Abstract
Prostate cancer is the most common cancer among men and the second leading cause of cancer-related deaths in the United States. CpG island methylation causes gene silencing and could be decisive in prostate carcinogenesis and progression. Its role was investigated at multiple gene sites during prostate carcinogenesis. Methylation-specific polymerase chain reaction (MS-PCR) was used to analyze 4 interest gene promoter status in 12 patients with adenocarcinoma, 7 patients with prostate intraepithelial neoplasia, 3 patients with peritumor tissues and 15 healthy patients, so a total of 37 prostate biopsy samples constituted the cohort of the study. Despite the biopsy histology, the results have confirmed that BRCA1, RASSF1, GSTP1 and EPHB2 promoter methylation was found in each sample, except two.
Prostate cancer is the second leading cause of cancer-related deaths in the United States. The American Cancer Society estimates that there were over 186,320 cases of new prostate cancer cases in 2008. Prostate cancer is the most commonly diagnosed malignancy among males in Western countries (1) and represented 40,000 new cases in France in 2000 (2). The net risk of developing prostate cancer before the age of 75 years for a patient born in 1943 was multiplied by 3 compared to those born in 1928 (3). Researches have focused on candidate genes to assess their involvement in the predisposition of prostate cancer. Since available screening methods show poor sensitivity and specificity, the development of new molecular markers is warranted. Epigenetic alterations, mainly promoter hypermethylation of cancer-related genes, are common events in prostate cancer and might be used as cancer biomarkers.
Epigenetic changes, particularly the DNA methylation, are found to be involved in a variety of cancers (4). DNA methylation refers to a covalent chemical modification resulting from the addition of a methyl (CH3) group at the C-5 position of the Cytosine ring in the DNA (5). The human genome is not uniformly methylated. “CpG islands” are small regions within the genome that are rich in Cytosine and Guanine bases and are largely unmethylated epigenetics target this region thereby affecting gene expression (4). Epigenetic changes are early event in cancer development and can be used to assess the risk of developing cancer. Epigenetic changes in prostate cancer are being studied extensively and genome wide screening will lead to development of novel epigenetic markers. The aim of this study was to determine the methylation promoter status of four cancer-related genes in the prostate according to the degree of malignancy. BRCA1, RASSF1, GSTP1 and EPHB2 methylation was explored on prostate biopsies. These four genes of interest are known to be cancer-related genes. The aim was to examine their promoter methylation status and to explore their relationship with histological degree of malignancy.
Patients and Methods
Data collection. This study was conducted with the participation of 37 men which had a prostate biopsy to establish their diagnostic by an urologist. They were hospitalized at the CHU of Clermont-Ferrand and, in a letter of consent, they accepted to give a sample for research. This procedure was advised by the PPC (People Protection Committee) within the framework of the constitution of collection of biological samples. A transrectal ultrasound-guided prostate biopsy was made with a spin and in sextant. This exam was made with local anaesthesia on the recumbent patient. The identity of each patient was confidential and through the anatomopathological exam, the stage of cancer development was diagnosed and could be correlated.
The cohort of 37 patients presented 15 non-malignant (NM) biopsies, 7 prostate intraepithelial neoplasia (PIN), 3 peri tumor tissues (PTT) and 12 adenocarcinomas (ADC).
DNA extraction. Prostate biopsies were disrupted with a scapel and DNA extraction was performed with Non-Organic DNA Extraction Kit® (Chemicon, International, S4520, USA) according to the manufacturer's protocol. First, the cells were lysed for 15 min in a Wash Solution, after centrifugation at 1000g for 20 min, the tissue sample was resuspended in a 1X Suspension Buffer I and the DNA was deproteinated with 50 μL of Protein Digesting Enzyme and 1 ml of Protein Precipitating solution. Protein contaminants were removed and the DNA was precipitated with two volumes of absolute ethanol and resuspended in 150 μL of Suspension Buffer II. Each sample was assayed with Nanodrop 8000 (LabTech®), measuring optical density ratio at 260/280 nm.
MS-PCR. Interest gene promoter methylation was determined by MS-PCR with bisulfite-converted DNA. The procedure takes avantage of the bisulfite-mediated chemical conversion of cytosine to uracil followed by PCR using primers designed to distinguish methylated DNA from unmethylated DNA (6). Bisulfite modification of DNA to convert unmethylated cytosine residues to uracil was carried out using MethylDetector Bisulfite Modification Kit (Active Motif®) following the protocol from the manufacturer. Sequences of the primers were designed by MethPrimer software (Table I). Each modification was checked using a verification method prescribed by the manufacturer.
PCR was carried out in a total volume of 30 μL per reaction containing 0.5 μL of AmpliTaq (Applied Biosystems®), 3 μL of gene probes, 0.5 μL of dNTP, 3 μL of PCR buffer (10X), 19.3 μL of water and 4 μL of DNA sample. Each PCR reaction underwent initial denaturation at 95°C for 10 min, and 40 cycles of the following profile: 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The PCR products were then analyzed by electrophoresis on a 2.5% agarose gel and stained with ethidium bromide and visualized by UV transillumination. A 50 bp DNA Ladder (Invitrogen®) was used. DNA was considered methylated if a PCR product using unmethylated-specific primers was absent. When PCR product was present using both unmethylated-specific primers and methylated-specific primers, it meant that between 25% and 75% of the gene promoter could be considered methylated. The assay was successfully completed for the four genes of the 37 subjects.
Results
Grading and pathologic evaluation. Tissue sections fixed in 10% buffered formalin that were routinely processed, whole-mount-embedded, and hematoxylin and eosin-stained were reviewed. The tumor foci was identified, circled in ink, and graded. The cohort of 37 patients presented 15 non-malignant biopsies, 7 prostate intraepithelial neoplasia, 3 peri-tumor tissues and 12 adenocarcinomas. The Gleason score grading system was used to assign a tumor grade (7). Among the twelve adenocarcinoma, 2 had a Gleason score of 6, 9 had a Gleason score of 7 and 1 had a Gleason score of 8 (Table II).
Analyses of BRCA1, RASSF1, GSTP1 and EPHB2 promoter status. It was confirmed that only modified samples presented a band. Each modification was consequently approved and successfully performed (Figure 1) for each sample. In case of RASSF1, GSTP1 and EPHB2, each sample revealed a promoter methylation estimated at more than 75%. This was because only the PCR product corresponding to methylated primer was present. For BRCA1, 35 out of 37 samples presented two bands. The PCR product was present using both unmethylated-specific primers and methylated-specific primers, and between 25% and 75% of the gene promoter could be considered methylated (Figure 2). The two other samples were detected as unmethylated and it is worth noting that these 2 samples were non-malignant biopsies (Table III).
Discussion
Epigenetics is one of the most rapidly expanding fields in cancer related research. Recent studies have shown that epigenetics plays an important role in cancer biology, somatic gene therapy, viral infections and genomic imprinting (5). CpG island hypermethylation is an indicator of prostate carcinogenesis and prognostic information increases if multiple gene loci are investigated simultaneously (8-10). In this study, it was attempted to identify the BRCA1, GSTP1, RASSF1 and EPHB2 promoter methylation both in healthy and in malignant prostate tissues with different clinical characteristics including Gleason score.
BRCA1, located on chromosome 17q21, encoded a multifunctional protein involved in DNA repair, control of cell-cycle checkpoints, protein ubiquitination and chromatin remodelling (11). It was found that in the major samples, between 25% and 75% of the gene promoter could be considered methylated. However, two samples were determined to have an unmethylated promoter status, and both samples were non-malignant. These results were corroborated by Xu et al. who demonstrated that BRCA1 promoter methylation was shown to be associated with increased mortality among women with breast cancer (12). Wilcox et al. screened 50 primary epithelial ovarian tumors for BRCA1 promoter hypermethylation using MS-PCR and the BRCA1 promoter was hypermethylated in 16% of tumors (13).
Pi-class glutathione-S-transferase (GSTP1), located on chromosome 11q13, encodes a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. GSTP1 plays an important role in protecting cells from cytotoxic and carcinogenic agents and is expressed in normal tissues at variable levels in different cell types (14). Methylation of CpG islands in the promoter of the Pi class of glutathione S-transferase occurs in prostatic intraepithelial neoplasia and cancer (15). Recent reports indicate that higher levels of GSTP1 promoter methylation were associated with the transition from prostatic intraepithelial neoplasia to carcinoma (16). It was confirmed that in studied prostate tissues, GSTP1 promoter was methylated, however differences between the degrees of malignant biopsies could not be detected.
The RAS-association domain family 1, isoform A (RASSF1A) is located in the 3p21.3 region (17), and is a well-known tumor-suppressor gene. The alternative transcript has been shown to be inactivated by hypermethylation in several human malignancies, including breast and prostate (18). The purpose of this study was to evaluate the methylation status of RASSF1 in human prostate tissues according to the degree of malignancy. In this study, it was found that all samples had a positive RASSF1 methylation status in prostate tissues but with no distinction between histological degrees.
The exact function of the gene Ephrine-B2 (EPHB2) is unknown, but evidence suggests that EPHB2 may be a tumor suppressor gene. The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/β catenin. Moreover Alazzouzi et al. found EPHB2 promoter hypermethylation in 53% of colorectal tumors studied (19). All of the studied samples presented an EPHB2 promoter methylation in prostate tissues.
The BRCA1, RASSF1, GSTP1 and EPHB2 methylation promoter status was determined with MS-PCR but it was difficult to estimate differences between the degrees of malignancy for which Methylight will be used in future studies.
This study concluded that BRCA1, RASSF1, GSTP1 and EPHB2 promoters were methylated in prostate tissues. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation, will be a promising approach for using DNA-based markers for the early detection of human cancers. It is quite important to choose a suitable method to minimize the influence of the mentioned limitations on the interpretation of data and its evaluation. MS-PCR lead to the determination of the BRCA1, RASSF1, GSTP1 and EPHB2 methylation promoter status.
Acknowledgements
N. Rabiau is recipient of a grant “CIFRE” from Soluscience S.A., Clermont-Ferrand, France.
This study was supported by “La Ligue Nationale Française de Lutte Contre le Cancer” (Puy-de-Dôme, Allier et Cantal). We thank T.H. Gunnels for assisting with the English translation of this study.
- Received December 24, 2008.
- Revision received January 19, 2009.
- Accepted February 23, 2009.
- Copyright © 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved