Cysteine dioxygenase type 1 (CDO1) gene promoter methylation during the adenoma-carcinoma sequence in colorectal cancer

Abstract

Background: Progression of colorectal cancer (CRC) has been explained by genomic abnormalities along with the adenoma-carcinoma sequence theory (ACS). The aim of our study is to elucidate whether the promoter DNA methylation of the cancer-specific methylation gene, cysteine dioxygenase 1 (CDO1), contributes to the carcinogenic process in CRC.

Methods: The study group comprised 107 patients with CRC who underwent surgical resection and 90 adenomas treated with endoscopic resection in the Kitasato University Hospital in 2000. We analyzed the extent of methylation in each tissue using quantitative TaqMan methylation-specific PCR for CDO1.

Results: The methylation level increased along with the ACS process (p < 0.0001), and statistically significant differences were found between normal-appearing mucosa (NAM) and low-grade adenoma (p < 0.0001), and between low-grade adenoma and high-grade adenoma (p = 0.01), but not between high-grade adenoma and cancer with no liver metastasis. Furthermore, primary CRC cancers with liver metastasis harbored significantly higher methylation of CDO1 than those without liver metastasis (p = 0.02). As a result, the area under the curve by CDO1 promoter methylation was 0.96, 0.80, and 0.67 to discriminate cancer from NAM, low-grade adenoma from NAM, and low-grade adenoma from high-grade adenoma, respectively.

Conclusions: CDO1 methylation accumulates during the ACS process, and consistently contributes to CRC progression.

Fig 1. Standard light microscopy findings on hematoxylin-eosin-stained specimens of colorectal adenoma (40×, magnification) and ROC curves for distinguishing between the two tissues.

A: mild atypia. B: moderate atypia. C: severe atypia. D: ROC curve of CDO1 promoter methylation for distinguishing between cancer tissue and NAM. When the cut-off value was 15.6, the AUC was 0.96, sensitivity was 95%, and specificity was 90%. E: ROC curve for distinguishing between low-grade adenoma and NAM. F: ROC curve for distinguishing between low-grade adenoma and high-grade adenoma.

Fig 2. Associations of clinicopathological factors with CDO1 TaqMeth V in cancerous tissue.

The relationship between CDO1 and A: age, B: histological type, C: tumor diameter, D: liver metastasis, E: pT, F: v, G and H: Dukes classification.

Fig 3. Results of the anchorage-independent colony formation assay.

A: Expression of CDO1 cDNA by RT-PCR. The positive control was HepG2 cells, and the negative control was DLD1 cells [8, 16, 18]. In HCT116 cells, expression of CDO1 was not observed. B: Control, not transfected; mock, mock treatment; CDO1 transfection, CDO1 cells transfected with pcDNA 3.1-CDO1. The colonies were photographed under UV after staining with ethidium bromide staining. The number of colonies was small in the cells transfected with CDO1. C: Colony formation of each experimental condition. Colonies were photographed under phase-contrast microscope. The image is magnified 100 times. D: The numbers of colonies are shown. Cell proliferation by HCT116 cells was suppressed by transfection of CDO1.

Fig 4. Results of CDO1 immunostaining.

A: IHC score = 0. B: IHC score = 1. C: IHC score = 2. D: Results of IHC scoring in cancer tissue and NAM are shown. Intense staining was observed in NAM, and less intense staining was seen in cancer tissues. A significant difference in scoring was seen between the groups.

Fig 5. Log-rank plot analysis and Kaplan-Meier survival curves for OS and RFS in CRC patients according to CDO1 TaqMeth V.

A, B: Log-rank plot analysis for OS. C: Kaplan-Meier survival curves for OS comparing CRC patients with CDO1 TaqMeth V equal to or below 20.5 and those with CDO1 TaqMeth V over 20.5. D, E: Log-rank plot analysis for RFS. F: Kaplan-Meier survival curves for RFS comparing CRC patients with CDO1 TaqMeth V equal to or below 44.8 and those with CDO1 TaqMeth V over 44.8.

Fig 6. Analysis of CDO1 TaqMeth V derived from NAM, adenoma, and cancerous tissue.

A: Adenoma is classified into three categories: mild atypia, moderate atypia, and severe atypia. B: Adenomas were divided into low-grade adenoma and high-grade adenoma.