Regulation of estrogen receptor beta by 5' untranslated regions in breast carcinogenesis

Background

Estrogen receptor (ER) expression is a key determinant of breast tumour behaviour. While the role of ERα in carcinogenesis is relatively well understood, the role of ERβ, the more recently identified receptor, remains uncertain. This is partly because analyses have been confused by a consistent discrepancy between ERβ expression at mRNA and protein levels [1]. Recently, evidence has accumulated that deregulation of gene-specific translation occurs during carcinogenesis in breast and other tissues. Regulation of ERβ translation could therefore be responsible for nonconcordance of its mRNA and protein levels, and could provide an important level of modulation of ER activity during breast cancer development.

Regulation of translation occurs mainly during initiation. Most initiation occurs by cap-dependent scanning, which requires binding of the initiation complex to the mRNA cap and recruitment of other proteins. These scan along the 5' untranslated region (UTR) of the mRNA to the reading frame, where they recognise an initiation codon, recruit more factors, and initiate protein synthesis. 5' UTRs vary greatly in length and sequence with some containing elements that allow regulation of factor recruitment or scanning, and thereby allow regulation of translation of their specific mRNAs [2]. It is thought that deregulation of translation, via 5'-UTR sequences, is responsible for a significant proportion of the expression changes in cancer cells and that this has a role in carcinogenesis.

We identified three alternative 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 [3] and EST databases (Figure 1). Our hypothesis is that these alternative 5' UTRs allow differential post-transcriptional regulation of ERβ expression, thereby providing critical regulation of ER function.

Figure 1

5' end of the human ERβ gene (14q23) aligned with mRNAs containing different ERβ 5' UTRs. UTR exons (filled boxes), transcriptional (black arrows) and translational (ATG) start sites, intron sizes, and primers used for PCR analysis (grey arrows, specific UTRs; open arrows, exon 1) are shown. Sequences strongly suggestive of translational regulation are described: uORFs and stable RNA structure, quantified as change in free energy, ΔG; for comparison, ΔG of the nonregulatory β-actin 5' UTR is only -24 kcal/mol.

Methods

We investigated the properties of these three ERβ 5' UTRs using established reporter assays [4]; each 5' UTR was cloned upstream of a GFP reporter. Breast cell lines (MCF7, MDA-MB-453, MDA-MB-231, BT-20 and HB2) were transiently transfected with either an unmodified GFP reporter as a control (this is 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 quantitative PCR, respectively. Semi-quantitative PCR was also used to analyse ERβ 5' UTRa and UTRb expression in matched normal/tumour breast tissues.

Results

Our results are the first to show that these alternative 5' UTRs do, in fact, allow the differential regulation of ERβ translation. The UTRa and UTRa long 5' UTRs strongly inhibited translation of the GFP reporter whilst UTRb had little effect. In addition, our preliminary data suggest that these alternative 5' UTRs are differentially expressed between breast normal and tumour tissue. The expression of UTRa mRNA was found to be upregulated in a panel of breast tumours compared with matched normal tissue. This may have important implications in breast cancer development. Work is currently ongoing to investigate the stability of these mRNA messages and to identify important regulatory sequences.

Conclusion

Post-transcriptional regulation plays an important role in determining the level of ERβ protein expression and may therefore have an influence on overall estrogen receptor activity. This may have important implications on our understanding of breast cancer biology and treatment.