Functional Epigenomics Identifies Genes Frequently Silenced in

Functional Epigenomics Identifies Genes Frequently Silenced in

Prostate Cancer

Dimitri Lodygin,1Alexey Epanchintsev,

1Antje Menssen,

1Joachim Diebold,

2and Heiko Hermeking

1

1Molecular Oncology Group, Max-Planck-Institutes of Biochemistry, Martinsried and 2Institute of Pathology,Ludwig-Maximilians University, Munich, Germany

Abstract

In many cases, silencing of gene expression by CpGmethylation is causally involved in carcinogenesis. Further-more, cancer-specific CpG methylation may serve as a tumormarker. In order to identify candidate genes for inactivationby CpG methylation in prostate cancer, the prostate cancercell lines LNCaP, PC3, and Du-145 were treated with 5-aza-2Vdeoxycytidine and trichostatin A, which leads to reversionof epigenetic silencing. By microarray analysis of 18,400individual transcripts, several hundred genes were found tobe induced when compared with cells treated with trichos-tatin A. Fifty re-expressed genes were selected for furtheranalysis based on their known function, which implied apossible involvement in tumor suppression. Twelve of thesegenes showed a significant degree of CpG methylation intheir promoters. Six genes were silenced by CpG methylationin the majority of five analyzed prostate cancer cell lines,although they displayed robust mRNA expression in normalprostate epithelial cells obtained from four different donors.In primary prostate cancer samples derived from 41 patients,the frequencies of CpG methylation detected in the promoterregions of these genes were: GPX3 , 93%; SFRP1 , 83%; COX2 ,78%; DKK3 , 68%; GSTM1 , 58%; and KIP2/p57 , 56%. Ectopicexpression of SFRP1 or DKK3 resulted in decreased prolif-eration. The expression of DKK3 was accompanied byattenuation of the mitogen-activated protein kinase pathway.The high frequency of CpG methylation detected in thepromoters of the identified genes suggests a potential causalinvolvement in prostate cancer and may prove useful fordiagnostic purposes. (Cancer Res 2005; 65(10): 4218-27)

Introduction

Prostate cancer is the second leading cause of cancer deaths inmen in the U.S. For the year 2004, it has been estimated that 230,000new cases of prostate cancer will be diagnosed and 30,000 deathsrelated to prostate cancer will occur (1). The molecular basis ofprostate cancer initiation and progression is still poorly understood,particularly due to the heterogeneity of primary tumors.Inactivation of tumor-suppressive genes by either genetic or

epigenetic mechanisms contributes to cancer formation. Epigeneticinactivation of genes in cancer cells is largely based ontranscriptional silencing by aberrant CpG methylation of CpG-richpromoter regions (2, 3). Epigenetic silencing has been reported for a

number of genes in prostate cancer. For example, aberrant CpGmethylation in prostate cancer was found for GSTP1 in f90%, forRASSF1A in f63%, and for RARb2 in f79% of the analyzedsamples (4–6). Other examples of genes frequently silenced inprostate cancer are APC , MGMT, and MDR1 (7–9). Inhibition ofDNA-methyltransferase activity by 5-aza-2Vdeoxycytidine leads toreversion of CpG-methylation and re-expression of silenced genes.As transcriptional silencing mediated by CpG methylation involvesthe recruitment of histone deacetylase activity (10), the effect of5-aza-2Vdeoxycytidine is augmented by the histone deacetylase–inhibitor trichostatin A. Silenced genes which are induced by acombined treatment with 5-aza-2Vdeoxycytidine and trichostatin Acan be identified by microarray analysis in a genome-wide manner.The identification of genes specifically silenced by CpG methylationmay contribute to a better understanding of the etiology of prostatecancer. Furthermore, CpG methylation promises to be useful as adiagnostic tool because the detection and quantification of specificCpG methylation patterns of DNA in biopsies or body fluids isfeasible (11). The use of a panel of CpG methylation markers incombination with standard histologic review of needle biopsies wasshown to increase the sensitivity of prostate cancer diagnosis (12).Moreover, aberrant CpG methylation may potentially be used as

a prognostic marker to identify prostate cancer, which will progressto symptomatic or metastatic disease. Here, we describe the resultsof a microarray-based, genome-wide screen for genes epigeneticallysilenced by CpG methylation in prostate cancer.

Materials and Methods

Cell culture and tissue samples. The cell lines Du-145, LNCaP, PC3,

PPC1, and TSU-Pr1 were maintained in RPMI 1640 supplemented with

10% fetal bovine serum (FBS) and antibiotics (Invitrogen, Karlsruhe,Germany). The TSU-Pr1 cell line was originally thought to be derived

from a metastatic prostate carcinoma, but was later shown to be a

variant of the bladder carcinoma cell line T24 (13). The cell line LAPC-4was kept in RPMI 1640 in the presence of 20% FBS. Human benign

prostate hyperplasia (BPH) cells immortalized with SV40 large T antigen

(BPH1) were obtained from the German Collection of Microorganisms

and Cell Cultures and cultured in RPMI 1640 supplemented with 20%FBS, 20 ng/mL testosterone, 50 Ag/mL transferrin, 50 ng/mL sodium

selenite, 50 Ag/mL insulin, and a mixture of trace elements (Invitrogen).

Human prostate epithelial cells (Cambrex Bio Science, Walkersville, MD)

were cultured in prostate epithelial cell growth medium (PrEGM,Cambrex Bio Science) on dishes coated with collagen type I (BioCoat,

BD Falcon, Heidelberg, Germany). HCT116 colon cancer cells were

maintained in 10% FBS McCoy’s medium (Invitrogen). Archival samplesof primary prostate carcinoma (Gleason Sum 5-10) and cancer-free

samples of prostate were obtained from the Institute of Pathology,

Ludwig-Maximilians University, Munich, and represent consecutive cases

from the year 2001. The samples had been fixed in formalin andembedded in paraffin. All patients had undergone surgery at the same

institution. In all cases, two board-certified pathologists agreed on the

diagnosis of prostate cancer.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Heiko Hermeking, Molecular Oncology Group, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Munich, Germany.Phone: 49-(0)-89-8578-2875; Fax: 49-(0)-89-8578-2540; E-mail: [email protected].

I2005 American Association for Cancer Research.

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Laser-assisted tissue microdissection. Archival specimens of primary

prostate cancer and tumor-free prostate tissue were deparaffinized in

xylene and briefly stained with H&E. Microdissection and laser-pressure

catapulting was done using a MicroBeam system (PALM, Bernried,

Germany). Material obtained from two to three parallel sections

(f103 cells) was pooled for subsequent DNA isolation.

Microarray analysis. LNCaP, PC3, and Du-145 cells were seeded at low

density 24 hours before treatment with 1 Amol/L 5-aza-2Vdeoxycytidine(Sigma, Munich, Germany) and/or 300 nmol/L trichostatin A (Sigma). Total

RNA was isolated from cell lines using the RNAgent kit (Promega, Madison,

WI). Biotin-labeled cRNA (15 Ag) was hybridized to U133A oligonucleotide

arrays, analyzed with a GeneChip Scanner 3000 and differential gene

expression identified with the Microarray Suite 4.0 software (Affymetrix,

Santa Clara, CA).RT-PCR analysis. Total RNA (5 Ag) was reverse-transcribed using the

SuperScript kit (Invitrogen). Quantitative PCR of EF1a was used to

normalize the employed cDNAs (data not shown). Two units Platinum

Taq polymerase (Invitrogen) were used per reaction. Primer sequencesand RT-PCR conditions are provided in Supplemental Table 3.

Isolation and bisulfite treatment of genomic DNA. Genomic DNA

was isolated by overnight incubation in 100 Ag/mL proteinase K (Sigma)and 0.1% SDS (Sigma) at 55jC with subsequent phenol/chloroform

extraction and isopropanol precipitation. Herring sperm DNA (1 Ag;Promega) was added as a carrier to DNA obtained from laser-

microdissected tissue. DNA (2 Ag) was denatured in 0.2 mol/L NaOHfor 10 minutes at 37jC in 50 AL total volume. 30 AL of 10 mmol/L

hydroquinone (Sigma) and 520 AL of 3.5 mol/L sodium bisulfite (pH 5.0;

Sigma) was added. After 16 hours at 50jC, the DNA was purified and

incubated in 0.3 mol/L NaOH for 5 minutes at room temperature. Afterethanol precipitation, the DNA was dissolved in 40 AL Tris-EDTA. For

methylation-specific PCR, 2 AL or amplification of templates for bisulfite-

sequencing 5 AL of this solution were used.Genomic bisulfite sequencing. Bisulfite-treated genomic DNA was used

as a template to amplify fragments of 400 to 1,000 bp with a high CpG-

content around the transcription start site using oligonucleotides listed in

Supplemental Table 1. After 5 minutes’ incubation at 95jC, 39 to 41 PCR-cycles were done for 20 seconds at 95jC, 30 seconds at annealing

temperature, and 60 to 90 seconds at 72jC. Five units of Platinum Taq

polymerase (Invitrogen) were used per 100 AL reaction. Gel-purified PCR-

products were subcloned in a TOPO-TA vector (Invitrogen). For each gene,at least six individual clones were sequenced on both strands using M13

primers and BigDye terminator, and analyzed on a 3700 capillary sequencer

(Applera, Darmstadt, Germany).

Methylation-specific PCR-analysis. Methylation-specific PCR was donein a total volume of 20 AL using 3 units Platinum Taq-polymerase

(Invitrogen) per reaction and oligonucleotides listed in Supplemental Table

2 (14). After denaturation at 95jC for 5 minutes, 40 PCR-cycles were donewhen genomic DNA obtained from cell lines and 45 cycles when

microdissected DNA was used as a template. Amplified fragments were

separated by 8% polyacrylamide gel electrophoresis and detected by

ethidium bromide staining.Immunohistochemistry. Six-micrometer sections were deparaffinized

in xylene, rehydrated in a decreasing ethanol series and boiled for 30

minutes in ProTaq IV buffer (Biocyc, Luckenwalde, Germany) for antigen

retrieval. Anti-SFRP1 antibodies (Santa Cruz, Heidelberg, Germany) wereused with Vectastain Elite avidin-biotin complex method kit (Vector

Laboratories, Burlingame, CA). After counterstaining with hematoxylin,

the images were acquired on an Axiovert 200 M microscope (Carl Zeiss,Oberkochen, Germany) coupled to a DXC-390P CCD camera (Sony,

Tokyo, Japan) using a PALMRobo V2.1.1 software (PALM).

Protein detection by immunofluorescence. Cells were cultured on

CELLocate slides (Eppendorf, Hamburg, Germany) and fixed in 3.7%paraformaldehyde solution for 20 minutes, permeabilized in 0.2% Triton X

(Sigma) in PBS for 15 minutes, and blocked in FBS for 30 minutes.

Primary antibodies specific for h-catenin (clone 19, Transduction

Laboratories, Lexington, KY) diluted in PBS with 10% FBS and 0.05%Tween 20 were added for 1 hour and detected with a Cy3-conjugated

donkey anti-mouse antibody (Jackson ImmunoResarch Laboratories, WestGrove, PA). The images were acquired using a fluorescent Axiovert 200 M

microscope (Carl Zeiss) and Metamorph software (Universal Imaging,

Downingtown, PA).

Western blot analysis. Western blotting analysis was done as describedpreviously (15). Antibodies used were directed against a-tubulin (Santa

Cruz), vesicular stomatitis virus (Sigma), and phospho-ERK1/2 and ERK1/2

(Cell Signaling Technology, Frankfurt, Germany). Secondary horseradish

peroxidase–conjugated anti-mouse and anti-rabbit antibodies (Promega)were used at a dilution of 1:5,000.

Transfection and luciferase reporter assay. The plasmids pGL3-OT,

pGL3-OF, and pcDNA3.1-His-WNT1 have been described previously (16).

LNCaP, PC3, Du-145 and HCT116 cells were plated at medium density in 12-well plates 24 hours before transfection. Three constructs were cotransfected

using Lipofectamine 2000 reagent (Invitrogen): (a) 0.5 Ag of pGL3-OT or

pGL3-OF; (b) 0.5 Ag of pcDNA3.1-His-WNT1 or pcDNA3.1-His-A (Invitrogen),(c) 50 ng of pCMV-h-gal (Promega). Transfections were done in triplicate.

After 36 hours, cells were assayed for luciferase activity using a Luciferase

Assay System kit (Promega) and for h-galactosidase activity with a Galacto-

Light kit (Tropix, Bedford, MA) on a MicroLumatPlus LB96V luminometer(EG&G Berthold, Bad Wildbad, Germany).

Generation and analysis of transgenic cell lines. For stable

expression of Dickkopf 3 (DKK3) or SFRP1 , the retroviral vector pLXSN

(BD Clontech, Heidelberg, Germany), which was modified by insertion ofan IRES-EGFP fragment derived from the plasmid pIRES-EGFP2 (BD

Clontech), was used. PC3 cells were retrovirally infected using pLXSN-

IRES-EGFP2, pL-DKK3vsv-IRES-EGFP2, or pL-SFRP1-IRES-EGFP2. Seventy-two hours after infection, green fluorescent protein–positive cells were

sorted by fluorescence-activated cell sorting and expanded. For the

assessment of colony formation, cells were seeded at low density in six-

well plates (2,000 cells per well) and grown for 10 days. Cells were fixedin 1% formaldehyde and stained with crystal violet. Apoptosis was

assessed by propidium iodide staining and flow cytometry as described

previously (15).

Results

Epigenetic analysis of prostate cancer cell lines. Conditionsfor optimal transcriptional induction after combined treatmentwith5-aza-2Vdeoxycytidine and trichostatin A were determined bymethylation-specific PCR of the promoter regions of GSTP1 ,RASSF1A , and p16 in several prostate cancer cell lines. Thereby,the metastatic prostate cancer cell lines PC3, LNCaP, and DU-145were identified as most suitable for this analysis (data not shown).For re-expression of silenced genes, the cell lines were exposed to1 Amol/L 5-aza-2Vdeoxycytidine for 72 hours and to 300 nmol/Ltrichostatin A for the final 24 hours. Treatment with 300 nmol/Ltrichostatin A for 24 hours served as a control. Total RNA wasisolated, converted to biotinylated cRNA and hybridized tooligonucleotide arrays representing f18,400 individual transcripts.Each microarray analysis was done in duplicate. Efficient demethy-lation of CpG-dinucleotides was confirmed by methylation-specificPCR analysis of selected promoters (Fig. 1A). The microarrayanalysis revealed that several hundred transcripts were induced inthe cells exposed to 5-aza-2Vdeoxycytidine and trichostatin A whencompared with cells treated with trichostatin A alone (data notshown). GSTP1 , a gene previously shown to be silenced by CpGmethylation in prostate cancer (4), was induced 1.87-fold in LNCaPcells. Therefore, an induction of at least 1.8-fold was chosen as theminimal requirement for further analysis of candidate genes. Theinduction of GSTP1 was confirmed by RT-PCR (Fig. 1B) andNorthern blot analysis (data not shown). Exemplary confirmationsof results obtained by microarray analysis were done by RT-PCR for10 different genes (Fig. 1B). In addition, the expression of RASSF1A

Epigenetic Analysis of Prostate Cancer

www.aacrjournals.org 4219 Cancer Res 2005; 65: (10). May 15, 2005



Figure 1. Analysis of CpG methylation in prostate cancer cell lines. A, pharmacologic reversion of epigenetic silencing in prostate carcinoma cell lines. LNCaP,PC3, and Du-145 prostate cancer cell lines were treated with 5-aza-2Vdeoxycytidine for 72 hours and with trichostatin A for the final 24 hours, with trichostatin Aonly for 24 hours, or left untreated. After bisulfite treatment, genomic DNA was subjected to methylation-specific PCR analysis with primers specific for the indicatedgenes. The PCR-products labeled with ‘‘M’’ were generated by methylation-specific primers, and those labeled with ‘‘U’’ by primers specific for unmethylated DNA.B, mRNA expression of the indicated genes was analyzed by semiquantitative RT-PCR. As a loading control and expression standard, amplification of thehousekeeping gene EF1a was used. C, comparison of GSTM1 and DKK3 CpG methylation pattern in cell lines versus primary tumors. Tumor cells were isolatedfrom paraffin-embedded tumor sections using laser microdissection and genomic DNA was isolated. Results of a parallel methylation-specific PCR analysis of thegenomic DNAs employed for bisulfite sequencing are depicted. D, determination of CpG methylation patterns of genes potentially silenced in prostate cancer.Bisulfite sequencing was done with genomic DNA derived from LNCaP (for COX2 , DDB2 , GSTM1 , and HPGD genes), PC3 (CUTL2 , DKK3 , GPX3 , and p57) andDu-145 (RIS1 ) cells or primary prostate epithelial cells (each gene). CpG-distribution and CpG methylation are shown. The depicted areas correspond to genomic DNAsequences of 2.5 kbp. Vertical bars , CpG dinucleotides; arrow , position of the transcription start site; horizontal , black rectangles , areas which were amplified andsubcloned after bisulfite treatment. Results of sequencing of at least six individual subclones for each area are shown: gray shaded chart areas , frequencies ofmethylated CpG dinucleotides within the respective fragments in prostate cancer cell lines; black shaded areas , methylation pattern detected in primary prostateepithelial cells. The y-axis corresponds to the relative abundance of methylation of the CpG-dinucleotide at the indicated relative positions. The exact location ofamplified fragments and CpG-dinucleotides is given in Supplemental Table 1. For RIS1 and p57 , results are provided at a higher resolution in Supplemental Fig. 1.

Cancer Research




and p16 , which are known to be induced after demethylation, wasanalyzed by RT-PCR (Fig. 1B). We detected the re-expression ofseveral imprinted genes (e.g., IGF2) and of genes silenced by CpGmethylation in somatic tissues (e.g., MAGE). Furthermore, IFN-responsive genes, which have been previously reported to beactivated by 5-aza-2Vdeoxycytidine treatment (17), were found tobe induced (data not shown). Genes belonging to these three classeswere excluded from further analysis. Re-expressed genes with knownputatively tumor-suppressive functions (e.g., involvement in DNA-repair, negative cell cycle regulation, induction of apoptosis,detoxification, differentiation, or transcriptional regulation) wereexamined for the presence of CpG islands in their promoters. Intotal, 50 genes met these criteria (listed in Table 1).The CpG methylation pattern of candidate promoters. The

CpG methylation pattern of GSTM1 and DKK3 was determinedin the cell lines LNCaP and PC3, respectively, and comparedwith the pattern found in primary prostate cancer cells isolatedby laser microdissection of paraffin-embedded prostate sectionsfrom two patients (Fig. 1C). The CpG methylation pattern foundin prostate cancer cell lines reflected the pattern found inprimary tumors and is therefore appropriate for the design ofmethylation-specific PCR primers used in the analysis of primarytumors. In total, the promoters of 44 induced genes weresubjected to analysis by bisulfite sequencing in prostate cancercell lines which have been used for the microarray analysis(Table 1). Extensive CpG methylation was detected in the 5Vregions of DDB2 , DKK3 , GPX3 , GSTM1 , and HPGD , whereascyclooxygenase 2 (COX2), CUTL2 , p57 , and RIS1 displayed focalCpG methylation (Fig. 1D). In nontransformed, primary prostateepithelial cells, these genes did not display significant CpGmethylation (Fig. 1D). Thirty-five of the 44 genes analyzed bybisulfite sequencing did not show detectable CpG methylation,although these genes were significantly up-regulated after thecombined 5-aza-2Vdeoxycytidine/trichostatin A treatment (listedin Table 1). Presumably, these genes were induced secondary tothe up-regulation of other genes silenced by CpG methylation ordue to nonspecific activation of transcriptional pathways by thecombined 5-aza-2Vdeoxycytidine/trichostatin A treatment (18, 19).It is remotely possible that the CpG dinucleotides responsible for thesilencing of these genes were not in the region analyzed by bisulfitesequencing in this study. However, as indicated in Fig. 1D , theanalyzed regions covered several hundred base pairs around thetranscription start site, which display a high CpG content and areexpected to be the main targets for CpG methylation.Validation of candidate genes for epigenetic silencing. The

obtained CpG methylation patterns were then used to assign thepositions of methylation-specific PCR primers (indicated inFig. 1D). The respective methylation-specific PCR primers weretested for their specificity and sensitivity (data not shown). Reliablemethylation-specific PCR conditions could not be established forCUTL2 only. For analysis of genes previously known to be silencedby CpG methylation in other tumor types, the respective publishedmethylation-specific PCR primers were tested and used formethylation-specific PCR analysis. Thirteen genes, which displayedsignificant CpG methylation as determined by bisulfite sequencingor methylation-specific PCR in one of the cell lines, PC3, LNCaP, orDu-145, were examined by methylation-specific PCR in a panel offive prostate cancer cell lines, one cell line established from BPH1,in primary prostate epithelial cells and in the bladder carcinomacell line (TSU-Pr1; Fig. 2, left). This analysis revealed that the genesinitially identified in selected prostate cancer cell lines also

displayed CpG methylation in other prostate cancer cell linesand occasionally in the cell line BPH1. Eight of the 13 analyzedgenes did not display CpG methylation in primary prostateepithelial cells. Therefore, CpG methylation of these eight genesis a specific feature of cancerous prostate epithelial cells. APOD ,DDB2 , GSTM1 , and RIS1 displayed partial CpG methylation inprostate epithelial cells (Fig. 2). However, the degree of CpGmethylation of DDB2 , APOD , and GSTM1 seemed to be significantlyelevated in most of the prostate cancer cell lines when comparedwith normal prostate epithelial cells (Fig. 2), suggesting a prostatecancer–specific increase in CpG methylation of these genes andpotential subsequent silencing. CpG methylation of SFRP1 , GPX3 ,p57 , HPGD , GSTM1 , and APOD was also detected in the bladdercarcinoma cell line TSU-Pr1.In order to determine whether CpG methylation correlates

with reduced or absent expression of the respective genes, weanalyzed the expression levels of nine genes which showedselective or preferential CpG methylation in prostate cancer. RT-PCR analysis of cDNAs obtained from normal prostate epithelialcells derived from four healthy donors and four prostate cancercell lines revealed several distinct patterns of mRNA expression(Fig. 2, right). The CpG methylation interrogated by themethylation-specific PCR primers used here largely correlatedwith reduced gene expression in the case of SFRP1 , DKK3 , GPX3 ,COX2 , GSTM1 , APOD , and p57 , whereas the detected CpGmethylation of DDB2 and HPGD was not accompanied bydecreased gene expression. In the case of HPGD , the expressionwas even induced in three prostate cancer samples, which clearlyshowed CpG methylation. These results indicate that CpGmethylation of a promoter should not be interpreted as proofof its transcriptional repression.Analysis of CpG methylation in primary prostate cancer

samples. The CpG methylation status of the genes showing CpGmethylation–mediated silencing in prostate cancer cell lines wasdetermined in 41 primary prostate cancer samples obtained afterradical (37 cases) or transurethral (4 cases) resection. In addition,the genes DDB2 and HPGD were included in this analysis, althoughwe had not detected a correlation between CpG methylation anddown-regulation of mRNA expression for these genes. Nonetheless,the detection of prostate cancer-specific CpG methylation in thepromoter of these genes may be useful for diagnostic applications.Prostatic tissue samples contain several cell types. Non-neoplasticepithelial cells, stromal cells, lymphocytic infiltrates, and bloodcells are present in close proximity to prostate cancer cells.Therefore, laser microdissection was employed to isolate prostatecancer cells. Similarly, non-neoplastic prostate epithelial cells wereisolated from samples obtained from 9 patients with benignprostate hyperplasia, which did not present prostate cancer andwere in the same age group as the 41 prostate cancer patients.Genomic DNA was isolated from these samples and subjected tomethylation-specific PCR analysis (Fig. 3A and B). The analyzedgenes showed CpG methylation at medium to high frequencies inprostate cancer cells. CpG methylation was detected for SFRP1 in34 (83%), COX2 in 32 (78%), DKK3 in 28 (68%), GPX3 in 38 (93%),p57 in 23 (56%), HPGD in 30 (73%), and DDB2 in 34 (83%) of the 41prostate cancer samples analyzed. Predominant methylation ofGSTM1 was detected in 24 (58%) of 41 cases. We had also identifiedconsistent silencing of 14-3-3r in prostate cancer in this screenwhich was described elsewhere (20). The silencing of 14-3-3r in theset of 41 prostate cancers analyzed here is depicted for comparison(Fig. 3B). For SFRP1 and COX2 , no CpG methylation was detected





Table 1. Candidate genes for epigenetic silencing identified by microarray analysis

Gene Symbol Chromosomal Induced by Function Methylation detected by

location 5-aza-2Vdeoxycytidine Bisulfite-sequencing

in cell linesMethylation-specificPCR

in celllines

in primarytumors

14-3-3j (Stratifin) SFN 1p35 L G2-M transition yes 41 of 41Secreted

frizzled-related

protein 1

SFRP1 8p12 P Wnt signaling yes 34 of 41

Apolipoprotein D APOD 3q26 L, P HDL component yes n.d.Tissue factor

pathway

inhibitor 2

TFPI2 7q22 P, D ECM protease

inhibitor

No

Thrombospondin 1 THBS1 15q15 L, P, D angiogenesisinhibitor

No

Retinoblastoma

protein-interactingzinc finger

RIZ1 1p36 P methyltransferase No

Caspase 7 CASP7 10q25 P, D apoptosis no

Apoptotic protease

activating factor

APAF1 12q23 P, D apoptosis no

Apoptosis-inducing

protein D

APPD 19q11 D apoptosis no

Tumor necrosis factor

receptor 10b

TNFRSF10B 8p22 D apoptosis no

Cyclin-dependent

kinase inhibitor

1C (p57, Kip2)

CDKN1C 11p15 L, P cdk inhibitor yes yes 23 of 41

Cyclin-dependent

kinase inhibitor

2D (p19)

CDKN2D 19p13 P cdk inhibitor no

Cyclin-dependentkinase inhibitor

1A (p21, Cip1)

CDKN1A 6p21.2 L, P, D cdk inhibitor no

Retinoblastoma-like 2 (p130) RBL2 16q12 D cell cycle no

Glutathione S-transferase M1 GSTM1 1p13 L detoxification yes yes 24 of 41*Glutathione peroxidase 3 GPX3 5q23 L, P detoxification yes yes 38 of 41

Hydroxyurea-sensitive 1 HUS1 7p13 D DNA damage

response

no

Meiotic recombination 11 MRE11A 11q21 D DNA damageresponse

no

Xeroderma pigmentosum,

complementation group C

XPC 3p25 L DNA repair no

Damage-specific

DNA binding protein 2

DDB2 11p12 L DNA repair yes yes 34 of 41

Postmeiotic segregation

increased 2

PMS2 7p22 D DNA repair no

Breast cancer 2, early onset BRCA2 13q12.3 D DNA repair,

transcription

no

Cylindromatosis

(turban tumor syndrome)

CYLD 16q11 P deubiquitination no

Bridging integrator 1 BIN1 2q14 D differentiation low n.d. n.d.

Growth arrest and

DNA-damage-inducible, a

GADD45A 1p31.2 P, D growth arrest low n.d. n.d.

Connective tissue growth factor CTGF 6q23 P, D growth factor no

Nerve growth factor receptor NGFR 17q21 P, D growth factor

receptor

no

(Continued on the following page)

Cancer Research




in BPH derived from nine different patients, suggesting that theCpG methylation of these genes is specific for neoplastic prostateepithelial cells. CpG methylation was detected for GPX3 in two andfor DKK3 in one of nine analyzed BPH samples (Fig. 3B). ForGSTM1 CpG methylation was detected in the five BPH samplesanalyzed (Fig. 3B). However, the CpG methylation of GSTM1 waselevated in the majority of prostate cancer samples, whereas in thenon-neoplastic BPH cells, equal signals for the PCR productsrepresenting methylated and unmethylated GSTM1 alleles weredetected. Furthermore, the nonmethylated GSTM1 allele was not

detected in several samples of prostate cancers. No obviouscorrelation between staging information and CpG methylation wasdetected by cluster analysis (data not shown).In order to evaluate the tumor-specificity of the CpG methylation

detected in prostate cancer, we examined the CpG methylationstatus in human diploid fibroblasts derived from neonatal skin, instroma isolated from five cancer-free prostate specimens, and inperipheral blood mononuclear cells from six individuals (Fig. 3B).The p57 gene did not show CpGmethylation in stromal cells. PartialCpG methylation of SFRP1 and COX2 was evident in only one of five

Table 1. Candidate genes for epigenetic silencing identified by microarray analysis (Cont’d)

Gene Symbol Chromosomal

location

Induced by Function Methylation detected by

5-aza-2Vdeoxycytidine Bisulfite-sequencing

in cell linesMethylation-specificPCR

in celllines

in primarytumors

IFN regulatory factor 1 IRF1 5q31 P, D IFN response noIFN regulatory factor 7 IRF7 11p15 L, P, D IFN response no

Hydroxyprostaglandin

dehydrogenase 15-(NAD)

HPGD 4q34 L, P prostaglandin

signaling

yes yes 30 of 41

Prostaglandin E receptor4 (subtype EP4)

PTGER4 5p13 L, P, D prostaglandinsignaling

no

Cyclooxygenase 2 PTGS2 1q25 L, P prostaglandin

signaling

yes yes 32 of 41

Sequestosome 1 SQSTM1 5q35 L, P proteindegradation

no

Dual specificity

phosphatase 1

DUSP1 5q34 P, D signaling no

Jun B proto-oncogene JUNB 14q32 P, D transcription

factor

no

Cut-like 2 CUTL2 12q24 P transcription

factor

yes n.d. n.d.

Zinc finger protein 36,

C3H type, homologue

ZFP36 19q13 L, D transcription

factor

no

Cbp/p300-interacting

transactivator

CITED2 6q23 P, D transcriptional

control

no

SWI/SNF related regulator

of chromatin A1

SMARCA1 Xq25 D transcriptional

control

no

Inhibitor of DNA binding 3 ID3 1p36 L, P transcriptionalcontrol

no

Serum-inducible kinase SNK 5q12 L, P signaling no

Serum/glucocorticoid

regulated kinase

SGK 6q23 L, P, D signaling no

Serine/threonine kinase 38 like STK38L 12p12.3 L signaling no

Growth arrest-specific 2 like 1 GAS2L1 22q12.2 L unknown no

Deleted in liver cancer 1 DLC1 8p22 D unknown no

Ras-induced senescence 1 RIS1 3p.21 D unknown yes yes n.d.B cell translocation gene 1 BTG1 12q22 D unknown no

B cell translocation gene 3 BTG3 21q21 P, D unknown no

Dickkopf homologue 3 DKK3 11p15 P Wnt signaling yes yes 28 of 41

Dickkopf homologue 1 DKK1 10q11 D Wnt signaling no

NOTE: Experimental results for 50 selected, CpG island–containing genes induced upon treatment with 5-aza-2Vdeoxycytidine and trichostatin A in

prostate carcinoma cell lines are summarized. Bold gene symbols, genes known to be silenced by CpG methylation in other types of human cancer (not

prostate cancer); underlined chromosomal locations, regions of frequent loss of heterozygosity in prostate cancer; boldfaced cell line abbreviations,

genomic DNA used for bisulfite sequencing; L, LNCaP; P, PC3; D, Du-145.*Methylation-specific PCR product dominant over PCR product corresponding to unmethylated allele.





analyzed stroma samples. One of six peripheral blood mononuclearcell samples showed partial methylation of SFRP1 and p57 genes. Bycontrast, DKK3 and GPX3 displayed CpG methylation in most of thestromal samples and blood cells.Loss of SFRP1 expression in prostate cancer. In order to

determine whether the CpG methylation of SFRP1 affects theexpression of the respective gene product in primary tumors, thelevel of SFRP1 protein expression was determined by immunohis-tochemistry in prostate cancer samples derived from 39 differentpatients (representative example shown in Fig. 4A). In non-neoplastic prostate glands, most of the luminal cells were positivefor SFRP1 with a characteristic granular cytoplasmic and apicalmembrane staining, whereas prostate cancer cells were devoid ofSFRP1 staining. A prominent down-regulation (>50% of reduction)or complete loss of SFRP1 protein was detected in 29 of 39 prostatecancer samples (data not shown).Functional analysis of DKK3 and SFRP1 . Epigenetic inactiva-

tion of the members of the SFRP gene family presumablycontributes to activation of WNT signaling in colorectal cancer(16). DKK3 negatively regulates the h-catenin pathway in osteo-sarcoma cells (21). Therefore, we asked whether silencing of SFRP1and/or DKK3 expression in prostate carcinoma cells is associatedwith constitutive activation of WNT/h-catenin signaling. Unexpect-edly, h-catenin was localized at the cell membrane and absent fromthe nucleus in PC3 and Du145 cells (Fig. 4B), which show significantsilencing of DKK3 and SFRP1 (Fig. 2). As nuclear h-catenin is ahallmark of an activated WNT/h-catenin pathway, this pathway ispresumably not active in prostate cancer. Furthermore, cotransfec-tion with a WNT1 expression construct did not result in activationof a TCF-reporter in PC3 and Du145 cells (Fig. 4C). In contrast,HCT116 colon cancer cell lines, which harbor an activated WNT/APC/TCF4 pathway (22), showed nuclear h-catenin localization(data not shown) and strong activation of the wild-type but not themutant TCF reporter by WNT1 coexpression (Fig. 4C).Recently, it has been reported that WNT1 activates the mitogen-

activated protein kinase (MAPK) pathway (23). Therefore, we tested

whether the WNT antagonists, DKK3 and SFRP1, inhibit the MAPKpathway. In exponentially growing PC3 cells ectopically expressingDKK3, the level of ERK1 and ERK2 phosphorylation, which isindicative of MAPK activity, was diminished (Fig. 5A and B).Furthermore, PC3 cells ectopically expressing DKK3 or SFRP1showed a significant decrease in colony formation (Fig. 5C), whichwas due to a decrease in colony size. The rate of spontaneousapoptosis was not affected by ectopic expression of DKK3 or SFRP1as determined by flow cytometry (data not shown). Therefore, lossof DKK3 and SFRP1 expression by epigenetic silencing presumablypromotes the proliferation of prostate epithelial cells.

Discussion

The high frequency of CpG methylation we observed in primarytumors suggests that the differences in CpG methylation whichwere initially detected in prostate cancer cell lines did not occurduring in vitro cultivation of prostate cancer cell lines. Whether theCpG methylation of distinct genes is the cause or consequence oftheir down-regulation during prostate cancer initiation or pro-gression remains to be shown. In addition, the functional relevanceof the down-regulation of these genes has to be determined in thefuture. However, the high frequency of the epigenetic silencingevents identified in this study suggests that these genes may becausally involved in prostate cancer development. In the followingparagraphs, we will discuss potential functional consequences ofthe epigenetic silencing events detected here.SFRP1 negatively regulates the WNT pathway, which is fre-

quently activated in cancer by mutations in the APC and b-cateningenes (24, 25). Recently, SFRP1 was shown to undergo both geneticand epigenetic alterations in colon and bladder cancers (26, 27).Frequent hypermethylation of SFRP1 was recently detected incolorectal cancer (28), where the loss of SFRP1 correlated with anincreased h-catenin/TCF4 activity and ectopic expression of SFRP1led to reduced h-catenin/TCF4 activity. However, in prostate cancer,we could not detect activation of the h-catenin/TCF4 pathway.

Figure 2. Comparative analysis of CpGmethylation and gene expression. Left ,methylation-specific PCR analysis of aseries of prostate cancer cell lines andprimary prostate epithelial cells.Methylation-specific PCR analysis with twoprimers sets (M , methylated;U , unmethlyated) specific for the indicatedgenes; (BPH1 ) benign prostatehyperplasia cells immortalized with SV40large T antigen. Right , RT-PCR analysis ofthe indicated genes in nontransformed andtumor cells. D1-D4 samples representprimary prostate epithelial cells from fourdifferent donors. h-Actin and g-tubulin(TUBG2 ) were used as additionalstandards.

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Therefore, loss of SFRP1 may contribute to activation of otheroncogenic signaling pathways in prostate cancer. We havepreviously observed an induction of SFRP1 mRNA in human pri-mary fibroblasts undergoing replicative senescence (15), suggestinga possible role of SFRP1 in the terminal proliferation arrest of sene-scent cells. While this paper was in preparation, CpG methylationof SFRP1 in <5% of prostate cancers was reported (7), whereas wedetected CpGmethylation of SFRP1 in 83% of prostate cancers. Thisdifference may potentially be caused by the different types ofmaterials thatwere analyzed. Florl et al. (7) isolated DNA from piecesof tissue containing prostate cancer and other cell types. Here, pro-state cancer cells isolated by laser microdissection were analyzed.DKK3 negatively regulates the WNT-pathway. The zonal

distribution of DKK3 expression in the adrenal gland suggeststhat DKK3 is involved in zonal differentiation or growth (29). DKK3was identified as a gene which is down-regulated upon immortal-ization of primary human cells (30). Reduced expression of DKK3was also found in non–small cell lung cancer and renal clear cellcarcinoma (31, 32). Overexpression of DKK3 inhibited growth,invasion, and motility of Saos-2 osteosarcoma cells by modulatingWNT-signaling (21). Interestingly, the DKK3 transcript was shownto be up-regulated in senescent primary prostate epithelial cells(33). The epigenetic silencing of DKK3 by CpG methylationobserved here might therefore contribute to de-differentiationand immortalization of prostate cancer cells.Our data suggest that the WNT/h-catenin pathway is not

activated in prostate cancer cell lines (Du-145 and PC3) withsilenced SFRP1 and DKK3 genes. This is in agreement with a recentcomprehensive study of 101 cases of primary prostate cancer: noneof the tumors showed nuclear h-catenin staining (34). However,genetic alterations of b-catenin or APC were detected in a subset ofadvanced prostate cancers and were associated with a resistance toapoptosis (35, 36). Loss of SFRP1 and DKK3 expression mayactivate alternative signaling pathways. Recently, it was reportedthat transactivation of the EGF receptor by WNT ligands, whichresults in MAPK activation, is inhibited by SFRP1 and DKK1 (23).Our data indicate that DKK3 may also have an inhibitory effect onMAPK signaling.p57/KIP2 belongs to a family of conserved CDK inhibitors, which

negatively regulate the cell cycle. Ectopic expression of p57suppresses cell transformation, whereas cells lacking p57 showincreased cell proliferation and decreased differentiation (37–39).p57 expression is decreased in prostate cancer cell lines and primaryprostate epithelial cells immortalized by HPV16 E7 (40). Expressionof p57 induces a senescence-like phenotype in prostate cancer cells

(40), suggesting that down-regulation of p57 may be required forimmortalization of prostate cells. The p57 gene is located onchromosome 11p15.5, a region implicated in both sporadic cancersand the Beckwith-Wiedemann cancer syndrome. Mutations of p57have rarely been detected in human tumors (41). Epigeneticsilencing of p57 was also detected in gastric, hepatocellular,pancreatic carcinomas, and acute myeloid leukemia (42, 43). Thedown-regulation of p57 in bladder cancer involves several mecha-nism including loss of heterozygosity and hypermethylation (44).

Figure 3. Detection of CpG methylation after laser microdissection.A, methylation-specific PCR analysis of in vivo CpG methylation after lasermicrodissection. Tumor cells or normal prostate epithelial cells were isolatedfrom paraffin-embedded tumor sections derived from 41 different patients(pc01-pc41 ) using laser-pressure catapulting. Representative examples ofmethylation-specific PCR analysis of five tumor cell samples and five samplesderived from normal epithelial cells (BPH ) for the indicated genes are shown.B, summary of methylation-specific PCR results. Gene names are indicatedon the top. Each row represents a primary prostate cancer tumor (pc01-41 ),non-neoplastic prostate epithelial cells (BPH1-9 ), or prostate stromasamples (str1-5) isolated by laser microdissection. Human diploid fibroblasts(HDF ) derived from skin and peripheral blood mononuclear cell (PBMC ) werecultured in vitro . The numbering is not meant to indicate that the differentcell types were obtained from the same patient. Color coding: white , nosignificant CpG methylation detected; gray , PCR-product representing CpGmethylation has a similar intensity as the PCR specific for unmethylated allele;black , allele with CpG methylation dominant over unmethylated alleleor unmethylated allele absent.





Glutathione peroxidase 3 (GPX3) catalyzes the reduction ofperoxides by glutathione and protects cells against oxidativedamage. The prostate cancer–specific silencing of GPX3 may leadto an impaired defense against endogenous and exogenousgenotoxic compounds, which could potentially result in anincreased rate of mutation in critical genes. Down-regulation ofGPX3 expression upon transition from normal to neoplasticprostate tissue was recently detected by microarray analysis ofmicrodissected primary prostate cancer (45). Presumably, thisdown-regulation of GPX3 in prostate cancer is caused by theepigenetic silencing of GPX3 detected here.Glutathione S-transferases are active in the detoxification of a

wide variety of toxins and carcinogens. The common null-alleleof GSTM1 shows a weak association with lung cancer (reviewedin ref. 46). However, no significant association of GSTM1 poly-morphisms or deletion with prostate cancer have been reported.The tumor-specific hypermethylation of GSTM1 identified heremay explain the decreased expression of GSTM1 in prostate cancerdetected in three previous studies (45, 47, 48).COX2 catalyzes the synthesis of prostaglandin H2, a precursor of

other prostanoids, and has been implicated in inflammation andcarcinogenesis (reviewed in ref. 49). While this manuscript was inpreparation, CpG-methylation of the COX2 gene in prostate cancerwas reported independently (8, 9). In the study by Kang et al. (8),the CpG methylation of COX2 was detected in 22% of primaryprostate cancer, whereas Yegnasubramanian et al. (9) detectedhypermethylation of COX2 in 88% of prostate cancer when theyapplied a quantitative method to analyze a large collection ofsamples. Interestingly, the hypermethylation of COX2 was associ-ated with a higher risk of recurrence of prostate cancer (9).The lack of a correlation between CpG methylation and grade or

stage of the prostate cancer is in agreement with other recentstudies (7–9). These studies and our data suggest that silencing ofgenes by CpG methylation occurs at an early stage of prostate

Figure 4. Expression of SFRP1 inprostate cancer and analysis ofthe h-catenin/TCF4 pathway.A, immunohistochemical detection ofSFRP1 (red) in primary prostate cancer.(N) non-neoplastic prostate epithelial cells,(PCa ) prostate cancer cells. B, detection ofh-catenin in PC3 and Du-145 prostatecancer cells by immunofluorescence.C, analysis of TCF/LEF reporter activity inprostate cancer (PC3 and Du145 ) andcolon (HCT116 ) cancer cell lines. Cellswere transfected with a pGL3-OT (OT )TCF/LEF reporter construct, or withpGL3-OF (OF ), a negative controlcontaining a mutated TCF binding site.A WNT1 expression construct or itsbackbone construct were cotransfected.Luciferase activity was measured 36 hoursafter transfection. Transfection efficiencywas normalized by cotransfection of ah-galactosidase encoding plasmid.The assays were done in triplicate(SD indicated). The values obtained fortransfection of the OF-plasmid alone wereset to 1 to visualize the differences amongthe three different cell lines.

Figure 5. Effects of ectopic DKK3 and SFRP1 expression in prostate cancercell lines. A, ectopic expression of DKK3 and SFRP1 in prostate carcinoma cells.PC3 cells were infected with bicistronic retroviruses encoding EGFP,DKK3 + EGFP or SFRP1 + EGFP; green fluorescent protein-positive cellswere isolated by flow cytometry and total protein lysates were subjected toWestern blot analysis with anti–vesicular stomatitis virus or anti-SFRP1antibodies. Equal loading was confirmed by detection of a-tubulin. B, inhibition ofERK1/2 phosphorylation in PC3 cells ectopically expressing DKK3 . Total proteinlysates were analyzed using antibodies specific for ERK1 and ERK2phosphorylated at the Thr202 and Tyr204 residues or anti-ERK1/2 antibodies.C, inhibition of cellular proliferation by DKK3 and SFRP1. Equal number of PC3cells transduced with the indicated constructs were plated at low density insix-well plates, cultured for 10 days and colonies were stained with crystal violet.The analysis was done in duplicate yielding identical results (data not shown).

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cancer development. This may have implications for the use ofthese CpG methylation events for detection of early prostate cancerlesions. The detection of aberrant CpG methylation has severalsignificant advantages when compared with protein- or RNA-basedtumor markers (50). According to our analysis, the CpGmethylation of SFRP1 , COX2 , and p57 has the highest specificityfor prostate cancer and is therefore of higher relevance forpotential diagnostic applications. However, this conclusionrequires validation in larger cohorts of patients in the future.

Acknowledgments

Received 12/10/2004; revised 2/17/2005; accepted 3/8/2005.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Reinhard Hoffman for help with the microarray analysis and AnjaHeyer for expert assistance with immunohistochemistry, Wolfgang Klinkert forassistance with flow cytometry, Hiromu Suzuki for WNT1 and SFRP1 encodingconstructs, and Axel Ullrich for antibodies and cell lines. Heiko Hermeking issupported by the Max-Planck-Society, the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung and the Rudolf-Bartling-Stiftung.

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2005;65:4218-4227. Cancer Res Dimitri Lodygin, Alexey Epanchintsev, Antje Menssen, et al. Silenced in Prostate CancerFunctional Epigenomics Identifies Genes Frequently

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