ANTICANCER RESEARCH 29: 4397-4400 (2009)

Changes in UNC5C Gene Methylation during Human Gastric

KENJI HIBI, MAKIKO SAKATA, KAZUMA SAKURABA, YO-HEI KITAMURA, ATSUSHI SHIRAHATA, TETSUHIRO GOTO, HIROKI MIZUKAMI, MITSUO SAITO, KAZUYOSHI ISHIBASHI, GAKU KIGAWA, HIROSHI NEMOTO and YUTAKA SANADA

Department of Surgery, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227-8501, Japan

Abstract. Background: UNC5C, one of the netrin-1 gastric cancer, the inactivation of hMLH1 (human mutL receptors, belongs to the functional dependence homolog 1), MGMT (O-6-methylguanine-DNA methyl- family, members of which share the ability to induce transferase), TIMP-3 (tissue inhibitor of metalloproteinase in the absence of their ligands. Recently, aberrant 3) and p16 by promoter hypermethylation has been methylation of the UNC5C gene was found in 34 out of 49 demonstrated (4-7). There has been substantial interest in (69%) primary colon carcinomas. Materials and Methods: attempting to adapt such cancer-associated aberrant gene The methylation status of the UNC5C gene was examined in methylation for clinical use. primary carcinomas and the corresponding normal tissues UNC5C, one of the netrin-1 receptors, belongs to the derived from 36 patients with gastric cancer using functional dependence receptor family, members of which quantitative methylation-specific polymerase chain reaction, share the ability to induce apoptosis in the absence of their and the correlation between the methylation status and the ligands (8-10). Such a trait has been hypothesized to confer clinicopathological findings was evaluated. Results: tumor-suppressor activity. Indeed, the loss of UNC5C Aberrant methylation of the UNC5C gene was detected in 9 expression is particularly prominent in colorectal cancer (11). out of the 36 (25%) primary gastric carcinomas. A However, the molecular mechanisms responsible for the loss significant difference was observed in regard to the TNM of UNC5C expression are poorly understood. Recently, two stage (p=0.0455). Conclusion: UNC5C methylation was reports have indicated that UNC5C methylation was closely observed in the course of gastric carcinogenesis and associated with loss of gene expression in colorectal cancer disappeared in highly advanced gastric carcinomas. (12, 13). We have also examined the methylation status of the UNC5C gene (14), and aberrant methylation of the UNC5C Gastric cancer is one of the most common malignancies gene was detected in 34 out of the 49 (69%) primary colon worldwide (1). This highly malignant type of cancer is carcinomas investigated, suggesting that it was frequent in usually treated with surgery and subsequent chemotherapy, colorectal cancer. These results prompted us to examine the and radiotherapy. methylation status of the UNC5C gene in surgically removed Accumulating evidence indicates that gastric cancer is the primary gastric carcinomas. result of various genetic and epigenetic alterations of The correlation between the methylation status and the oncogenes, tumor suppressor genes, DNA repair genes, cell- clinicopathological findings was also evaluated. cycle regulators, and cell adhesion molecules (2). Aberrant methylation of CpG-rich sequences (CpG islands) is an Materials and Methods epigenetic change that is common in human cancer (3). In Sample collection and DNA preparation. Thirty-six primary tumor and corresponding normal tissue specimens were collected at Showa University Fujigaoka Hospital from gastric cancer patients Correspondence to: Kenji Hibi, Department of Surgery, Showa during gastric surgery. All the tissue specimens were confirmed University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama histologically. Written informed consent, as required by the 227-8501, Japan. Tel: +81459711151, Fax: +81459717125, e-mail: Institutional Review Board, was obtained from all the patients. [email protected] The samples were stored immediately at –80˚C until analysis. The DNA was prepared as described elsewhere (15). The Key Words: UNC5C, quantitative methylation-specific PCR, gastric clinicopathological profiles of the patients enrolled in the study cancer. are shown in Table I.

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Sodium bisulfite modification. One μg of the genomic DNA this was the first such report. Moreover, a significant extracted from the tumor and the corresponding normal Gstric tissue difference was observed in regard to the TNM stage specimens was subjected to bisulfite treatment using an Epitect (p=0.0455), thus indicating that UNC5C methylation was Bisulfite Kit (Qiagen, Hilden, Germany). observed in the course of gastric carcinogenesis and qMSP. The bisulfite-treated DNA was amplified with quantitative disappeared in highly advanced gastric carcinomas. In a methylation-specific polymerase chain reaction (qMSP) conducted previous study, 25 out of 76 colorectal (33%), 11 out of 65 in a Thermal Cycler Dice® Real-time System TP800 (Takara Bio gastric (17%) and 1 out of 40 esophageal (3%) carcinomas Inc., Otsu, Japan). Thermocycling was carried out in a final volume demonstrated abnormal methylation of the helicase-like of 25 μL containing 1.0 μL of the DNA sample, 100 nM each of the transcription factor (HLTF) promoter (16). This result UNC5C or β-actin primers (forward and reverse) and 12.5 μL of suggested that HLTF might play a variety of roles depending SYBR Premix Ex Taq II (Takara Bio Inc.), which consists of Taq on the tissue type. Subsequently, we found 5 out of 37 DNA polymerase, reaction buffer and deoxynucleotide triphosphate mixture. The qPCR primer sequences for UNC5C were: UNC5C MS esophageal (14%) and 23 out of 66 gastric (35%) carcinomas (sense), 5’-CGACTAAAACTTTACCGACGCGACG-3’ and UNC5C demonstrated abnormal methylation of the cadherin 13 MAS (antisense), 5’-CGGGGGCGGGAGTACGTTCGTTC-3’. The (CDH13) promoter (17). Abnormal CDH13 methylation was PCR amplification consisted of 40 cycles (95˚C for 5 sec and 60˚C frequently found in gastric cancer at all clinical stages just for 30 sec) after an initial denaturation step (95˚C for 10 sec). The as E-cadherin methylation, another of the cadherin family, bisulfite-treated DNA obtained from L132 cells that was fully suggesting that these types of cancer could be methylated at methylated by SssI methylase was used as a positive control. To an early stage. Thus CDH13 might also play a variety of correct for differences in both quality and quantity between samples, β-actin was used as an internal control. The targets were obtained roles depending on the tissue type. from the same bisulfite-treated DNA. In another previous study (14), aberrant methylation of the UNC5C gene was detected in 34 out of 49 (69%) primary UNC5C methylation scores. The relative levels of UNC5C colon carcinomas, suggesting that this was frequent in methylated DNA in the gastric carcinomas and the corresponding colorectal cancer. Furthermore, a significantly greater normal tissues normalized to the internal control β-actin were proportion of cases with methylated UNC5C was found in calculated. The UNC5C methylation score in each tissue was Dukes’ stage C (p=0.0380) than in earlier stages, indicating defined as: relative level of UNC5C in tumor/average relative level of UNC5C in all corresponding normal tissues. UNC5C methylation that UNC5C might act as a tumor suppressor and UNC5C was considered as being positive when the methylation score was methylation might provide a malignant potential in colorectal more than 1.5. cancer. In the present study, UNC5C methylation in gastric cancer (25%) was less frequent than in colorectal cancer Statistical analysis. The associations between UNC5C methylation and (69%). Additionally, although a significant difference was clinicopathological parameters were analyzed using Chi-square test or observed in TNM stage, UNC5C methylation disappeared in Student’s t-test. A p-value <0.05 indicated statistical significance. highly advanced gastric carcinomas. Taken together, these Results results suggested that the methylation status of the UNC5C gene might depend on the type of primary carcinomas. Aberrant methylation of the UNC5C gene was detected in 9 Because of the frequent methylation of the UNC5C gene out of the 36 (25%) primary gastric carcinomas. and the high sensitivity of qMSP, UNC5C methylation in No significant correlations were observed between the clinical samples, serum could potentially be used for the presentation of abnormal methylation in the gastric carcinomas detection and monitoring of gastric carcinoma as suggested and patient gender or age, maximal tumor size, tumor extent, in colorectal carcinoma (18, 19). In conclusion, the UNC5C tumor histology, lymph node , peritoneal methylation occurs in gastric carcinomas. dissemination or distant metastasis (Table I). A significant References difference was observed in regard to the TNM stage (p=0.0455) (Table I), thus indicating that UNC5C methylation 1 Fuchs CS and Mayer RJ: Gastric carcinoma. N Engl J Med 333: was observed in the course of gastric carcinogenesis and 32-41, 1995. disappeared in highly advanced gastric carcinomas. 2 Yasui W, Yokozaki H, Fujimoto J, Naka K, Kuniyasu H and Tahara E: Genetic and epigenetic alterations in multistep Discussion carcinogenesis of the stomach. J Gastroenterol 35: 111-115, 2000. The identification of the genetic alterations as a new 3 Chen W-D, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, Platzer P, Lu S, Dawson D, Willis J, Pretlow TP, Lutterbaugh J, parameter to estimate the process of the neoplastic process Kasturi L, Willson JKV, Rao JS, Shuber A and Markowitz SD: is important to improve the success of treatment. Detection in fecal DNA of colon cancer-specific methylation of In the present study, aberrant methylation of UNC5C was the nonexpressed vimentin gene. J Natl Cancer Inst 97: 1124- shown in gastric cancer, and to the best of our knowledge, 1132, 2005.

4398 Hibi et al: UNC5C Methylation in Gastric Cancer

Table I. Clinicopathological features and UNC5C methylation in human gastric cancer.

Clinicopathological Variable No. of UNC5C methylation p-value feature cases +–

Gender male 29 7 22 0.8081 female 7 2 5 Age 50-82 36 71.9±9.03 68.9±9.8 0.4292 Maximal tumor size (mm) 10-130 36 65.0±24.53 59.6±26.5 0.5952 Extent of tumor ≤mt4 5 2 3 0.4041 mt< 31 7 24 Histology well5, mod6 14 2 12 0.2361 por7 22 7 15 Lymph node metastasis + 23 6 17 0.8411 –13310 Peritoneal dissemination + 9 2 7 0.824 –27720 Distant metastasis + 5 0 5 0.164 –31922 TNM stage 1-3 27 9 18 0.04551 4909

Total no. of cases 36 9 27

1Chi-square test; 2Student’s t-test; 3mean±S.D; 4mt, muscular tunic. 5Well: well-differentiated adenocarcinoma, 6mod: moderately-differentiated adenocarcinoma, 7por: poorly-differentiated mucinous or signet ring cell adenocarcinoma according to Japanese criteria.

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