Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers
Byonggu An
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Surgery, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu City, Shiga 520-2192, Japan
Search for more papers by this authorCorresponding Author
Yutaka Kondo
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Tel.: +81-52-764-2993, Fax: +81-52-764-2993
Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, JapanSearch for more papers by this authorYasuyuki Okamoto
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKeiko Shinjo
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorYukihide Kanemitsu
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKoji Komori
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorTakashi Hirai
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorAkira Sawaki
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorMasahiro Tajika
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorTsuneya Nakamura
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKenji Yamao
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorYasushi Yatabe
Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorMakiko Fujii
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorHideki Murakami
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorHirotaka Osada
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorTohru Tani
Department of Surgery, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu City, Shiga 520-2192, Japan
Search for more papers by this authorKeitaro Matsuo
Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorLanlan Shen
Department of Leukemia, The University of Texas at M.D. Anderson Cancer Center, Houston, TX 77030, USA
Search for more papers by this authorJean-Pierre J. Issa
Department of Leukemia, The University of Texas at M.D. Anderson Cancer Center, Houston, TX 77030, USA
Search for more papers by this authorYoshitaka Sekido
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorByonggu An
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Surgery, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu City, Shiga 520-2192, Japan
Search for more papers by this authorCorresponding Author
Yutaka Kondo
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Tel.: +81-52-764-2993, Fax: +81-52-764-2993
Division of Molecular Oncology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, JapanSearch for more papers by this authorYasuyuki Okamoto
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKeiko Shinjo
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorYukihide Kanemitsu
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKoji Komori
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorTakashi Hirai
Department of Gastroenterological Surgery, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorAkira Sawaki
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorMasahiro Tajika
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorTsuneya Nakamura
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorKenji Yamao
Department of Gastroenterology, Aichi Cancer Center Central Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorYasushi Yatabe
Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorMakiko Fujii
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorHideki Murakami
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorHirotaka Osada
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorTohru Tani
Department of Surgery, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu City, Shiga 520-2192, Japan
Search for more papers by this authorKeitaro Matsuo
Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Search for more papers by this authorLanlan Shen
Department of Leukemia, The University of Texas at M.D. Anderson Cancer Center, Houston, TX 77030, USA
Search for more papers by this authorJean-Pierre J. Issa
Department of Leukemia, The University of Texas at M.D. Anderson Cancer Center, Houston, TX 77030, USA
Search for more papers by this authorYoshitaka Sekido
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464-8681, Japan
Department of Cancer Genetics, Program in Function Construction Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, 466-8550, Japan
Search for more papers by this authorAbstract
Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP-negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal-appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP-negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal-appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray-based genome-wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP-negative distal CRCs and CIMP-positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP-negative distal CRCs were also methylated in the normal-appearing mucosae, indicating that hypermethylation in CIMP-negative distal CRCs is more closely associated with age-related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP-positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
| Filename | Description |
|---|---|
| IJC_25225_sm_supinfo.pdf833.6 KB | Supporting Information 1 |
| IJC_25225_sm_supfig1.eps341.4 KB | Supplementary Fig. 1 Confirmation of methylation status of CIMP-positive proximal and CIMP-negative distal CRC cases. A, Levels of methylation of normal-appearing mucosae or CRCs measured by bisulfite pyrosequencing methylation analysis in CIMP-positive proximal and CIMP-negative distal CRCs. Each circle represents the methylation level of normal-appearing mucosae and cancerous tissues from CIMP-positive proximal (white) or CIMP-negative distal (black) CRC cases. Y-axis indicates the levels of methylation of each gene. Horizontal bars denote median methylation levels for each group. *, P<0.05; **, P<0.01. B, Correlation analysis between methylation levels of three genes (MGMT, RASSF1A, and SFRP1) and patient age in the normal-appearing mucosae from CIMP-positive proximal and CIMP-negative distal CRCs. Upper right boxes indicate correlations for proximal colon samples, and lower left boxes indicate correlations for distal colon samples. r indicates Pearson's correlation coefficients. Colored boxes indicate a significant correlation (<0.01 or P<0.05). C, Scatter plots of LINE-1 methylation level versus patient age in normal-appearing mucosa from CIMP-positive proximal (white circles) or CIMP-negative distal CRC cases (black circles). |
| IJC_25225_sm_supfig2.eps180.7 KB | Supplementary Fig. 2 Kaplan-Meier analysis of the probability of disease-free survival (DFS) in two subgroups according to the methylation status of three genes (RASSF1A, SFRP1, and MGMT) in CRCs. CRCs with simultaneous methylation in RASSF1A, SFRP1, and MGMT genes CRCs (broken line, n=5) show significantly shorter DFS rates than the other CRCs (solidl line, n=67, P=0.03) |
| IJC_25225_sm_supfig3.eps290.2 KB | Supplementary Fig. 3 A, CpG island diagrams of the RASSF1A and MGMT promoters. Each vertical line represents a single CpG site. Arrows denote putative transcription initiation sites. Thick black and gray bars denote the regions amplified by the bisulfite sequencing analysis and pyrosequencing analysis primers, respectively. B, Bisulfite sequencing of the RASSF1A and MGMT promoter CpG islands in normal-appearing mucosae from CRC patients. DNA from six samples of normal-appearing mucosa was subjected to bisulfite sequencing. Methylation levels assayed via pyrosequencing analysis are also shown. Each row represents an individual clone that was sequenced following sodium bisulfite DNA modification. Circles represent CpG sites, and their spacing accurately reflects the CpG density of the region. Black circle, methylated CpG site; white circle, unmethylated CpG site. |
| IJC_25225_sm_suptable1.doc50.5 KB | Supporting Table 1 |
| IJC_25225_sm_suptable2.doc65.5 KB | Supporting Table 2 |
| IJC_25225_sm_suptable3.doc38.5 KB | Supporting Table 3 |
| IJC_25225_sm_suptable4.doc31.5 KB | Supporting Table 4 |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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