Abstract
Background/Aim: The current study aimed at evaluating the contribution of IL-13 promoter rs1881457 and rs1800925 genotypes to the risk of breast cancer in Taiwan. Materials and Methods: A total of 1,232 breast cancer cases and 1,232 age-matched controls were genotyped by typical polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Results: As for IL-13 rs1881457, the rates of AA, AC and CC genotypes were 54.8, 37.9 and 7.3% among the cases, and 53.8, 38.7 and 7.5% among the healthy controls, respectively; there were no statistically significant differences between the two groups (p for trend=0.8889). Also, regarding IL-13 rs1800925, there were no statistically significant differences between the two groups either (p for trend=0.6803). Furthermore, the allelic frequencies for IL-13 rs1881457 and rs1800925 were not differentially distributed between the case and control groups (p=0.6515 and 0.8753, respectively). Conclusion: The rs1881457 and rs1800925 IL-13 promoter polymorphisms may not serve as breast cancer susceptibility determinants for Taiwanese.
Breast cancer is the leading female malignant disease worldwide. In both genders combined in 2018, lung cancer was the most commonly diagnosed cancer (11.6% of the total cancer cases) and the leading cause of cancer-related death (18.4% of the total cancer deaths) followed by female breast cancer (11.6%) (1). Epidemiological studies indicate that breast cancer is caused by several environmental factors, including imbalanced diet, excessive alcohol drinking and cigarette smoking, and insufficient physical exercise (2, 3). In addition, genetic factors are also involved in breast cancer susceptibility, progression and prognosis (4-7).
The gene of the immune-regulatory cytokine interleukin-13 (IL-13), cloned in 1993, is located on chromosome 5q31 and has a length of approximately 1.4 kb (8). The 13 kDa protein IL-13 is secreted predominantly by Th2 lymphocytes and natural killer T cells (9, 10). The role of IL-13 in tumor progression is complex, and it seems to function like a double-sided blade. Regarding tumor promotion, it has been previously reported that the decrease in IL-13 levels in CD1-deficient mice lacking natural killer T cells may inhibit tumor recurrence (11-15). In addition, there is evidence that IL-13 may directly block apoptosis and promote proliferation of tumor cells in B chronic lymphocytic leukemia and Hodgkin disease (16-19). Furthermore, a study has also indicated that CD4-positive T cell-derived IL-13 can stimulate breast cancer cells and enhance tumor progression (20). However, other studies have indicated the anti-tumor activities for IL-13 in mice (12). At the cellular level, accumulating evidence has shown that IL-13 can inhibit the proliferation of a panel of human cancer cell lines, including renal cell carcinoma (21), B lineage acute lymphoblastic leukemia (22), and most of all, breast cancer cells (23, 24).
Among the IL-13 polymorphisms, those located in the promoter region are most likely to directly affect its expression levels and associate with cancer susceptibility. For instance, the IL-13 rs1800925 (C−1055T) polymorphism is adjacent to the nuclear factor of activated T cells (NFAT) binding site and some reports have indicated that it regulates IL-13 production (25-28). Furthermore, IL-13 promoter polymorphism rs1881457 has been investigated for its association with renal cell carcinoma (29), coronary artery disease (30), lung cancer (31, 32), and chronic obstructive pulmonary disease (33).
Based on the previous studies and its function in immuneregulation during carcinogenesis, it is worth examining the association between the rs1800925 and rs1881457 polymorphisms in IL-13 gene and breast cancer in Taiwan.
Materials and Methods
The study population. The study population consisted of breast cancer patients and age-matched healthy control subjects. The study was reviewed and approved by the Institutional Review Board of China Medical University Hospital (DMR99-IRB-108). Briefly, 2,464 subjects were recruited, including 1,232 female patients diagnosed with breast cancer and 1,232 age-matched healthy female visiting China Medical University Hospital as described in our previous publications (34, 35). They were all Taiwanese covered by the national insurance health system. The exclusion criteria for control recruiting included any metastatic cancer of another origin, previous malignancy, and hereditary or genetic disease. Each participant in this study was carefully oriented by well-trained nurses and/or doctors to complete a questionnaire and donate 3 to 5 ml of his peripheral blood samples. The contents of the questionnaire included questions focused on personal medical history and personal habits and diets such as alcohol consumption and cigarette smoking, and are listed in Table I. The informed consents of all the enrolled subjects in this study were collected and kept in the Tissue bank of China Medical University Hospital.
Demographics and lifestyles of the 1,232 breast cancer patients and the 1,232 healthy control Taiwanese women.
DNA preparation and IL-13 genotyping. DNA was extracted from peripheral leukocytes using conventional QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC), and genotyped with polymerase chain reaction-based restriction fragment length polymorphism methodology (PCR–RFLP) as we previously published (36-38). In this study, two IL-13 promoter polymorphic sites were genotyped. The primer sequences for IL-13 rs1881457 were: forward 5’-TTG CAC AGA CCA AGG TAG TT-3’ and reverse 5’-CTC ACA AGG CAT GCT GGA TT-3’. The primer sequences for IL-13 rs1800925 genotyping were: forward 5’-ACT TCT GGG AGT CAG AGC CA -3’ and reverse 5’-CCT TTT CCT GCT CTT CCG TC -3’; the under-lined G was specifically designed for enzyme cutting. The routine PCR cycling conditions were as follows: 1) one cycle at 94°C for 5 min; 2) 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 3) a final extension at 72°C for 10 min. The PCR fragments for IL-13 rs1881457 and rs1800925 were subjected to Aci I and Hpy99 I digestion at 37°C overnight and the bands were detected on ethidium bromide-stained 3% DNA agarose gel electrophoresis under UVC light. We summarized the information about primer sequences and genotype assay conditions in Table II.
Sequences of designed primers, restriction enzymes and polymerase chain reaction products for interleukin-13 genotyping.
Statistical analysis. To ensure that the control subjects were representative of the Taiwanese population and exclude genotyping errors, the fitness for the distributions of IL-13 genotypes at rs1881457 and rs1800925 in the healthy control group with Hardy-Weinberg equilibrium (HWE) were evaluated via the goodness-of-fit examination. Pearson’s chi-square was adopted to examine the distributions of IL-13 rs1881457 and rs1800925 genotypes among the subgroups of the investigated subjects. The associations between each IL-13 genotype and breast cancer risk were estimated via the adjusted odds ratios (ORs) together with the 95% confidence intervals (CIs) by unconditional logistic regression after being adjusted for confounding factors such as age, cigarette smoking and alcohol drinking behaviors.
Results
The demographic characteristics of the investigated population including 1,232 breast cancer patients and 1,232 healthy controls are systematically shown and compared in Table I. First, there was no difference between the two groups regarding age, age at menarche, age at birth of first child, or age at menopause (all p>0.05). Second, regarding personal behaviors including cigarette smoking and alcohol drinking, there were more smokers and alcohol drinkers among the breast cancer patients (13.8 and 13.1%) than healthy controls (7.0 and 7.4%) (both p<0.0001) (Table I). The significances may indicate that cigarette smoking and alcohol drinking are risk behaviors for breast cancer for females in Taiwan.
The genotypic distributions for IL-13 rs1881457 and rs1800925 among the breast cancer cases and controls are presented and analyzed in Table III. The two polymorphisms both followed HWE well in the control group (both p>0.05). Noticeably, there is neither association between the genotype of IL-13 rs1881457 with breast cancer susceptibility for Taiwan women nor that for IL-13 rs1800925 (the p for trend for IL-13 rs1881457 and rs1800925 are 0.8889 and 0.6803, respectively) (Table III). Even after taking in consideration and adjusting for confounding factors including age, smoking and alcohol drinking habits, the heterozygous and homozygous variant genotypes at either IL-13 rs1881457 or rs1800925 were still not associated with elevated or decreased breast cancer risk (Table III).
Distributions of interleukin-13 genotypic frequencies among 1,232 breast cancer cases and 1,232 healthy controls in Taiwan.
We further analyzed the allelic frequency distribution of the two IL-13 promoter polymorphisms, and the results are shown in Table IV. Consistent with the findings that none of the genotypes at IL-13 rs1881457 or rs1800925 was associated with breast cancer risk in Taiwan, none of the variant alleles at IL-13 rs1881457 or rs1800925 was significantly associated with breast cancer risk (both p>0.05). Overall, all findings presented in Tables III and IV indicate that neither the IL-13 rs1881457 genotype nor the IL-13 rs1800925 genotype can serve as a predictive marker for breast cancer risk in Taiwan.
Allele frequencies of interleukin-13 among 1,232 breast cancer cases and 1,232 healthy controls in Taiwan.
Discussion
In this study, we firstly revealed the genetic distribution patterns of IL-13 rs1881457 and rs1800925 genotypes among breast cancer and healthy control females in Taiwan. Noteworthy findings are that the variant genotypes or alleles at either IL-13 rs1881457 or rs1800925 are not major determinants of breast cancer risk in Taiwan (Tables III and IV). In 2010, it was reported that the IL-13 rs1800925 genotypes were associated with serum levels of IL-13 and risk for breast cancer among New Mexican females (39). The genotypes of IL-13 rs1800925 or rs1881457 have not reported be to be associated with breast cancer risk in two genome-wide association studies examining novel biomarkers for breast cancer (40, 41), and were not found to be predictors of poor survival rates among Chinese women (42). The results of these genome-wide association studies conducted in mainland China are consistent with ours. In 2019, Schuetz and his colleagues revalidated the genome-wide association studies and proposed the possibility that different genetic backgrounds and environmental factors may combine to influence one’s susceptibility to breast cancer and contribute to the differences between Eastern and Western populations (43). In 2013, Murray and his colleagues reported that IL-13 rs1800925 was associated with a shorter time to recurrence among those with early stage breast cancer (44). In the current study, we stratified the subjects according to their combined genotypes of IL-13 rs1881457 and rs1800925 and examined their combined effects. The results showed that there was no significant association between the combined genotypes of IL-13 rs1881457 plus rs1800925 and breast cancer risk (data not shown).
The interactions between environmental and genetic factors are also of great interest when investigating their association with cancers. As for breast cancer, in addition to imbalanced diet, excessive alcohol drinking, cigarette smoking, and insufficient physical exercise (2, 3) mentioned in introduction, there are some reports strengthening the concept that other environmental factors and life styles contribute to our personal susceptibility for breast cancer. First, cereal lignans have been reported to elevate the production of not only enterolactone and butyric acid, but also the cellular anticancer capacity (45). Second, soybean can significantly reduce the growth rates of both MCF-7 and MDA-MR-231 breast cancer cells (46). Third, 27-hydroxycholesterol (metabolite of pork cholesterol) can enhance the xenograft growth rates of MCF-7 cells in mice, and increase the risk of ER positive breast cancer (47). Fourth, increased levels of serum cholesterol have been associated with higher risk of breast cancer (48). Fifth, elevated nitrite formation due to improper storage of overnight food can serve as a contributor to higher risk for breast cancer (49). In the current study, we investigated the interaction between IL-13 genotypes and environmental factors including cigarette smoking and alcohol drinking. The results showed that there were neither additive nor synergistic effects of IL-13 (rs1881457 and rs1800925) genotypes and cigarette smoking or alcohol drinking on elevated breast cancer risk (data not shown).
Individuals carrying the T allele of rs1800925 have been reported to have higher levels of serum IL-13 than C allele carriers (50-52). This may be due to the fact that rs1800925 contains a putative binding site for NFAT and the change from C to T at this polymorphic site may increase the binding affinity of nuclear proteins and consequent IL-13 production (25-28).
It has been reported that local IL-13 expression at breast tumor sites directly affects breast tumor cells and promotes carcinogenesis (20). In contrast, it has also been reported that IL-13 is able to suppress the growth rates of several breast cancer cell lines (23, 24). These controversial findings indicate the complex role of IL-13 in breast cancer development and the need for further investigations. The current study did not find a correlation of IL-13 genotypes with breast cancer, but is of great value for determining the involvement of IL-13 in breast cancer progression.
There are other IL-13 polymorphic sites, not located in the promoter region as rs1881457 and rs1800925, that are worth of investigating to reveal the role for IL-13 in breast carcinogenesis. For instance, the polymorphism G+2044A that is located in exon 4 of IL-13 gene and results in a change from arginine to glutamine at the 130th codon, has been reported to correlate with the expression levels of IL-13 in serum. The A allele carriers have been previously reported to have higher serum IL-13 levels than the G allele carriers (25, 27, 53, 54).
In conclusion, the current study provided solid evidence showing that the IL-13 rs1881457 and 1800925 genotypes alone or in combination may not directly contribute to the susceptibility of Taiwanese women to breast cancer. Further investigations about other polymorphic sites of IL-13 and other immune-regulating genes may reveal predictors of breast cancer susceptibility and prognosis outcomes.
Acknowledgements
The Authors thank the Tissue Bank of China Medical University Hospital, and all the participants. The expert genotyping assistance was also kindly provided by Yu-Chen Hsiau, Kai-Cheng Chan, Yu-Ting Chin, Tai-Lin Huang, Yi-Ru Huang and Yu-Hsin. This study was funded mainly by Taichung Veterans General Hospital, (TCVGH-1094801B) and partially by the China Medical University Hospital (DMR-109-143).
Footnotes
↵* These Authors contributed equally to this study.
Author’s Contributions
Chung-Lin Tsai, Chia-Wen Tsai and Wen-Shin Chang designed the overall research; Chen-Hsien Su, Liang-Chih Liu and Hwei-Chung Wang collected samples and summaries questionnaire; Wen-Shin Chang and Yun-Chi Wang performed the genetic and experimental research; Hsu-Tung Lee, Chun-Ming Yen, Jaw-Chyun Chen and Chao-Hsuan Chen contributed to statistical analysis, while Chia-Wen Tsai and Da-Tian Bau contributed to the writing of the article. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received October 10, 2020.
- Revision received November 4, 2020.
- Accepted November 5, 2020.
- Copyright © 2020 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
