Identification of Lineage-Specific Epigenetic Regulators FOXA1 and GRHL2 through Chromatin Accessibility Profiling in Breast Cancer Cell Lines

Medicine Cell Biology Biochemistry Oncology Life Sciences Immunology
Breast Cancer Epigenetics Chromatin Accessibility FOXA1 GRHL2 Cell Lines Heterogeneity

Identification of Lineage-Specific Epigenetic Regulators FOXA1 and GRHL2 through Chromatin Accessibility Analysis of Breast Cancer Cell Lines

Background

Breast cancer is a highly heterogeneous disease currently classified clinically based on gene expression patterns, intrinsic molecular subtypes, and the expression of hormone receptors/human epidermal growth factor receptor 2 (HER2). However, studies have shown that breast tumors exhibit significant heterogeneity beyond gene expression. For example, the estrogen response element accessibility may be reduced in some estrogen receptor-positive (ER+) breast cancers, potentially leading to poor prognosis. Thus, identifying epigenetic states is crucial for a more comprehensive understanding of breast cancer heterogeneity.

Breast cancer cell lines are important models for studying breast cancer, typically categorized based on intrinsic subtypes and/or receptor status. However, the transcriptional epigenetic heterogeneity hidden within these cell lines has not been fully elucidated. Therefore, to more accurately simulate breast cancer and clarify its key regulatory mechanisms, it is essential to analyze the epigenetic landscape of breast cancer cell lines.

Source of Study

This study, conducted by Liying Yang, Kohei Kumegawa, Sumito Saeki, Tomoyoshi Nakadai, and Reo Maruyama, is affiliated with the Tokyo Cancer Research Foundation in Japan. The paper was published in the 2024 edition of the journal Cancer Gene Therapy.

Research Process

Analysis Process

The study first performed a whole-genome chromatin accessibility analysis on 23 breast cancer cell lines. These cell lines included 2 ER+/HER2- types, 3 ER+/HER2+ types, 3 HER2+ types, and 15 triple-negative breast cancer (TNBC) cell lines. The chromatin accessibility analysis divided these cell lines into three groups: the receptor-positive group (Group-P), the basal-like TNBC group (Group-B), and the mesenchymal-like TNBC group (Group-M).

Using Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) technology, the researchers identified 140,246 reproducible cis-regulatory elements (CREs), a large portion of which are located in distal regions and intronic regions, with promoter elements accounting for only 21.7% of total CREs, consistent with previous studies.

Next, through an analysis of the nucleosome periodic fragment pattern and transcription start site (TSS) enrichment scores, the researchers classified these cell lines and confirmed three chromatin accessibility groups: Group-P, Group-B, and Group-M.

Motif Enrichment Analysis

To understand the differences between these three subgroups, the researchers performed an enrichment analysis of the transcription factor (TF) binding motifs in the chromatin accessible regions of all cell lines. The results showed that the receptor-positive group (Group-P) exhibited co-enrichment of the FOXA1 and GRHL2 motifs, while the basal-like group (Group-B) displayed enrichment of only the GRHL2 motif, and Group-M had no motif enrichment.

Gene Ontology Analysis

Gene ontology analysis indicated that the specific accessible regions of Group-B and Group-P are related to their unique lineage characteristics. Furthermore, gene ontology analysis revealed distinctive epigenetic landscapes between different groups.

Knockdown Experiments

To investigate the roles of FOXA1 and GRHL2 in regulating chromatin accessibility, the researchers conducted knockdown experiments targeting FOXA1 and GRHL2 and performed ATAC-seq analysis. The results showed that FOXA1 maintains the accessibility of specific regions in Group-P while inhibiting the accessibility of Group-B specific regions. Conversely, GRHL2 maintains the accessibility of the shared regions between Group-P and Group-B in Group-B cells.

Research Results

Through chromatin accessibility analysis, the researchers found that the 23 tested breast cancer cell lines could be divided into three major subgroups based on accessibility patterns:

  • Group-P includes ER+ and/or HER2+ cell lines such as T47D and MCF7.
  • Group-B mainly consists of basal-like TNBC cell lines such as HCC1937 and MDA-MB-468.
  • Group-M includes mesenchymal-like and mesenchymal stem-like cell lines such as BT549 and MDA-MB-436.

Based on the enrichment patterns of FOXA1 and GRHL2 motifs, significant differences were observed between Group-P and Group-M. FOXA1 is specifically enriched in Group-P, while GRHL2 is enriched in Group-B.

Further analysis revealed that cell lines in Group-P exhibit considerable chromatin accessibility at the transcription start site (TSS) of FOXA1 and its flanking regions, suggesting the presence of enhancer elements. Although Group-B shows moderate accessibility at the TSS region of FOXA1, it lacks the enhancer region accessibility observed in Group-P.

By knocking down FOXA1 and GRHL2, the researchers discovered that FOXA1 maintains the accessibility of specific CREs in Group-P cells while inhibiting the accessibility of Group-B specific CREs; conversely, GRHL2 maintains the accessibility of shared CREs between Group-P/B in Group-B cells. RNA-seq analysis showed that knocking down FOXA1 results in the downregulation of cell cycle-related genes in T47D cells, while knocking down GRHL2 upregulates genes related to epithelial-mesenchymal transition (EMT).

Conclusion

The results indicate that FOXA1 and GRHL2 play crucial roles in maintaining specific chromatin accessibility in breast cancer cell lines. FOXA1 primarily regulates Group-P cells, while GRHL2 plays different roles in basal-like and mesenchymal-like subgroups. These findings provide new insights into understanding the epigenetic heterogeneity of breast cancer and its potential regulatory mechanisms.

Research Highlights

The highlights of this study include:

  1. Revealing the pivotal role of FOXA1 as a pioneer factor in ER+ breast cancer cell lines.
  2. Identifying the significant role of GRHL2 in basal-like TNBC and how it maintains epigenetic characteristics in different subgroups.
  3. Providing detailed analysis results of chromatin accessibility in breast cancer cell lines, serving as a valuable reference for future breast cancer research.
  4. Discovering the complementary roles of FOXA1 and GRHL2 in regulating the epigenetic features of breast cancer cells.

This study uncovers the crucial roles of FOXA1 and GRHL2 in maintaining chromatin accessibility and lineage characteristics in breast cancer cell lines, offering new perspectives and potential therapeutic targets, laying the foundation for further research into the mechanisms underlying breast cancer heterogeneity.