MOLECULAR CARCINOGENESIS
Genetic Polymorphisms in the microRNA Binding-Sites of the Thymidylate Synthase Gene Predict Risk and Survival in Gastric Cancer Rong Shen,1, 2 Hongliang Liu,3 Juyi Wen,1 Zhensheng Liu,3 Li-E Wang,1 Qiming Wang,1 Dongfeng Tan,4 Jaffer A. Ajani,5* and Qingyi Wei1* 1
Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Department of Chemotherapy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China 3 Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina 4 Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 5 Department of GI Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 2
Thymidylate synthase (TYMS) plays a crucial role in folate metabolism as well as DNA synthesis and repair. We hypothesized that functional polymorphisms in the 30 UTR of TYMS are associated with gastric cancer risk and survival. In the present study, we tested our hypothesis by genotyping three potentially functional (at miRNA binding sites) TYMS SNPs (rs16430 6bp del/ins, rs2790 A>G and rs1059394 C>T) in 379 gastric cancer patients and 431 cancer-free controls. Compared with the rs16430 6bp/6bp þ 6bp/0bp genotypes, the 0bp/0bp genotype was associated with significantly increased gastric cancer risk (adjusted OR ¼ 1.72, 95% CI ¼ 1.15–2.58). Similarly, rs2790 GG and rs1059394 TT genotypes were also associated with significantly increased risk (adjusted OR ¼ 2.52, 95% CI ¼ 1.25–5.10 and adjusted OR ¼ 1.57, 95% CI ¼ 1.04–2.35, respectively), compared with AA þ AG and CC þ CT genotypes, respectively. In the haplotype analysis, the T-G-0bp haplotype was associated with significantly increased gastric cancer risk, compared with the C-A-6bp haplotype (adjusted OR ¼ 1.34, 95% CI ¼ 1.05–1.72). Survival analysis revealed that rs16430 0bp/0bp and rs1059394 TT genotypes were also associated with poor survival in gastric cancer patients who received chemotherapy treatment (adjusted HR ¼ 1.61, 95% CI ¼ 1.05–2.48 and adjusted HR ¼ 1.59, 95% CI ¼ 1.02–2.48, respectively). These results suggest that these three variants in the miRNA binding sites of TYMS may be associated with cancer risk and survival of gastric cancer patients. Larger population studies are warranted to verify these findings. © 2014 Wiley Periodicals, Inc. Key words: gastric cancer; genetic susceptibility; microRNA; polymorphism; thymidylate synthase; survival
INTRODUCTION Gastric cancer is the fourth common type of cancer worldwide and remains the second most frequent cause of cancer-related death, although many new drugs and therapeutic methods are available [1]. Clinically, approximately 20–30% of the patients are inoperable at the time of diagnosis [2]. As a complex and multi-factorial process, gastric carcinogenesis is still not fully understood. Epidemiological studies have demonstrated that Helicobacter pylori (HP) infection, tobacco smoke, alcohol use, dietary factors (e.g., salty foods and N-nitrosocompounds) [3] and family cancer history [4] remain the main risk factors for gastric cancer. However, only a fraction of individuals exposed to these factors develop gastric cancer, suggesting that genetic factors also play an important role in environmentally induced gastric carcinogenesis. Folate is one of the constituents in fruits and vegetables, and folate metabolism is involved in DNA synthesis, methylation and repair in humans; therefore, folate deficiency may be associated with carcinogenesis of various tissues via alteration of nucleic acid methylation and disruption of DNA integrity and ß 2014 WILEY PERIODICALS, INC.
repair [5]. Thymidylate synthase (TYMS) is known to be involved in the folate metabolism, and it is a key enzyme that maintains a balanced supply of deoxynucleotides required for DNA synthesis and repair [6]. Thymidylate deficiency may result in chromosomal breakage and fragile sites, leading to individual susceptibility to gastric cancer [7]. As the most common chemotherapy agent used to treat gastric
Abbreviations: GC, gastric cancer; TYMS, thymidylate synthase; miRNA, microRNA; SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; UTR, untranslated regions. Rong Shen and Hongliang Liu contributed equally to this work. Grant sponsor: Texas 4000 Endowed Distinguished; Grant sponsor: Anderson Cancer Center; Grant number: CA 016672 *Correspondence to: Duke Cancer Institute, Duke University Medical Center, 905 Lasalle Street, Durham, NC 27710. **Correspondence to: Department of Gastrointestinal Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Received 7 January 2014; Revised 19 March 2014; Accepted 21 March 2014 DOI 10.1002/mc.22160 Published online in Wiley Online Library (wileyonlinelibrary.com).
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cancer, 5-fluorouracil (5-FU) is converted to 5fluorodeoxyuridylate and exerts its antitumor activity through the inhibition of TYMS and further DNA synthesis [8]. Increased levels of intratumoral TYMS expression have been associated with the observed tumor resistance to 5-FU and a poor clinical outcome in gastrointestinal cancers [9–11]. The reprogramming of the 30 untranslated regions 0 (3 UTRs) by various mechanisms is a common phenomenon in cancer, and disturbance of the 30 UTR regulatory mechanisms may increase the risk of cancer [12]. Recently, the polymorphism (rs16430) of a 6bp deletion/insertion at 1,494 bp in the 30 UTR of TYMS was investigated [13] and thought to influence TYMS mRNA expression and stability [13,14]. Although the rs16430 polymorphism is likely to be located in miRNA binding sites, the results of studies on the association between this TYMS 30 UTR polymorphism and risk and survival of cancer have been conflicting [15–19]. However, the associations of the other two SNPs (rs2790 and rs1059394) in the TYMS 30 UTR, which are also likely to be at the miRNA binding sites by hsa-miR-1248 and has-miR-892a, respectively, with risk and survival in gastric cancer patients have not been reported before. MATERIALS AND METHODS Study Population This study included 379 patients with newly diagnosed and histologically confirmed gastric cancer at The University of Texas MD Anderson Cancer Center (Houston TX) between February 1990 and April 2012, who were recruited for an ongoing case– control study. Meanwhile, an additional 431 cancerfree control subjects were also selected by using frequency matching on age, sex, and ethnicity from an ongoing molecular epidemiology study of the head and neck cancer in the nearly same period. These cancer-free control subjects were persons who were not seeking health care but accompanying the patients visiting the hospitals, who were genetically unrelated to the cases. Additional information about risk factors, such as smoke and alcohol use, family history in first-degree relatives with any cancer, was collected from each eligible subject who had provided a written informed consent. The study protocol was approved by the institutional review board (IRB) of M. D. Anderson Cancer center. Target Prediction and Selection of miRNA Binding Sites SNPs Four common SNPs (minor allele frequency > 0.05) in the miRNA binding sites with potentially functional significance according to NCBI dbSNPs and SNPinfo Web Server (http://snpinfo.niehs.nih.gov/snpinfo/ snpfunc.htm) were identified. These SNPs are as follows: rs1059394, rs699517, rs8423, and rs2790. But the first three are in LD with a high r2 (0.8), so we Molecular Carcinogenesis
chose rs1059394 to represent the other two SNPs. According to the prediction results of SNPinfo, compared with their wild alleles, rs2790 G allele and rs1059394 T allele increased the binding activity of hsa-miR-1248 and has-miR-892a, respectively. To compare with other published studies, the present study also included the well-studied indel variant rs16430 in the 30 UTR of TYMS, of which the 0bp allele was significantly associated with a higher rate of message degradation and a lower level of TYMS mRNA expression compared with the 6bp allele [20]. Based on the result from another prediction tool MirSNP (http:// cmbi.bjmu.edu.cn/mirsnp), we found that the 0bp allele of this variant would create new binding sites for three miRNAs (hsa-miR-142-5p, hsa-miR-340-5p, and hsa-miR-5590-3p). As a result, we selected and genotyped three potentially functional (at miRNA binding sites) SNPs (i.e., rs16430, rs2790, and rs1059394) in the 30 UTR of TYMS to evaluate their associations with risk and survival in gastric cancer patients. Genotyping Genomic DNA was extracted from the buffy coat fraction of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280 nm by UV spectrophotometer. PCR-based restriction fragment length polymorphism (PCR-RFLP) assays were used to identify the TYMS 30 -UTR 1,494 six bp insertion/deletion polymorphism. The PCR amplification parameters was as follows: 5-min denaturation cycle at 958C 35 cycles of 958C for 30 s, 588C for 45 s, and 728C for 1 min, and a final extension at 728C for 10 min. Amplified fragments were digested by DRAL (New England Biolabs). The 6bp containing 158-bp fragment was digested into 70-bp and 88-bp fragments, and the 152-bp fragment without the 6bp insertion was not digested. Genotyping of TYMS rs2790 A>G and rs1059394 C>T was performed by using the TaqMan methodology in 384-well plates, and the outputs were read with the Sequence Detection Software on an ABI-Prism 7900 instrument, according to the manufacturer’s instructions, Applied Biosystems (Foster City, CA), which also supplied primers and probes. The conditions of amplification were as follows: 508C for 2 min, 958C for 10 min followed by 40 cycles of 958C for 15 s, and 608C for 1 min. For each SNP, the genotyping success rate was >99%, and the results of repeated assays on 10% of the samples were 100% concordant. Statistical Analysis The x2 tests were performed to compare the distributions of demographic variables and selected risk factors, such as smoking status, alcohol use, and family history of cancer, between cases and controls.
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The Hardy–Weinberg equilibrium was tested by a goodness-of-fit x2 test to compare the observed genotype frequencies with the expected ones in cancer-free controls. The associations of TYMS SNPs with risk of gastric cancer were estimated by calculating the odds ratios (OR) and their 95% confidence intervals (CIs) using logistic regression modeling with or without adjustment for demographic variables and selected risk factors. Stratum-specific ORs were also estimated by stratified analysis based on these factors. The overall survival (OS) time was calculated from the date of registration at M.D. Anderson to the date of last contact or death. Patients who were still alive at the last contact were considered a censored event in the analysis. Kaplan–Meier method and log-rank test were applied to assess differences in OS by genotypes from each SNP. The hazard ratios (HR) and 95% CIs for each SNP for OS were estimated by applying the Cox proportional hazards model with adjustment for H pylori, histology, differentiation, stage, location, and application of surgery, chemotherapy and radiotherapy. The HAPLOTYPE procedure in SAS/Genetics software using the expectation-maximization (EM) algorithm was applied to generate maximum likelihood estimates of haplotype frequencies based on the observed genotypes. Those haplotypes with a frequency 0.05). Compared with the rs16430 6bp insertion allele, the 6bp deletion allele was associated with significantly increased gastric cancer risk in an allele dose-response manner (adjustMolecular Carcinogenesis
Table 1. Distribution of Selected Variables in Gastric Cancer Cases and Cancer-Free Controls in a North-American Population Variables
Cases (%)
All subjects 379 (100.0) Age, yr (Mean SD) 59.7 12.7 G, the GG genotype was associated with significantly increased gastric cancer risk compared with the AA genotype (adjusted OR ¼ 2.58, 95% CI ¼ 1.27–5.27, P ¼ 0.009). For the TYMS rs1059394 C>T, another TYMS SNP in the miRNA binding sites, the TT genotype was also associated with significantly increased gastric cancer risk compared with the CC genotype (adjusted OR ¼ 1.67, 95% CI ¼ 1.08–2.59, Ptrend ¼ 0.022). We further performed stratified analysis for the three TYMS SNPs by age, sex, ethnicity, smoking status, drinking status, and family history of cancer. The risk association with rs16430 remained significant in the subgroup of 59 years, females, non-white population, never smokers and never drinkers (P < 0.05), whereas the risk association with rs2790 remained statistically significant in the subgroup of 0.1). Because this non-white study population included three subpopulations, that is, Asian, African American and Hispanic population, we further evaluated the
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Table 2. Associations Between SNPs in TYMS and Gastric Cancer Susceptibility in a North-American Population Cases Genotypes rs16430 6bp/6bp 6bp/0bp 0bp/0bp Ptrend Dominant model Recessive model rs2790 AA AG GG Ptrend Dominant model Recessive model rs1059394 CC CT TT Ptrend Dominant model Recessive model a
Controls
n
%
n
%
144 163 72
38.0 43.0 19.0
192 190 49
44.5 44.1 11.4
222 130 26
148 164 67
58.7 34.4 6.9
39.0 43.3 17.7
272 146 12
192 190 49
P
Crude OR (95% CI)
Adjusted ORa (95% CI)
Pa
0.004 0.059 0.002
1.00 (Ref.) 1.14 (0.85–1.55) 1.96 (1.28–2.99) 1.33 (1.10–1.63) 1.31 (0.99–1.74) 1.83 (1.23–2.71)
1.00 (Ref.) 1.15 (0.85–1.56) 1.86 (1.20–2.86) 1.31 (1.07–1.60) 1.29 (0.97–1.72) 1.72 (1.15–2.58)
0.362 0.005 0.009 0.079 0.008
0.037 0.188 0.006
1.00 (Ref.) 1.09 (0.81–1.47) 2.65 (1.31–5.38) 1.29 (1.02–1.64) 1.21 (0.91–1.61) 2.57 (1.28–5.17)
1.00 (Ref.) 1.07(0.79–1.44) 2.58 (1.27–5.27) 1.27 (1.00–1.61) 1.19 (0.89–1.58) 2.52 (1.25–5.10)
0.659 0.009 0.054 0.247 0.010
0.017 0.114 0.011
1.00 (Ref.) 1.12 (0.83–1.51) 1.77 (1.16–2.72) 1.28 (1.05–1.55) 1.25 (0.95–1.66) 1.67 (1.13–2.49)
1.00 (Ref.) 1.13 (0.83–1.53) 1.67 (1.08–2.59) 1.25 (1.02–1.53) 1.24 (0.93–1.65) 1.57 (1.04–2.35)
0.435 0.022 0.034 0.146 0.031
63.2 34.0 2.8
44.5 44.1 11.4
Adjusted for age, sex, ethnicity, smoking status, drinking status, and family history of cancer.
associations between these three SNPs and gastric cancer risk in these subpopulations (Supplementary Table S2). Significant association with rs16430 was found in African American population and rs2790 in African American and Hispanic populations, although these subgroups had relatively small numbers of observations.
TYMS Haplotypes and Risk of Gastric Cancer LD analysis using the genotyping data from controls in the present study showed there were moderate to high LD between these three SNPs (Figure 1). Two SNPs (rs16430 and rs1059394) had high LD with each other (r2 ¼ 0.94, D0 ¼ 0.97) by using the genotyping data in controls. Therefore, we also explored the
Figure 1. Pairwise linkage disequilibrium (LD) between the three potentially functional SNPs in TYMS. These SNPs were predicted to affect the microRNA binding activity of the gene.
Molecular Carcinogenesis
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Table 3. Haplotype Analysis of the Three SNPs in TYMS and Gastric Cancer Susceptibility in a North-American Population Cases (N ¼ 758) Haplotypesa C-A-6bp T-G-0bp T-A-0bp Others
Controls (N ¼ 862)
n
%
n
%
ORcrude (95% CI)
P
ORadjusted (95% CI)b
Pb
448 181 114 15
59.1 23.9 15.0 2.0
568 167 116 11
65.9 19.4 13.4 1.3
1.00 (Ref.) 1.37 (1.08–1.75) 1.25 (0.94–1.66) 1.73 (0.79–3.80)
0.011 0.133 0.173
1.00 (Ref.) 1.34 (1.05–1.72) 1.20 (0.90–1.61) 1.73 (0.78–3.82)
0.019 0.215 0.176
a The order of SNPs was based on their physical positions at chromosome 18: rs1059394 C>T, rs2790 A>G and rs16430 6bp del/ins. b Adjusted for age, sex, race, smoking status, and drinking status and family history of cancer.
haplotypes in determining whether any particular haplotype may be responsible for the observed associations with gastric cancer risk. As shown in Table 3, three haplotypes were shown to have frequencies >5% among all the cases and controls, while other less common haplotypes (frequencies T, rs2790 A>G and rs16430 6bp del/ins (C-A-6bp, T-G-0bp and T-A-0bp) accounted for 98.0% and 98.7% of the chromosomes of the cases and controls, respectively. Compared with the C-A-6bp haplotype, T-G-0bp was associated with increased gastric cancer risks (crude OR ¼ 1.37, 95% CI ¼ 1.08– 1.75 and adjusted OR ¼ 1.34, 95% CI ¼ 1.05–1.72, respectively). We also performed stratified analysis of the haplotypes and found significant associations between variant haplotypes and gastric cancer risk within strata of males (P ¼ 0.048), females (P ¼ 0.040), nonwhites (P ¼ 0.004) and those without a family history of cancer (P ¼ 0.004) (Supplementary Table S3). Additional homogeneity tests indicated that there might be some significant sex difference in gastric cancer risk as wells (P ¼ 0.049). TYMS Genotypes and Haplotypes and Gastric Cancer Survival by Clinicopathological Characteristics We performed further analysis for associations of the three SNPs and their haplotypes with clinical outcomes, but did not find any statistically significant associations with OS of gastric cancer patients, nor for treatment. However, after adjustment for several potential prognostic factors (age, sex, ethnicity, tumor site, stage, histological grade), we found that rs16430 and its high LD SNP rs1059394 were significantly associated with poor survival in patients who received chemotherapy treatment (P ¼ 0.029 and 0.041, respectively) (Table 4). Compared with 6bp/ 6bp þ 6bp/0bp genotypes, the rs16430 0bp/0bp genotype was associated with poor survival of those patients who received chemotherapy treatment (adjusted HR ¼ 1.61, 95% CI ¼ 1.05–2.48). The SNP rs1059394 had a similar effect on survival of the patients who received chemotherapy treatment (adMolecular Carcinogenesis
justed HR ¼ 1.59, 95% CI ¼ 1.02–2.48 for the TT genotype compared with CC þ CT genotypes). We then evaluated the influence of these SNPs on other clinicopathological characteristics and found two SNPs (rs16430 and rs1059394) were significantly associated with tumor location under both additive and recessive models (Table 5). Considering multiple tests that had been performed in the present study, which might have increased the probability of false discoveries, we calculated the false positive report probability (FPRP) for these observed significant associations (Supplementary Table S4) [21]. When the assumption of prior probability was 0.25, the association between rs16430 and gastric cancer risk was still noteworthy (FPRP ¼ 0.026). DISCUSSION TYMS is an important enzyme involved in the folate metabolism, catalyzes the conversion of deoxyuridinemonophosphate (dUMP) to deoxythymidine monophosphate (dTMP) using the 5,10-methylenetetrahydrofolate as a methyl donor, and provides the sole de novo source of thymidine required for DNA synthesis and repair [6]. In this hospital-based case– control study, we assessed the associations of three miRNA binding site SNPs (i.e., rs16430, rs2790, and rs1059394) in TYMS with gastric cancer risk and patient survival. We found that the homozygous variants genotype of these SNPs (i.e., rs16430 0bp/ 0bp, rs2790 GG, and rs1059394 TT) were associated with a greater risk and a poor survival of gastric cancer. These findings were consistent with the results of bioinformatics predictions, which indicated that the variant alleles of these SNPs would increase the binding activity or create new binding sites for microRNAs. These data suggested that the three SNPs might be associated with high gastric cancer risk and poor survival by decreasing TYMS expression. The correlation between rs16430 and cancers susceptibility, including gastric cancer, has been investigated in many studies, but the results were still controversial. One study investigated the risk of gastric cancer associated with rs16430 in a Koreans population [19], and another studied associations of
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Table 4. Association Between SNPs in TYMS and Overall Survival of Gastric Cancer in Chemotherapy-Stratified Analysis in a North-American Population With chemotherapy treatment
Without chemotherapy treatment N (event/total)
MST (months)
HR (95% CI)a
rs16430 6bp/6bp 6bp/0bp
12/31 11/31
59.9 61.4
0bp/0bp
1/14
NA
1.00 (Ref.) 0.75 (0.31–1.80) 0.17 (0.02–1.30) 0.20 (0.03–1.56)
SNP
Recessive model rs2790 AA AG GG
15/43 9/29
71.7 61.4
0/4
NA
Recessive model rs1059394 CC CT TT Recessive model
1.00 (Ref.) 0.95 (0.37–2.45) NA NA
11/32 12/32
59.8 61.4
1/12
NA
1.00 (Ref.) 0.91 (0.38–2.17) 0.19 (0.02–1.60) 0.21 (0.03–1.59)
Pa
N (event/total)
MST (months)
HR (95% CI)a
0.517
113/57 132/72
23.9 22.8
0.099
58/29
17.6
1.00 (Ref.) 1.03 (0.84–1.26) 1.63 (1.02–2.62) 1.61 (1.05–2.48)
0.125
0.915
179/93 101/52
23.3 27.1
0.993
22/12
16.7
0.993
0.832
116/59 132/72
24.3 22.1
0.128
55/27
17.6
0.130
1.00 (Ref.) 0.89 (0.63–1.26) 1.71 (0.92–3.19) 1.78 (0.97–3.28) 1.00 (Ref.) 1.06 (0.75–1.50) 1.64 (1.01–2.67) 1.59 (1.02–2.48)
Pa
0.907 0.042 0.029
0.525 0.089 0.063
0.754 0.046 0.041
MST, median survival time. a Adjusted for age, sex, ethnicity, tumor site, stage, histological grade.
rs16430 with risk of esophageal squamous cell carcinoma (ESCC) and gastric cardiac adenocarcinoma (GCA) in a Chinese population [22] but did not observe an evidence of the 6bp deletion/insertion polymorphism as a marker for susceptibility to these cancers. There are two other studies that reported a significant association between this SNP and gastric cancer risk, but the effects were inconsistent in different populations: the 0bp allele showed a predictive effect in a Chinese population [23] but a risk effect in an Italy population [16]. In the present study, we found that the 6bp deletion genotypes were significantly associated with an increased gastric cancer risk in an allele dose-response manner, compared with the rs16430 6bp insertion homozygous genotype, and this finding was similar to the result in the Italy population [16]. The previously reported discrepancy might due to the difference in genotype frequencies between Caucasian and Asian populations. Studies found that Caucasian patients had a lower proportion of the favorable 0bp/0bp genotype in the 30 UTR (7.8–22.0%) [20,24,25], whereas Asian patients showed a much higher proportion of the favorable 0bp/0bp genotype (36.4–56.0%) [20,24,26]. These differences in frequencies between favorable and unfavorable genotypes Molecular Carcinogenesis
might contribute to different effects by ethnicity observed in association studies. For the TYMS rs2790 A>G SNP, we found that the GG genotype was associated with significantly increased gastric cancer risk, compared with the AA and AA þ AG genotypes, under the recessive model. However, there are reports of an opposite effect of this SNP on cancer risk. For example, Moore et al. [27] reported that the rs2790 GG genotype was associated with a lower risk of gastric cancer, compared to the AA genotype, when vegetable intake was low. Although we know little about the functionality of rs2790, it was in LD with the functional SNP (TSER, three or two repeats of a 28-bp sequence) in the TYMS gene (96 50 UTR). Another study has found a lower colon cancer risk to be associated with the 50 UTR functional TYMS variants [28]. These results were apparently not consistent with ours. It is likely that cancer cells harboring a variant TYMS gene encoding for an unstable protein might have higher or lower proliferation rates, depending on other possible confounders, compared to those with a normal gene. For example, increased or reduced thymidine synthesis may lead to the misincorporation of uracil into DNA, which may depend on folate intake. These speculations warrant further investigation.
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Table 5. TYMS rs16430 and rs2790 Polymorphisms and Clinicopathological Characteristics of Patients (N ¼ 379) in a North-American Population rs16430 Additive model Variables
6/6bp 6/0bp 0/0bp
H pylori No 85 Yes 8 Histology Intestinal 64 Signet ring 60 Other 20 Location Stomach 108 GEJ 36 Differentiationb Well 31 Moderate 15 Poor 95 Metastasis No 79 Yes 65 Stage I–II 56 III–IV 86 a
Pa
rs1059394
Recessive model 0/0bp 6/6 þ 6/0bp
Additive model Pa
CC
CT
TT
Recessive model Pa
TT
CC þ CT
Pa
104 18
44 10
0.196
44 10
189 26
0.215
85 10
105 17
43 0.499 9
43 9
190 27
0.355
81 62 20
30 33 9
0.774
30 33 9
145 122 40
0.627
68 60 20
79 64 21
28 0.889 31 8
28 31 8
147 124 41
0.614
129 34
66 6
0.015
66 6
237 70
0.006 111 37
130 34
62 0.012 5
62 5
241 71
0.005
43 6 114
15 9 48
0.095
15 9 48
74 21 209
0.270
32 16 95
42 6 116
15 0.109 8 44
15 8 44
74 22 213
0.418
90 73
44 28
0.646
44 28
169 138
0.351
81 67
91 73
41 0.656 26
41 26
172 140
0.364
58 103
31 39
0.489
31 39
114 189
0.303
58 86
56 106
31 0.179 34
31 34
114 194
0.109
Two-sided x2 test. Some cases data missed.
b
We also observed that the T-G-0bp haplotype was associated with an increased gastric cancer risk, compared with the common C-A-6bp haplotype. Since it has been reported that the 6bp deletion is associated with decreased mRNA stability in vitro and lower intra-tumoral TYMS expression in vivo [20], we speculated that the T-G-0bp haplotype may lead to a lower TYMS activity than the C-A-6bp haplotype, providing an insufficient supply of deoxynucleotides for DNA synthesis and repair in the epithelium cells and leading to a subsequent increase in the risk of gastric cancer. Again, this needs to be further investigated in the future studies. Previous studies indicated that cancer at the gastroesophageal junction (GEJ), which is different from distant gastric cancer, may be associated with a worse prognosis [29,30]. In the present study, patients with GEJ cancer had a poorer OS than patients with distant gastric cancer (MST 18 vs. 32 months); Apparently, distributions of the variant genotypes of rs16430 and rs1059394 were significantly different by GEJ and distant types of gastric cancer, and subjects with the homozygous TT variant genotype of rs1059394 had a greater risk of GEJ cancer. Given the previous concept that GEJ cancer may be different from other types of gastric cancer because of different cultural, epidemiologic/epigenetic factors or biomarkers involved [31,32], our results suggested that polymorphisms of TYMS might contribute to the risk of developing different types of gastric cancer. Molecular Carcinogenesis
TYMS is a target for chemotherapeutic drugs, such as 5-fluorouracil, and expression levels of TYMS mRNA and protein are thought to be prognostic indicators for certain cancers [8,11,33]. Several studies have reported an association between the TYMS rs16430 SNP and clinical outcomes of gastric cancer patients receiving fluorouracil-based chemotherapy, but controversial conclusions were also reported by different research teams. For example, Huang et al. [34] reported that patients with the 6bp/6bp genotype had a significant shorter OS of 20.7 months, compared with 29.8 and 41.0 months in those with the 6bp/0bp and 0bp/0bp genotype, respectively, in Chinese patients treated with fluorouracil-based adjuvant chemotherapy. However, a study conducted in Japanese patients with advanced gastric cancer treated with first-line fluorouracil-based chemotherapy indicated that patients carrying the 6bp/6bp genotype had a significantly better survival, compared with those carrying 0bp/0bp or 6bp/0bp genotypes [35]. Interestingly, Goekkurt et al. [36] reported that the 6bp insertion/deletion polymorphism was not associated with response to chemotherapy and OS in advanced gastric cancer patients treated with 5-Fu and cisplatin in a Germany study. In the present study, we found that the rs16430 0bp/0bp genotype was associated with a poor OS in patients received chemotherapy, which was consistent with the finding in Japanese populations. The reason for these reported contradictory results might be due to
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population difference in genetic background or confounding of other prognostic factors on cancer survival, which warrant additional investigations. The primary limitation of the present study is the lack of data on detailed dietary intake of folate, plasma folate levels and its precursors or metabolites, because the effect of genetic variations in the TYMS gene on cancer risk or survival will depend on folate intake status [37,38]. Therefore, the present study may have underestimated cancer risk in patients whose dietary folate intake was low and could not be evaluated for possible gene–nutrient interactions. Secondly, we only investigated three common, potentially functional TYMS SNPs in the 30 UTR, it is possible that some other untyped important but rare SNPs, which may also be associated with gastric cancer risk or survival, were not captured or that the observed associations may be due to other functional polymorphisms in LD with the SNPs we had studied. Thirdly, we cannot rule out false discoveries for our findings due to lack of validation in other independent samples or populations. To assess the false positive rate, we calculated the false positive report probability for those significant findings in the present study, in which the most noteworthy SNP was rs16430. Furthermore, we observed that genetic effects were more profound in never drinkers and never smokers in this study population, a phenomenon we also observed in other association studies, in which the risk associated with adverse genotypes appeared to be higher in non-exposed or less exposed subjects, suggesting a possible role of genetic susceptibility to cancer. Lastly, population stratification is one of the important influence factors for association studies, especially for SNPs whose frequencies may vary greatly across populations due to stronger evolutionary selection pressure. In the present study, we cannot exclude the potential influence of population stratification on the association results, since a possibly mixed study population had been used. Further work is needed to estimate the effect of population stratification on those identified associations, and additional replication or validation is needed as well. In conclusion, our findings support that the variant genotypes of three SNPs at the miRNA binding sites and the haplotype of TYMS may be associated with susceptibility to gastric cancer and survival. However, because we used a hospital-based case–control study design with a mixed population, the observed associations may have been biased or simply due to chances. Further larger, prospective studies with more diverse ethnicities are warranted to confirm our findings. ACKNOWLEDGMENTS The authors thank Margaret Lung and Jessica A Fiske for assistance in recruiting the subjects; Min Molecular Carcinogenesis
Zhao, Kejing Xu and Jianzhong He for laboratory assistance. This study was supported by funds from the Texas 4000 Endowed Distinguished professorship and M. D. Anderson Cancer Center (CA 016672). REFERENCES 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108. 2. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol 2006;24:2137– 2150. 3. Crew KD, Neugut AI. Epidemiology of upper gastrointestinal malignancies. Semin Oncol 2004;31:450–464. 4. Yaghoobi M, Bijarchi R, Narod SA. Family history and the risk of gastric cancer. Br J Cancer 2010;102:237–242. 5. Choi SW, Mason JB. Folate and carcinogenesis: An integrated scheme. J Nutr 2000;130:129–132. 6. Sharp L, Little J. Polymorphisms in genes involved in folate metabolism and colorectal neoplasia: A HuGE review. Am J Epidemiol 2004;159:423–443. 7. Lin D, Li H, Tan W, Miao X, Wang L. Genetic polymorphisms in folate-metabolizing enzymes and risk of gastroesophageal cancers: A potential nutrient–gene interaction in cancer development. Forum Nutr 2007;60:140–145. 8. Heidelberger C, Chaudhuri NK, Danneberg P, et al. Fluorinated pyrimidines, a new class of tumour-inhibitory compounds. Nature 1957;179:663–666. 9. Leichman L, Lenz HJ, Leichman CG, et al. Quantitation of intratumoral thymidylate synthase expression predicts for resistance to protracted infusion of 5-fluorouracil and weekly leucovorin in disseminated colorectal cancers: Preliminary report from an ongoing trial. Eur J Cancer 1995;31A:1306–1310. 10. Boku N, Chin K, Hosokawa K, et al. Biological markers as a predictor for response and prognosis of unresectable gastric cancer patients treated with 5-fluorouracil and cis-platinum. Clin Cancer Res 1998;4:1469–1474. 11. Shirota Y, Stoehlmacher J, Brabender J, et al. ERCC1 and thymidylate synthase mRNA levels predict survival for colorectal cancer patients receiving combination oxaliplatin and fluorouracil chemotherapy. J Clin Oncol 2001;19:4298–4304. 12. Li J, Lu X. The emerging roles of 30 untranslated regions in cancer. Cancer Lett 2013;337:22–25. 13. Ulrich CM, Bigler J, Velicer CM, Greene EA, Farin FM, Potter JD. Searching expressed sequence tag databases: Discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 2000;9: 1381–1385. 14. Chu J, Dolnick BJ. Natural antisense (rTSalpha) RNA induces site-specific cleavage of thymidylate synthase mRNA. Biochim Biophys Acta 2002;1587:183–193. 15. Chen J, Hunter DJ, Stampfer MJ, et al. Polymorphism in the thymidylate synthase promoter enhancer region modifies the risk and survival of colorectal cancer. Cancer Epidemiol Biomarkers Prev 2003;12:958–962. 16. Graziano F, Kawakami K, Watanabe G, et al. Association of thymidylate synthase polymorphisms with gastric cancer susceptibility. Int J Cancer 2004;112:1010–1014. 17. Gao J, He Q, Hua D, Mao Y, Li Y, Shen L. Polymorphism of TS 30 -UTR predicts survival of Chinese advanced gastric cancer patients receiving first-line capecitabine plus paclitaxel. Clin Transl Oncol 2012;15:619–625. 18. Zhou JY, Shi R, Yu HL, Zeng Y, Zheng WL, Ma WL. The association between two polymorphisms in the TS gene and risk of cancer: A systematic review and pooled analysis. Int J Cancer 2012;131:2103–2116. 19. Yim DJ, Kim OJ, An HJ, et al. Polymorphisms of thymidylate synthase gene 50 - and 30 -untranslated region and risk of gastric cancer in Koreans. Anticancer Res 2010;30:2325–2330.
GENETIC POLYMORPHISMS IN THE microRNA BINDING-SITES 20. Mandola MV, Stoehlmacher J, Zhang W, et al. A 6bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 2004;14:319– 327. 21. Wacholder S, Chanock S, Garcia-Closas M, El ghormli L, Rothman N. Assessing the probability that a positive report is false: An approach for molecular epidemiology studies. J Natl Cancer Inst 2004;96:434–442. 22. Zhang J, Cui Y, Kuang G, et al. Association of the thymidylate synthase polymorphisms with esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma. Carcinogenesis 2004;25:2479–2485. 23. Zhang Z, Xu Y, Zhou J, et al. Polymorphisms of thymidylate synthase in the 50 - and 30 -untranslated regions associated with risk of gastric cancer in South China: A case–control analysis. Carcinogenesis 2005;26:1764–1769. 24. Stoehlmacher J, Park DJ, Zhang W, et al. A multivariate analysis of genomic polymorphisms: Prediction of clinical outcome to 5-FU/oxaliplatin combination chemotherapy in refractory colorectal cancer. Br J Cancer 2004;91:344–354. 25. Ruzzo A, Graziano F, Kawakami K, et al. Pharmacogenetic profiling and clinical outcome of patients with advanced gastric cancer treated with palliative chemotherapy. J Clin Oncol 2006;24:1883–1891. 26. Lu JW, Gao CM, Wu JZ, Cao HX, Tajima K, Feng JF. Polymorphism in the 30 -untranslated region of the thymidylate synthase gene and sensitivity of stomach cancer to fluoropyrimidine-based chemotherapy. J Hum Genet 2006;51:155– 160. 27. Moore LE, Hung R, Karami S, et al. Folate metabolism genes, vegetable intake and renal cancer risk in central Europe. Int J Cancer 2008;122:1710–1715. 28. Curtin K, Ulrich CM, Samowitz WS, et al. Thymidylate synthase polymorphisms and colon cancer: Associations with tumor stage, tumor characteristics and survival. Int J Cancer 2007;120:2226–2232. 29. Siewert JR, Bottcher K, Stein HJ, Roder JD, Busch R. Problem of proximal third gastric carcinoma. World J Surg 1995;19: 523–531.
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Genetic Polymorphisms in the microRNA Binding-Sites of the Thymidylate Synthase Gene Predict Risk and Survival in Gastric Cancer Rong Shen,1, 2 Hongliang Liu,3 Juyi Wen,1 Zhensheng Liu,3 Li-E Wang,1 Qiming Wang,1 Dongfeng Tan,4 Jaffer A. Ajani,5* and Qingyi Wei1* 1
Department of Epidemiology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Department of Chemotherapy, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China 3 Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina 4 Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 5 Department of GI Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 2
Thymidylate synthase (TYMS) plays a crucial role in folate metabolism as well as DNA synthesis and repair. We hypothesized that functional polymorphisms in the 30 UTR of TYMS are associated with gastric cancer risk and survival. In the present study, we tested our hypothesis by genotyping three potentially functional (at miRNA binding sites) TYMS SNPs (rs16430 6bp del/ins, rs2790 A>G and rs1059394 C>T) in 379 gastric cancer patients and 431 cancer-free controls. Compared with the rs16430 6bp/6bp þ 6bp/0bp genotypes, the 0bp/0bp genotype was associated with significantly increased gastric cancer risk (adjusted OR ¼ 1.72, 95% CI ¼ 1.15–2.58). Similarly, rs2790 GG and rs1059394 TT genotypes were also associated with significantly increased risk (adjusted OR ¼ 2.52, 95% CI ¼ 1.25–5.10 and adjusted OR ¼ 1.57, 95% CI ¼ 1.04–2.35, respectively), compared with AA þ AG and CC þ CT genotypes, respectively. In the haplotype analysis, the T-G-0bp haplotype was associated with significantly increased gastric cancer risk, compared with the C-A-6bp haplotype (adjusted OR ¼ 1.34, 95% CI ¼ 1.05–1.72). Survival analysis revealed that rs16430 0bp/0bp and rs1059394 TT genotypes were also associated with poor survival in gastric cancer patients who received chemotherapy treatment (adjusted HR ¼ 1.61, 95% CI ¼ 1.05–2.48 and adjusted HR ¼ 1.59, 95% CI ¼ 1.02–2.48, respectively). These results suggest that these three variants in the miRNA binding sites of TYMS may be associated with cancer risk and survival of gastric cancer patients. Larger population studies are warranted to verify these findings. © 2014 Wiley Periodicals, Inc. Key words: gastric cancer; genetic susceptibility; microRNA; polymorphism; thymidylate synthase; survival
INTRODUCTION Gastric cancer is the fourth common type of cancer worldwide and remains the second most frequent cause of cancer-related death, although many new drugs and therapeutic methods are available [1]. Clinically, approximately 20–30% of the patients are inoperable at the time of diagnosis [2]. As a complex and multi-factorial process, gastric carcinogenesis is still not fully understood. Epidemiological studies have demonstrated that Helicobacter pylori (HP) infection, tobacco smoke, alcohol use, dietary factors (e.g., salty foods and N-nitrosocompounds) [3] and family cancer history [4] remain the main risk factors for gastric cancer. However, only a fraction of individuals exposed to these factors develop gastric cancer, suggesting that genetic factors also play an important role in environmentally induced gastric carcinogenesis. Folate is one of the constituents in fruits and vegetables, and folate metabolism is involved in DNA synthesis, methylation and repair in humans; therefore, folate deficiency may be associated with carcinogenesis of various tissues via alteration of nucleic acid methylation and disruption of DNA integrity and ß 2014 WILEY PERIODICALS, INC.
repair [5]. Thymidylate synthase (TYMS) is known to be involved in the folate metabolism, and it is a key enzyme that maintains a balanced supply of deoxynucleotides required for DNA synthesis and repair [6]. Thymidylate deficiency may result in chromosomal breakage and fragile sites, leading to individual susceptibility to gastric cancer [7]. As the most common chemotherapy agent used to treat gastric
Abbreviations: GC, gastric cancer; TYMS, thymidylate synthase; miRNA, microRNA; SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; UTR, untranslated regions. Rong Shen and Hongliang Liu contributed equally to this work. Grant sponsor: Texas 4000 Endowed Distinguished; Grant sponsor: Anderson Cancer Center; Grant number: CA 016672 *Correspondence to: Duke Cancer Institute, Duke University Medical Center, 905 Lasalle Street, Durham, NC 27710. **Correspondence to: Department of Gastrointestinal Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Received 7 January 2014; Revised 19 March 2014; Accepted 21 March 2014 DOI 10.1002/mc.22160 Published online in Wiley Online Library (wileyonlinelibrary.com).
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cancer, 5-fluorouracil (5-FU) is converted to 5fluorodeoxyuridylate and exerts its antitumor activity through the inhibition of TYMS and further DNA synthesis [8]. Increased levels of intratumoral TYMS expression have been associated with the observed tumor resistance to 5-FU and a poor clinical outcome in gastrointestinal cancers [9–11]. The reprogramming of the 30 untranslated regions 0 (3 UTRs) by various mechanisms is a common phenomenon in cancer, and disturbance of the 30 UTR regulatory mechanisms may increase the risk of cancer [12]. Recently, the polymorphism (rs16430) of a 6bp deletion/insertion at 1,494 bp in the 30 UTR of TYMS was investigated [13] and thought to influence TYMS mRNA expression and stability [13,14]. Although the rs16430 polymorphism is likely to be located in miRNA binding sites, the results of studies on the association between this TYMS 30 UTR polymorphism and risk and survival of cancer have been conflicting [15–19]. However, the associations of the other two SNPs (rs2790 and rs1059394) in the TYMS 30 UTR, which are also likely to be at the miRNA binding sites by hsa-miR-1248 and has-miR-892a, respectively, with risk and survival in gastric cancer patients have not been reported before. MATERIALS AND METHODS Study Population This study included 379 patients with newly diagnosed and histologically confirmed gastric cancer at The University of Texas MD Anderson Cancer Center (Houston TX) between February 1990 and April 2012, who were recruited for an ongoing case– control study. Meanwhile, an additional 431 cancerfree control subjects were also selected by using frequency matching on age, sex, and ethnicity from an ongoing molecular epidemiology study of the head and neck cancer in the nearly same period. These cancer-free control subjects were persons who were not seeking health care but accompanying the patients visiting the hospitals, who were genetically unrelated to the cases. Additional information about risk factors, such as smoke and alcohol use, family history in first-degree relatives with any cancer, was collected from each eligible subject who had provided a written informed consent. The study protocol was approved by the institutional review board (IRB) of M. D. Anderson Cancer center. Target Prediction and Selection of miRNA Binding Sites SNPs Four common SNPs (minor allele frequency > 0.05) in the miRNA binding sites with potentially functional significance according to NCBI dbSNPs and SNPinfo Web Server (http://snpinfo.niehs.nih.gov/snpinfo/ snpfunc.htm) were identified. These SNPs are as follows: rs1059394, rs699517, rs8423, and rs2790. But the first three are in LD with a high r2 (0.8), so we Molecular Carcinogenesis
chose rs1059394 to represent the other two SNPs. According to the prediction results of SNPinfo, compared with their wild alleles, rs2790 G allele and rs1059394 T allele increased the binding activity of hsa-miR-1248 and has-miR-892a, respectively. To compare with other published studies, the present study also included the well-studied indel variant rs16430 in the 30 UTR of TYMS, of which the 0bp allele was significantly associated with a higher rate of message degradation and a lower level of TYMS mRNA expression compared with the 6bp allele [20]. Based on the result from another prediction tool MirSNP (http:// cmbi.bjmu.edu.cn/mirsnp), we found that the 0bp allele of this variant would create new binding sites for three miRNAs (hsa-miR-142-5p, hsa-miR-340-5p, and hsa-miR-5590-3p). As a result, we selected and genotyped three potentially functional (at miRNA binding sites) SNPs (i.e., rs16430, rs2790, and rs1059394) in the 30 UTR of TYMS to evaluate their associations with risk and survival in gastric cancer patients. Genotyping Genomic DNA was extracted from the buffy coat fraction of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280 nm by UV spectrophotometer. PCR-based restriction fragment length polymorphism (PCR-RFLP) assays were used to identify the TYMS 30 -UTR 1,494 six bp insertion/deletion polymorphism. The PCR amplification parameters was as follows: 5-min denaturation cycle at 958C 35 cycles of 958C for 30 s, 588C for 45 s, and 728C for 1 min, and a final extension at 728C for 10 min. Amplified fragments were digested by DRAL (New England Biolabs). The 6bp containing 158-bp fragment was digested into 70-bp and 88-bp fragments, and the 152-bp fragment without the 6bp insertion was not digested. Genotyping of TYMS rs2790 A>G and rs1059394 C>T was performed by using the TaqMan methodology in 384-well plates, and the outputs were read with the Sequence Detection Software on an ABI-Prism 7900 instrument, according to the manufacturer’s instructions, Applied Biosystems (Foster City, CA), which also supplied primers and probes. The conditions of amplification were as follows: 508C for 2 min, 958C for 10 min followed by 40 cycles of 958C for 15 s, and 608C for 1 min. For each SNP, the genotyping success rate was >99%, and the results of repeated assays on 10% of the samples were 100% concordant. Statistical Analysis The x2 tests were performed to compare the distributions of demographic variables and selected risk factors, such as smoking status, alcohol use, and family history of cancer, between cases and controls.
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The Hardy–Weinberg equilibrium was tested by a goodness-of-fit x2 test to compare the observed genotype frequencies with the expected ones in cancer-free controls. The associations of TYMS SNPs with risk of gastric cancer were estimated by calculating the odds ratios (OR) and their 95% confidence intervals (CIs) using logistic regression modeling with or without adjustment for demographic variables and selected risk factors. Stratum-specific ORs were also estimated by stratified analysis based on these factors. The overall survival (OS) time was calculated from the date of registration at M.D. Anderson to the date of last contact or death. Patients who were still alive at the last contact were considered a censored event in the analysis. Kaplan–Meier method and log-rank test were applied to assess differences in OS by genotypes from each SNP. The hazard ratios (HR) and 95% CIs for each SNP for OS were estimated by applying the Cox proportional hazards model with adjustment for H pylori, histology, differentiation, stage, location, and application of surgery, chemotherapy and radiotherapy. The HAPLOTYPE procedure in SAS/Genetics software using the expectation-maximization (EM) algorithm was applied to generate maximum likelihood estimates of haplotype frequencies based on the observed genotypes. Those haplotypes with a frequency 0.05). Compared with the rs16430 6bp insertion allele, the 6bp deletion allele was associated with significantly increased gastric cancer risk in an allele dose-response manner (adjustMolecular Carcinogenesis
Table 1. Distribution of Selected Variables in Gastric Cancer Cases and Cancer-Free Controls in a North-American Population Variables
Cases (%)
All subjects 379 (100.0) Age, yr (Mean SD) 59.7 12.7 G, the GG genotype was associated with significantly increased gastric cancer risk compared with the AA genotype (adjusted OR ¼ 2.58, 95% CI ¼ 1.27–5.27, P ¼ 0.009). For the TYMS rs1059394 C>T, another TYMS SNP in the miRNA binding sites, the TT genotype was also associated with significantly increased gastric cancer risk compared with the CC genotype (adjusted OR ¼ 1.67, 95% CI ¼ 1.08–2.59, Ptrend ¼ 0.022). We further performed stratified analysis for the three TYMS SNPs by age, sex, ethnicity, smoking status, drinking status, and family history of cancer. The risk association with rs16430 remained significant in the subgroup of 59 years, females, non-white population, never smokers and never drinkers (P < 0.05), whereas the risk association with rs2790 remained statistically significant in the subgroup of 0.1). Because this non-white study population included three subpopulations, that is, Asian, African American and Hispanic population, we further evaluated the
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Table 2. Associations Between SNPs in TYMS and Gastric Cancer Susceptibility in a North-American Population Cases Genotypes rs16430 6bp/6bp 6bp/0bp 0bp/0bp Ptrend Dominant model Recessive model rs2790 AA AG GG Ptrend Dominant model Recessive model rs1059394 CC CT TT Ptrend Dominant model Recessive model a
Controls
n
%
n
%
144 163 72
38.0 43.0 19.0
192 190 49
44.5 44.1 11.4
222 130 26
148 164 67
58.7 34.4 6.9
39.0 43.3 17.7
272 146 12
192 190 49
P
Crude OR (95% CI)
Adjusted ORa (95% CI)
Pa
0.004 0.059 0.002
1.00 (Ref.) 1.14 (0.85–1.55) 1.96 (1.28–2.99) 1.33 (1.10–1.63) 1.31 (0.99–1.74) 1.83 (1.23–2.71)
1.00 (Ref.) 1.15 (0.85–1.56) 1.86 (1.20–2.86) 1.31 (1.07–1.60) 1.29 (0.97–1.72) 1.72 (1.15–2.58)
0.362 0.005 0.009 0.079 0.008
0.037 0.188 0.006
1.00 (Ref.) 1.09 (0.81–1.47) 2.65 (1.31–5.38) 1.29 (1.02–1.64) 1.21 (0.91–1.61) 2.57 (1.28–5.17)
1.00 (Ref.) 1.07(0.79–1.44) 2.58 (1.27–5.27) 1.27 (1.00–1.61) 1.19 (0.89–1.58) 2.52 (1.25–5.10)
0.659 0.009 0.054 0.247 0.010
0.017 0.114 0.011
1.00 (Ref.) 1.12 (0.83–1.51) 1.77 (1.16–2.72) 1.28 (1.05–1.55) 1.25 (0.95–1.66) 1.67 (1.13–2.49)
1.00 (Ref.) 1.13 (0.83–1.53) 1.67 (1.08–2.59) 1.25 (1.02–1.53) 1.24 (0.93–1.65) 1.57 (1.04–2.35)
0.435 0.022 0.034 0.146 0.031
63.2 34.0 2.8
44.5 44.1 11.4
Adjusted for age, sex, ethnicity, smoking status, drinking status, and family history of cancer.
associations between these three SNPs and gastric cancer risk in these subpopulations (Supplementary Table S2). Significant association with rs16430 was found in African American population and rs2790 in African American and Hispanic populations, although these subgroups had relatively small numbers of observations.
TYMS Haplotypes and Risk of Gastric Cancer LD analysis using the genotyping data from controls in the present study showed there were moderate to high LD between these three SNPs (Figure 1). Two SNPs (rs16430 and rs1059394) had high LD with each other (r2 ¼ 0.94, D0 ¼ 0.97) by using the genotyping data in controls. Therefore, we also explored the
Figure 1. Pairwise linkage disequilibrium (LD) between the three potentially functional SNPs in TYMS. These SNPs were predicted to affect the microRNA binding activity of the gene.
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Table 3. Haplotype Analysis of the Three SNPs in TYMS and Gastric Cancer Susceptibility in a North-American Population Cases (N ¼ 758) Haplotypesa C-A-6bp T-G-0bp T-A-0bp Others
Controls (N ¼ 862)
n
%
n
%
ORcrude (95% CI)
P
ORadjusted (95% CI)b
Pb
448 181 114 15
59.1 23.9 15.0 2.0
568 167 116 11
65.9 19.4 13.4 1.3
1.00 (Ref.) 1.37 (1.08–1.75) 1.25 (0.94–1.66) 1.73 (0.79–3.80)
0.011 0.133 0.173
1.00 (Ref.) 1.34 (1.05–1.72) 1.20 (0.90–1.61) 1.73 (0.78–3.82)
0.019 0.215 0.176
a The order of SNPs was based on their physical positions at chromosome 18: rs1059394 C>T, rs2790 A>G and rs16430 6bp del/ins. b Adjusted for age, sex, race, smoking status, and drinking status and family history of cancer.
haplotypes in determining whether any particular haplotype may be responsible for the observed associations with gastric cancer risk. As shown in Table 3, three haplotypes were shown to have frequencies >5% among all the cases and controls, while other less common haplotypes (frequencies T, rs2790 A>G and rs16430 6bp del/ins (C-A-6bp, T-G-0bp and T-A-0bp) accounted for 98.0% and 98.7% of the chromosomes of the cases and controls, respectively. Compared with the C-A-6bp haplotype, T-G-0bp was associated with increased gastric cancer risks (crude OR ¼ 1.37, 95% CI ¼ 1.08– 1.75 and adjusted OR ¼ 1.34, 95% CI ¼ 1.05–1.72, respectively). We also performed stratified analysis of the haplotypes and found significant associations between variant haplotypes and gastric cancer risk within strata of males (P ¼ 0.048), females (P ¼ 0.040), nonwhites (P ¼ 0.004) and those without a family history of cancer (P ¼ 0.004) (Supplementary Table S3). Additional homogeneity tests indicated that there might be some significant sex difference in gastric cancer risk as wells (P ¼ 0.049). TYMS Genotypes and Haplotypes and Gastric Cancer Survival by Clinicopathological Characteristics We performed further analysis for associations of the three SNPs and their haplotypes with clinical outcomes, but did not find any statistically significant associations with OS of gastric cancer patients, nor for treatment. However, after adjustment for several potential prognostic factors (age, sex, ethnicity, tumor site, stage, histological grade), we found that rs16430 and its high LD SNP rs1059394 were significantly associated with poor survival in patients who received chemotherapy treatment (P ¼ 0.029 and 0.041, respectively) (Table 4). Compared with 6bp/ 6bp þ 6bp/0bp genotypes, the rs16430 0bp/0bp genotype was associated with poor survival of those patients who received chemotherapy treatment (adjusted HR ¼ 1.61, 95% CI ¼ 1.05–2.48). The SNP rs1059394 had a similar effect on survival of the patients who received chemotherapy treatment (adMolecular Carcinogenesis
justed HR ¼ 1.59, 95% CI ¼ 1.02–2.48 for the TT genotype compared with CC þ CT genotypes). We then evaluated the influence of these SNPs on other clinicopathological characteristics and found two SNPs (rs16430 and rs1059394) were significantly associated with tumor location under both additive and recessive models (Table 5). Considering multiple tests that had been performed in the present study, which might have increased the probability of false discoveries, we calculated the false positive report probability (FPRP) for these observed significant associations (Supplementary Table S4) [21]. When the assumption of prior probability was 0.25, the association between rs16430 and gastric cancer risk was still noteworthy (FPRP ¼ 0.026). DISCUSSION TYMS is an important enzyme involved in the folate metabolism, catalyzes the conversion of deoxyuridinemonophosphate (dUMP) to deoxythymidine monophosphate (dTMP) using the 5,10-methylenetetrahydrofolate as a methyl donor, and provides the sole de novo source of thymidine required for DNA synthesis and repair [6]. In this hospital-based case– control study, we assessed the associations of three miRNA binding site SNPs (i.e., rs16430, rs2790, and rs1059394) in TYMS with gastric cancer risk and patient survival. We found that the homozygous variants genotype of these SNPs (i.e., rs16430 0bp/ 0bp, rs2790 GG, and rs1059394 TT) were associated with a greater risk and a poor survival of gastric cancer. These findings were consistent with the results of bioinformatics predictions, which indicated that the variant alleles of these SNPs would increase the binding activity or create new binding sites for microRNAs. These data suggested that the three SNPs might be associated with high gastric cancer risk and poor survival by decreasing TYMS expression. The correlation between rs16430 and cancers susceptibility, including gastric cancer, has been investigated in many studies, but the results were still controversial. One study investigated the risk of gastric cancer associated with rs16430 in a Koreans population [19], and another studied associations of
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Table 4. Association Between SNPs in TYMS and Overall Survival of Gastric Cancer in Chemotherapy-Stratified Analysis in a North-American Population With chemotherapy treatment
Without chemotherapy treatment N (event/total)
MST (months)
HR (95% CI)a
rs16430 6bp/6bp 6bp/0bp
12/31 11/31
59.9 61.4
0bp/0bp
1/14
NA
1.00 (Ref.) 0.75 (0.31–1.80) 0.17 (0.02–1.30) 0.20 (0.03–1.56)
SNP
Recessive model rs2790 AA AG GG
15/43 9/29
71.7 61.4
0/4
NA
Recessive model rs1059394 CC CT TT Recessive model
1.00 (Ref.) 0.95 (0.37–2.45) NA NA
11/32 12/32
59.8 61.4
1/12
NA
1.00 (Ref.) 0.91 (0.38–2.17) 0.19 (0.02–1.60) 0.21 (0.03–1.59)
Pa
N (event/total)
MST (months)
HR (95% CI)a
0.517
113/57 132/72
23.9 22.8
0.099
58/29
17.6
1.00 (Ref.) 1.03 (0.84–1.26) 1.63 (1.02–2.62) 1.61 (1.05–2.48)
0.125
0.915
179/93 101/52
23.3 27.1
0.993
22/12
16.7
0.993
0.832
116/59 132/72
24.3 22.1
0.128
55/27
17.6
0.130
1.00 (Ref.) 0.89 (0.63–1.26) 1.71 (0.92–3.19) 1.78 (0.97–3.28) 1.00 (Ref.) 1.06 (0.75–1.50) 1.64 (1.01–2.67) 1.59 (1.02–2.48)
Pa
0.907 0.042 0.029
0.525 0.089 0.063
0.754 0.046 0.041
MST, median survival time. a Adjusted for age, sex, ethnicity, tumor site, stage, histological grade.
rs16430 with risk of esophageal squamous cell carcinoma (ESCC) and gastric cardiac adenocarcinoma (GCA) in a Chinese population [22] but did not observe an evidence of the 6bp deletion/insertion polymorphism as a marker for susceptibility to these cancers. There are two other studies that reported a significant association between this SNP and gastric cancer risk, but the effects were inconsistent in different populations: the 0bp allele showed a predictive effect in a Chinese population [23] but a risk effect in an Italy population [16]. In the present study, we found that the 6bp deletion genotypes were significantly associated with an increased gastric cancer risk in an allele dose-response manner, compared with the rs16430 6bp insertion homozygous genotype, and this finding was similar to the result in the Italy population [16]. The previously reported discrepancy might due to the difference in genotype frequencies between Caucasian and Asian populations. Studies found that Caucasian patients had a lower proportion of the favorable 0bp/0bp genotype in the 30 UTR (7.8–22.0%) [20,24,25], whereas Asian patients showed a much higher proportion of the favorable 0bp/0bp genotype (36.4–56.0%) [20,24,26]. These differences in frequencies between favorable and unfavorable genotypes Molecular Carcinogenesis
might contribute to different effects by ethnicity observed in association studies. For the TYMS rs2790 A>G SNP, we found that the GG genotype was associated with significantly increased gastric cancer risk, compared with the AA and AA þ AG genotypes, under the recessive model. However, there are reports of an opposite effect of this SNP on cancer risk. For example, Moore et al. [27] reported that the rs2790 GG genotype was associated with a lower risk of gastric cancer, compared to the AA genotype, when vegetable intake was low. Although we know little about the functionality of rs2790, it was in LD with the functional SNP (TSER, three or two repeats of a 28-bp sequence) in the TYMS gene (96 50 UTR). Another study has found a lower colon cancer risk to be associated with the 50 UTR functional TYMS variants [28]. These results were apparently not consistent with ours. It is likely that cancer cells harboring a variant TYMS gene encoding for an unstable protein might have higher or lower proliferation rates, depending on other possible confounders, compared to those with a normal gene. For example, increased or reduced thymidine synthesis may lead to the misincorporation of uracil into DNA, which may depend on folate intake. These speculations warrant further investigation.
7
GENETIC POLYMORPHISMS IN THE microRNA BINDING-SITES
Table 5. TYMS rs16430 and rs2790 Polymorphisms and Clinicopathological Characteristics of Patients (N ¼ 379) in a North-American Population rs16430 Additive model Variables
6/6bp 6/0bp 0/0bp
H pylori No 85 Yes 8 Histology Intestinal 64 Signet ring 60 Other 20 Location Stomach 108 GEJ 36 Differentiationb Well 31 Moderate 15 Poor 95 Metastasis No 79 Yes 65 Stage I–II 56 III–IV 86 a
Pa
rs1059394
Recessive model 0/0bp 6/6 þ 6/0bp
Additive model Pa
CC
CT
TT
Recessive model Pa
TT
CC þ CT
Pa
104 18
44 10
0.196
44 10
189 26
0.215
85 10
105 17
43 0.499 9
43 9
190 27
0.355
81 62 20
30 33 9
0.774
30 33 9
145 122 40
0.627
68 60 20
79 64 21
28 0.889 31 8
28 31 8
147 124 41
0.614
129 34
66 6
0.015
66 6
237 70
0.006 111 37
130 34
62 0.012 5
62 5
241 71
0.005
43 6 114
15 9 48
0.095
15 9 48
74 21 209
0.270
32 16 95
42 6 116
15 0.109 8 44
15 8 44
74 22 213
0.418
90 73
44 28
0.646
44 28
169 138
0.351
81 67
91 73
41 0.656 26
41 26
172 140
0.364
58 103
31 39
0.489
31 39
114 189
0.303
58 86
56 106
31 0.179 34
31 34
114 194
0.109
Two-sided x2 test. Some cases data missed.
b
We also observed that the T-G-0bp haplotype was associated with an increased gastric cancer risk, compared with the common C-A-6bp haplotype. Since it has been reported that the 6bp deletion is associated with decreased mRNA stability in vitro and lower intra-tumoral TYMS expression in vivo [20], we speculated that the T-G-0bp haplotype may lead to a lower TYMS activity than the C-A-6bp haplotype, providing an insufficient supply of deoxynucleotides for DNA synthesis and repair in the epithelium cells and leading to a subsequent increase in the risk of gastric cancer. Again, this needs to be further investigated in the future studies. Previous studies indicated that cancer at the gastroesophageal junction (GEJ), which is different from distant gastric cancer, may be associated with a worse prognosis [29,30]. In the present study, patients with GEJ cancer had a poorer OS than patients with distant gastric cancer (MST 18 vs. 32 months); Apparently, distributions of the variant genotypes of rs16430 and rs1059394 were significantly different by GEJ and distant types of gastric cancer, and subjects with the homozygous TT variant genotype of rs1059394 had a greater risk of GEJ cancer. Given the previous concept that GEJ cancer may be different from other types of gastric cancer because of different cultural, epidemiologic/epigenetic factors or biomarkers involved [31,32], our results suggested that polymorphisms of TYMS might contribute to the risk of developing different types of gastric cancer. Molecular Carcinogenesis
TYMS is a target for chemotherapeutic drugs, such as 5-fluorouracil, and expression levels of TYMS mRNA and protein are thought to be prognostic indicators for certain cancers [8,11,33]. Several studies have reported an association between the TYMS rs16430 SNP and clinical outcomes of gastric cancer patients receiving fluorouracil-based chemotherapy, but controversial conclusions were also reported by different research teams. For example, Huang et al. [34] reported that patients with the 6bp/6bp genotype had a significant shorter OS of 20.7 months, compared with 29.8 and 41.0 months in those with the 6bp/0bp and 0bp/0bp genotype, respectively, in Chinese patients treated with fluorouracil-based adjuvant chemotherapy. However, a study conducted in Japanese patients with advanced gastric cancer treated with first-line fluorouracil-based chemotherapy indicated that patients carrying the 6bp/6bp genotype had a significantly better survival, compared with those carrying 0bp/0bp or 6bp/0bp genotypes [35]. Interestingly, Goekkurt et al. [36] reported that the 6bp insertion/deletion polymorphism was not associated with response to chemotherapy and OS in advanced gastric cancer patients treated with 5-Fu and cisplatin in a Germany study. In the present study, we found that the rs16430 0bp/0bp genotype was associated with a poor OS in patients received chemotherapy, which was consistent with the finding in Japanese populations. The reason for these reported contradictory results might be due to
8
SHEN ET AL.
population difference in genetic background or confounding of other prognostic factors on cancer survival, which warrant additional investigations. The primary limitation of the present study is the lack of data on detailed dietary intake of folate, plasma folate levels and its precursors or metabolites, because the effect of genetic variations in the TYMS gene on cancer risk or survival will depend on folate intake status [37,38]. Therefore, the present study may have underestimated cancer risk in patients whose dietary folate intake was low and could not be evaluated for possible gene–nutrient interactions. Secondly, we only investigated three common, potentially functional TYMS SNPs in the 30 UTR, it is possible that some other untyped important but rare SNPs, which may also be associated with gastric cancer risk or survival, were not captured or that the observed associations may be due to other functional polymorphisms in LD with the SNPs we had studied. Thirdly, we cannot rule out false discoveries for our findings due to lack of validation in other independent samples or populations. To assess the false positive rate, we calculated the false positive report probability for those significant findings in the present study, in which the most noteworthy SNP was rs16430. Furthermore, we observed that genetic effects were more profound in never drinkers and never smokers in this study population, a phenomenon we also observed in other association studies, in which the risk associated with adverse genotypes appeared to be higher in non-exposed or less exposed subjects, suggesting a possible role of genetic susceptibility to cancer. Lastly, population stratification is one of the important influence factors for association studies, especially for SNPs whose frequencies may vary greatly across populations due to stronger evolutionary selection pressure. In the present study, we cannot exclude the potential influence of population stratification on the association results, since a possibly mixed study population had been used. Further work is needed to estimate the effect of population stratification on those identified associations, and additional replication or validation is needed as well. In conclusion, our findings support that the variant genotypes of three SNPs at the miRNA binding sites and the haplotype of TYMS may be associated with susceptibility to gastric cancer and survival. However, because we used a hospital-based case–control study design with a mixed population, the observed associations may have been biased or simply due to chances. Further larger, prospective studies with more diverse ethnicities are warranted to confirm our findings. ACKNOWLEDGMENTS The authors thank Margaret Lung and Jessica A Fiske for assistance in recruiting the subjects; Min Molecular Carcinogenesis
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