Abstract
Objective
The molecular mechanism of prostate cancer is poorly understood. The aim of the study was to investigate the prevalence and prognostic value of promoter hypermethylation of retinoic acid receptor beta (RARB) and p16 among benign prostatic hyperplasia (BPH) and prostate cancer patients.
Methods
In this case-control study, 63 patients were included in three groups; 21 with BPH as the control group, 21 with prostate cancer and good prognostic factors (based on prostate-specific antigen, Gleason score and stage) as good prognosis group, and 21 with prostate cancer and poor prognostic features as poor prognosis group. The prostate biopsy specimen of each individual was examined for hypermethylation of RARB and p16 promoters by methylation specific PCR (MSPCR).
Results
Seven (33.3%) patients with good prognosis and 15 (71.4%) patients with poor prognosis were positive for RARB methylation, which were significantly higher than controls (P<0.0001). p16 promoter methylation was shown in 19.0% and 47.6% patients with good and poor prognosis, respectively. The RARB and p16 promoter methylation in the poor prognosis group was significantly higher than that in the good prognosis group (P =0.02 for RARB and P<0.0001 for p16).
Conclusion
Hypermethylation of RARB and p16 promoters may predict prognosis in prostate cancer.
Key words: Prostate cancer, CpG island, DNA methylation, Retinoic acid receptor beta (RARB), CDKN2 (p16/MTS1), Methylation Specific PCR
INTRODUCTION
As the most commonly diagnosed cancer (excluding skin cancer) among men, prostate cancer is a global public health problem. Previous studies showed about 27% of prostate cancer had poor prognosis and prostate cancer is the second leading cause of cancer-related deaths among men in North America and Western/Northern Europe[1-6]. While many prostate cancer patients present with advanced disease, early detection with highly specific and sensitive methods might be one of the main approaches of reducing mortality[5-7].
Currently, prostate-specific antigen (PSA), histological grade [Gleason score (GS)] and stage of disease are widely used to predict the prognosis[8-10], but the tumor behavior is not always predictable using these factors. Genetic mechanisms of malignant changes are the pioneer events during disease process, long before the clinical factors appear[11]. To date, more than 50 genes have been shown to be inactivated by promoter CpG island hypermethylation in prostate cancer[6].
In the recent years, a new group of cancer markers based on the characterization of epigenetic alterations are introduced to evaluate the tumors[12-15]. Investigations on these epigenetic markers in tissue samples are mainly restricted to the evaluation of retrospective series of just a fraction of all prostate tumors. Amongst them, promoter CpG island methylation is mainly emerged as a putative prognostic assessment tool to seek some biological markers for aggressive prostate cancer[5,16-21].
Retinoic acid receptor beta (RARB, OMIM: 180220), and cyclin-dependent kinase inhibitor 2a (CDKN2a, p16/Cdkn2ink4/ Mts1, OMIM: 600160) were widely reported to be hypermethylated in prostate cancer and some suggested their potential as diagnostic and prognostic markers[22-28].
Given the importance of finding a reliable prognostic marker, we investigated prognostic value of promoter hypermethylation of RARB and p16 genes in Iranian patients with prostate cancer with good and poor prognosis, in comparison to the patients affected with benign prostatic hyperplasia (BPH).
Materials and Methods
Patients and Samples
In this case-control study, all participants were enrolled from men referred to the Radiotherapy-Oncology Ward in Imam Hossein Hospital, Urology Ward in Shahid Modarres Hospital, and Shahid Labbafinezhad Hospital due to clinically suspected prostate cancer from 2007 to 2008.
Prostate biopsy specimens were collected by surgery. If the pathologic studies confirmed the diagnosis of prostate cancer, the patient was included in the study. Included patients signed the informed consent form if they were candidates for prostatectomy. A 5 ml blood sample was taken to measure PSA level through routine lab procedures before surgery. Histologic slides from formalin-fixed and paraffin- embedded tissue fragments were reviewed to confirm BPH or prostate cancer or to reassess GS of cancer cases by an expert in prostate diseases at the Department of Pathology, Shahid Labbafinezhad Hospital. Relevant clinical data, such as age, serum PSA level at diagnosis, and clinical stage of disease, were obtained from medical records.
Prostate cancer cases were divided into good and poor prognosis with respect to the serum PSA level, GS, and staging. PSA value more than 9, or GS summation equal or more than 7, or stage III and more, was considered as poor prognosis group, the rest with GS ≤6 and stage II, and PSA <10 were labeled as the good prognosis group.
Methods
The paraffin wax-embedded blocks, consisting of 42 cancerous and 21 BPH without cancer were prepared for methylation specific PCR (MSPCR) in our genetic laboratory to measure the degree of methylation. The genetic laboratory members were not informed of the prognostic situation of patients to which the paraffin blocks belong.
First, the DNA was extracted from tissue samples using the classical method of phenol/chloroform/isoamyl alcohol. Purified DNA samples were subjected to treatment with sodium bisulfite, which reacts with cytosine (C) bases in preference to methylated cytosine (5-mC) bases, facilitating the deamination of C to produce uracil (U) while 5-mC remains unchanged. Consequently, differences in DNA methylation become apparent as differences in DNA sequence. PCR primers specific for target sequences resulting from bisulfate modification of 5-mCpG-containing DNA are used for PCR to detect target methylated CpG island[29,30].
In brief, 40 µl of DNA (2 µg) was denatured at 97°C for 10 min, centrifuged briefly, and chilled on ice. Ten microlitres of 1 mol/L NaOH was then added and the mixture was stored at room temperature for 15 min. Then 500 μl of 3.5 mol/L sodium bisulphate and 1 mmol/L hydroquinone mixture was added to the denatured DNA, and stored at 55°C for 16 h. The treated DNA was purified using Wizard DNA purification resin (DNA clean up kit, Promega, Madison, WI, USA) according to the manufactures’ instruction and desulphonated with 0.3 mol/L NaOH at room temperature for 10 min. After adding 2.5 volumes of 100% cold ethanol and a two-thirds volume of 7.5 mol/L ammonium acetate and storing at −20°C for 12 h, the precipitated DNA was centrifuged. After washing in 70% ethanol and drying, DNA was dissolved in 10 mmol/L Tris buffer. This process was performed twice for each sample in order to increase the amount of remaining DNA after bisulfit treatment.
There were 63 paraffin blocks treated with bisulfit before MSPCR. Subsequent to purification of modified DNA, methylation specific amplification was performed for evaluating methylation of p16 and RARB using bisulphate-modified DNA (30–50 ng), primers specific for methylated and non-methylated cytosines (10 pmol each), dNTPs (each at 1 mmol/L), and 1× buffer [16.6 mmol/L (NH4)2SO4/67 mmol/L Tris/pH 8.8/6.7 mmol/L MgCl2/10 mmol/L β-mercaptoethanol] in a volume of 25 µl.
MSPCR
For each biopsy specimen, 6 PCRs were run (using methylation specific primers, non-methylation specific primers, and housekeeping primers) (although it was not required to use any housekeeping specific primers, it was used just in case that needed further studies). The products of all PCRs (7 µl) were electrophoresed on 2% agarose gels and visualized under UV illumination after staining with ethidium bromide.
The results were reported as positive or negative for p16 and RARB methylation. To confirm the positive results, the procedure was repeated one more time.
Statistical Analysis
To find out any correlation between p16 and RARB methylation and prognosis of prostate cancer, Chi-square (χ2(and Fisher exact tests were used to examine the results by SPSS software (version 11.5). P<0.05 was considered significant.
The whole study protocol was approved by the Ethic committee of Shahid Beheshty University of Medical Sciences.
RESULTS
We investigated the methylation profile of 63 subjects in three groups of controls (BPH), good and poor prognosis prostatic cancer, each consisted of 21 individuals. Data are provided in Tables 1 and 2.
Table 1. Clinical data of individuals in good prognosis group.
| Patient code | Age | PSA | GS | Stage | RATB2 methylation | P16 methylation |
|---|---|---|---|---|---|---|
| G1 | 58 | 2.5 | 6 | T2c | + | − |
| G2 | 64 | 8 | 6 | T2c | − | − |
| G3 | 63 | 9 | 6 | T2c | − | − |
| G4 | 65 | 6 | 6 | T2a | − | + |
| G5 | 68 | 9 | 6 | T2c | − | − |
| G6 | 63 | 6 | 6 | T2a | + | + |
| G7 | 58 | 7 | 6 | T2c | − | − |
| G8 | 63 | 9 | 6 | T2 | + | − |
| G9 | 71 | 2 | 6 | T2a | + | − |
| G10 | 63 | 2 | 5 | T2 | + | − |
| G11 | 65 | 9 | 5 | T2c | − | − |
| G12 | 67 | 3.5 | 5 | T2 | − | − |
| G13 | 67 | 5 | 6 | T2c | − | + |
| G14 | 63 | 8 | 5 | T2a | − | − |
| G15 | 80 | 3 | 5 | T2a | − | − |
| G16 | 70 | 7 | 6 | T2a | − | − |
| G17 | 60 | 6 | 6 | T2c | − | − |
| G18 | 59 | 9 | 5 | T2c | − | − |
| G19 | 60 | 4 | 6 | T2c | + | − |
| G20 | 68 | 6 | 6 | T2a | − | + |
| G21 | 55 | 8 | 6 | T2c | + | − |
| Mean | 64.3±5.5 | 6.1 | [+]:7(33.33%) | [+]: 4(19.04%) |
Table 2. Clinical data of individuals in poor prognosis group.
| Patient code | Age | PSA | GS | Stage | RARB2 methylation | P16 methylation |
|---|---|---|---|---|---|---|
| P1 | 47 | 36 | 7 | T2c | + | − |
| P2 | 73 | 8 | 9 | T3b | + | − |
| P3 | 60 | 23 | 7 | T2c | + | − |
| P4 | 64 | 6 | 8 | T3b | + | + |
| P5 | 53 | 15 | 6 | T3b | + | + |
| P6 | 58 | 6.1 | 7 | T3b | − | + |
| P7 | 56 | 19 | 7 | T2c | + | − |
| P8 | 69 | 13 | 7 | T2c | + | + |
| P9 | 49 | 10 | 9 | T4 | + | − |
| P10 | 56 | 20 | 9 | T2c | − | + |
| P11 | 74 | 25 | 8 | T3b | − | − |
| P12 | 56 | 24.5 | 6 | T3b | + | − |
| P13 | 59 | 10 | 8 | T2c | − | − |
| P14 | 71 | 15.6 | 7 | T3b | + | + |
| P15 | 64 | 38 | 7 | T2c | + | − |
| P16 | 69 | 25 | 7 | T2c | + | + |
| P17 | 63 | 12 | 9 | T3c | + | − |
| P18 | 72 | 12 | 7 | T3c | + | + |
| P19 | 69 | 16 | 8 | T4 | − | + |
| P20 | 50 | 31 | 7 | T2c | − | + |
| P21 | 53 | 8 | 8 | T2c | + | − |
| Mean | 61.19±8.44 | 17.77 | [+]:15(71.428%) | [+]:10(47.619%) |
The mean age was 61.5±5.5 in control group, 64.3±5.5 and 61.2±8.4 in good and poor prognosis groups respectively, without any significant difference. The mean PSA value was 6.14±2.48 and 17.77±9.46 in good and poor prognosis groups, respectively.
Table 3 shows RARB hypermethylation distribution and the two by two comparisons between the control, good, and poor prognosis groups. There was no RARB methylation positive subject in control group. In good prognosis group 33.3% were positive for RARB methylation, which was significant in comparison with control group (P<0.004). Fifteen (71.4%) of poor prognosis group were positive for RARB methylation that was significantly higher than the good prognosis patients (P<0.013). Compared with patients with good prognostic factors, those with poor prognostic factors were 5 times more likely to have RARB methylation [odds ratio (OR)=5; 95% confidence interval (95% CI): 1.34-18.55].
Table 3. The comparison of RARB methylation status between each two of study groups.
| RARB methylation | Poor prog. group | Good prog. group | Control group | |
|---|---|---|---|---|
| Pos. | 15 (71.4%) | 7 (33.3%) | 0 (0%) | |
| Neg. | 6 (28.6%) | 14 (66.7%) | 21 (100%) | |
| Total | 21 | 21 | 21 | |
| P | <0.001 | 0.004 |
Prog., Prognosis, Pos., Positive, Neg., Negative.
The p16 hypermethylation distribution of the three groups and the comparison are shown in Table 4. There was no p16 methylated subject in controls. Four patients in good prognosis group (19.04%) had p16 methylation that was significantly higher than controls (P<0.035). From patients with poor prognosis prostate cancer, ten (47.6%) had methylated p16 that was significant in comparison with controls (P<0.001).
Table 4. The comparison of p16 methylation status between each two of study groups.
| Group |
Group |
Group |
||||||
|---|---|---|---|---|---|---|---|---|
| Poor prog. | Control | Good prog. | Control | Good prog. | Poor prog. | |||
| P16 methylation | Pos. | 10 (47.6%) | 0 (0%) | 4 (19%) | 0 (0%) | 4 (19%) | 10 (47.6%) | |
| Neg. | 11 (52.4%) | 21 (100%) | 17 (81%) | 21 (100%) | 17 (81%) | 11 (52.4%) | ||
| Total | 21 | 21 | 21 | 21 | 21 | 21 | ||
| P | <0.001 | 0.035 | 0.05 | |||||
Prog.,Prognosis, Pos., Positive, Neg., Negative.
Patients with poor prognostic factors were more likely to have methylated p16 compared with patients with good prognostic factor. However the OR (equal to 3.8 with 95% CI of 0.967-15.44) has a trend to be significant.
Both p16 and RARB were methylated in six patients with poor prognostic features and just for one patient in good prognosis group (P<0.002). Individuals in poor prognosis group in comparison to those in good prognosis group were 33 times more likely to have both RARB and p16 hypermethylation (OR=33; 95% CI: 2.45-443.59).
DISCUSSION
Although the clinical and pathologic indices such as PSA values, grade and stage of the tumor predict the prognosis of the disease, they are products of gross cellular and tissue malfunction; therefore they will become measurable lately in the process of tumor development[8-10]. Finding prognostic factors in molecular and epigenetic level to predict the tumor behavior will probably help us to identify those patients who need more invasive approaches at earlier stage to save more lives[6,12-15,31].
At the time of diagnosis, prostate cancer cells contain many somatic mutations, gene deletions, gene amplifications, chromosomal rearrangements and changes in DNA methylation. These alterations probably accumulate over a period of several decades. Prostate cancer is associated with the greatest heritable risk of any human cancer and the molecular genetics underlying this disease displays a great deal of heterogeneity both between individuals as well as within an affected organ and sometimes between races. The diversity of currently identified somatic genetic abnormalities associated with prostate cancer suggests that there is not a single dominant molecular pathway required for prostate carcinogenesis. Prostate cancer development and metastasis are multistep processes that, among others, involve the inactivation of tumor suppressor genes. Aberrant DNA methylation of CpG (cytosine preceding guanosine) sites is among the earliest and most frequent alterations in cancer. CpG dinucleotides can be found in clusters called CpG islands often in promoter regions. CpG islands of many genes, including tumor suppressor genes, are unmethylated in normal tissues but are methylated to a varying degree in multiple types of cancer, causing silencing of gene transcription and inactivation.
Several studies have indicated that hypermethylation of one or several specific genes or the overall number of hypermethylation events might be useful to classify urothelial carcinomas. Specifically, hypermethylation has been reported to increase with grade, stage and histological subtype, to differ at different locations, and to yield prognostic information additional to that provided by histopathological parameters[32-36].
The use of DNA methylated genes as diagnostic biomarkers has potential application for distinguishing between normal and prostate cancer tissues and for identifying premalignant state. An ideal biomarker would have low levels of methylation in the normal tissue and be highly elevated in the cancer and premalignant state. Previous studies revealed that promoter hypermethylation of p16 and RARb2 was strongly correlated with the lack of protein expression, indicating promoter hypermethylation as the main inactivation mechanism of these genes in prostate carcinoma[22-28,37]. Furthermore, it has been shown that the number of hypermethylated genes increases as prostate cancer progresses; thus, the investigation of several genes may provide additional diagnostic information[6,12-15,29].
To our knowledge this is the first study on Iranian population investigating some epigenetic factors in prostate cancer. The association of p16 and RARB genes methylation with prostate cancer has been already examined in a number of studies[22-28,37].
In patients with good prognosis, 33.3% had RARB genes hypermethylation, and it was present in 71.4% of cases with poor prognosis. This was comparable with previous studies. Nakayama, et al. investigating RARB methylation in Japanese patients detected methylation in 79% of primary prostate cancers, 90% of hormone-refractory prostate cancers and 50% of prostate cancer cell lines, but not in any normal prostate samples[27]. Furthermore, in a study by Bastian, et al. on German patients, promoter methylation of RARB was present in 71% of prostate cancer patients but rarely or not in BPH[28].
While 19% of our patients with good prognosis had p16 gene hypermethylation, in cases with poor prognosis, 47.6% were hypermethylated. Our patients with poor prognosis prostate cancer were several times more likely to be positive for p16 promoter methylation compared with control group. Promoter methylation of RARB and p16 genes was previously reported to be associated with clinicopathologic parameters of prostate cancer progression.
We also investigated whether hypermethylation in prostate cancer is of any prognostic value. Although there is quite a few studies on prognosis evaluation, and the prior studies did not use a common prognosis definition of PSA, tumor stage, or tumor grade, our results are compatible with the preceding studies[6,14,15,25,29]. Most studies concur that the frequency of hypermethylated genes increases gradually with stage and grade in prostate carcinoma. Our study showed that hypermethylation of both RARB and p16 genes could be used as a prognostic factor in prostate cancer patients. This could mean that low stage and low grade tumors with multiple hypermethylated genes need to be regarded as high-risk cases. However, more studies are needed to have a better understanding of the molecular mechanisms of the prostate cancer to develop more effective prognostic and therapeutic approaches.
ACKNOWLEDGEMENTS
We would like to express our sincere gratitude to Sanofi-Aventis for the study support.
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