DLEC1 is a functional 3p22.3 tumour suppressor silenced by promoter CpG methylation in colon and gastric cancers

Abstract

Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours.

Figure 1

Identification of DLEC1 as a methylated gene in CRC (HCT116) cells. (A) Schematic illustration of the DLEC1 promoter and its CGI. Locations of the exon 1 (indicated with a long rectangle) and CpG sites (short vertical lines) in the CGI are shown. The transcription start site is indicated by a curved arrow. (B) Genetic demethylation reactivated DLEC1 expression in DKO cells. U: unmethylated; M: methylated. (C) Detailed BGS analysis confirmed the MSP results. Methylation status of each individual promoter allele was shown as a row of CpG sites sequenced from each bacterium colony. Filled circle, methylated; open circle, unmethylated.

Figure 2

Frequent methylation-associated silencing of DLEC1 in CRC and gastric cancer cell lines. (A) Expression profile of DLEC1 in human normal adult and foetal tissues by semi-quantitative RT–PCR, with GAPDH as a control. Sk M, skeletal muscle; BM, bone marrow; LN, lymph node. (B) Methylation status and expression levels of DLEC1 in a panel of CRC and gastric cancer cell lines. CCD-841 is an immortalised normal colon epithelial cell line. M, methylated; U, unmethylated. (C) Detailed BGS analysis confirmed the MSP results, as in Figure 1C. (D) Reactivation of DLEC1 by Aza treatment (+), accompanied with demethylation of its promoter CGI.

Figure 3

DLEC1 methylation in primary CRC and gastric tumours. (A) Representative results of DLEC1 methylation as detected by MSP in tumours (T), but not in the paired normal tissues (N). U: unmethylated; M: methylated. (B) High-resolution methylation mapping of individual CpG sites in the DLEC1 CGI by BGS. GsCa: gastric carcinoma.

Figure 4

Ectopic DLEC1 expression suppressed tumour cell clonogenicity of HCT116 and MKN45 cells. A representative inhibition of colony formation by DLEC1 through monolayer culture assay is shown in the left panel. Ectopic DLEC1 expression was determined by RT–PCR and quantitative analyses of colony numbers are shown in the right panel. Values are the mean±s.d. of three independent experiments. ^**P<0.01.