TIMP-2 gene rs4789936 polymorphism is associated with increased risk of breast cancer and poor prognosis in Southern Chinese women

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinase-2, and an important regulator of cancer growth and metastasis. Here, we evaluated the relationship between TIMP-2 gene rs4789936 polymorphism and breast cancer risk and prognosis in Southern Chinese women. Analysis of 480 breast cancer cases and 530 healthy controls revealed that compared to the CC genotype, the TT/CT genotypes increased the risk of breast cancer (CT: odds ratio (OR)=1.46, 95%CI: 1.11-1.92; TT: OR=2.57, 95%CI: 1.76-3.77). Stratified and cross-over analyses indicated that the increased risk of the rs4789936 polymorphism was associated with age, smoking, drinking, and the body mass index. The TT and CT genotypes were more common than the CC genotype in patients with lymph node metastases (TT: OR=2.54, 95%CI: 1.54-4.18; CT: OR=2.10, 95%CI: 1.40-3.15), advanced stage (TT: OR=1.91, 95%CI: 1.17-3.14; CT: OR=1.80, 95%CI: 1.20-2.70), or progesterone receptor positive cancer (TT: OR=2.02, 95%CI: 1.23-3.32). Multivariate cox regression indicated the CT and TT genotypes (HR=4.65, 95%CI: 1.26-17.18, P=0.021; HR=6.62, 95%CI: 1.51-29.11, P=0.012) were associated with poor prognosis in breast cancer patients. Thus, the TIMP-2 gene rs4789936 polymorphism is associated with elevated breast cancer risk and may be an independent prognosis factor in breast cancer patients.


INTRODUCTION
Breast cancer is the most common cancer in women worldwide [1], as well as in China [2]. The incidence of breast cancer in China continues to rise rapidly at a rate of 3% to 4% per year, which is double the global average [3]. Even though the breast cancer treatment in China has greatly improved, the overall survival rates are still lower than in other countries [4]. The increasing incidence of breast cancer, the huge population base, and the lower survival prognosis, represent a severe challenge for the prevention and control of breast cancer in China. Therefore, identifying the environmental and genetic risk factors of breast cancer in China is very important to reduce the incidence of breast cancer.
Occurrence and development of breast cancer are affected by many genetic and environmental factors and their interactions [5]. Many potential environmental risk factors of breast cancer have been identified, including smoking [6], drinking [7], diet [8], and hormone exposure [9]. In addition, genetic factors play a very important role in the development and progression of breast cancer [10]. Previous studies have suggested that the increased risk of breast cancer is associated with breast cancer susceptibility gene 1 [11], leptin G-2548A gene [12], interleukin 4 (IL-4) [13], and insulin-like growth factor 1 [14].
Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinase-2 AGING (MMP-2) [15], an important enzyme in the regulation of cancer cell proliferation and metastasis [16]. MMP-2 is over-expressed in cancer tissues, and its increased expression correlates with advanced cancer stage and poor prognosis. TIMP-2 forms a complex with MMP-2, thus regulating MMP-2 proteolytic activity. TIMP-2 mutations on chromosome 17q25 can affect its binding to MMP-2, resulting in cancer development [17]. Previous studies have indicated that TIMP-2 gene polymorphism is associated with gastric cancer [18], lung cancer [19], and prostate cancer [20]. In addition, TIMP-2 gene rs4789936/CT genotype is associated with an increased risk of colorectal cancer compared with CC genotype [21]. A recent study from Northern Chinese population has suggested that the mutation of TIMP-2 gene rs4789936 polymorphism is not associated with an increased risk of breast cancer [22]. However, the results remain controversial due to the different eating habits, environmental factors, and genetic mutation frequency. In this study, we evaluated the relationship between TIMP-2 gene rs4789936 polymorphism and the risk of breast cancer in Southern Chinese population. We also performed a follow-up study to determine the effect of rs4789936 polymorphism on breast cancer prognosis.

Characteristics of study population
The study included 480 breast cancer patients and 530 healthy controls; the main characteristics of the patient case group and the healthy control group are presented in Table 1. There were no significant differences in age (P=0.110), BMI (P=0.405), and drinking (P=0.283). Notably, the distribution of drinking population was significantly different between the case group and the control group (19.4% vs 13.6%, P=0.013). Among the case population, 52.1% of breast cancer patients had tumors with diameter ≥ 2cm, 54.2% of patients belonged to stage I/II, 42.7% of patients were ER positive, 48.5% were PR positive, and 67.5% were her-2 positive. Follow-up revealed that 33.8% of patients received radiation alone, and 66.3% received chemotherapy and radiotherapy.

TIMP-2 gene rs4789936 polymorphism and breast cancer risk
The genotype of control group did not exhibit a departure from Hardy-Weinberg equilibrium (P>0.05). The genotype distributions of case and control groups are presented in Table 2 Table 2. Stratified analysis (Table 3) indicated a significantly increased breast cancer risk in the homozygote model (TT vs CC) that was associated with age, smoking, drinking, and BMI. The heterozygote model (CT vs CC) was significant among population of <60 years, non-smokers and non-drinkers. For BMI>24, the dominant model showed an increased risk of breast cancer.

Gene-environment interactions and breast cancer risk
Using cross-over analysis, we evaluated the geneenvironment interactions (smoking and drinking) and the risk of breast cancer (Table 4). For non-smokers and non-drinkers, the TC genotype was not associated with an increased risk of breast cancer compared to the CC genotype. For smokers, the TC genotype increased the risk of breast cancer (OR=1.51, 95%CI: 1.12-2.02, P=0.006) compared to non-smokers carrying the CC genotype. For drinkers, an elevated risk of breast cancer was found in the TC genotype group compared to nondrinkers carrying the CC genotype (OR=1.53, 95%CI: 1.12-2.08, P=0.007). For non-smokers and nondrinkers, the TT genotype was associated with an increased breast cancer risk compared to the CC genotype, but smoking or drinking alone was not associated with an increased risk of breast cancer. However, drinkers and smokers carrying the TT genotype had a significantly increased risk of breast cancer compared to non-smokers and non-drinkers carrying the CC genotype (OR=5.56, 95%CI: 2.74-11.63, P<0.001; OR=5.12, 95%C: 2.37-11.06, P<0.001). These results suggest that the interactions between the genotype and smoking or drinking are associated with increased breast cancer risk in Southern Chinese women.

Association between rs4789936 polymorphism and clinical parameters of breast cancer patients
We further explored the association between clinical parameters and rs4789936 polymorphism among breast cancer patients (

TIMP-2 gene rs4789936 polymorphism and prognosis of breast cancer patients
Follow-up analysis of breast cancer patients showed a median survival time of 17.6 months. The Kaplan-Meier analysis indicated that the TT/CT genotypes had a poor survival prognosis compared to the CC genotype (CT vs CC: P=0.005; TT vs CC: P<0.001, Figure 1A). Patients with CT and TT genotypes also had poor survival rates (P<0.001, Figure 1B) (Table 6). Advanced stage, lymph node metastases, and Her-2 positive status were also predictors of a poor prognosis.

DISCUSSION
The main findings of this study are the following: (1) The TIMP-2 gene rs4789936 polymorphism is associated with an increased risk of breast cancer in Southern Chinese women; (2) There are interactions between SNP rs4789936 polymorphism, smoking, drinking, and breast cancer risk; (3) The rs4789936 TT and CT genotypes are more frequent in breast cancer patients with lymph node metastases, advanced stage, and PR positive status; (4) The rs4789936 TT and CT genotypes are associated with a poor prognosis in breast cancer patients.
Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of MMPs that are overexpressed in many tumors and stromal tissues [23]. The TIMPs have two functional domains: the N-terminal region containing the MMP binding site, and the C-terminal region [24]. TIMP-2 inhibits the MMP-mediated proteolysis, and TIMP-2 decreased expression has been associated with cancer progression. TIMP-2 forms a stable complex with MMP-1 precursor to prevent MMP-1 activation. In addition, TIMP-2 can bind to MMP and directly inhibit its catalytic activity [25]. A previous study has indicated that the dynamic balance between TIMP-2 and MMP-2 can affect tumor invasion and metastasis [26]. A decreased expression of TIMP in tumor tissues results in increased activity of MMP and its increased degradation of the extracellular matrix, thus promoting tumor cell invasion and metastasis [27,28].
The rs4789936 polymorphism is the transition from C to T that causes a synonymous amino acid change, which can result in dysregulated transcription of the TIMP-2 gene. TIMP-2 gene mutations have been associated with many diseases, including acne vulgaris [29], intracerebral hemorrhage [30], and cancer [31]. A previous study has indicated that TIMP-2 gene rs817990 and rs2277698 polymorphisms affect the risk of primary ovarian insufficiency in women [32]. In addition, two SNPs (rs7501477 and rs8136830) have been associated with an increased risk of breast cancer [33].
A recent case-control study to assess the association between TIMP-2 polymorphism and breast cancer in Northern Chinese population has suggested that the rs2277698 gene polymorphism is associated with a decreased risk of breast cancer [22]. This result is different from our study. Our results show that the T allele of rs4789936 is a risk factor for breast cancer in  Our cross-over analysis has demonstrated that compared to non-smokers and non-drinkers, women with TT/CT genotype, and drinking or smoking, have a significantly elevated risk for breast cancer, indicating a geneenvironment interaction. In addition, we have found that rs4789936 polymorphism is associated with an advanced stage, lymph node metastases, and a PR positive status, indicating that the rs4789936 polymorphism may affect the prognosis of breast cancer patients. A previous study has also suggested that TIMP-2 gene rsrs8136803 TT genotype is associated with a poor disease-free survival [33]. Our data show that the rs4789936 TT/CT genotype is associated with a poor overall survival prognosis compared to the CC genotype, and suggest that the TT/CT genotype might serve as an independent prognosis factor of breast cancer patients. The GTEx data indicate that the rs4789936 TT genotype is quite common in blood samples, and has higher expression levels than the CC genotype. These findings suggest that TIMP-2 regulates breast cancer progression, and that TIMP-2 mutation might result in a poor prognosis in breast cancer patients.
Some of the limitations of this study include the fact that the selected population was recruited from a hospital, and a selection bias may thus exist. Although we identified an association between TIMP-2 gene polymorphism and breast cancer, the potential molecular mechanisms are unclear. There are chances that this SNP has an impact on another gene in which it resides, CEP295NL, because it is position within its 5'UT (HapMap data). Or, probably the truth is that this SNP is in large linkage disequilibrium with a nearby, functional SNP, and further research are required. Besides, since this study only explored geneenvironment interactions, future studies should analyze AGING gene-gene interactions that might affect the susceptibility to breast cancer.
In conclusion, our data demonstrate that the TIMP-2 gene rs4789936 polymorphism is associated with an elevated risk of breast cancer in Southern Chinese women, and indicate that it might serve as an independent prognosis factor for breast cancer patients.

Patient population
The study consisted of a case-control population and a follow-up population of breast cancer patients. For casecontrol population, we enrolled 480 breast cancer patients and 530 healthy controls from the Department of Women's Cancer, Hunan Cancer Hospital (The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University). Criteria for enrollment were the following: All cases were newly diagnosed by pathology diagnosis; they were Han ethnic, and aged from 25 to 70 years old. Cases with other types of tumors, cardiovascular diseases, severe infectious disease, and immune disease were excluded. The control population was recruited from the physical examination center of the hospital during the same period. The follow-up population was from the case group.
General data of all human subjects were collected including age, height, and weight for calculating the body mass index (BMI), and the history of smoking and drinking. For the case group, we also collected the tumor size, lymph node metastases, stage, the status of estrogen receptor (ER), progesterone receptor (PR), Her-2, and treatment. A telephone follow-up was performed, and the primary outcome was overall survival (defined as the time from surgery to death). This study was approved by the ethics committee of Hunan Cancer Hospital. Written informed consent was obtained from all study subjects.

Blood sampling and genotyping
rs4789936 with minor allele frequency> 5% in East Asian population was selected for this study. 5 ml of fasting peripheral blood was collected from each participant, and was stored in anti-coagulative tubes with EDTA-disodium salt. The genomic DNA was extracted by TaKaRa Genome DNA Extraction Kit (Dalian Biological Engineering CO., LTD, China), and stored at -20°C.

AUTHOR CONTRIBUTIONS
LJY designed the study. LGM analyzed and interpreted the data, and wrote the manuscript. LJY proofread the final version. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
This study is supported by the Natural Science Foundation of Hunan Province (2020JJ8063).