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The Prostate Promoter Hypermethylation of GSTP1, AR, and14-3 -3s in Serum of Prostate Cancer Patients and its Clinical Relevance Jochen Reibenwein,1 Dietmar Pils,1 Peter Horak,1 Birgit Tomicek,2 Gregor Goldner,2 Nina Worel,3 Katarzyna Elandt,1 and Michael Krainer1*{ 1 Division of Oncology, Department of Internal Medicine I, Medical University of Vienna,Vienna, Austria 2 Department of Radiotherapyand Radiobiology, Medical University of Vienna,Vienna, Austria 3 Department of Bloodgroup Serologyand Transfusion Medicine, Medical University of Vienna,Vienna, Austria OBJECTIVE. Hypermethylation of tumor suppressor genes (TSG) is thought to play an important role in tumorigenesis of prostate cancer. The main focus of research was the detection of TSG hypermethylation in cancer tissue. Our aim was to evaluate the feasibility of detection of hypermethylated genes in serum of prostate cancer patients and its correlation with clinicopathological parameters. METHODS. One hundred twenty-five serum samples from 62 patients with hormone refractory prostate cancer (HRPC), 14 patients with early disease, and 49 healthy controls were examined. After DNA extraction and sodium-bisulfite treatment, conventional methylationspecific PCR (MSP) was performed for glutathione S-transferase P1 (GSTP1), androgen receptor (AR), and 14-3-3s. RESULTS. In serum of HRCP patients, frequency of GSTP1, AR, and 14-3-3s hypermethylation was 32.2, 40.3, and 86.6%, respectively. In serum of patients with early disease frequency of GSTP1, AR, and 14-3-3s, hypermethylation was 21.4, 35.7, and 85.7%. In healthy controls, frequency of GSTP1, AR, and 14-3-3s hypermethylation was 0, 26.5, and 55.1%, respectively. There was a significant increase of frequency of TSG hypermethylation for GSTP1 and 14-3-3s in HRPC patients, in comparison with healthy controls. GSTP1 hypermethylation in HRPC patients was significantly correlated with differentiation of cancer and metastatic disease. CONCLUSIONS. Hypermethylation of TSG can be detected in serum of prostate cancer patients. Some hypermethylated TSG can be detected in serum of healthy controls. GSTP1 was not detectable in controls and correlated significantly with Gleason score and stage of disease. Therefore, this gene may be a promising new tool in prostate cancer diagnosis. Prostate # 2006 Wiley-Liss, Inc. KEY WORDS: prostate cancer; hypermethylation; serum; CpG island INTRODUCTION Prostate cancer has become the most commonly diagnosed cancer among men and the third leading cancer-related cause of death after lung/bronchus and colon/rectum in western countries [1]. Detection of early-stage prostate cancer offers the opportunity to a therapy with curative intent, whereas for advanced stages only palliative treatment remains as available option. A mainstay in early prostate cancer detection is the measurement of PSA in serum. Since the beginning of the PSA era, a remarkable change in prostate cancer 2006 Wiley-Liss, Inc. { Professor of Medicine. *Correspondence to: Michael Krainer, MD, Department of Internal Medicine I, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: [email protected] Received 20 September 2006; Accepted 4 October 2006 DOI 10.1002/pros.20533 Published online in Wiley InterScience (www.interscience.wiley.com). 2 Reibenwein et al. detection has occurred, leading to a significant increase of newly diagnosed, nonpalpable cancers [2]. Nevertheless, an elevated PSA level is not specific to prostate cancer and can be caused by manifold nonmalignant pathologies like prostatic inflammation and benign prostatic hypertrophy [3]. Hence, an additional set of noninvasive screening markers is needed for the detection of early prostate cancer. Epigenetic changes like hypermethylation of CpG islands promoters of tumor suppressor genes (TSG) have been described as characteristics of malignant cells [4]. Hypermethylated tumor-specific DNA can be detected in body fluids like plasma, serum, urine, and ejaculate of prostate cancer patients [5–8]. The origin and the mechanism of release of this cell-free DNA is still not well known. Cell-free DNA may be released due to lysis of circulating cancer cells, tumor necrosis, tumor apoptosis, or active release, respectively [9]. One of the most frequent hypermethylated TSG found in serum of prostate cancer patients is GSTP1, which belongs to a family of detoxifier enzymes involved in DNA damage repair [10]. Hypermethylation of GSTP1 was detected by the use of conventional MSP in 36–72% of plasma/serum samples of prostate cancer patients, but it was not detectable in noncancerous controls [6,11]. Despite controversial data whether GSTP1 serves as a real TSG or just as a ‘‘caretaker’’ gene, leading to increased sensitiveness to oxidative DNA damage when loss of function [12], it is a promising marker for prostate cancer screening. Progress of prostate cancer is usually associated with loss of androgen sensitivity by the tumor. This represents a critical step in prostate cancer development. Hypermethylation of the androgen receptor (AR) and consequently, its downregulation is thought to be a possible mechanism for the loss of androgen sensitiveness [13]. Hypermethylation of the AR has already been described in prostate cancer tissue and cell lines [14,15], but there are no reports of detection of AR hypermethylation in body fluids of prostate cancer patients. 14-3-3s plays a critical role in signal transduction pathways and cell-cycle regulation [16]. In response to DNA damage, 14-3-3s enforces a G2/M arrest by inhibiting the cyclinB1-cdc2 complex from entering the nucleus. This allows DNA repair before cell-cycle progression [17]. Due to its function, 14-3-3s is considered as a TSG in different cancers, including prostate cancer [18]. Loss of or weak 14-3-3s expression was reported as a very frequent event in prostate cancer, seen in up to 97% of cases [19]. Downregulation of 14-3-3s expression seems to be significantly associated with 14-3-3s CpG methylation [20]. However, 14-3-3s hypermethylation can also be seen at a The Prostate DOI 10.1002/pros very high frequency in benign prostate pathologies like benign prostate hypertrophy (BPH) [21]. No reports of detection of 14-3-3s hypermethylation in body fluids of cancer patients have been published so far. The aim of our study was to detect hypermethylated TSG in serum of prostate cancer patients and to correlate the methylation status, with clinicopathological parameters. METHODS Patients and Sample Collection Serum samples from 62 patients with hormone refractory prostate cancer (HRPC) were examined in this study (HRPC group). All patients of the HRCP group were recruited consecutively from April 2003 to November 2005 for a prospective clinical trial for HRPC at the Department of Internal Medicine I, Division of Oncology, Medical University of Vienna, and received a chemotherapy, consisting of intravenous administration of docetaxel. Fourteen patients with histologically confirmed primary prostate cancer were additionally enrolled in this study (RT group). All RT patients were recruited consecutively from March 2006 to June 2006 at the Department of Radiooncology and Radiobiology, Medical University of Vienna, for radiation therapy of early-stage prostate cancer, and received a primary external beam radiotherapy with a maximum dose of 70–74 Gy (Table I). Forty-nine healthy male individuals served as controls. Serum samples were collected consecutively in May 2006 at the Department of Transfusion Medicine, Medical University of Vienna, during voluntary blood donation after informed consent was obtained. All patients gave informed consent, according to the criteria of the ethics committee of the Medical University of Vienna. One milliliter of serum was collected from each patient, prior to individual therapy. DNA was extracted from serum using the QIAamp1 DNA Mini Kit, according to the manufacturer’s instructions (Quiagen, Germany, Hilden). After extraction, the DNA level was measured using the PicoGreen1 dsDNA Quantification Kit (Molecular Probes, The Netherlands, Leiden). Bisulf|teTreatment In brief, after denaturation with 3 M NaOH, DNA was treated over 4 hr with 100 ml of a solution of 2.5 M sodium disulfite and 6.25 mM hydrochinone. For purification of bisulfite-treated DNA, the QIAquick1 PCR Purification Kit Protocol (Quiagen) was used according to the manufacturer’s instructions. Promoter Hypermethylation of GSTP1, AR, and14 -3 -3s TABLE I. Patient Characteristics Age (years) Median Range Gleason score 2–4 5–7 8–10 Missing Grade G1 G2 G3 Missing Gleason score/grade Valid Missing Pathologic stage T1 T2 T3 T4 Missing Bone metastasis Yes No Missing LN metastasis Yes No Missing LN and bone metastasis Yes No Missing PSA (ng/ml) Median Range DNA (ng/ml) Median Range HRPC group (n ¼ 62) RT group (n ¼ 14) 67 50–83 70 (ns) 58–76 1 12 18 31 0 12 1 1 1 8 6 47 0 1 0 13 45 17 14 0 4 1 13 11 11 9 5 0 0 0 44 11 7 0 14 0 19 24 19 0 14 0 13 42 7 0 14 0 57.9 0.01–3,779 6.0 (P < 0.001) 0.7–13.5 14.4 1.9–136.3 19.3 (ns) 6.4-32.7 ns, not significant. and negative controls were added to all MSP reactions. MSP products were loaded on 3% agarose gels, prestained with ethidium bromide, separated electrophoretically, and visualized under ultraviolet illumination. Statistical Analysis For comparison of age distribution, PSA level and DNA level between the different groups Mann– Whitney U tests were performed. Correlations between the TSG methylation status and clinicopathological parameters were analyzed using the chi-square test and Fisher exact test as appropriate. All statistical analyses were performed with SPSS 13.0 (SPSS, Inc., Chicago, IL, 2004). P-values below 0.05 were considered as statistical significant. P-values were corrected for multiple testing. RESULTS Our study population consisted of 62 patients with hormone refractory prostate cancer (HRPC group), 14 patients with early-stage prostate cancer (RT group), and 49 healthy male controls. Table I shows the clinicopathological and demographic features of the patients. Median PSA level was 57.9 ng/ml in the HRPC group and 6.0 ng/ml in the RT group (P < 0.001). In the RT group, all patients had early-stage prostate cancer, whereas metastatic disease was diagnosed in 81% of patients in the HRPC group. The remaining patients of the HRPC group had PSA rise only. No significant difference was seen between the two groups for age distribution and amount serum DNA level. Forty-nine healthy male served as controls. The methylation status of GSTP1, AR, and 14-3-3s in the serum of all three groups was assessed by conventional MSP (Table II). Representative examples of the results TABLE II. Frequencies of TSGPromoter Hypermethylation in Serum of HRCP Patients, RTX Patients, and Controls GSTP1 n/N Methylation-Specif|c PCR Methylation-specific PCR (MSP) was performed using primers and conditions selected to especially amplify bisulfite-treated promoter DNA for the gene of interest. All oligo sequences and MSP conditions are available upon request. The positive control was normal human DNA treated with Sss I methyl transferase before bisulfite modification. The negative control was distilled water without template. Positive The Prostate DOI 10.1002/pros 3 AR % a HRPC group 20/62 RT group 3/14b Controls 0/49 32.3 21.4 0 n/N 25/62 5/14 13/49 14-3-3s % 40.3 35.7 26.5 n/N % c 33/38 86.8 12/14 85.7 27/49 55.1 All P-values corrected for multiple testing. a Significant differences between HRPC group and controls (P < 0.001). b Significant differences between RT group and controls (P ¼ 0.03). c Significant differences between HRPC group and controls (P ¼ 0.006). 4 Reibenwein et al. of the electrophoresis of MSP products are shown in Figure 1. For GSTP1, the frequency of hypermethylation was 32.2, 21.4, and 0 in the HRCP group, RT group, and controls, respectively. A highly significant difference in the frequency of GSTP1 methylation was seen between the HRPC group and controls (P < 0.001) and between the RT group and controls (P ¼ 0.006), respectively. For AR, the incidence of methylation in the three groups was 40.3, 35.7, and 26.5%, respectively. No significant increase of AR methylation frequency between the groups was discovered. For 14-3-3s the frequency of methylation was 86.8, 85.7, and 55.1%, respectively. A significant difference in the methylation frequency of 14-3-3s was seen between the HRPC group and controls (P ¼ 0.03). GSTP1 hypermethylation was significantly associated with differentiation of cancer in the HRPC group. Eleven of 24 patients (46%) with Gleason score 8 or WHO Grade ¼ 3 showed GSTP1 hypermethylation, compared to 2/22 patients (9%) with Gleason score <8 or WHO Grade 2 (P ¼ 0.03). Moreover, a significant correlation of GSTP1 hypermethylation and presence of metastases were found in the HRPC group. Ten of 19 patients (53%) with lymph node metastases showed GSTP1 hypermethylation, in comparison to 7/38 patients (18%) without metastases of the lymph nodes (P ¼ 0.02). Furthermore, 8/13 patients (62%) with lymph node metastases as well as bone metastases presented GSTP1 hypermethylation, compared to 4/20 patients (20%) with bone metastases only (P ¼ 0.03). No correlation was found between the methylation status of the samples and age, pathologic stage, presence of bone metastases, response to treatment, overall survival, PSA level, and DNA level. Fig. 1. Representative examples of MSP analyses of the three methylatedgenes14 -3-3s, AR, and GSTP1in serum of nineprostate cancer patients. Amplification of the unmethylated form of14 -3-3s wasusedascontrolforDNAintegrity.Thepositive controlisnormal human DNA treated with Sss I methyl transferase before bisulfite modification. The negative control is distilled water without template. The Prostate DOI 10.1002/pros DISCUSSION We examined the methylation status of the three genes GSTP1, AR, and 14-3-3s in serum of prostate cancer patients. GSTP1 is an intensively investigated putative TSG in prostate cancer. Hypermethylation of GSTP1 is the most common somatic epigenetic alteration reported in prostate cancer. Frequency of GSTP1 hypermethylation in prostate cancer tissue varies from 36 to >90% [22–24]. In nonmalignant tissues, GSTP1 hypermethylation seems to be a very rare event. It was found to be hypermethylated in BPH in less than 5% [3,14,23]. In prostatic intraepithelial neoplasia, a potential precursor of prostate cancer, frequency varies between 30 and 70% [14,25]. Only a few reports about GSTP1 hypermethylation in serum of cancer patients have been published so far. The detection rate of GSTP1 hypermethylation in serum or plasma samples of patients, with clinically localized prostate cancer ranged from 12 to 36% [6,26]. In serum or plasma of a study cohort, including patients with clinically localized disease as well as advanced prostate cancer, GSTP1 hypermethylation was described in 72% [11]. No GSTP1 hypermethylation was found in serum of healthy controls or of patients with BPH [11,26]. In our study, GSTP1 hypermethylation was found in the HRPC group in 32% and in the RT group in 21% of serum samples. According to the literature, no GSTP1 hypermethylation was detectable in serum of controls. There are inconsistent reports about correlation of GSTP1 hypermethylation in prostate cancer with clinicopathological parameters. Maruyama et al. reports a significant correlation of GSTP1 hypermethylation in prostate cancer tissue with Gleason score >7 and with PSA levels >8 ng/ml [22]. Other investigators did not find such a correlation [23,24,27]. In a multivariate analysis of 55 men with clinically localized prostate cancer, GSTP1 hypermethylation in serum was the most significant predictor for PSA recurrence [26]. We found a significant correlation of GSTP1 hypermethylation with Gleason score 8 or WHO Grade ¼ 3 (P ¼ 0.03). Furthermore, a significant correlation with metastatic disease was seen (P ¼ 0.03). Moreover, the presence of lymph node metastases correlated significantly with GSTP1 hypermethylation (P ¼ 0.02). Bone metastases only did not correlate with GSTP1 hypermethylation. As far as we know, this is the first report about correlation of clinicopathological parameters, with methylation status in body fluids of prostate cancer patients. GSTP1 hypermethylation is not detectable in healthy controls. Hence, it seems to be a promising screening marker for prostate cancer in tissue and serum samples. Further efforts are needed to be done to establish GSTP1 as an Promoter Hypermethylation of GSTP1, AR, and14 -3 -3s additional diagnostic tool both in prostate tissue and body fluids. The progression from androgen sensitive to HRPC is a critical step in the natural course of prostate cancer and displays a fatal watershed in the individual medical history of most patients. Hypermethylation of the AR promoter downregulates AR expression and is thought to be one mechanism for development of HRPC, with a methylation frequency of 20% in primary prostate cancer tissues and of 28% in HRPC tissues [28]. Interestingly, Yamanaka et al. reported a decreasing AR methylation frequency of 50% for stage A, 24% for stage B, 10% for stage C, and 6% for stage D prostate cancer, respectively [14]. No reports about AR methylation in serum of prostate cancer patients have been published so far. In our present study, AR hypermethylation in the HRPC group, in the RT group, and in controls was seen in 40, 36, and 27%, respectively. Thus, because of the presence of AR methylation in healthy male controls, there must be other reasons for AR methylation beside carcinogenesis. It has been demonstrated that many genes are methylated as a function of age [29], but in our study, no association of methylation status and age was found (data not shown). Loss of 14-3-3s expression is a very frequent event, related to progression from normal epithelium to PIN and invasive prostate cancer. In normal prostate epithelium, expression of 14-3-3s was moderate or strong in all samples, whereas 90% of PIN and 97% of invasive prostate cancer showed negative or weak expression, respectively [19]. Downregulation of 14-33s expression was seen to be significantly correlated with CpG island methylation in prostate cancer [20]. However, 14-3-3s hypermethylation is not a carcinogenesis-specific event. Hypermethylation was detectable in 99% of prostate cancer, 100% of PIN, and 100% of BPH tissue samples, respectively [21]. Hypermethylation of 14-3-3s can also be seen in normal lymphoid cells [30]. In our present study, 14-3-3s hypermethylation was found in serum samples of the HRPC group, the RT group, and controls in 87, 86, and 55%, respectively. The difference between HRPC group and controls was statistically significant (P ¼ 0.006). 14-3-3s was not methylated as a function of age, so there must be other reasons for hypermethylation of 14-3-3s in healthy controls. Recapitulating, MSP is a feasible method for detection of hypermethylated TSG in serum of prostate cancer patients. GSTP1 seems to be a promising candidate for further development to an additional diagnostic tool for prostate cancer, based on prostate tissues and serum samples. 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