defects in many of the molecules that regulate the cell cycle have been implicated in cancer initiation and progression .
these include p53 , rb , p107 and prb2/p130 , and cdk inhibitors ( p15 , p16 , p18 , p19 , p21 , p27 ) , all of which act to prevent cell cycle progression until all repairs to damaged dna have been completed . in this section
, we will review tumor suppressor genes that regulate cell cycle and apoptosis that have been found to be methylated in gastric cancers .
tumor suppressor genes which are most recently found to be methylated in gastric cancers regulate cell cycle progression and apoptosis .
kim et al . reported that promoter methylation of early b - cell factor-3 ( ebf3 ) was detected in 40.4% ( 42/104 ) of gastric cancer tissues but not in normal gastric tissues .
the ebfs are a group of four highly conserved , dna - binding transcription factors with an atypical zinc - finger domain and a helix - loop - helix domain .
functional analysis demonstrates that ebf3 represses gastric cancer cell growth and migration but activates cell cycle arrest and apoptosis .
ebf3 up - regulates p21 and p27 in gastric cancer cells but represses cdk2 , cyclin d1 , cyclin d2 , and rb as previously reported in different types of cancers , .
interestingly , in addition to their central roles in apoptosis regulation , the bcl-2 family of proteins influences the cell cycle , specifically the transition between quiescence and proliferation .
bcl2l10 methylation was detected in 44.5% of gastric cancers and 21.34% of normal gastric mucosae ( p < 0.05 ) by bisulphite sequencing .
the pro - apoptotic effect of bcl2l10 and growth promotion by sirna targeting bcl2l10 in gastric cancer cells suggests that bcl2l10 may be a tumor suppressor by inducing apoptosis through mitochondrial pathways . the iroquois homeobox protein 1
( irx1 ) gene is located on chromosome 5p15.33 , which is a cancer susceptibility locus .
irx1 transcription was suppressed by hypermethylation in gastric cancer , and restoring irx1 expression in sgc-7901 and nci - n87 gastric cancer cells inhibited tumor growth , invasion , and tumorigenesis in vitro and in vivo .
irx1 directly targets bradykinin receptor b2 ( bdkrb2 ) , an angiogenesis - related gene , as well as histone h2b type 2-e ( hist2h2be ) and fibroblast growth factor 70 ( fgf7 ) , cell proliferation and invasion related genes .
hypermethylation of irx1 was detected not only in primary gastric cancer tissues but also in the peripheral blood cells of gastric cancer patients , suggesting that irx1 could potentially serve as a biomarker for gastric cancer .
the cklf - like marvel transmembrane domain - containing family ( cmtm ) is a novel family of proteins linking chemokines and the transmembrane-4 superfamily .
wang et al . reported that cmtm3 is frequently methylated in many carcinoma cell lines and some primary tumors including gastric cancer , resulting in loss of its expression at both the mrna and protein levels .
ectopic restoration of cmtm3 expression in tumor cells leads to the suppression of cell growth and induction of apoptosis with caspase-3 activation , suggesting that cmtm3 may function as a tumor suppressor .
hypermethylation signals were also observed in some cultured and primary gastric cancers with little or no expression of transcription factor sox2 , a sox transcription factor . among the 52 patients with advanced gastric cancers who were tested , those having sox2 gene methylation in their cancer tissues had a significantly shorter survival than did those without the methylation ( p = 0.006 )
. exogenous expression of sox2 inhibited cell growth through apoptosis and cell cycle arrest . sox2-overexpressing cells exhibited characteristics of apoptosis , such as dna laddering and caspase-3 activation .
cell cycle arrest was associated with decreased levels of cyclin d1 and phosphorylated rb as well as an increased p27 level .
reported that zinc - finger transcription factor znf382 functions as a tumor suppressor in many types of carcinomas including gastric cancer .
ectopic expression of znf382 in tumor cells in which the gene was silenced significantly reduced clonogenicity and proliferation and induced apoptosis .
further found that znf382 inhibited nf-b and ap-1 signaling and down - regulated the expression of multiple oncogenes , including myc , mitf , hmga2 , cdk6 , stat3 , stat5b , id1 , and ikbke , most likely through heterochromatin silencing . a , pyrosequencing ; b , q - msp ; c , high - resolution melting ( hrm ) analysis ; d , methylight ; e , bisulfit sequencing .
methylation of the ubiquitin carboxyl - terminal hydrolase l1 ( uchl1 ) was detected in primary digestive tumors , including 77% ( 53/69 ) of gastric carcinomas , but not in or occasionally in paired adjacent non - tumor tissues . restoring uchl1 expression in cell lines where the gene had been silenced significantly inhibited their growth and colony formation ability by inhibiting cell proliferation , causing cell cycle arrest in g2/m phase , and inducing apoptosis through the intrinsic caspase - dependent pathway
similarly , sdr - related gene product that binds to c - kinase ( hsrbc ) , another novel methylated tumor suppressor gene in gastric cancer , increased the stability of p53 and expression of p53 target genes , such as p21 , puma , and noxa .
hsrbc - mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function .
these results suggested that epigenetic inactivation of hsrbc contributes to the malignant progression of gastric tumors , in part , through attenuated p53 response to stress .
moreover , opioid binding protein / cell adhesion molecule - like gene ( opcml ) is also a stress- and p53-responsive gene , but this response is epigenetically impaired when the opcml promoter becomes methylated .
ecotopic expression of opcml led to significant inhibition of both anchorage - dependent and -independent growth of carcinoma cells in which the gene had been silenced .
as reviewed above , many methylated tumor suppressor genes in gastric cancer contribute to the dysregulation of the important aspects of tumorigenesis : cell cycle and apoptosis .
the understanding of these molecular mechanisms will provide accesses to novel molecular therapeutic strategies for inhibiting oncogenic activity of signaling pathways .
morbidity in most cancer patients is not due to primary cancer but to metastatic disease .
the high death rate of gastric cancer strikingly correlates with the high metastatic capacity of most gastric cancers .
thus , understanding tumor progression to the metastatic state and changes in highly aggressive cells is important for the development of novel approaches to diagnose and treat aggressive malignancies .
detachment of cells with increased motility from a primary tumor is the first step of cancer invasion and metastasis .
gastric cancer cells with fibroblastoid morphological changes show increased motility and invasiveness due to decreased cell - cell adhesion , which is reminiscent of epithelial - mesenchymal transition ( emt ) during embryonic development .
the dynamic regulation of cell - cell adhesion is crucial for developmental processes including tumorigenesis .
with reduced cell - cell adhesiveness , cancer cells can disobey the social order and result in destruction of the histological structure .
a common and early requirement for cell motility is actin polymerization , which drives the formation of cell protrusions that are used to adhere to the extracellular matrix , define the direction of migration and initiate cell crawling .
animal cells respond to signaling at the plasma membrane by remodeling their actin cytoskeleton . on the other hand ,
the movement of cells into a tightly woven extracellular matrix also require an active proteolytic system , which can cleave a path for cell migration .
so the genes regulating the host cell cytoskeleton rearrangement and proteolytic activity also have important functions in cell morphogenesis and motility .
our group reported that epigenetic inactivation of phospholipase c delta 1 ( plcd1 ) is common and tumor - specific in gastric cancer and that plcd1 acts as a functional tumor suppressor in gastric carcinogenesis .
located at the important tumor suppressor locus 3p22 , plcd1 encodes an enzyme that regulates energy metabolism , calcium homeostasis , and intracellular movements .
ectopic expression of plcd1 in gastric tumor cells with silenced plcd1 dramatically inhibited clonogenicity and migration , possibly through down - regulation of mmp7 expression and hampered cyto - skeletal reorganization via phosphorylation and inactivation of cofilin .
deleted in liver cancer 1 ( dlc1 ) is another tumor suppressor gene involved in the regulation of cytoskeletal organization and other functions and is frequently methylated in many cancers , including gastric cancer , .
recently , its new isoform , dlc1 isoform 4 ( dlc1-i4 ) , was also found to be silenced epigenetically , to have tumor inhibitory properties in multiple carcinomas , and to be regulated by p53 .
. the down - regulation of e - cadherin may favor dissociation of cancer cells from one another , facilitating their invasion through the basal membrane .
although mutation and allelic loss have been confirmed as major mechanisms for e - cadherin ( cdh1 ) gene inactivation in many malignancies , cdh1 promoter methylation could be frequently detected in gastric carcinoma . as a co - partner of e - cadherin , -catenin
not only is critical for cell adhesion in the membrane and cytoplasm of cells , but also plays a role as a transcription activating protein in nuclei .
aberrant activation of the wnt/-catenin signaling pathway is frequently found in many cancers , including gastric cancer , .
in addition to genetic deletions and point mutations , a number of negative regulators of wnt signaling , including secreted frizzled - related protein ( sfrp ) , dickkopf homolog 2 ( dkk2 ) , dickkopf homolog 2 ( dkk3 ) and wnt inhibitory factor 1 ( wif1 ) , were frequently methylated in gastric cancer. recently , other genes involving wnt pathway were also reported as methylated tumor suppressor genes in gastric cancer .
for example , sry - box containing gene 17 ( sox17 ) was reported to be indispensable for embryonic development and a candidate tumor suppressor gene that antagonizes the canonical wnt/-catenin signaling pathway in colorectal cancer .
treatment with a demethylating agent induced sox17 expression in gastric cancer cells , thus indicating the down - regulation of sox17 by methylation .
transgenic expression of sox17 suppressed dysplastic tumor development in the stomach of k19-wnt1/c2me mice by suppressing wnt activity .
these results suggested that sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis , and down - regulation of sox17 contributes to malignant progression through promotion of wnt activity .
the popeye domain - containing ( popdc ) genes bves , popdc2 , and popdc3 encode proteins that regulate cell - cell adhesion and cell migration during development .
bves and popdc3 were reported to be hypermethylated in 69% and 64% of the gastric cancer tissues , respectively .
promoter hypermethylation is a causal event for long - term repression of bves and popdc3 , whereas egf stimulation is an immediate repression mechanism for both genes in gastric cancer .
bves , popdc3 , and e - cadherin mrnas were down - regulated and snail mrna expression was up - regulated in egf - induced emt in snu-216 cells . interestingly , accumulating evidence suggests that a major subfamily within the cadherin superfamily , protocadherins , frequently act as tumor suppressor genes .
recent studies have shown that the main structural and functional properties of protocadherins are distinct from those of classical cadherins .
they may not have the strong cell - cell adhesion activity but do have other functions , such as mediating specificity of cell - cell interactions and signal transduction .
yu et al . reported that protocadherin 10 ( pcdh10 ) was silenced or down - regulated in 94% ( 16 of 17 ) of gastric cancer cell lines .
furthermore , pcdh10 methylation was detected in 82% ( 85 of 104 ) of gastric tumors compared with 37% ( 38 of 104 ) of paired non - tumor tissues ( p < 0.001 ) .
re - expression of pcdh10 reduced colony formation in vitro and tumor growth in vivo .
it also inhibited cell proliferation , induced cell apoptosis , and repressed cell invasion , possibly through up - regulation of the pro - apoptosis genes fas , caspase 8 , jun , and cdkn1a ; the anti - proliferation gene fgfr ; and the anti - invasion gene htatip2 .
in cancer cells , glucose is often converted into lactic acid , which is known as the  warburg effect . 
the reason that cancer cells have a higher rate of aerobic glycolysis , but not oxidative phosphorylation , remains largely unclear .
recently , fructose-1,6-bisphosphatase-1 ( fbp1 ) , which functions to antagonize glycolysis , was reported to be down - regulated through the nf-b pathway in ras - transformed nih3t3 cells .
fbp1 was hypermethylated in 57% ( 4/7 ) of gastric cancer cell lines and 33% ( 33/101 ) of gastric carcinomas .
restoration of fbp1 expression suppressed anchorage - independent growth , indicating the relevance of fbp1 down - regulation in carcinogenesis .
leucine - rich repeat - containing 3b ( lrrc3b ) is an evolutionary highly conserved leucine - rich repeat - containing protein , but its biological significance is unknown .
recently , lrrc3b was identified as a putative tumor suppressor gene and is reportedly silenced in gastric cancers by epigenetic mechanisms .
stable transfection of lrrc3b in gastric cancer cell line snu-601 inhibited anchorage - dependent and anchorage - independent colony formation . moreover , lrrc3b expression suppressed tumorigenesis in nude mice .
microarray analysis of lrrc3b - expressing xenograft tumors showed induction of immune response - related genes and ifn signaling genes .
hematoxylin and eosin ( h&e)-stained sections of lrrc3b - expressing xenograft tumors showed lymphocyte infiltration in the region .
these results suggested that lrrc3b silencing in cancer may play an important role in tumor escape from immune surveillance .
as reviewed above , aberrant dna methylation of promoter cpg islands is one of the major inactivating mechanisms of tumor suppressor genes and is deeply involved in gastric carcinogenesis .
moreover , in gastric cancer , dna methylation from the pre - malignant till the most aggressive stage of cancer can be defined .
for instance , many genes such as thbd , lox , hrasls , flnc , and hand1 were found to be infrequently methylated in non - cancerous gastric mucosae , in addition to their frequent methylation in cancers .
similar findings were reported for cdh1 , dapk , p14 , thbs1 , and timp-1 .
the presence of trace amounts of methylation in non - cancerous gastric mucosae suggested that some gastric carcinogens could have induced the methylation .
the most important gastric carcinogenic factor is helicobacter pylori ( h. pylori ) infection , which increases the risk of developing gastric cancers by 2.2- to 21.0-fold , .
recent studies to quantify dna methylation changes induced by the h. pylori infection in light of imflammatory reactions have been performed in both animal models and in the clinical setting .
the findings suggest that h. pylori infection induces aberrant methylation in gastric mucosae and that levels of accumulated methylation are associated with gastric cancer risk .
notably , although h. pylori infection is important to trigger inflammation capable of inducing aberrant dna methylation , some inflammation processes appear to be critical in induction of aberrant dna methylation. indeed , the inflammation induced by h. pylori infection , not h. pylori itself , was critically involved in methylation induction .
h. pylori may induce methylation of promoters containing cpg islands by release of reactive oxygen species ( ros ) and nitric oxide ( no ) and by activation of dna methyltransferase , .
more recently , sepulveda et al . suggested that permanent dna methylation after h. pylori eradication may occur in stem cells , thus promoting tumorigenesis .
considering the significant involvement of aberrant dna methylation of cpg islands in human cancers , further identification of the inducing factors and mechanisms can be expected to provide novel targets for cancer prevention .
in addition to regional hypermethylation , global hypomethylation is also purported to be a hallmark of cancer cells and is known to cause chromosomal instability as an early event during gastric carcinogenesis .
however , yoshida et al . reported that h. pylori infection potently induces alu and sata hypomethylation in gastric mucosae as an early event during gastric carcinogenesis , whereas global hypomethylation is present only in some individuals .
thus , the use of hypomethylation as a risk marker has not been considered realistic .
aberrant hypermethylation of promoter regions of specific genes is a key event in the formation and progression of cancer .
an accumulating number of studies have reported that gene silencing by dna methylation may be established at an early stage in the multistep process of carcinogenesis .
methylations of specific genes or methylation patterns of groups of genes were found to be associated with chemotherapy response and prognosis .
thus , aberrant dna methylation can be applied to cancer diagnostics in three ways : as a marker to detect cancer cells or cancer - derived dna , as a marker to predict prognosis , and as a biomarker for the assessment of therapeutic response .
methylation analysis has an advantage in that it can be performed using chemically stable dna ( compared to rna ) .
moreover , detecting gene methylation is easier than detecting gene mutation because the exact location of a mutation is usually unknown , making it difficult to specifically amplify dna molecules with an embedded mutation in excess of wild - type molecules .
detection of aberrant methylation can provide confirmation of the presence of intact cancer cells or cancer - derived dna in bodily fluids , such as blood , urine , sputum , saliva , and stools .
recently , the methylations of bnip3 , chfr , cyp1b1 , mint25 , sfrp2 , and rassf2 were analyzed in 107 specimens of peritoneal fluid by quantitative methylation - specific polymerase chain reaction ( msp ) .
the results showed that dna methylation in periotoneal fluid is a possible marker for detecting occult neoplastic cells on the peritoneum .
methylation analysis along with a cytological examination might , therefore , improve the positive detection of cancer cells in the peritoneal fluid of gastric cancer patients .
the development of the bisulfate conversion technique that reproducibly changes unmethylated cytosines to uracil but leaves methylated cytosines unchanged rapidly increased progress in dna methylation detection .
bisulfite sequencing , msp , and combined bisulfite restriction analysis ( cobra ) were all developed on the basis of bisulfite conversion among these technologies , msp is a subjective , gel - based assay and can not provide quantitative information . to date , several real - time msp methods , such as bisulfite treatment in combination with methylight , quantitative multiplex - msp(qm - msp ) , , or pyrosequencing , have been developed and used in dna methylation studies .
for example , a recent study showed that one pyrosequencing analysis of global and site - specific dna methylation in peripheral blood samples from 105 gastric cancer patients provides quantitative dna methylation values that may serve as important prognostic indicators .
however , prior to clinical application , these findings require validation in prospective clinical studies .
, an increasing number of drugs targeting dna methylation have been improved to increase efficacy and to decrease toxicity .
5-azacyti - dine , the most successful epigenetic drug to date , is currently recommended as the first - line treatment of high - risk myelodysplastic syndromes ( mds ) .
new methods for gene - specific epigenetic modification are being developed and tested in solid tumors .
gastric cancer is a biologically heterogeneous disease with various molecular tumor subsets that likely respond distinctly to therapy .
methylation of genes involved in dna repair and maintaining genome integrity ( e.g. mgmt , hmlh1 , wrn , and fancf ) , as well as genes involved in cell cycle checkpoints ( e.g. chfr , 14 - 3 - 3 , cdk10 , and p73 ) all reportedly influence sensitivity to chemotherapeutic drugs , suggesting that dna methylation could serve as a molecular marker for predicting tumor responsiveness to chemotherapy .
an investigation of dna methylation using specialized high - throughput platforms can potentially be applied to further stratify patients to individualized therapies .
methylated tumor suppressor genes are being intensively investigated in gastric cancer , but the underlying functions and mechanisms need to be carefully examined .
future investigations of the connections between h. pylori infection , gastric cancer , and epigenetic changes will greatly expand our understanding .
we believe that information obtained from studying dna methylation will have an impact on cancer prevention , diagnostics , and treatment , and will contribute to cancer elimination
. however , proper selection of methylation markers is crucial for sensitive and specific detection , and further technological advances are necessary in the future .
 gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide 
 . the pathogenesis mechanisms of gastric cancer are still not fully clear . 
 inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis . 
 accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes , particularly caused by hypermethylation of cpg islands in promoters , is critical to carcinogenesis and metastasis . here , we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer . 
 we also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications . 