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Methylation of the DFNA5 Gene Is Frequently Detected in Colorectal Cancer

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Methylation of the DFNA5 Gene Is Frequently Detected in Colorectal Cancer ANTICANCER RESEARCH 32: 1319-1322 (2012) Methylation of the DFNA5 Gene is Frequently Detected in Colorectal Cancer KAZUAKI YOKOMIZO, YOSHIKUNI HARADA, KAZUHIRO KIJIMA, KAZUKI SHINMURA, MAKIKO SAKATA, KAZUMA SAKURABA, YOUHEI KITAMURA, ATSUSHI SHIRAHATA, TETSUHIRO GOTO, HIROKI MIZUKAMI, MITSUO SAITO, GAKU KIGAWA, HIROSHI NEMOTO and KENJI HIBI Gastroenterological Surgery, Showa University Fujigaoka Hospital, Aoba-ku, Yokohama, Japan Abstract. Background: Recently, the human deafness, also found several other genes to be related to the autosomal dominant 5 gene, DFNA5, has frequently been pathogenesis of colorectal cancer (4-8). It is also known that detected in cancer tissues. The methylation status of the gene promoter hypermethylation is involved in the DFNA5 gene in colorectal cancer was examined and was development and progression of cancer (9). An investigation compared to the clinocopathological findings. Materials and of genetic changes is important in order to clarify the Methods: Eighty-five tumor samples and corresponding tumorigenic pathway of colorectal cancer (10). normal tissues were obtained from patients with colorectal The human deafness, autosomal dominant 5 gene (DFNA5) cancer who underwent surgery at our hospital. The at chromosome 7p15 causes an autosomal dominant form of methylation status of the DFNA5 gene in these samples was hearing impairment when mutated (11). DFNA5 encodes a examined by quantitative methylation-specific PCR (qMSP). protein of 496 amino acids that is found in human cochlea, Subsequently, the clinicopathological findings were brain, placenta and kidney cDNA preparation (12). An correlated with the methylation status of the DFNA5 gene. intronic insertion and/or mutation leads to premature Results: DFNA5 gene methylation was found in 29 (34%) out termination of the protein, resulting in a nonsyndromic of the 85 colorectal carcinomas, suggesting that it was progressive hearing loss (12). DFNA5 was also designated frequently observed in colorectal cancer. A significant ICERE-1 (inversely correlated with estrogen receptor correlation with methylation was observed for lymphatic expression) due to its lower expression in estrogen receptor vessel invasion and TNM stage (p=0.0268 and p=0.0189, (ER)-positive breast cancer compared to ER-negative tumors respectively). Conclusion: DFNA5 might act as a tumor (13). In addition, etoposide resistance in melanoma cells is suppressor gene and DFNA5 gene methylation might play an associated with reduced DFNA5 expression (14). Recently, important role in the development of colorectal cancer. Our p53 regulation of DFNA5, a role for DFNA5 in p53-mediated data implicate DFNA5 gene methylation as a novel cellular response to DNA damage and a pro-apoptotic effect molecular biomarker in colorectal cancer. of p53 were reported (15, 16). In the present study, the methylation status of the DFNA5 There is now solid evidence that a series of genetic gene was examined in primary tumors derived from 85 alterations in both dominant oncogenes and tumor suppressor patients with colorectal cancer and the correlation between genes are involved in the pathogenesis of human colorectal the DFNA5 gene methylation and the clinicopathological cancer. The activation of oncogenes, such as the RAS gene, findings was evaluated. and the inactivation of tumor suppressor genes, such as adenomatous polyposis coli (APC) and p53, have been Materials and Methods identified in colorectal cancer (1-3). In addition, we have Sample collection and DNA preparation. Eighty-five tumor samples were obtained at the time of surgical resection from patients with primary colorectal cancer at Showa University Fujigaoka Hospital Correspondence to: Kenji Hibi, Gastroenterological Surgery, Showa from 2007 to 2008. 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: +81 459711151, Fax: +81 459717125, e-mail: Institutional Review Board, was obtained from all the patients. All [email protected] the tumors and the corresponding normal tissue samples were immediately frozen and stored at –80˚C until analysis. The Key Words: DFNA5, quantitative methylation-specific PCR, clinicopathological findings of the patients enrolled in the study are colorectal cancer. shown in Table I. 0250-7005/2012 $2.00+.40 1319 ANTICANCER RESEARCH 32: 1319-1322 (2012) Table I. Clinicopathological findings and methylation of DFNA5 gene in colorectal tumors. Variable No. of cases DFNA5 gene methylation p-Value +– Gender Male 49 14 35 0.2094a Female 36 15 21 Age (years, mean±SD) 85 67.68±11.9 67.48±9.69 0.9314b Maximal tumor size (mm, mean±SD) 85 53.79±38.7 46.91±23.24 0.4599b CEA (ng/ml) <5 33 13 20 0.4153a ≥5 52 16 36 CA19-9 (U/ml) <37 15 7 8 0.2670a ≥37 70 22 48 Extent of tumor mt≤ 73 24 49 0.5567a <mt 12 5 7 Pathological type Well 68 21 47 0.2160a Poorly 17 8 9 Tumor site C, A, T 60 18 42 0.2195a D, S, R 25 11 14 Lymph vessel invasion ≥2 25 13 12 0.0268a <2 60 16 44 Venous invasion ≥2 23 8 15 0.9373a <2 62 21 41 Lymph node metastasis + 32 14 18 0.1478a –531538 Liver metastasis + 11 6 5 0.1356a –742351 Peritoneal dissemination + 5 2 3 0.7774a –802753 Distant metastasis + 5 2 3 0.7774a –802753 TNM stage ≥B 35 17 18 0.0189a <B 50 12 38 Total 85 29 56 aChi-square test; bStudent’s t-test; mt: muscular tunic; CEA: carcinoembryonic antigen; CA19-9: carbohydrate antigen 19-9; Well: well-differentiated adenocarcinoma (well- and moderately differentiated tubular adenocarcinoma); Poorly: poorly-differentiated adenocarcinoma and mucinous carcinoma; C: cecum; A: ascending colon; T: transverse colon; D: descending colon; S: sigmoid colon; R: rectum. Sodium bisulfite modification. One microgram of the genomic DNA TTTTTCGTTGCGCGGGATCG-3’. The PCR amplification consisted extracted from each tumor and the corresponding normal colorectal of 40 cycles (95˚C for 5 s and 60˚C for 30 s) after an initial tissue specimen was subjected to bisulfite treatment using an Epitect denaturation step (95˚C for 10 s). Bisulfite-treated DNA obtained Bisulfite Kit (Qiagen, Hilden, Germany). Briefly, the DNA was from L132 cells that were fully methylated by SssI methylase was denatured by NaOH and modified by sodium bisulfate. The DNA used as a positive control. To correct for differences in both quality samples were then purified using the kit-attached column, again treated and quantity between samples, β-actin was used as an internal control. with NaOH, precipitated with ethanol and resuspended in water. The targets were obtained from the same bisulfite-treated DNA. Quantitative methylation-specific polymerase chain reaction (qMSP). DFNA5 methylation score. The relative levels of DFNA5 gene- The bisulfite-treated DNA was amplified with qMSP, using a Thermal methylated DNA in the colorectal tumors and the corresponding Cycler Dice® Real-Time System TP800 (Takara Bio Inc., Otsu, normal tissues that were normalized to the internal control β-actin Japan). Thermocycling was performed in a final volume of 25 μl were calculated. The DFNA5 gene methylation score in each tissue containing 1.0 μl of the DNA sample, 100 nM each of the DFNA5 was defined as follows: relative level of methylated DFNA5 gene in gene or β-actin primers (forward and reverse) and 12.5 μl of SYBR tumor/relative level of methylated DFNA5 gene in all the Premix Ex Taq II (Takara Bio Inc.), which consists of Taq DNA corresponding normal tissues. DFNA5 gene methylation was defined polymerase, reaction buffer and a deoxynucleotide triphosphate as being positive when the methylation score was more than 15.0. mixture. The DFNA5 gene primers for qMSP have been described elsewhere (17) and were: DFNA5 MS (sense), 5’-CTAATTAAA Statistical analysis. The associations between DFNA5 gene CCGTAAAACCGCG-3’, and DFNA5 MAS (antisense), 5’- methylation and clinicopathological findings were analyzed using 1320 Yokomizo et al: DFNA5 Gene Methylation in Colorectal Cancer Chi-square tests or the Students’ t-test. A p-value <0.05 indicated increase of DFNA5 promoter methylation was observed in statistical significance in differences. tumors with lymphatic vessel invasion and high TNM stage. These data indicates that DFNA5 is a tumor suppressor gene Results with an important role in several frequent forms of cancer. Previously, we investigated the methylation status of the DFNA5 gene methylation was found in 29 (34%) out of the tissue factor pathway inhibitor-2 (TFPI2) gene and detected 85 colorectal carcinomas, suggesting that it was frequently it to be significantly associated with methylation in well observed in colorectal cancer. differentiated type (p=0.0053) and lymph node metastasis Correlation of the clinicopathological findings with the (p=0.0396) (22). We also investigated the methylation status methylation status of the DFNA5 gene is shown in Table I. of the E3 ubiquitin ligase (HACE1) gene and found No significant correlations were observed between the methylation to be significantly associated with maximal presence of methylation and patients’ gender, age, maximal tumor size (p=0.0304) (23). Moreover, a trend was shown tumor size, tumor markers, tumor extent, pathological type, towards a preferential development of lymph node metastasis tumor site, venous invasion, lymph node metastasis, liver in carcinomas with methylated HACE1 (p=0.0612) (23). metastasis, peritoneal dissemination or distant metastasis. Furthermore, the methylation of UNC5C gene, one of the Significant correlations were observed for lymphatic vessel Neutrin-1 receptors, was examined and a significantly greater invasion and TNM stage (p=0.0268 and p=0.0189 proportion of cases with methylated UNC5C gene was found respectively), thus indicating that DFNA5 is frequently in cases of Dukes’ stage C (p=0.0380) than in earlier stages methylated in advanced colorectal carcinoma. (24). Taking all these results together, the methylation status of genes in colorectal carcinoma was generally correlated Discussion with malignant parameters, such as lymphatic vessel invasion, high clinical stage, large tumor size, and lymph Colorectal cancer is the second leading cause of cancer and node metastasis.
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