Naoko Watanabe

Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers.

Lysyl oxidase (LOX) and HRAS-like suppressor (HRASLS) are silenced in human gastric cancers and are reported to have growth-suppressive activities in ras-transformed mouse/rat fibroblasts. Here, we analyzed whether or not LOX and HRASLS are tumor suppressor genes in human gastric cancers. Loss of heterozygosity and promoter methylation of LOX were detected in 33% (9 of 27) and 27% (26 of 96) of gastric cancers, respectively. Biallelic methylation and loss of heterozygosity with promoter methylation were also demonstrated in gastric cancers. Silencing of LOX was also observed in colon, lung, and ovarian cancer cell lines. As for mutations, only one possible somatic mutation was found by analysis of 96 gastric cancer samples and 58 gastric and other cancer cell lines. When LOX was introduced into a gastric cancer cell line, MKN28, in which LOX and HRASLS were silenced, it reduced the number of anchorage-dependent colonies to 57 to 61%, and the number of anchorage-independent colonies to 11 to 23%. Sizes of tumors formed in nude mice were reduced to 19 to 26%. Growth suppression in soft agar assay was also observed in another gastric cancer cell line, KATOIII. On the other hand, neither loss of heterozygosity nor a somatic mutation was detected in HRASLS, and its introduction into MKN28 did not suppress the growth in vitro or in vivo. These data showed that LOX is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in gastric cancers, and possibly also in other cancers.

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

Hypomethylation of the global genome, considered to be composed mainly of repetitive sequences, is consistently observed in cancers, and aberrant hypo‐ and hypermethylation of CpG islands (CGIs) in promoter regions are also observed. Since methylation alterations in unique promoter sequences and in other genomic regions have distinct consequences, we analyzed the relationship between the global hypomethylation and the hypomethylation of unique promoter CGIs using human gastric cancers. Seven of ten gastric cancer cell lines showed marked decreases in 5‐methylcytosine content, which correlated with hypomethylation of the LINE1 repetitive sequence. Six of the seven cell lines showed hypomethylation in five or all of the six normally methylated CGIs in promoter regions of six genes, and this was associated with induction of aberrant expression. The remaining three cell lines without global hypomethylation showed promoter hypomethylation in one or none of the six CGIs. Frequent promoter hypomethylation, however, did not correlate with frequent promoter hypermethylation. In primary gastric cancers too, global hypomethylation was associated with hypomethylation of LINE1 repetitive sequence and promoter hypomethylation. Of 93 gastric cancers, 33 cancers with frequent promoter hypomethylation and 27 cancers with frequent promoter hypermethylation constituted different groups. These findings represent experimental evidence that frequent hypomethylation of normally methylated promoter CGIs is associated with global hypomethylation, and that these hypomethylations occur independently of frequent promoter CGI hypermethylation. (Cancer Sci 2004; 95: 58–64)

Identification of Genes Targeted by CpG Island Methylator Phenotype in Neuroblastomas, and Their Possible Integrative Involvement in Poor Prognosis

Background/Aims: CpG island (CGI) methylator phenotype (CIMP) is strongly associated with poor prognosis in neuroblastomas (NBLs; hazard ratios 7–22). Methylation of nonpromoter CGIs is useful to detect the presence of the CIMP, while the poor prognosis is considered to be caused by gene silencing due to promoter methylation. Here, promoter CGIs targeted by the CIMP were searched for. Methods: A genome-wide screening was performed by methylation-sensitive representational difference analysis of CIMP(+) and CIMP(–) NBLs. Results: Promoter CGIs of 9 genes were methylated in CIMP(+) NBL cell lines and caused silencing of their downstream genes. On analysis of 90 clinical specimens, CYP26C1,FERD3L (N-TWIST), CRYBA2 and PCDHGC4 were methylated at significantly higher incidences in CIMP(+) NBLs than CIMP(–) NBLs, while the difference was unclear for NPY, SPAG6, DDIT4L, CHR3SYT and C6Orf141. Methylation of CYP26C1 and FERD3L was significantly associated with poor prognosis, but weaker than the presence of the CIMP. Treatment of an NBL cell line with a demethylating agent caused demethylation of multiple promoter CGIs, and enhanced 13-cis-retinoic acid-induced neuronal differentiation. Conclusion: Our results indicate that the CIMP causes poor prognosis of NBLs by inducing methylation of multiple promoter CGIs with various incidences.

Revisit of Field Cancerization in Squamous Cell Carcinoma of Upper Aerodigestive Tract: Better Risk Assessment with Epigenetic Markers

We quantified field cancerization of squamous cell carcinoma in the upper aerodigestive tract with epigenetic markers and evaluated their performance for risk assessment. Methylation levels were analyzed by quantitative methylation-specific PCR of biopsied specimens from a training set of 255 patients and a validation set of 224 patients. We also measured traditional risk factors based on demographics, lifestyle, serology, genetic polymorphisms, and endoscopy. The methylation levels of four markers increased stepwise, with the lowest levels in normal esophageal mucosae from healthy subjects without carcinogen exposure, then normal mucosae from healthy subjects with carcinogen exposure, then normal mucosae from cancer patients, and the highest levels were in cancerous mucosae (P < 0.05). Cumulative exposure to alcohol increased methylation of homeobox A9 in normal mucosae (P < 0.01). Drinkers had higher methylation of ubiquitin carboxyl-terminal esterase L1 and metallothionein 1M (P < 0.05), and users of betel quid had higher methylation of homeobox A9 (P = 0.01). Smokers had increased methylation of all four markers (P < 0.05). Traditional risk factors allowed us to discriminate between patients with and without cancers with 74% sensitivity (95% CI: 67%–81%), 74% specificity (66%–82%), and 80% area under the curve (67%–91%); epigenetic markers in normal esophageal mucosa had values of 74% (69%–79%), 75% (67%–83%), and 83% (79%–87%); and both together had values of 82% (76%–88%), 81% (74%–88%), and 91% (88%–94%). Epigenetic markers done well in the validation set with 80% area under the curve (73%–85%). We concluded that epigenetics could improve the accuracies of risk assessment. Cancer Prev Res; 4(12); 1982–92. ©2011 AACR.

Single nucleotide instability: a wide involvement in human and rat mammary carcinogenesis?

Genetic instability plays important roles in carcinogenesis. In two cell lines which we established from mammary carcinomas induced in lacI-transgenic rats by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), spontaneous point mutation rates (MRs) of the endogenous hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and lacI transgene were found to be increased. The two rat mammary carcinoma cell lines lacked microsatellite instability (MSI), and nuclear extracts from them were proficient in G/T mismatch binding. The increase of spontaneous point MRs was considered to be due to a mechanism(s) different from mismatch repair insufficiency, and this type of genetic instability was termed as single nucleotide instability (SNI). SNI in the rat mammary carcinoma cell lines was characterized by the elevation of A:T to C:G transversions of the hprt and lacI genes, which were rarely observed in normal mammary epithelial cells. The elevation of A:T to C:G transversions was also present in the lacI gene of the primary carcinomas of the two cell lines, which suggested that the molecular abnormality present in the cell lines was already present in their primary carcinomas. Mth1 mutation, which is known to cause elevation of A:T to C:G transversions, was analyzed in the 2 cell lines and in 11 primary PhIP-induced mammary carcinomas, but no mutations were observed. Finally, spontaneous point MRs of the hprt gene were measured in six human breast cancer cell lines, and increase was found in five of them. These human breast cancer cell lines were proficient in G/T mismatch binding, and were reported to lack MSI. SNI was suggested to play a wide involvement in human and rat mammary carcinogenesis.

Stronger Prognostic Power of the CpG Island Methylator Phenotype than Methylation of Individual Genes in Neuroblastomas

OBJECTIVE The CpG island methylator phenotype is strongly associated with poor survival in neuroblastomas. Neuroblastomas with the CpG island methylator phenotype include almost all neuroblastomas with MYCN amplification, and, even among neuroblastomas without MYCN amplification, have worse prognosis. At the same time, methylation of individual tumor-suppressor genes is also reported to be associated with poor survival. The purpose of this study was to compare the prognostic power of the CpG island methylator phenotype with that of methylation of individual genes. METHODS Methylation-specific polymerase chain reaction was performed for five individual genes (CASP8, EMP3, HOXA9, NR1I2 and CD44) in 140 Japanese and 152 German neuroblastomas. Kaplan-Meier analysis and log-rank tests were conducted to compare the survival between groups defined by methylation status. RESULTS Among the five individual genes, only CASP8 methylation had a significant association with poor overall survival both in Japanese (hazard ratio = 3.1; 95% confidence interval = 1.5-6.4; P = 0.002) and German (hazard ratio = 4.8; 95% confidence interval = 2.1-11; P = 0.0002) neuroblastomas. HOXA9 and NR1I2 methylation were associated with poor survival only in German neuroblastomas. On the other hand, the CpG island methylator phenotype had a strong and consistent association in Japanese (hazard ratio = 22; 95% confidence interval = 5.3-93; P = 1.5 × 10(-5)) and German (hazard ratio = 9.5; 95% confidence interval = 3.2-28; P = 4.7 × 10(-5)) neuroblastomas. CONCLUSION The CpG island methylator phenotype is likely to have stronger prognostic power than methylation of individual genes in neuroblastomas.

Abstract 141: Identification of a gene silenced in rat mammary carcinomas by combination of DNA methylation microarray and chemical genomic screening

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and analyze molecular mechanisms of methylation induction, animal models are indispensable. However, genes methylation-silenced in the animal models of breast cancer are unknown, and identification of inducers and analysis of mechanisms are hampered. Here, to identify genes methylation-silenced in rat primary mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray and chemical genomic screening. MeDIP-CGI microarray analysis of a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) revealed that, among 4,680 promoter CGIs analyzed, 664 were methylated in the cell line. Sixty-two of the 664 genes were re-expressed by treatment of the cell line with 5-aza-2’-deoxycytidine and trichostatin A. An unmethylated status in normal mammary glands was confirmed for 31 of the 62 genes by MeDIP-CGI microarray analyses of a pool of 4 normal mammary glands. Among 13 primary mammary carcinomas induced by PhIP, five of the 31 genes were methylated in one carcinoma or more. Among 12 primary carcinomas induced by 7,12-dimethylbenz[a]anthracene, methylation of all the five genes was also observed. RT-PCR analysis revealed that one of the five genes had high expression in normal mammary glands, but low expression in carcinomas, showing that this gene was methylation-silenced in rat primary mammary carcinomas. In humans, the gene was expressed in mammary epithelial cells that had unmethylated DNA, and was down-regulated in three cancer cell lines that had methylated DNA. The gene was methylated in about 9% of 80 primary human breast cancers, and it was suggested that aberrant DNA methylation of this gene was a common event between rat and human breast cancers. This is the first study of genome-wide DNA methylation analysis using rat mammary carcinomas, and the methylation-silenced gene, along with the other methylated genes, is expected to be useful as a marker in identifying inducers of aberrant DNA methylation and analyzing its molecular mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 141.