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A yeast-based bioassay for the determination of functional and non-functional estrogen receptors
P Balmelli-Gallacchi1, F Schoumacher, J W Liu
1Biochemistry/Endocrinology and Biomolecular Tumordiagnostics, Department of Research, University Women's Clinic Basel and Stiftung Tumorbank Basel, CH-4031 Basel, Switzerland.
Abstract:
The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene ( ADE2 ) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER.
Insights
A novel yeast assay identifies functional estrogen receptor (ER) variants in breast cancer. This method distinguishes active, inactive, and wild-type ER, improving treatment predictability beyond standard hormone receptor status.
Area of Science:
- Molecular Biology
- Oncology
- Yeast Genetics
Background:
- Endocrine therapy response in breast cancer is complex and not fully predicted by hormone receptor status alone.
- Mutated or spliced variants of the estrogen receptor (ER) can exhibit altered biological activities, impacting treatment outcomes.
Purpose of the Study:
- To develop a functional assay in Saccharomyces cerevisiae to characterize diverse estrogen receptor (ER) forms in heterogeneous breast tumors.
- To enable the identification and functional assessment of individual ER variants within breast cancer samples.
Main Methods:
- Total RNA from breast cancer cells and specimens was used to amplify ER cDNA via reverse transcription and PCR.
- Cloning of ER cDNA into an expression vector using in vivo homologous recombination in yeast.
- A yeast reporter system utilizing an estrogen response element and the ADE2 gene to visually distinguish ER activity (white colonies) from inactivity (red colonies).
Main Results:
- Successful development of a functional yeast assay for ER activity.
- Demonstration of the assay's ability to differentiate between wild-type, constitutively active, and inactive ER variants.
- Facilitation of subsequent analysis of ER functionality through plasmid rescue and DNA sequencing.
Conclusions:
- The developed yeast functional assay provides a simple, visual method to assess ER activity in breast cancer.
- This assay can identify functionally distinct ER variants, potentially improving the prediction of endocrine therapy response.
- Characterizing ER heterogeneity offers new insights into breast cancer biology and therapeutic strategies.