Abstract
BackgroundOrphan nuclear receptors ERRα, ERRβ and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERRα, ERRγ and less about ERRβ is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer.MethodsExpression of ERRα, ERRβ, ERRγ and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERRα, ERRβ, ERRγ and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERRα, ERRβ, ERRγ by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR.ResultsWe found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERRα and ERRβ was observed in-vivo.ConclusionOur findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.
Highlights
Orphan nuclear receptors ERRα, ERRβ and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology
Expression of ERRα, ERRβ, ERRγ and Enhancer of zeste homolog 2 (EZH2) in primary breast tumors of different grades To study the expression of EZH2 and estrogen-related receptors (ERRs) in breast cancer patient samples, immunohistochemistry was performed simultaneously in four identical tissue arrays consisting nineteen cases with less histology variances
In comparison to normal breast epithelial cells ERRα, ERRβ, ERRγ and EZH2 differentially express in breast cancerous cells To investigate the expression pattern of EZH2 and orphan nuclear receptors in cell lines, we checked their expression using quantitative real time PCR & immunoblot and by analyzing online data as well
Summary
Orphan nuclear receptors ERRα, ERRβ and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Function and regulation of ERRα, ERRγ and less about ERRβ is reported, role of histone methylation in their altered expression in cancer cells is not studied. Based on the expression of estrogen/progesterone receptor and human epidermal growth receptor 2, there are four major molecular intrinsic subtypes of breast cancer- luminal A (ER+/HER−), luminal B (ER +/HER2-or HER2+), triple negative/basal type and HER2 type Apart from these receptors there are receptors called estrogen-related receptors (ERRs) which share about 68% sequence homology in DNA binding domain (DBD) and significant sequence homology in ligand binding domain (LBD) with estrogen receptor [1]. Our results identify EZH2 as a functional coregulator for estrogen-related receptors especially ERRα and ERRβ in breast carcinoma
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