Aberrant DNMT1-mediated DACH1 methylation is associated with colorectal adenoma-to-carcinoma progression

Abstract

Colorectal cancer (CRC) remains a major contributor to cancer-related morbidity and mortality. While Dachshund homolog 1 (DACH1) was recognized as a critical regulator in cancer progression, its role in promoting or suppressing tumor development remains a subject of ongoing debate. This study aimed to elucidate the role of DACH1 in CRC progression and its underlying regulation mechanisms. The expression levels of Methyltransferase 1 (DNMT1) and DACH1, as well as its methylation status were assessed through a combination of TCGA data analysis and experimental validation using immunohistochemistry, PCR, methylation-specific PCR, and bisulfite sequencing RCR on 120 clinical samples, comprising normal mucosa, adenomas, and adenocarcinomas. The relationships among them were evaluated using Pearson or Spearman correlation analysis. The associations between the DACH1 and DNMT1 levels and clinicopathological parameters were examined to determine their clinical relevance. A progressive decrease in DACH1 expression and a concomitant increase in DACH1 promoter methylation and DNMT1 expression were observed from normal mucosa to adenoma and adenocarcinoma tissues. Higher DNMT1 expression and lower DACH1 expression were associated with poorer clinical outcomes, including worse tumor differentiation, lymphatic metastasis, and advanced tumor stages. Paired analysis of tissues from the same patient further validated their inverse expression patterns during CRC progression. DNMT1-mediated DACH1 epigenetic silencing plays a critical role in CRC progression, suggesting that the DNMT1-DACH1 regulatory axis may serve as a potential biomarker and therapeutic target in CRC.

Keywords: DACH1; DNMT1; colorectal cancer; promoter methylation; tumor progression.

FIGURE 1

DACH1 expression in normal, colorectal adenoma, and adenocarcinoma tissues. Forty cases each of normal colorectal mucosa, colorectal adenoma, and colorectal adenocarcinoma tissues were collected from patients. (A) The DACH1 protein expression determined by immunohistochemical analysis in these tissues. Representative images were demonstrated by the magnification of 100 × and 400 ×. (B) Immunohistochemical staining intensity evaluated using semi-quantitative scoring for negative (−), weak (+), moderate (++), and high (+++) DACH1 expression across the “normal-adenoma-adenocarcinoma” progression. (C) Relative mRNA expression of DACH1 was measured by qRT-PCR. Data are presented as mean ± SEM. ***p < 0.001.

FIGURE 2

Relationship of DACH1 expression and promoter methylation cross colorectal cancer progression. (A) Correlation analysis of DACH1 promoter methylation levels and mRNA expression and (B) comparison of DACH1 methylation levels between the patients stratified by DACH1 median mRNA expression in colorectal tissues based on the TCGA database. (C) Genomic organization of the DACH1 promoter region and visualization of CpG island regions targeted by methylation-specific PCR. (D) The schematic diagram of the DACH1 promoter and the location of 15 CpG sites detected in this study. (E) Methylation profiling across normal mucosa, adenoma, and adenocarcinoma tissues and quantitative analysis of mean methylation rates across the three stages. (F) Luciferase reporter assay confirmed that DACH1 methylation regulates the transcriptional activity of its promoter. Data are presented as mean ± SEM. ***p < 0.001.

FIGURE 3

Relationship of DNMT1 expression with DACH1 expression and methylation in colorectal cancer. Correlation analysis of DNMT1 and (A) DACH1 mRNA expression as well as (B) DACH1 promoter methylation levels using TCGA database. (C) DACH1 methylation levels in patients divided based on median DNMT1 levels. Data are presented as mean ± SEM. *p < 0.05.

FIGURE 4

DACH1 expression was regulated by DNMT1-mediated methylation in vitro. After being exposed to a DNMT1 inhibitor (5-Aza-dC), HCT116 cells were collected for analyzing the (A) mRNA, (B) protein, and (C) methylation levels of DACH1. Data are presented as mean ± SEM. ***p < 0.001.

FIGURE 5

DNMT1 expression in colorectal cancer progression. (A) Immunohistochemical analysis of DNMT1 protein expression in normal colorectal mucosa, adenoma, and adenocarcinoma tissues of patients (magnifications used were 100 × and 400 ×) and (B) semi-quantitative scoring of staining defined as negative (−), weak (+), moderate (++), and high (+++) levels. (C) Quantitative PCR analysis of DNMT1 mRNA levels from normal mucosa to adenoma and adenocarcinoma tissues. (D) Correlation analysis of DNMT1 and DACH1 mRNA expression conducted by Pearson’s correlation analysis. Data are presented as mean ± SEM. ***p < 0.001.

FIGURE 6

DACH1 and DNMT1 protein expression patterns across normal, adenoma, and carcinoma tissues from the same patient. Three types of tissues were collected from a patient to evaluate the protein levels of (A) DACH1 and (B) DNMT1 by immunohistochemical analysis. The magnifications used were 100 ×. (C) The DACH1 and DNMT1 expressions were scored, and a correlation analysis was performed between them.