• Open Access

Isoflavone, polymorphisms in estrogen receptor genes and breast cancer risk in case-control studies in Japanese, Japanese Brazilians and non-Japanese Brazilians

Authors


To whom correspondence should be addressed. E-mail: moiwasak@ncc.go.jp

Abstract

Epidemiologic studies have shown an inverse association between isoflavones and breast cancer risk. Because isoflavones bind estrogen receptors, we hypothesized that polymorphisms in the estrogen receptor genes might modify the association between isoflavone intake and breast cancer risk. We conducted hospital-based case-control studies of patients aged 20–74 years with primary, incident, histologically confirmed invasive breast cancer, and matched controls from among medical checkup examinees in Nagano, Japan, and from cancer-free patients in São Paulo, Brazil. A total of 846 pairs (388 Japanese, 79 Japanese Brazilians and 379 non-Japanese Brazilians) completed validated food frequency questionnaires, and provided blood samples. Five single nucleotide polymorphisms in the estrogen receptor alpha (rs9340799, rs1913474, and rs2234693) and beta (rs4986938 and rs1256049) genes were genotyped. We found no consistent association between the five single nucleotide polymorphisms and breast cancer risk among the three populations. In analyses of combinations of isoflavone intake and single nucleotide polymorphisms, an inverse association between intake and risk was limited to women with the GG genotype of the rs4986938 polymorphism for postmenopausal Japanese (odds ratio for highest versus lowest tertile = 0.47; P for trend = 0.01), Japanese Brazilians (odds ratio for highest versus lowest median = 0.31) and non-Japanese Brazilians (odds ratio for consumers versus non-consumers = 0.37) (P for interaction = 0.11, 0.08, and 0.21, respectively). We found no remarkable difference for the other four polymorphisms. Our findings suggest that polymorphisms in the estrogen receptor beta gene may modify the association between isoflavone intake and breast cancer risk. (Cancer Sci 2009; 100: 927–933)

Abbreviations:
CI

confidence interval

CYP17

cytochrome P450c17α

CYP19

aromatase

CYP2E1

cytochrome P450 2E1

ESR1

estrogen receptor alpha

ESR2

estrogen receptor beta

FFQ

food-frequency questionnaire

NAT2

N-acetyltransferase 2

OR

odds ratio

SNP

single-nucleotide polymorphism

Soy foods are a traditional staple dish in Asian countries. They are a primary source of isoflavones such as genistein and daidzein, which are classified as phytoestrogens. Because breast cancer risk is substantially lower in Asian than Western countries,(1) the contribution of a high isoflavone intake to low breast cancer risk has been hypothesized.(2) A meta-analysis supported this hypothesis and found a small decrease in breast cancer risk with higher soy intake.(3) On the other hand, a more recent meta-analysis indicated that risk reduction was limited to Asian populations.(4) This discrepancy might reflect differences in exposure levels and genetic factors between Asian and Western populations.

Several mechanisms by which isoflavones may reduce the risk of breast cancer have been proposed.(5,6) The most prominent and thoroughly investigated are those mediated via estrogen receptors, which arise due to the similarity in chemical structures between isoflavones and human estrogen hormone, and the consequent binding affinity of isoflavones for estrogen receptors.(6,7) Isoflavones can therefore act as estrogen agonists and antagonists competing for estradiol at the receptor complex,(5) suggesting in turn that isoflavones might interact with estrogen receptor genes in the development of breast cancer. However, the possible joint effect of isoflavone intake and polymorphisms in the estrogen receptor genes on the risk of breast cancer has not been investigated.

Here, we conducted hospital-based case-control studies in Nagano, Japan and São Paulo, Brazil, targeting three populations with a substantially different intake of isoflavones and distribution of polymorphisms in the estrogen receptor genes: Japanese living in Japan, Japanese Brazilians living in São Paulo, and non-Japanese Brazilians living in São Paulo. In a previous report, we found a non-significant inverse association between isoflavone intake and the risk of breast cancer in postmenopausal Japanese women but a statistically significant inverse association in Japanese Brazilians and non-Japanese Brazilians.(8) Based on this finding, the present study tested the hypothesis that polymorphisms in estrogen receptor genes may modify the association between isoflavone intake and breast cancer risk.

Materials and Methods

Study subjects.  These multicenter, hospital-based case-control studies of breast cancer were designed to determine lifestyle factors and genetic susceptibility to the risk of breast cancer, and to compare potential risk factors among Japanese living in Nagano, Japan, and Japanese Brazilians and non-Japanese Brazilians living in São Paulo, Brazil. Eligible cases were a consecutive series of female patients aged 20–74 years with newly diagnosed and histologically confirmed invasive breast cancer. Patients with cancer were recruited between 2001 and 2005 at four hospitals in Nagano, and between 2001 and 2006 at eight hospitals in São Paulo, totaling 405 patients (98%) in Nagano, and 83 Japanese Brazilians (91%) and 389 non-Japanese Brazilians (99%) in São Paulo. In the Nagano study, eligible controls were selected from medical checkup examinees in two of the four hospitals and confirmed not to have cancer. One control was matched for each case by age (within 3 years) and residential area. Among potential controls, one examinee refused to participate and two refused to provide blood samples. Eventually, we obtained written informed consent from 405 matched pairs. In the study in São Paulo, eligible controls were preferentially selected from cancer-free patients who visited the same hospital as the index cases. One control was matched for each patient with cancer by age (within 5 years) and ethnicity. Among potential controls, 22 patients refused to participate (participation rate = 96%). Eventually, we obtained written informed consent from 472 matched pairs (83 for Japanese Brazilians and 389 for non-Japanese Brazilians). The study protocol was approved by Comissão Nacional de Ética em Pesquisa (CONEP), Brasília, Brazil and by the institutional review board of the National Cancer Center, Tokyo, Japan.

Questionnaire.  Participants in Nagano were asked to complete a self-administered questionnaire, while those in São Paulo were interviewed by trained interviewers using a structured questionnaire. The two questionnaires contained similar questions concerning demographic characteristics, medical history, family history of cancer, menstrual and reproductive history, anthropometric factors, physical activity and smoking habits. For dietary habits, we used a semiquantitative food frequency questionnaire (FFQ) (136 items for the Japanese version and 118 items for the Brazilian version), which was developed and validated in each population.(9–11) In the FFQ, participants were questioned on how often they consumed the individual food items (frequency of consumption), as well as relative sizes compared to standard portions. Daily food intake was calculated by multiplying frequency by standard portion and relative size for each food item in the FFQ. Daily intakes of genistein and daidzein were calculated using a food composition table of isoflavones developed previously.(12,13) Isoflavone intake was defined for this study as the sum of genistein and daidzein intake. Other nutrients were calculated using the Japanese Standard Tables of Food Composition for the Japanese version,(14) and the United States Department of Agriculture (USDA) food composition tables for the Brazilian version.(15) For some Japanese-specific foods in the Brazilian version, the Japanese Standard Tables of Food Composition(14) was used.

The validity of isoflavone intake estimated from the Japanese version of the FFQ was evaluated in a subsample of the Japan Public Health Center-based Prospective Study by comparing the estimated intake according to the FFQ to that in four consecutive seven-day dietary records, one conducted in each of the four seasons. Spearman's correlation coefficients between energy-adjusted genistein and daidzein intake estimated from the FFQ and from dietary records to be 0.59 for genistein and 0.60 for daidzein.(10) For the Brazilian version, the validity of isoflavone intake estimated from the FFQ was evaluated in a subsample of the control group of this case-control study by comparing the estimated intake according to the FFQ to that in two consecutive four-day dietary records, one each in two seasons. Spearman's correlation coefficients between energy-adjusted genistein and daidzein intake estimated from the FFQ and from dietary records were 0.76 for genistein and 0.76 for daidzein.(11)

Genotyping.  Genomic DNA samples were extracted from the peripheral blood using FlexiGene® DNA kits (Qiagen K.K., Tokyo, Japan) according to the manufacturer's protocol. We selected five single nucleotide polymorphisms (SNPs) in the estrogen receptor alpha (ESR1) gene (rs9340799, rs1913474, and rs2234693) and estrogen receptor beta (ESR2) gene (rs4986938 and rs1256049), which were the most frequently studied SNP in relation to breast cancer risk.(16–20) Genotyping of the five SNPs was performed by a commercial laboratory (Genetic Laboratory, Inc., Sapporo, Japan) using TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) (Table 1). Patients with cancer and matched controls were analyzed in the same well by laboratory personnel unaware of the case-control status. For quality control assessment, we genotyped six SNPs of four genes (N-acetyltransferase 2 [NAT2], cytochrome P450c17α[CYP17], aromatase [CYP19], and cytochrome P450 2E1 [CYP2E1]) in our laboratory using about 24% of the samples in the present study. However, SNPs used in the present study were not included. The concordance rates between Genetic Laboratory Inc. and our laboratory varied between 97.6 and 99.5% among the six SNPs.

Table 1. Single-nucleotide polymorphisms in estrogen receptor genes and their allele frequency
GeneSNP rs numberSynonymRegionMajor/minor alleleMinor allele frequency among control groups
Japanese living in Nagano, JapanJapanese Brazilians living in São Paulo, BrazilNon-Japanese Brazilians living in São Paulo, Brazil
  1. SNP, single-nucleotide polymorphism.

Estrogen receptor alpha geners9340799XbaIintron 1A/G0.190.200.31
rs1913474 intron 3C/T0.480.480.21
rs2234693PvuIIintron 1T/C0.450.450.42
Estrogen receptor beta geners4986938AulI3′-UTRG/A0.140.130.33
rs1256049RsaIexon 6G/A0.300.200.05

Statistical analysis.  Comparison of baseline characteristics between cases and controls was evaluated by the Mantel–Haenszel test using matched-pair strata in each population. Genotype frequencies were tested for deviation from the Hardy–Weinberg equilibrium with the χ2-test. Dietary intake of isoflavones was adjusted for total energy intake by the residual method and divided into median or tertile categories based on control distribution for Japanese and Japanese Brazilians, respectively. Because of the small proportion of consumers, non-Japanese Brazilians were categorized into non-consumers and consumers of isoflavones. Using a conditional logistic regression model, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) of breast cancer for isoflavone intake, SNPs, and the joint effect between isoflavone intake and genotypes. An unconditional logistic regression model was used for stratified analyses according to menopausal status. Linear trends for ORs were tested in the logistic regression model using the exposure categories as ordinal variables. Tests for the interaction were performed based on the difference between two likelihood ratios of the models with and without the interaction terms between isoflavone intake and the SNP of interest. Adjustments were made for the following variables, selected mainly on the basis of comparison of baseline characteristics between patients with cancer and controls, as potential confounders: menopausal status, number of births, family history of breast cancer, smoking status, moderate physical activity in the past 5 years and vitamin supplement use. We did not include a history of benign breast disease as a covariate since we regarded it as an intermediate variable in the causal pathway between isoflavone intake and breast cancer. All P-values reported are two-sided, and significance level was set at P < 0.05. All statistical analyses were performed with SAS version 9.1 software (SAS Institute, Inc., Cary, NC, USA).

Results

We excluded subjects who reported extremely low or high total energy intake (<500 or ≥4000 kCal) or had no DNA sample, leaving 388 pairs of Japanese, 79 pairs of Japanese Brazilians and 379 pairs of non-Japanese Brazilians for inclusion in the present analyses.

Characteristics of patients with cancer and controls are shown in a previous report (data not shown in table).(8) For Japanese women, the proportion of premenopausal women, current smokers, and vitamin supplement users was higher in cases than in controls, and patients with cancer tended to have a family history of breast cancer and history of benign breast disease. Patients with cancer were less likely than controls to breast-feed, be physically active and eat vegetables. For Japanese Brazilians, patients with cancer were less likely than controls to give birth and be physically active, and more likely to eat vegetables and fruits. For non-Japanese Brazilians, the proportion of premenopausal women and current smokers was higher in patients with cancer than controls, while the proportion of physically active women and vitamin supplement users was lower. Isoflavone intake substantially varied among populations, with mean intakes in control subjects of 46.2 mg/day for Japanese, 23.5 mg/day for Japanese Brazilians, and 4.4 mg/day for non-Japanese Brazilians.

The distributions of SNPs in the ESR1 gene (rs9340799, rs1913474 and rs2234693) and ESR2 gene (rs4986938 and rs1256049) are shown in Tables 1 and 2. No deviation from the Hardy–Weinberg equilibrium was observed among the controls in any population. The prevalence of the minor allele in the rs9340799 and rs4986938 polymorphisms was lower in the control group of Japanese and Japanese Brazilians than in that of non-Japanese Brazilians, while that of the minor allele in the rs1913474 and rs1256049 polymorphisms was higher in the control group of Japanese and Japanese Brazilians. We found a decreased risk of breast cancer among Japanese women with at least one minor allele of the rs9340799 or rs2234693 polymorphism in comparison with those with the major allele homozygote, but not among Japanese Brazilian and non-Japanese Brazilian women. This decrease was statistically significant for the rs2234693 polymorphism but not for the rs9340799 polymorphism. Stratified analyses by menopausal status showed that this decreased risk occurred primarily among postmenopausal Japanese for both SNPs (data not shown). In contrast, no association was observed for the rs1913474, rs4986938, or rs1256049 polymorphisms in the three populations, regardless of menopausal status.

Table 2. Odds ratios and 95% confidence intervals of breast cancer according to polymorphisms in estrogen receptor genes
 Japanese living in Nagano, JapanJapanese Brazilians living in São Paulo, BrazilNon-Japanese Brazilians living in São Paulo, Brazil
No.OR95% CINo.OR95% CINo.OR95% CI
CaseControlCaseControlCaseControl
  • Conditional model adjusting for menopausal status (premenopausal, postmenopausal), number of births (0, 1, 2, 3, 4, 5+), family history of breast cancer (yes, no), smoking status (never, past, current smokers), moderate physical activity in the past 5 years (no, less than 3 days/month, 1–4 days/week, more than 5 days/week), and vitamin supplement use (yes, no). ORs and 95% CIs with statistical significance are written in bold letter. CIs, confidence intervals; OR, odds ratio.

Estrogen receptor alpha gene (rs9340799)
AA2732561 54501 1611821 
AG1031190.68(0.45–1.02)22260.75(0.31–1.84)1751611.16(0.84–1.59)
GG12130.75(0.28–1.98)330.68(0.10–4.57)43361.27(0.78–2.07)
AG + GG1151320.69(0.47–1.02)25290.74(0.31–1.79)2181971.18(0.88–1.59)
Estrogen receptor alpha gene (rs1913474)
CC1001131 25241 2372391 
CT1921761.19(0.81–1.76)39341.24(0.55–2.81)1271221.09(0.80–1.49)
TT96991.08(0.70–1.66)15210.79(0.28–2.20)14180.80(0.38–1.67)
CT + TT2882751.15(0.80–1.64)54551.07(0.51–2.27)1411401.05(0.78–1.42)
Estrogen receptor alpha gene (rs2234693)
TT1441151 25221 1071221 
TC1801960.70(0.49–0.995)39430.66(0.29–1.47)1871940.99(0.68–1.43)
CC64770.64(0.40–1.02)15140.93(0.31–2.86)85631.51(0.98–2.31)
TC + CC2442730.68(0.49–0.96)54570.71(0.32–1.54)2722571.15(0.83–1.61)
Estrogen receptor beta gene (rs4986938)
GG2892811 59601 1691761 
GA941020.88(0.59–1.31)17171.32(0.53–3.31)1631541.09(0.78–1.51)
AA551.53(0.39–6.07)320.71(0.09–5.57)47490.93(0.59–1.47)
GA + AA991070.91(0.62–1.34)20191.22(0.51–2.93)2102031.05(0.77–1.42)
Estrogen receptor beta gene (rs1256049)
GG2031821 47481 3423451 
GA1611780.79(0.56–1.10)26300.95(0.46–1.98)36321.21(0.71–2.04)
AA24280.84(0.44–1.60)614.80(0.50–46.19)120.54(0.04–6.53)
GA + AA1852060.79(0.57–1.09)32311.04(0.50–2.13)37341.16(0.70–1.94)

Analyses of combinations of isoflavone intake and the rs4986938 polymorphism in the ESR2 gene revealed that the risk of breast cancer significantly decreased with increasing isoflavone intake only among women with the GG genotype among postmenopausal Japanese (OR for highest versus lowest tertile = 0.47; 95%CI 0.27–0.84; P for trend = 0.01), Japanese Brazilians (OR for highest versus lowest median = 0.31; 95%CI 0.12–0.78), and non-Japanese Brazilians (OR for consumers versus non-consumers = 0.37; 95%CI 0.16–0.85) (P for interaction = 0.11, 0.08 and 0.21, respectively) (Tables 3 and 4). Moreover, we found no remarkable difference in the association between isoflavone intake and breast cancer risk by the four other polymorphisms.

Table 3. Odds ratios and 95% confidence intervals of breast cancer for combinations of dietary intake of isoflavones and polymorphisms in estrogen receptor genes among Japanese
 All subjectsPremenopausal womenPostmenopausal women
Isoflavone intake (mg/day), tertile categoryP for trendIsoflavone intake (mg/day), tertile categoryP for trendIsoflavone intake (mg/day), tertile categoryP for trend
123123123
  • No. of patients with cancer/No. of controls.

  • Conditional model adjusting for menopausal status (premenopausal, postmenopausal), number of births (0, 1, 2, 3, 4, 5+), family history of breast cancer (yes, no), smoking status (never, past, current smoker), moderate physical activity in the past 5 years (no, less than 3 days/month, 1–4 days/week, more than 5 days/week), and vitamin supplement use (yes, no). For stratified analyses according to menopausal status, an unconditional model adjusting for age, area, number of births (0, 1, 2, 3, 4, 5+), family history of breast cancer (yes, no), smoking status (never, past, current smoker), moderate physical activity in the past 5 years (no, less than 3 days/month, 1–4 days/week, more than 5 days/week), and vitamin supplement use (yes, no). ORs and 95% CIs with statistical significance are written in bold letter. CIs, confidence intervals; OR, odds ratio.

Estrogen receptor alpha gene (rs9340799)
AA
No.109/8376/9088/83 54/4130/3133/19 55/4246/5955/64 
OR10.730.780.3210.681.130.9610.750.640.15
(95% CI) (0.45–1.18)(0.47–1.29)  (0.34–1.35)(0.53–2.39)  (0.41–1.34)(0.36–1.15) 
AG + GG
No.42/4642/3931/47 25/2522/1212/7 17/2120/2719/40 
OR0.520.680.510.750.641.381.130.540.590.560.380.15
(95% CI)(0.27–0.99)(0.37–1.24)(0.26–1.01) (0.31–1.35)(0.59–3.23)(0.39–3.30) (0.26–1.32)(0.26–1.20)(0.18–0.79) 
 P for interaction = 0.39P for interaction = 0.15P for interaction = 0.87
Estrogen receptor alpha gene (rs1913474)
CC
No.41/3832/4227/33 20/1616/1213/4 21/2216/3014/29 
OR10.680.760.6211.152.390.0810.600.470.09
(95% CI) (0.34–1.36)(0.37–1.59)  (0.40–3.26)(0.61–9.30)  (0.24–1.47)(0.18–1.21) 
CT + TT
No.110/9186/8792/97 59/5036/3132/22 51/4150/5660/75 
OR0.970.970.840.330.910.860.970.991.201.080.800.14
(95% CI)(0.54–1.74)(0.55–1.72)(0.45–1.55) (0.41–2.02)(0.37–2.04)(0.39–2.44) (0.56–2.59)(0.51–2.29)(0.38–1.68) 
 P for interaction = 0.69P for interaction = 0.58P for interaction = 0.73
Estrogen receptor alpha gene (rs2234693)
TT
No.58/3638/4148/38 33/2112/1621/11 25/1526/2527/27 
OR10.550.680.5410.411.150.7710.790.580.28
(95% CI) (0.26–1.16)(0.32–1.43)  (0.15–1.12)(0.43–3.10)  (0.32–1.92)(0.24–1.40) 
TC + CC
No.93/9380/8871/92 46/4540/2724/15 47/4840/6147/77 
OR0.510.520.420.460.640.990.860.390.540.420.350.14
(95% CI)(0.28–0.96)(0.28–0.98)(0.21–0.82) (0.31–1.34)(0.45–2.16)(0.35–2.15) (0.24–1.21)(0.19–0.92)(0.16–0.76) 
 P for interaction = 0.37P for interaction = 0.08P for interaction = 0.97
Estrogen receptor beta gene (rs4986938)
GG
No.115/8688/9686/99 57/4639/3232/21 58/4049/6454/78 
OR10.740.650.0610.961.030.9410.600.470.01
(95% CI) (0.47–1.16)(0.39–1.07)  (0.51–1.83)(0.49–2.15)  (0.33–1.07)(0.27–0.84) 
GA + AA
No.36/4330/3333/31 22/2013/1113/5 14/2317/2220/26 
OR0.570.780.900.230.800.911.990.200.470.800.620.49
(95% CI)(0.31–1.08)(0.40–1.50)(0.45–1.82) (0.37–1.72)(0.35–2.34)(0.62–6.46) (0.21–1.06)(0.36–1.75)(0.28–1.35) 
 P for interaction = 0.17P for interaction = 0.48P for interaction = 0.11
Estrogen receptor beta gene (rs1256049)
GG
No.85/6259/6259/58 43/3228/2023/12 42/3031/4236/46 
OR10.740.820.1611.050.980.8010.560.510.08
(95% CI) (0.43–1.27)(0.46–1.48)  (0.49–2.27)(0.39–2.47)  (0.28–1.13)(0.26–1.01) 
GA + AA
No.66/6759/6760/72 36/3424/2322/14 30/3335/4438/58 
OR0.700.740.610.930.790.781.310.200.500.600.410.35
(95% CI)(0.41–1.19)(0.43–1.27)(0.35–1.07) (0.39–1.58)(0.36–1.69)(0.55–3.08) (0.24–1.03)(0.31–1.18)(0.21–0.80) 
 P for interaction = 0.63P for interaction = 0.65P for interaction = 0.31
Table 4. Odds ratios and 95% confidence intervals of breast cancer for combinations of dietary intake of isoflavones and polymorphisms in estrogen receptor genes among Japanese Brazilian and non-Japanese Brazilian subjects
 Japanese Brazilians living in São Paulo, BrazilNon-Japanese Brazilians living in São Paulo, Brazil
Isoflavone intake (mg/day), median categoryIsoflavone intake (mg/day)
12Non-consumersConsumers
  • No. of patients with cancer/No. of controls.

  • Conditional model adjusting for menopausal status (premenopausal, postmenopausal), number of births (0, 1, 2, 3, 4, 5+), family history of breast cancer (yes, no), smoking status (never, past, current smokers), moderate physical activity in the past 5 years (no, less than 3 days/month, 1–4 days/week, more than 5 days/week), and vitamin supplement use (yes, no). ORs and 95% CIs with statistical significance are written in bold letter. CIs, confidence intervals; OR, odds ratio.

Estrogen receptor alpha gene (rs9340799)
AA
No.31/2123/29145/15716/25
OR10.3610.68
(95% CI) (0.14–0.95) (0.32–1.43)
AG + GG
No.15/1810/11198/16120/36
OR0.440.341.230.61
(95% CI)(0.14–1.45)(0.09–1.32)(0.89–1.68)(0.33–1.13)
 P for interaction = 0.36P for interaction = 0.52
Estrogen receptor alpha gene (rs1913474)
CC
No.13/1212/12213/20424/35
OR10.7610.65
(95% CI) (0.18–3.18) (0.36–1.19)
CT + TT
No.33/2721/28129/11412/26
OR1.250.551.130.49
(95% CI)(0.42–3.72)(0.17–1.78)(0.82–1.56)(0.24–1.01)
 P for interaction = 0.52P for interaction = 0.40
Estrogen receptor alpha gene (rs2234693)
TT
No.17/128/1097/10610/16
OR10.4110.57
(95% CI) (0.10–1.65) (0.22–1.47)
TC + CC
No.29/2725/30246/21226/45
OR0.650.361.200.65
(95% CI)(0.23–1.84)(0.12–1.08)(0.84–1.71)(0.37–1.15)
 P for interaction = 0.71P for interaction = 0.94
Estrogen receptor beta gene (rs4986938)
GG
No.38/3021/30156/14813/28
OR10.3110.37
(95% CI) (0.12–0.78) (0.16–0.85)
GA + AA
No.8/912/10156/17023/33
OR0.620.970.970.68
(95% CI)(0.16–2.35)(0.31–3.01)(0.70–1.35)(0.37–1.24)
 P for interaction = 0.08P for interaction = 0.21
Estrogen receptor beta gene (rs1256049)
GG
No.27/2320/25308/28634/59
OR10.4910.55
(95% CI) (0.21–1.17) (0.35–0.90)
GA + AA
No.19/1613/1535/322/2
OR0.970.531.100.84
(95% CI)(0.36–2.58)(0.18–1.58)(0.64–1.87)(0.10–6.97)
 P for interaction = 0.89P for interaction = 0.78

Discussion

In these case-control studies of Japanese, Japanese Brazilians, and non-Japanese Brazilians, we found that a statistically significant inverse association between isoflavone intake and breast cancer risk appeared only among women with the GG genotype of the rs4986938 polymorphism in the ESR2 gene, but the interaction was not statistically significant. Our findings support the hypothesis that polymorphisms in the ESR2 gene may modify the association between isoflavone intake and breast cancer risk.

To date, many studies investigating the possible effect of SNPs in the ESR2 gene on breast cancer risk have focused on the rs4986938 and rs1256049 polymorphisms, although their functional importance has yet to be clarified. Here, we found no association between either SNP and the risk of breast cancer, which is in general agreement with most previous studies.(16,17) In contrast, we did see an inverse association between isoflavone intake and breast cancer risk with the rs4986938 polymorphism in three populations, but only among women with the GG genotype. We also saw a suggestive interaction in the case-control studies of Japanese and Japanese Brazilians but not in the case-control study of non-Japanese Brazilians. Although the reason for the inconsistency in interactions among populations remains unclear, it might reflect the amount of intake, on the basis that the findings were relatively consistent among the populations with a high intake (Japanese and Japanese Brazilians). Moreover, the prevalence of the GG genotype of the rs4986938 polymorphism among the control group was higher in Japanese (72.4%) and Japanese Brazilians (75.9%) than in non-Japanese Brazilians (46.4%). This might partly explain the previous inconsistencies in results for isoflavone exposure and breast cancer risk between Asian and Western populations.(4)

To our knowledge, only two studies have investigated interactions between phytoestrogen exposure and polymorphisms in the ESR2 gene in the risk of hormone-related diseases.(21,22) Hedelin et al. reported a significant interaction between phytoestrogen intake and a promoter SNP in the ESR2 gene (rs2987983) in the risk of prostate cancer in a population-based case-control study in Sweden.(22) Tsuchiya et al. reported a significant interaction between urinary genistein level and RsaI polymorphism in the ESR2 gene in the risk of advanced endometriosis among infertile Japanese women.(21) These findings suggest that isoflavones may reduce the risk of hormone-related diseases via a mechanism that involves estrogen receptor beta. Considering that functional data are not presently available, our finding suggests that the rs4986938 polymorphism, or some other genetic variants in strong linkage disequilibrium with this SNP, modify the protective effect of isoflavones on breast cancer. In this regard, we provide further evidence for a role of isoflavones in the development of breast cancer.

We found a decreased risk of breast cancer among Japanese women with at least one minor allele of the rs9340799 or rs2234693 polymorphism in comparison with those with the major allele homozygote. Although these are the most frequently studied SNPs, results have been inconsistent.(18–20) Most studies have shown no association between the rs2234693 polymorphism and breast cancer risk.(18–20) On the other hand, several but not all studies have reported that the G allele of the rs9340799 polymorphism was associated with a decreased risk of breast cancer,(18,20) which is consistent with our findings in Japanese women. Since we failed to observe an overall consistency of findings in our three populations, however, our findings in Japanese women might be merely due to chance.

Although interactions between phytoestrogen exposure and polymorphisms in the ESR1 gene in the risk of breast cancer have not been investigated, we are aware of two studies examining interactions on circulating sex hormone levels.(23,24) In their study of 125 postmenopausal women in the European Prospective Investigation of Cancer and Nutrition–Norfolk cohort, Low et al. reported that urinary and serum isoflavones were negatively correlated with plasma estradiol among women with the CC genotype for PvuII polymorphism in the ESR1 gene, but not those with other genotypes.(23) Moreover, they reported a significant interaction between urinary lignans and rs9340835 polymorphism in the ESR1 gene, affecting plasma estrone levels in a cross-sectional study of 1988 healthy postmenopausal women from the same cohort.(24) Although these studies imply the presence of gene–nutrient interaction, we found no remarkable difference in the association between isoflavone intake and breast cancer risk by polymorphisms in the ESR1 gene. Further studies based on a comprehensive evaluation of this gene would clarify this gene–nutrient interaction.

Our study has methodological advantages over studies conducted previously. First, and unique to this study, we assessed gene–nutrient interactions using three populations with substantially different isoflavone intakes and allele frequencies of SNPs. For example, isoflavone intake differed considerably among the three populations, with median levels (interquartile range) in the control group of (mg/day) 40.7 (25.8–61.4) among Japanese, 13.4 (7.9–31.1) among Japanese Brazilians, and 0 (0–0) among non-Japanese Brazilians. In addition, allele frequency also differed among the populations, such as that of the G allele of the rs4986938 polymorphism in the ESR2 gene, at 0.86 for Japanese, 0.87 for Japanese Brazilians, and 0.67 for non-Japanese Brazilians. Second, the overall consistency of findings in the three populations could allow the results to be more generalized than those from a single population.

Several limitations of the study also warrant mention. First, dietary intake of isoflavones was assessed after the diagnosis of breast cancer, and therefore, is sensitive to recall bias. Second, although the substantially high participation rates among both eligible patients with cancer and controls minimized potential biases related to control selection, the use of controls from medical checkup examinees and cancer-free patients, whose dietary habits may differ from those of the general population due to health consciousness or disease, might have lead to selection bias. For example, isoflavone intake was higher among women aged 50–69 years in the control group of the Nagano study (median intake = 46.3 mg/day) than in participants aged 50–69 years living in Nagano in the 10-year follow-up survey of the Japan Public Health Center-based Prospective Study (median intake = 38.8 mg/day), which used a similar FFQ and had a high response rate. Third, the evaluation of gene–nutrient interactions was performed in a relatively small number of patients with cancer. The interpretability of our results might therefore be limited.

Allowing for these methodological issues, we found a suggestive interaction between isoflavone intake and the rs4986938 polymorphism of the ESR2 gene in the risk of breast cancer in case-control studies of Japanese and Japanese Brazilians. Our findings support the hypothesis that polymorphisms in the ESR2 gene may modify the association between isoflavone intake and breast cancer risk. Further, they provide additional evidence that the mechanisms by which isoflavones may reduce the risk of breast cancer might involve estrogen receptor beta.

Acknowledgments

This study was supported by a Grant-in-Aid for Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare of Japan, and Grants-in-Aid for Scientific Research on Priority Areas (17015049) and for Young Scientists (B) (17790378 and 19790415) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Society for the Promotion of Science.

We are grateful to the members of the São Paulo-Japan Breast Cancer Study Group: S. Ikeda and C. Nishimoto (Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo); C.I. Yamaguchi, C.M. Kunieda, and S.S. Sugama (Nikkei Disease Prevention Center, São Paulo); C.K. Taniguchi and J.A. Marques (Departamento de Ginecologia, Hospital Pérola Byington, São Paulo); M.R. Eichhorn (Departamento de Nutrição, Hospital Pérola Byington, São Paulo); H. Iyeyasu, M.S. Maciel, S.M.T. Carvalho, J.B.D. Collins, and C.E.M. Fontes (Departamento de Mastologia, Hospital A.C. Camargo, São Paulo); L.P. Kowalski and J.M.F. Toyota (Departamento de Cirurgia de Cabeça e Pescoço e Otorrinolaringologia, A.C. Camargo Hospital, São Paulo); E.M. Barbosa (Departamento de Mastologia, Instituto Brazileiro de Controle ao Câncer, São Paulo); O. Ferraro (Departamento de Mastologia, Hospital do Servidor Público Estadual Francisco Morato de Oliveira, São Paulo); R. Anzai (Departamento de Mastologia, Hospital Santa Cruz); E.H. Hotta and D.A. Petti (Instituto de Ginecologia e Mastologia, Hospital Beneficencia Portuguesa); and S. Mendes (Instituto Brazileiro de Mastologia e Ginecologia, Hospital Beneficencia Portuguesa).

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