Estrogen receptor β1 (ERβ1) downregulation occurs in many breast cancers, but the responsible molecular mechanisms remain unclear. Here, we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumors confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-untranslated region (UTR) of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer. Cancer Res; 70(11); 4778-84. ©2010 AACR.

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

Gene expression is regulated at multiple levels, including transcriptional silencing by methylation. ERβ1 is downregulated in breast cancer compared to normal breast and mechanisms surrounding this are unclear. Two novel ERβ mRNA isoforms with distinct 5'-UTRs have been identified, ERβ-0K-1 and ERβ-0N-1. The aim of study was to examine whether methylation at these promoter regions plus a novel promoter identified by our group were involved in ERβ1 regulation (figure 1).Bisulfite modification and direct sequencing analysis were performed for promoters 0K, 0N, and a novel mini CpG island upstream of ERβ exon1 in two ERβ1- and one ERβ1+ breast cancer cell lines. Distinct methylation patterns were observed. Promoter 0N was completely methylated in BT20, partially methylated in MDAMB453 and unmethylated in T47D, while the mini CpG island was methylated in all 3 cell lines. In contrast, promoter 0K was unmethylated. Furthermore, a negative correlation between ERβ1 mRNA expression and the methylation status of promoter 0N was observed in breast cells. To further investigate whether methylation of the ERβ1 promoter was responsible for the loss of ERβ1 expression, BT20 and MDAMB453 cells were treated with either 5-aza-dC, TSA or both, concentrations of which had been previously optimised for each cell line. In BT20 cells which had complete methylation of 0N, both agents were required for induction of ERβ1 and ERβ-0N-1 but not ERβ-0K-1 expression, both of which are part of the 5'UTR region. However in MDAMB453 treatment with 5-aza-dC was sufficient to induce ERβ1 and ERβ-0N-1 expression with no additional re-expression seen with TSA and no effects on ERβ-0K-1. This suggests that promoter 0N is responsible for driving the transcription of ERβ1. On going work is confirming these observations in clinical samples. Our results suggest that promoter 0N plays an important role in regulation of ERβ1 mRNA expression in breast cancer. Our results add to growing literatures which demonstrate ERβ1 is regulated at multiple levels in breast cancer and that DNA methylation is an important mechanism for silencing ERβ1 gene expression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1148.

The human C-type lectin domain family 7 member A (CLEC7A) gene encodes a Dectin-1 protein that recognizes beta-1,3-linked and beta-1,6-linked glucans, which form the cell walls of pathogenic bacteria and fungi. It plays a role in immunity against fungal infections through pathogen recognition and immune signaling. This study aimed to explore the impact of nsSNPs in the human CLEC7A gene through computational tools (MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and PredictSNP) to identify the most deleterious and damaging nsSNPs. Further, their effect on protein stability was checked along with conservation and solvent accessibility analysis by I-Mutant 2.0, ConSurf, and Project HOPE and post-translational modification analysis using MusiteDEEP. Out of the 28 nsSNPs that were found to be deleterious, 25 nsSNPs affected protein stability. Some SNPs were finalized for structural analysis with Missense 3D. Seven nsSNPs affected protein stability. Results from this study predicted that C54R, L64P, C120G, C120S, S135C, W141R, W141S, C148G, L155P, L155V, I158M, I158T, D159G, D159R, I167T, W180R, L183F, W192R, G197E, G197V, C220S, C233Y, I240T, E242G, and Y3D were the most structurally and functionally significant nsSNPs in the human CLEC7A gene. No nsSNPs were found in the predicted sites for post-translational modifications. In the 5′ untranslated region, two SNPs, rs536465890 and rs527258220, showed possible miRNA target sites and DNA binding sites. The present study identified structurally and functionally significant nsSNPs in the CLEC7A gene. These nsSNPs may potentially be used for further evaluation as diagnostic and prognostic biomarkers.

<div>Abstract<p>Estrogen receptor β1 (ERβ1) downregulation occurs in many breast cancers, but the responsible molecular mechanisms remain unclear. Here, we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumors confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-untranslated region (UTR) of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer. Cancer Res; 70(11); 4778–84. ©2010 AACR.</p></div>