. This androgen receptor pathway gene in prostate cancer that results in fusion of TMPRSS2 with ETS signature was then used for profiling prostate cancer xeno- transcription factor genes explains this conversion (3). The ETS grafts and patient-derived samples. Approximately half of family members ERG and ETV1 are normally not or lowly the androgen receptor pathway genes were up-regulated in expressed in prostate tissues. Fusion of these genes to the strong well-differentiated prostate cancer compared with normal androgen-regulated TMPRSS2 promoter results in androgen- prostate. Functionally distinct parts of the androgen receptor induced expression of these proto-oncogenes, which likely cause pathway were specifically down-regulated in high-grade can- prostate tumorigenesis. cers. Unexpectedly, metastases have down-regulated the vast That enhanced androgen receptor activity of itself is unlikely majority of androgen receptor pathway genes. The signifi- to be sufficient for causing aggressive growth could already be cance of this progressive down-regulation of androgen deduced from our knowledge on androgen receptor function in the receptor pathway genes was shown for a few androgen normal prostate. In vitro experiments indicated that androgens receptor–regulated genes. Lower mRNA expression of HER- induce differentiation and thereby inhibit growth of prostate epi- PUD1, STK39, DHCR24, and SOCS2 in primary prostate tumors thelial cells (4, 5). In a transgenic mouse model, overexpression of was correlated with a higher incidence of metastases after the wild-type (WT) androgen receptor in prostatic epithelial cells radical prostatectomy. HERPUD1 mRNA expression predicted resulted in intraepithelial neoplasia but not in carcinomas (6). The the occurrence of metastases almost perfectly. In vitro growth-stimulating effect of androgens on normal epithelial cells experiments showed that overexpression of the stress response is at least partly mediated by the prostatic stromal cells, which gene HERPUD1 rapidly induces apoptosis. Based on the func- produce and secrete growth factors, the andromedins, in response tions of the genes within the distinct subsets, we propose to androgen exposure (1, 2, 7). During prostatic carcinogenesis, the following model. Enhanced androgen receptor activity the balance between the differentiation- and proliferation-inducing is involved in the early stages of prostate cancer. In well- functions of the androgen receptor might be disturbed. Such a differentiated prostate cancer, the androgen receptor acti- transition would likely be reflected by changes within the andro- vates growth-promoting as well as growth-inhibiting and gen receptor pathway genes. cell differentiation genes resulting in a low growth rate. The Therefore, we studied the expression levels of androgen receptor progression from low-grade to high-grade prostate carcinoma pathway genes during progression of prostate carcinoma. Many and metastases is mediated by a selective down-regulation gene expression profiling studies have been published for prostate of the androgen receptor target genes that inhibit prolifera- carcinoma as well as for other types of cancers. This has led to the tion, induce differentiation, or mediate apoptosis. (Cancer Res identification of gene sets specific for localized prostate carcinoma 2006; 66(10): 5012-20) and metastases or related to the occurrence of relapse after radical prostatectomy (8–14). The functions of the genes within these Introduction sets are very diverse. Although these gene sets might have diag- The androgen receptor plays a pivotal role in the growth and nostic and prognostic prospects, their effect on our understanding survival of both normal prostate epithelium and prostate of the biological mechanisms involved in cancer progression is carcinoma. Both normal and malignant prostate tissues regress limited. The use of ‘‘functional gene sets,’’such as target genes from on androgen deprivation, although this effect on prostate a transcription factor, might be a more valuable tool to enhance this understanding. This was tested in the present study using an androgen receptor pathway gene signature. Note: Supplementary data for this article are available at Cancer Research Online To identify androgen receptor pathway genes, expression mi- (http://cancerres.aacrjournals.org/). Requests for reprints: Guido Jenster, Department of Urology, Josephine Nefkens croarray analyses were done on the prostate carcinoma cell line Institute Be362a, Erasmus Medical Center, P.O. Box 3000 DRRotterdam, LNCaP and on a panel of 13 prostate carcinoma xenografts. In the Netherlands. Phone: 31-10-408-7672; Fax: 31-10-408-9386; E-mail: g.jenster@ total, 200 androgen receptor pathway genes were identified. To erasmusmc.nl. I2006 American Association for Cancer Research. test whether the androgen receptor pathway is changed during doi:10.1158/0008-5472.CAN-05-3082 prostate carcinoma progression, the expression of these 200 genes

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Androgen Receptor Pathway in Prostate Cancer was assessed in both xenografts and clinical prostate carcinoma (Invitrogen). Xenograft cDNA was labeled with Cy3 and cohybridized specimens. The results indicate that prostate carcinomas down- with Cy5-labeled cDNA prepared from RNA extracted from 13 cell lines, regulate part of the androgen receptor pathway before acquiring including the 2 prostate carcinoma cell lines Du145 and LNCaP. Four the ability to metastasize. This finding was further supported by microarrays were used per xenograft on tissue samples collected in separate experiments. Two samples were taken from intact male mice and another quantitative reverse transcription-PCR(RT-PCR)on a selection of two after castration. genes on an independent set of prostate carcinoma samples. The normalization and flagging procedure is provided in Supplementary Data. Materials and Methods The programs Cluster and Treeview were used for hierarchical clustering (20). Comparisons with other microarray databases were done using Prostate carcinoma xenografts, cell lines, and patient-derived tissues. Thirteen prostate carcinoma xenografts have been established at Sequence Retrieval System 7 (Lion Bioscience AG, Heidelberg, Germany) our laboratory (15–17), and their main characteristics are given in Table 1. as published recently (21). Quantitative real-time RT-PCR analysis. Additional information about the xenografts is provided in Supplementary Quantitative real-time RT- PCRanalysis was done with a ABI Prism 7700 Sequence Detection System Data. Xenografts were collected either from intact male mice or 7 to 14 days after castration. Tissues were frozen at 80jC. The xenograft PC329 has using AmpliTaq Gold according to the manufacturer’s specifications been lost, and tissues taken from castrated mice are not available anymore. (Applied Biosystems, Foster City, CA). The probes and primers were validated with Taqman Gene Expression Assays (Applied Biosystems). The The prostate carcinoma cell line LNCaP has been well described (18). The LNCaP cell line was maintained in RPMI 1640 with 5% FCS and penicillin/ assay identification numbers and PCRsettings are given in Supplementary streptomycin (Invitrogen, Merelbeke, Belgium). Before R1881 treatment, Data. The amount of target gene expressed was normalized to an endo- cells were androgen deprived for 72 hours in medium containing 5% genous reference and relative to a calibrator. The endogenous reference was glyceraldehyde-3-phosphate dehydrogenase; a mixture of cDNAs of the dextran-filtered, charcoal-stripped FCS with a medium replacement after 36 hours. After androgen deprivation, the medium was supplemented for prostate carcinoma xenografts was used as the calibrator. Criteria for up-regulation and down-regulation in the microarray 2, 4, 6, or 8 hours with 1 nmol/L R1881 or ethanol vehicle. analyses and statistics. Normal and tumor specimens from patients used for quantitative real- For the LNCaP time series, spots were considered to be up-regulated or down-regulated by R1881 when both dye swaps gave a time RT-PCR analysis were obtained from the frozen tissue bank of the Erasmus Medical Center (Rotterdam, the Netherlands). The specimens were ratio >1.62 (2 log 0.7) for at least one time point. For the xenografts, spots collected between 1984 and 2001. Additional information about these were considered to be up-regulated or down-regulated by castration when the average ratios of two experiments between the intact and castrated specimens is provided in Supplementary Data. RNA amplification, cDNA labeling, and cDNA microarray hybrid- mice were >2 (2 log 1). Additionally, both individual comparisons between izations. Total RNA (3 Ag) was used for a T7-based linear mRNA xenografts grown on an intact mouse versus castrated mouse had to yield a amplification protocol (19). Amplified RNA (2 Ag) was used to produce Cy3- ratio >1.62 (2 log 0.7). The significant analysis of microarrays method (SAM; version 1.21; ref. 22) was used to detect significantly differentially expressed or Cy5-labeled cDNA. The cDNA microarrays were manufactured at the Central Microarray Facility at the Netherlands Cancer Institute (Amster- spots between normal prostate and prostate carcinoma samples of patients. m2 dam, the Netherlands) and contained >18,000 features that have been analyses were used for comparisons of proportions as indicated in selected from the Research Genetics Human Sequence Verified Library Results. The significance of overlap between the recurrence-associated gene set published by Henshall et al. (14) and our androgen receptor pathway genes was determined by Venn Mapper (23). Box plots, Mann-Whitney U test, Kaplan-Meier test, log-rank test, and Table 1. Panel of prostate cancer xenografts Cox regression analyses on quantitative real-time RT-PCR results were done using SPSS11 software (Chicago, IL). Ps < 0.05 were considered significant. Tumor model Androgen dependence AR status* Origin HERPUD1 overexpression. LNCaP and the liver cancer cell line Hep3B were transfected with HERPUD1 constructs using Fugene (Roche, PC82 Androgen dependent AR+ Prostate Mannheim, Germany) according to the manufacturer’s specifications. The PC295 Androgen dependent AR+ Lymph node HERPUD1 expression constructs are described in Supplementary Data. PC310 Androgen dependent AR+ Prostate HERPUD1-transfected cells were detected with either anti-HERPUD1 (24) or PC329 Androgen dependent AR+ Prostate FITC-labeled anti-MYC-Tag (Invitrogen). A rabbit anti-caspase-3 active was PC346 Androgen responsive AR+ TURP obtained from R&D Systems (Minneapolis, MN). Nuclei of the cells were PC346B Androgen independent AR+ TURP counterstained with 0.5 Ag/mL Hoechst 33342 (Invitrogen). The terminal PC346I Androgen independent AR+ TURP deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) PC346BI Androgen independent AR+ TURP reaction was done on pDsRed2-mHERPf transiently transfected cells using PC374 Androgen independent AR+ Skin the DeadEnd Fluorometric TUNEL kit (Promega, Madison, WI). To PC133 Androgen independent AR Bone determine the proportion of apoptotic cells, at least 100 transfected cells PC135 Androgen independent AR Prostate c per treatment were counted. The transfection experiments were done in PC324 Androgen independent AR TURP triplicate. PC339 Androgen independent AR TURP

Results Abbreviations: AR, androgen receptor; TURP, transurethral resection of the prostate. Using expression microarray analyses, we followed two strategies *Xenografts are designated as AR+ when the androgen receptor is to identify androgen receptor pathway genes. First, LNCaP cells detectable by immunoblotting and the xenograft expresses prostate- were treated with R1881 to detect genes that were up-regulated specific antigen. within 8 hours by androgen. Secondly, we assessed which genes cPC324 shows some androgen receptor expression but does not were down-regulated by androgen deprivation in androgen express prostate-specific antigen. Although no mutations in the receptor–expressing prostate carcinoma xenografts. androgen receptor have been detected, PC324 is considered to have a R1881-induced genes in LNCaP. LNCaP cells were treated nonfunctional androgen receptor pathway. with R1881 for short times (2, 4, 6, and 8 hours) to enrich for genes directly regulated by the androgen receptor. RNA from www.aacrjournals.org 5013 Cancer Res 2006; 66: (10). May 15, 2006

R1881-treated and control cells were compared directly by hybridization to the same microarray. This was done in duplicate with reversed Cy dye labeling. Spots were considered to be up- regulated or down-regulated by R1881 when both dye swaps gave a ratio >1.62 (2 log 0.7) for at least one time point. One hundred thirty-six up-regulated genes (represented by 163 spots) and 60 down-regulated genes (represented by 60 spots) were detected (Supplementary Fig. S1; Supplementary Table S1). Genes affected by castration in prostate carcinoma xenografts. We assessed the expression profiles of 12 xenografts collected either from intact male mice or 7 to 14 days after castration. Eight xenografts have functional androgen receptor expression. Three of these xenografts depend on androgens for growth, whereas four xenografts grow androgen independently (Table 1; see Materials and Methods). One xenograft (PC346) is hormone responsive. It shows reduced albeit continuous growth after castration, which is stimulated by administration of androgens. Four other xenografts lack expression of a functional androgen receptor and grow independently of androgens. We did two castration experiments per xenograft yielding two samples taken from intact male mice and another two after castration. Based on the selection criteria given in Materials and Methods, 293 spots were up-regulated or down-regulated by castration in at least 1 of the 12 xenografts. A hierarchical clustering of these 293 spots showed the overlap in castration- affected genes among the xenografts (Fig. 1A). A high number of genes were up-regulated or down-regulated in the androgen- dependent and hormone-responsive xenografts. Few genes were affected by castration in the androgen receptor–expressing, androgen-independent xenografts. As expected, very few genes were affected by castration in the xenografts lacking androgen receptor expression. Another observation is that a subset of the genes are up-regulated or down-regulated in the majority of androgen receptor–expressing xenografts, whereas other genes are castration responsive in only one or two xenografts. Evidently, xenograft-specific androgen receptor pathway genes exist in addition to common androgen receptor pathway genes. Because androgen-dependent xenografts regress after androgen deprivation, castration was expected to affect both cell cycle– related and androgen-regulated genes. We tested this assumption by selecting all spots down-regulated by castration in at least one androgen-dependent xenograft (n = 132; Fig. 1A) and clustered these spots based on expression levels in the xenografts (Fig. 1B). The clustering discriminated two groups of genes. The genes of group I (n = 65; 71 spots) showed highest expression in the androgen-dependent xenografts and also, for a part, in androgen receptor–expressing, androgen-independent xenografts. In contrast, the genes of group II (n = 59; 61 spots) showed low expression levels in androgen-dependent xenografts compared with all others. The two groups clearly differed in proportion of spots that were up- regulated by R1881 in LNCaP: 19 from 71 in group I and 4 from 61 Figure 1. Castration-affected genes in xenografts. A, hierarchical clustering m2 of castration-affected spots. A total of 293 spots were affected by castration in in group II (P = 0.002, analysis). To acquire more insight in the at least one xenograft. The 293 spots were clustered on the differences in functions of these two groups of genes, we made use of a data set of expression levels between intact and castrated mice. Red, down-regulation genes that are differentially expressed during the cell cycle in HeLa by castration; green, up-regulation by castration; blue box, a group of 132 spots taken for Fig. 2B. B, expression of castration-down-regulated genes cells and for which the expression is related to the cell proliferation in prostate cancer xenografts. Genes were selected on down-regulation by rate (25). The proportion of cell cycle–related genes was much castration in androgen-dependent (A-dep) xenografts (blue box in A). higher within group II [(26 of 59 genes (44%)] than within group I Hierarchical clustering on expression in xenografts separated these genes 6 2 in two groups with high (Group I; n = 71) and low (Group II; n = 61) [4 of 65 genes (6%); P =10 , m analysis; Fig. 1B). The genes of expression levels in the androgen-dependent xenografts. Right, up-regulation group II therefore very likely represent cell proliferation–related (red) or down-regulation (green) of these genes in LNCaP. Red, cell cycle–associated expression based on results published by Whitfield et al. genes that are down-regulated as a second-term effect of castration (25). AI, androgen independent; HR, hormone responsive; AR, androgen due to the cease of cell division. Their higher expression levels in receptor.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Androgen Receptor Pathway in Prostate Cancer hormone refractory than androgen-dependent xenografts simply reflect the higher proliferation rate of hormone refractory xenografts. Group I likely represents genes that are more directly associated with the androgen pathway. Forty-nine genes of group I were not present in the set of 136 R1881 up-regulated genes in LNCaP and included in the androgen receptor pathway signature set. Fifteen additional genes were selected based on their down- regulation by castration in the androgen receptor–expressing, androgen-independent xenografts of the PC346 group whose growth is unaffected by castration. Combining this gave a set of 200 androgen receptor pathway genes. We investigated the expression of these genes in xenografts and clinical prostate carcinoma samples to assess the changes of the androgen receptor pathway during prostate carcinoma progression. The complete microarray results have been submitted to GEO (accession GSE4048). Expression levels of the androgen receptor pathway genes in prostate carcinoma xenografts. The 200 androgen receptor pathway genes were hierarchically clustered on expression in the xenografts (Fig. 2). As expected, most androgen receptor pathway genes were much higher expressed in the androgen-dependent xenografts than in the androgen-independent xenografts lacking androgen receptor expression. The androgen receptor–expressing, androgen-independent xenografts showed high expression for only a fraction of the androgen receptor pathway genes, indicating inter- ference of other pathways/mechanisms that prevent transcriptional activation of the majority of these genes by the androgen receptor. Expression levels of the androgen receptor pathway signature genes in primary prostate carcinoma and metastases in patients. Expression levels of the androgen receptor pathway genes were also evaluated in clinical tumor samples. From the 200 androgen receptor pathway genes, 171 were represented in a microarray study on 41 normal prostate specimens, 62 primary prostate tumors, and 9 lymph node metastases published by Lapointe et al. (13). The prostate carcinoma samples in this study contained at least 90% tumor tissue. The metastases have not been subjected to androgen ablation therapy. The 171 androgen receptor pathway genes were sufficient to separate Figure 2. Expression of androgen receptor pathway genes in prostate cancer normal prostate, low-grade prostate carcinoma, and high-grade xenografts. Two hundred genes were selected on up-regulation in LNCaP and/or prostate carcinoma and metastases in discrete groups by down-regulation in androgen receptor–expressing xenografts by castration. unsupervised hierarchical clustering (Fig. 3A). Data reported by Genes (n = 165) with unflagged expression value for at least 50% of the xenografts were hierarchically clustered on expression in xenografts grown on Stuart et al. (26) were used to indicate genes for which the intact mice. Thereafter, expression values of xenografts 1 to 2 weeks after expression levels are associated with the content of either stroma, castration were added. benign prostatic hyperplasia (BPH), or tumor tissue. Subsets of androgen receptor pathway genes were differentially expressed The expression of the set of 200 androgen receptor pathway among the sample groups (Fig. 3A). One subset was higher genes in prostate carcinoma samples was also assessed using expressed in normal prostate (Fig. 3A, green), and nearly all genes microarray data published by Dhanasekaran et al. (8). Again, within this subset were stroma associated. Approximately half of approximately half of the androgen receptor pathway genes were the 171 androgen receptor pathway genes were highly expressed in up-regulated in primary prostate carcinoma, whereas only few well-differentiated prostate carcinoma (Fig. 3A, blue and red), and genes were relatively high expressed in metastases (Supplementary most of these genes were tumor associated. This implies that the Fig. S2). All androgen receptor pathway genes that according to up-regulation of these androgen-up-regulated genes in prostate SAM were differentially expressed between primary prostate carcinoma is due not only to a higher content of epithelial cells but carcinoma and metastases in Lapointe et al. and/or Dhanasekaran also to an increased expression in tumor cells compared with et al. are listed in Supplementary Table S2. Fifty-nine genes were normal epithelial cells. A subset of these genes is down-regulated in more highly expressed in primary prostate carcinoma than in high-grade prostate carcinoma (Fig. 3A, red). Only a few genes metastases, whereas only two genes were more highly expressed in remain relatively highly expressed in metastases (Fig. 3A, orange). metastases (lymph node) than in primary prostate carcinoma. The metastases showed also low expression for the well-known Taken together, this confirms our results with the xenografts that androgen receptor target gene prostate-specific antigen (KLK3). main parts of the androgen receptor pathway are down-regulated In marked contrast, the androgen receptor itself is higher expressed in more advanced stages of prostate carcinoma. in the metastases than in the majority of primary prostate We verified this analysis with alternative sets of androgen carcinomas (Fig. 3A). receptor pathway genes that were selected on up-regulation by www.aacrjournals.org 5015 Cancer Res 2006; 66: (10). May 15, 2006

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research androgens at later time points in either LNCaP or other androgen- receptor pathway genes in metastases is intriguing. We addressed dependent prostate carcinoma cell lines (27). The expressions of the important issue whether this down-regulation occurs after these gene sets in normal prostate and prostate carcinoma sam- development of the metastasis or that the primary prostate ples were essentially the same as that of our set of 200 genes (see carcinoma down-regulates the androgen receptor pathway genes Supplementary Fig. S3). We then confirmed that the expression before acquiring the ability to metastasize. Support for the latter patterns of the androgen receptor pathway genes did not simply hypothesis was found in the results published previously by reflect the general variation in gene expression. Compared with all Henshall et al. (14) who compared the expression profiles of spots of the Lapointe database, the androgen receptor pathway primary prostate carcinomas that did or did not relapse after gene sets selected on prostate carcinoma cell lines had much radical prostatectomy. For 152 genes represented by 181 spots, higher proportions of spots (P <10 6) that were either (a) up- relatively low expression in primary prostate carcinoma was found regulated in primary prostate carcinoma versus normal prostate, to be associated with fast relapse (14). Ten of these genes were (b) down-regulated in lymph node metastases versus primary present within our set of 200 androgen-up-regulated genes, which prostate carcinoma or normal prostate, or (c) down-regulated in was significantly (P <10 5) more than expected by random chance. high-grade prostate carcinoma versus low-grade prostate carcino- Nine of these 10 genes were included in the data set of Lapointe et ma (see Supplementary Fig. S4). al. and all up-regulated in the majority of primary prostate Down-regulation of androgen receptor pathway genes in carcinoma compared with normal prostate (Fig. 3B). Interestingly, primary prostate carcinoma predicts development of distant most of these genes had low expression levels in metastases metastases. The down-regulation of the majority of androgen (Fig. 3B). Taken together, this indicates down-regulation of part

Figure 3. Expression of androgen receptor pathway genes in prostate cancer specimens from patients. A, expression data were taken from Lapointe et al. (13), which included 171 genes from our androgen receptor pathway signature set. A hierarchical clustering was done on both genes and samples. Bottom, androgen receptor and KLK3; right, genes significantly up-regulated (red)or down-regulated (green) according to SAM. Association with proportion of stroma, BPH, or tumor as published by Stuart et al. (26). PC I, II, and III, three clustering groups from primary prostate carcinoma (PC) samples on 5,153 variably expressed genes as described by Lapointe et al. (13). PC II and III, relatively high-grade and advanced-stage tumors. All prostate carcinoma samples with early recurrence of metastases after radical prostatectomy (n = 7) clustered within this high-grade PC II and III group. B, selection of nine genes included in (A) for which down-regulation in primary prostate cancer has been reported to be associated with fast relapse after radical prostatectomy (14). LN metast, lymph node metastases; normal, normal prostate.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Androgen Receptor Pathway in Prostate Cancer of the androgen pathway to be associated with an enhanced metas- cytoplasm induces apoptosis, which is mediated by the ubiquitin- tatic potential. like domain. We confirmed that HERPUD1 induced cell death via To verify these findings, we did quantitative real-time RT-PCR for activation of apoptosis. HERPUD1 overexpression resulted in the five androgen receptor pathway genes on normal prostate and activation of caspase-3 (Fig. 5C and D) and DNA degradation as prostate carcinoma specimens obtained at our institute. STK39, detected by the TUNEL assay (data not shown). SOCS2, HERPUD1, and 24-dehydrocholesterol reductase (DHCR24) or Seladin-1 were selected based on down-regulation in high-grade prostate carcinomas and lymph node metastases within the Discussion Lapointe data set. STK39, SOCS2, and DHCR24 were among the The current study shows that focusing on a set of target genes 10 genes for which down-regulation was correlated with fast tumor from one transcription factor can be a very valuable tool in relapse according to Henshall et al. (Fig. 3B). The fifth androgen unraveling the biological changes occurring during cancer receptor pathway gene, a-methylacyl-CoA racemase (AMACR), is a progression. The focus on the expression of androgen receptor well-known marker for prostate carcinoma for which a reduced pathway genes yielded new insights in the role of the androgen expression in primary prostate carcinoma has been correlated with receptor in the progression of prostate carcinoma. Our results recurrence before (28). We included AMACR together with another indicate that changes in the androgen receptor pathway rather well-known prostate carcinoma marker Hepsin as positive con- than a generally enhanced androgen receptor activity play a critical trols. Compared with normal prostate and nonmetastatic primary role in the development of more aggressive prostate carcinoma. prostate carcinoma, HERPUD1, STK39, and SOCS2 were signifi- Specific sets of androgen receptor pathway genes are down- cantly down-regulated in metastatic prostate carcinoma (Fig. 4A). regulated during the progression from well-differentiated prostate The expression of HERPUD1, but not STK39, remained low in carcinoma to high-grade prostate carcinoma, and this might be lymph node metastases. AMACR and Hepsin were the only two a prerequisite to acquire the ability to metastasize. Only few genes with significantly higher expression in prostate carcinoma androgen receptor pathway genes remained highly expressed in than normal prostate. The Kaplan-Meier results of the quartiles for metastases compared with primary prostate carcinoma. the occurrence of metastases after radical prostatectomy are Adaptations of the androgen receptor pathway between shown in Fig. 4B.ForHERPUD1 and STK39, none of the primary normal prostate and primary prostate carcinoma. We selected prostate carcinomas within the three or two quartiles with highest 200 androgen receptor pathway genes up-regulated by androgens expression were metastatic (Fig. 4B). When the most optimal in the prostate cancer cell line LNCaP and/or down-regulated on threshold level per gene was selected on receiver operator castration in prostate cancer xenografts. We then assessed the characteristic curves, low expression levels in primary prostate expression of these genes in clinical prostate carcinoma samples. carcinoma were significantly correlated with an increased rate of A proportion of the androgen receptor pathway genes were stroma development of a distant metastasis for all five androgen receptor associated. Because prostate carcinoma contains less stromal cells pathway genes but not for Hepsin (P < 0.00005 for HERPUD1, than normal prostate, these stroma-associated genes were down- P = 0.0001 for STK39, P =0.02forDHCR24 and SOCS2, P = 0.03 regulated in prostate carcinoma. The majority of the genes up- for AMACR, and P =0.11forHepsin, log-rank test). Low or high regulated in primary prostate carcinoma were tumor associated. This HERPUD1 expression almost perfectly predicted recurrence or implies that these androgen receptor pathway genes are up-regulated no recurrence. The HERPUD1 expression value correctly classified in the malignant epithelial cells rather than simply reflecting the the 2 of 24 prostate carcinomas with Gleason score 6 and 2 of 14 increase of percentage of epithelial cells (26). Approximately half of prostate carcinomas with Gleason score 7 that developed metas- the androgen receptor pathway genes are up-regulated in prostate tases. In addition, it also correctly classified the 2 of 8 prostate carcinoma, indicating an enhanced androgen receptor activity that carcinomas with Gleason score 8 that did not develop metastases. however does not up-regulate all target genes. Apparently, prostate Peculiarly, the only misclassified tumor had a Gleason score 10 carcinoma cells activate androgen receptor pathway genes that are from which a metastasis became apparent 4 months after radical less or not androgen responsive in normal prostate epithelial cells. prostatectomy already. The hazard ratio (and 95% confidence Adaptations of the androgen receptor pathway between interval) was 1.13 (0.22-5.88; P = 0.88) for the Gleason score and low- and high-grade primary prostate carcinoma. The expres- 0.26 (0.11-0.64; P = 0.03) for HERPUD1 mRNA expression (Cox mul- sion levels of the androgen receptor pathway signature within the tivariate regression analysis), indicating HERPUD1 to be a better data set of Lapointe et al. clearly differed between low- and high- predictor for recurrence than the Gleason score. grade prostate carcinoma (13). A subset of 33 androgen receptor Overexpression of HERPUD1 induces apoptosis. HERPUD1 pathway genes was higher expressed in low-grade prostate (alias HERP, Mif1, SUP, and KIAA0025) is an endoplasmic reticulum carcinoma than in most of the high-grade tumors. A function (ER)–resident protein that is up-regulated during a variety of stress has been ascribed to 28 from the 33 genes (see Supplementary conditions (24, 29). We selected HERPUD1 to test whether Table S3). Thirteen genes have been described to affect cell growth overexpression affects cell viability. Overexpression of HERPUD1 from which 6 act as proliferation inhibitors, 1 as proliferation efficiently induced cell death (apoptotic nuclei) in both LNCaP and inducer, and 3 as mediators of apoptosis. Six genes are a marker or Hep3B cells (Fig. 5A). The presence of apoptotic nuclei co-occurred inducer of differentiation, whereas two genes are involved in cell with a localization of HERPUD1 in the entire cytoplasm instead of adhesion. Down-regulation of these 33 genes therefore is expected the ER(Fig. 5 A). Two deletion mutants of HERPUD1 were tested as to be beneficial for cell proliferation and prevention of apoptosis, comparison. HERPUD1 lacking a transmembrane domain induced and this likely explains the difference in proliferation rate between apoptosis even more rapidly than WT HERPUD1 (Fig. 5B). In low- and high-grade prostate carcinoma. contrast, HERPUD1 lacking the ubiquitin-like domain had a de- Adaptations of the androgen receptor pathway between creased ability to induce apoptosis (Fig. 5B). These findings point high-grade primary prostate carcinoma and metastases. to a mechanism, in which release of HERPUD1 from the ER to the Fifteen genes up-regulated in low-grade prostate carcinoma www.aacrjournals.org 5017 Cancer Res 2006; 66: (10). May 15, 2006

Figure 4. Expression of five androgen receptor pathway genes as measured by quantitative real-time RT-PCR on an independent set of prostate cancer samples. A, box plots, expression of five androgen receptor pathway genes in prostate cancer samples. Well-known prostate cancer marker gene Hepsin was included as comparison. Top, significant differences (P < 0.05, Mann-Whitney U test); boxes, interquartile range, which contains 50% of values; o or *, whiskers extending from the highest to lowest values, excluding outliers; line across the box, median; PC ! no MET or PC ! MET, primary prostate cancers that did or did not develop distant metastases; LN-MET, prostate cancer metastases in the lymph node. Expression in lymph node metastases was not assessed for SOCS2, DHCR24, and Hepsin. X axis, number of patients per sample group. B, Kaplan-Meier curves for the metastasis-free time of patients with different expression levels in the primary prostate cancer. 1st quartile, comprises the 25% of the patients with the lowest expression. RP, radical prostatectomy. remained highly expressed in high-grade prostate carcinoma and (TPD52) has been reported previously to be up-regulated in were down-regulated in metastases (see Supplementary Table S3). prostate carcinoma (31, 32). Apparently, high TPD52 expression is Twelve of these 15 genes have a known function, and most are not beneficial for growth of metastases. involved in metabolism, exocytosis, transport, protein folding, or Eleven androgen receptor pathway genes remained highly signal transduction. DHCR24 has recently been shown to be a key expressed in lymph node metastases as indicated by their higher mediator of Ras-induced senescence (30). Tumor protein D52 expression levels in these samples compared with normal prostate

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the androgen receptor as an important inducer for their expression in prostate carcinoma. Only two genes, NOL8 and G1P2, are higher expressed in lymph node metastases than in primary prostate carcinoma. However, both genes are highly expressed in lymphocytes, and their high expression in lymph node metastases might therefore be due to contaminating lymphocytes. Down-regulation of androgen receptor pathway genes is correlated to the ability to metastasize. Ten of the androgen receptor pathway genes with high expression in primary prostate carcinoma and low expression in metastases were included in a set of 152 genes (181 spots) for which down-regulation in primary prostate carcinoma was associated with fast relapse after radical prostatectomy (14). We confirmed this correlation for five androgen receptor pathway genes using quantitative real-time RT-PCR on an independent set of prostate carcinoma samples. For AMACR, this correlation has been reported recently using immunohistochemistry on tissue microarrays as well (28). In our experiments, HERPUD1 and STK39 were better predictors for development of metastases than AMACR. When using the most optimal threshold level of 3, low or high expression of HERPUD1 mRNA in primary prostate carcinomas correlated perfectly with the occurrence or nonoccurrence of distant metastases with the exception of one prostate carcinoma with Gleason score 10. None of the prostate carcinomas with STK39 mRNA relative expression higher than 7.8 developed a metastasis. Both HERPUD1 and STK39 are stress response proteins and have been reported before to be up-regulated by androgens in LNCaP cells (24, 35–37). HERPUD1 is an ER-resident protein that is up-regulated during the unfolded protein response and after cellular stresses, such as amino acid deprivation and oxidative stress. The unfolded protein response results in a temporary inhibition of protein synthesis. For chronic stress, this leads to activation of apoptotic pathways (38, 39). Interestingly, the HERPUD1 gene is localized at chromosome 16q, a region with high frequency of copy number losses in prostate carcinoma and other types of cancer (40). HERPUD1 is considered to be a protein that protects against apoptosis because knockout of this gene causes cells to become less tolerant to stress (39). Our transfection experiments in both LNCaP and Hep3B cells indicate that also high expression of HERPUD1 is not favorable to cells. HERPUD1 expression induces apoptosis, and this apoptotic potential is affected by domain mutations of HERPUD1, showing a functional significance. Therefore, we expect that HERPUD1 acts as an apoptosis inhibitor on a shorter term and as an apoptosis inducer on a longer term. In summary, our results point out that prostate carcinoma cells disable the HERPUD1- and STK39-related stress responses to Figure 5. Overexpression of HERPUD1 induces apoptosis. A, green, Hep3B cells transiently transfected for 1 day with HERPUD1; arrows, condensed acquiring the potency to metastasize. The HERPUD1 status apoptotic nuclei in the cells with HERPUD1 distributed over the entire cytoplasm promises to have high clinical utility as a biomarker to predict instead of localized at the ER. B, deletion of the transmembrane domain (del tumor recurrence. Its expression level might become a valuable TM) accelerates induction of apoptosis, whereas deletion of the ubiquitin-like domain (del Ub-like domain) slows down induction of apoptosis by HERPUD1 criterion for decisions about ‘‘watchful-waiting’’ protocols or overexpressed in Hep3B cells. Representative of three experiments. a, b, and c, aggressiveness of therapy. Because down-regulation can only be significant differences daily between a and b, b and c, and a and c (P < 0.01, detected when occurring in the majority of the cells, our findings Bonferroni). C, overexpression of HERPUD1 (green) results in activation of caspase-3 (red). Hep3B cells were transiently transfected for 2 days with on HERPUD1 also supports the earlier proposed view that the HERPUD1. Left, counterstaining of the nuclei with Hoechst 33342. D, activation characteristics of the bulk of the tumor rather than that of a small of caspase-3 in HERPUD1-transfected Hep3B cells during time. a and b, significant differences between days (P < 0.01, Bonferroni). subset of cells determine the metastatic capability (41). Evolution of the androgen receptor pathway during prostate carcinoma progression. Our results lead to the fol- (see Supplementary Table S2). SIM2 and AMACR have been lowing model. In nonmalignant prostatic epithelial cells, the an- reported to positively affect prostate carcinoma growth (33, 34). drogen receptor regulates genes that are mainly involved in the Within our xenograft panel, both genes have a low expression level differentiation into a secretory epithelial cell and in this cell’s in all xenografts lacking functional androgen receptor, suggesting function of production of prostate fluid with all its components, www.aacrjournals.org 5019 Cancer Res 2006; 66: (10). May 15, 2006

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research such as vesicles, proteins, and metabolites. Cell growth and the mechanisms that mediate the selective down-regulation of apoptosis of normal epithelium are likely regulated indirectly by the differentiation-promoting and proliferation-inhibiting genes. stromal-epithelial interactions. Tumorigenesis and androgen- Eventually, this may lead to development of drugs that prevent or dependent growth of prostate cancer are acquired traits often reverse the down-regulation of differentiation-promoting and cell caused by chromosomal rearrangements leading to a fusion gene growth–inhibiting genes. of the androgen-regulated TMPRSS2 promoter to the oncogenic ETS family members (3). Androgen regulation in early-stage cancer cells will drive not only growth but also differentiation and normal Acknowledgments prostate functions. These cells will become more aggressive by Received 8/29/2005; revised 3/8/2006; accepted 3/17/2006. selective down-regulation of androgen receptor pathway genes that Grant support: Dutch Cancer Society (Amsterdam, the Netherlands) grant DDHK inhibit proliferation, induce differentiation, or mediate stress 2001-2455. responses and apoptosis. This down-regulation results in a higher The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance proliferation rate and enhances the potency to metastasize. with 18 U.S.C. Section 1734 solely to indicate this fact. The present results provide novel insight and are expected to We thank Drs. Hans C. Romijn and Theo H. van der Kwast for their participation in the development of the xenografts; Drs. Marcel Smid, Marion Meijer-van Gelder, have important consequences for the direction of future research. and Mark Wildhagen for the support with statistical analyses; the Central Micro- Taking advantage of the differentiation-promoting and cell growth– array Facility (http://microarrays.nki.nl/) of the Dutch Cancer Institute for providing inhibiting effects of the androgen receptor might be a more pro- the expression microarrays; Wilma Teubel, Monique Oomen, and Dr. Peter H.J. Riegman for providing the prostate carcinoma tissues (Erasmus Medical Center fro- mising route to therapy development than attempts to fully block zen tissue bank); and Dr. Michael J. Moorhouse and his mother for careful editing of androgen receptor activity. For this, it will be essential to reveal the article.

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