Cell Discovery: Epigenetic regulatory mechanisms in pancreatic ductal adenocarcinoma

Pancreatic cancer is one of the common digestive tract malignancies, and is known as the king of cancer because of its extremely high mortality rate.

In order to comprehensively reveal the molecular characteristics of pancreatic ductal adenocarcinoma tumor cells, the research group of Professor Tang Fuchou from Beijing Future Gene Diagnosis Advanced Innovation Center and Peking University Biomedical Frontier Innovation Center cooperated with the research group of Professor Hao Jihui from Tianjin Medical University Cancer Hospital.

Figure 1.

The main findings of the study are:

(1) There is a high proportion of normal pancreatic epithelial cells in the primary tumor tissue of pancreatic ductal adenocarcinoma, which provides a precise control for the study of tumor cell characteristics

In this study, the researchers optimized the single-cell multi-omics sequencing technology developed in the laboratory.

Figure 2.

(2) In pancreatic ductal adenocarcinoma tumor cells, DNA methylation in the promoter regions of hundreds of genes was strongly up-regulated, and the corresponding chromatin changed from an open state to a closed state, and showed a high degree of consistency in all patients

Although the overall DNA methylation level of pancreatic ductal adenocarcinoma tumor epithelial cells was reduced, the results of differential methylation analysis of gene promoter regions showed that the number of genes whose DNA methylation was up-regulated in gene promoter regions was much higher in tumor cells.

Figure 3.

(3) In pancreatic ductal adenocarcinoma tumor cells, the DNA methylation of gene promoters was negatively correlated with the expression of corresponding genes, while the DNA methylation of gene body was enhanced with the expression of corresponding genes.

Through the correlation analysis of transcriptome and DNA methylome in the same single cell, the researchers found that compared with normal epithelial cells, the DNA methylation and RNA expression of the gene body of tumor cells were higher.

Figure 4.

(4) Genome-wide large-scale DNA demethylation in pancreatic ductal adenocarcinoma tumor cells is strongly enriched in heterochromatin regions, indicating that heterochromatin instability is an important molecular feature of pancreatic ductal adenocarcinoma tumorigenesis

Compared with normal epithelial cells, pancreatic ductal adenocarcinoma tumor cells from different patients experienced different degrees of genome-wide DNA demethylation (except for the P05 patient)

Figure 5.

(5) Combined multi-omics analysis revealed key transcription factors that play an important role in the tumorigenesis of pancreatic ductal adenocarcinoma represented by FOSL2 (function enhancement) and ASCL1 (function reduction)
.

The researchers conducted a correlation analysis between the degree of chromatin openness and the level of gene expression, and found that the chromatin state of the promoter region was positively correlated with the level of gene expression (Figure 6)
.
64,339 and 47,622 open chromatin regions were identified in tumor cells and normal epithelial cells, respectively, of which about 30% were tumor cell-specific
.
Through the enrichment analysis of their specific open region transcription factor binding sequences, the researchers identified a family of transcription factors that were specifically enriched in pancreatic ductal adenocarcinoma tumor cells, such as the FOSL2 gene, which showed higher expression levels in tumor cells and regulatory activity, and higher expression levels indicate worse prognosis, indicating that enhanced regulatory activity may play an important role in pancreatic ductal adenocarcinoma tumorigenesis
.
In contrast, nervous system-related transcription factors, such as ASCL1, are significantly enriched in normal epithelial cells, exhibit lower expression levels and regulatory activity in tumor cells, and lower expression levels indicate worse prognosis, These results indicated that the reduced regulatory activity may play an important role in the tumorigenesis of pancreatic ductal adenocarcinoma
.

Figure 6.
Enrichment analysis of chromatin open regions and transcription factor binding sequences specific for pancreatic ductal adenocarcinoma tumor cells and normal epithelial cells

(6) The prognostic candidate markers of pancreatic ductal adenocarcinoma represented by ZNF667 and ZNF667-AS1 were screened by combined analysis of DNA methylome and transcriptome
.

Through combined analysis of DNA methylation and gene expression, the researchers found 77 differentially methylated genes of candidate pancreatic ductal adenocarcinoma prognostic and diagnostic molecular markers
.
Among them, ZNF667 and ZNF667-AS1 are marker molecules that have been reported in cancers such as laryngeal squamous cell carcinoma and non-small cell lung cancer
.
Through quantitative analysis of ZNF667 protein in tumor tissues of 98 PDAC patients, the researchers found that patients with high expression of ZNF667 protein had significantly longer survival time
.
In addition, the researchers found that the overexpression of ZNF667 and ZNF667-AS1 genes in various pancreatic cancer cell lines can inhibit tumor cell proliferation, but not affect tumor cell apoptosis, indicating that these two genes are mainly inhibited by inhibiting cell proliferation.
Oncogenesis of pancreatic ductal adenocarcinoma
.

Figure 7.
Bioinformatics analysis and functional validation reveal ZNF667 and ZNF667-AS1 as candidate prognostic markers for pancreatic ductal adenocarcinoma

In summary, this study used high-precision single-cell multi-omics sequencing analysis to deeply analyze the molecular characteristics of pancreatic ductal adenocarcinoma tumor cells in different omics dimensions and their interrelationships.
A series of potential molecular markers for the prognosis and diagnosis of pancreatic ductal adenocarcinoma, represented by FOSL2, ASCL1, ZNF667 and ZNF667-AS1, were characterized and provided a reference scheme for the diagnosis and treatment of pancreatic ductal adenocarcinoma
.

Researcher Fan Xiaoying from Guangzhou Laboratory, Lu Ping, a doctoral student from the School of Life Sciences of Peking University, Dr.
Wang Hongwei from Tianjin Medical University Cancer Hospital, Researcher Bian Shuhui from Nanjing Medical University, and Associate Professor Wu Xinglong from Hebei Agricultural University are the co-first authors of the paper
.
Tang Fuchou and Hao Jihui are the co-corresponding authors of the paper
.
This research project is supported by the National Natural Science Foundation of China, the Beijing Municipal Science and Technology Commission and the Beijing Future Gene Diagnosis Advanced Innovation Center
.