The Mechanism of Action of Specific Reprogramming of Histone H3 Lysine 9 Acetylation (H3K9ac) in the Progression and Metastasis of Esophageal Squamous Cell Carcinoma (ESCC)

Background

Esophageal cancer is one of the most prevalent and invasive malignant tumors globally, causing over 500,000 cancer-related deaths annually, ranking sixth among cancer-related causes of death. Esophageal squamous cell carcinoma (ESCC) accounts for nearly 90% of all esophageal cancer cases, primarily affecting populations in East Asia and Africa. Despite significant advancements in treatment over the past few decades, the overall 5-year survival rate for ESCC remains below 20%. This is mainly due to the asymptomatic early stages of ESCC, with most patients being diagnosed at a locally advanced stage or with regional lymph node metastasis, leading to high recurrence or distant metastasis, eventually causing morbidity.

Currently, it is known that most human cancers result from the combined effects of gene mutations and epigenetic changes, including DNA methylation and histone post-translational modifications (PTMs). The genomic abnormalities of ESCC have been widely studied, but driver mutations are lacking, resulting in the limited implementation of genome-guided therapeutic strategies. In contrast, the plasticity and reversibility of epigenetic modifications make them ideal drug targets in anti-cancer strategies. Therefore, accurately assessing epigenetic reprogramming patterns in the progression and metastasis of ESCC is of great significance.

Research and Occurrence

This paper, co-authored by researchers from the School of Biomedical Engineering at Wenzhou Medical University, the First Affiliated Hospital of Wenzhou Medical University, and the Ninth People’s Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, was published in the journal “Cancer Gene Therapy.” The main purpose of this study is to analyze the mechanism of the influence of H3K9ac in the progression and metastasis of ESCC and explore its potential value as a therapeutic target.

This study focuses on the genomic distribution characteristics of H3K9ac, and compares it with another active marker, H3K27ac. The research involves three ESCC patients, extracting samples from their paired adjacent normal tissue (nor), primary carcinoma tissue (ec), and metastatic lymph node tissue (lnc), and mapping their H3K9ac distribution via chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). The data were integrated and compared with previously generated H3K27ac data, combined with whole-genome transcriptome data and single-cell RNA sequencing (scRNA-seq) data, to reveal the characteristics of H3K9ac in the progression and metastasis of ESCC and validate its regulatory pattern.

Research Methods and Process

Experimental Process

This study mainly includes the following steps:

1. Sample Collection and Processing:

   • Selected three ESCC patients, extracting paired adjacent normal tissue, primary carcinoma tissue, and metastatic lymph node tissue.
   • The samples were subjected to ChIP-seq to map the distribution of H3K9ac and H3K27ac.

2. Data Processing and Analysis:

   • Quality control and alignment were performed on the H3K9ac and H3K27ac sequencing data.
   • Standardization methods were employed to eliminate batch effects, ensuring comparability between different datasets.

3. Peak Analysis and Region-Specific Study:

   • Peak overlap analysis was performed to identify specific regions occupied by H3K9ac and H3K27ac.
   • DNA motif analysis was conducted to predict transcription factors enriched in H3K9ac or H3K27ac regions.

4. Identification of Differential Peaks:

   • Differential peaks of H3K9ac and H3K27ac in EC and LNC samples were identified.
   • Unsupervised hierarchical clustering analysis was performed to determine specific groups of H3K9ac and H3K27ac signals.

5. Functional Relevance Analysis:

   • Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to understand the function of genes associated with H3K9ac and their role in cancer.
   • Gene Set Enrichment Analysis (GSEA) validated the functional differences between different gene groups.

6. In Vitro Experimental Validation:

   • Gene knockout experiments were used to validate the regulatory role of H3K9ac on representative target gene msi1.
   • CCK-8 and Transwell migration assays evaluated the impact of H3K9ac knockout on ESCC cell proliferation and migration.

Main Results

1. Region-Specific Occupancy of H3K9ac and H3K27ac:

   • H3K9ac and H3K27ac occupy different regions in paired normal tissues, with H3K9ac occupying significantly more unique regions than H3K27ac. Motif analysis indicated that H3K9ac-specific regions are rich in important transcription factors that regulate cell growth and differentiation.

2. Epigenetic Characteristics of H3K9ac Reprogramming in ESCC:

   • ChIP-seq data analysis revealed differences in H3K9ac-specific regions between primary cancer and metastatic lymph node samples. Specific regions were enriched in cancer driver genes, such as msi1.

3. Transcriptome Data Supporting H3K9ac Regulatory Patterns:

   • Combined transcriptome data showed that genes in regions enriched with H3K9ac are upregulated, while genes in regions with H3K9ac loss are downregulated.
   • Gene expression levels were analyzed in each group, verifying the significant positive correlation between H3K9ac and gene transcription activity.

4. Functional Relevance of H3K9ac-Specific Reprogramming to Tumorigenesis and Metastasis:

   • Clarified the role of H3K9ac-specific reprogramming in tumor signaling pathways, especially in cell polarity establishment and maintenance, junction assembly, and amoeboid cell migration.
   • Single-cell RNA sequencing revealed the heterogeneity of ESCC cells in different pathological states, with g2 and g5 gene groups being enriched in highly migratory mesenchymal cells.

Experimental Validation

a) In Vitro Experiments: Using shRNA interference to downregulate H3K9ac expression in ESCC cells, a significant decrease in the expression of target gene msi1 was observed, thus inhibiting the proliferation and migration ability of ESCC cells.

b) Clinical Relevance: High expression of msi1 was detected in The Cancer Genome Atlas (TCGA) database, significantly associated with lymph node metastasis and poor survival prognosis in ESCC patients.

Research Conclusions and Value

This study demonstrated the unique role of H3K9ac-specific reprogramming in the development of ESCC, revealing its close association with transcriptome abnormalities, highlighting its potential therapeutic value in tumor epigenetic regulation.

Scientific Value: - The study enriches the understanding of the role of H3K9ac in cancer epigenetic regulation, revealing for the first time its specific reprogramming pattern distinct from another active marker, H3K27ac.

Application Value: - In-depth research on H3K9ac epigenetic regulation provides new targets and strategies for precise treatment and prognosis evaluation of ESCC.

Research Highlights: - Comprehensive revelation of H3K9ac’s role in ESCC progression and metastasis through integration of ChIP-seq, RNA-seq, and scRNA-seq data. - In vitro experiments confirmed the regulatory role of H3K9ac on the representative target gene msi1, providing a solid foundation for future research.

Despite the limited number of research samples, the results provide important insights for further exploration of H3K9ac in ESCC treatment, potentially opening new pathways for cancer therapy through epigenetic regulation.