Integrated Single-Cell Analysis Reveals Distinct Epigenetic-Regulated Cancer Cell States and a Heterogeneity-Guided Core Signature in Tamoxifen-Resistant Breast Cancer

Academic Background

Breast cancer is one of the most common cancers in women, and endocrine therapy (e.g., tamoxifen) is the standard treatment for estrogen receptor α (ER)-positive breast cancer patients. Although endocrine therapy significantly reduces the risk of recurrence, approximately one-third of patients eventually develop endocrine resistance and relapse. Inter- and intra-tumor heterogeneity is considered a significant factor contributing to endocrine resistance. Recent advances in single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) have enabled the exploration of tumor heterogeneity at single-cell resolution. However, such integrated single-cell analysis has not yet been applied to characterize the transcriptome and chromatin accessibility in breast cancer endocrine resistance.

Source of the Paper

This study was conducted by Kun Fang, Aigbe G. Ohihoin, Tianxiang Liu, Lavanya Choppavarapu, and others, with contributions from multiple institutions including the Medical College of Wisconsin, Luxembourg Institute of Health, and Duke University. The paper was published in Genome Medicine in 2024.

Research Process

1. Sample Collection and Single-Cell Isolation

The study utilized eight human breast tissue samples, including two normal tissues (NTs), three primary tumors (PTs), and three tamoxifen-treated recurrent tumors (RTs). Approximately 82,400 cells were isolated from these tissues using single-nucleus isolation techniques, followed by scRNA-seq and scATAC-seq analysis.

2. Single-Cell RNA Sequencing and ATAC Sequencing

Library preparation for scRNA-seq and scATAC-seq was performed using the 10x Genomics Chromium Single Cell 3’ Library and ATAC Solution Kit, respectively. Sequencing data were preprocessed and analyzed using software such as Seurat and Signac.

3. Cell Type Annotation and Tumor Heterogeneity Assessment

Through scRNA-seq data, researchers identified various cell types in breast tumor tissues and defined PT- and RT-specific cancer cell states (CSS). By integrating scRNA-seq and scATAC-seq data, a heterogeneity-guided core signature (HCS) of 137 genes was identified.

4. Functional Validation

The researchers validated the oncogenic role of BMP7, a key gene within the core signature, in tamoxifen-resistant breast cancer cells through in vitro experiments. Knockdown of BMP7 using siRNA significantly inhibited cancer cell proliferation and restored sensitivity to tamoxifen. Further mechanistic studies revealed that BMP7 exerts its oncogenic effects by modulating the MAPK signaling pathway.

Main Results

1. Single-Cell Heterogeneity in Breast Cancer Tissues

The study revealed significant inter- and intra-tumor heterogeneity, particularly between PTs and RTs. Using scRNA-seq data, researchers defined nine cancer cell states (CSS), including five PT-specific, three RT-specific, and one PT-RT shared CSS.

2. Epigenetic-Regulated Cancer Cell States

Through scATAC-seq data, researchers identified open chromatin regions in RT-specific CSS and found that these regions were closely associated with the expression of core signature genes. Further analysis suggested that the epigenetic regulation of these genes may be achieved through enhancer-promoter interactions.

3. Functional Validation of the Core Signature

The researchers functionally validated the role of BMP7, a key gene in the core signature, through in vitro experiments. Knockdown of BMP7 significantly inhibited the proliferation of tamoxifen-resistant breast cancer cells and restored their sensitivity to tamoxifen. Mechanistic studies indicated that BMP7 exerts its oncogenic effects by modulating the MAPK signaling pathway.

Conclusions

This study, through integrated single-cell analysis, revealed the epigenetic regulatory mechanisms underlying endocrine resistance in breast cancer and identified a heterogeneity-guided core signature. These findings provide important scientific insights into understanding tumor heterogeneity and developing new therapeutic strategies.

Research Highlights

1. Single-Cell Resolution Analysis of Tumor Heterogeneity: The study is the first to comprehensively analyze the transcriptome and chromatin accessibility in breast cancer endocrine resistance at single-cell resolution.
2. Identification and Functional Validation of the Core Signature: The study identified a core signature of 137 genes and functionally validated the role of the key gene BMP7.
3. Revealing Epigenetic Regulatory Mechanisms: The study highlighted the critical role of epigenetic factors in breast cancer endocrine resistance, providing a theoretical foundation for developing new therapeutic targets.

Significance and Value

This study not only deepens our understanding of the mechanisms underlying endocrine resistance in breast cancer but also provides new insights for developing personalized therapeutic strategies targeting tumor heterogeneity. Through integrated single-cell analysis, the study demonstrated the critical role of epigenetic regulation in tumor progression, opening new avenues for future cancer research.