Dynamic Regulation of Estrogen Receptor-β Expression by DNA Methylation During Prostate Cancer Development and Metastasis

Abstract

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Differential expression of steroid receptors in prostate tissues before and after radiation therapy for prostatic adenocarcinoma

“…We propose that ER-b may have a role in tissue repair and healing after radiation therapy. Although most authors describe low levels of ER-b expression in LNCaP, in some studies, including our current results and a recent study by Zhu et al 40 no mRNA levels could be detected. Zhu et al 40 actually demonstrated that this lack or low expression of ER-b in LNCaP is the result of epigenetic silencing and found a progressive decrease in ER-b gene expression from early passages (<7 passages) to high passages (>25 passages).…”

“…Although most authors describe low levels of ER-b expression in LNCaP, in some studies, including our current results and a recent study by Zhu et al 40 no mRNA levels could be detected. Zhu et al 40 actually demonstrated that this lack or low expression of ER-b in LNCaP is the result of epigenetic silencing and found a progressive decrease in ER-b gene expression from early passages (<7 passages) to high passages (>25 passages). In addition to the number of passages, some other variation in culture conditions may be responsible, but variation in the sensitivity of RT-PCR studies should not be excluded a priori.…”

AP-2 regulates the transcription of estrogen receptor (ER)-β by acting through a methylation hotspot of the 0N promoter in prostate cancer cells

“…No information is available about how ERb is regulated in PCa cells except for our recent study suggesting that changes of ERb expression during prostate carcinogenesis in vivo could be regulated by DNA methylation (Zhu et al, 2004). In this study, we identified one composite CpG island (CGI) spanning the proximal promoter and the untranslated 0N exon of the ERb gene (Figure 1).…”

“…The relative position of the 46 CpG sites in the three methylation centers identified in our previous study (Zhu et al, 2004) are shown in Figure 1. We used the 0N-Luc (581 bp) as a template and performed serial-and center-specific deletions to characterize the role of the sequences in these methylation hotspots of the 0N promoter.…”

DNA demethylation and histone deacetylation inhibition co‐operate to re‐express estrogen receptor beta and induce apoptosis in prostate cancer cell‐lines

“…It contains a typical CpG island within its promoter (5), and has been shown to be re-expressed in prostate cancer cell-lines treated with DNMT inhibitors such as 5-aza-2'deoxycytidine (6)(7)(8). Furthermore, methylation-specific PCR studies have shown that the promoter region of the ERβ gene is hypermethylated in prostate cancer tissue studies (6,8,9). The loss of ERβ in prostate cancer cell-lines and in tissue studies adds to an accumulating body of evidence supporting an antiproliferative role for ERβ in the prostate.…”