Alternative 5' Untranslated Regions Regulate Estrogen Receptor Beta Expression In Breast Carcinogenesis

Abstract Abstract #604 Background: Estrogen receptor (ER) expression is a key determinant of breast tumour behaviour, although the role of ERβ, unlike the well-studied ERα, remains uncertain. This is partly because analyses have been confused by discrepancies between ERβ mRNA and protein levels. We have identified 3 alternative 5'-untranslated regions (5'UTRs) for ERβ: UTRa (including upstream exon 0K), UTRa long (UTRa containing an additional 5' sequence) and UTRb (including upstream exon 0N) from the literature and EST databases. We demonstrate that these alternative 5'UTRs allow differential post-transcriptional regulation of ERβ expression, which may be responsible for non-concordance of mRNA and protein and could provide an important level of modulation of ER activity.
 Methods and Results: Semi-quantitative PCR was used to show that ERβ 5'UTRs are differentially expressed between breast normal and tumour tissues. We investigated the properties of these 5'UTRs using established reporter assays, in which each 5'UTR was cloned upstream of a GFP reporter. A panel of breast cell lines were transiently transfected with either an unmodified GFP reporter as a control (this was identical to experimental vectors except for its non-specialised 5'UTR), or with equal copy numbers of specific 5'UTR reporters. Effects of each 5'UTR on translation were assessed by measurement of relative GFP protein and mRNA expression from each plasmid using flow cytometry and qPCR. Our results are the first to show that each 5'UTR has a profound and differential influence on mRNA translational efficiency. We also investigated the sequence determinants of these effects by mutating the AUG start codon of each upstream open reading frame (uORF) within these 5'UTRs to UUG or AUC by site-directed mutagenesis. We determined that the presence of uORFs partly mediates inhibition of translation. In addition, using gene-specific primers for cDNA synthesis we enriched for ERβ1, ERβ2 and ERβ5; isoforms of ERβ that are particularly relevant in breast cancer. Interrogation of the 5' ends by qPCR revealed that each 5'UTR is preferentially associated with mRNA variants containing different 3' splicing patterns, which code for different biological functions. Furthermore, using a 424-case breast cancer tissue microarray (TMA), we identified a positive correlation between the expression of ERβ1 and ERβ2, and eIF4E; a basal translation factor that enhances the translation of mRNAs with highly structured 5'UTRs. Finally, an over-expression of eIF4E de-repressed translation of GFP in cells transfected with the UTRb-specific GFP reporter. This indicates that secondary structure within UTRb also mediates inhibition of translation and suggests a role for cross-talk between 5'UTRs and cellular factors in defining ERβ expression.
 Discussion: Post-transcriptional regulation plays an important role in determining the level of ERβ protein expression and may therefore have an influence on overall ER activity. This may have important implications on our understanding of breast cancer biology and treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 604.