DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells ''the cancer methylome'' would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.

p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63 ؉/؉ mouse, it is undetectable in these tissues in the p63 ؊/؊ mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.p63 is a member of the p53 tumor suppressor gene family. Similar to p53, p63 is a transcription factor that activates target genes through sequence-specific DNA binding (35,41,43,52,56). It has been shown that expression of p21 waf-1 , MDM2, and BAX are induced by TAp63s through binding to p53 response elements (p53-REs) (45). In spite of their structural similarities, p63 functions differ greatly from those of p53. The most striking difference is the apparent involvement of p63 in skin and limb development. The p63 knockout mouse exhibits skin and limb defects as well as craniofacial abnormalities (29, 57). On the other hand, the p53 knockout mouse develops normally but is prone to suffering from various cancers from an early age (7). Heterozygous p63 germ line mutations cause several skin and other developmental disorders (1,3,17,28,53). On the other hand, germ line mutations of p53 cause Li-Fraumeni syndrome, in which affected individuals are exceptionally prone to developing cancer (26). p63 complements p53-dependent apoptosis induced by DNA damage. However, p63 itself induces apoptosis to a lesser extent than p53 (12, 42).These differences may be due to the differential regulation of target genes by p53 and p63. The p53 and p63 proteins can bind to two or more tandem repeats of RRRCWWGYYY (p53-RE) or some other motifs and subsequently activate target gene expression (5, 9, 54, 56). In the case of the 14-3-3 promoter, p53 and p63 differentially bind to two distinct response elements (55). Until now, a number of genes have been reported to be targets of p63 and its close relative, p73, such as JAG1, JAG2, IL4R, ⌬Np73, AQP3, and REDD1 (11,30,39,40,59). However, p63-specific response elements (p63-REs) have not yet been defined. Thus, the specific mechanism of gene activation exhibited by p63 and its distinction from that exhibited by p53 remain unclear.In order...

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Promoter hypermethylation accompanied by gene silencing is a common feature of human cancers. We identified previously several new tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of reexpression on microarray analysis. In this study, we modified the selection of candidates from our previous microarray data by excluding genes that showed basal expression in cancer cell lines. With the new method, we found novel methylated genes with 90% accuracy. Among these 33 novel methylated genes that we identified in esophageal squamous cell carcinoma (ESCC) cell lines, N-methyl-D-aspartate receptor type 2B (NMDAR2B) was of particular interest. NMDAR2B was methylated in 95% of primary human ESCC tissue specimens and 12 ESCC cell lines by sequence analysis. NMDAR2B expression was silenced in all 12 ESCC cell lines and was reactivated by the demethylating agent 5-aza-2V-deoxycytidine. Moreover, reintroduction of the gene was accompanied by marked Ca 2+ -independent apoptosis in ESCC cell lines, suggesting that NMDAR2B can suppress tumor growth. Thus, NMDAR2B promoter methylation is common in ESCC, abrogating gene transcription and leading to cellular resistance to apoptosis.

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

PGP9.5/UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family with a potential role in carcinogenesis. We previously identified PGP9.5 as a putative tumor-suppressor gene and methylation of the promoter as a cancer-specific event in primary cancer tissues. In this current study, we analyzed PGP9.5 methylation in 50 esophageal squamous cell carcinoma (ESCC) primary tumors with well characterized clinicopathologic variables including patient outcome. Two independent modalities for methylation analysis (TaqMan methylation-specific PCR and combined bisulfite restriction analysis) were used to analyze these samples. The two data sets were consistent with each other, as the 21 patients (42%) with highest methylation levels by TaqMan analysis all showed visible combined bisulfite restriction analysis bands on acrylamide gels. Using an optimized cutoff value by TaqMan quantitation, we found that patients with higher PGP9.5 methylation ratios in the primary tumor showed poorer 5-year survival rates than those without PGP9.5 methylation (P = 0.01). A significant correlation was also seen between PGP9.5 promoter methylation and the presence of regional lymph node metastases (P = 0.03). Multivariate analysis subsequently revealed that PGP9.5 methylation was an independent prognostic factor for ESCC survival (P = 0.03). These results suggest that PGP9.5 promoter methylation could be a clinically applicable marker for ESCC progression. (Cancer Res 2005; 65(11): 4963-8)

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2 ¶-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPA and HOPB, respectively), and HOPA and HOPB were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPB silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPA silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.

Diffuse-type gastric cancer (DGC) is the most deadly form of gastric cancer and is frequently accompanied by peritoneal dissemination and metastasis. The specific molecular events involved in DGC pathogenesis remain elusive. Accumulating evidence of epigenetic inactivation in tumor suppressor genes led us to conduct a comprehensive screen to identify novel methylated genes in human cancers using pharmacologic unmasking and subsequent microarray analysis. We compared differential RNA expression profiles of DGC and intestinaltype gastric cancer (IGC) cell lines treated with 5-aza-2V -deoxycytidine using microarrays containing 22,284 genes. We identified 16 methylated genes, including many novel genes, in DGC cell lines and studied PGP9.5 with particular interest. In primary gastric cancers, PGP9.5 was found to be more frequently methylated in DGCs (78%) than in IGCs (36%; DGC versus IGC, P < 0.05). Furthermore, real-time methylationspecific PCR analysis of PGP9.5 showed relatively higher methylation levels in DGC than in IGC. Our data thus implicate a molecular event common in the DGC phenotype compared with IGC. (Cancer Res 2006; 66(7): 3921-7)