Cancer epigenetics is rapidly moving into a translational phase, and knowledge on how aberrant DNA methylation is induced is becoming important. Aging, chronic inflammation, and viral infections are known to promote methylation of non-core regions of promoter CpG islands (CGI). The non-core methylation and 'seeds of methylation', scattered methylation in a CGI, are considered to serve as triggers for dense methylation of a promoter CGI, which permanently represses expression of its downstream gene. Decreased gene transcription is an important factor that promotes induction of dense methylation. The presence of the CGI methylator phenotype (CIMP), in which methylation of multiple CGI was observed, is under dispute. Some gastric cancer cell lines have increased rates of de novo methylation, and neuroblastoma cases with CIMP show qualitatively different prognosis from those without. This strongly supports the presence of CIMP, but it seems to contain multiple entities. Limited knowledge is available for epimutagens, the chemicals that induce DNA demethylation or methylation. We have developed an assay system to detect demethylating agents, and an assay system for methylating agents is necessary. Efforts in the field on how aberrant methylation is induced will lead to new cancer prevention, diagnostics, and therapeutics. (1) The first example was identified for the RB gene in sporadic retinoblastomas in 1993 (2,3) followed by VHL, (4) CDKN2A ( p16 ), (5,6) CDH1 (E-cadherin), (7,8) and hMLH1.(9) Now, many tumorsuppressor genes are known to be inactivated by methylation of their promoter CGI in a wide variety of cancers.(1) Methylation of a promoter CGI excludes some methylation-sensitive transcription factors, such as CTCF, and recruits methyl-CpG binding proteins, such as MeCP2 and MBD1-MBD3.(10) These methyl-CpG binding proteins further recruit histone deacetylases, histone methyltransferases, and heterochromatin proteins.(11) It is believed that changes in chromatin structure will block the access of transcription complex to DNA, and repress transcription.In parallel with the mechanistic studies on how DNA methylation leads to gene silencing, the search for genomic regions aberrantly methylated in cancers has also made a lot of progress. (12) In the late 1990s, before the human genome sequence was available, several genome-wide screening methods were developed, such as restriction landmark genomic scanning-methylation, methylation-sensitive-representational difference analysis (MS-RDA), methylation-sensitive-arbitrarily primed PCR, and methylated CpG island amplification-RDA.(13 -17) These methods revealed that cancers harbor many aberrantly methylated genomic regions. Now, owing to completion of the sequencing of the human genome, it has become evident that, even if limited to CGI in promoter regions or putative promoter regions (5′ regions) of genes, most cancers have multiple aberrant methylations. (18)(19)(20)(21) These aberrant methylations are considered to provide a good source of tumor markers,and targets ...

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2 0 -deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

Toll-like receptors (TLRs) are key regulators of innate immune responses, and their dysregulation is observed in numerous inflammation-associated malignancies, including gastric cancer (GC). However, the identity of specific TLRs and their molecular targets which promote the pathogenesis of human GC is ill-defined. Here, we sought to determine the clinical utility of TLR2 in human GC. TLR2 mRNA and protein expression levels were elevated in >50% of GC patient tumors across multiple ethnicities. TLR2 was also widely expressed among human GC cell lines, and DNA microarray-based expression profiling demonstrated that the TLR2-induced growth responsiveness of human GC cells corresponded with the up-regulation of six anti-apoptotic (BCL2A1, BCL2, BIRC3, CFLAR, IER3, TNFAIP3) and down-regulation of two tumor suppressor (PDCD4, TP53INP1) genes. The TLR2-mediated regulation of these anti-apoptotic and tumor suppressor genes was also supported by their increased and reduced expression, respectively, in two independent genetic GC mouse models (gp130 and Gan) characterized by high tumor TLR2 expression. Notably, enrichment of this TLR2-regulated gene signature also positively correlated with augmented TLR2 expression in human GC tumors, and served as an indicator of poor patient survival. Furthermore, treatment of gp130 and cell line-derived xenograft (MKN1) GC mouse models with a humanized anti-TLR2 antibody suppressed gastric tumor growth, which was coincident with alterations to the TLR2-driven gene signature. Collectively, our study demonstrates that in the majority of GC patients, elevated TLR2 expression is associated with a growth-potentiating gene signature which predicts poor patient outcomes, thus supporting TLR2 as a promising therapeutic target in GC.

Tumor suppressors with extracellular function are likely to have advantages as targets for cancer therapy, but few are known. Here, we focused on angiopoietin-like 4 (ANGPTL4), which is a secreted glycoprotein involved in lipoprotein metabolism and angiogenesis, is methylation-silenced in human cancers, but has unclear roles in cancer development and progression. We found a deletion mutation in its coiled-coil domain at its N-terminal in human gastric cancers, in addition to hypermethylation of the ANGPTL4 promoter CpG islands. Forced expression of wild-type ANGPTL4, but not ANGPTL4 with the deletion, at physiological levels markedly suppressed in vivo tumorigenicity and tumor angiogenesis, indicating that the latter caused the former. Tumor-derived ANGPTL4 suppressed in vitro vascular tube formation and proliferation of human umbilical vascular endothelial cells, partly due to suppression of ERK signaling. These showed that ANGPTL4 is a genetically and epigenetically inactivated secreted tumor suppressor that inhibits tumor angiogenesis.

Tumor-suppressor genes on chromosome X can be inactivated by a single hit, any of the point mutations, chromosomal loss and aberrant DNA methylation. As aberrant DNA methylation can be induced frequently, we here aimed to identify a tumor-suppressor gene on chromosome X inactivated by promoter DNA methylation. Of 69 genes on chromosome X upregulated by treatment of a gastric cancer cell line with a DNA-demethylating agent, 5-aza-2'-deoxycytidine, 11 genes had low or no expression in the cell line and abundant expression in normal gastric mucosae. Among them, FHL1 was frequently methylation-silenced in gastric and colon cancer cell lines, and methylated in primary gastric (21/80) and colon (5/50) cancers. Knockdown of the endogenous FHL1 in two cell lines by two kinds of shRNAs significantly increased cell growth in vitro and sizes of xenografts in nude mice. Expression of exogenous FHL1 in a non-expressing cell line significantly reduced its migration, invasion and growth. Notably, a somatic mutation (G642T; Lys214Asn) was identified in one of 144 colon cancer specimens, and the mutant FHL1 was shown to lack its inhibitory effects on migration, invasion and growth. FHL1 methylation was associated with Helicobacter pylori infection and accumulated in normal-appearing gastric mucosae of gastric cancer patients. These data showed that FHL1 is a methylation-silenced tumor-suppressor gene on chromosome X in gastrointestinal cancers, and that its silencing contributes to the formation of an epigenetic field for cancerization.