Biomedical Research

Abstract

Objective: We performed a case-control study to evaluate the role of IL-18-07C/A (rs1946518) and -137G/C (rs187238) polymorphisms in the development of breast cancer.

Methods: A total of 305 breast cancer patients and 305 healthy controls were recruited between May 2015 and May 2016. The genotyping of IL-18 -607C/A and -137G/C polymorphisms was carried out by an iPlex GOLD SNP genotyping analysis using the Sequenom MassARRAY® System.

Results: We observed that the CC genotype of IL-18 -607C/A was associated with breast cancer risk (OR=1.80, 95%CI=1.06-3.07), when compared with the CC+CA genotype. The CC genotype of IL-18 -137G/C had a 2.90 fold risk of breast cancer as compared to the CC genotype, and the CC genotype also showed an increased risk of breast cancer compared with the GG+GC genotype (OR=2.94, 95%CI=1.41-6.11). A completely linkage disequibrium was found between IL-18 rs1946518 and rs187238 (D’=1.00, r2=0.19). The A-C (OR=1.52, 95%CI=1.11-2.08) and C-G (OR=0.79, 95%CI=0.62-0.99) haplotypes were associated with breast cancer risk.

Conclusion: We found that IL-18 -607C/A and -137G/C polymorphisms and haplotypes contribute to increase the breast cancer risk.

Keywords

IL-18, Polymorphism, Haplotype, Breast cancer

Introduction

Breast cancer is the most common cancer among women worldwide and is the second leading cause of cancer-related death in women, after lung cancer, in developed countries [1]. This cancer is among the top five most common cancers in Iran and ranks first among cancers diagnosed in women [2]. Breast cancer has a diverse etiology and several risk factors contribute to its development [3,4], such as a family history of cancer, lack of exercises, obesity, alcohol drinking, early menarche, late menopause, age at the birth of first child, number of months of breastfeeding and hormone uses and reproductive history [5-7]. However, the prevalence of breast cancer showed differences across different populations if they exposed similar lifestyles and environmental factors, suggesting that the hereditary factors may contribute to the development of this disease. Previous studies have reported that many genetic factors are involved in the risk of breast cancer, such as tumor necrosis factor receptor 2, ABCB1, insulin-like growth factor-I [8-10].

Chronic systemic inflammation has emerged as an important factor in the pathogenesis of chronic diseases such as diabetes, spontaneous abortion and of certain types of cancer [11-13]. There is convincing evidence describing the influence of lowgrade inflammation and cytokines in breast carcinogenesis [14,15]. IL-18 is an 18-kDa cytokine, which belongs to the interleukin-1 (IL-1) superfamily [16]. Previous studies have shown that IL-18 modulates the immune system for attacking cancer cells through inhibition of breast cancer cell proliferation and potential invasion [14]. The single nucleotide polymorphic position of IL-18 -607C/A and -372C/G have confirmed impact on IL-18 activity and expression in tissues [17,18]. Currently, many studies have explored the association between IL-18 -607C/A and -137G/C and risk of several kinds of cancers, such as non-small cell lung cancer, acute myeloid leukemia, hepatocellular carcinoma and gastric cancer [19-22]. However, only several studies investigated the association of IL-18 -607C/A with risk of breast cancer [23-26], but no correlation between IL-18 -137G/C polymorphism and risk of breast cancer in Chinese population. In the present study, we performed a case-control study to evaluate the role of IL-18 -607C/A (rs1946518) and -137G/C (rs187238) polymorphisms in the development of breast cancer.

Materials and Methods

Subjects

This case-control population is composed of 610 Chinese unrelated Han adults who were enrolled from the Affiliated Hospital of Xinxiang Medical University between May 2015 and May 2016. A total of 305 breast cancer patients were collected prior to receiving any anti-cancer treatment, and all these patients were confirmed to be breast cancer by pathologic examination. The exclusion criteria for breast cancer patients were those with a history of other malignant tumors, recurrent or metastasis breast cancer.

Meanwhile, one healthy control subject was selected and matched with one patient with age ( ± 5 years). A total of 305 healthy controls were recruited from physical examination center and outpatient clinics of the Affiliated Hospital of Xinxiang Medical University. All the controls were confirmed to be free of cancers by health examination. Those who had a history of malignant tumor, serious liver and kidney diseases, and autoimmune diseases. The mean ages of patients and controls were 57.46 ± 9.74 and 58.29 ± 8.81 years, respectively; the mean BMI values were 23.38 ± 3.06 and 22.03 ± 3.19, respectively.

The demographic and lifestyle characteristics of respondents were collected from a face-to-face questionnaires or medical records, including age, body mass index (BMI), age of menarche, menopausal status, age at first live birth, nulliparous, breastfeeding, months of breastfeeding, tobacco smoking, alcohol drinking, history of hormone uses, family history of cancer in the first relatives, and history of benign breast diseases. The BMI was calculated as body weight (kg) divided by square of the body height. All investigated subjects volunteered for the study and signed informed consents, and the research proposal was approved by the ethics committee of the Affiliated Hospital of Xinxiang Medical University.

Genotyping assays

A total of five ml peripheral blood samples were collected from each respondent, and kept in tubes with 5% EDTA. The genomic DNA was extracted using TIANamp Blood DNA Kit (Tiangen, Beijing, China) according to the instruction. The genotyping of IL-18 -607C/A and -137G/C polymorphisms was carried out by an iPlex GOLD SNP genotyping analysis using the Sequenom MassARRAY® System (Sequenom, San Diego, USA). The primers used for amplification of IL-18 -607C/A and -137G/C were designed by Sequenom Assay Design 3.1 software. Then the SAP and iPLEX reactions were carried out to analyze the PCR amplification products. The PCR products are then desalted and crystallized, and then analyzed by SpectroCHIP and MALDI-TOF MS reaction. The mass spectra peak and genotypes of IL-18 -607C/A and -137G/C was analyzed by Typer 4.0 software.

Statistical analysis

Chi-square (χ²) test was used to compare the categorical variables between the two study groups, such as lifestyle characteristics and genotypes of IL-18 -607C/A and -137G/C. Student t test was taken to compare the continued variables. The χ² test with one degree of freedom was used to estimate whether the IL-18 -607C/A and -137G/C genotype distributions conformed to the Hardy-Weinberg equilibrium (HWE) in controls. The correlation between IL-18 -607C/A and -137G/C polymorphisms and breast cancer risk was analyzed by conditional multivariate logistic regression, and the results were expressed by odds ratio (OR) and 95% confident intervals (95% CI) and adjusted for potential confounding factors. The linkage disequilibrium and haplotype analyses of three SNPs were estimated by SHEsis software. All the statistical analyses were analyzed by IBM SPSS Statistics for Windows, Version 21.0. (IBM Corp, Armonk, NY, USA). Two sided P-value <0.05 was regarded as statistically significance.

Results

When compared the baseline characteristics of controls, we found that patients with breast cancer were more likely to have higher BMI, earlier age of menarche, less months of breastfeeding, more history of hormone uses and history of benign breast diseases (Table 1). The genotype distributions of IL-18 -607C/A and -137G/C were shown in Table 2. The GG, GC and CC genotypes of IL-18 -137G/C were significantly different between patients with breast cancer and controls (χ²=11.57, P=0.003), while the CC, CA and AA genotypes of IL-18 -607C/A did not show significant difference (χ²=2.65, P=0.27).

Table 1. Baseline characteristics of patients with breast cancer and controls.

Table 2. The genotype distribution of IL-18 -607C/A and -137G/C genetic polymorphisms in the study subjects.

Using conditional logistic regression analysis, we observed that the CC genotype of IL-18 -607C/A was associated with breast cancer risk (OR=1.80, 95%CI=1.06-3.07), when compared with the CC+CA genotype (Table 3). The CC genotype of IL-18 -137G/C had a 2.90 fold risk of breast cancer as compared to the CC genotype, and the CC genotype also showed an increased risk of breast cancer compared with the GG+GC genotype (OR=2.94, 95%CI=1.41-6.11). A completely linkage disequibrium was found between IL-18 rs1946518 and rs187238 (D’=1.00, r2=0.19). The A-C haplotype was correlated with an increased risk of breast cancer (OR=1.52, 95%CI=1.11-2.08), while the C-G haplotype showed a reduced risk (OR=0.79, 95%CI=0.62-0.99) (Table 4).

Table 3. Association between IL-18 genetic polymorphisms and risk of breast cancer.

Table 4. Haplotype analysis of IL-18 rs1946518- rs187238 with breast cancer risk.

Discussion

In the current study, we investigated the association of IL-18 -607C/A AA genotype and -137G/C CC genotype were associated with risk of breast cancer, and a completely linkage disequibrium was observed between rs1946518 and rs187238. Cytokines are reported to be a protein family of regulatory factors derived from tumors and their environmental components that are involve in the onset, growth, invasion and metastasis of breast cancer [27]. Srabović et al. revealed that IL-18 expression was significantly elevated in breast cancer tumor tissue in comparison to its expression in surrounding unchanged tissue of the same patients [28]. Jung indicated that IL-18 was associated with inflammation and was up-regulated in breast cancer tissues in mice [29]. Expression of IL-18 equally showed in inflammatory and non-inflammatory locally advanced breast cancer, and was correlated with complete response to neoadjuvant chemotherapy [29]. Therefore, expression of IL-18 was associated with the development and outcome of breast cancer. IL-18 is located on chromosome 11q22.2-q22.3, and single nucleotide polymorphic position of -607C/A (rs1946518) and -137G/C (rs187238) have impact on IL-18 activity and expression in tissues [17,18]. Currently, many studies indicated that IL-18 polymorphisms were associated with many kinds of cancers [19,20].

Similarly, several studies reported the association between IL-18 polymorphisms and risk of breast cancer in several kinds of populations [26]. However, some studies reported no association between them. Back LK et al. reported that IL-18 -607A/C and -137G/C polymorphisms contributed to increase the breast cancer risk in co-dominant and recessive genetic models [26]. Khalili-Azad et al. indicated that the CC genotype of IL-18 -137G/C was significantly associated with metastasis of breast cancer patients, but not associated with onset of breast cancer [24]. A recent meta-analysis reported that IL-18 -607C/A polymorphism is associated with increased susceptibility to breast cancer in Asian and Mixed population [23]. Our study reported the similar results with previous ones. In addition, we observed that the IL-18 rs1946518- rs187238 A-G and C-G haplotypes were associated with breast cancer risk. However, some studies reported inconsistent results. Taheri et al. conducted a study with 72 breast cancer patients and 93 cancer free women, and found that IL-18 -607A/C polymorphism was not associated with breast cancer in a sample of Iranian population [26].

Our study firstly reports that the AA genotype of IL-18 -607C/A and CC genotype of IL-18 -137G/C are associated with the development of breast cancer, and a completely linkage disequibrium was observed between the two SNPs and A-C and C-G haplotypes also play an important role in the risk of this disease. Our findings should be confirmed in further studies with large sample size and more ethnic groups.

Acknowledgement

We thank for the funding from National Natural Science Foundation of China (No. 81502313) and Doctoral Scientific Research Activation Foundation of Xinxiang Medical University (No. XYBSKYZZ201603).

References

1. Khazaee-Pool M, Montazeri A, Majlessi F, Rahimi Foroushani A, Nedjat S, Shojaeizadeh D. Breast cancer-preventive behaviors: exploring Iranian women's experiences. BMC Womens Health 2014; 14: 41.
2. Almasi Z, Mohammadian-Hafshejani A, Salehiniya H. Incidence, mortality, and epidemiological aspects of cancers in Iran; differences with the world data. J BUON 2016; 21: 994-1004.
3. Almendro V, Fuster G. Heterogeneity of breast cancer: etiology and clinical relevance. Clin Transl Oncol 2011; 13: 767-773.
4. Hiatt RA, Haslam SZ, Osuch J. The breast cancer and the environment research centers: transdisciplinary research on the role of the environment in breast cancer etiology. Environ Health Perspect 2009; 117: 1814-1822.
5. Zakhari S, Hoek JB. Alcohol and breast cancer: reconciling epidemiological and molecular data. Adv Exp Med Biol 2015; 815: 7-39.
6. Shaukat U, Ismail M, Mehmood N. Epidemiology, major risk factors and genetic predisposition for breast cancer in the Pakistani population. Asian Pac J Cancer Prev 2013; 14: 5625-5629.
7. Engel C, Fischer C. Breast cancer risks and risk prediction models. Breast Care (Basel) 2015; 10: 7-12.
8. Yang F, Zhao Z, Zhao N. Clinical implications of tumor necrosis factor receptor 2 in breast cancer. Oncol Lett 2017; 14: 2393-2398.
9. Sharif A, Kheirkhah D, Reza Sharif M, Karimian M, Karimian Z. ABCB1-C3435T polymorphism and breast cancer risk: a case-control study and a meta-analysis. J BUON 2017; 22: 355-364.
10. Bai X, Xie J, Sun S, Zhang X, Jiang Y, Pang D. The associations of genetic polymorphisms in CYP1A2 and CYP3A4 with clinical outcomes of breast cancer patients in northern China. Oncotarget 2017; 8: 38367-38377.
11. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420: 860-867.
12. Duncan BB, Schmidt MI, Pankow JS, Ballantyne CM, Couper D, Vigo A, Hoogeveen R, Folsom AR, Heiss G. Low-grade systemic inflammation and the development of type 2 diabetes: the atherosclerosis risk in communities study. Diabetes 2003; 52: 1799-1805.
13. Vahid F, Shivappa N, Hekmatdoost A, Hebert JR, Davoodi SH, Sadeghi M. Association between Maternal Dietary Inflammatory Index (DII) and abortion in Iranian women and validation of DII with serum concentration of inflammatory factors: case-control study. Appl Physiol Nutr Metab 2017; 42: 511-516.
14. Nicolini A, Carpi A, Rossi G. Cytokines in breast cancer. Cytokine Growth Factor Rev 2006; 17: 325-337.
15. Porta C, Larghi P, Rimoldi M, Totaro MG, Allavena P, Mantovani A, Sica A. Cellular and molecular pathways linking inflammation and cancer. Immunobiology 2009; 214: 761-777.
16. Dinarello CA. IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J Allergy Clin Immunol 1999; 103: 11-24.
17. Kalina U, Ballas K, Koyama N, Kauschat D, Miething C, Arnemann J, Martin H, Hoelzer D, Ottmann OG. Genomic organization and regulation of the human interleukin-18 gene. Scand J Immunol 2000; 52: 525-530.
18. Giedraitis V, He B, Huang WX, Hillert J. Cloning and mutation analysis of the human IL-18 promoter: a possible role of polymorphisms in expression regulation. J Neuroimmunol 2001; 112: 146-152.
19. Gan WY, Li HM, Zhang YG, Li CM, Wang Y. Association between IL18-607C/A and -137G/C polymorphisms and susceptibility to non-small cell lung cancer in a Chinese population. Genet Mol Res 2016.
20. Wang H, Hua M, Wang S, Yu J, Chen C, Zhao X, Zhang C, Zhong C, Wang R, He N, Hou M, Ma D. Genetic polymorphisms of IL-18 rs1946518 and IL-1beta rs16944 are associated with prognosis and survival of acute myeloid leukemia. Inflamm Res 2017; 66: 249-258.
21. Lau HK, Hsieh MJ, Yang SF, Wang HL, Kuo WH, Lee HL, Yeh CB. Association between Interleukin-18 Polymorphisms and Hepatocellular Carcinoma Occurrence and Clinical Progression. Int J Med Sci 2016; 13: 556-561.
22. Yao J, Li ZH, Li YX, Zhang R, Zhang DG, Xu ZL, Wang LS, Wang JY. Association between the -607 C > A polymorphism in interleukin-18 gene promoter with gastrointestinal cancer risk: a meta-analysis. Genet Mol Res 2015; 14: 16880-16887.
23. Li X, Ren D, Li Y, Xu J, Liu C, Zhao Y. Increased cancer risk associated with the -607C/A polymorphism in interleukin-18 gene promoter: an updated meta-analysis including 12,502 subjects. J BUON 2015; 20: 902-917.
24. Back LK, Farias TD, da Cunha PA, Muniz YC, Ribeiro MC, Fernandes BL, Fernandes CK, de Souza IR. Functional polymorphisms of interleukin-18 gene and risk of breast cancer in a Brazilian population. Tissue Antigens 2014; 84: 229-233.
25. Khalili-Azad T, Razmkhah M, Ghiam AF, Doroudchi M, Talei AR, Mojtahedi Z, Ghaderi A. Association of interleukin-18 gene promoter polymorphisms with breast cancer. Neoplasma 2009; 56: 22-25.
26. Taheri M, Hashemi M, Eskandari-Nasab E, Fazaeli A, Arababi F, Bahrani-Zeidabadi M, Bahari G. Association of -607 C/A polymorphism of IL-18 gene (rs1946518) with breast cancer risk in Zahedan, Southeast Iran. Prague Med Rep 2012; 113: 217-222.
27. Jung MY, Kim SH, Cho D, Kim TS. Analysis of the expression profiles of cytokines and cytokine-related genes during the progression of breast cancer growth in mice. Oncol Rep 2009; 22: 1141-1147.
28. Srabovic N, Mujagic Z, Mujanovic-Mustedanagic J, Muminovic Z, Cickusic E. Interleukin 18 expression in the primary breast cancer tumour tissue. Med Glas (Zenica) 2011; 8: 109-115.
29. Aguiar MAN, Wanderley CWS, Nobre LMS, Alencar MRM, Saldanha MDPS, Souza AM, Wong DVT, Barros PG, Almeida PRC, Lima-Júnior RCP, Ribeiro RA. Interleukin-18 (IL-18) is equally expressed in inflammatory breast cancer and noninflammatory locally advanced breast cancer: A possible association with chemotherapy response. Asia Pac J Clin Oncol 2017.