Abstract PD7-02: Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model

Abstract

Background: Estrogen such as estradiol (E2) is known to promote ER⁺ breast cancer. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER⁺ postmenopausal breast cancer. The objective of this study is to uncover the mechanisms of E2-induced tumor regression, leading to an unconventional treatment of AI resistance. Methods: An E2-suppressive patient-derived xenograft model (named GS3) was established from an AI resistant ER⁺/PR^-/HER2^- brain metastatic breast cancer. Placebo or E2 pellets were implanted in mice carrying GS3 for evaluating the effects of E2. Immunohistochemistry (IHC) and RNA sequencing of GS3 were conducted to decipher molecular changes after E2 treatments. Since the cancer tissue has a heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist, not by ERβ antagonist. IHC showed that ER, Ki-67 and CEA expressions decreased and PR expression appeared after E2 treatment. Gene set enrichment analysis (GSEA) using RNAseq results showed that the E2 response gene sets were significantly up-regulated after E2 treatment. However, the cell cycle gene sets and the TNFA/NFKB gene set were down-regulated. GS3 gained an E2 independence after three cycles of intermittent E2 treatment (E2 pellet on/off every 4 weeks; Int-E2). Interestingly, the cell cycle and TNFA/NFKB gene sets were up-regulated after Int-E2 treatment. Single-cell RNAseq analysis revealed that cells from one-week E2-treated and Placebo-treated GS3 were placed in different clusters based on principle component analysis of Highly Variable Genes. Although E2 response genes were up-regulated, the percent of ESR1 ⁺ cells decreased after E2 treatment (41.3% vs. 31.5%). The number of cells arrested at the G1 phase increased (+12.5%) after E2 treatment. GSEA using genes expressed in only ESR1 ⁺ cells showed that the TNFA/NFKB gene set was significantly down-regulated after E2 treatment. Meanwhile, GSEA using genes expressed in only ESR1 ^– cells showed that cell cycle gene sets were significantly down-regulated. Single-cell trajectory analysis disclosed three major branches; 1) common E2 and placebo, 2) E2, and 3) placebo. In the E2 only branch, the cell cycle arrested at the G1 phase, the E2 response gene sets were up-regulated, and the NFKB gene set was down-regulated in ESR1 ⁺ cells. Significantly, E2 response gene sets were also up-regulated and cell cycle genes were down-regulated even in ESR1 ^– cells. In the placebo branch, E2 response gene sets were not up-regulated and cell cycle genes were not down regulated. A group of MKI67 ⁺ cells (at G2M phase), including some ESR1⁺ cells, were present in both E2-treated and placebo-treated tumors. Conclusions: E2-induced suppression is an unexpected outcome of AI resistance. In these cases, elimination of estrogen by AI results in maintaining tumor growth. Analysis of GS3 PDX has revealed that estrogen can induce cell cycle arrest and the expression of estrogen-regulated genes. Our results also suggest the cross-talk between ESR1⁺ and ESR1 ^- cells as well as potential roles of the TNFA/NFKB pathway. Our findings point out the need of markers for such patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not sufficient. An intermittent treatment strategy does not sustain the effect of estrogen-mediated suppression. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-02.