Estrogen-Induced Cell Cycle Arrest and Apoptosis as an Unexpected Outcome of Aromatase Inhibitor-Resistance in ER+ Breast Cancer

Abstract

Background: Estrogen is known to promotes hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ breast cancer. Considering potential impact of such clinical observation, we decided to determine the mechanisms of estrogen-induced tumor regression. Methods: A unique estrogen-inhibitory patient-derived xenograft (PDX) tumor, GS3, was established from an AI resistant ER+/HER2– brain metastatic breast cancer. In vivo estrogen suppression was confirmed through experiments by implanting 17β-estradiol (E2) pellets in mice carrying GS3, and then the single-cell analysis was performed using GS3 tumors. In vitro E2 suppression analysis was carried out using organoids from GS3.Results: The E2-induced suppression of GS3 involves ERα, which was wild-type and not amplified. Single cell RNA sequencing analysis of this PDX has revealed that E2 treatment (for 1 week) induces cell cycle arrest in both ESR1+ cells and ESR1– cells, demonstrating the unexpected influence of estrogen on ESR1– cells in ER+ breast cancer. E2 upregulated the expression of estrogen-regulated genes, including a tumor suppressor gene, IL24, and lower levels of IL24 were linked to estrogen independence, after three rounds of intermittent E2 treatment. IL24+ cells included more G1 phase cells of cell cycle compared to IL24– cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. The number of apoptotic cells was significantly increased after long term (for 4 weeks) E2 treatment. Western blotting analysis demonstrated that long term E2 treatment induced expression of apoptosis-associated protein cleaved-PARP and reduction of the pro-survival protein Bcl-xl level.Conclusions: There is the need of markers for patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not enough. Analysis of GS3 PDX has revealed that estrogen induces cell cycle arrest and apoptosis. Our study has revealed the cross-talk between ESR1+ and ESR1– cells as well as potential roles of IL24 in estrogen-suppressive tumors.