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1.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
2.  Profiling of matrix metalloproteinases and tissue inhibitors of metalloproteinases proteins in bladder urothelial carcinoma 
Oncology Letters  2010;1(4):691-695.
We investigated the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) proteins in transitional cell carcinoma (TCC) cell lines and surgical specimens of the bladder neoplasm. The expression level was correlated to the degree of cellular differentiation and invasiveness of bladder cancer. Panels of six TCC cell lines with different degrees of differentiation were tested with monoclonal antibodies (mAbs) to MMP-1, MMP-2, MMP-3, MMP-9a, MMP-9b, TIMP-1 and TIMP-2 by immunocytochemistry. Gelatin zymography was also conducted on the cell lines for MMP-2 and -9. In addition, immunohistochemistry with the mAbs to MMP and TIM molecules was performed on 30 TCC specimens. We found that TCC cell lines were stained positively for MMP-1 (6/6), weakly for MMP-9a (2/6), MMP9b (5/6) and TIMP-1 (3/6), and negatively for MMP-2 (3/6) and MMP-3 (3/6). Zymographic analysis of the cell lines showed a high level of MMP2 in the MGH-U4 cell line. In bladder cancer surgical specimens, all specimens were positive for MMP1 (30/30), 19 were positive for MMP-2 (63.3%), 21 positive for MMP-9a (70%) and 15 positive for MMP-9b (50%). The expression of MMP-2 was found to be positively correlated with higher-grade tumors (p=0.036) and the expression of MMP-9a and -9b was found to be positively correlated with tumor stage (p=0.012 and 0.023, respectively). However, the expression of MMP-1, MMP-3, TIMP-1 and TIMP-2 was not correlated with either tumor staging or grading. In conclusion, the expression of MMP-2 and -9 was correlated with high-grade or high-stage bladder tumors, respectively. However, this correlation was not observed with TCC cell lines in which high- and low-grade tumors are included. Immunohistochemical results on tumor lesions may have more clinical relevance, since in a given tumor microenvironment the interaction among tumor cells in situ and tumor-associated cells, such as neutrophils, macrophages, lymphocytes and endothelial cells, as well as environmental factors (hypoxia and pH), cytokines and growth factors released by these cells may be required for TCC to express selective MMPs and TIMPs. The selective expression of these molecules then regulates tumor progression.
PMCID: PMC3436408  PMID: 22966365
bladder neoplasm; metastasis; matrix metalloproteinases; cell line; tissue inhibitors of metalloproteinases
3.  Expression of matrix metalloproteinases and their inhibitors in different immunohistochemical-based molecular subtypes of breast cancer 
BMC Cancer  2014;14:959.
Metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) are involved in several key pathways of tumor growth, invasion and metastasis, but little is known about their expression according to different molecular subtypes of breast cancer. The aims of this study were to assess the prevalence and clinical significance of MMP and TIMP expression in invasive breast cancer and to determine its association with immunohistochemical-based molecular classification.
Tissue microarray sections were immunostained for estrogen receptor-α (ER-α), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin (CK) 5/6, epidermal growth factor receptor (EGFR) and with specific antibodies against MMP-1, 2, 7, 9, 11, 13, and 14 and TIMP-1, 2, and 3. Based on the immunostaining data from five of the markers used (ER-α, PR, HER2, EGFR and CK5/6), three major subtypes (123 luminal A, 31 basal-like, and 17 HER2-overexpressing) were selected.
Statistically significant differences in the expression of MMPs and TIMPs among the three subtypes were found in tumoral MMP7 (P = 0.005), tumoral MMP-9 (P = 0.000), tumoral MMP-13 (P = 0.016) and stromal MMP-13 (P = 0.016). The incidence of tumoral MMP-9 expression in the HER2-overexpressing subtype was significantly higher than in the luminal A subtype (P = 0.021). Tumoral MMP-9 and stromal MMP-13 expression were significantly higher in the HER2-overexpressing subtype than in the basal-like subtype (P = 0.000 and P = 0.016, respectively). Tumoral MMP-7 expression was significantly higher in the basal-like subtype compared to luminal A (P = 0.007) and HER2-overexpressing subtype (P = 0.004). Tumoral MMP-13 showed a higher expression in the basal-like subtype than in the HER2-overexpressing subtype (P = 0.010). In multivariate analysis, stage and stromal MMP-1 expression were significantly related to overall survival. Stage was of independent prognostic significance for disease-free survival.
We found some variations in MMP and TIMP expression among the immunohistochemical-based molecular subtypes of breast carcinomas, suggesting differences in their tumor pathophysiology. Additional studies are needed to determine the mechanisms underlying the differences of MMP and TIMP expression in the molecular subtypes for the development of specific therapeutic targets for breast cancer subtypes.
PMCID: PMC4301952  PMID: 25510449
Breast cancer; Molecular subtype; Immunohistochemistry; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase
4.  Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential 
BMC Cancer  2009;9:20.
The metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including breast cancer. The role of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in the metastatic process has previously been established. However, in all published studies only a limited number of MMPs/MMP inhibitors was analyzed in a limited number of cell lines. Here, we propose a more comprehensive approach by analyzing the expression levels of several MMPs (MMP-2, MMP-9 and MMP-14) and MMP inhibitors (TIMP-1, TIMP-2 and RECK) in different models (five human breast cancer cell lines, 72 primary breast tumors and 30 adjacent normal tissues).
We analyzed the expression levels of MMP-2, MMP-9 and MMP-14 and their inhibitors (TIMP-1, TIMP-2 and RECK) by quantitative RT-PCR (qRT-PCR) in five human breast cancer cell lines presenting increased invasiveness and metastatic potential, 72 primary breast tumors and 30 adjacent normal tissues. Moreover, the role of cell-extracellular matrix elements interactions in the regulation of expression and activity of MMPs and their inhibitors was analyzed by culturing these cell lines on plastic or on artificial ECM (Matrigel).
The results demonstrated that MMPs mRNA expression levels displayed a positive and statistically significant correlation with the transcriptional expression levels of their inhibitors both in the cell line models and in the tumor tissue samples. Furthermore, the expression of all MMP inhibitors was modulated by cell-Matrigel contact only in highly invasive and metastatic cell lines. The enzyme/inhibitor balance at the transcriptional level significantly favors the enzyme which is more evident in tumor than in adjacent non-tumor tissue samples.
Our results suggest that the expression of MMPs and their inhibitors, at least at the transcriptional level, might be regulated by common factors and signaling pathways. Therefore, the multi-factorial analysis of these molecules could provide new and independent prognostic information contributing to the determination of more adequate therapy strategies for each patient.
PMCID: PMC2631003  PMID: 19144199
5.  Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma 
AIM: The expressive balance between matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor of metalloproteinase-1 (TIMP-1) plays a critical role in maintaining the degradation and synthesis of extracellular matrix. Loss of such balance is associated with invasion and metastasis of tumors. This study aimed to determine the expression of MMP-9 and TIMP-1 in gastric carcinoma, and the association of the expressive imbalance between MMP-9 and TIMP-1 with the invasion and metastasis and prognosis of gastric carcinoma.
METHODS: We used immunohistochemistry to determine the expressions of MMP-9, TIMP-1 and proliferating cell nuclear antigen Ki-67 in the gastric specimens taken from 256 patients with primary gastric carcinoma. The patients were followed-up for up to 96 months.
RESULTS: No association between the expression of MMP-9 and TIMP-1 and patients’ sex and age, tumor size and location of gastric carcinoma was observed. The incidence of the positive expression of MMP-9 in cases with tumors invasion to muscularis propria and visceral peritoneum (70.13% and 69.09%, respectively) was significantly higher than that in cases with tumor invasion only to lamina propria or submucosa (42.50%, P = 0.0162). The positive correlation between MMP-9 expression and the depth of tumor invasion was observed (Pearson correlation coefficient = 0.2129, P = 0.016). Along with the increase of the metastatic station of lymph nodes, the incidence of the MMP-9 expression was increased by degrees; a positive correlation between them was observed (Pearson correlation coefficient = 0.2910, P = 0.0001). There was also a significant correlation between MMP-9 expression and the TNM stage in gastric carcinoma (Pearson correlation coefficient = 0.3027, P < 0.0001). The incidence of MMP-9 expression in stage II and III/IV (75.00% and 76.15%, respectively) was significantly higher than those in stage I (46.15%, P < 0.0001). A negative correlation between TIMP-1 immunoreactivity and the depth of invasion, status of lymph node metastasis and TNM stage was observed (Pearson correlation coefficient = -0.1688, -0.3556 and -0.3004, P = 0.023, < 0.0001 and < 0.0001, respectively). Four types of co-expression of MMP-9 and TIMP-1 were observed; i.e. MMP-9 positive but TIMP-1 negative (n = 115), both positive (n = 52), both negative (n = 62) and MMP-9 negative but TIMP-1 positive (n = 27). The frequency of serosal invasiveness was significant higher in patients with MMP-9 but without TIMP-1 expression than those with other types of the co-expression (P = 0.0303). The incidence of lymph node metastasis was highest in patients with MMP-9 but without TIMP-1 expression, and lowest in those with TIMP-1 but without MMP-9 expression (P < 0.0001). The survival rate in patients with MMP-9 but without TIMP-1 expression was lower than that in those with TIMP-1 but without MMP-9 expression (P = 0.0014).
CONCLUSION: Our results in gastric carcinoma demonstrated a significant positive association of MMP-9 over-expression with proliferation of tumor cells, the depth of invasiveness, lymph node metastasis and TNM stage, suggesting MMP-9 can serve as a molecular marker of tumor invasion and metastasis. We also demonstrate a significant negative relationship of TIMP-1 expression with the depth of invasiveness and lymph node metastasis, which provide a new idea in the tumor biological and genetic treatment. The interaction between MMP-9 and TIMP-1 in the processes of tumor invasion and metastasis is that MMP-9 mainly promotes tumor invasion and metastasis and TIMP-1 inhibits functions of MMP-9. The imbalance between MMP-9 and TIMP-1 expression may suggest the occurrence of tumor invasion and metastasis, predict poor prognosis. For patients with imbalanced MMP-9 and TIMP-1 expression, the optimal treatment scheme needs to be selected.
PMCID: PMC4611394  PMID: 12717827
6.  Matrix metalloproteinase-9, -10, and tissue inhibitor of matrix metalloproteinases-1 blood levels as biomarkers of severity and mortality in sepsis 
Critical Care  2009;13(5):R158.
Matrix metalloproteinases (MMPs) play a role in infectious diseases through extracellular matrix (ECM) degradation, which favors the migration of immune cells from the bloodstream to sites of inflammation. Although higher levels of MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) have been found in small series of patients with sepsis, MMP-10 levels have not been studied in this setting. The objective of this study was to determine the predictive value of MMP-9, MMP-10, and TIMP-1 on clinical severity and mortality in a large series of patients with severe sepsis.
This was a multicenter, observational, and prospective study carried out in six Spanish Intensive Care Units. We included 192 (125 surviving and 67 nonsurviving) patients with severe sepsis and 50 age- and sex-matched healthy controls in the study. Serum levels of MMP-9, MMP-10, TIMP-1, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-10 were measured in patients with severe sepsis at the time of diagnosis and in healthy controls.
Sepsis patients had higher levels of MMP-10 and TIMP-1, higher MMP-10/TIMP-1 ratios, and lower MMP-9/TIMP-1 ratios than did healthy controls (P < 0.001). An association was found between MMP-9, MMP-10, TIMP-1, and MMP-9/TIMP-1 ratios and parameters of sepsis severity, assessed by the SOFA score, the APACHE-II score, lactic acid, platelet count, and markers of coagulopathy. Nonsurviving sepsis patients had lower levels of MMP-9 (P = 0.037), higher levels of TIMP-1 (P < 0.001), lower MMP-9/TIMP-1 ratio (P = 0.003), higher levels of IL-10 (P < 0.001), and lower TNF-α/IL-10 ratio than did surviving patients. An association was found between MMP-9, MMP-10, and TIMP-1 levels, and TNF-α and IL-10 levels. The risk of death in sepsis patients with TIMP-1 values greater than 531 ng/ml was 80% higher than that in patients with lower values (RR = 1.80; 95% CI = 1.13 to 2.87;P = 0.01; sensitivity = 0.73; specificity = 0.45).
The novel findings of our study on patients with severe sepsis (to our knowledge, the largest series reporting data about MMP levels in sepsis) are that reduced MMP-9/TIMP-1 ratios and increased MMP-10 levels may be of great pathophysiologic significance in terms of severity and mortality, and that TIMP-1 levels may represent a biomarker to predict the clinical outcome of patients with sepsis.
PMCID: PMC2784384  PMID: 19799791
7.  Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion 
Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion.
Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided.
Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04).
This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.
PMCID: PMC2242664  PMID: 17922906
8.  TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells 
BMC Cancer  2012;12:26.
Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.
The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays.
In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors.
Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.
PMCID: PMC3277461  PMID: 22260435
9.  Clinical significance and prognostic value of S100A4 and matrix metalloproteinase-14 in patients with organ-confined bladder cancer 
Various therapeutic modalities are available for treatment of bladder cancer, and their effectiveness and patient outcome often depend on cancer cell invasiveness. However, the mechanisms underlying the early steps of bladder cancer cell invasion remain unknown. This study aimed to clarify the relationships between S100A4 expression and bladder cancer invasion of surrounding muscles, prognosis and expression of matrix metalloproteinase (MMP)-14 in patients with organ-confined bladder cancer. S100A4 and MMP-14 expression was analyzed in 85 cases of organ-confined (pTa, pT1 and pT2) bladder cancer using immunohistochemical technique. The expression levels were compared among the pTa, pT1 and pT2 tumors. In addition, the predictive values of S100A4 or MMP-14 expression for muscle invasion, metastasis and survival were investigated, as was the possible correlation between the expression of the two proteins. The proportion of S100A4-positive cancer cells in pT2 tumors (53%) was significantly higher (p<0.001) than in pTa (38.7%) or pT1 (40.9%) tumors; there was no difference between pTa and pT1. The results were similar for MMP-14 expression, which was significantly correlated with S100A4 expression (r=0.360, p<0.001). S100A4 expression predicted metastasis-free survival (p=0.009), but not cause-specific survival. The results implicated S100A4 in the early steps of muscle invasion via MMP-14, but not for mucosal invasion. S100A4 is therefore a potential therapeutic target for bladder cancer, and its expression is a risk factor for muscle invasion in patients with superficial tumors. In addition, S100A4 expression may be a useful prognostic factor for metastasis in patients with organ-confined bladder cancer.
PMCID: PMC3490373  PMID: 23136588
bladder cancer; S100A4; matrix metalloproteinase-14; muscle invasion; disease-specific survival
10.  Expression of the Matrix Metalloproteases 2, 14, 24, and 25 and Tissue Inhibitor 3 as Potential Molecular Markers in Advanced Human Gastric Cancer 
Disease Markers  2014;2014:285906.
Background. During progression of gastric cancer (GC), degradation of the extracellular matrix is mediated by the matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs): changes in the expression of these have been related to unfavorable prognosis in GC. Objective. To analyze the expression of certain MMPs and TIMPs in chronic superficial gastritis (SG) and GC. Methods. The expression of MMPs and TIMPs was determined using qRT-PCR; the expression was classified, using threshold cycle (CT) values, as very high (CT ≤ 25), high (CT = 26–30), moderate (CT = 31–35), low (CT = 36–39), or not detected (CT = 40). Strength of association was estimated between the proteins, which were detected by Western blot, and the risk of developing GC. Results. We found a high expression of MMP1, MMP2, MMP14, TIMP1, and TIMP3; moderate one of MMP9 and MMP25, and low one of MMP13 and MMP24 in both tissues. In absolute mRNA levels, significant differences were found in expression of MMP2, MMP24, and MMP25, which are overexpressed in GC compared with SG. The presence of the proteins MMP-14 and TIMP-3 was associated with the risk of developing GC. Conclusions. We consider that MMP2, MMP24, and MMP25 and the proteins MMP-14 and TIMP-3 could be candidates for prognostic molecular markers in GC.
PMCID: PMC3942291  PMID: 24669030
11.  Tumor-derived Matrix Metalloproteinase-13 (MMP-13) correlates with poor prognoses of invasive breast cancer 
BMC Cancer  2008;8:83.
Experimental evidence suggests that matrix metalloproteinase-13 (MMP-13) protein may promote breast tumor progression. However, its relevance to the progression of human breast cancer is yet to be established. Furthermore, it is not clear whether MMP-13 can be used as an independent breast cancer biomarker. This study was conducted to assess the expression profile of MMP-13 protein in invasive breast carcinomas to determine its diagnostic and prognostic significance, as well as its correlation with other biomarkers including estrogen receptor (ER), progesterone receptor (PR), Her-2/neu, MMP-2, MMP-9, tissue inhibitor of MMP-1 and -2 (TIMP-1 and TIMP-2).
Immunohistochemistry (IHC) was performed on paraffin-embedded tissue microarray containing specimens from 263 breast carcinomas. The intensity and the extent of IHC were scored by pathologists in blind fashion. The correlation of the gene expression profiles with patients' clinicopathological features and clinical outcomes were analyzed for statistical significance.
MMP-13 protein was detected in the cytoplasm of the malignant cells and the peritumoral stromal cells. MMP-13 expression by tumor cells (p < 0.001) and stromal fibroblasts (p <0.001) both correlated with carcinoma infiltration of lymph nodes. MMP-13 also correlated with the expression of Her-2/neu (p = 0.015) and TIMP-1 (p < 0.010), respectively in tumor cells. Tumor-derived, but not stromal fibroblast-derived, MMP-13 correlated with aggressive tumor phenotypes. Moreover, high levels of MMP-13 expression were associated with decreased overall survival. In parallel, the prognostic value of MMP-13 expressed by peritumoral fibroblasts seems less significant. Our data suggest that lymph node status, tumor size, Her-2/neu expression, TIMP-1 and MMP-13 expression in cancer cells are independent prognostic factors.
Tumor-derived, but not stromal fibroblast-derived, MMP-13 correlated with aggressive tumor phenotypes, and inversely correlated with the overall survival of breast cancer patients. MMP-13 may serve as an independent prognostic factor for invasive breast cancer patients. MMP-13 may be particularly useful as a prognostic marker when evaluated along with Her-2/neu and lymph node status.
PMCID: PMC2362123  PMID: 18373849
12.  The biochemical value of urinary metalloproteinases 3 and 9 in diagnosis and prognosis of bladder cancer in Egypt 
Matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis. Few studies are available that describe this association with bladder cancer either related or unrelated to schistosoma infection.
Evaluating the urinary levels of MMP3 and MMP9 as diagnostic and prognostic biomarkers in different stages of schistosomal and non schistosomal bladder cancer was the aim of the present study.
Urine samples were collected from 70 patients with schistosomal and non schistosomal bladder cancer at early and advanced stages and also from12 healthy volunteers as controls. Urinary levels of MMP-3 and MMP-9 was measured by ELISA technique. Sensitivity and specificity of both markers were determined.
Urinary levels of both MMP-3 and MMP-9 were significantly elevated in all bladder cancer patients compared with controls. MMP-3 started to elevate in early stages of schistosomal bladder cancer ( 0.173 ng/ml) and non-schistosomal bladder cancer patients (0.308 ng/ml) compared to control (0.016 ng/ml) and remained elevated in advanced stages (0.166, 0.235 ng/ml) of both types of bladder cancer patients. In contrast, MMP-9 showed a significant elevation in advanced stages only of both schistosomal and non schistosomal bladder cancer patients (10.33, 21.22 ng/ml) compared to control (0.409 ng/ml) and this elevation of both markers was much higher in non schistosomal bladder cancer. Both Metalloproteinases were specific for the diagnosis of the disease but MMP-3 was more sensitive and this sensitivity was evident in the early stage (84.85% for MMP3, 27.28% for MMP9).
MMP3 may be the recommended urinary metalloproteinases as early diagnostic biomarker in the early stages of both types of bladder cancer although both MMP9 and MMP3 can be used in the diagnosis of advanced stages. Further studies are required on large number of urine samples to confirm these results.
PMCID: PMC4237805  PMID: 25135219
Bladder cancer; Schistosoma; MMP3,MMP9
13.  Expression of Matrix Metalloproteinases and Their Inhibitors in Squamous Cell Carcinoma of the Tonsil and Their Clinical Significance 
To investigate expressions of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in squamous cell carcinoma of the tonsil and to correlate expression profiles with clinicopathological characteristics.
Paraffin blocks were obtained from 45 tonsil squamous cell carcinoma (SCC) patients, who underwent surgery as an initial treatment between 1994 and 2004, and from 20 normal controls. Expressions of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 were investigated immunohistochemically.
The expressions of MMPs (except MMP-2) and TIMPs were found to be significantly different in tonsil SCC and normal control tissues. Furthermore, MMP-13 expression was found to be correlated with tumor invasion (P=0.05), and the expressions of MMP-9 and TIMP-1 with nodal metastasis (P=0.048, 0.031). No relation was found between MMP or TIMP expression and recurrence. However, MMP-9 expression was found to be significantly associated with 5-year survival in tonsil SCC patients by multivariate analysis (hazard ratio, 3.853; P=0.013).
Significant overexpressions of multiple MMPs and TIMPs were found in tonsil SCC tissues. Furthermore, our findings suggest that MMP-9 expression might be a useful prognostic factor.
PMCID: PMC3109333  PMID: 21716956
MMP; TIMP; Tonsil; Squamous cell carcinoma; Prognosis
14.  Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in pediatric human sarcoma cell lines  
International Journal of Oncology  2013;43(4): 1027 - 1035 .
Pediatric sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on activity of u-PA, MMPs and TIMPs in various human pediatric sarcomas. Human osteosarcoma MNNG-HOS, osteosarcoma U-2OS and rhabdomyosarcoma RD cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 μ g/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All sarcoma cell lines studied expressed u-PA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, osteosarcoma MNNG-HOS showed a band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. U-2OS osteosarcoma cells showed strong bands corresponding to inactive MMP-2 and MMP-9 and faint bands corresponding to active MMP-2 and MMP-9 dimer; PMA treatment enhanced MMP-9 and MMP-9 dimer activity. Rhabdomyosarcoma showed MMP-2 and faint MMP-9 bands; PMA treatment enhanced MMP-9 expression. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of pediatric sarcomas.
doi: 10.3892/ijo.2013.2031
PMCID: PMC3829799  PMID: 23900236
osteosarcoma MNNG-HOS and U-2OS ;  rhabdomyosarcoma RD ;  urokinase plasminogen activator ;  matrix metalloproteinase-2 ;  matrix metalloproteinase-9 ;  tissue inhibitor of metalloproteinase-2 ;  PMA ;  nutrient mixture
15.  miR-21 may acts as an oncomir by targeting RECK, a matrix metalloproteinase regulator, in prostate cancer 
BMC Urology  2012;12:14.
Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line.
Materials and methods
To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR).
The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025).
Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.
PMCID: PMC3431982  PMID: 22642976
Prostate cancer; Prognosis; RECK; Micro RNA; Metaloproteinases
16.  Expression of MMP-2, −7, −9, MT1-MMP and TIMP-1 and −2 has no prognostic relevance in patients with advanced epithelial ovarian cancer 
Oncology Reports  2012;27(4):1049-1057.
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumor invasion, but their prognostic significance is still under discussion. We set out to analyze the epithelial and stromal expression of MMP-2, MMP-7, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 in advanced epithelial ovarian cancers and to assess their prognostic value. A tissue microarray of malignant ovarian tumors from 69 patients was constructed. Immunostaining results were scored using the HSCORE and assessed by univariate analysis with Bonferroni correction and classical multidimensional scaling (CMDS). Kaplan-Meier survival curves calculated with regard to patient and tumor characteristics were compared by the log-rank test. Patients treated by primary surgery (n=43) had a higher tumor size and a trend toward higher epithelial MMP and TIMP expression than those treated by interval surgery (n=26). Optimal cytoreduction (residue ≤1 cm) was obtained in 27 and 18 patients, respectively. Clinical and histological characteristics were not different in patients with optimal cytoreduction and those with suboptimal cytoreduction. The expression of epithelial MMP-9 (P=0.002) and TIMP-2 (P=0.026) were higher in the latter group. CMDS failed to demonstrate any influence of MMP and TIMP expression with regard to cytoreduction outcome. MMP and TIMP expression did not influence survival. Their prognostic values were outweighed by histological type, lymph node involvement and cytoreduction. Standard statistical analysis adjusted after Bonferroni correction and CMDS reduced the relevance of MMPs and TIMPs in the prognosis of patients with advanced ovarian cancer.
PMCID: PMC3583568  PMID: 22200690
matrix metalloproteinase; prognosis; ovarian neoplasms
17.  Expression of tissue levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in renal cell carcinoma 
Matrix metalloproteinases (MMPs) are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis and are inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). {AU Query: Please verify that corrections made to previous sentence did not alter intended meaning}. In this study, we examined the expression of MMP-2, MMP-9, membrane-type 1 (MT1)-MMP, TIMP-1, and TIMP-2 in renal tissue samples of renal cell cancer and examined the correlation between their expression and clinicopathological parameters.
Renal tissue samples from 76 patients with renal cell carcinoma were available for this study. To determine the expression of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out on tumor and normal tissues.
Mean MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 mRNA expression in the renal cell carcinomas was significantly higher than in the normal renal tissue (P <0.05). The RT-PCR data of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 did not show any significant correlation with tumor type or pathologic grade of renal cell carcinoma. MMP-2, MMP-9 and MT1-MMP mRNA expression increased significantly with the TNM stage of the tumor.
Mean MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 mRNA expression in the renal cell carcinomas was significantly higher than in the normal renal tissue.
PMCID: PMC3548713  PMID: 23281640
Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Semi-quantitative RT-PCR; Renal cell carcinoma
18.  HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis 
PLoS ONE  2012;7(3):e33585.
Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.
PMCID: PMC3306414  PMID: 22438955
19.  Changes in the expression of MMP2, MMP9, and ColIV in stromal cells in oral squamous tongue cell carcinoma: relationships and prognostic implications 
Type IV collagen (ColIV) is the most important scaffold for the basement membrane (BM) proteins, and plays an important role in regulating and limiting tumour invasion and metastasis.
Here, we observed the changes in morphology and distribution of type IV collagen (ColIV) in the basement membrane (BM) surrounding nests of carcinoma in 48 patients with oral tongue squamous cell (OTSCC). We examined the correlation between the expressions of ColIV, MMP-2 and MMP-9 and the prognosis of OTSCC patients. The intensity and patterns of expression were assessed immunohistochemically using anti-human mouse monoclonal MMP-2, MMP-9 and Col IV antibodies. Statistical analyses were performed to determine the prognostic correlations of ColIV, MMP-2, and MMP-9 levels.
MMP-2 and MMP-9 expressions in OTSCC were higher than those in normal oral mucosa and dysplastic oral mucosa group(MMP-2 iOD: 66.40 ± 24.20, 134.69 ± 37.08, and 357.79 ± 116.78; MMP-9 iOD: 88.05 ± 23.85, 307.13 ± 93.22, and 791.31 ± 260.52; in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01); however, ColIV immunoreactivity was lower (ColIV iOD: 406.87 ± 62.95, 247.83 ± 42.30, and 151.92 ± 38.17 in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01). High tumour and stromal MMP-2 and MMP-9 expression was significantly associated with positive lymph node status. Col IV expression was associated with positive lymph node status (P < 0.05), and have negatively correlated with the expression of MMP-2 and MMP-9. Overall survival was significantly shorter in patients with high tumour and stromal MMP-2 and MMP-9 expression, and tended to be shorter in patients with low ColIV expression.
Degradation of ColIV was closely related to increased MMP-2 and MMP-9 expression; MMP-9 have more important function than MMP-2 during the cancer development. Monitoring changes in the expression of ColIV, MMP-2, and MMP-9 may be a useful technique for assessing prognoses in OTSCC patients.
PMCID: PMC3490717  PMID: 23107277
Oral tongue squamous carcinoma; MMPs; ColIV; Immunohistochemistry; Prognosis
20.  Urinary high molecular weight matrix metalloproteinases as non-invasive biomarker for detection of bladder cancer 
BMC Urology  2013;13:25.
Matrix Metalloproteinases (MMPs) are key molecules for tumor growth, invasion and metastasis. Over-expression of different MMPs in tumor tissues can disturb the homeostasis and increase the level of various body fluids. Many MMPs including high molecular weights (HMWs) were detected in the urine of prostate and bladder cancer patients. Our aim here is to assess the usefulness of HMW MMPs as non invasive biomarkers in bilharzial bladder cancer in Egyptian patients.
The activity of different MMPs including HMW species was determined using zymographic analysis technique in the urine samples procured from sixty six bladder cancer patients (bilharzial and non-bilharzial) as well as hundred healthy control subjects. Also, the correlation between these HMW MMPs activities and different clinico-pathological parameters was investigated.
High frequency of urine MMPs (uMMPs) activity was determined in 63.6% of examined tumor cases, however, none of the control cases showed any uMMPs activity. MMP-9 had the highest activity (62%) followed by MMP9/NGAL (60%), MMP-2 (54.5%), MMP-9 dimer (53%), ADAMTS (25.6%), and the lowest one was MMP-9/TIMP-1 (12%) only. There was no correlation between uMMPs and any of clinico-pathological parameters including age, gender, tumor size and type, bilharziasis, grade, lymph node involvement, and invasion to the prostate. A significant correlation was established only between MMP-9/TIMP-1 activities with the tumor size.
This study revealed that the detection of urinary MMPs including HMWs activity might be sensitive biomarkers for prediction of bladder cancer. It is also demonstrate that the detection of these urinary HMW gelatinases could not differentiate between bilharzial and non bilharzial bladder cancer subtypes.
PMCID: PMC3656799  PMID: 23672427
Bladder cancer; High molecular weight matrix metalloproteinases; Early detection; Biomarkers
21.  Overexpression of MMP Family Members Functions as Prognostic Biomarker for Breast Cancer Patients: A Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(8):e0135544.
Matrix metalloproteinases (MMPs) are regarded to be relevant to the prognosis of breast cancer. Numerous studies have confirmed the association between MMPs and tumor growth, invasion and metastasis in breast cancer. However, their prognostic values for survival in patients with breast cancer remain controversial. Hence, a meta-analysis was performed to clarify a more accurate estimation of the role of MMPs on prognosis of breast cancer patients.
A systemic electronic search was conducted in PubMed, Embase and Web of science databases to identify eligible studies, which were associated with the relationship between MMPs and prognosis of breast cancer. The correlation in random-effect model was evaluated by using the hazard ratios (HRs) and 95% confidence intervals (CIs).
A total of 28 studies covering 4944 patients were included for meta-analysis. A summary hazard ratio (HR) of all studies was calculated, as well as the sub-group HRs. The combined HRs calculated by either univariate or multivariate analysis both suggested that overexpression of MMPs had an unfavorable impact on overall survival (OS) (HR = 1.694, 95%CI: 1.347–2.129, P < 0.001; HR = 1.611, 95%CI: 1.419–1.830, P < 0.001, respectively). And the univariate analysis showed that patients with overexpression of MMPs had worse relapse-free survival (RFS) (HR = 1.969, 95%CI: 1.460–2.655, P < 0.001) in all eligible studies. In the sub-group analyses, HRs of MMP-9 positivity with poor OS were 1.794 (95%CI: 1.330–2.420, P < 0.001) and 1.709 (95%CI: 1.157–2.526, P = 0.007) which were separately evaluated by univariate and multivariate analysis. A small number of articles demonstrated that MMP-2 overexpression was not related with shorter OS (HR = 1.400, 95%CI: 0.610–3.029, P = 0.427). Four studies included in the OS analysis of MMPs expression in serum suggested that positive expression of serum MMPs may be an unfavorable factor (HR = 1.630, 95%CI: 1.065–2.494) for breast cancer patients. No publication bias was observed in the current meta-analysis.
Our findings suggested that MMPs overexpression (especially MMP-9, MMP-2, MMPs overexpression in serum) might indicate a higher risk of poor prognosis in breast cancer. Larger prospective studies are further needed to estimate the prognostic values of MMPs overexpression.
PMCID: PMC4535920  PMID: 26270045
22.  The Behavior of Matrix Metalloproteinases and Their Inhibitors in Colorectal Cancer 
Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix components crucial for tumor growth, invasion and metastasis. MMPs are controlled by natural inhibitors called tissue inhibitors of metalloproteinases (TIMPs). We and others have demonstrated that MMPs and TIMPs are especially important in the process of tumor invasion, progression and the metastasis of colorectal cancer (CRC). It has been proposed that MMPs and TIMPs might play a part not only in tumor invasion and initiation of metastasis but also in carcinogenesis from colorectal adenomas. Several recent studies demonstrated that high preoperative serum or plasma MMP-2, MMP-9 and TIMP-1 antigen levels are strong predictive factors for poor prognosis in patients with CRC and their determination might be useful for identification of patients with higher risk for cancer recurrence. MMP-9 and TIMP-1 have significant potential tumor marker impact in CRC. Their diagnostic sensitivity is consistently higher than those of conventional biomarkers. The pharmacological targeting of CRC by the development of a new generation of selective inhibitors of MMPs, that is highly specific for certain MMPs, is a promising and challenging area for the future.
PMCID: PMC3497324  PMID: 23202950
matrix metalloproteinase; tissue inhibitor of matrix metalloproteinase; colorectal cancer; adenoma; tumor progression; invasion; metastasis; prognosis; biomarker
23.  Androgen receptor expresion in breast cancer: Relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibitors 
BMC Cancer  2008;8:149.
In the present study we analyze, in patients with breast cancer, the tumor expression of androgen receptors (AR), its relationship with clinicopathological characteristics and with the expression of several matrix metalloproteases (MMPs) and their inhibitors (TIMPs), as well as with prognosis.
An immunohistochemical study was performed using tissue microarrays and specific antibodies against AR, MMPs -1, -2, -7, -9, -11, -13, -14, and TIMPs -1, -2 and -3. More than 2,800 determinations on tumor specimens from 111 patients with primary invasive ductal carcinoma of the breast (52 with axillary lymph node metastases and 59 without them) and controls were performed. Staining results were categorized using a score based on the intensity of the staining and a specific software program calculated the percentage of immunostained cells automatically.
A total of 83 cases (74.8%) showed a positive immunostaining for AR, but with a wide variation in the staining score values. There were no significant associations between the total immunostaining scores for AR and any clinicopathological parameters. However, score values for MMP-1, -7 and -13, were significantly higher in AR-positive tumors than in AR-negative tumors. Likewise, when we considered the cellular type expressing each factor, we found that AR-positive tumors had a higher percentage of cases positive for MMP-1, -7, -11, and TIMP-2 in their malignant cells, as well as for MMP-1 in intratumoral fibroblasts. On the other hand, multivariate analysis demonstrated that patients with AR-positive tumors have a significant longer overall survival than those with AR-negative breast carcinomas (p = 0.03).
Our results confirm that AR are commonly expressed in breast cancer, and are correlated with the expression of some MMPs and TIMP-2. Although we found a specific value of AR expression to be a prognostic indicator in breast cancer, the functional role of AR in these neoplasms is still unclear and further data are needed in order to clarify their biological signification in breast cancer.
PMCID: PMC2416360  PMID: 18507821
24.  Evaluation of MMP-2, MMP-9, TIMP-1, TIMP-2, NGAL and MMP-9/NGAL complex in urine and sera from patients with bladder cancer 
Oncology Letters  2015;10(4):2527-2532.
The identification of biomarkers in urine or serum samples from patients with bladder cancer is urgently required for the development of non-invasive methods for the diagnosis of bladder carcinoma and to facilitate follow-up surveillance, to combat the high progression and recurrence rates of this type of cancer. The current study measured the content of matrix metalloproteinase (MMP)-2 and -9, as well as tissue inhibitor of metalloproteinase (TIMP)-1 and -2 in the urine and sera of 41 patients with bladder cancer by ELISA. The association between levels of MMP-2 and -9 and TIMP-1 and -2, and tumor grade and stage were investigated to verify whether these molecules are involved in tumor differentiation. Statistical analysis of the data revealed that urinary TIMP-1 levels were significantly higher in the high grade group compared with those of the low grade samples (P=0.022). The results also revealed a significantly differing distribution of TIMP-1 expression between Ta and T1 stage specimens (P=0.040). The corresponding area under the curves (AUCs) were 0.72, with a sensitivity of 0.70 and specificity of 0.75. In addition, neutrophil gelatinase-associated lipocalin (NGAL) and MMP-9/NGAL complex levels in the sera were measured. All molecules evaluated were detected in the sera of the patients studied. In particular, tumors staged as non-muscle invasive (Ta and T1), demonstrated significantly higher NGAL levels compared with those of muscle invasive (>T1) bladder cancer (32.8 ng/ml vs. 16.2 ng/ml; P=0.029). The discriminatory ability of NGAL expression was confirmed by receiver operating characteristic curve analysis that revealed an AUC of 0.75, a sensitivity of 0.88 and a specificity of 0.67. These data indicated that urinary TIMP-1 and serum NGAL may be useful non-invasive biomarkers to provide clinical information for bladder cancer disease management. Multicenter, prospective studies are required to confirm these preliminary results.
PMCID: PMC4580016  PMID: 26622883
matrix metalloproteinase-2; matrix metalloproteinase-9; tissue inhibitor of metalloproteinase-1; sera; tissue inhibitor of metalloproteinase-2; urine; neutrophil gelatinase-associated lipocalin; bladder carcinoma
25.  Expression of MMP-2, TIMP-2 protein and the ratio of MMP-2/TIMP-2 in gallbladder carcinoma and their significance 
World Journal of Gastroenterology  2002;8(6):1138-1143.
AIM: To study the correlation between expression of MMP-2, TIMP-2 protein and the ratio of MMP-2/TIMP-2 and clinical-pathological parameters of patients with gallbladder carcinoma.
METHODS: Carcinomas (n = 45) and polypoid lesions (n = 15) of the gallbladder were studied for the expression of MMP-2 and TIMP-2 protein by immunohistochemical avidin-biotin-complex method and image analysis. Clinical-pathological data of patients with gallbladder carcinoma such as histological type, grade of differentiation, level of infiltration, liver invasion and lymph node involvement, etc, were recorded.
RESULTS: There was significant difference between the average level (1.123 ± 0.108 vs 1.030 ± 0.054, P = 0.002) of MMP-2, the ratio (1.050 ± 0.013 vs 0.937 ± 0.078, P = 0.003) of MMP-2/TIMP-2 in gallbladder carcinomas and in polypoid lesions of the gallbladder. Significant difference was found between the expression of MMP-2 in early stage and advanced tumors, but there was no correlation between MMP - 2 protein expression and histological type, differentiation degree, infiltration level, lymph node involvement or liver invasion. Although no difference was observed between TIMP-2 expression and histological type or differentiation degree, significant difference was found between TIMP-2 expression and different Nevin stage, infiltration level, local lymph node involvement or liver invasion (1.168 ± 0.067 vs 1.048 ± 0.075, 1.170 ± 0.062 vs 1.039 ± 0.069, 1.039 ± 0.076 vs 1.147 ± 0.083, 1.048 ± 0.074 vs 1.103 ± 0.095, P < 0.05). MMP-2/TIMP-2 ratio did not correlate with histological type, grade of differentiation and liver invasion, but significant differences were found between MMP-2/TIMP-2 ratio and different Nevin stage, infiltration level and lymph node involvement in patients with carcinoma of gallbladder.
CONCLUSION: TIMP-2 and MMP-2/TIMP-2 ratio could reflect more accurately biological characteristic of gallbladder carcinoma and MMP-2/TIMP-2 ratio might be a new significant marker in early diagnosis, in the judgment of invasion or metastasis and the estimate of prognosis in patients with gallbladder carcinomas.
PMCID: PMC4656396  PMID: 12439941

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