Functional Epigenetic Analysis of Prostate Carcinoma: A Role for Seryl-tRNA Synthetase?

Abstract

Transcriptional silencing, as a result of aberrant promoter hypermethylation, is a common mechanism through which genes in cancer cells become inactive. Functional epigenetic screens using demethylating agents to reexpress transcriptional silenced genes may identify such inactivated genes for needing further evaluation. We aimed to identify genes so far not known to be inactivated by promoter hypermethylation in prostate cancer. DU-145 and LNCaP cells were treated with the DNMT inhibitor zebularine. Expression changes of total RNA from treated and untreated cells were compared using an RNA expression microarray. Genes upregulated more than 2-fold were evaluated by RT-qPCR in 50 cases of paired normal and tumor tissues of prostate cancer patients. SARS was found to be downregulated in prostate cancer in 42/50 cases (84%). In addition, GADD45A and SPRY4 showed a remarkable diminished expression (88% and 74%, resp.). The gold standard for promoter hypermethylation-inactivated genes in prostate cancer (GSTP1) was repressed in 90% of our patient samples. ROC analyses reported statistically significant AUC curves in SARS, GADD45A, and GSTP1 and positive Spearman correlations were found between these genes. SARS was discovered to be a novel gene that is repressed in prostate cancer and could therefore be recommended for its involvement in prostate carcinogenesis.

Figure 1

Number of shared ≥1.5-fold upregulated genes in Venn diagrams in three independent biological experiments in PCa cell lines DU-145 (a) and LNCaP (b) after treatment with the demethylating agent zebularine.

Figure 2

Expression of downregulated candidate genes SPRY4, SARS, GADD45A, and GSTP1 in prostate nonmalignant and malignant tissue samples. RT-qPCR was performed from 50 paired prostate tissue samples. Values are given in boxes (white: nonmalignant; black: malignant) that represent lower and upper quartiles with medians as horizontal line. Whiskers depict the 10–90 percentiles. Considered significances (P < 0.05) are calculated with Wilcoxon signed rank test for all genes. Data are given in Table 3.

Figure 3

Receiver operating characteristic (ROC) curve for the significantly downregulated candidate genes GADD45A, SARS, SPRY4, and GSTP1 to discriminate between tumor and adjacent normal samples. Comparison of area under the receiver operating characteristic curve (AUC) of candidate genes with AUC of GSTP1. Data are given in Table 4.