HMGA1a induces aberrant splicing of estrogen receptor α in MCF-7 breast cancer cells

Abstract

AbstractThe well known transcription factor, HMGA1a, also known as an oncogene, causes aberrant splicing of Presenilin-2 found in sporadic Alzheimer's disease, through sequence specific RNA binding. Since HMGA1 protein levels correlate with tumorigenic malignancy, we are interested in the potential of HMGA1a as an aberrant splicing factor in cancer. The N-terminal truncated isoform of Estrogen Receptor alpha, ERα46, is known to increase expression when breast cancer cells (MCF-7) become hyperconfluent. Transiently expressed HMGA1a induced ERα46 mRNA expression. Plasmid driven micro RNA containing the presenilin-2 HMGA1a RNA binding site and a micro RNA nuclear localization signal (miRHMGBSNLS), inhibited ERα46 mRNA expression, acting as a decoy. Re-expressing HMGA1a restored ERα46 mRNA expression. An HMGA1a binding site was found in the skipped exon 1A, 33 bp upstream the authentic 5’ splice site, adjacent a pseudo 5’ splice site. RNA gel shift assay showed binding of HMGA1a to this site. Exon skipping by HMGA1a and exon inclusion by the HMGA1a binding site decoy could be reproduced in vitro in a heterologous context. Psoralen UV crosslinking showed HMGA1a anchored U1 snRNP to the upstream pseudo 5’ splice site. Psoralen UV crosslinking combined with antisense DNA oligo / RNaseH protection comfirmed U1 snRNP anchored to the upstream pseudo 5’ splice site by HMGA1a. This is the second example of the PSI model found in Drosophila, but the first in human. Finally, the stable transfectant of the decoy, miRHMGBSNLS (MCF-7 cells), which shows decreased ERα46 protein level, was prone to apoptosis by high dose estrogen. We hope this decoy, miR_HMGBS_NLS, in combination with high dose estrogen, will lead to development of a novel breast cancer therapy, an alternative of conventional estrogen deprivation.