Expression profiling after induction of demethylation in MCF-7 breast cancer cells identifies involvement of TNF-α mediated cancer pathways

Abstract

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethylation by 5-Aza-2'-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confirmed by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change ≥ 2), respectively, in common in cells treated with 5 and 10 μM of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-α in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.

Fig. 1.

Induction of demethylation by Aza in MCF-7 cells. The methylation status of CpG sites at the SULT1A1 promoter was determined by sequencing after subcloning of the bisulfate-PCR product into the pGEM-T plasmid. Schematic diagram of the CpG sites at the promoter is drawn on the top. Methylated and unmethylated CpGs are indicated as black and open circles, respectively.

Fig. 2.

Highest confidence network of genes displaying altered expression levels in response to demethylation in MCF-7 cells. According to IPA, the network is relevant to ‘Cellular Movement, Cellular Development, Cancer’. Genes that were upregulated in the Aza-treated MCF-7 cells are red, while those that were downregulated are green, and the intensity of the color reflects the magnitude of expression change. Each interaction is supported by at least one literature reference, with solid lines representing direct interactions, and dashed lines representing indirect interactions.

Fig. 3.

Pathways most strongly associated with the significantly altered genes in the Aza-treated MCF-7 cells. (A) Top functional categories. (B) Canonical pathways. The Ingenuity software assigns a P-value based on the likelihood of obtaining the observed number of category or pathway-related molecules in a given data set by chance alone. The threshold line denotes the P = 0.05 level. The line graph represents the ratio of affected genes to the total number of genes in a pathway.

Fig. 4.

Differential methylation and expression of TNF-α in normal and cancer cell lines. Real-time MSP (A) and real-time RT-PCR analysis (B) of TNF-α were performed in the normal cell line, MCF-10A, and the cancer cell line, MCF-7. Each sample was examined in duplicate and the average relative methylation and expression levels are presented.

Fig. 5.

Gene expression profiles of genes with altered expression in response to treatment of the MCF-7 cells with Aza. Heatmap analysis was conducted for the 59 genes that were included in the top two networks of IPA pathway analysis. Each row represents a gene, and each column represents the MCF-7 cells treated with 5 or 10 μM of Aza. As shown in the color bar, black represents no change, red represents upregulation, and green represents downregulation of gene expression.

Fig. 6.

MSP analysis of selected genes displaying altered expression in response to demethylation in the MCF-7 cells. Seven of the upregulated and six of the downregulated genes were randomly selected from the top network of IPA pathway and their methylation was examined by methylation-specific PCR. Each sample was analyzed in duplicate and the average relative methylation level is presented.