Influence of Estrogen Treatment on ESR1(+) and ESR1(−) Cells in ER(+) Breast Cancer: Insights from Single-Cell Analysis of Patient-Derived Xenograft Models

SIMPLE SUMMARY: The benefit of endocrine therapy is normally observed for cancers with 10% or more of cells positive for ER expression. We compared the gene expression profiles in both ESR1(+) and ESR1(–) cells in ER(+) tumors following estrogen treatment. Our single-cell RNA sequencing analysis of estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft models offered an unprecedented opportunity to address the molecular and functional differences between ESR1(+) and ESR1(–) cells. While estrogen should activate ERα and stimulate ESR1(+) cells, our findings regarding ESR1(–) cells were important, indicating that the proliferation of ESR1(–) cells in ER(+) cancer is also influenced by estrogen. Another valuable finding from our studies was that estrogen also upregulated a tumor-suppressor gene, IL-24, only in GS3. Estrogen increased the percentage of cells expressing IL-24, associated with the estrogen-dependent inhibition of GS3 tumor growth. ABSTRACT: A 100% ER positivity is not required for an endocrine therapy response. Furthermore, while estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced tumor suppression in ER(+) breast cancer has been clinically observed. With the success in establishing estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft (PDX) models, single-cell RNA sequencing analysis was performed to determine the impact of estrogen on ESR1(+) and ESR1(–) tumor cells. We found that 17β-estradiol (E2)-induced suppression of GS3 transpired through wild-type and unamplified ERα. E2 upregulated the expression of estrogen-dependent genes in both SC31 and GS3; however, E2 induced cell cycle advance in SC31, while it resulted in cell cycle arrest in GS3. Importantly, these gene expression changes occurred in both ESR1(+) and ESR1(–) cells within the same breast tumors, demonstrating for the first time a differential effect of estrogen on ESR1(–) cells. E2 also upregulated a tumor-suppressor gene, IL-24, in GS3. The apoptosis gene set was upregulated and the G2M checkpoint gene set was downregulated in most IL-24(+) cells after E2 treatment. In summary, estrogen affected pathologically defined ER(+) tumors differently, influencing both ESR1(+) and ESR1(–) cells. Our results also suggest IL-24 to be a potential marker of estrogen-suppressed tumors.