Abstract
Estrogen receptor β (ERβ) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. In contrast to ERα, its closest homolog, ERβ shows significant estrogen-independent activities, including the ability to inhibit cell cycle progression and regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERβ in BC MCF-7 and ZR-75.1 cells by means of microRNA (miRNA) sequencing, we identified 30 miRNAs differentially expressed in ERβ+ versus ERβ- cells in the absence of ligand, including up-regulated oncosuppressor miRs such miR-30a. In addition, a significant fraction of >1,600 unique proteins identified in MCF-7 cells by iTRAQ quantitative proteomics were either increased or decreased by ERβ, revealing regulation of multiple cell pathways by ligand-free receptors. Transcriptome analysis showed that for a large number of proteins regulated by ERβ, the corresponding mRNAs are unaffected, including a large number of putative targets of ERβ-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERβ. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERβ in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration was significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERβ on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation might represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.
Highlights
From the ‡Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery, University of Salerno, 84081 Baronissi (SA), Italy; §Department of Science and Technologies, University of Sannio, 82100 Benevento, Italy; ¶IRGS, Biogem, Via Camporeale, 83031 Ariano Irpino, Avellino, Italy; ʈProtein Chemistry Unit, Biomedicum Helsinki, University of Helsinki, 00290 Helsinki, Finland; **Institute of Biotechnology, University of Helsinki, 00790 Helsinki, Finland
To explore this possibility in the case of hormone-responsive breast cancer (BC), we investigated here the ability of unliganded Estrogen receptors (ERs) to control BC cell functions in a model represented by MCF-7 cell clones stably expressing fulllength human ER carrying a TAP tag at the N terminus (Nt-ER) or C terminus (Ct-ER), which has been shown to be a good model for studying the activity of this ER subtype in hormone-responsive BC cells [8, 12, 28]
When considering the decline in estrogen levels during menopause or locally induced by endocrine therapies based on the use of aromatase inhibitors [67], a better characterization of the cellular pathways influenced by unliganded ER in hormone-responsive BC cells becomes important
Summary
In order to identify experimentally the mRNA targets of this miRNA in MCF-7 cells, we transiently transfected a synthetic miR-30a-5p mimic and performed a gene expression profiling analysis of transfected cells with oligonucleotide microarrays to detect significant changes in mRNA concentration induced by the transfected oligonucleotide.
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