Phytoestrogen coumestrol as an anti-cancer therapy against triple-negative inflammatory breast cancer

Abstract

Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which Cou can exert its effect in the most aggressive breast cancer subtype, triple-negative inflammatory breast cancer (TN-IBC), is still unknown. Studies show that TN-IBC cell lines are unresponsive to hormonal therapies, but E2 can activate signaling pathways involved in pro-oncogenic phenotypes such as motility and invasion by activating a rapid estrogen-dependent non-genomic signaling. Therefore, the objective of this study was to determine the anti-cancer effect of Cou in TN-IBC cell lines (using 2D and 3D culture models) and the molecular mechanism Cou exerts for its anti-cancer activity. Dose-response curves of Cou in 2D and 3D culture models of TN-IBC cell lines were generated to determine the half-maximal inhibitory concentration (IC₅₀). Relative cell viability was measured in ER-positive, triple-negative, TN-IBC, and HER2-amplified IBC cell lines. Functional assays were performed in TN-IBC cell lines to determine the effect of E2, Cou, and E2/Cou combination treatments on cell viability, migration, invasion, proliferation, and tumor emboli growth. Additionally, the effect of Cou and E2 treatments on phosphorylation of downstream kinases was analyzed by a proteome profile human phospho-kinase array. RNA-seq was performed in TN-IBC cell lines to identify transcriptome changes after Cou treatment. Our data demonstrated that Cou treatment using the IC₅₀ (13µM in 2D models and 50µM in 3D models) decreased cell viability in TN-IBC cell lines. Similarly, Cou treatment reduces triple-negative non-IBC cell viability. In comparison with E2, Cou decreases migration, invasion, proliferation, and tumor emboli growth in TN-IBC cell lines. Finally, Cou treatment reduces the phosphorylation of kinases MAPK/ERK and PI3K/AKT that promote pro-oncogenic phenotypes and upregulate genes such as TIPARP, recently involved in breast cancer phenotype suppression. In summary, this study guide us in elucidating a signaling pathway affected by Cou’s anti-cancer activity. More importantly, it opens the opportunities to design more effective targeted therapeutic strategies to improve the prognosis and survival rates of IBC patients.