Estrogen-Metabolizing Gene Polymorphisms in the Assessment of Female Hormone-Dependent Cancer Risk

Estrogen-metabolizing gene polymorphisms in the assessment of female hormone-dependent cancer risk

ON Mikhailova1, LF Gulyaeva1, Allelic variants of cytochrome P450: CYP1A1, CYP1A2, CYP19 (Aromatase) 1 2 and II-phase enzyme Sulfotransferase (SULT1A1) genes are associated with a AV Prudnikov , AV Gerasimov high risk of hormone-dependent cancers. We estimated a frequency of these 2 and SE Krasilnikov allelic variants in the female Caucasian population of the Novosibirsk region of Russia and their association with the elevated risk of ovarian and 1Institute of Molecular Biology and Biophysics, Novosibirsk, Russia and 2Regional Clinical endometrial cancer. A DNA bank of gynecologic oncology patients, patients Oncological Hospital, Novosibirsk, Russia with benign gynecologic diseases and healthy women was created, and the following single nucleotide polymorphisms (SNPs) were examined: CYP1A1 Correspondence: M1 polymorphism, that is, T264-C transition in the 30-noncoding region; Dr ON Mikhailova, Department of Molecular CYP1A2*1F polymorphism, that is, C734-A transversion in CYP1A2 gene; Mechanisms of Carcinogenesis, Institute of - Molecular Biology and Biophysics, Siberian C T transition (Arg264Cys) in exon 7 of CYP19; SULT1A1*2 polymorphism, Branch of the Russian Academy of Medical that is, G638-A transition (Arg213His) in SULT1A1 gene. A positive Sciences, Timakov Str. 2, Novosibirsk, correlation of C allele of CYP1A2*1F and G allele of SULT1A1*2 with 630117, Russia. hormone-dependent cancers in women was found. Thus, these genes are E-mail: [email protected] appropriate candidates for studying the contribution of genetic factors to endocrine disorder and environmentally determined diseases susceptibility. In contrast, no association of CYP19 and CYP1A1 polymorphisms with increased cancer risk was revealed. The Pharmacogenomics Journal (2006) 6, 189–193. doi:10.1038/sj.tpj.6500365; published online 10 January 2006

Keywords: SNP; drug metabolizing enzymes; polymorphism; endometrial cancer; ovarian cancer

Introduction

Genetic polymorphisms in drug-metabolizing enzymes contribute to susceptibility to a number of diseases. Identification of certain allelic variants of drug- metabolizing enzymes as genetic susceptibility markers can provide an important tool in early diagnosis and prevention. Recently, a worldwide increase in the frequency of female hormone-dependent cancers has been observed, primarily due to endometrial, ovarian and breast cancers. Endometrial cancer (EC) is the most common gynecological cancer. Statistics indicate the case rate of EC in Russia in 1970 was 6.4 per 100 000 women. In 2001, this number has increased to 19.49, with an increase of 8–10% annually.1 In the Novosibirsk region of Russia, the annual rate of increase is 9.4%, and for the last 20 years the case rate of EC has doubled, with the rate among women of reproductive age increasing from Received 5 May 2005; revised 21 November 2 2005; accepted 21 November 2005; published 7 to 15 per 100 000 women. Ovarian cancer (OC) is another type of hormone- online 10 January 2006 dependent cancer. In Russia, the case rate of this disease has increased for the last Estrogen-metabolizing gene polymorphisms ON Mikhailova et al 190

20 years. The mortality rate is high; the main reason is an CYP1A2*1F in cancer patients compared to controls advanced stage of the disease at initial diagnosis. (P ¼ 0.0003) and these results were still statistically signifi- Identified risk factors for hormone-dependent cancers cant after Bonferroni correction for multiple testing. We include endocrine disorders, environmental factors and found no statistical differences (P ¼ 0.097) between patients genetic predisposition. The genetic nature of hormone- with benign tumors and the control group. CYP1A2 AA dependent cancer is supported by the fact that in the homozygotes exhibited a reduction of malignant tumor risk Novosibirsk region there is an increase in the number of (P ¼ 0.0003) and these results were also significant after first-degree relatives diagnosed with cancer (e.g., mother Bonferroni correction. CYP1A2 CA heterozygotes and CC with EC, daughter with breast cancer, son with colon homozygotes exhibited an increase of tumor risk (P ¼ 0.0027 cancer) as well as an increase of the number of patients and P ¼ 0.0139, respectively), but these results did not previously living in areas with radiation (Semipalatinsk, remain statistically significant after Bonferroni correction. Northern Kazakhstan, Altai region) in excess of back- Significant differences were found in the genotype and allele ground.2 distribution for SULT1A1 polymorphism between patients The risk of hormone-dependent cancer is directly or with malignant tumors and controls as well as between indirectly related to reproductive hormones, in particular, patients with benign tumors and controls. Comparison of estrogens.3 The main reason for this is thought to be allele frequencies revealed a deficiency of allele A of estrogen stimulation of cell proliferation in the target SULT1A1*2 in patients with benign tumors (P ¼ 0.0014) organs. The whole process of estrogen metabolism is quite and with malignant tumors (P ¼ 0.0192) compared to complex, but it can be divided in three main stages: controls. However, when a Bonferroni correction was synthesis, metabolism and detoxification. The key enzyme applied the differences between patents with malignant in the complex chain of biochemical reactions during tumors and controls were not statistically significant. the synthesis of estrogens from androgens is the cytochrome SULT1A1 AA homozygotes exhibited a reduction of malig- P450, subfamily 19 (CYP19 or aromatase).4 Estrogens nant tumor risk (P ¼ 0.0068) and benign tumor risk are metabolized by several enzymes, including cytochrome (P ¼ 0.000004) while AG heterozygotes exhibited an increase P450 1A1 and 1A2 (CYP1A1 and CYP1A2), through hydro- of benign tumor risk (P ¼ 0.0061). However, after correction xylation resulting in reactive metabolites.5–8 Further degra- for multiple comparisons only results of reduction of benign dation of these metabolites via detoxification pathways, tumor risk for SULT1A1 AA homozygotes remained statisti- involves participation of sulfotransferase 1A1 (SULT1A1).9 cally significant. This enzyme catalyzes the sulfate conjugation of estrogens metabolites, forming hydrophilic sulfates of estrogens which are excreted in the urine. Endocrine system dis- Discussion turbances may occur through changes in the activities of enzymes involved. The CYP1A1, CYP1A2, CYP19 as well as Female hormone-dependent tumors, such as endometrial SULT1A1 phenotypes vary between human individuals, and ovarian carcinomas, are among the most common suggesting the possibility of genetic component in the malignancies afflicting women from both developing and activities of these enzymes. This relationship has been developed countries.3,16 A preponderance of evidence confirmed by several studies.10–15 indicates that these cancers are multifactorial diseases, with The purpose of our study was to estimate a frequency of genetic susceptibility, environment, nutrition and lifestyle allelic variants of CYP19, CYP1A1, CYP1A2 and SULT1A1 risk factors playing important etiologic roles. Currently, a and their association with the elevated risk of hormone- prevailing concept in the mechanisms of female hormone- dependent cancer in the female population of the Novosi- dependent cancer appearance is the impairment of estrogen birsk region of Russia. metabolism in general, and 17-beta estradiol (E2), in particular. This impairment results in the increased concentra- tion of hormones circulating in blood, or a local increase of Results their content in target tissues.17 The genetic nature of such disorders may also contribute to disease development. For For the detection of germline mutations in genes encoding example, genetic variations of estradiol metabolism have four selected estrogen-metabolizing enzymes, the PCR-RFLP been described as significant contributors to disease suscept- method was used. The genotype and allele distributions for ibility, with variations depending on ethnic background. CYP1A1, CYP1A2, CYP19 and SULT1A1 polymorphisms for In the field of obstetrics and gynecology, the genetic both patients and controls are given in Table 1. There was contribution of polymorphic markers to a series of disorders not a significant difference in both genotype and allele has been characterized.18 Identification of at-risk groups distributions for CYP1A1 and CYP19 polymorphisms in for developing hormone-dependent tumors is one of the patients with benign or malignant tumors. In contrast, most promising approaches to ultimately preventing these significant differences in the genotype and allele distribu- diseases. tions for CYP1A2 polymorphism between patients with In the current work, we report the genetic polymorphism malignant tumors and controls were found. Comparison of of selected estrogen-metabolizing enzymes. A significant allele frequencies revealed a deficiency of A allele of difference in genotypes and allele distributions was obtained

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Table 1 Genotype and alleles frequencies of CYP1A1, CYP1A2, CYP19 and SULT1A1 polymorphisms

Malignant tumor cases Benign tumor cases Controls

n OR (95% CI) P-valuea n OR (95% CI) P-valuea

CYP1A1 Alleles frequencies T 300 312 296 C 36 0.88 (0.53–1.47) 0.715 28 0.66 (0.39–1.14) 0.126 40 Genotype frequencies T/T 132 0.696 142 0.104 128 T/C 36 0.87 (0.51–1.50) 0.696 28 0.63 (0.36–1.12) 0.104 40 C/C 0 1.000 0 1.000 0 Total 168 170 168

CYP1A2 Alleles frequencies C 94 74 56 A 238 0.50 (0.34–0.74) 0.0003 (0.0096)b 266 0.71 (0.47–1.06) 0.097 284 Genotype frequencies C/C 6 0.0139 (0.4448)b 4 0.1228 0 C/A 82 0.0027 (0.0864)b 66 0.3089 56 A/A 78 0.0003 (0.0096)b 100 0.1442 114 Total 166 170 170

CYP19 Alleles frequencies C 310 326 326 T 22 1.28 (0.65–2.56) 0.5152 18 1.00 (0.49–2.05) 1.000 18 Genotype frequencies C/C 146 0.2789 154 0.5782 158 C/T 18 1.95 (0.82–4.70) 0.1148 18 1.85 (0.78–4.45) 0.1666 10 T/T 2 0.54 (0.01–3.85) 0.6850 0 0.1228 4 Total 166 172 172

SULT1A1 Alleles frequencies G 206 222 180 A 126 0.69 (0.50–0.95) 0.0192 (0.6144)b 118 0.60 (0.43–0.82) 0.0014 (0.0448)b 160 Genotype frequencies G/G 68 0.3126 68 0.4334 60 A/G 70 1.03 (0.61–1.73) 0.2184 86 1.26 (0.76–2.10) 0.0061 (0.1952)b 60 A/A 28 0.49 (0.27–0.92) 0.0068 (0.2176)b 16 0.28 (0.14–0.57) 0.000004 (0.00013)b 50 Total 166 170 170 n ¼ number of individuals in each category. Bold values are statistically significant. aFisher exact two-tailed P-values. bValues in parentheses are Bonferroni corrected P-values (32 tests).

for two genes studied. The cytochrome P450 1A2 allele CYP1A2*1F was higher in women with hormone-dependent variants can be considered as a risk factor for many human cancer, in agreement with a previous study.21 These results cancers. This is a major phase I enzyme, and accounts for suggest that women with C allele of CYP1A2*1F, which is about 15% of total liver P450 content.19 This enzyme is most associated with a decrease in CYP1A2 enzyme activity, have active in catalyzing 2-hydroxylations of 40–50% of circulat- a higher risk of developing endometrial or OCs. ing estrogens.7,8 Increased formation of 2-hydroxylated A major pathway for estrogen metabolism in humans is estrogens would be consistent with a reduced risk for breast sulfate conjugation catalyzed by SULT enzymes, which cancer under a ‘receptor-mediated’ hypothesis, because 2- enhance the solubility and renal excretion of steroids. Thus, hydroxyestrogens can bind only weakly to the estrogen steroid sulfonation contributes to the control of levels of receptor.20 In our study, the frequency of C allele of biologically active hormone in target tissues. Inherited low

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activity of SULT may increase the risk of breast cancer.22 For Clinical Oncological Hospital of Novosibirsk. Prior to study the SULT1A1 a nucleotide substitution G638-A has been enrollment, the aims of the study were fully explained and identified, resulting in an Arg213His substitution accom- informed consent was obtained from each patient. The panied by a decrease in activity.15 study protocol was reviewed and approved by the appro- In contrast to expected results, patients with benign and priate Institutional Review Boards. malignant gynecological tumors in the Novosibirsk region Genotyping of the studied population utilized the poly- had a higher frequency of G allele of SULT1A1*2 compared merase chain reaction-restriction fragment length poly- to healthy women. This may indicate that women with morphism (PCR-RFLP) method. Exfoliated buccal cell tumors have increased SULT1A1 activity compared to samples were collected from patients as a source of genomic healthy women. This can be related to the dual function- DNA for genotyping assays. DNA was isolated from buccal ality of the SULT family: inactivation of estrogens in epithelium using the MasterAmp Buccal Swab DNA Extrac- addition to activation of procarcinogens. To date, little tion Kit (BIOzym, Germany). Following precipitation, DNA information is known about the balance between these samples were dissolved in 10 mM Tris/1 mM EDTA, pH 8.0 functions and risk factors that these enzymes play. In our and stored at 41C. case, we can suggest a potential environmental component Respective PCR reactions for each gene were performed in in hormone-dependent carcinogenesis, since SULT1A1 con- a total volume of 50 ml of buffer solution containing PCR verts not only estrogens but also heterocyclic amines and buffer (10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2;50mM KCl), aromatic amines into reactive metabolites.23 It follows from 0.2 mM of each deoxynucleotide triphosphate; 20 pmol of this that the role of SULT1A1 in estrogen inactivation is not each primer (in the primer pair), 100 ng of genomic DNA essential. A study of SULT1E1, the major estrogen-inactivat- and 2 U of Taq DNA polymerase (purchased in Medigen, ing enzyme, would help clarify the role of sulfonation in Novosibirsk, Russia). The PCR cycling conditions were as hormonal carcinogenesis. The recently discovered poly- follows: denaturation at 951C for 3 min followed by 35 morphism of this enzyme24 is a subject of further investiga- cycles of denaturation at 951C for 30 s, annealing at 621C for tion in our laboratory. 30 s and extension at 721C for 1 min. A final extension step Our data did not indicate any significant differences in at 721C for 10 min was followed by cooling to 41C. The genotypes and allele frequencies both for CYP1A1 and oligonucleotide primers for CYP1A1, CYP1A2, CYP19 and CYP19 polymorphisms in patients compared to healthy SULT1A1 were synthesized by Medigen company, Novo- controls. These results are in agreement with the recent sibirsk, Russia (http://www.medigen.ru) according to sequen- publication, which showed only a weak association of MspI ces chosen using the PCR primer design software GeneR- polymorphism in CYP1A1 gene with hormone-dependent unner (available at http://www.generunner.com) to ensure cancer.25 The absence of correlation for CYP19 polymorph- specific and efficient amplification of target sequences. The ism and cancer in our case is supported by other investiga- oligonucleotide primer sequences were: tors.26 This might be explained by the fact that contribution CYP1A1 0 0 of genetic component in aromatase activity is not the only U, 5 -TAGGAGTCTTGTCTCATGCC-3 ; factor for cancer development. The concept of local estrogen CYP1A1 R, 50-GCACTTAAGCAGTCTGTTTGAG-30; biosynthesis due to increased aromatase activity is one of CYP1A2 U, 50-TGAGGCTCCTTTCCAGCTCTCA-30; the leading factors for the development of estrogen- CYP1A2 R, 50-AGAAGCTCTGTGGCCGAGAAGG-30; depending tumors for today.27 According to this concept 0 0 the local synthesis of estrogens in single cells resulting in the CYP19 U, 5 -CGCTAGATGTCTAAACTGAG-3 ; variation of estrogen content and, as a consequence, CYP19 R, 50-CATATGTGGCATGGGAATTA-30; 28,29 enhancement of proliferation processes was accepted. SULT1A1 U, 50-GGGTCTCTAGGAGAGGTGGC-30; In conclusion, the results of our study indicate that SULT1A1 R, 50-GCTGTGGTCCATGAACTCCT-30. women with C allele of CYP1A2*1F or G allele of SULT1A1 in the Novosibirsk region of Russia have an increased risk of endometrial or OC. We studied the following single nucleotide polymorphisms (SNPs): CYP1A1 M1 polymorphism, that is, T264-C in the 30-noncoding region giving rise to an MspI restriction site; Materials and methods C734- A transversion in CYP1A2 gene abolishing an ApaI restriction site; C-T transition (Arg264Cys) in exon 7 of A DNA bank of cancer patients and healthy women of CYP19 gene which creates a recognition site for the SfaNI Novosibirsk region was created, utilizing two experimental restriction enzyme; and a G638-A transition (Arg213His) groups – patients with different stages of endometrial or in SULT1A1 gene that eliminates an HhaI restriction site. ovarian tumor malignancy (n ¼ 168, 19–55 years old) and The amplified DNA fragments were digested with MspI patients with benign gynecological tumors (n ¼ 172, 19–55 (for CYP1A1), ApaI (for CYP1A2), SfaNI (for CYP19), HhaI years old) – and a group of healthy controls over the age of (for SULT1A1) restriction enzymes (purchased from Siben- 55 with an uncomplicated obstetric and gynecological zyme, Novosibirsk, Russia) and subjected to electrophoresis history (n ¼ 172). Clinical diagnosis was performed by on a 8% polyacrylamide gel followed by staining with board-certified gynecologic-oncologists from the Regional ethidium bromide for UV visualization. Fragment sizes were

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estimated by comparison to standard DNA molecular 12 Eaton DL, Gallagher EP, Bammler TK, Kunze KL. Role of cytochrome ladders. P4501A2 in chemical carcinogenesis: implications for human vari- Statistical analysis was performed with GraphPad (http:// ability in expression and enzyme activity. Pharmacogenetics 1995; 5: 259–274. www.graphpad.com) and SISA (http://home.clara.net/sisa/) 13 Christiansen L, Bygum A, Jensen A, Thomsen K, Brandrup F, Horder M on-line calculators. The genotype data were analyzed using et al. Association between CYP1A2 polymorphism and susceptibility the Fisher exact test. A Bonferroni correction for multiple to porphyria cutanea tarda. Hum Genet 2000; 107: 612–614. testing was applied. All polymorphisms examined in the 14 Goodman MT, McDuffie K, Kolonel LN, Terada K, Donlon TA, Wilkens LR et al. 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