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Lymph Node Metastasis Is Infrequent in Patients with Highly-Methylated Colorectal Cancer

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Lymph Node Metastasis Is Infrequent in Patients with Highly-Methylated Colorectal Cancer ANTICANCER RESEARCH 26: 55-58 (2006) Lymph Node Metastasis is Infrequent in Patients with Highly-methylated Colorectal Cancer KENJI HIBI1 and AKIMASA NAKAO2 1Department of Surgery, Seirei Hospital, 56 Kawanayama-machi, Showa-ku, Nagoya 466-8633; 2Gastroenterological Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan Abstract. In this study, the combined methylation status of p16, was found to harbor promoter hypermethylation p16, p14, HLTF, CDH13 and RUNX3 was examined in 59 associated with the loss of protein expression in cancer cells resected primary colorectal cancers using methylation-specific (7). Though homozygous deletions of the p16 locus were not PCR and the methylation status was correlated with the present (8), p16 promoter methylation was detected in clinicopathological features of the affected patients. A colorectal cancer (9). Subsequently, it has been found that significant difference was found in lymph node metastasis human p14 was also silenced by promoter hypermethylation (p=0.0359) on comparing the methylation status in in colorectal cancer (10). p14 interacts with the MDM2 colorectal cancers with lymph node metastasis to those protein and neutralizes the MDM2-mediated degradation without. There was also a significant gender difference of p53. Thus, p14 acts as a tumor suppressor gene via (p=0.0248) when the methylation status was compared. inhibition of p53 degradation. These studies indicated that Colorectal cancer without lymph node metastasis frequently p16 and p14 inactivation due to promoter methylation was exhibited aberrant methylation of the p16, p14, HLTF, important for colorectal tumorigenesis. We previously CDH13 and RUNX3 genes. examined the methylation status of these genes in 86 primary colorectal cancers using methylation-specific PCR There is now strong evidence that a series of genetic (MSP) (11). Aberrant promoter methylation of the p16 and alterations in both dominant oncogenes and tumor suppressor p14 genes was detected in 43 out of 86 (50 %) and 25 out genes are involved in the pathogenesis of human colorectal of 86 (29%) colorectal cancers, respectively. cancer. The activation of oncogenes such as the ras gene and The loss of expression of a helicase-like transcription inactivation of tumor suppressor genes such as the APC and factor (HLTF), a SWI/SNF family member gene, p53 genes have been identified (1-3). In addition, we found accompanied by HLTF promoter methylation was reported that several other genes are related to the pathogenesis of in colon cancer (12). In this report, all colon cancer cell colorectal cancer (4, 5), and investigation of genetic changes is lines that lacked HLTF gene expression demonstrated important to clarify its tumorigenic pathway (6). methylation of CpG sites within the putative HLTF Several tumor suppressor genes contain CpG islands in promoter, while methylation was not detected in the HLTF- their promoters, prompting many investigations into the expressing cell lines. Moreover, HLTF methylation was role of methylation in silencing these genes. Many tumor detected in 25 out of 76 primary colon cancers, suggesting suppressor genes show evidence of methylation silencing, that HLTF is a common target for methylation and providing a potential new pathway for the deactivation of epigenetic gene silencing in colon cancer and qualifies as a tumor suppressor genes. At first, a tumor suppressor gene, potential colon cancer suppressor gene (13). Subsequently, it has also recently become clear that CDH13 (H-cadherin, T-cadherin) expression is frequently silenced by aberrant methylation in colorectal cancer and Correspondence to: Kenji Hibi, Department of Surgery, Seirei adenoma (14, 15). CDH13 encodes a protein belonging to Hospital, 56 Kawanayama-machi, Showa-ku, Nagoya 466-8633, the cadherin family of cell surface glycoproteins responsible Japan. Tel: +81-52-744-2245, Fax: +81-52-744-2255, e-mail: [email protected] for selective cell recognition and adhesion (16). Ubiquitous methylation of CDH13 in colorectal cancer and adenoma Key Words: Methylation, colorectal cancer, p16, p14, HLTF, indicated that such methylation occurs at an early stage in CDH13, RUNX3. the multistage process of oncogenesis. 0250-7005/2006 $2.00+.40 55 ANTICANCER RESEARCH 26: 55-58 (2006) Recently, the loss of expression of RUNX3, a as described previously (11,13,15,18). The methylation transcription factor containing a highly conserved domain, status of the p16, p14, HLTF, CDH13 and RUNX3 genes designated as a ‘runt domain’, accompanied by RUNX3 gene were originally examined in 86, 86, 76, 84 and 92 methylation was reported in gastric cancer (17). In this colorectal cancer specimens, respectively. Fifty-nine report, between 45% and 60% of human gastric cancer cells specimens were tested for the methylation status of all 5 did not significantly express RUNX3 due to hemizygous genes. Aberrant methylation of the p16, p14, HLTF, deletion and hypermethylation of the RUNX3 gene. CDH13 and RUNX3 genes was detected in 34 (58%), 20 Tumorigenicity of human gastric cancer cell lines in nude (34%), 18 (31%), 22 (37%) and 22 (37%) of 59 colorectal mice was inversely related to their level of RUNX3 cancer specimens, respectively (Figure 1). All tumors also expression, suggesting that RUNX3 is a common target for exhibited non-methylation of each gene, which might be methylation and epigenetic gene silencing in gastric cancer the result of the contamination of the tumor specimens and qualifies as a potential gastric cancer suppressor gene. with non-tumor cells. Otherwise, it might be possible that Aberrant promoter methylation of the RUNX3 gene was these gene expressions had not been completely inhibited detected in 31 out of 92 (34%) colorectal cancers (18). in these cancer specimens. In this study, the combined methylation status of p16, The correlation between the methylation status and p14, HLTF, CDH13 and RUNX3 was examined in 59 clinicopathological data was analyzed (Table I). Colorectal resected primary colorectal cancers using methylation- cancers with more than 3 methylated genes were included specific PCR (MSP). To determine whether the methylation in the highly-methylated group (HMG), and colorectal status could be a marker for the malignancy of colorectal cancers with less than 2 methylated genes in the low- cancer, the methylation status was correlated with the methylated group (LMG). There was a significant difference clinicopathological features of the affected patients. in lymph node metastasis (p=0.0359) when the methylation status in colorectal cancers with and without lymph node Materials and Methods metastases was compared, indicating that the colorectal cancers without lymph node metastasis were highly- Sample collection and DNA preparation. Fifty-nine primary tumors methylated. This result suggested that the aberrant and corresponding normal tissues were collected consecutively at methylation of these genes occurred early in the Nagoya University Hospital, Japan, from colorectal cancer patients. carcinogenesis of colorectal cancer, although it was not All tissues were confirmed histologically. The collected samples often shown in advanced colorectal cancer with lymph node were stored immediately at –80ÆC until analysis. DNA was prepared, as described previously (19). metastasis. There was also a significant gender difference (p=0.0248) on comparing the methylation status, indicating Sodium bisulfite modification. One Ìg of genomic DNA extracted that the colorectal cancer were highly-methylated in males. from tumors and corresponding normal colorectal tissues was No other factors such as age, tumor size, extent of tumor, subjected to bisulfite treatment, as described previously (20). histology, or tumor site correlated with the number of Briefly, alkali-denatured DNA was modified by 2.1 M sodium methylated genes. bisulfite / 0.5 mM hydroquinone at pH 5.0. The bisulfite-reacted DNA was then treated by NaOH, purified using the Wizard DNA Clean-Up System® (Promega, Madison, WI, USA), precipitated Discussion with ethanol and resuspended in distilled water. Our results indicated that colorectal cancer without lymph Methylation-specific PCR. The bisulfite-treated DNA was amplified node metastasis exhibited a higher number of methylated with MSP. The primers and PCR conditions were described genes including p16, p14, HLTF, CDH13 and RUNX3. previously (7,10,12,14,17). Ten ml of each PCR product was loaded Therefore, the methylation of these genes is a low directly onto non-denaturing 8% polyacrylamide gels, stained with malignancy factor, as for lymph node metastasis. Shannon ethidium bromide and visualized under UV illumination. et al. also investigated the methylation status of the hMLH1, Statistical analysis. The possible association between the number of p16 and MDR1 genes in 58 colorectal cancers (21). They methylated genes and clinicopathological features was examined found methylation of the hMLH1, p16 and MDR1 genes in using the Chi-squared test or Student’s t-test. The computed p-values 23, 29 and 28% of the colorectal cancers, respectively. As a were two-tailed. Statistical significance was considered to be p<0.05. result, these 3 genes were significantly methylated in MSI+ colorectal cancers compared to MSI– cancers. Additionally, Results they were significantly associated with poor histological differentiation. Although the results of our studies differed The methylation status of the p16, p14, HLTF, CDH13 from
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