Associations between estrogen receptor genetic polymorphisms, smoking status, and prostate cancer risk: a case–control study in Japanese men
Environ Health Prev Med
Associations between estrogen receptor genetic polymorphisms, smoking status, and prostate cancer risk: a case-control study in Japanese men
Xi Lu 0 1 2
Yuko Yamano 0 1 2
Hiroyuki Takahashi 0 1 2
Masahide Koda 0 1 2
Yuki Fujiwara 0 1 2
Aya Hisada 0 1 2
Wataru Miyazaki 0 1 2
Takahiko Katoh 0 1 2
0 Department of Hygiene and Preventive Medicine, Showa University School of Medicine , Tokyo , Japan
1 Department of Public Health, Faculty of Life Sciences, Kumamoto University , 1-1-1 Honjo, Chuo-Ku, Kumamoto 860-8556 , Japan
2 Department of Pathology, The Jikei University School of Medicine , Tokyo , Japan
Objective Prostate cancer (PCa) is one of the major causes of death among men. Our study investigated the association of ESR1 and ESR2 genotypes with susceptibility to PCa in relation to smoking status in Japanese. Method A case-control study was performed with 750 Japanese prostate cancer patients and 870 healthy controls. After age-matching in case-controls, 352 controls and 352 cases were enrolled in this study. By using logistic regression analysis, the different genotypes from ESR1 and ESR2 were analyzed according to case/control status. Result ESR2 rs4986938 AG and AG ? AA genotypes were associated with significantly decreased risk of PCa (AG: OR = 0.68, 95 % CI 0.47-0.97, P \ 0.05 and AG ? AA: OR = 0.67, 95 % CI 0.47-0.94, P \ 0.05). However, there was no significant association between ESR1 rs2234693 and PCa risk. When patients were grouped according to smoking status, the ESR2 rs1256049 AA genotype (OR = 0.48, 95 % CI 0.25-0.95, P \ 0.05) and ESR2 rs4986938 AG ? AA genotype (OR = 0.64, 95 % CI 0.41-1.00, P \ 0.05) showed significantly decreased PCa risk in the ever-smoker group. Conclusion Our results suggest that the estrogen receptor ESR2 has a very important function to predict PCa and that
Prostate cancer; Estrogen receptor; Polymorphism; Genetic association studies; Smoke
different SNPs have different predictive values. Smoking
may influence estrogenic activity and may influence PCa
together with the estrogen receptor.
Prostate cancer (PCa), also known as carcinoma of the
prostate, is the development of cancer in the prostate, a
gland in the male reproductive system [
]. The number of
clinical cases has been increasing annually. In Japan, PCa
deaths accounted for 3.5–4 % of the total cancer deaths and
it is predicted that mortality will increase to 10 % of the
total cancer deaths, with more than 80,000 males suffering
from PCa in 2020 [
One of the hypothesized risk factors of PCa is older age.
Almost all prostate cancers are detected in men aged
[50 years; asymptomatic patients are usually identified
through screening programs and symptomatic individuals
present at outpatient clinics. About 70 % of deaths due to
prostate cancer are observed in patients C75 years old in
the USA [
] and Japan [
Smoking is also a risk factor for PCa. An association with
smoking could have a hormonal basis; male smokers were
found to have elevated levels of circulating androsterone and
testosterone, which may increase PCa risk or contribute to
cancer progression [
]. Twenty-four prospective cohort
studies showed increased risk of incident PCa for smokers
], though five studies found no positive association
between smoking and PCa incidence in Japan [
Although the roles of estrogen in the pathogenesis of
PCa remains poorly understood, estrogens have been
implicated in the stimulation of aberrant prostate growth,
control of cell growth, and programmed cell death in PCa
]. Despite the controversy surrounding the exact
role of estrogens on the prostate epithelium, estrogens have
been used in the treatment of PCa because of their
growthinhibitory effects . Sex steroids (estrogen and
progesterone) could play a key pathophysiological role in the
development PCa. The effects of estrogen are mediated by
two estrogen receptors (ERs), the ER-1, and the ER-2 [
These belong to a superfamily of nuclear receptors that are
ligand-dependent transactivators [
]. The ESR1 gene is
located on chromosome 6q25.1 and the ESR2 gene is
located on chromosome 14q23.1. The ESR1 gene intron 1
contains a single-nucleotide polymorphisms (SNPs) named
the PvuII (T/C) (rs2234693) [
]. The 50 and 30 regions of
the ESR2 gene have two common SNPs: a silent 1082 G/A
transition in exon 5 (RsaI, rs1256049), and G/A exchange at
nucleotide 1730 in the 30 untranslated region in exon 8
(AluI, rs4986938) [
]. Both receptors have been detected
in human prostate normal mucosa [
]. The ESR1 gene
is expressed in the stroma and at low levels in basal
epithelial cells of normal prostate [
]; the ESR2 gene is
highly expressed in the prostate epithelium, signifying a
direct effect of estrogen on the prostate [
]. Imamov and
Cheng’s studies have shown antiproliferative and
anti-invasion properties of estrogen acting through ERS2 [
The correlation between PCa and estrogen receptors ESR1
and ESR2 according to smoking status is unclear.
We studied a common SNP (rs2234693) in the ESR1
gene and two in the ESR2 gene (rs1256049 and
rs4986938). To the best of our knowledge, no study has
reported the association of ESR1 and ESR2 genotypes with
susceptibility to PCa in relation to age and smoking status
in Japanese men.
Materials and methods
DNA samples were obtained from participants at Jikei
University Hospital (Tokyo, Japan) and Mitsui Memorial
Hospital (Tokyo, Japan), the ethical approval and informed
consent of this study were obtained from all participants.
Case subjects in this study were 750 men of Japanese
ancestry who were diagnosed with histologically confirmed
PCa at Jikei University Hospital from April 1, 2005 to
December 31, 2006. Men of Japanese ancestry who were
undergoing health screening at Mitsui Memorial Hospital
during the same period were asked to participate as control
subjects. Although a total of 870 subjects were included,
due to the fact that age can have a great impact on prostate
cancer, we selected an age-adjusted control group using the
case group’s average age. Thus, we enrolled 352 controls
and 352 cases in this study. All subjects were classified into
two groups according to smoking status by self-report: the
‘‘never’’ group composed of non-smokers and the ‘‘ever’’
group composed of both current smokers and ex-smokers.
The blood samples were collected during the protocol
period before each procedure was performed. We stored
buffy coats immediately after blood collection at -80 C
until we isolated DNA for genotyping of all case patients
and control subjects. The Ethic Review boards at both
Miyazaki University and Kumamoto University approved
this study on April 1, 2005 (approval number 180) and
April 26, 2012 (approval number 209). All participants
were given an explanation of the nature of this study.
Common SNPs in ESR1 (rs2234693: T[C) and ESR2
(rs4986938: G[A, rs1256049: G[A) genes, previously
associated with alteration in receptor expression were
selected for the purpose of the current study. Pre-validated
allelic discrimination TaqMan real-time PCR assays
(Applied Biosystems, USA) were used for detection of the
respective SNPs in ESR1 and ESR2 genes. The reaction
solution (9 lL) was placed into each well of a 48-well
reaction plate (the remainder of the reaction solution was
used to prevent experimental errors), and 1 lL of DNA
sample or water control was added to each tube. DNA
samples with homozygous ESR1 mutant T/T and ESR2
mutant G/G were used as control samples in each array
The Student’s t test and Chi-squared test were used to
compare genotype and smoking status between the patients
and control subjects. The Pearson’s Chi-squared test was
also used for evaluating the probability of
Hardy–Weinberg equilibrium. Relative associations between the two
groups were assessed by calculating odds ratios (ORs) from
contingency tables. In logistic regression analysis, the OR
with corresponding 95 % confidence intervals (CI) were
calculated. All statistical tests were based on two-tailed
probability and P values of \0.05 were considered to be
significant. Statistical analyses were carried out using SPSS
Ver. 20 (SPSS Inc., Chicago, IL).
The general characteristics of the cases and the controls are
shown in Table 1. This study involved 352 patients with
pathologically confirmed PCa and 352 controls, aged from
Controls N = 352 (%)
Case N = 352 (%)
50 to 79. The average ages of the case and control groups
were both 65.0 years. No significant differences were
observed between the cases and controls in terms of
smoking status and pack-year status.
The genotype frequency distributions are shown in
Table 2; SNP genotyping completion rates were 100 %.
Hardy–Weinberg equilibrium was confirmed for the ESR1
rs2234693 genotype (P = 0.95), ESR2 rs1256049
genotype (P = 0.41), and ESR2 rs4986938 genotype
(P = 0.99) in the controls. For the ESR1 rs2234693 SNP,
there were no statistically significant differences in risk for
PCa between the case and control groups according to
genotype. On the other hand, for the ESR2 rs1256049 SNP,
the AA carriers showed decrease risk of PCa (OR = 0.58,
95 % CI 0.34–1.00, P \ 0.05). For the ESR2 rs4986938
SNP, AG and AG ? GG genotypes also showed decrease
risk of PCa (OR = 0.68, 95 % CI 0.47–0.97, P \ 0.05 and
OR = 0.67, 95 % CI 0.47–0.94, P \ 0.05, respectively).
In order to check the PCa risk associated with genotypes
in combination with smoking status, we classified all
individuals in this study group as either never smokers or
ever-smokers (Table 3). In the never-smoker group, there
was no significant association between different genotypes
and risk of PCa. In the ever-smoker group, the ESR2
rs1256049 AA genotype (OR = 0.48, 95 % CI 0.25–0.95,
P \ 0.05) and the ESR2 rs4986938 AG ? AA genotype
In the last decade, a large number of studies have attempted
to unravel the genetic basis of PCa. Evidence points to
genetic factors, such as variations in genes involved in
hormone pathways, as the key players in PCa development
]. In this study, we investigated the associations
between genetic polymorphisms of the ESR1 and ESR2
estrogen receptors, smoking status, and PCa risk in a case–
control study in Japanese men.
Comparison of ESR1 rs2234693 genotype frequencies
between the PCa patient and control groups did not show
statistically significant differences. In previous studies,
however, further subgroup analyses based on country
suggested that ESR1 rs2234693 (C/T) may be associated
with increased risk of PCa among Indian and Iranian
populations (ORs ranging from 1.93 to 4.46) [
Another study failed to confirm these findings among
American or Japanese populations  and our findings are
consistent with this study.
Regarding the ESR2 rs1256049, those with the AA
genotype had significantly reduced risk for PCa. Chen et al.
reported that the ESR2 rs1256049 was not associated with
PCa risk in either all subjects (Prostate Cancer Cohort
Consortium) or only Caucasians [
], but to our
knowledge, there were no data regarding the ESR2 rs1256049
SNP and PCa in Japanese men. Another finding of this
study was a significant association between ESR2
rs4986938 and PCa; men with ESR2 rs4986938 AG and
AG ? AA genotypes were less likely to have PCa.
Although no significant association was observed for the
AA genotype, there was a trend towards decreased chance
of having PCa, which is consistent with some previous
Bergner et al. tested the ESR1 and ESR2 genes in two
human PCa cell lines. In their study, polymorphisms were
found in both ESR1 and ESR2 genes and these may
contribute to the genetic factors that influence the risk for
developing PCa [
]. In another study, the author reported
that high intake of phytoestrogens substantially reduced
PCa risk among men with specific polymorphic variation in
the promoter region of the ESR2 gene [
]. In a large
population-based case–control study (1415 cases and 801
controls), 28 SNPs spanning the entire ESR2 gene were
]. There was a statistically significant
difference in allele frequency between cases and controls only
for one of the typed htSNPs (rs2987983). Different ESR1
and ESR2 genotypes may exert their effects on PCa via
different serum levels of reproductive hormones. Both
androgens and estrogens play significant roles in the
prostate. Specifically, it is a balance between their actions
that is critically important in maintaining normal prostate
]. All of these experimental studies and our
results suggest that the ESR1 and ESR2 genetic
polymorphisms examined in this study have functional significance,
and thus modulate PCa risk.
In terms of smoking status, there was no significant
difference observed between the cases and the controls in
terms of smoking status (P = 0.37) and we also found no
significant association between different genotypes in the
never-smoker group. In the ever-smoker group, however,
the ESR2 rs1256049 AA genotype and the ESR2
rs4986938 AG ? AA genotype carriers showed
significantly decreased PCa risk. Our findings agree with a study
by Takamura et al. [
], which demonstrated that smoking
may influence estrogenic activity and that these two factors
may influence PCa together. Dai et al. found an association
with smoking could also have a hormonal basis. Male
smokers were found to have elevated levels of circulating
androsterone and testosterone, which may increase PCa
risk or contribute to cancer progression [
]; however, they
did not report on the possible interaction between smoking
and ESR genotype.
In interpreting the results of the current study, some
limitations need to be addressed. First, the sample size was
relatively small and may not provide sufficient power to
estimate the association between gene polymorphisms and
prostate cancer risk. Second, we did not account for the
potential effects of diet in our study; for instance, some
studies have reported that high intake of isoflavone, which
is mainly found in soybeans and soy products, may reduce
the risk of PCa. Third, all subjects were classified into
either the ‘‘never’’ group, composed of non-smokers, or the
‘‘ever’’ group, composed of both current smokers and
exsmokers; thus, we could not analyze the impact of smoking
burden in terms of either the number of cigarettes smoked
or pack-years. In spite of these limitations, our study still
had some merits and values. To the best of our knowledge,
this is the first study to analyze the relationship between
estrogen receptor gene polymorphisms, smoking status,
and PCa risk in Japanese men.
In summary, our results demonstrated that the ESR2
rs1256049 and rs4986938 genotypes, but not ESR1
rs2234693 genotype, had significant associations with risk
for PCa in Japanese men. Our findings suggest that estrogen
receptor genotype may be an independent risk factor for
PCa and may also play a modulatory role in the metabolism
of tobacco smoke components. The genotypes of estrogen
receptor SNPs may differentially predict PCa risk.
Conflict of interests The authors have declared that no competing
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