Urological Oncology 95Urology Journal Vol 4 No 2 Spring 2007 Role of PTEN Gene in Progression of Prostate Cancer Gholamreza Pourmand,1 Abed-Ali Ziaee,2 Amir Reza Abedi,1 Abdolrasoul Mehrsai,1 Hossein Afshin Alavi,3 Ali Ahmadi,1 Hamid Reza Saadati2 Introduction: The aim of this study was to clarify the role of PTEN gene in progression of prostate cancer. Materials and Methods: A total of 51 formalin-fixed paraffin-embedded specimens of prostate cancer were analyzed for PTEN mutations. Tissue microdissection and polymerase chain reaction/single-strand conformation polymorphism methods were used. Clinical and pathologic data of the patients were reviewed with regard to PTEN mutation. Results: The Gleason score (GS) was less than 7 in 29 (56.8%), 7 in 11 (21.6%), and greater than 7 in 11 (21.6%). Tumor stage was IIa, IIb, IIc, and IV in 14 (27.4%), 4 (7.8%), 21 (41.2%), and 12 (23.6%) patients, respectively. Eleven of 12 stage IV tumors had metastases at the time of presentation. Six of 51 cases (11.6%) showed mutation in PTEN which had involved exones 1, 2, and 5. Two of these cases had localized and the others had advanced prostate cancer. One case of the tumors with PTEN mutation had a GS of 7 and 5 had GSs greater than 7. Patients with a positive mutation of PTEN had a significantly greater GS (P < .001), lower survival rate (P = .001), higher tendency to metastasis (P = .002), and higher prostate-specific antigen (P = .03). Cox proportional hazard model showed that only GS was significantly correlated with mortality (P = .03). Conclusion: Patients with prostate cancer who had PTEN mutation had also a significantly greater GS, poorer prognosis, and higher rate of metastasis. However, this mutation cannot predict the prognosis and the GS is a more precise factor. Urol J. 2007;4:95-100. www.uj.unrc.ir Keywords: prostatic neoplasms, PTEN, mutations, Gleason score, prostate-specific antigen, Iran 1Urology Research Center, Tehran University of Medical Sciences, Tehran, Iran 2Institute of Biophysics and Biochemistry, University of Tehran, Tehran, Iran 3Department of Pathology, Day Hospital, Tehran, Iran Corresponding Author: Gholamreza Pourmand, MD Urology Research Center, Sina Hospital, Hasanabad Sq, Tehran, Iran Tel: +98 21 6671 7447 Fax: +98 21 6671 7447 E-mail: gh_pourmand@yahoo.com Received September 2006 Accepted April 2007 INTRODUCTION Prostate adenocarcinoma is one of the most commonly diagnosed malignancies affecting the men in both the United States and Europe.(1) The prognostic factors in patients with prostate cancer who undergo radical prostatectomy are pathological stage and Gleason score (GS).(2) Prostate cancer is a heterogeneous disease and identifying factors associated with a poor outcome at the time of radical prostatectomy is challenging.(2) The molecular mechanisms of prostate carcinogenesis are unknown. PTEN/ MMAC1 is a tumor suppressor gene located on 10q23.(3,4) PTEN that encodes a dual-specificity phosphatase is a tumor suppressor gene whose inactivation has been associated with many different types of cancers including glioma, melanoma, and carcinoma of the endometrium, kidney, breast, lung, upper respiratory tract, and prostate.(5) The tumor suppressor activity of PTEN is thought to be primarily due to its ability to dephosphorylate phosphoproteins PTEN and Prostate Cancer—Pourmand et al 96 Urology Journal Vol 4 No 2 Spring 2007 or phospholipids and negatively regulate the activity of the phosphatidylinositol 3-kinase pathway. It is shown that expression of PTEN can inhibit cell cycle progression, induce a G1 arrest, inhibit cell migration, and induce cell cycle arrest and apoptosis.(6-8) Loss of PTEN activity as a tumor suppressor gene enhances cell proliferation and tumor angiogenesis and decreases apoptosis.(9) Mutations in PTEN have often been detected in metastases of prostate cancer; however, lower rates of mutations have been found in localized tumors (0 to 20% in different studies).(2) The rate of PTEN mutations in prostate cancer has not been adequately studied in Asia. One study based on 32 Chinese men with prostate cancer showed a 16% rate of PTEN mutations.(5) We analyzed prostate cancers of 51 Iranian patients to scrutinize the role of PTEN mutations in tumor progression. MATERIALS AND METHODS Tumor Specimens This study was performed in accordance with the declaration of Helsinki and subsequent revisions and approved by ethics committee at Tehran University of Medical Sciences. We used 51 paraffin- embedded prostate cancer specimens archived in the departments of pathology of Day and Sina hospitals between 1997 and 2005. Twelve specimens were of patients who had undergone transurethral resection and the remaining had been obtained by radical prostatectomy. The prostatectomy procedures had been performed by 3 surgeons over a period of 8 years. All specimens were collected from the archived paraffin blocks used for routine diagnosis of cancer. Follow-up data were available for all of the cases in the database with a mean patient follow-up period of 48 months. The initial values of prostate-specific antigen (PSA) and GS were recorded. For every case, a representative paraffin block was selected that contained both tumor and benign prostate tissue. DNA Extraction Formalin-fixed paraffin samples were cut into 10-μm sections. The sections were pulverized under liquid nitrogen condition using microdismembrator (B Braun Melsungen AG, Melsungen, Germany). Of each sample, 0.1 g of pulverized tissue powder was resuspended in 1 mL of xylene and left for 15 minutes at 55°C. The suspension was then centrifuged at 14 000 g for 5 minutes. The pellet was suspended in 0.1 mL of xylene and processed as above for the second time. The resulted sediment was mixed with 100% ethanol and processed with xylene lysis buffer (Tris, sodium dodecyl sulfate, ethylenediamine tetraacetic acid [EDTA]). A lysis buffer containing 300 μg/mL of proteinase-k was added to the pellet, mixed and incubated at 55°C for an overnight period. The DNA was extracted following the use of phenol-chloroform procedure, then dissolved in TE buffer (Tris-HCl and EDTA) and stored at 4°C. Polymerase Chain Reaction Analysis For polymerase chain reaction (PCR) application (Genius, Boehringer-Mannheim, Indianapolis, USA), increasing concentrations of extracted DNA of each specimen was tested to find out the optimum dose that resulted in good amplicon product. Each primer pair of the selected exons was used for mutation detection of PTEN/MMAC1 following the PCR for the single-strand conformation polymorphism (PCR- SSCP). The PCR protocol was carried out as outlined in Table 1, and primers used for each PTEN exon were as follows: PTEN 1F 5’-AGTCGCTGCAACCATCCA PTEN 1R 5’-GATATTTGCAAGCATACAAA PTEN 2F 5’-GTTTGATTGCCATATTTCAG PTEN 2R 5’-GGCTTAGAAATCTTTTCTAAATG PTEN 5F 5’-GCAACATTTCTAAAGTTACCTACTTG PTEN 5R 5’-CATATCATTACACCAGTTCG Table 1. Polymerase Chain Reaction Protocol Denaturation Annealing Extension Exon Temperature, °C Times Temperature, °C Times Temperature, °C Times PTEN exon 1 95 40 60 45 72 60 PTEN exon 2 95 30 60 45 72 60 PTEN exon 5 95 40 56 60 72 60 PTEN exon 8 95 40 58 45 72 60 PTEN and Prostate Cancer—Pourmand et al Urology Journal Vol 4 No 2 Spring 2007 97 PTEN 8F 5’-CATTATAAAGATTCAGGCAATG PTEN 8R 5’-GACAGTAAGATACAGTCTATC Any shift in the pattern of single-strand migration in the gel electrophoresis was considered as mutated exon. The PCR product was mixed with an SSCP denaturating buffer (98% foramide, 20 mM EDTA, 0.05% xylene cynol, 0.05% bromophenol blue, 0.05 M NaOH) and heated at 98°C for 8 minutes. The heated mixture was subsequently loaded on polyacrylamide gel and electrophoresed at 250 V for 6 to 8 hours at room temperature. The electrophoresed gel was processed, stained, and developed using Silver staining method. Statistical Analyses The t test and Mann-Whitney U test were used to compare the PSA and GS between the patients with and without PTEN mutation, respectively. The chi-square test was used to evaluate metastasis in the patients with and without PTEN mutation. To analyze survival of the patients and the prognostic variables, Kaplan-Meier method, log-rank test, and Cox proportional hazards regression model were used. Data analyses were performed by the SPSS software (Statistical Package for the Social Sciences, version 13.0, SPSS Inc, Chicago, Ill, USA). A value of P less than .05 was considered significant. RESULTS Fifty-one formalin-fixed paraffin-embedded prostate cancer specimens of the patients were used in this study. Radical prostatectomy had been performed in 39 localized prostate cancers and transurethral resection of prostate plus adjuvant therapy in 12 advanced cancer cases. The mean age of the patients was 69.1 ± 7.9 years (range, 57 to 82 years). Of the patients, 29 (56.8%), 11 (21.6%), and 11 (21.6%) had GSs less than 7, equal to 7, and greater than 7, respectively. Twenty-eight of 39 localized prostate cancers (71.8%) had GSs of less than 7, while 9 out of 12 advanced prostate cancers (75.0%) had GSs greater than 7. Preoperative serum PSA level was less than 4 ng/mL in 9 patients with localized prostate cancer (23.1%), greater than 10 ng/mL in 3 (7.7%), and between 4 ng/mL and 10 ng/mL in 27 (69.2%). In advanced cancer cases, all of the patients had a preoperative PSA level greater than 10 ng/mL. There was a correlation between the PSA level and the GS (P = .001; Figure 1). Eleven of 12 advanced prostate cancers had metastases at the time of presentation. The prostate cancer stage was IIa in 14 patients with radical prostatectomy (35.9%), IIb in 4 (10.3%), and IIc in 21 (53.8%). The PCR-SSCP analyses of the specimens revealed band shifts in 6 tumors (2 in exon 1, 1 in exon 2, and 3 in exon 5) which indicated the existence of possible sequence alterations within these sites. Two of these cases had localized and the others had advanced prostate cancer. One case of the tumors with PTEN mutation had a GS of 7 and 5 had GSs greater than 7 (Table 2). All of the tumors with a positive mutation of PTEN and advanced prostate cancer were associated with metastasis, whereas 1 of the tumors with a positive mutation and localized prostate cancer was metastatic. During the follow-up, Table 2. Frequency of PTEN Gene Mutation in Different Gleason Score Categories PTEN Gleason Score Negative Positive Total < 7 29 (100.0) 0 29 7 10 (90.9) 1 (9.1) 11 > 7 6 (54.6) 5 (45.4) 11 Total 45 (88.2) 6 (11.8) 51 Figure 1. The correlation of preoperative serum PSA level with Gleason score. PTEN and Prostate Cancer—Pourmand et al 98 Urology Journal Vol 4 No 2 Spring 2007 5 of 6 patients with PTEN mutation had died as a result of metastases. Patients with a positive mutation of PTEN had a significantly greater GS (P < .001), lower survival rate (P = .001; Figure 2), and higher tendency to metastasis (P = .002). The mean PSA value was 21.42 ± 14.60 ng/mL in the mutation-positive patients and 11.25 ± 12.93 ng/mL in the mutation- negative ones (P = .03; Figure 3). Cox proportional hazard model was used and the variables including age, GS, PTEN mutation, and PSA value entered in the model (Table 3). Only GS was significantly correlated with mortality (P = .03; Figure 4). DISCUSSION Prostate cancer is the most common form of malignancy in men in the western countries and is the second most common cause of cancer deaths in the United States.(1) Quantitative and structural genetic alterations can cause the development and progression of prostate cancer. Detection of these genes will be a key role for improving the treatment of prostate cancer. The frequency of PTEN inactivation coincide with the progression of prostate cancer.(1) The PTEN tumor suppressor gene is frequently inactivated in human tumors including glioma, melanoma, and carcinoma of the endometrium, kidney, breast, lung, upper respiratory tract, and prostate.(10) Our finding of PTEN mutations in 6 of 51 prostate cancer specimens, 5 of which being high grade, confirms that PTEN is a major gene in progression of prostate cancers. The frequency of PTEN mutation in prostate cancer differs between studies published to date, most probably because of differences in tumors’ grade and stage in the study populations. In one study of 37 tumors with 20 (54.1%) high-grade and 17 (45.9%) low-grade tumors, 5 cases had PTEN mutations, 4 of which were high-grade tumors.(11) In another study of 45 prostate cancers that were mainly low grade (67%), no PTEN mutations were found.(12) In a study on 32 cases of prostate cancer (70% with a GS of 8 to 10), PTEN mutations were detected in 5 (15.6%).(5) Summarizing 5 studies on PTEN in prostate cancer, 51 of 192 high-grade tumors (26.6%) showed Figure 2. Patients with positive mutation had worse prognoses. Figure 4. High grade tumors have worse prognosis. Figure 3. The frequency of PTEN gene mutation in each preoperative PSA level category. 0 5 10 15 20 25 30 < 4 4 to 10 > 10 PSA, ng/m L N um be r o f P at ie nt s PTEN Negative PTEN Positive PTEN and Prostate Cancer—Pourmand et al Urology Journal Vol 4 No 2 Spring 2007 99 mutations in the PTEN, while only 3 of 95 low-grade cases (3.2%) showed mutations (Table 4).(2,5,11-13) We found PTEN mutations in 6 of 51 (11.8%) Iranian patients; Five of the 6 cases with mutations were high-grade tumors and the patients died as a result of metastasis. These studies indicate that PTEN mutations occur more often in tumors with greater GSs. Orikasa and associates examined 45 primary prostate cancer specimens. Loss of heterozygosity at the PTEN locus was observed in 2 out of 18 tumors (11.1%). However, no mutations were observed in any of the primary prostate cancers. These data propose that mutation of the PTEN gene does not play an important role in prostate carcinogenesis of Japanese patients.(12) In another study, the PTEN appeared to be the most commonly mutated gene in metastases of prostate cancer occurring in at least 1 metastatic site in 12 of 19 (63%) patients with multiple metastases.(14) Mutations of PTEN in localized prostate cancers have been detected at lower rates (2.5% to 5%).(15-17) These results show a role for the PTEN in the progression of prostate cancer. In our study, the variables which showed correlation with mortality in univariate analyses (PSA and PTEN), did not correlate with mortality in multivariate analysis. Only GS was significantly correlated with mortality (P = .03). CONCLUSION Patients with prostate cancer who had PTEN mutation had also a significantly greater GS, poorer prognosis, and higher rate of metastasis. The increase in the GS was associated with PTEN gene mutation and increase in the mortality. The same condition exists about the PSA value. As a result, in multivariate analysis, only GS was significantly correlated with mortality. CONFLICT OF INTEREST None declared. REFERENCES 1. Deocampo ND, Huang H, Tindall DJ. The role of PTEN in the progression and survival of prostate cancer. Minerva Endocrinol. 2003;28:145-53. 2. McMenamin ME, Soung P, Perera S, Kaplan I, Loda M, Sellers WR. Loss of PTEN expression in paraffin- embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer Res. 1999;59:4291-6. 3. Gray IC, Phillips SM, Lee SJ, Neoptolemos JP, Weissenbach J, Spurr NK. Loss of the chromosomal region 10q23-25 in prostate cancer. Cancer Res. 1995;55:4800-3. 4. Arps S, Rodewald A, Schmalenberger B, Carl P, Bressel M, Kastendieck H. Cytogenetic survey of 32 cancers of the prostate. Cancer Genet Cytogenet. 1993;66:93-9. 5. Dong JT, Li CL, Sipe TW, Frierson HF Jr. Mutations of PTEN/MMAC1 in primary prostate cancers from Chinese patients. Clin Cancer Res. 2001;7:304-8. 6. Furnari FB, Huang HJ, Cavenee WK. The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells. Cancer Res. 1998;58:5002-8. 7. Davies MA, Koul D, Dhesi H, et al. Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN. Cancer Res. 1999;59:2551-6. Table 3. Cox Regression Hazard Model for Survival of Patients With Prostate Cancer Variable Hazard Ratio Standard Error z P 95% Confidence Interval Age 1.068 0.048 1.46 0.14 0.977 1.167 PSA 1.007 0.026 0.27 0.79 0.956 1.060 GS 2.347 0.915 2.19 0.03 1.092 5.041 PTEN 1.699 1.698 0.53 0.60 0.239 12.051 Table 4. Summary of 5 Studies on PTEN Mutations and Gleason Scores in Prostate Cancer Patients High-grade Tumor Low-grade Tumor Authors Total PTEN Mutation Total PTEN Mutation Total PTEN Mutation McMenamin and colleagues(2) 109 17 (15.6) 79 17 (21.5) 30 0 Dong and colleagues(5) 38 7 (18.4) 27 6 (22.2) 12 1 (8.3) Gray and colleagues(11) 37 5 (13.5) 20 4 (20.0) 17 1 (5.9) Orikasa and colleagues(12) 45 0 15 0 30 0 Leube and colleagues(13) 57 22 (38.6) 51 21 (41.1) 6 1 (16.7) PTEN and Prostate Cancer—Pourmand et al 100 Urology Journal Vol 4 No 2 Spring 2007 8. Persad S, Attwell S, Gray V, et al. Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci U S A. 2000;97:3207-12. 9. Backman SA, Ghazarian D, So K, et al. Early onset of neoplasia in the prostate and skin of mice with tissue- specific deletion of Pten. Proc Natl Acad Sci U S A. 2004;101:1725-30. 10. Cooney KA, Tsou HC, Petty EM, et al. Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer. Clin Cancer Res. 1999;5:1387-91. 11. Gray IC, Stewart LM, Phillips SM, et al. Mutation and expression analysis of the putative prostate tumour- suppressor gene PTEN. Br J Cancer. 1998;78:1296- 300. 12. Orikasa K, Fukushige S, Hoshi S, et al. Infrequent genetic alterations of the PTEN gene in Japanese patients with sporadic prostate cancer. J Hum Genet. 1998;43:228-30. 13. Leube B, Drechsler M, Muhlmann K, et al. Refined mapping of allele loss at chromosome 10q23-26 in prostate cancer. Prostate. 2002;50:135-44. 14. Suzuki H, Freije D, Nusskern DR, et al. Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues. Cancer Res. 1998;58:204-9. 15. Facher EA, Law JC. PTEN and prostate cancer. J Med Genet. 1998;35:790. 16. Dong JT, Sipe TW, Hyytinen ER, et al. PTEN/MMAC1 is infrequently mutated in pT2 and pT3 carcinomas of the prostate. Oncogene. 1998;17:1979-82. 17. Pesche S, Latil A, Muzeau F, et al. PTEN/MMAC1/ TEP1 involvement in primary prostate cancers. Oncogene. 1998;16:2879-83.