Introduction

Breast cancer (BC) is the most common cancer among women worldwide. Many factors such as age, sex, genetic background, lifestyle, and ethnicity are associated with the risk of developing breast cancer. However, prolonged exposure to estrogens is considered to be the main factor associated with both risk and prognosis of disease development [1].

Classical estrogen pathways include ligand binding to the estrogen receptor (ERα) in the cytoplasm, after which the receptor dimerizes, translocates to the nucleus, and binds to estrogen response elements (EREs) located near the promoters of genes of phase I metabolism [2]. On the other hand, polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbons receptor (AhR) and estrogen receptors (ERs). Their potential endocrine-disrupting activities may depend on both inhibitory AhR–ER crosstalk and AhR-dependent metabolic production of estrogenic PAH metabolites. After binding its ligand, AhR translocates into the nucleus and dimerizes with an AhR nuclear translocator (ARNT) [3]

Activation of ERs and AhRs includes relevant proposed mechanisms of crosstalk between their signaling pathways from the step of heterodimerization (for AhR with ARNT) or homodimerization (for ERs). AhR has been reported to inhibit ER activity through a combination of different mechanisms: direct inhibition by the activated AhR/ARNT heterodimer through binding to inhibitory xenoestrogen receptor (iXRE) present in ER target genes and squelching of shared coactivators, including ARNT.

Inside the cell, estrogens and xenobiotics, after binding to their specific receptors, are transformed by enzymes of phase I metabolism (CYP1A1 and CYP1B1) and phase II metabolism [glutathione S-transferases (GSTs) and catechol O-methyl transferase (COMT)], generating products that could interact with DNA to form adducts. These adducts can induce mutations that may initiate cancer processes. However, there are enzymes capable of reverting the damage caused to the DNA, which are known as repair enzymes, such as methylguanine-DNA methyltransferase (MGMT) [4] and X-ray repair cross-complementing (XRCC1) [5].

The genes involved in the biotransformation of estrogens and xenobiotics and the metabolism of agents used in the treatment of breast cancer have allelic variants with modified enzymatic activities (Table 1). These alleles have been widely studied in different populations, including the Mexican population. In 2013, our work group determined the associations of the polymorphisms CYP1A1 rs1048943, CYP1B1 rs1056836, COMT rs4680, GSTT1 null, and GSTM1 null, finding a significant association (OR = 1.95, CI 1.13–3.36) between CYP1A1 rs1048943 polymorphism and risk of breast cancer. The results showed that breast cancer risk significantly increases in women with three to six risk polymorphisms [6]. Due to this important association between breast cancer risk and the studied genes, we decided to include the AhR rs2066853, ERα rs2234693 MGMT rs12917, and XRCC1 rs25487 polymorphisms to provide further information about the importance of genes of the catechol pathway and determine whether these nine polymorphisms are associated with breast cancer risk, clinicopathological characteristics, and survival in our sample of Mexican women.

Table 1 Genetic polymorphisms involved in estrogen and xenobiotic metabolism
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Materials and methods

Subjects

We included 150 Mexican Mestizo women who attended the Instituto Nacional de Cancerología (Mexico City) from 2006 to 2007. These patients were diagnosed with in situ (38 cases) or invasive (112 cases) breast carcinoma. All patients were over 30 years old and had no history of hereditary BC syndrome, according to the specifications of the US National Cancer Institute and the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology. The clinicopathological and survival data were taken from the clinical records of the Instituto Nacional de Cancerología.

We included 150 controls. Healthy Mexican Mestizo women were selected from blood donors who attended the Hospital “20 de Noviembre” in Mexico City from October 2001 to November 2004. Women filled out a questionnaire, which included data about age, birthplace, parents’ and grandparents’ birthplace, and lifestyle. Controls were healthy age-paired women without diagnosis of BC.

Informed consent was obtained from all participants, who also answered a questionnaire about risk factors for BC; all participants and their parents and grandparents were born in Mexico.

The research protocol was approved by the Bioethics Committees of the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and the Instituto Nacional de Cancerología (Mexico City). The Hospital “20 de Noviembre” gave permission to use the buffy coat of blood bank samples.

DNA samples

DNA samples used for genotyping analyses were extracted from blood samples collected and stored at −20°C until use. Mononuclear white cells and genomic DNA were isolated as described by Lahiri and Cols [7].

Genotyping

The CYP1A1 rs1048943, CYP1B1 rs1056836, COMT rs4680, and XRCC1 rs25487, GSTT1 and GSTM1 polymorphisms were determined as mentioned before [5, 6]. TaqMan assays were used for genotyping of AhR rs2066853 and ERα rs2234693 according to manufacturer specifications. The polymorphism MGMT rs12917 was determined by high-resolution melting (HRM) analysis. The sequences of primers were as follows: 5-CTGCTAATGCCTATTTCC-3 (forward primer) and 5-AACACCGCAGATGGCTTAGT-3 (reverse primer). PCR consisted of initial denaturing at 94 °C for 5 min, followed by 35 cycles of denaturing at 94 °C for 20 s, annealing at 62 °C for 20 s, and extension at 72 °C for 20 s, with final extension at 72 °C for 10 min. HRM analysis was performed using temperatures in the range from 80–90 °C in 0.1 °C increments. PCR amplifications using EvaGreen were carried out in 10 µL volumes containing 2× HRM master mix (contains HotStar Taq Plus DNA Polymerase, EvaGreen dye, 10 µM each primer, and 10 ng template DNA). PCR and subsequent melt curve analysis was carried out in a Rotor-Gene 6000 (Corbett Research). The increase and decrease in fluorescence of EvaGreen during PCR and the melt phase was acquired on the HRM of the Rotor-Gene.

Statistical Analyses

Hardy–Weinberg equilibrium (HWE) was tested separately in cases and controls for each polymorphism, when applicable, using statistical package GenePop version 4.0.10 (http://genepop.curtin.edu.au).

Epidemiological risk factors, risk of developing BC, and the associations between polymorphisms and the clinicopathological characteristics of patients were examined using the chi-square test with Yates correction for data n ≤ 5 to calculate the crude odds ratio (OR) with 95% confidence interval (CI). The former two tests were carried out using GraphPad Prism 5 software. Assuming a biological activity gradient (codominant model), the heterozygous and homozygous variant genotypes of these genes were compared with the homozygous wild-type genotype. Groups having the lowest risk were used as reference.

Survival distributions were estimated by the Kaplan–Meyer method and compared by log-rank test.

Statistical differences were considered significant for p ≤ 0.05. All p-values reported are two tailed.

Results

The genotypes tested were under HWE. Table 2 presents the allelic and genotypic frequencies of the nine polymorphisms studied.

Table 2 Allele/genotype frequency of polymorphisms from breast cancer patients and control women, and the association with risk of breast cancer
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A statistically significant negative association with BC risk was found for AhR A (OR = 0.55; p = 0.01) and AhRA/G (OR = 0.50; p = 0.03). On the other hand, a positive association with BC risk was observed for the polymorphisms ER alpha C (OR = 1.98; p =  < 0.0001), ERαC/C (OR = 3.2; p = 0.0006), CYP1A1G/G (OR = 1.95; p = 0.01), CYP1B1 G (OR = 1.48; p = 0.02),COMT A (OR = 1.42; p = 0.04), MGMT T (OR = 1.42; p = 0.04), MGMT T/T (OR = 3.77; p =  < 0.0001), XRCC1 G (OR = 1.92; p = 0.0004), and XRCC1 G/G (OR = 3.85; p = 0.0006). It is important to clarify that Table 2 shows data previously published by our group, which are highlighted in bold.

The clinicopathological characteristics of breast cancer patients are presented in Table 3. Most breast cancer patients included were over 35 years old. With respect to tumor stage, 37.3% were classified as IIIA and 30.6% as IIIB, 74.6% showed invasive carcinomas, 26% showed lobular histopathological type, and 39.3% were triple negative.

Table 3 Clinicopathological features of breast cancer patients
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To determine whether the polymorphisms have an effect on the different clinical features of breast cancer patients, we analyzed associations between the nine polymorphisms and different clinical parameters (age at diagnosis, tumor stage, malignant lesions, tumor size, lymph node status, distant metastasis, histological type of cancer, estrogen receptor status, progesterone receptor status, HER-2/neu status, and triple-negative status); we mention only the associations that were statistically significant.

We found a negative association between CYP1B1 G/G polymorphism and tumor size > T2 (OR = 0.40; p = 0.05) and lymph node status N2 + N3 (OR = 0.34; p = 0.04) an,d between AhR G/A polymorphism and lymph node status N2 + N3 (OR = 0.28; p = 0.02) (Table 4).

Table 4 Associations between polymorphisms and tumor size and lymph node status
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When we examined the association between polymorphisms and negative status of estrogen receptor, we only found a negative association for the CYP1B1 rs1056836 polymorphism (CYP1B1C/G OR = 0.39; CYP1B1G/G OR = 0.29) (Table 5).

Table 5 Associations between polymorphisms and estrogen receptor status
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The associations between polymorphisms and triple-negative status showed statistical significance for CYP1B1G/G (OR = 3.9; p = 0.01) (Table 6).

Table 6 Associations between polymorphisms and triple-negative status
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Interestingly, we found that CYP1B1G/G was associated negatively with survival ≤ 5 years (OR = 0.07; p = 0.01) (Table 7).

Table 7 Associations between studied polymorphisms and survival
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In addition, we plotted Kaplan–Meier curves for survival versus time (months) for all polymorphisms included in this study, finding a significant relationship for the CYP1B1C/C genotype (Fig. 1).

Fig. 1
figure 1

Effect of CYP1B1 rs1056836 on survival proportion

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Discussion

The allelic and genotypic frequencies determined in our study for AhR rs2066853 are similar to those reported for a population of Mexican women by Sierra-Martinez in 2016 [8]. However, they did not find any association with breast cancer risk, while in our study we found significant negative associations, both with the heterozygous variant and the homozygous mutant. These differences may be due to the smaller size used: They had 96 cases and 111 controls [8], while we used 150 cases and 150 controls. On the other hand, Sierra-Martinez does not report an association between ERα rs2234693 polymorphism and the risk of breast cancer in postmenopausal women [8], while we found a risk association with the T allele (OR = 1.98), and the C/C genotype (OR = 3.2).

When we compared the alleles of CYP1B1 we found a positive association between breast cancer risk and the Val allele, which may be because the C4326G transition (CYP1B1*3 C/G) leading to the corresponding amino acid transition is associated with increased catalytic activity. A possible cause of this increase in catalytic activity might be changes in the tertiary (or quaternary) structure of the CYP1B1 protein, as the CYP1B1*3 polymorphism is located near a catalytically important heme-binding domain in CYP1B1 [9]. Furthermore, the CYP1B1*3 transition is also responsible for significant increases in AhR-mediated CYP1B1 gene expression during AhR-mediated signaling event.

We found that homozygous mutants MGMT T/T are associated with risk of BC, and this could be related to the Leu84Phe polymorphism in the MGMT gene, since it has been reported in other populations that such polymorphism affects DNA repair capability and enzymatic activity, thereby leading to the risk of breast cancer [14].

The relationship between breast cancer risk and XRCC1 polymorphism rs25487 could be because the XRCC1 gene is necessary for maintenance of genetic stability, and its DNA repair capacity can be reduced due to the change of Arg for Gln, which could lead to accumulated DNA damage, mutations, and subsequently development of diseases such as cancer. On the other hand, it has been indicated that polymorphisms in XRCC1 play a contributing role in the formation of DNA adducts and an increased risk of developing cancer [12].

We found a negative association between CYP1B1 G/G polymorphism and tumor size, lymph node status, and estrogen receptor status. These associations may be due to increased activity of CYP1B1 because of polymorphism rs1056836, and increased catabolism of estradiol reflected by the decrease in estradiol levels and induction of ERα, favoring reduction of tumor size and lymph node status. This theory can be supported by reports describing a close relationship between this polymorphism and clinicopathological characteristics leading to poor prognosis [9, 13].

The negative relationship between CYP1B1*3 polymorphism and clinicopathological characteristics of poor prognosis may be congruent with the positive association between this polymorphism and the increase in survival of patients with the G/G genotype.

Another possible explanation for the association between the genotype and an increase in breast cancer patients’ survival may be that CYP1B1 is involved in the metabolism of some clinically relevant anticancer agents used in the treatment of cancers [9].

Although the sample included in this study is adequate, when dividing it by clinicopathological characteristics, it may be insufficient, but the data obtained show statistically significant associations between the polymorphisms included and the prognosis and survival of Mexican women with breast cancer.

Conclusions

We demonstrate, for the first time, that genes involved in phase I and II metabolism of xenobiotics and estrogens are associated not only with the risk of breast cancer but also with the clinicopathological characteristics of poor prognosis of patients with breast cancer, highlighting the important role of CYP1B1G/G, as it has also been shown to be associated with longer survival in Mexican Mestizo women with breast cancer.