Dynamic Changes in Estrogen Receptor Alpha (ERα) Promoter Methylation and Drug Resistance in Breast Cancer Cells

Background

Breast cancer is one of the most common cancers among women worldwide, with approximately 75% of breast cancer patients expressing estrogen receptor alpha (ERα, gene symbol ESR1). ERα is not only an important prognostic marker for breast cancer but also a key target for endocrine therapy. Commonly used endocrine therapies include aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs) such as tamoxifen, and selective estrogen receptor degraders (SERDs) such as fulvestrant. However, prolonged use of these drugs often leads to the development of drug resistance, resulting in treatment failure. The emergence of resistance is typically associated with changes in ERα expression, although the exact mechanisms remain incompletely understood.

In recent years, epigenetic mechanisms, particularly DNA methylation, have been recognized as important regulators of gene expression. DNA methylation typically occurs at CpG sites in gene promoter regions and can influence transcription factor binding, thereby regulating gene expression. In breast cancer, the methylation status of the ERα promoter region may be closely related to its expression levels, affecting the tumor’s response to endocrine therapy. Therefore, studying the role of ERα promoter methylation in breast cancer cell resistance is crucial for understanding resistance mechanisms and developing new therapeutic strategies.

Source of the Paper

This paper was co-authored by Juliane Albrecht, Mirjam Müller, Völundur Hafstað, Kamila Kaminska, Johan Vallon-Christersson, Gabriella Honeth, and Helena Persson from the Department of Oncology at Lund University in Sweden. The paper was accepted on July 26, 2024, and published online on August 6, 2024, in the journal Molecular Oncology (DOI: 10.¹⁰⁰²⁄₁₈₇₈-0261.13713).

Research Process and Results

1. Study Design and Cell Models

The research team used six ERα-positive breast cancer cell lines (CAMA-1, HCC1428, MCF7, T-47D, ZR-75-1, and EFM-19) and developed resistant sublines (Fulvestrant-Resistant Sublines, FR) by gradually increasing the concentration of fulvestrant. Additionally, the researchers cultured resistant sublines in the absence of fulvestrant (FR-F) to study the stability of resistance. These cell lines exhibited significant differences in resistance, ERα expression levels, and responses to other drugs.

2. DNA Methylation Analysis

To investigate the methylation status of the ERα promoter region, the research team used bisulfite sequencing to analyze the methylation levels of 108 CpG sites across 12 ERα promoter regions. These regions are located near different ERα promoters and cover transcription start sites of multiple alternative first exons. Through high-throughput sequencing (Illumina MiSeq), the researchers obtained methylation fractions for each CpG site and conducted detailed data analysis.

3. Relationship Between Methylation and ERα Expression

The study found significant differences in methylation patterns across different cell lines. In particular, CpG sites near the main promoter region and the first coding exon exhibited high methylation in long-term stable resistant cell lines (e.g., ZR-75-1 FR-F and CAMA-1 FR-F), while these regions were almost completely demethylated in other cell lines. Additionally, methylation levels were negatively correlated with the expression of alternative first exons, suggesting that methylation may regulate ERα expression by affecting local chromatin accessibility and transcription factor binding.

4. Alternative Promoter Usage and Breast Cancer Prognosis

The research team also analyzed the expression of alternative first exons in breast cancer tumors. By analyzing RNA sequencing data from the SCAN-B breast cancer cohort, the researchers found that tumors could be clustered based on the expression patterns of alternative first exons. Higher expression of upstream promoters (e.g., PromC and PromF) was associated with worse prognosis, particularly in postmenopausal ERα-positive breast cancer patients receiving endocrine therapy. This finding suggests that alternative promoter usage may influence tumor biology and clinical outcomes.

5. CTCF Binding Sites and ERα Expression Regulation

The study also identified a region within an ERα gene intron (Region 6) containing multiple CTCF (CCCTC-binding factor) binding sites. The CpG methylation levels in this region were negatively correlated with ERα expression. CTCF is an important transcriptional insulator protein that can regulate gene expression through chromatin remodeling. The study hypothesized that this region may influence ERα expression status by modulating chromatin structure.

Conclusions and Significance

This study reveals the dynamic changes in ERα promoter methylation in breast cancer cell resistance and elucidates the relationship between methylation and alternative promoter usage. The findings suggest that changes in methylation status may regulate ERα expression, affecting the tumor’s response to endocrine therapy. Additionally, alternative promoter usage is closely related to breast cancer patient prognosis, indicating its potential clinical application in diagnosis and treatment.

Research Highlights

1. Dynamic Methylation Changes: The study systematically reveals the dynamic changes in ERα promoter methylation in breast cancer cell resistance, particularly the hypermethylation state in long-term stable resistant cell lines.
2. Alternative Promoters and Prognosis: The study finds that alternative promoter usage is closely related to breast cancer patient prognosis, providing new prognostic markers for clinical use.
3. Role of CTCF Binding Sites: The study highlights the potential role of CTCF binding sites in regulating ERα expression, offering new directions for further research into chromatin remodeling mechanisms in breast cancer resistance.

Additional Valuable Information

The research team also developed a high-throughput sequencing-based methylation analysis method capable of precisely detecting methylation status at individual CpG sites. This method provides a powerful tool for future epigenetic research.

This study not only deepens our understanding of breast cancer resistance mechanisms but also provides important theoretical foundations for developing new therapeutic strategies.