Estrogen-related receptors alpha, beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma

Abstract

Background: 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. 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.

Methods: Expression 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.

Results: We 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.

Conclusion: Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.

Keywords: Breast cancer; EZH2; Orphan nuclear receptors.

Fig. 1

Negatively correlated expression of ERRα, ERRβ & ERRγ and EZH2 in primary breast tumor tissues. a (i), Expression of ERRα, ERRβ, ERRγ and EZH2 in three different grades of breast tumors was visualized by immunohistochemistry (Magnification 4X (Scale bar: 500 μm), 40X (Scale bar: 50 μm)). (ii), Graphs show the Q-score for the expression of genes in the breast tissues. b, Relative expression of ERRα, ERRβ, ERRγ and EZH2 in different grades of breast tumor as observed in MERAV online expression dataset. One-way ANOVA was used for statistical analysis for experiments done in triplicate.* P < 0.05, ** P < 0.005, *** P < 0.001

Fig. 2

Differential expression of ERRα, ERRβ, ERRγ and EZH2 in normal and cancerous breast cell lines. a, Scatter plot shows relative expression of estrogen-related receptors and EZH2 in breast cancerous and non-cancerous cell line dataset of MERAV. b, mRNA expression level of orphan nuclear receptors in breast cancer cell lines including normal breast epithelial cells. c, Immunoblot shows the protein expression of the genes in different breast cells. Graphs were plotted with SD, which is calculated from three independent experiments. One-way ANOVA was used for statistical analysis for experiments done in triplicate.* P < 0.05, ** P < 0.005, *** P < 0.001

Fig. 3

Orphan nuclear receptors shares a negative correlation with EZH2 in breast cancer. Correlation of EZH2 with ERRα(i), ERRβ(ii) and ERRγ(iii) in normal breast epithelial cells (a), breast cancerous cells (b), normal breast tissues (c) and primary breast tumor (d) as evidenced in MERAV expression dataset. Graph was plotted and Pearson correlation coefficient was computed using GraphPad Prism software

Fig. 4

EZH2 overexpression in normal breast epithelial cells affects the expression of orphan nuclear receptors. a, Western blot shows the alteration in the expression of ERRs upon ectopic expression of EZH2 in (i), MCF-7 cells and in normal breast epithelial (ii) MCF10A cells. b, Transcript level of ERRs in (i) MCF-7 and (ii) MDA-MB-231 upon transfection of cDNA construct of EZH2 (iii). Graphs were plotted with SD, which is calculated from three independent experiments. One-way ANOVA was used for statistical analysis for experiments done in triplicate. ** P < 0.005, *** P < 0.001

Fig. 5

Silencing of EZH2 enhances the expression of ERRα, ERRβ, ERRγ and EZH2 in breast cancerous cell lines. a, Western blot shows the enriched expression of ERRs upon EZH2 silencing in (i), MCF-7 and (ii) MDA-MB-231 cancerous cells. b, Graphs show the transcript level of ERRs in (i) MCF-7 and (ii) MDA-MB-231 cells upon transfection of EZH2si (iii). Graphs were plotted with SD, which is calculated from three independent experiments. One-way ANOVA was used for statistical analysis for experiments done in triplicate.* P < 0.05, ** P < 0.005, *** P < 0.001

Fig. 6

EZH2 interacts with ERRα and ERRβ in-vivo. a, Diagram shows the putative EZH2 binding sites on ERRα promoter. b, Graphs show the fold enrichment of EZH2 at two binding sites at ERRα promoter in MCF-7(i) and MDA-MB-231(ii) cells. c. Diagram shows the putative EZH2 binding sites on ERRβ promoter and downstream genomic loci. D, Graphs show the fold enrichment of EZH2 at two binding sites at in-gene region of ERRβ in MCF-7(i) and MDA-MB-231(ii) cells