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Identification of a dominant negative form of the human estrogen receptor
1Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Abstract:
Experiments were undertaken to characterize mRNAs coding for the estrogen receptor (ER) in the human breast cancer cell line T47D. We report here the isolation of cDNAs corresponding to three isoforms of this receptor in addition to a majority of wild-type clones. Sequence analysis showed that these isoforms are generated through alternative splicing. None of the alternatively spliced isoforms of ER is able to bind to an estrogen-responsive element (ERE) in a gel mobility shift assay in vitro or to activate transcription of a reporter gene containing an ERE in vivo. One isoform, ER delta E3, which harbors a deletion of exon 3 encoding the second zinc finger of the DNA-binding domain, inhibits estrogen-dependent transcription activation in a dominant negative fashion when it is cotransfected with the wild-type ER and reporter plasmid. It also inhibits DNA binding of wild-type ER in a gel mobility shift assay in vitro. Since ER delta E3 is not able to bind to its response element, the observed inhibitory effect probably occurs through protein-protein interactions. This could involve the formation of a heterodimer between mutant and wild-type receptors, competition for a limiting transcription factor, or both. These results may have implications for understanding the loss of estrogen responsiveness that frequently occurs in breast cancer.
Insights
Researchers identified three estrogen receptor (ER) mRNA isoforms in breast cancer cells, generated by alternative splicing. One ER isoform, ER delta E3, inhibits estrogen-dependent transcription and DNA binding, potentially impacting breast cancer progression.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Estrogen receptor (ER) plays a crucial role in breast cancer development and progression.
- Alternative splicing of ER mRNA can lead to various receptor isoforms with potentially altered functions.
- Understanding ER isoforms is vital for developing targeted therapies.
Purpose of the Study:
- To characterize estrogen receptor (ER) mRNA isoforms in the T47D human breast cancer cell line.
- To investigate the functional consequences of identified ER isoforms on estrogen-responsive element (ERE) binding and transcriptional activity.
- To explore the mechanism by which a dominant-negative ER isoform inhibits wild-type ER function.
Main Methods:
- Isolation and sequencing of complementary DNAs (cDNAs) encoding ER isoforms.
- Gel mobility shift assays to assess ER binding to estrogen-responsive elements (EREs) in vitro.
- Reporter gene assays to evaluate transcriptional activation in vivo.
- Co-transfection experiments to study dominant-negative effects of ER isoforms.
Main Results:
- Three novel ER mRNA isoforms were identified in T47D cells, alongside wild-type ER.
- These isoforms are generated through alternative splicing of the ER gene.
- None of the alternatively spliced ER isoforms could bind to EREs or activate transcription independently.
- ER delta E3, lacking a key DNA-binding domain component, exhibited dominant-negative inhibition of wild-type ER-mediated transcription and DNA binding.
- The inhibitory effect of ER delta E3 likely involves protein-protein interactions with wild-type ER.
Conclusions:
- Alternative splicing generates functionally distinct ER isoforms in breast cancer cells.
- The ER delta E3 isoform acts as a dominant-negative inhibitor, potentially contributing to loss of estrogen responsiveness in breast cancer.
- These findings offer insights into the molecular mechanisms underlying endocrine resistance in breast cancer.