Volume 131, Issue 3, Pages 797-808 (September 2006) A Genome-Wide Search Identifies Epigenetic Silencing of Somatostatin, Tachykinin-1, and 5 Other Genes in Colon Cancer  Yuriko Mori, Kun Cai, Yulan Cheng, Suna Wang, Bogdan Paun, James P. Hamilton, Zhe Jin, Fumiaki Sato, Agnes T. Berki, Takatsugu Kan, Tetsuo Ito, Carmit Mantzur, John M. Abraham, Stephen J. Meltzer  Gastroenterology  Volume 131, Issue 3, Pages 797-808 (September 2006) DOI: 10.1053/j.gastro.2006.06.006 Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 1 Outline of search for colon cancer-specific methylation target genes. This figure is an outline of our experiments, which consisted of 2 parts: the gene filtering for candidate methylation targets and the epigenetic validation. The gene-filtering part consisted of 2 sets of cDNA microarray experiments and a 2-step in silico genetic search. The first set of cDNA microarray experiments was performed to identify genes that were down-regulated in primary colon cancers relative to normal colonic mucosae. The second set of cDNA microarray experiments was to identify genes that were up-regulated in colon cancer cell lines after 5-aza-dC treatment (PostAzaC) relative to cells before the treatment (PreAzaC). Genes that were identified in both sets of cDNA microarray experiments were subjected to the subsequent selection using in silico search. The in silico search consisted of screenings for genes possessing (1) CpG island overlapping the 5′-UTR or the first exon and (2) putative tumor suppressive function. Candidate methylation targets meeting both of these selection criteria were then validated for the presence of promoter methylation in colon cancer cell lines and primary colon cancers using real-time quantitative MSP. Numbers in this figure represent the actual number of genes that fell into each of the corresponding categories. Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 2 SST and TAC1 gene promoter regions were hypermethylated in primary colon cancers. (A) These box plots represent SST- and TAC1-promoter methylation index (MI) for 17 normal colonic mucosae (highlighted in light grey) or 34 primary cancers (highlighted in dark grey). Methylation index (MI) represents the ratio of densely methylated DNA in the sample at the target sequence relative to the fully methylated positive control DNA. Primary colon cancers were significantly more methylated than were normal colonic mucosae for both SST and TAC1 (P < .001 and P = .01, Mann–Whitney U test). Circle, sample belonging to over the 90th or below the 10th percentile range; whisker, 10th or 90th percentile value; rectangle box, range for 25th to 75th percentile; black square, median value. Measurement of MI was performed using real-time quantitative MSP, and a CpG-free β-actin genomic sequence was used for normalization. (B) These line graphs display MI at the SST- and TAC1-promoter regions for matching normal colonic mucosae (left-side data point) and colon cancers (right-side data point) from 6 patients. An increased promoter methylation level was observed for SST in all 6 (100%) and TAC1 in 4 (67%) of 6 cases. Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 3 SST and TAC1 promoter methylation were significantly associated with colon tumor characteristics. These plots demonstrate the significant association between tumor MSI status and SST promoter methylation (left panel) as well as Duke’s stage and TAC1 promoter methylation (right panel). Each box plot represents the methylation index (MI) of each gene for each tumor group. SST methylation was significantly higher in 7 MSI-L cancers than in 27 non-MSI-L cancers (12 MSI-H plus 15 MSS; P = .02, Mann–Whitney U test). Similarly, TAC1 methylation was significantly higher in 13 Duke’s A/B cancers than in 20 Duke’s C/D cancers (P = .01, Mann–Whitney U test). Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 4 SST and TAC1 demonstrated decreased promoter methylation and up-regulated mRNA expression in colon cancer cell lines after 5-aza-dC treatment. These line graphs demonstrate mRNA up-regulation in association with decreased promoter methylation for SST and TAC1 induced by 5-aza-dC treatment in 2 colon cancer cell lines: HCT116 and HT29. Dashed and solid plots represent expression index (EI) and methylation index (MI), respectively. EI is mRNA expression level relative to a normal colonic mucosa specimen. Red and blue plots represent data for HCT116 and HT29, respectively. PreAzaC: before 5-aza-dC treatment; PostAzaC: after 5-aza-dC treatment. MI and EI data were measured using real-time quantitative MSP and real-time quantitative RT-PCR, respectively. Data normalization was performed using a CpG-free genomic sequence (MSP) and mRNA sequence (RT-PCR) of the β-actin gene. Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 5 Promoter regions of NELL1, CAV1, AKAP12, ENG, and MAL were hypermethylated in primary colon cancers. Each box plot represents the methylation index (MI) at each gene promoter region for 17 normal colonic mucosae (highlighted in light grey) or 34 primary cancers (highlighted in dark grey). Primary colon cancers were significantly more methylated than normal colonic mucosae for NELL1 (P < .001), CAV1 (P < .001), AKAP12 (P = .01), ENG (P < .01), and MAL (P < .01). P values were calculated by the Mann–Whitney U test. Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

Figure 6 NELL1, CAV1, AKAP12, ENG, and MAL demonstrated decreased promoter methylation and up-regulated mRNA expression in colon cancer cell lines after 5-aza-dC treatment. These plots demonstrate mRNA up-regulation in association with decreased promoter methylation for each gene induced by 5-aza-dC treatment in 2 colon cancer cell lines: HCT116 and HT29. Dashed and solid plots represent expression index (EI) and methylation index (MI), respectively. Red and blue plots represent data for HCT116 and HT29, respectively. PreAzaC: before 5-aza-dC treatment; PostAzaC: after 5-aza-dC treatment. A cell line was eliminated from analysis when the target gene promoter region was not methylated prior to 5-aza-dC treatment or failed to be demethylated by 5-aza-dC treatment. Gastroenterology 2006 131, 797-808DOI: (10.1053/j.gastro.2006.06.006) Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions