DNMT1-Induced Downregulation of CBX7 Inhibits ERK Phosphorylation and Promotes Pancreatic Ductal Adenocarcinoma Progression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types, characterized by an alarmingly low 5-year survival rate. DNA methylation has been implicated in the progression of various tumors, with DNA methyltransferase 1 (DNMT1) being the most extensively studied enzyme in this context. However, the expression patterns and underlying mechanisms of DNMT1 in PDAC remain poorly understood. The levels of DNMT1 and CBX7 in PDAC tissues and cells were determined by IHC and Western blot. ChIP and dual-luciferase reporter assays confirmed the interaction between DNMT1 and the CBX7 promoter. Cellular functions were evaluated through CCK-8, wound healing, and transwell assays. The expression of MAPK-related proteins was analyzed by Western blot. DNMT1 expression was upregulated in PDAC tissues and cell lines, whereas CBX7 expression was downregulated. Silencing DNMT1 inhibited cell proliferation, migration, and invasion in PDAC by modulating CBX7 expression. Moreover, DNMT1 methylates the CBX7 promoter region, leading to increased ERK phosphorylation, which subsequently drives tumorigenesis and metastasis in PDAC. DNMT1 promotes the malignant progression of PDAC through the CBX7/ERK pathway. Our study provides evidence for potential therapeutic targets for the comprehensive treatment of PDAC.

Keywords: CBX7; DNMT1; pancreatic ductal adenocarcinoma; promoter methylation.

FIGURE 1

DNMT1 expression is upregulated in PDAC tissues and cells, and is associated with the malignant progression of PDAC cells. (A) IHC examined DNMT1 expression in PDAC tissues and adjacent normal pancreatic tissues. The scale bar indicated in panel (B) was 100 and 10 μm, respectively. (B) The IHC scoring was performed, and the correlation between IHC score of CBX7 and cancer stage was assessed. The scale bar indicated in panel (B) was 100 and 10 μm, respectively. (C) DNMT1 expression in the human pancreatic duct epithelial cell line (HPDE) and a panel of PDAC cell lines was measured by western blot analysis. (D) PANC‐1 and ASPC‐1 cells were transfected with si‐NC, siDNMT1‐1, or siDNMT1‐2 for 48 h. Then, cells were collected for western blot analysis. (E–G) PANC‐1 and ASPC‐1 cells were infected with indicated si‐NC, siDNMT1‐1, or siDNMT1‐2 for 48 h. Cells were harvested for CCK‐8 assay, transwell assay, and wound healing assay. Data presented as the mean ± SD of three independent experiments. ***p < 0.001.

FIGURE 2

Integrative Bioinformatics and Experimental Analysis Reveal CBX7 as a Key DNMT1‐Associated Gene in PDAC. (A) A total of 5048 genes were identified as strongly associated with DNMT1 through a correlation analysis conducted on DNMT1 and all mRNAs in the TCGA‐ICGC dataset. (B) Correlation analysis of DNMT1‐HRGs and EZH2 identified 159 genes with a negative correlation, as calculated using the method described above. (C) Correlation analysis identified mRNAs highly correlated with DNMT1 and EZH2 expression (|correlation coefficient| > 0.6), followed by PPI network analysis using STRING (https://cn.string‐db.org/). (D) PANC‐1 cells were transfected with si‐NC, siDNMT1‐1, or siDNMT1‐2 for 48 h. Then, cells were collected for western blot analysis. (E) CBX7 mRNA expression between PDAC tissues and the normal tissues in TCGA and GTEx. (F) Kaplan‐Meier plot of overall survival of PDAC patients in TCGA_ICGC, GSE71729, GSE21501, and GSE62452. (G‐H) IHC examined CBX7 expression in PDAC tissues and adjacent normal pancreatic tissues. The scale bar indicated in panel (B) was 100 and 10 μm, respectively. ***p < 0.001.

FIGURE 3

CBX7 overexpression inhibits PDAC cell proliferation, migration, and invasion, while promoting apoptosis. (A, B) CBX7 expression in the HPDE cell line and a panel of PDAC cell lines was measured by (A) western blot analysis and (B) qRT‐PCR analysis. (C) PANC‐1 and ASPC‐1 cells were transfected with the pCDH‐CBX7 expression vector for 48 h, and CBX7 expression was measured by western blot. (D–F) PANC‐1 and ASPC‐1 cells were transfected with the pCDH‐CBX7 expression vector for 48 h. Cells were harvested for CCK‐8 assay, transwell assay, and wound healing assay. Data presented as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001.

FIGURE 4

DNMT1 suppresses CBX7 expression by promoting methylation of the CBX7 promoter CpG island region. (A) Schematic representation of CBX7 promoter CpG islands and bisulfite sequencing region in the CBX7 promoter. (B) The UALCAN (http://ualcan.path.uab.edu/index.html) website was used to analyze the correlation between promoter methylation and expression of CBX7 in PDAC. (C)Methylation status of the CBX7 promoter was evaluated in DNMT1 knockdown cells using methylated (M) and unmethylated (U) primers. Methylation was determined by PCR agarose gel electrophoresis. (D) Luciferase reporter assays detected the CBX7 promoter region activity after ectopic expression of DNMT1 in PANC‐1 cells. (E) ChIP assays on the CBX7 promoter were done using anti‐DNMT1 antibody in PANC‐1 cells and were analyzed by qRT‐PCR. DNA fragments obtained without antibody served as input controls. Histone H3 antibodies were used as the positive control in ChIP assays, with IgG as the negative control. ***p < 0.001.

FIGURE 5

DNMT1 promotes the viability of PDAC cells via the CBX7/ERK signaling pathway. (A) This heatmap shows the clustering of transcriptomes from two groups: CBX7‐overexpressing (oe‐CBX7) PANC‐1 and control cells (NC), each with three biological replicates (oe‐CBX7: Oe‐1, oe‐2, oe‐3; NC: NC‐1, NC‐2, NC‐3). (B) KEGG enrichment analysis of RNA‐seq data between the oe‐CBX7 and NC groups. p‐values as indicated. (C) GSEA analysis of RNA‐seq data of CBX7 from the PANC‐1 cells. p‐values as indicated. (D) PANC‐1 and ASPC‐1 cells were transfected with pCDH‐CBX7 expression vector for 48 h. Cells were harvested for western blot analysis. (E) PANC‐1 cells were transfected with si‐DNMT1 + si‐NC, si‐DNMT1 + si‐CBX7, si‐NC + si‐CBX7, or si‐NC + si‐NC for 48 h. Then, cells were harvested for western blot analysis. (F) A working model of the DNMT1/CBX7/ERK axis in PDAC progression by Figdraw.