N-acetyltransferase-2 gene polymorphism as a possible biomarker for prostate cancer in Japanese men

Authors


Takashi Hamasaki md phd, Department of Urology, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan. Email: t-hama@fukushima-rosai.jp

Abstract

Background: The purpose of this study was to determine the frequency of a polymorphism of the candidate metabolic enzyme N-acetyltransferase-2 (NAT2) in Japanese prostate cancer patients and Japanese non-cancer controls, in order to determine if an association exists between NAT2 genotype and the occurrence, clinical stage and grade of prostate cancer.

Methods: In the present case-control study, 111 patients with prostate cancer and 152 controls were genotyped for the NAT2 polymorphism using the polymerase chain reaction-based restriction fragment length polymorphism method. The NAT2 genotypes (slow or rapid acetylator genotype) were determined by the combination of three known NAT2 mutant alleles (M1, M2, M3) and the wild-type allele.

Results: The NAT2 slow acetylator genotype was statistically higher among prostate cancer patients (17.1%) compared with controls (8.6%) (Odds ration (OR) = 2.21; 95% confidence interval (CI), 1.04–4.69; P = 0.0289). In addition, there was a statistically increased risk of prostate cancer among smokers with the NAT2 slow genotype (OR = 3.78: 95% CI, 1.48–9.66; P = 0.0041). Furthermore, the NAT2 slow acetylator genotype was significantly higher among prostate cancer patients with locally advanced and metastatic disease (22.7%) compared with controls (8.6%) (OR = 3.14; 95% CI, 1.40–7.06; P = 0.0051). Lastly, the NAT2 slow acetylator genotype was significantly higher among prostate cancer patients with high-grade tumors (31.4%) compared with controls (8.6%) (OR = 4.90; 95% CI, 1.97–12.20; P = 0.0010).

Conclusion: These data demonstrate that the NAT2 slow acetylator genotype plays an important role in determining the risk of developing prostate cancer in Japanese men and is also associated with more clinically advanced and pathologically aggressive disease. Furthermore, a possible interaction between the NAT2 slow acetylator genotype and smoking status was suggested.

Introduction

Prostate cancer is the most common cancer affecting men and is a major medical problem. Several factors are associated with an increased risk for prostate cancer, including ethnicity, environmental factors, age, family history and lifestyle. African-American men have the highest rate of prostate cancer in the world (incidence approximately 149 per 100 000 person-year). Caucasians living in the USA have an intermediate incidence (107 per 100 000 person-year), whereas Japanese men have the lowest incidence (39 per 100 000 person-year).1,2 However, the incidence of prostate cancer in Japanese immigrants to the USA is markedly higher than the incidence in Japanese living in Japan, although the rate on the west coast of the USA is still significantly less than that among Caucasians.1,2 These epidemiologic studies suggest that the development of prostate cancer is influenced by environmental factors, includ-ing UV irradiation,3 smoking habit4 and diet, with the most important environmental factor being dietary fat.5,6 However, recent molecular epidemiologic studies have demonstrated an association of steroid hormones with prostate cancer. Prostate cancer cell division is influenced by the steroid hormones, testosterone and vitamin D. These two steroid hormones exert their biological activity by binding to their respective receptors; the androgen receptor and vitamin D receptor. Recent studies have demonstrated an association of androgen receptor and vitamin D receptor gene polymorphisms with the development of prostate cancer.7–9 However, the majority of prostate cancers cannot be explained by the biological actions of single steroid hormones.

Many chemical and dietary carcinogens, such as nitrosamines and arylamine amines derived from dietary fat, as well as tobacco smoke products, require bioactivation or inactivation by enzymes. This suggests that polymorphisms of genes encoding metabolic enzymes may represent potential risk factors.10 Recent molecular epidemiologic studies have analyzed the relationship between various metabolic enzymes, for example N-acetyltransferase (NAT), cytochrome P450 (CYP) and glutathione S-transferase (GST), and prostate cancer to determine if they represent possible biomarkers.5,11,12 The genetically variable NAT, CYP and GST enzymes metabolize drugs, carcinogens and natural products and are therefore of interest as candidate genes for cancer susceptibility.10,13 NAT metabolizes the carcinogen arylamine. In humans, hereditary differences in N-acetylation activity have led to a phenotypic classification of individuals as rapid or slow acetylators, with resultant differing rates of metabolism of arylamines. Recent epidemiological studies have demonstrated that the NAT slow acetylator genotype is associated with an increased risk of uroepithelial cancer (transitional cell carcinoma)14,15 oral cancer (squamous cell carcinoma),16 and colorectal cancer (adenocarcinoma).17

It is known that humans express two forms of NAT: NAT1 and NAT2. Recent studies have not supported a relationship between NAT1 genotype and N-acetylation activity.18,19 Furthermore, NAT2 has been reported to exhibit a polymorphism,15,20 resulting in the potential expression of four mutant alleles (M1, M2, M3 and M4), which can be identified by restriction fragment length polymorphism (RFLP) analysis following NAT2 amplification by the polymerase chain reaction (PCR). NAT2 activity is predicted from the detected combination of these NAT2 alleles. The presence of at least one wild-type (WT) allele results in a rapid acetylator genotype, whereas the carriage of two mutant alleles results in a slow acetylator genotype.15,21–23 There are significant interethnic differences in certain NAT2 allele frequencies.15,21,22

In this study, we examined the NAT2 polymorphism and tested the hypothesis that the NAT2 slow acetylator genotype is associated with an increased risk of prostate cancer in Japanese men compared with the NAT2 rapid acetylator genotype. We also examined the associations between the NAT2 polymorphisms and the clinical stage and pathological grade of prostate cancer.

Methods

Subjects

We studied a total of 111 patients with prostate cancer and 173 benign male urology clinic patients. Blood samples were obtained between March 1996 and September 2001 from both patient groups and patient data were obtained from the medical records of patients. This study was approved by the ethics committee of medical care and research of the University of Occupational and Environmental Health (UOEH) under the guidelines of the Ministry of Education, Culture, Sports, Science and Technology. All patients gave their informed consent to participate in this study. The diagnosis of prostate cancer was confirmed histologically in the study group. The 173 patients consisted of male urology clinic patients with benign retroperitoneal and genitourinary diseases. Physical (digital rectal examination), serological (prostate-specific antigen) and radiological examinations were performed in all 173 patients in order to exclude the possibility of prostate cancer and other malignant disease. Among 173 patients, a total of 21 patients with an elevated serum prostate-specific antigen level (≥ 4.0 ng/mL by the Tandem-R assay; 16 patients) and with a palpable nodule by digital rectal examination (five patients) underwent a transrectal needle prostate biopsy and excluded prostate cancer. The number of biopsy-negative cases was not so high, however, 21 patients were excluded from controls because of the possibility of subclinical prostate cancer. Lastly, in the present case-control study, a total of 152 benign male urology clinic patients as controls and 111 patients with prostate cancer were analyzed. There was no statistical difference in the age at diagnosis between the groups (68.5 ± 5.3 vs 66.9 ± 6.7 for prostate cancer patients and controls, respectively; P = 0.21, unpaired Student's t-test).

Genotyping

The NAT genotypes were determined using the PCR-RFLP method as described previously by Inatomi et al.15 Genomic DNA was isolated from peripheral leukocytes by proteinase K digestion and phenol/chloroform extraction. An 815-bp DNA fragment was generated by PCR. The primer sequences were 5′-ctt ctc ctg cag gtg acc at-3′ (forward) and 5′-agc atg aat cac tct gct tc-3′ (reverse). Genomic DNA (0.5 µg) was added to a PCR mix composed of 50 pmol of each primer, 200 µmol dNTPs, 2.5 units Taq polymerase (Ampli Taq Gold; Perkin-Elmer, Ltd, Norwalk, USA) and PCR buffer composed of 10 mol/mL Tris-HCl (pH 8.3), 50 mol/mL KCl and 2.5 mol/mL MgCl2 in a volume of 50 µL. We used a PC-800 Programmable Temp Control System (ASTEC, Fukuoka, Japan) with a preincubation set up comprising 95°C for 9 min The 35 cycles were performed at denaturation (94°C, 1 min), annealing (57°C, 1 min) and extension (72°C, 1 min). Following PCR, 10 µL of the PCR products were removed and digested with three separate restriction enzymes including KpnI (M1 allele) at 37°C for 3 h, TaqI (M2 allele) at 65°C for 3 h and BamHI (M3 allele) at 37°C for 3 h (restriction enzymes from Takara Shuzou, Kyoto, Japan). Products were then run on 4% nusieve agarose gels.15

If the allele could not be identified as either M1, M2 or M3 after digestion with KpnI, TaqI and BamHI, then the remaining allele(s) were assigned as WT since the WT polymorphic allele possesses all of these restriction sites. The genotype of each individual was classified as WT/WT, WT/M1, WT/M2, WT/M3, M1/M1, M1/M2, M1/M3, M2/M2, M2/M3 and M3/M3.15 The M4 allele was not determined in this study because this allele was reported to be present only in populations of African origin.22 The NAT rapid acetylator genotypes are WT allele homo/heterozygotes (WT/WT, WT/M1, WT/M2 and WT/M3), whereas the slow acetylator genotypes are those with two mutant alleles (M1/M1, M1/M2, M1/M3, M2/M2, M2/M3 and M3/M3).15

Clinical staging and pathological grading in prostate cancer patients

Clinical staging and histological study of patients with prostate cancer at diagnosis were evaluated according to the General Rule for Clinical and Pathological Studies on Prostate Cancer in 2001.24

Statistical analysis

The relative associations between prostate cancer patients and controls were assessed by calculating the odds ratios (OR) from contingency tables and 95% confidence intervals (CI) were calculated. The variables were analyzed with the χ2 test. Statistical significance was defined as a P value of less than 0.05.

Results

NAT2 allele frequency of Japanese controls and other ethnic populations (%)

The NAT2 allele frequencies of the control group in this study and previously reported ethnic populations are shown in Table 1. There are significant interethnic differences in the NAT2 allele frequencies. The M1 allele is the most common of the NAT2 variants in the Caucasian-American population, but is rare in Japanese, whereas the M4 allele is only found in the African-American population.15,22,25 The NAT2 allele frequency in our control group was not significantly different from another Japanese population (Inatomi et al.: χ2 = 2.64, P = 0.4493, 3 df;15 Deguchi et al.: χ2 = 1.40, P = 0.7065, 3 df25).

Table 1.  The N-acetyltransferase-2 (NAT2) allele frequency of Japanese controls and other ethnic populations (%)
 Wild
type
M1M2M3M4
  1. The overall allele frequencies are not significantly different from our controls and Japanese (Inatomi et al.: χ2 = 2.64, P = 0.4493, 3 df;15 Deguchi et al.: χ2 = 1.40, P = 0.7065, 3 df25). †NAT2 allele frequency of controls from our control group.

Controls (n = 173)66.1 1.019.713.2
Japanese15 (n = 146)70.6 0.722.9 5.8
Japanese25 (n = 145)70.0 1.021.0 8.0
Caucasian-American22
 (n = 372)
25.045.028.0 2.00.0
African-American22
 (n = 128)
36.030.022.0 2.09.0

Frequency of the NAT2 genotype in prostate cancer patients and controls

The first major finding of this study is that the NAT slow acetylator genotype is associated with development of prostate cancer. The frequency of the NAT slow acetylator genotype was statistically higher among prostate cancer patients (17.1%) compared with controls (8.6%) and exhibited a 2.2-fold increased risk of prostate cancer (OR = 2.21; 95% CI, 1.04–4.69; P = 0.0289) (Table 2).

Table 2.  Frequency of the N-acetyltransferase-2 (NAT2) genotypes in prostate cancer patients and controls
 NAT2 genotype (%)Risk
Odds ratio (95% confidence interval)
RapidSlow
  1. Rapid genotypes, NAT2 wild-type homozygote or wild/mutant heterozygote; slow genotype, NAT2 mutant type homozygote.

Controls (n = 152)139 (91.4)13 (8.6)1
Prostate cancer (n = 111) 92 (82.9)19 (17.1)2.21 (1.04–4.69)
P = 0.0289

Risk of prostate cancer according to the combination of NAT2 genotype and smoking status

We analyzed the effect of combining NAT2 genotype and smoking status (past and current smokers) on the risk of developing prostate cancer. Using non-smokers with the NAT2 rapid acetylator genotype as the reference group, there was no apparent increased risk for individuals who were NAT2 rapid acetylator genotype/smokers (OR = 1.51; 95% CI, 0.89–2.57; P = 0.0838) or NAT2 slow acetylator genotype/non-smokers (OR = 1.14; 95% CI, 0.26–5.02; P = 0.5717). However, smokers with the slow acetylator genotype exhibited a statistically increased risk of prostate cancer (OR = 3.78; 95% CI, 1.48–9.66; P = 0.0041) (Table 3). These results suggest a possible interaction between the NAT2 slow acetylator genotype and smoking status.

Table 3.  Risk of prostate cancer according to the combination of N-acetyltransferase-2 (NAT2) genotype and smoking status
 Risk/combination of NAT2 genotype and smoking status
Rapid, non-smokerRapid, smokerSlow, non-smokerSlow, smoker
  1. The NAT2 rapid acetylator genotypes and non-smokers combination was used as a reference group to calculate the odds ratio and 95% confidence interval of another combination of cases.

Odds ratio (95% confidence interval)11.51 (0.89–2.57)1.14 (0.26–5.02)3.78 (1.48–9.66)
P-value 0.08380.57170.0041
No. cases/no. of controls37/7055/693/516/8

Frequency of NAT2 genotypes of prostate cancer patients categorized by stage of disease

In order to analyze the risk factors associated with advanced and lethal prostate cancer, we divided the clinical stages into two groups; the organ-confined group (intraprostate disease: T1a-c/T2a-b, N0, M0; 45 patients) and the locally advanced or metastatic group (extra-prostate disease: T3a-b/T4/N1/M1a-c; 66 patients). In patients with prostate cancer, the NAT2 slow acetylator genotype was significantly higher in the locally advanced or metastatic group (22.7%) compared with controls (8.6%) (OR = 3.14; 95% CI, 1.40–7.06; P = 0.0051). However, there was no statistically significant difference in the distribution of the NAT2 slow acetylator genotype between the organ-confined group (8.9%) and controls (OR = 1.04; 95% CI, 0.32–3.37; P = 0.5735) (Table 4).

Table 4.  Frequency of N-acetyltransferase-2 (NAT2) genotypes of prostate cancer patients categorized by stage of disease
 NAT2 genotype (%)Risk
Odds ratio (95% confidence interval)
RapidSlow
  1. TNM classification: T, tumor extension (T1–T4); N, lymph node metastasis (N0/N1); M, distant metastasis (M0/M1). †Organ-confined group (intra-prostate disease); ‡Locally advanced or metastatic group (extra-prostate disease).

Controls (n = 152)139 (91.4)13 (8.6)1
Prostate cancer (n = 111)
 T1a-c/T2a-b N0 M0 (n = 45) 41 (91.1) 4 (8.9)1.04 (0.32–3.37) P = 0.5735
 T3a-b/T4/N1/M1a-c (n = 66) 51 (77.3)15 (22.7)3.14 (1.40–7.06) P = 0.0051

Frequency of NAT2 genotypes of prostate cancer patients categorized by pathological grade of tumor

We also divided the pathological findings into two groups; the low and intermediate-grade group (Well and Mod: 76 patients) and the high-grade group (Por: 35 patients). The NAT2 slow acetylator genotype was significantly higher among the high-grade group (31.4%) compared with controls (8.6%) (OR = 4.90; 95% CI, 1.97–12.20; P = 0.0010). However, there was no statistical difference in the distribution of the NAT2 slow acetylator genotype between the low and intermediate-grade group (10.5%) and controls (OR = 1.26; 95% CI, 0.50–3.18; P = 0.3962) (Table 5). Therefore, the second major finding of this study is that the NAT2 slow acetylator genotype is associated with a more advanced clinical stage and pathological grade of prostate cancer, which is associated with a higher mortality rate.

Table 5.  Frequency of N-acetyltransferase-2 (NAT2) genotypes of prostate cancer patients categorized by pathological grade of tumor
 NAT2 genotype (%)Risk
Odds ratio (95% confidence interval)
RapidSlow
  1. Pathological finding is defined as †low and intermediate-grade group (well and moderately differentiated adenocarcinoma); ‡high-grade group (poorly differentiated adrenocarcinoma).

Controls (n = 152)139 (91.4)13 (8.6)1
Prostate cancer (n = 111)
 Wel and Mod (n = 76) 68 (89.5) 8 (10.5)1.26 (0.50–3.18)
P = 0.3962
 Por (n = 35) 24 (68.6)11 (31.4)4.90 (1.97–12.20)
 = 0.0010

Discussion

Prostate cancer exhibits a genetic component. In addition to genetic factors, sporadic prostate cancer appears to be dependent on the interplay between endogenous steroid hormones and environmental influences, particularly dietary fat.5,6 Most molecular epidemiological studies of the relationships between gene polymorphisms and prostate cancer have analyzed steroid hormone receptor gene polymorphisms (androgen receptor and vitamin D receptor).7–9 However, a recent study reported that diet can alter steroid hormone profile and modify prostate cancer risk.26,27 It has been suspected that heterocyclic amines and polycyclic hydrocarbons, which are produced by cooking meat at a high temperature, act as carcinogens,28 and it is therefore of considerable interest that food mutagens can induce prostate cancer in rats.29 Since many chemical and dietary carcinogens, such as nitrosamines and arylamine amines derived from dietary fat and tobacco products, require bioactivation or inactivation by enzymes, it has been proposed that metabolic enzyme gene polymorphisms may represent potential risk factors.5,10

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is a heterocyclic amine identified in human dietary fat and tobacco smoke. PhlP is bioactivated by CYP enzymes to N-hydroxylated metabolites that undergo further activation by conjugating enzymes, including the N-acetyltransferase, NAT1 and NAT2.30 Leff et al. reported that PhlP produced prostate cancers in a transgenic mouse model.30 Furthermore, they demonstrated prostate-specific NAT2 expression in PhlP-induced prostate cancer in the mouse, but absent NAT1 expression. Wang et al. demonstrated NAT expression and DNA binding of PhlP in human prostate epithelium.31 These results suggest that PhlP is a potential carcinogen for prostate cancer, since dietary fat and tobacco smoke, which contain heterocyclic amines, have been associated with prostate cancer.

The metabolic enzyme NAT2 plays a significant role in the development of uroepithelial cancer in Japanese populations.14,15 However, differences in the frequency of NAT2 slow acetylator genotype between prostate cancer patients and controls have not previously been published. We tested the hypothesis that the NAT2 slow acetylator genotype would be associated with an increased risk of prostate cancer as well as uroepithelial cancer in Japanese populations. In this study, the NAT2 slow acetylator genotype was significantly higher among prostate cancer patients compared with controls (OR = 2.21). Moreover, the occurrence of the NAT2 slow acetylator genotype was significantly associated with a more advanced stage of disease (T3/T4/N1/M1; OR = 3.14) and a higher pathological grade of tumor (por; OR = 4.90). Wadelius et al. analyzed NAT2 acetylator genotype frequency in prostate cancer patients and controls in Swedish and Danish populations and reported the absence of an association between NAT2 slow acetylator genotype and the development of prostate cancer.5 How can we explain this discrepancy? The NAT2 allele frequencies in our control group are comparable with those previously published among Japanese populations.15,25 However, there are significant interethnic differences in the NAT2 allele frequencies. In Caucasian populations, the NAT2 M1 allele is the most frequent allele (40–50%), in contrast to Asian populations, which exhibit a very low frequency (0–5%).15,22,25,32,33 Wadelius et al. reported that the frequency of the NAT2 slow acetylator genotype was almost the same in prostate cancer patients (61%) and controls (61.7%) in the Swedish and Danish populations.5 In contrast to our study, the NAT2 slow acetylator genotype was significantly less frequent in prostate cancer patients and controls, occurring with a frequency of 17.1% and 8.6%, respectively. The difference of NAT2 genotype frequencies may well be secondary to differences in the populations under study since the prostate cancer patients and controls in the previous study5 were of different ethnicity (Swedish and Danish populations) to our study, which was performed in a Japanese population.

Currently, there is a scarcity of data regarding the actual associations of NAT2 acetylator genotype with tobacco smoking in prostate cancer patients. The combination of the NAT2 slow acetylator genotype and tobacco smoking has previously been implicated as a risk factor for the development of uroepithelial cancer.14,15 Smokers with the NAT2 slow acetylator genotype exhibited a statistically increased risk of prostate cancer (OR = 3.78) when the NAT2 rapid genotypes/non-smokers were used as the reference group. This suggested a possible interaction between NAT2 slow acetylator genotype and smoking status.

Recent molecular epidemiologic studies have analyzed the relationship between the metabolic enzymes, CYP and GST, with prostate cancer. Regarding CYP polymorphisms, Murata et al. demonstrated that the CYP1A1 mutant genotype (valine/valine), but not CYP1A2 and CYP2E1 polymorphisms, was associated with an increased risk of prostate cancer (OR = 2.4) in Japanese populations.11 Wadelius et al. found no association between prostate cancer and the CYP2D6 and CYP2C19 polymorphisms in Swedish and Danish populations.5 Concerning GST polymorphisms, Rebbeck et al. demonstrated that the GSTT1 mutant genotype (GSTT1-0), but not the GSTM1 polymorphism, has an increased risk of prostate cancer (OR = 1.83) in Caucasian, African and other racial populations living in the USA.34 However, Kote-Jarai et al. found no significant difference in the frequency of GST mutant genotypes (GSTT1 and GSTM1) between prostate cancer patients and controls in Caucasian populations living in the UK.12 These results suggest that the associations of metabolic enzyme gene polymorphisms with prostate cancer may differ according to the metabolic enzyme (NAT, CYP or GST) and ethnic population under study. Further work is required in this field, including large-scale analysis of different metabolic enzyme gene polymorphisms in different ethnic populations.

In conclusion, this study indicates that the NAT2 acetylator genotype exhibits a significant association with the risk of developing prostate cancer. Furthermore, NAT2 acetylator genotypes play an important role in determining the clinical stage and pathological grade of prostate cancer in Japanese. The NAT2 gene polymorphism therefore represents a potential biomarker for prostate cancer.

Ancillary