The Association of Retinoic Acid Receptor Beta2(RARβ2) Methylation Status and Prostate Cancer Risk: A Systematic Review and Meta-Analysis

The retinoic acid receptor beta2(RARβ2) is a type of nuclear receptor that is activated by both all-trans retinoic acid and 9-cis retinoic acid, which has been shown to function as a tumor suppressor gene in different types of human tumors. Previous reports demonstrated that the frequency of RARβ2 methylation was significantly higher in prostate cancer patients compared with controls, but the relationship between RARβ2 promoter methylation and pathological stage or Gleason score of prostate cancer remained controversial. Therefore, a meta-analysis of published studies investigating the effects of RARβ2 methylation status in prostate cancer occurrence and association with both pathological stage and Gleason score in prostate cancer was performed in the study. A total of 12 eligible studies involving 777 cases and 404 controls were included in the pooled analyses. Under the random-effects model, the pooled OR of RARβ2 methylation in prostate cancer patients, compared to non-cancer controls, was 17.62 with 95%CI = 6.30–49.28. The pooled OR with the fixed-effects model of pathological stage in RASSF1A methylated patients, compared to unmethylated patients, was 0.67 (95%CI = 0.40–1.09) and the pooled OR of low-GS in RARβ2 methylated patients by the random-effect model, compared to high-GS RARβ2 methylated patients, was 0.54 (95%CI = 0.28–1.04). This study showed that RARβ2 might be a potential biomarker in prostate cancer prevention and diagnosis. The detection of RARβ2 methylation in urine or serum is a potential non-invasive diagnostic tool in prostate cancer. The present findings also require confirmation through adequately designed prospective studies.


Introduction
Prostate cancer is the most commonly diagnosed noncutaneous neoplasia in the world. The disease predominantly affects men after the 6th decade of life and is associated with considerable morbidity and mortality [1]. Curative treatment entails radical prostatectomy or radiotherapy, and the best outcome is seen in patients with the earliest stage disease [2,3]. Patients with locally advanced or systemic disease carry a poor long-term prognosis because of the notable lack of curative therapy [4].
Recently, methylation of CpG islands within the promoter and/ or 59 regions of genes is recognized as a common alteration in cancer-related genes often associated with partial or complete transcriptional disruption [5]. This epigenetic alteration provides an alternative pathway to gene silencing in addition to gene mutation or deletion, which is suggested to be a new molecular marker for early cancer detection [6]. The retinoic acid receptor (RAR) is a type of nuclear receptor that is activated by both alltrans retinoic acid and 9-cis retinoic acid [7]. There are three retinoic acid receptors (RAR), RARa, RARb, and RARc, which are differentially expressed during development and in adult life, and there is strong evidence that RARb plays a central role in growth regulation of epithelial cells and in tumorigenesis [8][9][10]. The human RARb gene generates multiple isoforms by use of promoters P1 and P2 and alternative splicing [11,12]. P1 directs the transcription of isoform RARb1, whereas P2 promotes the transcription of isoforms RARb2 and RARb4 [13].
The RARb2 is mapped to chromosomal region 3p24 (2477/ +392, GenBank accession numbers S82362 and M96016), which is expressed in most tissues and has been shown to function as a tumor suppressor gene in lung, breast, and gynecological neoplasia [14][15][16][17]. The RARb2 promoter is characterized by a CpG (cytidine phosphate guanosine)-rich region, the CpG island [18], which is located in the 59-untranslated region, along with several motifs that are potential binding sites for transcription factors such as AP-1, AP-2, and Sp1. Moreover, RARb2 was shown recently to be frequently hypermethylated in several primary human neoplasms, including prostate cancer [19][20][21]. All of these findings suggested that it might play a pivotal role in the development of human cancer.
To date, RARb2 methylation has been proved in a number of individual studies, which is detected not only in tissue samples but also in urine and serum samples. The prognostic value of RARb2 methylation status in prostate cancer patient's diagnosis and the relationship between RARb2 methylation and pathological stage of prostate cancer and Gleason score remains unclear. Therefore, a systematic review was performed of the literature with metaanalysis to obtain a more accurate evaluation of the role of RARb2 methylation in prostate cancer management.

Publication selection
Studies were identified via an electronic search of PubMed and Google Scholar using the following key words: prostate cancer, PCa, retinoic acid receptor b2, RARb2, RARbeta2, methylation. We also manually searched the references of these publications in order to retrieve additional studies. Only those published as fulltext articles and in English were included as candidates. The search updated on 28, December,2012.

Inclusion and exclusion criteria
Studies were selected for analysis if they met the following criteria: 1) they were original epidemiological studies on the correlation between RARb2 promoter methylation and the prognosis of prostate cancer patients; 2) RARb2 methylation status was examined using methylation-specific PCR (MSP) or quantitative MSP (QMSP); 3)the subjects in every study comprised patients and non-cancer controls; 3) studies should be with full text not only abstracts for relevant information extraction; 4) when the same patient population reported in several publications, only the most recent report or the most complete one was included in this analysis to avoid overlapping between cohorts; 5) the numbers of patients and controls in each study should be more than 5 respectively.

Data collection
For each eligible study, we collected information regarding authors, year and source of publication, country of origin, inclusion criteria, exclusion criteria, pathological stage, Gleason score, RARb2 methylation frequencies in non-cancer controls and patients of prostate cancer and the method for methylation detection. All included studies used non-cancer people as a control group, though some of them did not provide the definition of noncancer. In studies defining non-cancer people, there are two definitions: (1) normal healthy person; (2) people with benign hyperplasia prostate. Since it is impossible to redefine non-cancer people on a unified standard, we combined non-cancer people in our meta-analysis according to their original group in each individual study. Of these studies, pathological stage # T2 was defined as low-stage, and pathological stage $ T3 was defined as high-stage which were defined by clinical differentiation. Gleason score$7 was defined as high-GS and Gleason score#6 was defined as low-GS. The final eligible articles selected for further meta-analysis were evaluated independently by two reviewers. Minor discrepancies were resolved by the authors' discussion.

Meta-analysis and statistical analysis
The foremost analysis examined the differences in the frequency of RARb2 methylation between prostate cancer patients and noncancer people by odds ratio (OR) with the corresponding 95% CI. Moreover, the strength of association between RARb2 methylation and patients' pathological stage and tumor Gleason score were also assessed by OR with the corresponding 95% CI. To assess heterogeneity across the studies, the statistics analysis for heterogeneity was performed [22]. If the studies were shown to be homogeneous with P.0. 05 for the Q-statistics, the summary of OR was calculated by a fixed-effects model (the Mantel-Haenszel method) when between-study heterogeneity was absent [23]. Otherwise, a random-effects model (the DerSimonian and Laird method) was selected [24]. In addition, stratified analyses were also performed by material and method. Furthermore, a sensitivity analysis, by which a single study in the meta-analysis was deleted each time to determine the influence of the individual data set to the overall pooled OR, was performed to assess the stability of the results. The potential publication bias was examined visually in a funnel plot of log [OR] against its standard error (SE), and the degree of asymmetry was tested by Egger's test [25]. This metaanalysis was performed using the software STATA version 12. 0. All P-values were based on two-sided tests and a P-value of less than 0. 05 was considered statistically significant.

Meta-analysis
In general, the frequencies of RARb2 methylation were tested in 12 reliable studies. The main results were summarized in Table 2. Under the random-effects model, the pooled OR of RARb2 methylation in prostate cancer patients, compared to noncancer controls, was 63.44 with 95%CI = 23.94-168.11. In the stratified analysis by material, significantly increased risks were found in tissues samples in detection RARb2 methylation in prostate cancer(OR = 67.85, 95%CI = 33.75-136.42) and in nontissues (OR = 46.76, 95%CI = 2.68-817.26). As stratified analysis by method, significantly increased risks were also found in MSP(OR 89. 15, 95% CI = 31.93-248.90) and QMSP (OR = 47.10, 95%CI = 10.59-209.53). In the evaluating the association between RARb2 methylation and pathological stage in prostate cancer, study was carried out in five studies by fixedeffect model. The pooled OR of low-stage in RARb2 methylated patients, compared to high-stage RARb2 methylated patients was 0.67 (95%CI = 0.40-1.09, table3). The relationship between RARb2 methylation and tumor Gleason score was also compared by random-effect model. The pooled OR of low-GS in RARb2 methylated patients, compared to high-GS RARb2 methylated patients was 0.54(95%CI = 0.28-1.04, table3).

Publication bias
As shown in Figure 1A, the shape of the funnel plots seemed asymmetrical in the methylation comparison between prostate cancer patients and non-cancer controls, suggesting the presence of publication bias. Then, the Egger's test provides statistical evidence of funnel plot asymmetry (t = 2. 38, P = 0. 038). To adjust this bias, a trim-and-fill method developed by Duval and Tweedie [38] was implemented ( Figure 1B). The pooled OR of RARb2 methylation in prostate cancer patients, compared to non-cancer controls, was 17.62 with 95%CI = 6.30-49.28 by trim-and-fill method (table2). Meta-analysis with or without the trim-and-fill method did not draw different conclusions, indicating that our results were statistically robust. Funnel plot and Egger's test were performed to assess the publication bias in studies of association between RARb2 methylation and pathological stage/Gleason score, The shape of the funnel plot did not indicate any evidence of obvious asymmetry (figure 2, 3) and the Egger's test suggested the absence of publication bias (P.0.05).

Discussion
The results of our systematic review showed that RARb2 methylation in prostate cancer was associated with tumor risk as either detected in urine, serum or tissue by MSP or QMSP. However, the RARb2 methylation was not associated with increased risk for developing pathological stage or Gleason score of prostate cancer in comparison between RARb2 methylated bladder cancer patients and unmethylated patients.
Hypermethylations of the RARb2 gene having been reported in many studies declared that the frequency of RARb methylation was found to be significantly higher in patients group compared with controls [21,[26][27][28][29][30][31][32][33][34][35][36][37]. Previous reports also demonstrated that genetic variations of RARb affect prostate cancer susceptibili-  ty [39].To further confirm RARb2 promoter methylation status in prostate cancer patient' s diagnosis, we carried out a meta-analysis of 12 studies involving 777 cases and 404 controls to derive a more precise estimation of the association. The analysis showed that RARb2 methylation in prostate cancer patients, compared to noncancer controls, was 17.62 times higher than that in non-cancer people after the trim-and-fill method which further confirmed RARb2 methylation was a potential risk factor for prostate cancer as detected both in urine, serum and tumor tissues. MSP is a nonquantitative nonfluorometric PCR method to investigate promoter methylation. This method may fail to detect low concentrations of methylated alleles, unlike QMSP which can detect up to 1/1000 methylated alleles [40]. In this meta-analysis, both MSP and QMSP have the same effect in RASSF1A methylation detection. However, Carmen Jerónimo's study suggested that RARb2 methylation levels correlated with pathological tumor stage but not with Gleason score [32]. R. Dumache's study demonstrated that RARb2 methylation was correlated not only pathological tumor stage but also Gleason score [26]. But in Woodson K's study, methylation of RARb2 was proved to correlate with tumor grade but not pathological stage [41]. To resolve the conflicting results, we carried out a meta-analysis which indicated that the RARb2 methylation status did not correlate with either the pathological stage or Gleason score of prostate cancer patients, suggested that inactivation of RARb2 may be an early event in prostate carcinogenesis. Early diagnosis of prostate cancer currently relied on transrectal ultrasound guided needle biopsy (10 to 12 cores) in men with increased total PSA (greater than 4.0 ng/ml) and/or abnormal DRE findings [42]. However, 65% to 70% of men with total PSA in the 4.0 to 10.0 ng/ml range had a negative prostate biopsy result [43]. In addition, more than 20% of men with PSA in the 2.0 to 4.0 ng/ml range were found to have cancer when evaluated by prostate biopsy [44]. More troublesome, there was no conclusive evidence that screening based on PSA decreases prostate cancer mortality [45]. Early detection of prostate cancer may be made more effective and efficient as RARb2 may be an early biomarker in prostate carcinogenesis diagnosis.
Cancer is not a single cell disease. Aberrant cancer cells and their interactive microenvironment are needed for cancer to progress to androgen independence and distant metastasis [46]. Tumor heterogeneity in methylation patterns may be influenced by response to the microenvironment and local expression of genes, hormones, oxidative stress, or some other factor that can  RARb2 Methylation Status and Prostate Cancer Risk PLOS ONE | www.plosone.org modulate methylation. Tsuyoshi Nakayama'study indicated that three CpG sites (numbers 20 to 22) near the bRARE region were consensus regions of methylation in PCa, which might be critical for the silencing of the gene by blocking access of liganded RAR/ RXR heterodimers and other cis-acting transcription factors to their binding sequences [35].
RARb2 might be silenced not only by DNA methylation but also by histone deacetylation. Acetylation and deacetylation on lysine residues of histone amino-terminal tails had profound effects on gene transcription [47]. The RARb promoter was under the control of a high-affinity retinoic acid response element itself. Thus, once silencing of RARb had occurred, the lack of RAR-beta might reinforce the inactive silent state at its own promoter probably promoting methylation as a secondary repression mechanism [48]. Fuks F's study demonstrated that RARb methylation-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both H3 and H4. Combined TSA and all-trans retinoic acid treatment after 5-azacytidine treatment increases the accumulation of acetylated histones, leading to reactivation of the methylated RARb promoter and subsequently the expression of RARb [49].
In conclusion, our meta-analysis suggested that detection of RARb2 methylation might be a potential biomarker diagnostic tool in prostate cancer. The detection of RARb2 methylation in urine or serum is a potential non-invasive diagnostic tool in prostate cancer. It is necessary to conduct large sample size studies of the association between RARb2 methylation and prostate cancer risk, eventually leading to our better understanding.

Author Contributions
Conceived and designed the experiments: TG SW. Performed the experiments: TG LG. Analyzed the data: TG BH YP. Contributed reagents/materials/analysis tools: LC ZN RL YX. Wrote the paper: TG.