Human Cancer Biology ssDNA-Binding 2 Is Frequently Hypermethylated and Suppresses Cell Growth in Human Prostate Cancer Jun-Wei Liu,1Jatin K. Nagpal,1 Wenyue Sun,1Juna Lee,1Myoung Sook Kim,1Kimberly L. Ostrow,1 Shaoyu Zhou,1Carmen Jeronimo,2 Rui Henrique,2 Wim Van Criekinge,3 Chu So Moon,1 Joseph A. Califano,1Barry Trink,1and David Sidransky1

Abstract Purpose: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. Experimental Design: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray.We then did quantitative methyl- ation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. Results: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2¶-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. Conclusion: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer ^ specific DNA marker, especially in high stages of human prostate cancer.

In 2007, it is estimated that more than 27,000 deaths will be Unlike genetic alterations, promoter methylation, one due to prostate cancer and more than 200,000 new cases will be type of epigenetic change, is commonly found in prostate diagnosed. Prostate cancer is the most common malignant cancer (3). More than 30 , including GSTP1, APC, neoplasm and the second cause of cancer death among men in RASSF1, and RARB, have been reported to be hypermethylated the United States (1). The current screening method for prostate in prostate cancer (3–7). Promoter methylation contributes to cancer relies predominantly on the serum level of the prostate- tumorigenesis through silencing of tumor suppressor genes by specific antigen. However, its poor specificity limits this down-regulating gene expression (8). However, many of the approach; about one third of patients with an elevated specific molecular events involved in prostate cancer progres- prostate-specific antigen undergo further unnecessary medical sion still remain elusive. procedures as they do not have a malignant form of the disease Sequence-specific ssDNA-binding protein, the chicken ortho- (2). Therefore, it is urgent to discover novel molecular markers logue of human SSBP, shares 97% identity with both mouse for prostate cancer to augment current detection approaches. and human expressed sequence tags (9). Human ssDNA- binding protein 2 (SSBP2) was identified as a target of unbalanced translocations between 5q and 17p in acute myeloid leukemia (10, 11). A recent study on SSBP2 has Authors’ Affiliations: 1Division of Head and Neck Cancer Research, Department shown that it is a novel regulator of hematopoietic growth and of Otolaryngology, Johns Hopkins University School of Medicine, Baltimore, differentiation (12). However, the role of SSBP2 in human Maryland; 2Departments of Genetics and Pathology, Portuguese Oncology Institute-Porto, Porto, Portugal; and 3OncoMethylome Sciences S.A., Liege, solid tumors remains unexplored. In this article, we report that Belgium SSBP2 is hypermethylated in human prostate cancer and that Received 10/29/07; revised 3/4/08; accepted 3/6/08. this epigenetic alteration is closely related to SSBP2 protein The costs of publication of this article were defrayed in part by the payment of page expression. Forced SSBP2 expression was able to inhibit cell advertisement charges. This article must therefore be hereby marked in accordance growth through cell cycle arrest in prostate cancer cells. with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: David Sidransky, Division of Head and Neck Cancer Research, Department of Otolaryngology, Johns Hopkins University, 1550 Orleans Materials and Methods Street, Baltimore, MD 21231. Phone: 410-502-5152; Fax: 410-614-1411; E-mail: [email protected]. F 2008 American Association for Cancer Research. Cell lines and primary tissues. Four prostate cancer cell lines, doi:10.1158/1078-0432.CCR-07-4763 DU145, 22Rv1, LNCaP, and PC3, were included in the study. All cell

Clin Cancer Res 2008;14(12) June 15, 2008 3754 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Hypermethylation of SSBP2 in Human Prostate Cancer lines were obtained from the American Type Culture Collection. Cells DNA was done as described (13). Briefly, 2 Ag of genomic DNA were were maintained in RPMI 1640 supplemented with 10% fetal bovine used for chemical treatment. Then, the sample was purified using the serum. Wizard DNA Clean Up System (Promega) followed by incubation with Eighty-eight primary prostate adenocarcinomas with no metastatic sodium hydroxide and ammonium acetate separately. The sample was lesions detected were obtained from patients who were consecutively precipitated in 100% ethanol. The treated DNA was resuspended in diagnosed and treated primarily with radical prostatectomy at the 100 AL LoTE [10 Amol/L Tris-HCl (pH 8), 2.5 Amol/L EDTA (pH 8)] Portuguese Oncology Institute-Porto, Porto, Portugal. Thirty paired and stored at -80jC for further analysis. high-grade prostatic intraepithelial neoplasia (PIN) lesions identified in PCR amplification of bisulfite-treated DNA and sequencing analysis. 30 of these specimens were also collected. Twenty-three randomly Bisulfite-modified genomic DNA was amplified using primer sets that selected patients with benign prostatic hyperplasia (BPH) who were designed to recognize DNA alterations after bisulfite treatment. underwent transurethral resection of the prostate were used as controls. The primer sequences of SSBP2 for bisulfite-treated DNA were The patient characteristics, such as Gleason score and grade, are 5¶-TGGGTGGATTAGGAGGAATG-(649-668)-3¶ and 5¶-TGTGAA- shown in Table 1. All tissue specimens were obtained immediately GGGCTGACTCACTG-(785-804)-3¶. A touchdown PCR program was after surgical resection and stored at -80jC for further analysis. Then, used to amplify the promoter region of SSBP2. All PCR products were 5-Am-thick sections were cut from frozen tissue fragments in cryostat to gel extracted (Qiagen) and sequenced using the ABI BigDye cycle identify areas of adenocarcinoma and high-grade PIN in the radical sequencing kit (Applied Biosystems). prostatectomy specimens, and BPH in the control tissue samples was Real-time quantitative methylation-specific PCR. Bisulfite-modified obtained from transurethral resections of the prostate after H&E genomic DNA was amplified using Taqman methylation-specific PCR staining. The areas of interest were identified and the frozen tissue (MSP) primer sets that were designed based on the sequencing results of block was trimmed to allow the specific collection of the selected areas the SSBP2 promoter in tumor and BPH. The Taqman MSP primer [containing at least 70% neoplastic cells (either PIN or adenocarcino- sequences of SSBP2 were 5¶-ATTTTTGCGGTCGTAGCGGT-(694-714)-3¶ ma)]. An average of fifty 12-Am-thick sections was then cut in the (forward) and 5¶-TTCTACGACAAATCTAACGAA-(763-784)-3¶ (reverse). cryostat and processed for DNA extraction (4). The fluorescent probe was designed to hybridize to a region of SSBP2 5-Aza-2¶-deoxycytidine treatment of prostate cancer cells. Cells were promoter that was determined previously to be methylated in prostate  6 seeded in T-75 flasks at a density of 1 10 per flask at 37jC with 5% cancer cell lines by sequencing. The probe sequence of SSBP2 is 6FAM- A ¶ CO2. The cells were treated with 5 mol/L 5-aza-2 -deoxycytidine 5¶-ATATCCAAAACGCCGCGAAACTCC-3¶-TAMRA. The annealing tem- (5-Aza-dC) every 24 h for 4 d. The cells were harvested for DNA and perature is 60jC. h-Actin was used as a control (13). For all MSP RNA preparation. reactions, 3 AL of bisulfite-treated DNA were added to a final volume of RNA extraction and reverse transcription-PCR. RNA extraction and 20 AL. All reactions were done in duplicate. Serial dilutions of in vitro reverse transcription-PCR were done as described previously (13). Four methylated human leukocyte genomic DNA were used to construct a micrograms of total RNA were reverse transcribed with SuperScript II calibration curve. The methylation ratio was defined as the Taqman reverse transcriptase (Invitrogen) and amplified by PCR. The primer value derived from SSBP2 promoter amplification divided by sequences of SSBP2 were 5¶-GCCTGAGCGACAAAAAGTTC-3¶ (forward) Taqman value from h-actin amplification and multiplied by 100 and 5¶-CATCTCCCCATCTCCAAAGA-3¶ (reverse). The PCR conditions (Taqman methylation value: TaqMeth V). were 1 min at 95jC, 1 min at 58jC, and 1 min at 72jC for 30 cycles. Construction of recombinant mammalian expression vector of h-Actin was used as a control, and its primer and amplification were SSBP2. A human cDNA clone of SSBP2 was purchased from OriGene. described previously (13). The full length of SSBP2 was amplified using primer sets that were Bisulfite treatment of DNA. Genomic DNA was extracted using the designed to cover the whole open reading frame. The primers used were phenol chloroform method, and bisulfite modification of genomic 5¶-TCGGCACAAGCATGTACGGCAA-3¶ (forward) and 5¶-TGGATCA- CACGCTCATTGTCA-3¶ (reverse). The PCR amplifications were done as follows: 1 min at 95jC, 1 min at 54jC, and 1 min at 72jC for 40 cycles followed by a 5-min denaturation at 95jC. A 7-min elongation Table 1. Clinicopathologic variables and SSBP2 step at 72jC completed the PCR amplification. The PCR products were methylation in primary prostate cancer gel extracted and cloned into a PCR2.1-TOPO TA cloning vector (Invitrogen) through TA ligation. Then, the fragment of SSBP2 was SSBP2 Taqman P subcloned into a pcDNA3.1 vector in the sense direction using methylation analysis restriction enzymes HindIII and XholI whose sites flanked the fragment. TaqMeth V >120 TaqMeth V <120 The sequence and direction of the insert was confirmed during each step by ABI BigDye cycle sequencing. n =54 n =34 Transfection and colony formation assay. Three prostate cancer cell Age, 65.5 (53-74) 64.5 (51-72) NS lines (DU145, 22Rv1, and PC3) were transfected with SSBP2 expression median (range) plasmids using Fugene6 reagent following the manufacturer’s protocol PSA (ng/mL), 10.5 (3.11-25.3) 9.11 (3.71-48.3) NS (Roche). After 24 h, the medium was changed to complete RPMI 1640 median, (range) (Invitrogen). Forty-eight hours after transfection, cells were selected Gleason score NS using G418 (1.2 mg/mL). Colonies were photographed 2 wk after 6 21 (55%) 17 (45%) selection. 7 29 (63%) 17 (37%) A stable cell line of PC3 overexpressing SSBP2 was generated and 8 4 (100%) 0 (0%) Tumor staging 0.016 maintained in complete RPMI 1640 containing G418 (1.2 mg/mL). The VpT3a 46 (57.5%) 34 (42.5%) overexpression of SSBP2 was then confirmed by Western blot analysis. zpT3b 8 (100%) 0 (0%) Cell cycle analysis. A stable cell line overexpressing SSBP2 was Relapse NS maintained in complete medium containing 1.2 mg/mL G418. For flow Yes 13 (68%) 6 (32%) cytometry analysis, after the cells were synchronized for 48 h in No 31 (66%) 26 (34%) medium containing 1% serum, the cells were then cultured in complete Lost 10 2 medium. After 24 h, the cells were harvested and washed in PBS. Then, they were fixed in 70% ethanol overnight at 4jC. Subsequently, Abbreviations: NS, no significance; PSA, prostate-specific antigen. the cells were stained with propidium iodide (20 mg/mL) solution for 30 min at room temperature. DNA content was measured by flow

www.aacrjournals.org 3755 Clin Cancer Res 2008;14(12) June 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology cytometry (BD FACSCalibur), and data were analyzed using CellQuest Pro Software (BD Technology). Microarray analysis. Ten micrograms of total RNA from separate stable PC3 cell line overexpressing SSBP2 or pcDNA3.1 were used for microarray analysis on the U133 plus gene chip (Affymetrix). The cDNA preparation, RNA labeling, hybridization, and data analysis were done per the Affymetrix protocol. Only genes with a 1.5-fold increase or decrease in expression were considered for further analysis. The genes associated with cell cycle pathways and apoptosis, such as p15, p16, p27, and Fas, were presented. Western blotting. For Western blotting, whole-cell lysates were prepared in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Sigma). Protein concentrations were measured by MicroBCA kit (Pierce). For each sample, 30 Ag of whole- cell lysate were loaded onto a 10% precast SDS-polyacrylamide gel (Bio- Rad). After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-SSBP2 polyclonal antibody (Geneway), mouse anti-c-Myc monoclonal antibody (BD PharMingen), and anti- actin monoclonal antibody. Secondary antibodies used were goat anti- rabbit or anti-mouse IgG conjugated to horseradish peroxidase. Protein bands were displayed by enhanced chemiluminescence. Tissue microarrays and immunohistochemical analysis. Prostate adenocarcinoma tissue microarrays were purchased from U.S. Biomax, Inc. The arrays had 40 prostate mucinous adenocarcinomas (pT1-pT4) along with 8 nonmalignant normals and tumor adjacent normals. Immunohistochemical analysis was done as described previously (13). Briefly, the tissue arrays were deparaffinized, rehydrated, and micro- waved. Then, the tissue array was incubated with rabbit anti-SSBP2 polyclonal antibody (1:100; Geneway) for 16 h at 4jC followed by biotinylated secondary antibody and peroxidase-labeled streptavidin complex detection (Dako). The slides were then analyzed by Fig. 1. Methylation analyses of SSBP2 promoter in prostate cancer cell lines and A, microscope and quantified based on the intensity of the staining with primary prostate tissues. methylation of SSBP2 promoter in cell lines, primary prostate cancer tissues, and BPH by direct sequencing of bisulfite-treated DNA. Mayer’s hematoxylin. Parallel sections with nonimmune rabbit IgG of Gray boxes, methylation of the promoter region; white boxes, no methylation in the the same isotype were examined as negative control. The slides were promoter region. B, direct sequencing of the SSBP2 promoter after bisulfite examined under microscope and the pictures were taken by Nikon treatment in representative primary prostate cancer tissues showed complete ACT-1 software. methylation in the tested region but not in BPH. Arrows, all guanines present after sequencing that are complementary to methylcytosines on the opposite DNA Statistical analysis. Statistical analyses were done using Statistical strand. Stars, all adenines present after sequencing that are complementary to Analysis System software. The significance level used P V 0.05. thymines converted from cytosines on the opposite DNA strand.

Results treated DNA (Materials and Methods). The primers were located in a region 694 to 784 bp upstream of the transcription SSBP2 is identified as a candidate methylation gene in prostate start site in the SSBP2 promoter (Fig. 2A). Both the primers and cancer. Our laboratory has previously described a whole- probe sequences detected CpG methylated sites in cancer but genome approach to identify methylated genes using probabi- not in BPH. listic search algorithms in combination with an established To confirm that SSBP2 methylation is actually cancer cell pharmacologic unmasking strategy (14). We found that the specific, we did quantitative MSP in 88 samples from prostate SSBP2 promoter was methylated in DU145, 22Rv1, and PC3 cancer lesions with tumor grades from pT2a to pT4 as well as 23 cell lines but not in LNCaP by direct sequencing of bisulfite- benign prostatic lesions from BPH patients that were used as treated DNA (Fig. 1A). Therefore, DU145, 22Rv1, and PC3 were normal control. We also analyzed SSBP2 promoter methylation used in subsequent experiments. To examine whether the levels in 30 paired PIN samples that were microdissected from methylation of SSBP2 is tumor specific, we tested tissues from the same set of 88 prostate cancer patients. The Taqman five primary human prostate cancers and six BPH. SSBP2 methylation values in primary prostate cancer were significantly promoter methylation was observed in four of five (80%) higher than in BPH and PIN (Fig. 2B). The frequency of primary prostate cancers compared with zero of six (0%) BPH hypermethylation in primary prostate cancer was 61.4% (54 of (Fig. 1A). The representative sequencing data of bisulfite-treated 88 cases; cutoff value of 120) compared with 0% (0 of 23 cases) DNA from primary prostate cancer and BPH were shown in Fig. in BPH. PIN showed an intermediate hypermethylation 1B. The cytosines of the SSBP2 promoter in DNA from BPH frequency (30%, 9 of 30 cases), consistent with its premalig- were converted to thymines after bisulfite treatment, whereas nant characteristics. The average methylation level of the SSBP2 the cytosines in DNA from prostate cancer were protected due promoter was much higher in prostate cancer than in BPH or to methylation (Fig. 1B). PIN (Fig. 2C). The above data suggest that hypermethylation of SSBP2 is hypermethylated in prostate cancer but not in SSBP2 is cancer cell specific in prostate cancer. BPH. To determine the quantity of SSBP2 methylation, we SSBP2 hypermethylation is associated with advanced prostate designed MSP primers and a Taqman probe conjugated with cancer. Based on our quantification data, of the 88 cases of specific fluorescence based on the sequencing data of bisulfite- prostate cancer, there were 54 cases with a higher SSBP2

Clin Cancer Res 2008;14(12) June 15, 2008 3756 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Hypermethylation of SSBP2 in Human Prostate Cancer methylation level and 34 cases with a lower SSBP2 methylation The reverse transcription-PCR data also showed reduced mRNA level than the cutoff value of 120. Statistical analysis showed levels of SSBP2 in prostate cancer cell lines (Fig. 3B). We treated that SSBP2 hypermethylation status was closely related to the three SSBP2 methylated prostate cancer cell lines (DU145, prostate tumor staging (P = 0.016; Table 1). All patients with 22Rv1, and PC3) with the demethylating agent 5-Aza-dC and tumor staging higher than pT3b (100%, eight of eight) were found that SSBP2 could be robustly reactivated (Fig. 3B). The detected as hypermethylated (Table 1). No significance was above results suggest that SSBP2 is down-regulated through found between SSBP2 methylation and other clinicopathologic promoter hypermethylation. factors, such as age, serum prostate-specific antigen level, or SSBP2 inhibits prostate cancer cell growth through suppression Gleason score. of cell cycle. To examine whether SSBP2 has the ability to SSBP2 expression is down-regulated in primary prostate cancer suppress cell proliferation in prostate cancer cell lines, we did cells and can be reactivated by 5-Aza-dC. To examine the the colony formation assay separately in all three prostate expression pattern of SSBP2 in primary prostate tissues, we did cancer cell lines (DU145, 22Rv1, and PC3) in which immunohistochemistry on a prostate adenocarcinoma tissue endogenous SSBP2 was methylated and its expression was array with an anti-SSBP2 polyclonal antibody. The immuno- silenced. The results showed that the colony formation ability histochemical results of primary prostate tissue showed that was significantly suppressed by ectopic expression of SSBP2 in SSBP2 was predominantly expressed in normal prostate all of the above prostate cancer cell lines (Fig. 4A). We then epithelial cells (Fig. 3A). All eight normal prostate epithelium established a stable cell line overexpressing SSBP2 in PC3 cells. examined showed intense SSBP2 expression, whereas SSBP2 We did cell cycle analysis in PC3 cells stably expressing SSBP2 was expressed at much lower levels in epithelial cells from and found that the cells with overexpressing SSBP2 were prostate cancer. Of 40 prostate cancer samples, only 5 (12.5%) arrested in the G1 phase. The percentage of cells in S and G2-M showed the same expression level as normal prostate, whereas phase was decreased from 18.69% to 11.55% and 23.31% to the remaining 35 (87.5%) showed absent or lower expression 11.62% separately by SSBP2 (Fig. 4B). Overexpression of SSBP2 (Table 2). Interestingly, all six tumors examined with staging in stable PC3 cell lines was confirmed by Western blotting pT3 and pT4 showed absent or decreased expression compared (Fig. 4C). with normal prostate (Table 2). This expression pattern was To explore the molecular events involved in cell cycle consistent with hypermethylation in advanced prostate cancers. regulation by SSBP2, we did microarray analysis with total

Fig. 2. Quantitative MSP (qMSP) analyses of the SSBP2 promoter in primary prostate tissues. A, positions of CpGs in a region upstream of the transcription start site (TSS) of the SSBP2 promoter. Gray areas, CpG islands.The region analyzed by MSP for bisulfite-treated DNA is 694 to 784 bp upstream of the transcription startsiteintheSSBP2 promoter. B, scatter plot of quantitative MSP analyses of the SSBP2 promoter in primary prostate tissues, including 88 prostate cancer, 23 BPH, and 30 PIN patients. Methylation was detected in 61.4% (54 of 88) of prostate cancer (PCa) compared with 0% (0 of 23) of BPH and 30% (9 of 30) of PIN. Horizontal bar, a cutoff value of 120was assigned based on the highest methylation level in BPH (Results). C, quantitative average methylation levels of SSBP2 in prostate cancer tissues are much higher than in BPH and PIN.

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Fig. 3. A, immunohistochemical analysis of SSBP2 in primary human prostate tissues. Representative immunohistochemical picture showed that SSBP2 was expressed intensely in normal prostate epithelium (normal), although its expression level was absent or reduced in epithelium of prostate cancer. Magnification, Â200. The intensity of SSBP2 was scored as follows: (-), no detectable immunostaining or basal immunostaining in <10% cells; (+), mild immunostaining in 10% to 30% of cells; (++), moderate immunostaining in 30% to 50% of cells; (+++), intense immunostaining in 50% to 80% of cells; and (++++), intense immunostaining in >80% of the cells showing cytoplasmic staining. B, reactivation of SSBP2 by 5-Aza-dCin prostate cancer cell lines. SSBP2 expression was lost or reduced in DU145, 22Rv1, and PC3cells.The expression was reactivated by 5 Amol/L 5-Aza-dC( 5A) in all of three cell lines. M, mock.

RNA from SSBP2-transfected PC3 stable cell line using PC3 that SSBP2 was physiologically inactivated through epigenetic cells transfected with pcDNA3.1 alone as a control. From the silencing in prostate cancer cell lines. We further examined microarray data, we found that genes that inhibit cell cycle SSBP2 expression pattern in an independent set of primary progression (such as p15, p16, p27, GDF15, and CCNG2) were human prostate tissue from cancer patients and nonneoplastic up-regulated at the transcriptional level in SSBP2-overexpressed tissues using immunohistochemistry. Our results showed that PC3 cells. On the other hand, genes that promote cell cycle SSBP2 was significantly down-regulated in most of the primary (such as CDC25A, CDC20, MCM4, and MCM5) were down- prostate cancers (35 of 40) compared with normal prostate regulated (Table 3). Previous work in leukemia cells suggested tissues. This down-regulation was again more obvious in decreased c-Myc protein as a target of cell cycle arrest induced cancers with higher tumor stages. by SSBP2 (12). We also found that the c-Myc protein level was SSBP2 belongs to a gene family that shares high identity in its reduced in overexpressed PC3 cells by Western blot (Fig. 4C), open reading frames (9). SSBP2 localizes to whereas microarray data showed minimal changes at the 5q13.1, which was found to be translocated and deleted in mRNA level. The above data suggest that SSBP2 acts as a myelodysplasia and acute myelogenous leukemia (10). A recent tumor suppressor gene in prostate cancer cells and that one study showed that SSBP2 might be a candidate tumor mechanism of suppression is due to its inhibition of normal suppressor gene in hematopoietic malignancies. It is a regulator cell cycle progression. of cell growth and differentiation in myeloid leukemia cells (12). Our functional analysis showed that SSBP2 contained the Discussion ability to suppress cell proliferation in prostate cancer cells, showing its tumor-suppressive activity in solid tumor as well as Here, we show that SSBP2 may be a potential DNA marker in in hematopoietic malignancy. prostate cancer. We confirmed SSBP2 promoter methylation in Further experiments revealed that SSBP2 induced cell cycle 88 primary prostate cancers by quantitative MSP analysis. Of 88 arrest in PC3 cells. The cell cycle arrest induced by SSBP2 cases, 54 (61.4%) were hypermethylated in the SSBP2 seemed to be through inhibition of progression from the G1 to promoter region, whereas it was not found to be hyper- methylated in BPH (0 of 23), confounding condition that can mimic prostate cancer (Fig. 2B). Additionally, we found that the methylation levels of PIN were between those of tumor and Table 2. Expression of SSBP2 in normal prostate BPH, consistent with its pathologic characteristics as a and prostate cancer preinvasive form of prostate cancer. This finding is different from other reported methylation genes, such as GSTP1, APC, No. specimens SSBP2 SSBP2 SSBP2 V RASSF1,andMGMT, which were found to have similar ( +) (++, +++) (++++) methylation levels in PIN and prostate cancer (4, 7). Normal prostate 0 0 8* Interestingly, statistical analysis revealed that SSBP2 hyper- Prostate cancer methylation was correlated with tumor stage in prostate cancer. pT1 1 0 1 pT2 8 20 4 Our quantitative and clinical data suggest that SSBP2 promoter pT3 1 3 0 methylation is a specific and frequent event in prostate cancer. pT4 0 2 0 SSBP2 was found to be hypermethylated in three prostate cancer cell lines (DU145, 22Rv1, and PC3) and reactivated after *P < 0.0001. treatment with the demethylating agent 5-Aza-dC, indicating

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SandG2-M phase in prostate cancer cells. In contrast, antiapoptosis, cell adhesion promotion, and Wnt signaling overexpression of SSBP2 in leukemia cells (U937) induced a were down-regulated (Table 3). The reduction of c-Myc protein predominantly S-phase change. The difference between cell level in SSBP2-overexpressed PC3 cells as well as in hemato- cycle arrest effects by SSBP2 on prostate and hematopoietic cells poietic cells indicates that SSBP2 may share a common indicates that the effects of SSBP2 might be cell type dependent. molecular target in both cell types. The cross-talk between Our microarray data revealed that several genes involved in cell SSBP2 and the above gene products needs to be further cycle progression were dysregulated, confirming the cell cycle explored. arrest caused by SSBP2 overexpression in PC3 cells. We also We thus report that the SSBP2 promoter is hypermethylated observed changes in expression of genes involved in apoptosis, in primary human prostate cancers compared with nonneo- Wnt signaling, and cell adhesion. Generally, genes associated plastic prostate lesions (BPH). To our knowledge, this is the with proapoptosis and cell adhesion inhibition were up- first report that SSBP2 expression is down-regulated in primary regulated in SSBP2-transfected cells, whereas genes related to prostate cancer and cancer cell lines and is tightly correlated

Fig. 4. A, colony formation assay of SSBP2 in prostate cancer cell lines. Prostate cancer cell lines were transiently transfected with SSBP2. Colonies were formed after 2-week selection with G418 (1.2 mg/mL) in 22Rv1, DU145, and PC3 cells. Significant decrease of colony formation was seen in various prostate cancer cell lines with forced SSBP2 expression. B, cell cycle inhibition by SSBP2 in prostate cancer cells stably expressing SSBP2. Stable PC3 cell line transfected with SSBP2 was generated (Materials and Methods). A decrease in S and G2-M phases was observed in cells overexpressing SSBP2. C, Western blotting showed the overexpression of SSBP2 and reduction of c-Myc expression in PC3 cells transfected with pcDNA3.1 empty vector (Vector ) or SSBP2-pcDNA3.1 (SSBP2). h-Actin was used as loading control. *, P < 0.01.

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Table 3. Genes differentially expressed in SSBP2-overexpressed PC3 cells

Gene function Up-regulated genes Ratio change Down-regulated genes Ratio change Cell cycle GDF15 4 RBL1 -2.17 CDKN2B/p15 3.34 MCM4 -2 CDKN2A/p16 1.86 CDC25A -1.92 CCNG2 1.78 MCM5 -1.64 CDKN1B/p27 1.55 CDC20 -1.54 DNA replication GMNN -1.69 Apoptosis Fas 2.1 BCL2A1 -2.5 TP73 1.61 PAK4 -1.59 PRF1 1.54 BNIP3L 1.52 Cell adhesion ITGB3 4.72 DOCK1 -2.33 PELO 2.1 PAK4 -1.59 ITGB5 2 ITGB1 1.7 Wnt signaling WNT3 -10 WNT6 -10 FOSL1 -2.33 WNT10A -2.17 Other MMP23A 14.5 IGF1 -14.3 LHX2 9.1 FOSL1 -2.33 TIMP4 1.6 RARA -2 EIF5 -1.72 with its promoter hypermethylation. Moreover, we found that Disclosure of Potential Conflicts of Interest SSBP2 plays a role in tumor suppression through inhibition of cell cycle progression, revealing clues to its role in tumorigen- Under a licensing agreement between OncoMethylome Sciences, S.A. and the esis. Our findings identify SSBP2 as a novel biomarker in Johns Hopkins University, Dr. Sidransky is entitled to a share of royalty received by prostate cancer and further support its role through inactivation the University on sales of products described in this article. Dr. Sidransky owns in human prostate tumorigenesis. SSBP2 hypermethylation and OncoMethylome Sciences, S.A. stock, which is subject to certain restrictions under University policy. Dr. Sidransky is a paid consultant to OncoMethylome Sciences, its expression need to be tested in a larger numbers of patients S.A. and is a paid member of the company’s Scientific Advisory Board. The term of of prostate cancer to see if it predicts poor outcome in a future this arrangement is being managed by the Johns Hopkins University in accordance study. with its conflict of interest policies.

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Jun-Wei Liu, Jatin K. Nagpal, Wenyue Sun, et al.

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