Abstract
MMP-2 and MMP-9 genes have been suggested to play a role in breast cancer. Their functions have been associated with invasion and metastasis of breast cancer; however, their involvement in the development of the disease is not well-established. Herein, we reviewed the literature investigating the association between circulating levels and polymorphisms of MMP-2 and MMP-9 and breast cancer risk. Various studies report conflicting results regarding the relationship of polymorphisms in MMP-2 and MMP-9 and breast cancer risk. Nevertheless, it appears that the T allele in rs243865 and rs2285053 in MMP-2 are associated with reduced risk of breast cancer. In addition, high levels of latent form and low levels of active form of MMP-2 were observed in breast cancer patients compared to controls. For MMP-9, high latent levels and low total levels were found in breast cancer patients compared to controls. Additional studies are needed to comprehend the role of these genes in breast carcinogenesis.
Matrix metalloproteinases (MMP) are a family of Zn2+-dependent endopeptidases responsible for cleaving components of the extracellular matrix (1). They are classified into several families according to their structural differences (1, 2). MMP-2 and -9 comprise the gelatinase family that possesses three fibronectin repeats allowing for degradation of denatured collagen (gelatin) and collagens IV and V (2, 3). These gelatinases degrade collagen in the basement membrane (4), as well as other extracellular matrix components, thus promoting extracellular matrix remodeling and consequently play a key role in several physiological processes, such as tissue repair, wound healing, and cell differentiation (5, 6).
Gelatinases could be involved in carcinogenesis processes, including cell proliferation, angiogenesis, and tumor metastasis through their proteolytic function (7). Indeed, the literature suggests their involvement in several pathological processes critical for cancer development, including inflammation, angiogenesis, and cell proliferation, as well as in tumor progression (8, 9). More specifically, the biological functions of MMP-2 and -9 proteins have been associated with invasive and metastatic stages of breast cancer (10, 11); however, their involvement in breast cancer development is unclear.
Herein, we aimed to review and discuss articles which studied the association between gelatinases and breast cancer risk. For this purpose, an electronic search of the MEDLINE (PubMed) database was performed to identify all published studies that evaluated the association of polymorphisms or circulating levels of MMP-2 and MMP-9 with breast cancer risk.
Matrix Metalloproteinase-2 (MMP-2)
MMP-2 is located on chromosome 16 and codes for gelatinase A. The substrates for this enzyme include gelatin, collagen V, and collagen VI (12). The MMP-2 gene has been studied in several abnormal physiological processes, such as obesity and cancer (13). Polymorphisms that alter the function and efficacy of this protein could be associated with breast cancer risk. Several studies have evaluated this association (14-23).
Circulating Levels of MMP-2 and Breast Cancer Risk
MMP-2 exists in three forms: latent, active and total. These forms vary in their molecular weight, making it possible to measure them in the blood. Enzymatic techniques such as zymography (12, 24) allow quantifying the presence of these different MMP-2 forms in the blood. However, there are other less specific methods to measure circulating levels. Studies that have examined circulating levels of MMP-2 and breast cancer risk have measured either the latent, active or concentrations of both combined (latent plus active) (25-30), but two studies did not specify the form (31, 32). Eight research articles on circulating levels of MMP-2 are presented in Table I.
Latent form (pro-MMP-2). MMP-2 is expressed in its latent form and is activated in the extracellular matrix. This latent form can be measured in either serum or plasma. The molecular weight of pro-MMP-2 is 72 kDa (33). La Rocca et al. (25) found that the serum levels of the pro-MMP-2 form were higher in breast cancer patients than in healthy patients (p<0.0001). The authors used the zymography technique to quantify the enzyme in the study.
Active MMP-2 form (aMMP-2). Active MMP-2 results from the activation of the pro-MMP-2 form by proteolytic cleavage of the N-terminus (34). The molecular weight of this active species is 63 kDa (33). This functional form of MMP-2, as measured in plasma, was reportedly lower in breast cancer patients than in healthy people (100 pg/mg vs. 130 pg/mg, p=0.038) (26). The concentrations of aMMP-2 in serum were also lower in breast cancer patients than in healthy groups (375 pg/mg vs. 725 pg/mg, p<0.001) (27). Thus, circulating levels of aMMP-2 appear to be lower in breast cancer patients than healthy people.
Total MMP-2 (tMMP-2). This form of MMP-2 includes both the pro-enzyme and the active enzyme. Somiari et al. (26) found that the plasma concentrations of tMMP-2 were higher in patients with breast cancer than healthy individuals (1350 pg/mg vs. 900 pg/mg, p=0.002). However, Aroner et al. (30) and Kim et al. (29) did not find any association between plasma levels of tMMP-2 in breast cancer cases and healthy individuals. In addition, two studies revealed that tMMP-2 concentrations, when assessed in the serum, did not differ between breast cancer patients and healthy low risk women (p=0.926) (27) or healthy women (p>0.05) (28). Thus, tMMP-2 does not appear to be associated with breast cancer. These results for tMMP-2 levels are not surprising since opposite associations have been observed for pro-MMP-2 and aMMP-2 levels with the risk of breast cancer.
No specific form. Other authors have studied circulating levels of MMP-2 in breast cancer without identifying the form. Two studies found that serum mean levels of MMP-2 are higher in patients with breast cancer than in healthy patients (694.3 ng/ml vs. 593.3 ng/ml (31), 806.5 ng/ml vs. 771.2 ng/ml (32), p<0.05). These results are nonetheless concordant with those observed between pro-MMP-2 levels and breast cancer risk.
MMP-2 Gene Polymorphisms and Breast Cancer Risk
A total of 37 polymorphisms located on the MMP-2 gene were studied, with most located in the promoter region. The main studied polymorphisms were rs243865, rs2285053, rs243866 and rs243864. Studies of MMP-2 polymorphisms are described in Table II.
rs243865. The rs243865 polymorphism in MMP-2 is a common C→T transition at position -1306 in the promoter. This transition interrupts binding with stimulating protein 1 (Sp1), which is a transcription factor. It has been reported that the T allele reduces the expression of MMP-2 (35). Ten studies investigated the association between rs243865 and breast cancer risk, but the results were unclear (14-23). Several authors have hypothesized that this polymorphism may reduce the risk of breast cancer because of less protein expression. Three studies found a significant association between rs243865 in MMP-2 and risk of breast cancer in Chinese (OR=0.46; 95% CI=0.34-0.63; p=0.00001) (14), Mexican (OR=0.47; 95% CI=0.24-0.88; p=0.01) (17) and Tunisian (OR=0.39; 95% CI=0.25-0.72) (22) populations. These three studies used the dominant model and found that CT+TT genotypes reduced the risk of breast cancer compared to the CC genotype. However, Saeed et al. (19) also used this same dominant model in the Saudi population and found that CT+TT genotypes increased the risk of breast cancer compared to the CC genotype (OR=2.12; 95% CI=1.09-4.11; p=0.025). It is important to note that, deviation from Hardy-Weinberg equilibrium was observed for rs243865 in this study (19). Using a recessive model in a Caucasian-Hispanic population, Slattery et al. (21) observed that the association between TT carriers compared to CT+CC carriers was significant (OR=0.84; 95% CI=0.73-0.97) after adjustment for body mass index and other risk factors. Additionally, five studies analyzed the relation between rs243865 in MMP-2 and breast cancer risk in Caucasian (15, 20), Brazilian (16), Chinese (18) and Tunisian (23) populations, but did not find any association. In contrast with studies reporting decreased breast cancer risk with rs243865, these four former studies used an additive model. Increasing the number of copies of the T allele did not affect the risk of breast cancer. Habel et al. (23) compared the T allele to the C allele of rs243865 in MMP-2 in the Tunisian population and demonstrated that the T allele was not associated with the risk of breast cancer. Taken together, we cannot exclude the possibility that the T allele may reduce breast cancer risk in some populations.
rs243866. rs243866 is a G to A transition located in the MMP-2 promoter, at position -1575. MMP-2 is estrogen-responsive, but the -1575 G→A transition appears to be an incomplete palindromic binding site for estrogen receptor and the -1575A allele reduces the transcriptional activity of MMP-2 (36). According to two studies in Chinese (18) and Tunisian (23) populations, rs243866 was not associated with breast cancer risk (p>0.05). These results are consistent with the fact that the G and A alleles have similar allelic expression. The A allele is likely non-functional (35).
rs243864. This polymorphism is located in the MMP-2 promoter at position -790 and involves a transition of the common allele G to T. The functional significance of the wild T allele is unclear. The rs243864 polymorphism has been studied in Chinese (18) and Tunisian (23) populations, but none of these studies found an association between this polymorphism and breast cancer risk (p>0.05).
rs2285053. rs2285053 is located in the MMP-2 promoter at position -735 and implicates a transition of the common allele C to T. To our knowledge, the biological significance of the wild T allele is undefined. However, Yu et al. (37) have reported that according to bioinformatics analyses, rs2285053 in MMP-2 could alter a Sp1 binding site and influence MMP-2 transcription. Three studies were identified that investigated the association between rs2285053 in MMP-2 and breast cancer risk. Two studies showed that the rs2285053 T allele rather than the C allele reduced the risk of breast cancer in the Tunisian (OR=0.59; 95% CI=0.46-0.75) (23) and Iranian (OR=0.61; 95% CI=0.37-0.99; p=0.049) (38) populations. However, Beeghly-Fadiel et al. (18) did not find any association between CC, CT, TT genotypes of rs2285053 and breast cancer risk in additive models (p=0.436). Based on this evidence, the T allele may affect breast cancer risk.
Other polymorphisms in MMP-2. Thirty-three other polymorphisms in MMP-2 were also examined in two studies (18, 21). However, none of these polymorphisms was associated with breast cancer risk in the study populations, except for rs11541998 that CG+GG increased breast cancer risk compared to CC (OR=1.16; 95% CI=1.02-1.31) (21).
Haplotypes in MMP-2. Haplotype analyses have also been performed. Beeghly-Fadiel et al. (18) observed significant haplotype effects of rs11644561 and rs11643630 on breast cancer risk. The authors found that the haplotype with minor alleles (AG) for both SNPs was associated with reduced breast cancer risk (OR=0.6; 95% CI=0.4-0.8; p=0.003) compared to the haplotype with both major alleles (GT). In the same study, no significant haplotype effects for rs243865 and rs2285053 were observed. However, Habel et al. (23) found that patients who had GCTT and GTTC combinations of rs243866, rs243865, rs243864 and rs2285053 respectively had a lower risk of breast cancer (GCTT: OR=0.49, 95% CI=0.25-0.94; GTTC: OR=0.39, 95% CI=0.19-0.81) than those who had GCTC haplotypes. Several studies are needed to clarify the haplotype effect of polymorphisms located in MMP-2 gene on breast cancer risk.
Matrix Metalloproteinase-9 (MMP-9)
The MMP-9 gene is located on chromosome 20 and encodes the gelatinase B protein. MMP-9 expression is either lower or absent in normal tissues, and elevated in inflammation and wound healing (39). The main substrates for this enzyme include gelatin, collagen IV, and V (12).
Circulating Levels of MMP-9 and Breast Cancer Risk
Few studies have evaluated the role of circulating levels of MMP-9 in carcinogenesis. Similar to gelatinase A, gelatinase B is also translated into a pro-enzymatic form and activated in the extracellular space. Six studies of circulating levels of MMP-9 are presented in Table I.
Pro-MMP-9. This form is the latent form of MMP-9 and has a molecular weight of 92 kDa (33). In a study conducted by La Rocca et al., the serum concentrations of pro-MMP-9 were significantly higher in women with breast cancer than in healthy women (p<0.0001) (25).
Active MMP-9 (aMMP-9). This form of MMP-9 is the functional form of the enzyme, which binds to different substrates of MMP-9 for degradation. The molecular weight of aMMP-9 is 87 kDa (33). The plasma concentrations of aMMP-9 were found to be higher in breast cancer patients compared to healthy low risk participants (p=0.015) (26). However, serum aMMP-9 concentrations did not differ between women with breast cancer and healthy women (27). Therefore, the association between aMMP-9 and breast cancer has not been clarified yet; further studies are needed to clarify the role of aMMP-9 in breast cancer.
Total MMP-9. Total MMP-9 (tMMP-9) consists of the pro-MMP-9 and active MMP-9 forms. Plasma MMP-9 levels were found to be lower in breast cancer patients than in healthy low risk women (p=0.013) (26). In a small population, Katunina et al. (28) found that circulating levels of MMP-9 in serum were also lower in breast cancer cases than in controls (p<0.05). However, one study found no differences in serum MMP-9 levels between breast cancer patients and healthy women (p=0.177) (27). Taken together, it is possible that high tMMP-9 levels could be associated with lower breast cancer risk.
No specific form. Two other studies evaluated the circulating levels of MMP-9, though without specifying the form, in breast cancer (32, 40). These studies demonstrated that circulating serum levels of MMP-9 were higher in breast cancer patients than healthy women (p<0.05). These results are concordant with those observed for pro-MMP-9 levels and breast cancer risk.
Polymorphisms Located in the MMP-9 Gene and Breast Cancer Risk
Ten polymorphisms located in the MMP-9 gene have been studied in breast cancer risk, four of which have been the most investigated, namely: rs3918242, rs17576, rs2274756, rs2250889. Studies of the MMP-9 polymorphisms are described in Table II.
rs3918242. The rs3918242 in the MMP-9 promoter is the most studied polymorphism for its relation to breast cancer risk. This polymorphism involves a C to T transition. The presence of the T allele leads to the loss of a nuclear repressor protein binding site and increases the expression of gelatinase B (41). The relationship between rs3918242 and breast cancer risk was unclear in the literature. The majority of studies did not find an association between this polymorphism and breast cancer risk in Caucasian (15) and Brazilian (16, 42) populations (p>0.05). However, Chiranjeevi et al. (43) suggested a decreased risk of breast cancer in the Indian population using additive and recessive models, although deviation from Hardy-Weinberg equilibrium was observed for rs3918242 in this study. Similarly, Padala et al. (44) found that the TT genotype increased the risk of breast cancer, but also observed deviation from Hardy-Weinberg equilibrium for rs3918242. One study conducted in the Iranian population showed an increased risk of breast cancer using additive and dominant models (45). In total, literature evidence suggests that rs3918242 in MMP-9 does not influence breast cancer risk.
rs2274756. This polymorphism is a G→A transition in exon 12 of MMP-9 gene. In previous studies, it was not associated with breast cancer risk in Asian (p=0.056) (46) and Caucasian (p=0.19) (47) populations.
rs2250889. The rs2250889 polymorphism is a G→C transition located in exon 10. Two studies in the Asian population have demonstrated conflicting results. Beeghly-Fadiel et al. (46) did not find any association between rs2250889 and breast cancer risk. On the other hand, Chachil et al. (48) analyzed a small population and showed that the GG genotype increased the risk of breast cancer (OR=10.84; 95% CI=1.31-89.83) compared to CC.
rs17576. The rs17576 is an A→G transition located in exon 6. The G allele alters protein conformation and changes substrate-binding and enzyme activity (49). The GA and GG genotypes of rs17576 compared to AA were associated with increased breast cancer risk in Caucasian populations (GA: OR=1.21; 95% CI=1.04-1.40; GG: OR=1.46; 95% CI=1.08-1.96; p=0.01) (47). The GG genotype was also associated with increased breast cancer risk compared to AA in Asian populations (OR=4.73; 95% CI=1.44-15.54) (48), but deviation from Hardy-Weinberg equilibrium was observed in this study. However, conflicting evidence was presented in a study by Beeghly-Fadiel et al., who did not find any association between rs17576 and breast cancer risk (p=0.905) in an Asian population (46).
Other polymorphisms in MMP-9. Some other polymorphisms in MMP-9 [rs6065912 (46), rs4810482 (46), rs3918241 (46), rs3918249 (46), rs3918262 (46), rs3787268 (21)] have been investigated in relation to breast cancer risk. No evidence has been found to support an association between those polymorphisms and breast cancer risk.
Haplotypes in MMP-9. To our knowledge, one study performed haplotype analysis for polymorphisms (rs6065912, rs4810482, rs3918241, rs3918249, rs17576, rs2250889, rs2274756) in MMP-9 gene, but no significant associations were revealed (46).
Conclusion
Review of evidence on the relation between 37 polymorphisms located in MMP-2 gene and breast cancer risk showed that most of them were not associated with breast cancer risk. Among these polymorphisms, the T allele of rs243865 and rs2285053 was the only one that has been associated with reduced expression of MMP-2 as well as with decreased breast cancer risk. However, functional analyses of rs2285053 polymorphism are required. These observations are consistent with the results of circulating levels of latent or unspecified forms of MMP-2 that appear to be higher in breast cancer patients than in healthy women. Conversely, aMMP-2 levels appeared to be lower in breast cancer cases than in healthy women. Since it is unclear why levels of pro-MMP-2 and aMMP-2 are associated in opposite directions with breast cancer risk, it would be useful for future studies to clarify why the direction of this association depends on the forms of MMP-2.
Concerning the 10 studied polymorphisms located in MMP-9, only rs17576 G allele was reported to influence breast cancer risk. However, only a few studies have evaluated this association. Additional, functional analyses and studies with homogenous populations are required. Similar to MMP-2, circulating levels of latent or unspecified forms of MMP-9 were higher in breast cancer patients than healthy women, while the opposite was observed for total MMP-9 levels. Thus, several analyses are necessary to clarify why different forms of MMP-9 are differentially associated with breast cancer risk. Furthermore, most studies so far have measured MMP-9 in serum. However, the literature suggests that serum samples are not appropriate to assess MMP-9 concentrations (50, 51). Hence, future studies should focus on plasma levels in order to investigate the association between different forms of circulating MMP-9 and breast cancer risk.
Footnotes
Authors' Contributions
SGD and CD designed the review, wrote the manuscript and analyzed results of literature research. SGD, CD and SLC reviewed the article.
This article is freely accessible online.
Conflicts of Interest
The Authors declare that they have no competing interests.
- Received April 21, 2020.
- Revision received May 19, 2020.
- Accepted May 28, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved