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Aberrant Promoter Methylation Profile of Prostate Cancers and Its Relationship to Clinicopathological Features¹

Hamon Center for Therapeutic Oncology Research [R. M., S. T., K. O. T., A. K. V., S. Z-M., A. J. F., J. D. M., A. F. G.], the Departments of Pathology [A. K. V., A. F. G.], Internal Medicine [J. D. M., E. P. F.], Pharmacology [J. D. M.], and Urology [J. M.], and the Simmons Cancer Center [E. P. F.], University of Texas Southwestern Medical Center, Dallas Texas 75390

	ABSTRACT

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Purpose: We investigated the aberrant methylation profile of prostate cancers and correlated the data with clinical findings.

Experimental Design: Gene promoter methylation was analyzed in 101 prostate cancer samples. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 25 with benign disease, benign prostatic hypertrophy, or prostatitis, and 7 normal tissues adjacent to cancer. The methylation status of 10 genes was determined. The methylation index (MI) was calculated as a reflection of the methylated fraction of the genes examined.

Results: Methylation percentages of the genes tested in prostate cancers were: RARß, 53%; RASSF1A, 53%; GSTP1, 36%; CDH13, 31%; APC, 27%; CDH1, 27%; FHIT, 15%; p16^INK4A, 3%; DAPK, 1%; and MGMT, 0%. Methylation percentages in nonmalignant tissues were much lower. For clinicopathological correlations, we divided the cancer cases into low (6 or less) or high (7 or more) Gleason score (GS) groups, and into low (8 ng/ml or less) or high (greater than 8 ng/ml) preoperative serum prostate-specific antigen (PSA) groups. Methylation of RASSF1A, GSTP1, RARß, and CDH13 genes was significantly more frequent in the high GS group than in the low GS group. Methylation of RASSF1A, CDH1, and GSTP1 genes was significantly more frequent in the high PSA group than in the low PSA group. The median MIs were significantly higher in the high GS and the high PSA groups. According to the Spearman rank-correlation test, there was significant correlation between MI and GS (coefficient = 0.43, P < 0.0001) and the preoperative serum PSA (coefficient = 0.37, P = 0.0003).

Conclusions: Our results indicate that the methylation profile of prostate cancers correlates with clinicopathological features of poor prognosis.

	INTRODUCTION

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Prostate cancer is the most common malignancy and the second leading cause of cancer mortality among men in the United States (1) . Once the tumor has metastasized, the long-term prognosis is poor because no curative therapy is available (2) . Cancer development and metastasis are multistep processes that, among others, involve the inactivation of tumor suppressor genes. When the normal expression levels of these growth-inhibitory proteins are suppressed, uncontrolled cell cycling and growth can result. Identifying such specific molecular changes may contribute to prediction of the clinical state and aggressiveness of newly diagnosed prostate cancers.

DNA is methylated only at cytosines located 5' to guanosine in the CpG dinucleotide, and DNA methylation of CpG sites in the promoter regions of genes is a frequent acquired epigenetic event in the pathogenesis of many human cancers (3 , 4) . This modification has important regulatory effects causing loss of gene expression, when there is involvement of CpG-rich areas known as CpG islands. Promoter region DNA methylation is acquired during tumorigenesis but is not normally present in nontumor tissue. DNA methylation may provide an alternate pathway to gene deletion or mutation for the loss of tumor suppressor gene function. Methylated DNA markers also represent a promising avenue for monitoring the onset and progression of cancer. Aberrant promoter methylation has been described for several genes in various malignancies, and the spectrum of genes involved suggests that specific tumors may have their own distinct pattern of methylation (4 , 5) .

For these reasons, we examined 10 genes the expression of which is frequently silenced by aberrant methylation in other cancers to develop a methylation profile for prostate cancer. We used the MSP³ method (6) , analyzed the frequency and extent of methylation (by determining the MI) for the 10 selected genes in prostate cancer, and correlated our findings with clinicopathological features known to be important for prognosis of prostate cancer patients.

	MATERIALS AND METHODS

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Clinical Samples.
All prostate specimens were collected from 101 patients with prostate cancer, and adjacent nonmalignant tissue was available from 7 of these patients. In addition, we obtained nonmalignant tissues from 25 patients with benign prostatic hypertrophy or prostatitis. Patient operations were performed at Parkland Memorial or Zale-Lipshy University Hospitals, Dallas, TX, between 1994 and 2000, after Institutional Review Board-approved, signed consent was obtained. The patients underwent radical prostatectomy or transurethral resection. The tissue was maintained frozen in our urology tissue bank. The histological grading was according to the GS (7) , and the stage of the disease was by the clinical TNM classification of the American Joint Committee on Cancer (8) . The clinical characteristics of cancer patients are detailed in Table 1 .

For MSP assays, DNA was treated with sodium bisulfite as described previously (6) . Briefly, 1 µg of genomic DNA was denatured by incubation with 0.2 M NaOH for 10 min at 37°C. Aliquots of 10 mM hydroquinone (30 µl; Sigma Chemical Co., St. Louis, MO) and 3 M sodium bisulfite (pH 5.0, 520 µl; Sigma Chemical Co.) were added, and the solution (final volume 615.5 µl) was incubated at 50°C for 16 h. Treated DNA was purified by use of Wizard DNA Purification System (Promega Corp., Madison, WI), desulfonated with 0.3 M NaOH, precipitated with ethanol, and resuspended in water. Modified DNA was stored at -70°C until used. Gene location and primer sequences of all genes for both the methylated and the unmethylated forms, annealing temperatures, cycle number, and reference for methodology are summarized in Table 2 . Two sets of primers were used to amplify each region of interest: one pair recognized a sequence in which CpG sites are unmethylated (bisulfite modified to UpG), and the other recognized a sequence in which CpG sites are methylated (unmodified by bisulfite treatment). Negative control samples without DNA were included for each set of PCR. PCR products were analyzed on 2% agarose gels containing ethidium bromide.

Data Analysis.
A comparison of the proportion was done using the ² test or Fisher’s exact test. To compare the overall extent of methylation for the panel of genes examined, we calculated the MI for each case, as follows, and then determined the mean for the different groups.

Statistical analysis of MI between two variables was performed using the Mann-Whitney nonparametric U test. To examine the correlation between two variables, we used the Spearman rank-correlation test. A P less than 0.05 was defined as being statistically significant. All data were analyzed with the use of Survival Tools for StatView (Abacus Concepts Inc., Berkeley, CA).

	RESULTS

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Frequency of Methylation in Prostatic Tissues.
The frequencies of methylation of the 10 genes in 101 prostate cancers and 32 nonmalignant prostate tissues (7 normal prostate tissues and 25 prostate gland tissues with benign disease) are detailed in Table 3 . Methylation percentages in prostate cancers were: RARß, 53%; RASSF1A, 53%; GSTP1, 36%; CDH13, 31%; APC, 27%; CDH1, 27%; FHIT, 15%; p16^INK4A, 3%; DAPK, 1%; and MGMT, 0%. The percentages of methylation in nonmalignant tissues were similar for CDH1 and lower for the other genes. These differences were significant for RARß, RASSF1A, GSTP1, APC, and FHIT. There was no methylation of FHIT, p16^INK4A, or DAPK in nonmalignant tissues. Most nonmalignant tissue (18 of 32, 56%) lacked methylation of any gene, and none had methylation of four or more genes. By contrast, most cancers (81 of 101, 80%) had methylation of one or more genes, and 32 (32%) had methylation of four to seven genes. Fig. 1 illustrates representative examples of the methylation patterns in tumors of the four most frequently methylated genes. The unmethylated form of p16^INK4A, run as a control for DNA integrity, was present in all samples.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Representative examples of MSP analyses of the methylated form (M) of four genes frequently methylated in prostate cancers, RARß, RASSF1A, GSTP1, and CDH13. Amplification of the unmethylated form of p16INK4A (p16U) was used as a control for DNA integrity. T, tumor samples 1 through 10; N, negative control.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Correlation of methylation findings with the low and high groups for GS (A), preoperative serum PSA (B), and tumor stage (C). On the right side of each panel are illustrated the corresponding MIs.

Survival data were available for 44 patients with a median follow-up period of 27 months. Although methylation status did not correlate with survival, no deaths were noted until 48 months after surgery. Thus, a lengthy follow-up will be required to determine whether methylation is a predictive factor for survival.

	DISCUSSION

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Previous studies have described the importance of DNA methylation in human cancers and have focused on regions of the genome that may have functional significance resulting from the extinction of gene activity. Whereas most individual cancers have several, perhaps hundreds, of methylated genes (4) , the methylation profiles of individual tumor types are characteristic (4 , 5 , 11) .

Of the 10 genes studied, we found 6 genes (RARß, RASSF1A, GSTP1, CDH13, APC, and CDH1) that were selectively methylated in prostate cancer tissues at frequencies greater than 25%. One gene (FHIT) was occasionally methylated, whereas three genes (p16^INK4A, DAPK, and MGMT) were seldom if ever methylated. Of particular interest, the MI and the frequencies of methylation of three or four genes were significantly higher in tumors of patients with increased risk of death, namely high GS, high preoperative PSA, and high stage. Other studies have demonstrated that the preoperative serum PSA reflects both tumor grade (12) and tumor volume (13) .

Several reports indicate that GSTP1 methylation is present in prostate cancers at very high frequencies (9 , 14) . Our finding of a methylation rate of 36% in prostate tumors is lower than most other reports and may reflect the use of a higher annealing temperature in our MSP assay, which provides for specificity over sensitivity. Using our conditions, several control tissues from healthy individuals (data not shown) and all but one sample of nonmalignant prostate tissue were negative for methylation. In addition, there was a complete concordance between methylation and expression of the gene in eight tumor cell lines. Other reports have failed to find an association between GSTP1 methylation and markers of increased risk. In the high risk groups, the methylation frequencies of GSTP1 were between 44% and 53%. Our findings that frequent methylation of GSTP1 was correlated with markers of increased risk suggest that our stringent conditions may yield more clinically useful information.

Our finding of a low incidence of p16^INK4A methylation in prostate cancers is consistent with published data (15) . There is little published information about the other genes we studied. Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by RARß gene. Because the isoforms RARß2 and RARß4 are frequently methylated in lung and breast cancers (16) , we investigated methylation of their promoter, P2, and found frequent methylation in the prostate cancers (53%). Our gene panel included RASSF1, a recently identified tumor suppressor gene. There are two major RASSF1 gene products, RASSF1A and RASSF1C. Selective promoter methylation of the RASSF1A promoter, but not of RASSF1C, is frequent in many cancers including lung, breast, nasopharyngeal, ovarian, and renal carcinomas and mesotheliomas (17, 18, 19, 20, 21) . The RASSF1A promoter was methylated frequently in prostate cancers (53%), and methylation was associated with high GS and preoperative serum PSA. These findings indicate a potential role of RASSF1A in the pathogenesis and spread of prostate cancers.

Inactivation of CDH13 resulting from aberrant methylation and other mechanisms is frequent in some types of cancer (22 , 23) . We found methylation of CDH13 in 31% of prostate cancers and 20% of benign diseases of prostate. Our findings suggest that methylation of CDH13 is an early event during the pathogenesis of prostate cancer. Inactivation of the APC gene is frequent in colorectal and other gastrointestinal carcinomas, usually by truncating mutations (24) . An alternative method of inactivation of the gene in some gastrointestinal cancers is by promoter methylation (25) . Recently we reported that selective methylation and silencing of the APC 1A promoter and its specific products were frequent in lung and breast cancers (26) . We found methylation of APC 1A in 27% of prostate cancers. It has been demonstrated that decreased expression of CDH1 is associated with high grade, advanced stage prostate tumors (27) , and methylation of a subset of prostate cancer cell lines has been described (28) . We found methylation of CDH1 in 27% of prostate cancers, indicating that aberrant methylation may be the major mechanism of silencing of this gene. Guo et al. (29) demonstrated down-regulation of FHIT protein in more than half of the primary prostate cancers examined by immunostaining. However, another study failed to identify the mechanism of inactivation (30) . We found methylation of FHIT in 15% of prostate cancers, indicating that promoter methylation of the FHIT gene is one of the mechanisms involved in gene silencing.

Methylation of some genes (CDH1, CDH13, and RASSF1A) were occasionally methylated in nonmalignant prostatic tissues (benign prostatic hypertrophy and prostatitis). Of interest, GSTP1 was methylated in only 3% of these tissues. Methylation has been described in various nonmalignant tissues as an aging change or following chronic inflammation (31 , 32) .

We have identified several new genes methylated at relatively high frequencies in prostate cancer. Our results indicate that the methylation profile of prostate cancers correlates with clinicopathological features of poor prognosis. Although our findings need to be extended to a larger series, our results suggest that the methylation profile of genes may be a potential new biomarker for determining the prognosis of prostate cancer patients.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research was supported by Grant U01CA84971 from the Early Detection Research Network, National Cancer Institute, and awards from the Nasher Family Cancer Program and the Amon Carter Foundation.

² To whom requests for reprints should be addressed, at Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4921; Fax: (214) 648-4940; E-mail: adi.gazdar{at}utsouthwestern.edu

³ The abbreviations used are: MSP, methylation-specific PCR; MI, methylation index; GS, Gleason score; PSA, prostate-specific antigen; FHIT, fragile histidine triad; RASSF1A, RAS association domain family protein 1A; RARß, retinoic acid receptor ß; APC, adenomatous polyposis coli; DAPK, death-associated protein kinase; MGMT, O⁶-methylguanine-DNA-methyltransferase; GSTP1, glutathione S-transferase P1; CDH1, E-cadherin; CDH13, H-cadherin.

Received 9/ 4/01; revised 11/21/01; accepted 11/21/01.

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				R. Henrique, C. Jeronimo, M. R. Teixeira, M. O. Hoque, A. L. Carvalho, I. Pais, F. R. Ribeiro, J. Oliveira, C. Lopes, and D. Sidransky Epigenetic Heterogeneity of High-Grade Prostatic Intraepithelial Neoplasia: Clues for Clonal Progression in Prostate Carcinogenesis Mol. Cancer Res., January 1, 2006; 4(1): 1 - 8. [Abstract] [Full Text] [PDF]

				E. Rosenbaum, M. O. Hoque, Y. Cohen, M. Zahurak, M. A. Eisenberger, J. I. Epstein, A. W. Partin, and D. Sidransky Promoter Hypermethylation as an Independent Prognostic Factor for Relapse in Patients with Prostate Cancer Following Radical Prostatectomy Clin. Cancer Res., December 1, 2005; 11(23): 8321 - 8325. [Abstract] [Full Text] [PDF]

				M. O. Hoque, O. Topaloglu, S. Begum, R. Henrique, E. Rosenbaum, W. Van Criekinge, W. H. Westra, and D. Sidransky Quantitative Methylation-Specific Polymerase Chain Reaction Gene Patterns in Urine Sediment Distinguish Prostate Cancer Patients From Control Subjects J. Clin. Oncol., September 20, 2005; 23(27): 6569 - 6575. [Abstract] [Full Text] [PDF]

				H. Enokida, H. Shiina, S. Urakami, M. Igawa, T. Ogishima, L.-C. Li, M. Kawahara, M. Nakagawa, C. J. Kane, P. R. Carroll, et al. Multigene Methylation Analysis for Detection and Staging of Prostate Cancer Clin. Cancer Res., September 15, 2005; 11(18): 6582 - 6588. [Abstract] [Full Text] [PDF]

				P. J. Bastian, J. Ellinger, A. Wellmann, N. Wernert, L. C. Heukamp, S. C. Muller, and A. von Ruecker Diagnostic and Prognostic Information in Prostate Cancer with the Help of a Small Set of Hypermethylated Gene Loci Clin. Cancer Res., June 1, 2005; 11(11): 4097 - 4106. [Abstract] [Full Text] [PDF]

				A. Agathanggelou, W. N. Cooper, and F. Latif Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers Cancer Res., May 1, 2005; 65(9): 3497 - 3508. [Abstract] [Full Text] [PDF]

				G. P. Larson, Y. Ding, L. S-C. Cheng, C. Lundberg, V. Gagalang, G. Rivas, L. Geller, J. Weitzel, D. MacDonald, J. Archambeau, et al. Genetic Linkage of Prostate Cancer Risk to the Chromosome 3 Region Bearing FHIT Cancer Res., February 1, 2005; 65(3): 805 - 814. [Abstract] [Full Text] [PDF]

				L.-C. Li, P. R. Carroll, and R. Dahiya Epigenetic Changes in Prostate Cancer: Implication for Diagnosis and Treatment J Natl Cancer Inst, January 19, 2005; 97(2): 103 - 115. [Abstract] [Full Text] [PDF]

				C. An, I.-S. Choi, J. C. Yao, S. Worah, K. Xie, P. F. Mansfield, J. A. Ajani, A. Rashid, S. R. Hamilton, and T.-T. Wu Prognostic Significance of CpG Island Methylator Phenotype and Microsatellite Instability in Gastric Carcinoma Clin. Cancer Res., January 15, 2005; 11(2): 656 - 663. [Abstract] [Full Text] [PDF]

				S. Tommasi, R. Dammann, Z. Zhang, Y. Wang, L. Liu, W. M. Tsark, S. P. Wilczynski, J. Li, M. You, and G. P. Pfeifer Tumor Susceptibility of Rassf1a Knockout Mice Cancer Res., January 1, 2005; 65(1): 92 - 98. [Abstract] [Full Text] [PDF]

				C. M. Lewis, L. R. Cler, D.-W. Bu, S. Zochbauer-Muller, S. Milchgrub, E. Z. Naftalis, A. M. Leitch, J. D. Minna, and D. M. Euhus Promoter Hypermethylation in Benign Breast Epithelium in Relation to Predicted Breast Cancer Risk Clin. Cancer Res., January 1, 2005; 11(1): 166 - 172. [Abstract] [Full Text] [PDF]

				C. Jeronimo, R. Henrique, M. O. Hoque, E. Mambo, F. R. Ribeiro, G. Varzim, J. Oliveira, M. R. Teixeira, C. Lopes, and D. Sidransky A Quantitative Promoter Methylation Profile of Prostate Cancer Clin. Cancer Res., December 15, 2004; 10(24): 8472 - 8478. [Abstract] [Full Text] [PDF]

				P. M. Das and R. Singal DNA Methylation and Cancer J. Clin. Oncol., November 15, 2004; 22(22): 4632 - 4642. [Abstract] [Full Text] [PDF]

				G. Clement, F. T. Bosman, C. Fontolliet, and J. Benhattar Monoallelic Methylation of the APC Promoter Is Altered in Normal Gastric Mucosa Associated with Neoplastic Lesions Cancer Res., October 1, 2004; 64(19): 6867 - 6873. [Abstract] [Full Text] [PDF]

				J. V. Tricoli, M. Schoenfeldt, and B. A. Conley Detection of Prostate Cancer and Predicting Progression: Current and Future Diagnostic Markers Clin. Cancer Res., June 15, 2004; 10(12): 3943 - 3953. [Abstract] [Full Text] [PDF]

				C. Jeronimo, R. Henrique, M. O. Hoque, F. R. Ribeiro, J. Oliveira, D. Fonseca, M. R. Teixeira, C. Lopes, and D. Sidransky Quantitative RAR{beta}2 Hypermethylation: A Promising Prostate Cancer Marker Clin. Cancer Res., June 15, 2004; 10(12): 4010 - 4014. [Abstract] [Full Text] [PDF]

				S. Yegnasubramanian, J. Kowalski, M. L. Gonzalgo, M. Zahurak, S. Piantadosi, P. C. Walsh, G. S. Bova, A. M. De Marzo, W. B. Isaacs, and W. G. Nelson Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer Cancer Res., March 15, 2004; 64(6): 1975 - 1986. [Abstract] [Full Text] [PDF]

				S. Zheng, X. Ma, L. Zhang, L. Gunn, M. T. Smith, J. L. Wiemels, K. Leung, P. A. Buffler, and J. K. Wiencke Hypermethylation of the 5' CpG Island of the FHIT Gene Is Associated with Hyperdiploid and Translocation-Negative Subtypes of Pediatric Leukemia Cancer Res., March 15, 2004; 64(6): 2000 - 2006. [Abstract] [Full Text] [PDF]

				E. Dulaimi, R. G. Uzzo, R. E. Greenberg, T. Al-Saleem, and P. Cairns Detection of Bladder Cancer in Urine by a Tumor Suppressor Gene Hypermethylation Panel Clin. Cancer Res., March 15, 2004; 10(6): 1887 - 1893. [Abstract] [Full Text] [PDF]

				I. H. N. Wong, J. Chan, J. Wong, and P. K. H. Tam Ubiquitous Aberrant RASSF1A Promoter Methylation in Childhood Neoplasia1 Clin. Cancer Res., February 1, 2004; 10(3): 994 - 1002. [Abstract] [Full Text] [PDF]