Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model

Abstract

Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer-specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19(Arf), Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.

Figure 1. Global methylation analysis of AOM induced tumors

A.) DNA samples from normal colon (N1 and N2) and colon adenocarcinomas (T1–T5) were heat denatured, spotted on a nitrocellulose membrane in triplicate then immobilized with ultraviolet light. The blot was probed first with an antibody against methylcytosine, then with a secondary antibody conjugated to an infrared dye. Three replicates (Rep 1–3) were performed for each sample. The signal for each band was quantified and the average and standard errors were calculated for each sample. B.) The N1 and N2 samples were averaged to set a normal threshold. Each sample was then graphed as the Log₂ ratio of this normal threshold.

Figure 2. MSP analysis of representative candidate genes in AOM induced colon adenocarcinomas and normal colon mucosa

Representative images from MSP assays for Mgmt and Cdkn2A (p16^Ink4a) from colon neoplasms (T1–T4) and normal colon (N1–N4) are shown. Methylated genes (M) are present in the normal colon mucosa and colon tumor tissues. Unmethylated (U) genes were identified in both normal and tumor cells, which may represent contamination with heterogeneous tissue elements or a clonal mixture of cells. MSP reactions were performed using the primer sets and annealing parameters listed in Supplementary Table 1. Amplicon size (in bp) is shown. Sssi (methylation positive control) and WGA (methylation negative control) control DNA samples are not shown.

Figure 3. MSP and bisulfite sequencing forGja9 and Zik1

A1, B1. Schematic diagrams of the promoter region of Gja9 and Zik1, respectively. Location of the transcription start site (Tss) is shown with an arrow and location of MSP and bisulfite sequencing primers are shown with black and grey boxes, respectively. A2, B2. Results of MSP analysis for Gja9 and Zik1 for four AOM tumors (T1–T4) and normal colon samples (N1–N4). Methylated (M) and Unmethylated (U) genes are shown. A3, B3. Results of bisulfite sequencing analysis of the promoter region of Gja9 and Zik1. Ten clones for each gene were isolated and sequenced from a tumor that was shown to carry either methylated Gja9 or Zik1 by MSP. Empty and black circles represent unmethylated and methylated CpG dinucleotides, respectively. A subset of the sequenced clones demonstrates methylation for each gene.

Figure 4. qRT-PCR analysis of Zik1 expression

Normalized expression of Zik1 in AOM tumors T1–T4 and normal colonic epithelium N1–N2. The designation of the tumors corresponds to methylation analysis of tumors and normal tissue demonstrated in Figure 3.