Deregulation of an imprinted gene network in prostate cancer

Abstract

Multiple epigenetic alterations contribute to prostate cancer progression by deregulating gene expression. Epigenetic mechanisms, especially differential DNA methylation at imprinting control regions (termed DMRs), normally ensure the exclusive expression of imprinted genes from one specific parental allele. We therefore wondered to which extent imprinted genes become deregulated in prostate cancer and, if so, whether deregulation is due to altered DNA methylation at DMRs. Therefore, we selected presumptive deregulated imprinted genes from a previously conducted in silico analysis and from the literature and analyzed their expression in prostate cancer tissues by qRT-PCR. We found significantly diminished expression of PLAGL1/ZAC1, MEG3, NDN, CDKN1C, IGF2, and H19, while LIT1 was significantly overexpressed. The PPP1R9A gene, which is imprinted in selected tissues only, was strongly overexpressed, but was expressed biallelically in benign and cancerous prostatic tissues. Expression of many of these genes was strongly correlated, suggesting co-regulation, as in an imprinted gene network (IGN) reported in mice. Deregulation of the network genes also correlated with EZH2 and HOXC6 overexpression. Pyrosequencing analysis of all relevant DMRs revealed generally stable DNA methylation between benign and cancerous prostatic tissues, but frequent hypo- and hyper-methylation was observed at the H19 DMR in both benign and cancerous tissues. Re-expression of the ZAC1 transcription factor induced H19, CDKN1C and IGF2, supporting its function as a nodal regulator of the IGN. Our results indicate that a group of imprinted genes are coordinately deregulated in prostate cancers, independently of DNA methylation changes.

Keywords: DNA methylation; ZAC1; cancer epigenetics; differentially methylated region; imprinted gene network; imprinted genes; prostate cancer; pyrosequencing.

Figures

Figure 1. Expression of imprinted genes in prostate benign and cancer tissues. The mRNA expression of the indicated imprinted genes relative to TBP in benign (n = 13) and tumor (n = 45) prostate tissues was assessed by qRT-PCR and is represented as boxplots. Mann-Whitney-U test was used to evaluate the differences between the two groups. The significance (p) is shown above the bracket in each individual panel (* P < 0.05, ** P < 0.01).

Figure 2. Representation of the significant correlations among expression of imprinted genes (A) and between imprinted genes and EZH2, ERG and HOXC6 genes expression (B) in prostate cancer tissues. Spearman correlation analysis was performed in SPSS software using gene expression data (obtained by qRT-PCR) for 45 prostate cancers. Thicker lines represent correlations with P < 0.01, thinner lines –correlations with P < 0.05; positive correlations are shown in blue, negative ones - in red. For exact values see Table 1.

Figure 3. Methylation of imprinted DMRs in benign and tumor prostatic tissues. DNA methylation (%) of the indicated imprinted DMRs in benign and tumor prostatic tissues was quantitated by bisulfite pyrosequencing. The boxplots represent the mean methylation values of all assessed CpG positions for the indicated region for benign or tumor samples. Mann-Whitney-U test was used to evaluate the differences between the two sample groups; none of the differences was significant at P < 0.05. Note the exceptionally large variation in the H19 DMR.

Figure 4. Biallelic expression of PPP1R9A in prostate benign and cancer tissues. Heterozygosity at the SNP rs854544 in the coding sequence of PPP1R9A was determined by SNP pyrosequencing analysis using genomic DNA (gDNA). Biallelic expression of the gene is evident from the presence of two peaks for the polymorphism A/G, as examined by pyrosequencing analysis of PCR products using cDNA as a template from benign (“pN”s in upper panel) and tumor (“pTu”s in lower panel) heterozygous samples.

Figure 5. Influence of androgens on imprinted genes expression. Expression of the indicated imprinted genes was measured relative to TBP mRNA by qRT-PCR in LNCaP cells treated for 3, 8 or 24 h with 10 nM synthetic androgen R1881 (black boxes) or with solvent only (gray circles). Treatments were performed in biological duplicates and qRT-PCR was performed in duplicate for each sample, whereby less than 10% variation between duplicates was accepted. The data points with error bars represent mean values from the biological duplicates normalized to the expression of the untreated control at 3 h that was set to 100%. Standard deviation is presented as error bars. *Significant changes (P < 0.05) as calculated by T-Test.

Figure 6. Induction of imprinted genes by transient ZAC1 overexpression. mRNA expression of the indicated imprinted genes was measured by qRT-PCR in 22Rv1 cells transfected for 48 h with an expression vector containing cDNA encoding the full-length ZAC1 protein (ZAC1) or the shorter ZAC1 protein isoform (ZAC1Δ). As controls, the empty vector (vector) or the same vector containing the lacZ gene (lacZ) were employed. Transfections were performed in duplicates, PCR for each of which was performed in duplicates. The data presented are mean values from the biological duplicates (bars) of the mRNA expression relative to TBP including standard deviation (error bars). Notably, transfection of the short ZAC1Δ form resulted in higher ZAC1 protein amounts than transfection of the full-length ZAC1 form, as assessed with western blot analysis (insert in the first panel). α-Tubulin was detected as a loading control. The protein samples were loaded in the same sequence as shown in the panel