E-cadherin gene (CDH1) promoter methylation as the second ... - Nature

Oncogene (2001) 20, 1525 ± 1528 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

E-cadherin gene (CDH1) promoter methylation as the second hit in sporadic di€use gastric carcinoma JC Machado*,1, C Oliveira1,2, R Carvalho1, P Soares1, G Berx3, C Caldas2, R Seruca1, F Carneiro1,4 and M Sobrinho-SimoÄes1,4 1

IPATIMUP, 4200 Porto, Portugal; 2Department of Oncology, University of Cambridge, Cambridge CB2 2XY, UK; 3Department of Molecular Biology, VIB-Ghent University, K.L., B-9000 Ghent, Belgium; 4Faculty of Medicine, University of Porto, 4200 Porto, Portugal

In di€use gastric carcinoma, despite common E-cadherin gene (CDH1) mutations, tumors show absence of CDH1 loss of heterozigosity (LOH) in most cases. This observation challenges the classical two-hit model of tumor suppressor gene inactivation. In order to investigate whether or not CDH1 promoter methylation may function as the second hit we analysed a series of 23 sporadic gastric carcinomas for the presence of CDH1 mutations, CDH1 promoter methylation, LOH and Ecadherin expression. CDH1 mutations were detected in nine of the 16 (56.3%) di€use gastric carcinomas and in none of the seven intestinal gastric carcinomas. In di€use gastric carcinomas harboring CDH1 mutations, LOH was observed in a single case. Loss of plasma membrane E-cadherin expression was consistently found in all nine cases with CDH1 mutation, suggesting that tumors inactivated the remaining CDH1 allele via a di€erent mechanism. CDH1 promoter methylation was observed in nine of the 16 (56.3%) di€use-type gastric carcinoma cases, including six of the nine cases (66.7%) harboring CDH1 mutations. CDH1 promoter methylation was also seen in two (28.6%) intestinal-type cases. Our results show that CDH1 promoter methylation is the second hit in more than half of the sporadic di€use gastric carcinoma cases harboring CDH1 mutations. Oncogene (2001) 20, 1525 ± 1528. Keywords: E-cadherin; mutation; methylation; gastric cancer; stomach; di€use cancer

E-cadherin is a member of the cadherin family of calcium dependent cell adhesion molecules and plays a major role in the process of intercellular adhesion between epithelial cells (Takeichi, 1991). It has been demonstrated that loss of E-cadherin function is pivotal in both sporadic and hereditary forms of di€use gastric tumorigenesis (Becker et al., 1994;

*Correspondence: JC Machado, IPATIMUP, Rua Roberto Frias s/n, 4200 Porto, Portugal Received 23 October 2000; revised 5 December 2000; accepted 5 January 2001

Gayther et al., 1998; Guilford et al., 1998; Machado et al., 1999). The absence of loss of heterozigosity (LOH) at the CDH1 locus in the majority of di€use gastric cancer cases with CDH1 mutation challenges the classical twohit model of tumor suppressor gene inactivation (Becker et al., 1994; Guilford et al., 1998; Machado et al., 1999). This contrasts to what happens in other tumor models (Berx et al., 1996, 1998). Alternative mechanisms of CDH1 inactivation have been analysed and it has been shown that methylation of the CDH1 promoter leads to E-cadherin expression silencing (Hennig et al., 1995; Gra€ et al., 1995). In di€use gastric carcinoma CDH1 promoter methylation was reported in 83% of cases, but CDH1 mutation status was not assessed in this series (Tamura et al., 2000). More recently CDH1 promoter methylation was demonstrated in di€use gastric cancers from carriers of germline CDH1 mutations (Grady et al., 2000). These results suggest that promoter methylation may function as the second hit in sporadic cases with CDH1 mutation. In order to investigate this hypothesis we analysed a series of 23 sporadic gastric carcinomas for the presence of CDH1 mutations (some of the cases were previously known to harbor CDH1 mutations (Machado et al., 1999)) and CDH1 promoter methylation. CDH1 mutation-positive cases were also examined for the presence of 16q22.1 LOH and E-cadherin protein expression. CDH1 mutations were found in nine of the 23 sporadic gastric carcinomas (39.1%) (Table 1). All nine mutant cases were part of the group of 16 di€use gastric carcinomas (56.3%) (Table 1). These results further con®rm the observation that CDH1 mutations are a common event in di€use gastric carcinomas but do not occur in intestinal gastric carcinomas (Becker et al., 1994; Machado et al., 1999). Among the CDH1 mutations (Table 2) we observed ®ve missense mutations, causing single aminoacid substitutions (cases 2, 4, 5, 6 and 9). A G to A transition in the last nucleotide of intron 7, resulting in the loss of the acceptor splice site, was identi®ed in cases 1 and 7. One nonsense mutation and one frameshift deletion were detected in cases 3 and 6, respectively. Finally, one 16 bp deletion encompassing

E-cadherin methylation in diffuse gastric carcinoma JC Machado et al

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the intron 4 donor splice site was observed in case 8 (Figure 1a,b). Case 6 harbors two di€erent CDH1 mutations (Table 2); due to RNA unavailability we could not elucidate whether these mutations are located in di€erent alleles or in the same allele. LOH analysis was performed using two ¯anking microsatellite markers and CDH1 polymorphisms as intragenic markers. All nine cases bearing CDH1 mutations were informative for at least one of the polymorphic markers used for LOH analysis (Figure 1c). Only case 4 (11.1%) showed LOH for markers D16S301 and CDH1+2076T (Table 2). In summary, only two cases with CDH1 mutation appear to ful®l the two-hit genetic inactivation model: case 6 presents two somatic CDH1 mutations; and case

Table 1

Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

CDH1 mutation and promoter methylation status in a series of 23 gastric carcinomas

Histologic type

Mutation status

Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse Intestinal Intestinal Intestinal Intestinal Intestinal Intestinal Intestinal

+ + + + + + + + + 7 7 7 7 7 7 7 7 7 7 7 7 7 7

CDH1 Methylation status + + + 7 7 7 + + + + + + 7 7 7 7 + + 7 7 7 7 7

Surgical specimens from 23 gastric carcinomas resected and diagnosed at Hospital S. JoaÄo/Medical Faculty and IPATIMUP, Porto, Portugal, were studied. 7, negative; +, positive

Figure 1 Di€use gastric carcinoma (case 8) displaying a CDH1 mutation, no CDH1 LOH, lack of membrane E-cadherin expression and CDH1 promoter methylation. (a) CDH1 exon 4 PCR/SSCP (polymerase chain reaction/single strand conformation polymorphism) analysis showing a regular band pattern in the normal DNA (N) and several band shifts (arrows) in the tumoral DNA (T). Method: PCR/SSCP analysis was performed as described previously (Berx et al., 1995; Machado et al., 1999). Primers were designed to individually amplify each CDH1 exon, including exon/intron boundaries (Berx et al., 1995; Machado et al., 1999). All cases presenting band shifts were submitted to a second analysis (new PCR ampli®cation and SSCP analysis), and only reproducible bands were considered. (b) Sequencing analysis of the abnormal bands represented in A, revealed a 16 bp deletion encompassing the CDH1 intron 4 donor splice site. Method: Automatic ¯uorescent sequencing analysis (Perkin-Elmer) was performed on both DNA strands using the PCR/SSCP primers. (c) No CDH1 LOH could be observed with the intragenic marker CDH1+2076T in the comparison between normal DNA (N) and tumoral DNA (T). Method: Microsatellite markers D16S265 and D16S301 (Isogen, Netherlands) and intragenic CDH1 polymorphisms were compared in tumoral and matched constitutional DNA. (d) Lack of E-cadherin IHC expression at the plasma membrane of the neoplastic cells. Method: Monoclonal antibody HECD-1 (Zymed) was used for the IHC expression study of E-cadherin according to the method described previously (Machado et al., 1998). (e) Methylation-speci®c PCR for the CDH1 promoter shows the presence of both methylated (M) and unmethylated (U) alleles in the tumoral DNA

Table 2 CDH1 mutations, 16q22.1 LOH status, E-cadherin expression and CDH1 promoter methylation status in nine sporadic di€use gastric carcinomas Case 1 2 3 4 5 6 7 8 9 a

Nucleotide changea 1009-1G?A 1742T?C 1057G?T 1108G?C 1436A?G 1204G?A 1027delC 1009-1G?A 521del10+1del6 1000G?C

Mutation Predicted protein change In frame deletion L581P E353X D370H D479G D402N Frameshift In frame deletion Disturbed splicing D334H

LOH status

IHC expression

Methylation status

7 7 7 + 7 7

7 C 7 C C C

+ + + 7 7 7

7 7 7

7 7 C

+ + +

Numbering is according to the cDNA starting at the A in the start codon (GenBank Accession No. Z13009). LOH, loss of heterozigosity; IHC, immunohistochemistry; 7, negative; +, positive; C, cytoplasmic staining

Oncogene

E-cadherin methylation in diffuse gastric carcinoma JC Machado et al

Figure 2 Methylation-speci®c PCR of the CDH1 promoter in di€use gastric carcinoma cases harboring CDH1 mutations. Cases 1, 2, 3, 7, 8 and 9 display CDH1 promoter methylation. Normal gastric mucosa and cases 4, 5 and 6 show only unmethylated alleles. C7, negative control; N, normal gastric mucosa; 1 ± 9, cases 1 to 9, respectively. Method: CDH1 promoter methylation analysis was performed using methylation-speci®c PCR according to the method described by Herman et al. (1996) and using primers described by Gra€ et al. (1997) for CpG island 3 of the CDH1 promoter. Brie¯y, this assay entails initial modi®cation of DNA by sodium bisulphite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent ampli®cation with primers speci®c for methylated versus unmethylated DNA

4 with a CDH1 mutation in one allele and deletion (LOH) of the second allele. However, upon immunohistochemical (IHC) examination of E-cadherin expression in the remaining seven cases bearing CDH1 mutations, we consistently found loss of E-cadherin at the plasma membrane of the tumor cells (Table 2) (Figure 1d). Aberrant ectopic E-cadherin expression in the cytoplasm of tumor cells, observed in some cases, is thought to be the result of missense mutation (Handschuh et al., 1999; Grady et al., 2000; Luber et al., 2000). These results suggest that the remaining CDH1 allele is inactivated via a mechanism other than allelic deletion. Our next step was to evaluate whether there was any relationship between CDH1 mutation and promoter methylation status. CDH1 promoter methylation was detected in 11 cases (47.8%): in nine of the 16 di€usetype cases (56.3%), and in two of the seven intestinaltype cases (28.6%) (Table 1). In normal gastric mucosa the CDH1 promoter was always unmethylated (Figure 2). This result shows that the promoter methylation is tumor cell-speci®c.

Among the nine di€use-type cases harboring CDH1 mutations, CDH1 promoter methylation was detected in six cases (66.7%), all of them corresponding to tumors in which no second genetic hit had been identi®ed (Table 2) (Figures 1e and 2). Noteworthy, the two tumors (cases 4 and 6) which already carried two hits were negative for CDH1 promoter methylation (Table 2) (Figure 2). In methylation positive cases we also detected unmethylated alleles (Figure 2). Because RNA is not available it is not possible to determine with certainty whether these are the mutant allele or normal cell contamination. In case 5 no CDH1 promoter methylation was observed, suggesting that a yet unidenti®ed mechanism is ivolved in inactivation of the second allele. We also observed CDH1 promoter methylation in a fraction (28.6%) of intestinal-type carcinomas, similarly to what Tamura et al. (2000) described. The signi®cance of this ®nding is uncertain. However, it is admissible that CDH1 promoter methylation might provide a molecular mechanism whereby intestinal-type carcinomas may temporarily downregulate E-caherin expression. It has been demonstrated that the pattern of E-cadherin expression in breast cancers re¯ects a dynamic CDH1 promoter methylation (Gra€ et al., 2000). In conclusion, our results suggest that CDH1 inactivation in sporadic di€use gastric tumorigenesis is biallelic and show that, in more than half of the cases harboring CDH1 mutations, the second hit is achieved by CDH1 promoter methylation. These results o€er an example of promoter methylation providing a key event in gastric carcinogenesis and add to the growing list of tumor suppressor genes silenced by promoter methylation in gastric carcinoma, e.g. P16(INK4A) (Lee et al., 1997), hMLH1 (Leung et al., 1999), P57 (Shin et al., 2000) and TGFBRI (Kang et al., 1999). As pointed out by Grady et al. (2000) promoter methylation might provide, due to its reversible nature, an attractive target for the development of new anti-cancer therapies.

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References Becker KF, Atkinson MJ, Reich U, Becker I, Nekarda H, Siewert JR and Ho¯er H. (1994). Cancer Res., 54, 3845 ± 3852. Berx G, Becker KF, Ho¯er H and van Roy F. (1998). Hum. Mutat., 12, 226 ± 237. Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M, Cornelisse C and van Roy F. (1995). EMBO J., 14, 6107 ± 6115. Berx G, Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy F and Cornelisse C. (1996). Oncogene, 13, 1919 ± 1925. Gayther SA, Gorringe KL, Ramus SJ, Huntsman D, Roviello F, Grehan N, Machado JC, Pinto E, Seruca R, Halling K, MacLeod P, Powell SM, Jackson CE, Ponder BAJ and Caldas C. (1998). Cancer Res., 58, 4086 ± 4089.

Grady WM, Willis J, Guilford PJ, Dunbier AK, Toro TT, Lynch H, Wiesner G, Ferguson K, Eng C, Park JG, Kim SJ and Markowitz S. (2000). Nat. Genet., 26, 16 ± 17. Gra€ JR, Gabrielson E, Fujii H, Baylin SB and Herman JG. (2000). J. Biol. Chem., 275, 2727 ± 2732. Gra€ JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE and Baylin SB. (1995). Cancer Res., 55, 5195 ± 5199. Gra€ JR, Herman JG, Myohanen S, Baylin SB and Vertino PM. (1997). J. Biol. Chem., 272, 22322 ± 22329. Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P, Taite H, Scoular R, Miller A and Reeve AE. (1998). Nature, 392, 402 ± 405.

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1528

Oncogene

Handschuh G, Candidus S, Luber B, Reich U, Schott C, Oswald S, Becke H, Hutzler P, Birchmeier W, Ho¯er H and Becker KF. (1999). Oncogene, 18, 4301 ± 4312. Hennig G, Behrens J, Truss M, Frisch S, Reichmann E and Birchmeier W. (1995). Oncogene, 11, 475 ± 484. Herman JG, Gra€ JR, Myohanen S, Nelkin BD and Baylin SB. (1996). Proc. Natl. Acad. Sci. USA, 93, 9821 ± 9826. Iida S, Akiyama Y, Nakajima T, Ichikawa W, Nihei Z, Sugihara K and Uasa Y. (2000). Int. J. Cancer, 87, 654 ± 658. Kang SH, Bang YJ, Im YH, Yang HK, LeeDA, Lee HY, Lee HS, Kim NK and Kim SJ. (1999). Oncogene, 18, 7280 ± 7286. Lee YY, Kang SH, Seo JY, Jung CW, Lee KU, Choe KJ, Kim BK, Kim NK, Koe‚er HP and Bang YJ. (1997). Cancer, 80, 1889 ± 1896. Leung SY, Yuen ST, Chung LP, Chu KM, Chan AS and Ho JC. (1999). Cancer Res., 59, 159 ± 164.

Luber B, Candidus S, Handschuh G, Mentele E, Hutzler P, Feller S, Voss J, Ho¯er H and Becker KF. (2000). Cell Adhes. Commun., 7, 391 ± 408. Machado JC, Carneiro F, Beck S, Rossi S, Lopes J, TaveiraGomes A and Cardoso-Oliveira M. (1998). Int. J. Surg. Pathol., 6, 135 ± 144. Machado JC, Soares P, Carneiro F, Rocha A, Beck S, Blin N, Berx G and Sobrinho-SimoÄes M. (1999). Lab. Invest., 79, 459 ± 465. Shin JY, Kim HS, Park J, Park JB and Lee JY. (2000). Cancer Res., 60, 262 ± 265. Takeichi M. (1991). Science, 251, 1451 ± 1455. Tamura G, Yin J, Wang S, Fleisher AS, Zou T, Abraham JM, Kong D, Smolinski KN, Wilson KT, James SP, Silverberg SG, Nishizuka S, Tershima M, Motoyama T and Meltzer SJ. (2000). J. Natl. Cancer Inst., 92, 569 ± 573.