A functional polymorphism (347 G
GA) in the E-cadherin gene is associated with colorectal cancer
Yong Shin1,
Il-Jin Kim1,
Hio Chung Kang1,
Jae-Hyun Park1,
Hye-Won Park1,
Sang-Geun Jang1,
Min Ro Lee2,
Seung-Yong Jeong3,
Hee Jin Chang3,
Ja-Lok Ku1 and
Jae-Gahb Park1,4
1 Korean Hereditary Tumor Registry, Laboratory of Cell Biology, Cancer Research Institute and Cancer Research Center, Seoul National University, Korea, 2 Department of Surgery, Seoul National University Hospital, Seoul, Korea and 3 Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi, Korea
4 To whom correspondence should be addressed Email: park{at}ncc.re.kr
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Abstract
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E-cadherin, the main adhesion molecule of epithelial cells, has been implicated in carcinogenesis because its expression is frequently lost in human epithelial cancers. E-cadherin protein expression is significantly reduced in sporadic colorectal cancers (CRC), but apparently not as a consequence of allele loss or somatic mutation. We reported recently that a single nucleotide polymorphism (347 G
GA) in the E-cadherin promoter suppressed E-cadherin expression and was associated with familial gastric cancer. Here we sought to investigate whether the functional polymorphisms of E-cadherin might affect CRC. We genotyped 407 individuals (260 CRC patients and 147 normal controls) for the 347 G
GA promoter polymorphism of E-cadherin using denaturing high-performance liquid chromatography and direct sequencing. We also measured the activity of promoters harboring the polymorphism by dual luciferase reporter assay in several CRC cell lines. We found that the E-cadherin GA genotype (G/GA heterozygous and GA homozygous) was more common in CRC patients than in normal controls (P = 0.011). Subjects with the E-cadherin GA genotype had an overall 1.75-fold increased risk of CRC. We also observed an increased risk association between the E-cadherin GA genotype and both proximal colon (P = 0.019) and distal CRC (P = 0.036). Interestingly, the GA allele decreased transcriptional efficiency by 12-, 9- and 10-fold compared with the G allele in SNU-C4, SNU-C5 and SNU-1033 cell lines, respectively. Additionally, we examined whether there was a correlation between the E-cadherin promoter polymorphism and microsatellite instability status, and found no such correlation. Taken together, our results suggest that the E-cadherin 347 G
GA polymorphism may be associated with CRC.
Abbreviations: CRC, colorectal cancer; MSI, microsatellite instability; MSS, microsatellite stability
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Introduction
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Colorectal cancer (CRC) is a major cause of cancer death in Western populations, and is becoming more prevalent in Asian countries such as Korea (1,2). Genetic and epigenetic factors affecting DNA methylation and gene expression are known to be involved in the development of CRC (3), but the full range of genetic alterations and many key genes involved in the pathogenesis of CRC remain to be identified (4). E-cadherin, the main adhesion molecule of epithelial cells, has been implicated in carcinogenesis because its expression is frequently lost in human epithelial cancers (5). E-cadherin protein expression is significantly reduced in sporadic CRCs, but apparently not as a consequence of allele loss or somatic mutation (3). 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 within the 5'-promoter region have been known to change the transcriptional efficiency of a variety of genes (6,7). Two functional promoter polymorphisms have been identified within the E-cadherin promoter, at nucleotides 347 G
GA (6) and 160 C
A (7). First, the E-cadherin 160 C
A polymorphism was shown to suppress E-cadherin expression (7), and has been associated with gastric (810), breast (11), colon (12), bladder (13) and prostate (14) cancers. Secondly, the E-cadherin 347 G
GA polymorphism was found to have no effect on transcriptional activity in a previous report (15), but we recently used a dual luciferase reporter assay to show that the 347 G
GA E-cadherin polymorphism affected transcriptional activity under our study conditions (6). Additionally, in a small sample population, we found an association between the 347 G
GA E-cadherin polymorphism and familial gastric cancer (6), indicating that single nucleotide polymorphisms within the E-cadherin promoter may be associated with cancer.
Here, we screened CRC patients to examine whether the functional polymorphisms (347 and 160) of E-cadherin might affect CRC risk. To begin investigating possible consequences of these polymorphisms, we measured the activity of 347 G
GA polymorphism-containing promoter using a dual luciferase reporter assay in CRC cell lines. Furthermore, in CRC, microsatellite instability (MSI) status is biologically significant; it is well established that CRC patients with MSI have a better prognosis (16). Because MSI CRCs have distinct characteristics compared with microsatellite stability (MSS) CRCs, it is clinically important to classify CRCs based on the absence or presence of MSI (17). Accordingly, we examined the relationship between E-cadherin polymorphisms 347 and 160 and MSI status in CRC.
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Materials and methods
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Samples and DNA extraction
This study included 260 sporadic CRC patients (110 proximal colon and 150 distal CRC patients) and 147 healthy normal controls were collected from Seoul National University Hospital and National Cancer Center, Korea. Informed consent was obtained from all participants prior to testing. 260 CRC patients were enrolled from Seoul National University Hospital and National Cancer Center after we confirm that these patients were not familial cases. 147 normal controls without any apparent cancer phenotype or history were collected from Seoul National University Hospital. 260 CRC patients (mean ages 54 ± 13.3, 158 males and 99 females) were sporadic cases. Out of 147 cancer-free controls, 94 (32 males and 62 females) were available for the characteristics information. We did not observe any characteristic difference in association between the cases and controls. DNA was extracted from normal colorectal tissues from CRC patients, and from peripheral blood lymphocytes. Total genomic DNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
Genotyping of E-cadherin promoter polymorphisms
We screened the 347 G
GA E-cadherin polymorphism using denaturing high-performance liquid chromatography (DHPLC) (WAVE, Transgenomic, Omaha, NE), and direct sequencing (ABI 3100 DNA sequencer, Perkin-Elmer, Foster, CA) in 407 individuals. DNA fragments containing the promoter region were amplified and screened by DHPLC as described previously (6). Briefly, PCR amplification for DHPLC analysis was carried out in a final volume of 25 µl 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, Munster, Germany). The 160 C
A E-cadherin polymorphism was analyzed as described previously (6). Briefly, PCR products were amplified as above and digested with HincII. The A allele yielded two fragments, whereas the C allele was visualized as a single band. These results were confirmed by direct sequencing.
Transient transfection and dual luciferase reporter assay
To examine the potential effect of the 347 G
GA polymorphism on E-cadherin gene transcription in SNU-C4, SNU-C5 and SNU-1033 CRC cell lines, we performed a dual luciferase reporter assay as described previously (6). Briefly, the G and GA alleles were amplified from DNA samples, digested with KpnI and BglII, and cloned into the promoterless pGL3 enhancer plasmid vector. Three different luciferase reporter plasmids were generated: pGL3-G (containing the G allele), pGL3-GA (containing the GA allele) and pGL3-control (Promega, Madison, WI), which contains SV40 promoter and enhancer sequences. SNU-C4, SNU-C5 and SNU-1033 CRC cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Luciferase activity was measured with a luminometer (Promega) and normalized against the activity of the Renilla luciferase gene. All experiments were repeated three times.
MSI analysis
Five microsatellite markers (BAT-25, BAT-26, D2S123, D5S346 and D17S250) were amplified using the previously described primers (2). Briefly, PCR reactions were performed as follows: 5 min at 94°C; 30 s at 55°C (BAT-25, D2S123 and D5S346), 50°C (BAT-26) or 52°C (D17S250); and 1 min at 70°C for 35 cycles, followed by a final elongation of 7 min at 70°C. Of 260 CRC cases, 204 subjects had both CRC tissues and matched normal tissues available for MSI analysis.
Statistical analysis
The
2 or Fisher's exact tests were used to assess differences in genotypic distribution. The genotypic-specific risks were estimated as odds ratios (OR) with associated 95% confidence intervals (CI) by unconditional logistic regression (6). The ORs were adjusted for age and sex. The observed genotype frequencies were compared with a
2 test to determine whether they were in HardyWeinberg equilibrium. All tests were performed with the STATISTICA software package (StatSoft, Galvaniweg, OK). A P value <0.05 was considered statistically significant.
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Results
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Genotyping the 347 G
GA polymorphism
We screened the polymorphic region of the E-cadherin promoter in 260 CRC patients and 147 normal controls. The clinical characteristics for the 260 CRC patients in relation to their E-cadherin genotypes are summarized in Table I. Of the CRC patients, 155 (59.6%) were homozygous for the G allele, 93 (35.8%) were heterozygous (G/GA) and 12 (4.6%) were homozygous for the GA allele. In the normal controls, 106 (72.1%) were homozygous for G, 41 (27.9%) were heterozygous (G/GA) and 0 (0%) were homozygous for GA. We observed a significantly increased risk associated with the E-cadherin GA genotype (G/GA heterozygous and GA homozygous) in CRC patients (OR, 1.75; 95% CI, 1.1312.712; P = 0.011) compared with normal controls (Table II). In addition, we observed an increased risk association between the E-cadherin GA genotype and both proximal colon (OR, 1.85; 95% CI, 1.1023.135; P = 0.019) and distal CRC (OR, 1.68; 95% CI, 1.032.728; P = 0.036). Separated genotype frequencies of G/GA heterozygous and GA homozygous were also compared (Table III). No significant association was observed. The allele and genotype frequencies among controls were consistent with the HardyWeinberg equilibrium values and the observed effects were not influenced by other potential predictors of CRC risk such as age, gender and TNM stage.
Dual luciferase reporter assay of the 347 G
GA polymorphism-containing promoter in CRC cell lines
To begin assessing possible mechanisms for the observed association between the E-cadherin 347 GA promoter allele and CRC, we measured promoter activity with a Dual Luciferase Reporter Assay System (Promega) and compared the activities of the 347 G and 347 GA alleles by transient transfection assay in SNU-C4, SNU-C5 and SNU-1033 CRC cell lines. As shown in Figure 1, significantly lower luciferase activities were generated by the pGL3-GA construct as compared with the pGL3-G construct. In SNU-C4 cells, the GA allele decreased transcriptional efficiency by 12-fold (P = 0.00021) compared with the G allele. Similar results were obtained in SNU-C5 and SNU-1033 cells (9- and 10-fold decreases, respectively).

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Fig. 1. Dual luciferase reporter assay of the activity of the 347 G GA polymorphism-containing E-cadherin promoter in colorectal cancer cell lines. The human E-cadherin gene promoter was cloned from homozygous (G) and heterozygous (GA) CRC patients, inserted upstream of the luciferase reporter gene in plasmid pGL3 and transiently transfected into SNU-C4 (A), SNU-C5 (B) and SNU-1033 (C) cells (bars indicate the means of three independent experiments).
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Genotyping the 160 C
A polymorphism
Of the 260 CRC patient samples, 187 (71.9%) were homozygous for the C allele, 66 (25.4%) were heterozygous (C/A) and seven (2.7%) were homozygous for the A allele. Of the 147 normal controls, 114 (77.6%) were homozygous for the C allele, 32 (21.8%) were heterozygous (C/A) and one (0.6%) was homozygous for the A allele (Table II). We did not observe a significant difference in risk associated with the E-cadherin 160 genotype in CRC patients (OR, 1.35; 95% CI, 0.8412.163; P = 0.213) compared with normal controls, nor did we observe any difference in risk associated between the E-cadherin 160 genotype and tumor localization (proximal colon and distal CRC). In addition, we did not observe any joint effect between the E-cadherin 347 and 160 genotypes.
MSI status and E-cadherin promoter polymorphisms
MSI analysis was performed in 204 CRC patients with available matched tumor and normal samples (103 proximal colon and 101 distal CRC). Of the 103 proximal colon patients, 38 (36.9%) had MSI-H (or
2 of the five markers) tumors, and 65 (63.1%) had MSS (MSS or MSI in only one of the five markers) tumors. Of the 101 patients with distal CRC, only five (5%) showed MSI-H. The frequency of the E-cadherin GA genotype was not statistically different in MSI-H compared with MSS tumors (P = 0.964; Table II). Similarly, no significant association was observed between MSI status and the 160 C
A polymorphism (P = 0.818).
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Discussion
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Single nucleotide polymorphisms are common DNA sequence variations among individuals (8) that may significantly advance our ability to understand and treat human disease, including cancer. Single nucleotide polymorphisms, particularly in gene promoters and protein encoding regions, may be important to gene function and/or transcriptional efficiency (8). Recently, we reported that a single nucleotide polymorphism (347 G
GA) in the E-cadherin promoter weakens the binding affinity for transcription factors and thus results in a striking 10-fold decrease in the transcriptional efficiency of the GA allele compared with the G allele and was associated with familial gastric cancer (6). Here, we sought to determine whether there is an association between CRC and the 347 G
GA polymorphism in the E-cadherin promoter. We found that in CRC patients, the frequency of the E-cadherin GA genotype (G/GA heterozygous and GA homozygous) was significantly greater than in normal controls (P = 0.011). To address possible mechanisms for this effect, we used a dual luciferase reporter assay to examine the promoter activity of the 347 G
GA polymorphism in CRC cell lines. The GA allele decreased the transcriptional efficiency of the E-cadherin gene promoter in all tested CRC cell lines, suggesting that down-regulation of E-cadherin expression in individuals harboring the GA allele might be a predisposing factor for CRC. We also investigated the 160 C
A polymorphism of the E-cadherin promoter, the A allele of which had been shown previously to have lower transcriptional activity in comparison with the C allele (7). Many studies have examined the relationship between the E-cadherin 160 C
A promoter polymorphism and gastric (810), breast (11), colon (12), bladder (13) and prostate (14) cancers. The results of most of these studies, however, remain controversial. Consistent with a previous report (12), we found no significant difference between the E-cadherin 160 genotype frequencies in CRC patients and normal controls. Additionally, we examined whether there was any correlation between the E-cadherin promoter polymorphisms (347 and 160) and MSI status, and found no such correlation.
In conclusion, we found that the E-cadherin GA genotype (G/GA heterozygous and GA homozygous) was more common in CRC patients than in normal controls (P = 0.011), and that the GA allele was associated with significant suppression of E-cadherin promoter activity in CRC cell lines. Taken together, our results suggest that the E-cadherin 347 G
GA polymorphism may be associated with CRC.
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Notes
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The first two authors contributed equally to this article.
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Acknowledgments
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This work was supported by a research grant from the National Cancer Center, Korea, and the BK21 project for Medicine, Dentistry, and Pharmacy.
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Received April 6, 2004;
revised June 18, 2004;
accepted June 22, 2004.