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Updated: Dec 8, 2025
Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer
Published on: March 17, 2016
Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click
Quynh-Trang Do1, Ting-En Huang1, Yi-Chen Liu1
1Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan.
Abstract:
Catechol estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of an experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment) were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and redigestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CE-adducted peptides. A total of 310 protein targets and 40 adduction sites were repeatedly (n ≥ 2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may downregulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CE-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data will provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.
Insights
Catechol estrogens (CEs) are toxic breast cancer initiators. This study identifies protein targets of CEs in cancer cells, revealing potential links to disease progression and biomarkers.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Biology
Background:
- Catechol estrogens (CEs) are toxic metabolites implicated in breast cancer initiation through DNA adduct formation.
- While CEs can react with proteins, their specific cellular targets have remained largely uncharacterized.
Purpose of the Study:
- To identify protein targets of catechol estrogens (CEs) in the cytosol of estrogen-sensitive breast cancer cells.
- To develop and validate an improved proteomics workflow for detecting CE-protein adducts.
Main Methods:
- Utilized a click chemistry-based workflow coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Employed multiple comparative proteomics with stable isotope dimethyl labeling and a 4-hydroxyethynylestradiol (4OHEE2) probe.
- Incorporated on-bead digestion and redigestion for enhanced recovery of adducted peptides.
Main Results:
- Identified 310 protein targets and 40 adduction sites with high confidence (D/H ratio >4, n ≥ 2), demonstrating a low background (<1 in controls).
- Observed downregulation of target proteins involved in metabolism and detoxification, suggesting a negative correlation between CE adduction and cellular defense mechanisms.
- The developed workflow significantly improved the identification of CE-adducted peptides compared to control experiments.
Conclusions:
- The study successfully identified novel protein targets of catechol estrogens in breast cancer cells.
- The findings suggest a mechanism where CE adduction may impair cellular detoxification pathways, potentially contributing to disease progression.
- The validated proteomics method and identified targets offer new avenues for studying estrogen metabolism-derived diseases and discovering biomarkers.
