Investigation of MMP-9 rs3918242 and TIMP-2 rs8179090 polymorphisms in renal cell carcinoma tissues

Introduction

Kidney cancer is the seventh common cancer type among men (2.7% of all cancer cases) in Turkey [1]. Renal cell carcinoma (RCC) accounts for 90–95% of kidney-forming neoplasms and shows resistance to radiation and chemotherapy [2]. According to the worldwide incidence of renal cell carcinoma, about 209,000 new cases and 102,000 deaths occur each year [3]. The use of molecular markers is important for early diagnosis and prognosis because of the high mortality rate of kidney cancer [4].

Matrix metalloproteinases (MMPs) are extracellular proteases, composed of about 28 enzymes, and play a crucial role in physiological and pathological tissue destruction [5]. Tissue inhibitors of metalloproteinases (TIMPs) inhibit the activation of the latent enzyme form of MMPs and the maintenance of catalytic activity by binding irreversibly and non-covalently to the MMPs. The four types of TIMPs are identified as TIMP-1, TIMP-2, TIMP-3 and TIMP-4 [6], [7], [8]. The disruption of the balance between MMP and TIMP can lead to the pathological processes [9].

MMP-9 known as gelatinase B or type 4 collagenase has proteolytic activity against type 4 collagen which is the major constituent of the membrane base [10]. It is thought that MMP-9 is also effective in early stages of carcinogenesis because of the roles in invasion and metastasis [11]. MMP-9 was found associated with bladder, breast and prostate cancers [12], [13], [14]. A genetic polymorphism in the promoter region (C1562T, rs3918242) of MMP-9 gene causes increased expression levels [15].

TIMP-2 was found associated with prostate cancer, pancreatic cancer, colorectal cancer and gastric cancer [5], [16], [17], [18. It is thought that G418C (rs8179090) polymorphism in the promoter region of the TIMP-2 gene occurs in the Sp-1 binding site and reduces the transcription levels [18]. Thus, we conducted a case-control study for the first time in the renal cell carcinoma tissues to evaluate the relation between MMP-9 (rs3918242) and TIMP-2 (rs8179090) polymorphisms and RCC.

Materials and methods

Results

One hundred tumor tissues and adjacent healthy tissues from the patients with renal cell cancer were included in the study. The mean age of the patient was 59.12±5.89 and the mean tumor diameter was 5.89±2.65 with minimum 2.5 and maximum 13 (data not shown).

MMP-9 rs3918242 polymorphism was not in the HWE (p<0.05), while TIMP-2 rs8179090 was in the HWE (Table 1) (p>0.05). The genotype and allele distributions of the tumor and adjacent tissue are identified in Table 2. There was a significant difference in MMP-9 rs3918242 genotypes and alleles between the tumor and adjacent tissues (p<0.01). MMP-9 rs3918242 T allele was higher in tumor tissues (36.5%) than adjacent tissues (17%) and T allele carrier renal cell cancer tissues had 2.8 odds ratio compared to the adjacent tissues (p<0.001). Tumors with homozygous TT genotype had a significantly higher frequency (21%) than the adjacent tissues (10%, p=0.004). Odds ratio values were found 3.325 in the carriers of TT genotype and 3.5 in the carriers of CT genotype to the carriers of CC genotype (p<0.01).

The polymorphism of TIMP-2 rs8179090 was not found statically significant in tumor tissues than adjacent tissues (p>0.05). The frequency of individuals with C alleles was found higher in the tumor tissues (16.5%) than the adjacent tissues (13.5) (p>0.05).

The relation between MMP-9 rs3918242, TIMP-2 rs8179090 and tumor diameters were analyzed, no correlation was observed (data not shown).

Discussion

We conducted a case-control study in a 100 patients with renal cell cancer. DNA was obtained from renal cancer cells and adjacent healthy kidney tissues and PCR-RFLP method was used. Our results showed that MMP-9 rs3918242 genotypes and alleles between the tumor and adjacent tissues were statistically different (p<0.01). Odds ratio values were 3.325 in TT genotype and 3.5 in CT genotype to the CC genotype (p<0.01). However, TIMP-2 rs8179090 was not found statically significant in tumor tissues than adjacent tissues (p>0.05).

MMP family proteolytic enzymes have been upregulated in almost all types of cancer. Although MMPs are known for their role in cancer invasion and metastasis in terms of degrading extracellular matrix and basement membrane [19], they were also found associated with cancer development stages such as modulation of cell proliferation, apoptosis and angiogenesis [21], [22]. The balance between MMPs and TIMPs levels regulates the extracellular matrix turnover and cell invasion process [23]. MMP-9 rs3918242 polymorphism was not found associated with increased risk for development of osteosarcoma and cervical cancer [24], [25]. However, we found that MMP-9 rs3918242 TT genotype was significantly associated with tumor invasion and increased MMP-9 expression in the previous study. MMP-9 and TIMP-2 expression levels were also found upregulated in urinary bladder tumors [20]. MMP-9 rs3918242 polymorphism was identified by various studies as a risk factor for certain types of cancer. The literature findings about the relation between MMP-9 rs3918242 polymorphism and renal cell carcinoma are limited. Awakura et al. analyzed MMP-9 rs3918242 polymorphism in the genomic DNA from the blood samples in the renal cell carcinoma in Japanese population and did not find any significant association with the risk of RCC [11]. In our study, we found rs3918242 TT genotype and mutant T allele were higher in tumor tissue than adjacent tissue (p<0.01). Our results may not be similar to the study of Awakura et al. since we did not evaluate blood samples, we used the somatic DNA from tumor and adjacent healthy tissue samples from Turkish patients in our study [11].

Chiranjeevi et al. showed that MMP-9 1562C>T polymorphism the T allele was higher in breast cancer [26]. Another study showed that MMP-9 1562C>T CT+TT genotypes were associated with higher serum levels of MMP-9, lower event-free survival and prognosis of the T-cell acute lymphoblastic leukemia [27]. MMP-9 CC genotype was found associated with increased esophageal squamous cell carcinoma (ESCC) and increased risk of death from ESCC [28]. Additionally, in a meta-analysis study, TT genotype was found significantly associated with increased risk of breast cancer and suggested as a risk factor for breast cancer [13]. In our study, we found rs3918242 T allele was higher in renal cell cancer tissues (p<0.001). Furthermore, Odds ratio values were found 3.325 in the carriers of TT genotype and 3.5 in the carriers of CT genotype to the carriers of CC genotype (p<0.01). These odds ratio values detected in cancer tissue compared to the may give us an idea that this somatic change may provide some advantages in environmental adaptation for carcinogenic profile. Thus, T allele may contributes to the renal cell carcinogenesis. Demacq et al. reported that C1562T polymorphism has no effect on MMP-9 activity in plasma [15]. However, Zhang et al. showed that C1562T polymorphism site is important for binding a transcription repressor protein and C-to-T substitution results higher promoter activity. Thus, a transcription repressor protein might bind to C allelic promoter rather than T allelic promoter which mean the activity of transcription has an allele-specific manner [29]. We did not show the MMP-9 rs3918242 effects on metastasis in our study because of the lack of these samples. Although we did not demonstrate MMP-9 gene expression in our study, high T allele and TT+CT genotypes in renal cell cancer tissues may increase carcinogenic profile via increasing MMP-9 activity.

The polymorphism of TIMP-2 418G>C was not found associated with endometrial carcinoma, cervical cancer and bladder cancer [20], [30], [31]. On the other hand, this polymorphism was found associated with prostate, gallbladder and colorectal cancer [32], [33], [34]. Our results demonstrated that there is no statistically significant difference between the genotypes of TIMP-2 in the samples, but the frequency of C allele was found higher in the tumor tissues (16.5%) than the adjacent tissues (13.5) (p>0.05). On the other hand, TIMP-2 CC+GC genotypes were found associated with a moderately increased risk of the head and neck cancer [35]. Yang et al. found TIMP-2 CC+GC genotypes increased risk of gastric cancer by 51% [36]. However, we did not find this relation in renal cell cancer.

Conclusion

This study was the first MMP-9 (rs3918242) and TIMP-2 (rs8179090) polymorphism research in Turkish renal cell carcinoma patients. Although these polymorphisms have been studied in genomic DNA from renal cell carcinoma patients [11], they have not been studied in tissues before. In conclusion, MMP-9 rs3918242 polymorphism T allele may have a role in the carcinogenesis of renal cell carcinoma via increasing gene expression. MMP-9 rs3918242 T allele, TT and CT genotypes can be used as screening biomarker for the detection of renal cell carcinoma. However, our results should be supported by larger sample groups. Moreover, to elucidate the putative roles in early stage of renal cell carcinoma, the gene expression and protein levels of MMP-9 should be investigated. If similar results are demonstrated by these analyses, MMP-9 inhibitors such as Minocycline, DP-b99, MMP-9 neutralizing antibodies and MMP-9 siRNA may be provide a potential strategy for treating renal cell carcinoma [37].