Carcinogenesis Advance Access published January 16, 2004 Carcinogenesis 2004 # Oxford University Press; all rights reserved.

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The E-cadherin -347G→GA promoter polymorphism and its effect on transcriptional regulation

Yong Shin1,3, Il-Jin Kim1,3, Hio Chung Kang1, Jae-Hyun Park1, Hye-Rin Park1, HyeWon Park1, Mi Ae Park2, Jong Soo Lee2, Kyong-Ah Yoon1, Ja-Lok Ku1 and Jae-Gahb Park1,2,4

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Korean Hereditary Tumor Registry, Cancer Research Center and Cancer Research Institute, Seoul National University and 2Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi, Korea

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These authors (Y. S and I-J. K) contributed equally to this work.

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To whom requests for reprints should be addressed at, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi, 411-764, Korea. Phone: 82-31-920-1501; Fax: 82-31-920-1511; E-mail: [email protected]

Abbreviations: FGC, familial gastric cancer; SNP, single nucleotide polymorphism; DHPLC, denaturing high-performance liquid chromatography; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; EMSA, electrophoretic mobility shift assay.

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Abstract E-cadherin plays a critical role in epithelial cell-cell adhesion and maintenance of tissue architecture. Loss of E-cadherin expression in humans has been associated with cancer, and a number of cancer-related mutations have been identified. Here, we sought to investigate whether the -347G→GA single nucleotide polymorphism affects the transcriptional activity of the E-cadherin gene. First, we measured the promoter activity of the -347G→GA polymorphism using a dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The dual luciferase reporter assay showed that the GA allele decreased the transcriptional efficiency by 10-fold (p< 0.001) compared to the G allele. Similarly, EMSA revealed that the GA allele had a weak transcription factor binding strength compared to the G allele. We then examined the frequency of this polymorphism in familial gastric cancer (FGC) patients by denaturing high-performance liquid chromatography (DHPLC). We found that the E-cadherin genotype (-347G/GA heterozygous or GA homozygous) was associated with FGC patients (p< 0.05) compared with the G homozygous genotype. Taken together, these results suggest that the GA allele may cause weak transcription factor binding affinity and low transcriptional activity in E-cadherin expression.

Introduction Gastric cancer is one of the most common cancers worldwide. Although the occurrence rate of gastric cancer has decreased in recent years, the incidence of the disease is still high in Asian countries such as Korea and Japan (1). However, relatively little is known regarding genetic susceptibility in the pathogenesis of gastric cancer (2). Mutations in the calcium-dependent cell adhesion molecule, E-cadherin, have been associated with the early development of gastric cancer (3). E-cadherin germline mutations were first identified in New Zealand Maori families with early-onset diffuse gastric cancer; since then, the majority of E-cadherin germline mutations have been reported in diffuse type gastric cancer (4-7). Recently, we reported a MET germline mutation as well as Ecadherin germline mutations in the diffuse type of FGC (6, 8). E-cadherin is found predominantly in epithelial cells and plays a pivotal role in maintaining tissue integrity (9). A large number of reports have identified down-regulation of E-cadherin expression in human carcinomas, and E-cadherin function is lost during the development of most epithelial cancers. Indeed, it is thought that loss of E-cadherin function in cancer cells likely plays an important role in tumor development and progression (10). However, it is not yet understood how these losses of expression are governed. Just as nucleotide variations in the coding region of a gene can alter protein function, polymorphisms

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within the 5’-promoter region have been known to change the transcriptional efficiency of a variety of genes (11, 12). Recently, two frequent polymorphisms in human cancers have been identified in the promoter region of the E-cadherin gene. The first is a C→A single nucleotide polymorphism -160 nt from the transcriptional start site of the Ecadherin gene promoter; transcription of the A allele is 68% less efficient than that of the C allele (13). The second reported promoter variant is a G→GA single nucleotide polymorphism -347 nt from the transcriptional start site of the E-cadherin gene. The original report suggested that this polymorphism had no effect on transcriptional activity (14). In this study, we sought to better understand the mechanisms of altered Ecadherin expression by investigating -347 G→GA polymorphism effects on E-cadherin transcriptional activity.

Materials and methods DNA isolation from blood samples Blood samples of 28 cases from 27 FGC families without germline mutations in the Ecadherin coding sequence (8) and 142 normal control individuals were collected from the Seoul National University Hospital, Korea. Informed consent was obtained from all participants prior to testing. Twenty-seven Korean families affected with familial gastric cancer were investigated for genotyping of -347G→GA promoter polymorphism of Ecadherin gene. Criteria for family inclusion were at least two first or second degree relatives affected with gastric cancer, at least one of whom was diagnosed with cancer before the age of 50 (8). Out of 27 probands (range 22-69 ages), twelve represented families suffering from diffuse types of gastric cancer, four represented families suffering from intestinal types, and histological data for the type of the remaining eleven were not available. The classification of HDGC (hereditary diffuse gastric cancer) or HIGC (hereditary intestinal gastric cancer) was not possible in these families owing to the lack of histological information. The normal control population was randomly selected from 142 healthy Korean individuals. Peripheral blood lymphocytes were isolated from samples using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. Total genomic DNA was extracted with the Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. DNA analysis of the E-cadherin promoter regions We screened each of the above samples for the -347G→GA E-cadherin polymorphism using DHPLC (WAVE®, Transgenomic Inc., NE, USA). DNA fragments containing the promoter region of interest were amplified with the following primers: forward, 5’-

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CGCCCCGACTTGTCTCTCTAC-3’; reverse, 5’-GGCCACAGCCAATCAGCA-3’. PCR amplification for DHPLC analysis was carried out in a volume of 25 ul containing 100 ng genomic DNA, 10 pmol of each primer, 0.25 mM each dNTP, 0.5 U of Taq polymerase and the provided reaction buffer (Genecraft Ltd, Munster, Germany). Reactions were carried out in a programmable thermal cycler (MWG Biotech AG, Ebersberg, Germany) as follows: denaturation for 5 min at 94 ℃, followed by 5 cycles of 94 ℃ for 30 s, 65 ℃ for 30 s, 72 ℃ for 1 min, then 30 cycles of 94 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 1 min, followed by 5 cycles of 94 ℃for 30 s, 55 ℃ for 30 s, 72 ℃ for 1 min, with a final extension of 10 min at 72 ℃. DHPLC was performed as previously described (15, 16). For heteroduplex formation, PCR products were denatured at 95 ℃ for 5 min followed by gradual cooling to 25 ℃ over a period of 1 hour. All samples were investigated by DHPLC and direct sequencing. Direct sequencing was carried out using a Big-dye terminator cycle sequencing kit and an ABI 3100 DNA sequencer (Perkin-Elmer, CA, USA). PCR-RFLP To investigate the frequency of the -160C→A polymorphism, we performed PCR-RFLP analysis. PCR primers and conditions were the same as above. PCR products were digested with HincⅡ, and separated on a 3% agarose gel. The A allele yielded two fragments (369 and 79 bp), whereas the C allele was visualized as a single band (448 bp). The results were confirmed by direct sequencing as above. Transient transfection and dual luciferase reporter assay To examine the potential effect of the -347G→GA polymorphism on E-cadherin gene transcription, a 794 bp promoter region of the E-cadherin gene (from -647 to +147) carrying either the G or GA allele was inserted upstream of the firefly luciferase gene in the pGL3 Enhancer plasmid vector (Promega Corp., WI, USA). The G and GA alleles were amplified from DNA samples taken from FGC patients, digested with KpnⅠand BglⅡ, and cloned into the promoterless pGL3 enhancer plasmid vector. Three different luciferase reporter plasmids were generated: pGL3-G (containing the G allele), pGL3GA (containing the GA allele), and pGL3-control (Promega), which contains SV40 promoter and enhancer sequences. Each construct was confirmed by sequencing. We performed transient transfections in CV-1, HeLa, SNU-719, AGS and KatoⅢ cells obtained from Korean Cell Line Bank. HeLa, SNU-719, AGS and KatoⅢ cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, while CV-1 cells were cultured in DMEM supplemented with 10% heat-inactivated

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fetal bovine serum. Approximately 5×104 cells/well in a 24-well plate were inoculated and cultured for 24h before transient transfections were performed with the Effectene reagent (Qiagen, CA, USA). As an internal standard, all plasmids were co-transfected with pRL-TK, which contains the Renilla luciferase gene. Cells were collected 48h after transfection, and cell lysates were prepared according to Promega’s instruction manual. Luciferase activity was measured with a luminometer (Promega) and normalized against the activity of the Renilla luciferase gene. The pGL3-basic vector (Promega), which lacks both promoter and enhancer, was used as a negative control in each of the transfection experiments. Independent triplicate experiments were performed for each plasmid. EMSA (electrophoretic mobility shift assay) EMSA was performed using the Gel Shift Assay System (Promega). Complementary oligonucelotide pairs corresponding to the E-cadherin promoter sequence were synthesized (Bioneer, Seoul, Korea) as follows (bold letters indicate polymorphism): 347G (containing the G allele), 5’-GGGTGAAAGAGTGAGCCCCATCTCCAAAAC3’, -347GA (containing the GA allele), 5’GGGTGAAAGAGTGAGACCCCATCTCCAAAAC-3’. The oligonucleotide pairs were annealed and labeled with [γ-32P] ATP (Amersham Biosciences, Buckinghamshire, UK). Binding reactions were carried out with HeLa nuclear extracts (Promega) according to the manufacturer’s instructions. One ul of 32P-labeled probe was incubated with 5 ug of HeLa nuclear extract for 20 min at room temperature. DNA-protein binding specificity was tested by competition assays in which the binding reactions were preincubated with 10-, 50- and 100-fold excesses of unlabeled specific or nonspecific competitor oligonucleotides prior to the addition of the labeled probe. After the binding reaction was complete, the DNA-protein complexes were resolved by electrophoresis in a 4% nondenaturing acrylamide gel. After electrophoresis, the gels were transferred onto 3M Whatman paper, dried, and autoradiographed. Statistical analysis The chi-square test for association was used to assess the differences in genotype distribution. The genotypic-specific risks were estimated as odds ratios (OR) with associated 95% confidence intervals (CI) by unconditional logistic regression (17). The observed genotypes frequencies were compared using a chi-square test to determine if they were in Hardy-Weinberg equilibrium. All tests were performed with the STATISTICA software package (StatSoft Inc, OK, USA). A p value less than 0.05 was

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considered statistically significant.

Results Effect of the -347G→GA polymorphism on promoter activity To examine the effect of -347G→GA promoter region polymorphism on transcription of the E-cadherin gene, we measured promoter activity with a Dual Luciferase Reporter Assay System (Promega) and compared the activities of the -347GA and -347G alleles by transient transfection assay in CV-1, HeLa, SNU-719, AGS and KatoⅢ cells. As shown in Figure 1, significantly lower luciferase activities were generated by the pGL3GA construct as compared with the pGL3-G construct. In CV-1 cells, the GA allele decreased the transcriptional efficiency by 10 fold (p = 0.000261) compared with the G allele. Similar results were obtained in HeLa, SNU-719, AGS and KatoⅢ cells (12-, 8-, 9-, 13-fold decrease, respectively). Effect of the -347G→GA polymorphism on the binding activity of nuclear factors To determine whether the -347G→GA polymorphism affects the binding activity of nuclear factors, synthetic -347G and -347GA oligonucleotides were incubated with HeLa cell nuclear extracts and subjected to EMSA. The -347GA oligonucleotide showed weak DNA-protein binding, whereas the -347G oligonucleotide showed stronger DNA-protein binding (Figure 2). To verify the DNA-protein complex, competition assays were performed with specific and nonspecific oligonucleotides (Figure 2). When a -347G oligonucleotide was used to compete with a -347GA oligonucleotide, it totally disrupted the -347GA oligonucleotide binding with nuclear protein. However, when a -347GA oligonucleotide was used to compete with a -347G oligonucleotide, it was not as effective as a -347G oligonucleotide in disrupting oligonucleotide binding with nuclear protein. Allele frequencies in FGC samples and normal controls. To determine whether there is a correlation between the promoter polymorphisms and FGC, we screened a 448 bp region (-529 to -82 from the transcriptional start site) of the E-cadherin promoter in 28 cases of FGC and 142 normal controls using PCR-RFLP or DHPLC (Figure 3). We identified the previously reported -160C→A and -347G→GA polymorphisms (13, 14), and noted a positive association between the -347GA allele and FGC. Eleven (39.4%) of 28 FGC samples were heterozygous at this locus, as compared to 39 (27.5%) of 142 normal controls. Individuals with the E-cadherin genotype (-347G/GA heterozygous or GA homozygous) had an increased risk

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(p=0.03059) for FGC (TableⅠ). The distribution of genotypes was in Hardy-Weinberg equilibrium. In contrast, we did not identify a positive or negative association between the -160C→A polymorphism and FGC (TableⅠ).

Discussion In the present study, we focused on the effect of the -347G→GA E-cadherin polymorphism on transcriptional activity. Several major cis-acting elements have been identified within a short section of the proximal promoter of the E-cadherin gene, including two E boxes, a CAAT box, and a GC rich-box SP1 binding site (14, 18). The E-cadherin gene promoter thus exhibits a modular structure, suggesting that the strict control of epithelium-specific E-cadherin expression might result from interactions among the various regulatory elements (19). Our results demonstrate that the -347 single nucleotide polymorphism has a significant effect on transcriptional activity in transient transfection experiments. We performed transient transfections in CV-1, HeLa, SNU-719, AGS and KatoⅢ cells, because it was previously reported that the -347 G→GA promoter polymorphism of E-cadherin gene had no effect on transcriptional activity in CV-1 cells (14). In contrast to previous reported result, our study showed that in CV-1 cells, the GA allele of this polymorphism decreased the transcriptional efficiency by 10 fold (p = 0.000261) compared with the G allele (Fig. 1). Similar results were obtained in HeLa, SNU-719, AGS and KatoⅢ cells. The molecular mechanism of this difference may relate to differences in the affinity of DNA-binding proteins to the two alleles of the E-cadherin promoter. We searched for putative transcriptional factors that might bind with the -347 single nucleotide polymorphism, using the Ds gene software package (Accelrys Inc, CA, USA). We identified four putative transcription factors (Site_C2, ZESTE_CS, T-Ag-SV40.3, T-Ag-EP) with similarities to sequences near the E-cadherin -347G→GA promoter polymorphism. These putative transcription factors are not well characterized, and their in-depth study may be a target of future work. To investigate binding between the alleles and nuclear factors in general, we performed EMSA, which revealed that the -347GA allele bound nuclear factors more weakly than did the -347G allele. In competition assay, the -347G allele was able to disrupt -347GA-protein binding, whereas the -347GA allele was less able to disrupt nuclear protein binding to the -347G allele. Although further work will be necessary to investigate the exact molecular mechanism by which activity of E-cadherin is affected by the allelic variation, our results suggest that the -347GA polymorphism may negatively impact transcription factor binding, leading to a decrease in E-cadherin expression. Lastly, we examined whether the -347G→GA promoter polymorphism of

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the E-cadherin gene was associated with FGC. Individuals with the E-cadherin genotype (-347G/GA heterozygous or GA homozygous) had an increased risk for FGC. In the case of the -160C→A polymorphism, several reports have investigated the correlation between the -160C→A polymorphism of the E-cadherin and gastric cancer (2, 17, 20). However, the correlation between the -160C→A polymorphism of the Ecadherin and gastric cancer is still controversial. We found that the genotype frequency of -160C→A polymorphism did not differ between the normal controls and FGC patients (Table Ⅰ). However, the sample number was too small to determine the statistical significance of these differences. In the future, larger population studies will be required to confirm whether these variants increase the risk of cancer in Korean and other ethnic groups. In summary, we investigated the importance of the -347G→GA polymorphism in the promoter region of the E-cadherin gene. The GA allele was associated with significant suppression of E-cadherin transcription in CV-1, HeLa, SNU-719, AGS and KatoⅢ cells. Additionally, EMSA revealed that the GA allele had a weak transcriptional factor binding strength compared to the G allele. Therefore, it seems that -347G→GA polymorphism may affect the expression of E-cadherin, possibly increasing the cancer risk.

Acknowledgements This work was supported by a research grant from the National Cancer Center, Korea, grant HMP-99-M-03-0001 from the Ministry of Health and Welfare, and the BK21 project for Medicine, Dentistry, and Pharmacy.

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and Ladner,RD. (2003) A novel single nucleotide polymorphism within the 5’ tandem repeat polymorphism of the Thymidylate Synthase gene abolishes USF-1 binding and alters transcriptional activity. Cancer Res., 63, 2898-2904. 13. Li,LC., Chui,RM., Sasaki,M., Nakajima,K., Perinchery,G., Au,HC., Nojima,D., Carroll,P., and Dahiya,R. (2002) A single nucleotide polymorphism in the E-cadherin gene promoter alters transcriptional activities. Cancer Res., 60, 873-876. 14. Nakamura,A., Shimazaki,T., Kaneko,K., Shibata,M., Matsumura,T., Nagai,M., Makino,R., and Mitamura,K. (2002) Characterization of DNA polymorphisms in the E-cadherin gene(CDH1) promoter region. Mutation Res., 502, 19-24. 15. Kang,HC., Kim,IJ., Park,JH., Kwon,HJ., Won,YJ., Heo,SC., Lee,SY., Kim,JH., Shin,Y., Noh,DY., Yang,DH., Choe,KJ., Lee,BH., Kang,SB., and Park,JG. (2002) Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families. Hum. Mutat., 20, 235, 2002. 16. Park,JH., Kim,IJ., Kang,HC., Lee,SH., Shin,Y., Kim,KH., Lim,SB., Kang,SB., Lee,KU., Kim,SY., Lee,MS., Lee,MK., Park,JH., Moon,SD., and Park,JG. (2003) Germline mutations of the MEN1 gene in Korean families with multiple endocrine neoplasia type 1 (MEN1) or MEN1-related disorders. Clin. Genet., 64, 48-53. 17. Pharoah,PD., Oliveira,C., Machado,JC., Keller,G., Vogelsang,H., Laux,H., Becker,KF., Hahn,H., Paproski,SM., Brown,LA., Caldas,C., and Huntsman. (2002) CDH1 c-160a promoter polymorphism is not associated with risk of stomach cancer. Int. J. Cancer, 101, 196-197. 18. Giroldi,LA., Bringuier,PP., de,Weijert,M., Jansen,C., van,Bokhoven,A., and Schalken,JA. (1997) Role of E boxes in the repression of E-cadherin expression. Biochem. Biophys. Res. Commun., 241, 453-458. 19. Behrens,J., Lowrick,O., Klein-hitpass,L., and Birchmeier,W. (1991) The E-cadherin promoter: functional analysis of a G.C-rich region and an epithelial cell-specific palindromic regulatory element. Proc. Natl. Acad. Sci. USA., 88, 11495-11499. 20. Park,WS., Cho,YG., Park,JY., Kim,CJ., Lee,JH., Kim,HS., Lee,JW., Song,YH.,

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Park,CH., Park,YK., Kim,SY., Nam,SW., Lee,SH., Yoo,NJ., and Lee,JY. (2003) A single nucleotide polymorphism in the E-cadherin gene promoter –160 is not associated with risk of Korean gastric cancer. J. Korean Med. Sci. 18; 501-504

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Tables Table Ⅰ. Genotype and allele frequencies of E-cadherin polymorphisms Polymorphisms

-347

-160

a

Normal

ORa

FGC

(n=142)

(n=28)

G

103 (72.5%)c

16 (57.1%)

G/GA

39 (27.5%)

11 (39.3%)

GA

0 (0%)

1 (3.6%)

C

110 (77.5%)

21 (75%)

C/A

31 (21.8%)

6 (21.4%)

A

1 (0.7%)

1 (3.6%)

b

(95% CI )

1.815 (0.7-4.2)

1.01 (0.3-2.7)

p-value

6.97

0.03059

1.65

0.43726

5.2 (0.3-87.1)

Odds ratio compared to homozygous individuals (GG or CC)

b 95

% Confidence Interval (CI) ; OR and 95% CIs were calculated by logistic regression

with the GG or CC genotype as the reference group. c

χ2

Allele frequency

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Legends to figures Fig. 1. Dual luciferase reporter assay of the -347G→GA polymorphism. The human E-cadherin gene promoter (nt –647 to +147 relative to the transcription initiation site) was cloned from homozygous (G) and heterozygous (GA) FGC patients. The fragment was inserted upstream of the luciferase reporter gene in plasmid pGL3 and transiently transfected into CV-1 (A), HeLa (B), SNU-719 (C), AGS (D) and KatoⅢ (E) cells. The luciferase activity of each construct was normalized against the activity of Renilla luciferase. Data are expressed as a percentage of the corrected luciferase activity of pGL3-control (bars indicate the means of three independent experiments). Fig. 2. EMSA with HeLa nuclear extract using -347G and -347GA oligonucleotides. Binding activity of the -347G and -347GA oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extract. Competition assays were performed with unlabelled -347G or -347GA oligonucleotides. Each binding reaction contained 5 ug of HeLa nuclear extract and labeled -347G (lanes 2-6) or -347GA (lanes 8-12) oligonucleotides. Excess unlabeled -347G or -347GA oligonucleotides (10, 50, and 100-fold) were included in the binding reactions as competitor (lanes 3-5 and lanes 9-11, respectively). In addition, 100-fold excesses of unlabeled -347GA and -347G oligonucleotides were used to compete with -347G (lane 6) and -347GA (lane 12) oligonucleotides. Arrows indicate DNA-protein complexes. Fig. 3. E-cadherin -347 G→GA polymorphism. A: -347G (containing the G allele); B: -347GA (containing the GA allele). The underline denotes the single nucleotide polymorphism (SNP) site; arrow indicates DHPLC chromatogram and matched sequencing chromatogram.

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Fig. 1.

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Fig. 2.

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Fig. 3.