Aberrant Epigenetic Modifications in the CTCF Binding Domain of the IGF2/H19 Gene in Prostate Cancer Compared with Benign Prostate Hyperplasia

Expression of the imprinted genes insulin-like growth factor 2 (IGF2) and H19 depends on the methylation pattern of their common imprinting control region (ICR) located on chromosome 11p15. As the somatic imprinting pattern may be lost during tumorigenesis due to epigenetic alterations, in the present study, we analyzed the DNA methylation and histone modifications in the differentially methylated region (DMR) of IGF2/H19 in benign prostate hyperplasia (BPH) and prostate carcinoma (PCa). Sodium bisulfite sequencing was performed on frozen tissue collected after radical prostatectomy. Thirty tumors and 17 non-cancerous tissue samples were analyzed. Histological diagnosis was, in addition, confirmed by amplification of the epithelial tumor marker alpha-methylacyl coenzyme-A racemase. Chromatin immunoprecipitation assay (ChIP) was carried out on sonificated chromatin from fresh tissue samples from 10 PCa, 10 BPH using antibodies against trimethyl histone H3K9, dimethyl histone H3K9, trimethyl H3K27 and acetyl H3K9. The methylation pattern of 17 CpGs within 227 bp of the H19 fragment was characterized from each DNA sample. All (BPH) samples demonstrated >80% methylation of CpGs. In contrast, we found 41% of CpGs methylated in 9 out of 30 PCa specimens. We observed statistically significant differences in the methylation state between PCa and BPH groups, especially in the DMR of H19 (p<0.0001) and in the ICR (p=0.0034), which corresponds to CTCF binding domain. ChIP assay revealed that dimethyl H3K9 is associated with the ICR of IGF2/H19 in BPH, but not in PCa (p<0.0001). Our data demonstrate that DNA methylation and histone methylation analysis of the ICR within the DMR of IGF2/H19 provides important insights into early steps of carcinogenesis and, therefore, may contribute to improving diagnosis of PCa.