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Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast
1Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903, USA.
Abstract:
We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.
Insights
Polyamines, such as spermidine, play a crucial role in regulating estrogen receptor (ER) protein interactions within breast cancer cells. Inhibiting polyamine synthesis affects these interactions, highlighting the pathway's importance in gene regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Estrogen receptor (ER) signaling is critical in breast cancer.
- Polyamines are essential for cell growth and proliferation.
- The interplay between polyamines and ER signaling is not fully understood.
Purpose of the Study:
- To investigate the impact of polyamine biosynthesis inhibitors on the estrogenic signaling pathway in MCF-7 breast cancer cells.
- To identify proteins interacting with the ER and how these interactions are modulated by polyamines and estradiol.
Main Methods:
- Utilized a protein-protein interaction system involving glutathione-S-transferase (GST)-linked ER.
- Examined the effects of difluoromethylornithine (DFMO) and CGP 48664, polyamine biosynthesis inhibitors.
- Analyzed protein-ER associations using GST-ER pull-down assays and Western blot analysis.
Main Results:
- ER specifically associated with a 45 kDa protein in control cells; estradiol treatment increased ER-associated proteins to nine.
- Polyamine inhibitors altered ER-protein binding, with estradiol and inhibitors decreasing these associations.
- Spermidine addition reversed the effects of DFMO, significantly increasing ER-protein interactions, particularly with the 45 kDa protein.
Conclusions:
- Intracellular polyamine concentrations, especially spermidine, critically influence ER protein associations.
- Multiple proteins interact with ER, and their binding is sensitive to polyamine levels.
- The polyamine pathway is vital for estradiol's gene regulatory function in breast cancer cells.