Genetic Polymorphism of Cytochrome P450–1B1 and Risk of Breast Cancer¹

CYP1B1³has been identified recently as one of the most important enzymes catalyzing the formation of 4-hydroxyestradiol (1, 2), a catechol estrogen metabolite that retains significant estrogenic activity (1, 2). This estrogen metabolite can also undergo metabolic redox cycling to generate potentially mutagenic free radicals that may damage DNA and other cell structures (2, 3). In the hamster kidney tumor model, 4-hydroxyestradiol has been shown to be carcinogenic, whereas 2-hydroxyestrogens, another major group of catechol estrogen metabolites, do not induce any tumor (2, 3). Although estrogen 4-hydroxylation is a relatively minor pathway in the liver for the formation of catechol estrogens,significant estrogen 4-hydroxylase activity has been observed in several extrahepatic estrogen target tissues (2, 3, 4, 5),including human mammary epithelial cells (2, 3, 4, 5). The activity of CYP1B1 was found to be higher in breast cancer than its adjacent normal tissue (6), suggesting that this enzyme may be involved in the pathogenesis of breast cancer.

Catechol estrogens, including 4-hydroxyestradiol, are metabolically inactivated by catechol-O-methyltransferase (COMT; Ref. 3). Recently, a variant COMT allele that is associated with reduced enzyme activity has been linked to elevated risk of breast cancer in several epidemiological studies (7, 8, 9), including our study among Chinese women in Shanghai (9). This suggests further that 4-hydroxyestradiol, and thus its metabolic enzyme CYP1B1, may be involved in the etiology of breast cancer.

In addition to catalyzing the formation of 4-hydroxyestradiol, CYP1B1 has also been shown to be involved in the metabolic activation of certain environmental procarcinogens, including polycyclic aromatic hydrocarbons and heterocyclic aromatic amines (10, 11),potent mammary carcinogens in experimental animals (12, 13, 14). In some experiments, CYP1B1 has been shown to be even more active than CYP1A1 in the activation of several polycyclic aromatic hydrocarbons to genotoxic intermediates (11). The expression of the CYP1B1 gene in human breast epithelial cells suggests that this enzyme may play an important role in the in situ activation of both environmental carcinogens and endogenous estrogens.

The CYP1B1 gene is located in chromosome 2p21-p22 and contains three exons (15, 16, 17). The entire coding sequence of the genes, however, is contained in exons 2 and 3 (15, 16, 17), and exon 3 encodes the heme-binding region of the enzyme (18). A G-to-C transversion at exon 3 was reported to result in a valine (GTG) to leucine(CTG) substitution in codon 432 (17). This change creates an Eco57I restriction site, making detection of this polymorphism in large epidemiological studies cost efficient. Prompted by the discovery of this easily identifiable polymorphism and findings from recent laboratory studies showing an important role of CYP1B1 in the metabolic activation of estrogens and environmental mammary carcinogens, we evaluated the relation between CYP1B1 genotype and breast cancer risk in a subset of women who participated in the Shanghai Breast Cancer Study.

The Shanghai Breast Cancer Study is a population-based case-control study conducted among Chinese women in Shanghai, the largest city on the east coast of China with a population of over six million residents (19). This study was designed to recruit all eligible breast cancer cases who were 25 to 64 years of age and newly diagnosed with breast cancer during the period of August 1996 to March 1998, as well as a representative random sample of controls from the general population. All cases and controls were permanent residents of urban Shanghai who had no prior history of cancer and were alive at the time of interview. Through a rapid case-ascertainment system,supplemented by the population-based Shanghai Tumor Registry, 1602 eligible breast cancer cases were identified during the study period,and in-person interviews were completed for 1459 (91%) of them. The major reasons for nonparticipation were refusal (109 cases, 6.8%),death prior to interview (17 cases, 1.1%), and inability to locate (17 cases, 1.1%). Cancer diagnoses for all patients were confirmed by two senior study pathologists through the review of tumor slides.

Controls were randomly selected from the female general population and frequency-matched to cases by age (5-year intervals). The number of controls in each age-specific stratum was determined in advance according to the age distribution of the incident breast cases reported to the Shanghai Tumor Registry during 1990–1993. The Shanghai Resident Registry, which keeps registry cards for all adult residents in urban Shanghai, was used to randomly select controls. For each age-predetermined control, a registry card identifying a potential control of the same 5-year age group was randomly selected. Only the women who lived at the address during the study period were considered to be eligible for the study. In-person interviews were completed for 1556 (90.3%) of the 1724 eligible controls identified. Reasons for nonparticipation included refusal (166 controls, 9.6%) and death or a prior cancer diagnosis (2 controls, 0.1%).

A structured questionnaire was used to elicit detailed information on demographic factors, menstrual and reproductive history, hormone use,dietary habits, prior disease history, physical activity, tobacco and alcohol use, weight, and family history of cancer. All participants were also measured for their current weight, circumferences of the waist and hip, and sitting and standing heights. Among those who completed the in-person interviews, blood samples, 10 ml from each woman, were collected using EDTA or heparin Vacutainer tubes from 1193(82%) cases and 1310 (84%) controls. These samples were processed on the same day at the Shanghai Cancer Institute, and buffy coats (WBCs)for each participant were aliquoted into two 2-ml vials and stored−70°C.

Only a subset of subjects (200 cases and 200 controls) was included in the current study to better use the limited funds available for this substudy. With this sample size, we had 85% statistical power to detect a 2-fold or higher risk of breast cancer for factors with a 20%frequency (type 1 error, 0.05, two-sided). The subjects included in this study were primarily from those who were recruited in the early phase of the Shanghai Breast Cancer Study, because the genotyping work was initiated prior to the completion of the main study. Fourteen patients with a diagnosis of benign breast disease were accidentally included in the initial case group and were subsequently excluded from the analysis. Genomic DNA for cases and controls was extracted from buffy coat fractions using the method described previously (20). Briefly, frozen WBCs were thawed at the room temperature and then digested overnight at 55°C in 500 μl lysis buffer [50 mm Tris-HCl (pH 8.5), 1 mm EDTA,0.2% SDS, and 200 μg/ml proteinase K]. The digestion was precipitated directly with isopropanol, and the pellets were washed with 70% ethanol. The genomic DNA pellets (50–100 μg) were dissolved in 300–800 μl of Tris-EDTA buffer. Genomic DNA(10–100 ng) was used for each PCR reaction.

CYP1B1 genotypes were determined using a PCR-RFLP-based assay. According to the published sequence of the human CYP1B1 gene, we designed two primers (forward,5′-TCACTTGCTTTTCTCTCTCC; reverse, 5′-AATTTCAG-CTTGCCTCTTG) to amplify a 650-bp fragment of exon 3. The PCR reactions were performed on Perkin-Elmer GeneAmp System 9700 according to the manufacturer’s protocol. Specifically, these reactions were carried out in 50 μl of volume of 20 mm Tris-HCl (pH 8.4), 50 mm KCl, 1.0 mmMgCl₂, 0.2 mmdeoxynucleotide triphosphate, 1 unit of Taq polymerase, and 0.4 μm of each oligonucleotide primer. The reactions were heated to 94°C for 1 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s. At the end, the reactions were extended for 7 min at 72°C. Each PCR product was subjected to Eco57I digestion prior to electrophoresis. The DNA fragments were then separated using 3% 2:1 Nusiev/SeaKern agarose gel. The allele types were determined as follows: two fragments of 310- and 340-bp for the Leu allele(CTG), and a single 650-bp fragment for the Valallele (GTG). Representative genotypes are shown in Fig. 1. To monitor the quality of laboratory work, a second tube of 20 study samples was included in the genetic assays. Laboratory staff were blinded to the identification of the quality control samples. The genotypes for 20 women that were determined from the first and second tubes were in complete agreement.

ORs were used to measure the strength of the association between CYP1B1 genotypes and cancer risk (18). Stratified analyses were conducted to examine the potential confounding or modifying effect of other risk factors in the CYP1B1-breast cancer association. Unconditional logistic regression was used to control for potential confounders and derive adjusted ORs and 95% CIs. All statistical tests were based on two-tailed probability.

Table 1 compares the distributions of cases and controls by selected demographic characteristics and major risk factors for breast cancer. Breast cancer cases and controls were comparable in age and education level. With the exception of family history of breast cancer, positive associations were observed for all major risk factors for breast cancer, although the ORs were statistically significant only for a prior history of breast fibroadenoma, no leisure physical activity, and late age at first live birth. These three factors were included in these multivariate analyses as potential confounders. The lack of statistically significant associations with other breast cancer risk factors may be attributable to the small sample size of this substudy.

The Leu allele frequency was 53% in cases and 46% in controls (P = 0.06). Twenty-six % of cases versus 15% of controls were found to have the Leu/Leu genotype in our study population, resulting in an OR of 2.3 (95% CI, 1.2–4.3) after adjusting for potential confounding factors (Table 2). This positive association was more pronounced among postmenopausal women (OR, 3.1; 95% CI, 1.0–9.1) than premenopausal women (OR, 1.9;95% CI, 0.8–4.3). Heterozygosity for the Leu allele was not found to be associated with breast cancer risk.

To evaluate the consistency of the association between the CYP1B1 genotype and breast cancer risk, stratified analyses were performed (Table 3). Because women with the Val/Leu genotype had similar risk of breast cancer as those with the Val/Val genotype, these two groups of women were combined to enhance the stability of risk estimate. With the exception of the women with a prior history of breast fibroadenoma, women with the Leu//Leu genotype had a higher risk of breast cancer than the group of women with combined Val/Val and Val/Leu genotypes in all strata of life-style factors presented in Table 3, indicating that the Leu/Leu genotype may be a risk factor independent of these life-style factors.

Despite strong evidence from animal studies and in vitro experiments (1, 2, 3, 4, 5, 6), the potential role of CYP1B1 in the etiology of human breast cancer has not been investigated adequately. To our knowledge, only one study published to date has evaluated the association of CYP1B1 genetic polymorphisms with breast cancer risk (21). No statistically significant association between the CYP1B1 polymorphisms and breast cancer risk, however, was observed in that study, a hospital-based case-control study involving 164 Caucasian and 59 African-American women with breast cancer who were treated at the Vanderbilt University Medical Center over a 14-year period from 1982 to 1996. Controls for that study were patients treated in the same hospital for diseases other than cancer. Participation rates for cases and controls were not provided, and information related to most demographic and risk factors for breast cancer was not collected. Therefore, potential biases associated with hospital-based case-control studies might be a concern for that study.

The functional significance of the Val⁴³²Leu polymorphism has not been studied adequately. Because exon 3 encodes for the heme-binding region of the CYP1B1 enzyme, nucleotide changes in this exon may be significant to the function of this enzyme. Bailey et al.(21) reported that Caucasian patients with the Val/Val genotype had a significantly higher percentage of breast cancer that were positive for ERs or PRs, suggesting that this polymorphism may be functionally important for the expression of these steroid receptors in breast cancer (21). Because the prognosis for ER/PR-positive cancer is better than ER/PR-negative cancer, case-control studies including prevalent cases may be subjective to survival bias. In other words, the Val/Valgenotype may be overrepresented in prevalent cases, which may attenuate the positive association, if any, with the Leu/Leu genotype. In our study, all breast cancer cases were newly diagnosed, and only 1.1% of eligible cases were missed because of death prior to interviews. Therefore, the potential for survival bias should be minimal in this study. We found that the Leu/Leu genotype was associated with an elevated risk of breast cancer. Furthermore, the positive association between the Leu/Leu genotype and breast cancer was more pronounced among postmenopausal women, among whom more breast cancers are reported to be positive for ERs and/or PRs than those diagnosed among premenopausal women. We were unable at present to evaluate directly the association of CYP1B1 genotypes with subtypes of breast cancer defined by steroid receptors, because no such data were collected.

In addition to estrogen 4-hydroxylation, estrogen hydroxylations also occur at C2, C6, C15, and C16 positions. Among them, estrogen C2 and C16 hydroxylation are of particularly biological and quantitative importance (2). Similar to 4-hydroxyestrogen,16α-hydroxyestrone has been shown to be genotoxic, reacting with DNA to form adducts (2). On the other hand, 2-hydroxyestrogen has no estrogenic effect, and in fact, 2-methoxyestrogen may act as an antiestrogen in estrogen-sensitive tissues (2, 3). CYP1B1 primarily catalyzes estrogen 4-hydroxylation (2). We found that the positive association of the CYP1B1-Leu allele with breast cancer was stronger among postmenopausal than premenopausal women. Because the estrogen level is higher among premenopausal than postmenopausal women, our findings suggest that this polymorphism might be more closely related to the activation of environmental mammary carcinogens than estrogen 4-hydroxylation. Very recent data from in vitro experiments suggested that the isozyme encoded by the Leu allele might have an elevated activity in the activation of mammary carcinogens but a reduced activity in estrogen 4-hydroxylation (22).

The methodological limitations of this study were few. The participation rate of this study was high, minimizing potential selection bias that is common to many case-control studies. Chinese women living in Shanghai are relatively homogeneous in ethnic backgrounds, because >98% of them are classified into a single ethnic group (Han Chinese). We have also compared the place of birth between cases and controls and found that these two groups of women were very similar. Therefore, the potential confounding effect by ethnicity in association studies of genetic biomarkers, may not be a major concern in our study. The sample size in this study was not large, which may have resulted in a relatively unstable estimate of risk, particularly in stratified analyses. Nevertheless, the results from this study are consistent with the observations from laboratory investigation,implicating a potential important role of the CYP1B1 enzyme in the etiology of breast cancer. Our findings are thus biologically plausible and merit further investigation with a larger sample size or in other ethnic populations.

We are grateful to Dr. Kim Creek for providing laboratory space and logistical support for this project. We also thank Dr. Zhong-Ling Deng for performing assays for part of the study samples,Dr. Bao-Tin Zhu for helpful comments and suggestions on the manuscript,and Kathy Coats for assistance in manuscript preparation.