Abstract P6-04-19: Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression

Background: Breast cancer (BC) is the most frequently diagnosed cancer and the primary leading cause of cancer deaths in women worldwide. Although the clinical outcome of BC patients has been considerably improved, resistance to endocrine and chemotherapy treatments contributes to BC relapse and mortality. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Recent studies suggested that estrogen receptor beta (ERβ) and its ligand S-Equol promote suppression of BC progression. However, the mechanisms by which S-equol inhibits growth of BC cells and enhance therapy response remains unknown. Here, we examined the mechanisms by which S-equol contribute to BC growth inhibition in hormone therapy sensitive and resistant BC cells. Methods: We evaluated the therapeutic efficacy of S-equol in therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells. Expression levels of target genes were evaluated in both cell lines by global RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses, qRT-PCR and Western blot. ERβ knock down cell lines were generated by CRISPR-Cas9 technology. Xenograft and patient derived BC explants (PDEX) were used for preclinical evaluation of the antiproliferative activity of S-equol. Results: S-equol reduced the growth of endocrine sensitive and resistant BC models in vitro and in vivo. Treatment of ER-positive PDEX with S-equol decreased the proliferation compared to untreated tumors. RNA-seq analysis of S-equol treated BC cells showed modulation of ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis showed that S-equol treatment upregulated 438 proteins and downregulated 429 proteins in MCF7-Aro cells compared to controls. Whereas, DIA analysis in Letrozole-resistant cells revealed that S-equol upregulated 572 proteins and downregulated 481 proteins compared to controls. Reactome pathway analysis revealed that S-equol treatment modulated the proteins involved in DNA replication, cell cycle, proteins regulating p53 activity and VEGF-VEGFR2 pathway. Our mechanistic studies confirmed that treatment with S-equol upregulated FOXO3 and downregulated FOXM1. Using CRISPR-Cas9 mediated ERβ knockout cells, we confirmed that S-equol could not modulate forkhead box transcription factors in BC cells lacking ERβ compared to parental cells. Conclusions: Collectively, our results suggest that S-equol exhibits its antitumor activity by targeting forkhead box transcription factors and that S-equol might be an effective treatment strategy in treating / preventing breast cancer recurrence. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294). Citation Format: Kumaraguruparan Ramasamy, Shaorong Chen, Suryavathi Viswanadhapalli, Rong Li, Ratna K Vadlamudi, Rajeshwar R Tekmal. Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-19.