2001;61:3225-3229.细数流年
Cancer Res
Manel Esteller, Paul G. Corn, Stephen B. Baylin, et al.
A Gene Hypermethylation Profile of Human Cancer
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[CANCER RESEARCH61,3225–3229,April15,2001]
Perspectives in Cancer Rearch
A Gene Hypermethylation Profile of Human Cancer1
Manel Esteller,2,3Paul G.Corn,2Stephen B.Baylin,and James G.Herman4 The Johns Hopkins Comprehensive Cancer Center,Baltimore,Maryland21231
Abstract
We are in an era where the potential exists for deriving comprehensive profiles of DNA alterations characterizing each form of human cancer. Such profiles would provide invaluable insight into mechanisms underly-ing the evolution of each tumor type and will provide molecular markers, which could radically improve cancer detection.To date,no one type of DNA change has been defined which accomplishes this purpo.Herein, by using a candidate gene approach,we show that one category of DNA alteration,aberrant methylation of gene promoter regions,can enor-mously contribute to the above goals.We have now analyzed a ries of promoter hypermethylation changes in12genes(p16INK4a,p15INK4b, p14ARF,p73,APC,5BRCA1,hMLH1,GSTP1,MGMT,CDH1,
TIMP3,and DAPK),each rigorously characterized for association with abnormal gene silencing in cancer,in DNA from over600primary tumor samples rep-renting15major tumor types.The genes play known important roles in process encompassing tumor suppression,cell cycle regulation,apopto-sis,DNA repair,and metastastic potential.A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific.The hypermethy-lation of the genes occurs independently to the extent that a panel of three to four markers defines an abnormality in70–90%of each cancer type. Our results provide an unusual view of the pervasiveness of DNA alter-ations,in this ca an epigenetic change,in human cancer and a powerful t of markers to outline the disruption of critical pathways in tumori-genesis and for derivation of nsitive molecular detection strategies for virtually every human tumor type.
Unfolding a Gene Hypermethylation Profile of
Human Cancer
Mutations in individual genes have outlined critical aspects of tumorigenesis,including disruption of the Rb/p16INK4a,APC/5-catenin/Tcf,and p53/p14ARF/MDM2pathways.Global genome screens,su
ch as for repeat microsatellite quence alterations and for gene expression changes by rial analysis of gene expression(1)or cDNA microarrays(2),have also provided important information about molecular events important for tumorigenesis.Despite the above studies,no detection of any one type of DNA alteration,either by candidate gene approach or by genomic screening techniques,has provided universal markers for all tumor types.In the prent study, we demonstrate how one single type of DNA alteration,aberrant methylation of gene promoters,can point to pathways disrupted in every type of cancer and can provide markers for nsitive detection of virtually all tumor types.
The growing list of genes inactivated by promoter region hyper-methylation provides an opportunity to examine the patterns of inac-tivation of such genes among different tumors(3,4).Recently,a global pattern of methylation events in tumors using restriction land-mark genomic scanning was reported(5).We have instead ud a candidate gene approach.We have studied multiple key cancer genes undergoing epigenetic inactivation in a large t of primary human tumors with the aim of obtaining a map of this alteration in malignant transformation.A total of12genes,including well-characterized tumor suppressor genes(p16INK4a,p15INK4b,p14ARF,p73,APC,and BRCA1),DNA repair genes(hMLH1,GSTP1,and MGMT),and genes related to metastasis and invasion(CDH1,TIMP
3,and DAPK)were included in the study.Each of the genes posss a CpG island in their5Јregion which is unmethylated in corresponding normal tissues, as expected for a typical CpG island(6).We and others have shown, in previous studies for such genes in individual tumor types,that when the CpG islands are hypermethylated in cancer cells,expression of the corresponding gene is silenced and the silencing can be partially relieved by demethylation of the promoter region(3,4).However,the scope of the changes has not been easy to obrve by examining each study individually.
The primary tumor samples examined in the prent study consti-tute over600specimens that cover15major tumor types(colon, stomach,pancreas,liver,kidney,lung,head and neck,breast,ovary, endometrium,kidney,bladder,brain,and leukemia and lymphomas). The profile of promoter hypermethylation for each of the above genes in each tumor type is shown in Fig.1.Important features of the data are as follows.First,one or more of the genes studied is hypermethy-lated in every tumor type.However,the profile of promoter hyper-methylation for the genes differs for each cancer type,providing a tumor-type and gene-specific profile.Some genes,such as the cell cycle inhibitor p16INK4a,are hypermethylated across many tumor types including colorectal,lung,and breast carcinomas as previously described(7–9).This alteration reflects the widespread contribution
of disruptions of the cyclinD-Rb cell cycle control pathway in human cancer.The extent of p16INK4a epigenetic silencing reported in the literature expands over the neoplasms described in Fig.1and also include bladder(10)and cervical tumors(11)or melanomas(12)and gliomas(13).
Other changes,such as for the DNA repair gene MGMT and DAPK, also have a wide distribution(14–17).Hypermethylation of p14ARF and APC are most prevalent in gastrointestinal ,colon and stomach)(18,19),whereas GSTP1is characteristic of steroid-related neoplasms such as breast,liver,and prostate(20,21).The mentioned spectrum of epigenetic inactivation we have obrved has been con-firmed in other laboratories studying a single tumor type such as MGMT(22)and APC(23)in colorectal,p14ARF in gastric tumors (24),DAPK in bladder tumors(25),and GSTP1in hepatic neoplasms (26).The aberrant methylation of certain genes reflects their very specific involvement in lected tumor types or groups of tumors. Thus,as might be predicted from the tumor types en in families who
Received10/27/00;accepted2/19/01.
The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertiment in accordance with 18U.S.C.Section1734solely to indicate this fact.
1Supported by NIH Grants CA43318and CA54396.J.G.H.is a Glick Scholar in Lung Cancer Rearch.
2M.E.and P.ibuted equally to this work.
3Prent address:Cancer Epigenetics Laboratory,Molecular Pathology Program, Centro Nacional de Investigaciones Oncologicas,Ctra.Majadahonda-Pozuelo,Km2, 28220Majadahonda,Madrid,Spain.
4To whom requests for reprints should be addresd,at The Johns Hopkins Compre-hensive Cancer Center,1650Orleans Street,Room543,Baltimore,MD21231.Phone: (410)955-8506;Fax:(410)614-9884;E-mail:hermanji@jhmi.edu.
5The abbreviations ud are:APC,adenomatous polyposis coli;CDH1,E-cadherin; DAPK,death-associated protein kina;GSTP1,glutathione S-transfera P1;MGMT, O6-methylguanine-DNA methyltransfera;TIMP3,tissue inhibitor of metalloprotein-a-3.
inherit mutations in BRCA1,we found hypermethylation of this gene only in breast and ovarian carcinomas (27),consistent with other reports (28),whereas hypermethylation of the mismatch repair gene hMLH1is restricted to the three sporadic tumor types characteristic of the hereditary nonpolypo
sis colorectal cancer syndrome:colorectal,endometrial,and gastric tumors with microsatellite instability (29–32).Similarly,hypermethylation of p73and p15INK4b is only obrved in hematological malignancies (33–37).
Another interesting point is raid by the fact that epigenetic inactivation may affect all of the molecular pathways involved in cell immortalization and transformation.We obrved promoter hyper-methylation-associated silencing in cell cycle (p16INK4a and p15INK4b ),DNA repair (hMLH1,MGMT ,and BRCA1),cell adherence and metastasis process (CDH1,TIMP3,DAPK ),p53network (p14ARF and p73),metabolic enzymes (GSTP1),and the APC/-catenin route (APC).Thus,in any given tumor it is possible to find simultaneous inactivation of veral pathways by aberrant methylation compromis-ing all of the described ,a colorectal tumor may have disruption of cell cycle,DNA repair,and metastasis-related process by hypermethylation of p16INK4a ,hMLH1,and TIMP-3,respectively,whereas a mammary tumor can accomplish similar objectives silenc-ing p16INK4a ,BRCA1,and CDH1and a lung tumor affecting p16INK4a ,MGMT ,and DAPK .风流人物的意思
If we look at our gene hypermethylation profile from the tumor type standpoint,the scenario is particularly interesting.Gastrointestinal tumors (colon and gastric)share a t of genes undergoing hyper-methylation characterized by p16INK4a ,p14ARF ,MGMT ,APC ,and hMLH1,whereas other ae
rodigestive tumor types,such as lung and head and neck,have a different pattern of hypermethylated genes including DAPK ,MGMT ,and p16INK4a ,but not hMLH1or p14ARF .Similarly,breast and ovarian cancers tend to methylate certain genes including BRCA1,GSTP1,and p16INK4a .This gene hypermethylation profile of human cancer that we report is consistent with the data of particular “methylotypes”propod for single tumor types including tumors originated from the pancreas,esophagus,stomach,colon,and leukemia (38–42).It is noteworthy that hematological malignancies have markedly different epigenetic alterations than do tumors origi-nating in solid organs.This is evident in the high frequency of p73and p15INK4b hypermethylation in the tumors while the genes are not altered in the epithelial tumors.
In each ca and tumor type,the epigenetic lesions occur in the abnce of a genetic lesion.A couple of illustrative examples are found in colorectal tumorigenesis.First,while homozygous deletion of the INK4a/ARF are common in other tumor types,this genetic abrogation is uncommon in colon tumors and instead this locus is commonly shutdown by simultaneous methylation of p16INK4a and p14ARF (8,9,18,43).Second,becau APC somatic mutation is very prevalent in the tumors,APC methylation is obrved at a low frequency,but other gastrointestinal tumors types,that usually do not harbor APC mutations,can disrupt the APC /-catenin pathway through APC hypermethylation (19,39).
Furthermore,the prence of the epigenetic lesion is often an
early
Fig.1.A ,depiction of the profile of gene promoter hypermethylation across human tumor types.All cas reprent random and unlected populations of each particular tumor type,except ء,where hMLH1methylation was determined in colorectal,endometrial,and gastric tumors enriched in microsatellite-unstable samples.Analysis of the methylation status was studied in most cas by sodium bisulfite modification of DNA and subquent PCR using primers designed for either methylated or unmethylated DNA (PCR conditions and quences are available upon request).Additional samples were analyzed by Southern blot with methyl-nsitive enzymes,restriction cut analysis,and bisulfite genomic quencing.B ,numerical distribution of promoter hypermethylation according to gene and tumor type.
event in the natural history of human cancer.Promoter hypermethy-lation affecting p16INK4a ,p14ARF ,MGMT ,and APC occurs in colo-rectal adenomas (15,18,19),p16INK4a hypermethylations is detecta-ble in basal cell hyperplasia squamous metaplasia and carcinoma in situ of the lung (44),and hMLH1epigenetic silencing can be dem-onstrated in endometrial hyperplasias (45)and ulcerative colitis (46),both precursor lesions of uterine and colorectal tumors.
This analysis of candidate genes can be en as only a partial picture of the methylation changes in cancer.First,there are certainly still numerous genes that undergo epigenetic inactivation waiting to be discovered.The completion of the human genome quence and the u of veral described techniques to find new genes with differential methylation,such as methylation-nsitive arbitrarily primed PCR (47),methylated CpG island amplification (48),restric-tion landmark genomic scanning (5,49),and differential methylation hybridization (50)will be extremely uful for this purpo.Examples of genes found by the and other approaches include genes such as TPEF (51)or the proapoptotic TMS1(52),and future studies will likely address their distribution and relevance in multiple tumor types.Insights into a Molecular Marker System for Cancer Bad on Aberrant Methylation
A major possibility raid by our current data are that promoter hypermethylation changes might provide a molecular marker system for the detection of the major forms of human cancer.This DNA change is obviously common to each tumor type studied and the frequency for hypermethylation of many of the genes,determined by tumor type,is often high.In fact,for each tumor type studied,three or more of the genes tested were hypermethylated in at least 5–10%of the samples tested and often many more.For ea of detection,the promoter hypermethylation may offer many advantages as co
mpared to other DNA alterations such as mutations.The latter changes often occur at different sites,even for point mutations within a given gene,between individual tumors even of the same type.Promoter hyper-methylation,in contrast,occurs over the same regions of a given gene in each form of cancer.Thus,one need not first test the methylation status of a given gene in tumor DNA to devi means for detecting the hypermethylation marker in DNA from a distal site.Finally,as compared to other frequent chromosome changes in cancer,such as allelic loss,the hypermethylation constitutes a positive signal,which is easier to detect against a background of normal DNA.With regard to all of the above points,a number of studies,using nsitive PCR strategies for detection of promoter hypermethylation changes in specific genes,provide proof of principle that the changes can be ud to detect cancer through analys of DNA from readily obtain-able sites such as rum and sputum (16,17,53).
To test the diagnostic potential of our findings for the 12genes under study,we picked a subt of genes,lected according to the frequency data in Fig.1,of hypermethylated genes for each of five tumor types.To test the feasibility of this approach,we first explored whether hypermethylation for each constitutive gene in the panel is an independent event (Fig.2).This would be necessary to obtain maxi-mum coverage of each cancer type using a minimum number of markers to assay.Indee
d,for each cancer type (Fig.2),the incidence of hypermethylation for zero,one,two,three,and four genes was not statistically different from the changes being randomly associated events (P ϭ0.38–0.97).Most important,we detected changes in at least one of the genes in approximately 80%or more of the samples from each tumor type (Fig.2).
The u of methylation markers for the detection of transformed cells is not only a black and white tool,but also a qualitative one.According to the profile of genes who hypermethylation-associated inactivation is detected,we may have important information about the biological behavior of that particular malignancy.Two ts of genes are noteworthy to mention:the DNA repair and the cell adherence group.First,we have previously demonstrated that transcriptional inactivation of MGMT by methylation occurs in a wide spectrum of human tumors (14).We have now shown that MGMT
epigenetic
Fig.2.Left ,depiction of coincident number of gene hypermethylation events in a defined tumor.Right ,predicted detection rate if the changes are independent events versus linked events and how this impacts on the percentage of a given tumor type that a panel of markers would pick up.
silencing in gliomas confers enhanced nsitivity to alkylating drugs (54).Similar results have also been suggested in the past to be related to the action of hMLH1and GSTP1in relation to other agents(55,56). Thus,the combined methylation analysis of the three genes may contribute to predict which chemotherapy would be more effective in each cancer patient.Second,the test of the epigenetic status of CDH1, TIMP3,and DAPK may provide us with a valuable measure of the metastastic potential of any given tumor(57).Becau aberrant meth-ylation can occur when the cancer cells have not yet spread,this knowledge could be ud to try treatments to prevent dismination in the aggressive tumors.
兵蚁Conclusions
Overall,our data demonstrate,using a candidate gene approach, that promoter hypermethylation of12genes involving important cellular pathways in tumorigenesis is a feature of each of15major hum
an tumor types studied.Moreover,although many tumors share this change for a given gene,unique profiles do exist for the tumor types.Finally,small panels of hypermethylated gene markers can detect a high percentage of each of the tumor types studied.Thus,the spectrum of epigenetic alterations for a relatively small subt of genes provides a potentially powerful system of biomarkers for de-veloping molecular detection strategies for virtually every form of human cancer.
Acknowledgments
We apologize to the authors and colleagues who work was not cited becau of limitations on the length of the article and number of references. S.B.B.and J.G.H.are entitled to sales royalties from Intergen,which is developing products related to rearch described in this report.The terms of the arrangement have been reviewed and approved by The Johns Hopkins University in accordance with its conflict of interest policies. References
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