To explore the potential role of CpG island methylation in the transcriptional silencing of the TNFRSF10C
gene, we checked the methylation status in 59 pairs of DNA samples isolated from fresh-frozen prostate cancer tissues and matched normal controls with bisulfite sequencing, which is the gold standard for mapping methylation across CpG sites. After subcloning of PCR product generated from bisulfite-treated DNA using a pCR4-TOPO vector, sequencing of each selected clone provided the precise methylation status for 13 CpG sites in and near the TNFRSF10C
promoter CGI (as seen in , 12 CpG sites within CGI and an additional CpG site located outside the CGI). Among these 13 CpG sites, six of them were also surveyed by another study [16
]. Overall, 48 of the 59 primary prostate cancer samples (81.4%) showed evidence of hypermethylation of the TNFRSF10C
promoter CGI (). Additionally, TNFRSF10C
promoter CGI hypermethylation in two of the matched normal prostate controls (2 we only detected.3%) (). The proportion of TNFRSF10C
promoter CGI hypermethylation in normal prostate tissue is consistent with two other studies [16
Figure 1 Schematic representation of TNFRSF10C promoter region. The TNFRSF10C transcription start site is marked with a +1 and an arrow. Amplicon (−257 ~ +30) for sequencing and CpG island (−130 ~ +413) are indicated by filled and stripped bars, (more ...)
Methylation and deletion status of TNFRSF10C in primary prostate cancer samples.
We then evaluated whether TNFRSF10C promoter CGI hypermethylation was associated with clinical/pathological features like Gleason score. As the data shown in , hypermethylation of the TNFRSF10C promoter CGI occurred equally frequently in both low-grade (Gleason score ≤7) and high-grade tumors (Gleason score ≥8) (81.1% vs. 81.0%).
Clinical characteristics of prostate cancer samples with TNFRSF10C methylation and deletion.
The deletion status of TNFRSF10C
in 59 pairs of samples was reanalyzed using our previous Affymetrix 100K and/or 500K SNP mapping array data [27
]. We used the same criteria to define copy number changes and found that among 59 prostate cancer samples, 44 (74.5%) showed hemizygous deletion of the TNFRSF10C
locus. No homozygous deletions of the TNFRSF10C
locus were detected. Similarly, no association between deletion and Gleason scores (73.0% vs.
76.2% between Gleason score ≤7 and Gleason score ≥8) was observed.
To test whether biallelic inactivation, with one allele deleted and the other allele inactivated by promoter CGI hypermethylation, occurred for TNFRSF10C, we compared the frequency of TNFRSF10C promoter CGI methylation between TNFRSF10C deleted and non-deleted groups. As shown in , TNFRSF10C promoter CGI methylation was observed in 81.8% of TNFRSF10C deleted tumors, compared to 80.0% in TNFRSF10C retained tumors. There was no obvious trend in the frequency of promoter CGI hypermethylation among tumors with TNFRSF10C deletion compared to tumors without TNFRSF10C deletion. Additionally, there is no significant difference in biallelic inactivation frequency of TNFRSF10C between low grade tumors and high grade tumors, although the total biallelic inactivation frequency is 61% (data not shown). Interestingly, the great majority of the tumors examined (94.9%, 56 out of 59) harbor either a hemizygous deletion or promoter CGI hypermethylation of TNFRSF10C. The three tumors without TNFRSF10C hemizygous deletion and promoter CGI hypermethylation are all low grade tumors.
We also analyzed TNFRSF10C promoter CGI methylation and deletion status in three prostate cancer cell lines (LNCaP, PC3 and DU145) using bisulfite sequencing of multiple subclones and by examination of previous Affymetrix 100K SNP array data. PC3 and LNCaP showed dense methylation of the TNFRSF10C promoter CGI and DU145 showed more heterogeneity in the TNFRSF10C promoter CGI methylation status, with low frequency of methylation in each CpG site (). Among these three prostate cancer cell lines, hemizygous deletion of the TNFRSF10C loci was only found in PC3 but not in the other two cell lines.
Figure 2 Reduction of TNFRSF10C hypermethylation and reactivation of TNFRSF10C expression in prostate cancer cells in vitro by treatment with a demethylating agent. A. Methylation status of TNFRSF10C in 3 prostate cancer cell lines was analyzed by bisulfite sequencing. (more ...)
Transcriptional silencing of TNFRSF10C by promoter CGI hypermethylation has not been reported in PCa. To evaluate the effect of promoter CGI hypermethylation on TNFRSF10C expression, the PC3 prostate cancer cell line was treated with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (decitabine). The methylation status of TNFRSF10C promoter CGI was analyzed using bislufite sequencing of multiple subclones. Bisulfite sequencing analysis of the TNFRSF10C promoter CGI in PC3 demonstrated that treatment of 5-aza-2’-deoxycytidine resulted in demethylation in the TNFRSF10C promoter CGI (). Real-time PCR results demonstrated that partial demethylation of TNFRSF10C promoter CGI was accompanied by a 29.6 fold increase in the transcriptional expression of TNFRSF10C (SD = 1.06, ). The data indicate that silencing of the TNFRSF10C gene was achieved through promoter methylation in this prostate cancer cell line.
To determine whether TNFRSF10C promoter CGI methylation and deletion affects gene expression, we performed real-time PCR to measure expression levels in a subset of tumor and normal samples for which RNA samples are also available. In total, there are 18 tumor samples (11 of them harboring both TNFRSF10C deletion and methylation, four of them harboring only TNFRSF10C deletion but not TNFRSF10C methylation, and the rest of the three tumors with TNFRSF10C methylation but not TNFRSF10C deletion). Differences in TNFRSF10C gene expression among these four groups were statistically significant (p=0.00012, General Linear Model test). There were also statistically significant differences between baseline group (normal tissue) and the tumor group harboring both deletion and methylation (mean difference in ΔCt for TNFRSF10C= 2.17, 95% CI= 1.21 to 3.13, p < 0.001), and between baseline group (normal tissue) and the tumor group harboring only promoter methylation but not deletion (mean difference in ΔCt for TNFRSF10C= 1.42, 95% CI= 0.023 to 2.82, p = 0.03) ().
Figure 3 Correlation of TNFRSF10C hypermethylation and deletion with TNFRSF10C gene expression in prostate tumors and normal tissues. The y-axis indicates ΔCt of TNFRSF10C. The x-axis indicates four groups of tissues in comparison: 1) normal tissues with (more ...)