Serum Matrix Metalloproteinase-2, -7 and -9 (MMP-2, MMP-7, MMP-9) levels as Prognostic Markers in Patients with Colorectal Cancer

Introduction : Matrix metalloproteinases are produced by tumour cells, hence, they may be associated with tumour progression including invasion, migration, angiogenesis and metastasis. Finding prognostic markers to better identify patients with higher risk for poor survival would be valuable in order to customize pre- and postoperative treatment as well as to enable closer follow-up of these patients. Aim of our study was to ex-amine MMP-2, MMP-7 and MMP-9 serum levels and correlated them with pathological data such as stage of the colorectal cancer (CRC) and outcome. Methods : The investigation included 82 patients with operable CRC without distant metastases, who had underwent blood tests in order to determine the MMP-2, MMP-7 and MMP-9 serum levels in the following time periods: preoperatively, 3, 6, 9 and 12 months postoperatively. Results : The values of the investigated MMPs decrease postoperatively and start to increase 6 month later in patients of all stages of the disease, reaching the highest value 12 month postoperatively with statistically important differences of MMP-2, MMP-7 and MMP-7 serum levels in terms of disease staging and deﬁ ned points of time. Analysis of the results showed that the MMP-2 serum levels obtained 3 and 12 months postoperatively, than MMP-7 serum levels 12 months postoperatively and the MMP-9 serum levels in all analyzed points in time were in signiﬁ cant association with the CRC patients’outcome. Conclusion : The MMP-2, MMP-7 and especially MMP-9 serum values could be important indicators for diagnosis of the patients with CRC and for monitoring of disease progression.


Introduction
Th e degradation of extracellular matrix (ECM) is a crucial step in tumour progression, aggressive growth and metastases. Th e invasion of cancer cells within the basement membrane depends on matrix metalloproteinases (MMPs) and their inhibition activities (1). MMPs are a family of extracellular structurally related zinc-dependent endoproteases capable of degrading all the ECM components. At present, 23 members of the human MMP gene family are known. Based on their structure and substrate specifi city, MMPs are classifi ed into subgroups of collagenases, stromelysins and stromelysinlike MMPs, matrilysins, gelatinases, membranetype MMPs (MT-MMPs) and other MMPs (1). MMPs play an important role in the physiologic degradation of ECM, e.g., in tissue morphogenesis, tissue repair and angiogenesis. MMPs have also important functions in pathologic conditions characterized by excessive degradation of ECM, such as rheumatoid arthritis, osteoarthritis, periodon-titis, autoimmune blistering disorders of the skin and in tumour invasion and metastasis. MMPs are produced by tumour cells; hence, they may be associated with tumour progression including invasion, migration, angiogenesis and metastasis (2,3). Among the MMPs, matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9), as members of gelatinases, plays important roles in the migration of malignant cells, because of their ability to degrade type IV collagen (4). Th e mechanisms of activation of these enzymes are diff erent. MMP-9 modulates permeability of the vascular endothelium, whereas MMP-2 promotes cleavage of extracellular matrix proteins and is intensively expressed by tumour and stromal components of cancer (5). Matrix metalloproteinase 7 (MMP-7) or matrilysin, as a member of stromelysins is able to induce cell apoptotic impairment. Matrilysin can regulate angiogenesis either by inducing a direct proliferative eff ect on vascular endothelial cells or producing angiogenesis inhibitors (angiostatin, endostatin and neostatin-7) or by enriching the variety of angiogenesis mediators, such as the soluble vascular endothelial growth factor (sVEGF) (6). Increased levels of matrix metalloproteinase in tumour tissues or in blood circulation have been found to correlate with many cancers, including colorectal cancer (CRC). Several previous studies have shown that MMPs may play an important role as an indicator for appearance of CRC and its progression (7,8,9). Colorectal cancer (CRC) is a common disease and it is one of the leading causes of cancer related deaths in developed countries (10). Despite improvements in surgical techniques, adjuvant and neo-adjuvant chemotherapy, the 5-year survival rate in patients with CRC ranges from 5-90% with tumour progression (stages: I: 90-95%, II: 75-85%, III: 50-60% and IV: 0-10%). Th e prognosis in patients without distant metastasis varies from 50-95% depending on the tumour stage (11). Th e correct staging of each CRC patient is crucial in order to plan an optimal treatment regimen. It is widely recognised that prognostic information based on clinical and histopathological investigation is insuffi cient, although tumour stage and lymph node involvement are the main prognostic tools in evaluating cancer specifi c survival. It is questionable to expose a large number of patients to adjuvant treatment with considerable side effects without indications that they will benefi t from such treatment. Finding prognostic markers to better identify patients with higher risk for poor survival (12,13) would be valuable in order to customise pre-and postoperative treatment as well as to enable closer follow-up of these patients. In our study, we examined MMP-2, MMP-7 and MMP-9 serum levels and correlated them with pathological data such as stage of the disease and the patients 'outcome.

Methods
Th e study included a total of 82 previously untreated CRC patients, 30 (36.58%) females and 52 (63.41) males (aged 43 to 75 years, mean age of 67.85; SD±9.67) with operable CRC, without detectable distant metastases, who respected the medical instructions and were available for follow-up. All patients underwent a surgical resection of the primary neoplasm at the University Clinic for Abdominal Surgery in Skopje in the period of 2 years (2007-2009). Blood samples from all patients were drawn before surgical treatment, as well as 3, 6, 9, and 12 months postoperatively in order to examine MMP-2, MMP-7 and MMP-9 serum levels. None of the CRC patients had received chemotherapy before blood sample collection. To standardize clotting conditions, all sera were separated within 1 h aft er blood collection, aliquoted and stored at −80°C until assayed. Serum levels of MMP-2, MMP-7 and MMP-9 were determined using a quantitative solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA) according to the manufacturer's instructions. MMP-2, MMP-7 and MMP-9 technique can detect both pro-and active forms of recombinant human MMP-2, MMP-7 and MMP-9. High concentrations of MMP-2, MMP-7 and MMP-9 were diluted with calibrator, to produce samples with values within the dynamic range of the assay. Th e surgically removed specimens were histopathologically analyzed at the Institute of Pathology of the Faculty of Medicine, Skopje, where the pathological stage was defi ned for every patient according to the International Union   Table 2, Figure 1 and Figure 2. Th e mean MMP-2 serum levels shown in Table 1 and Figure 1, in patients with stage I of the disease, decreased aft er the operation and started slightly to increase aft er the 3th month postoperatively. Th is might be due to the 6 patients with poor outcome in   Th e mean MMP-7 serum levels are separately shown in Figure 2, and they show a similar tendency of postoperative decreasing and permanent increasing aft er the 3 months, especially in stage III. We did not fi nd a signifi cant diff erence between the MMP-7 serum levels in stage I and stage II during each control points in time, but there was a signifi cant diff erence between the MMP-7 serum levels from stage I and II in terms of stage III (p<0.05). Th ere was a signifi cant diff erence between preoperative and postoperative MMP-7 serum levels (p<0.05). Th ere were signifi cant diff erences between MMP-9 serum levels in all stages (p<0.01), as well as between preoperative and postoperative serum levels in all defi ned points of time. Th e number of patients who received chemotherapy and the outcome of all included patients in the study are shown in Table 3. We found signifi cant diff erences in terms of    Table 4, where it is shown that MMP-2 serum levels preoperatively, at 3 and 12 months postoperatively, are in a signifi cant correlation with the lethal outcome of the CRC patients, than MMP-7 serum levels preoperatively and at 12 months, as well as MMP-9 preoperatively and at 3, 9 and 12 months postoperatively.

Discussion
MMP-2 is a collagenase discovered for the fi rst time in metastatic murine tumours and in cultured human melanoma cells. It is secreted by fi broblasts, endothelial cells, osteoblasts, keratocytes, macrophages and many malignant cells (14). MMP-2 is expressed in numerous normal tissues as the lungs, heart, kidneys, placenta, and the muscles. MMP-2 is synthesized and secreted as an inactive proenzyme, while as an active enzyme it degrades the type-IV collagen as well as the type I, V, VII and X, the laminin, the elastin, the fi bronectin and the proteoglycans (15,16). MMP-7 (matrilysin) is a proteolytic enzyme, which is expressed in glandular and ductal epithelium of many tissues. It degrades type IV and X collagen, the elastin, the fi bronectin, the laminin, the osteopontin, the proteoglycans, as well as numerous other substrates. MMP-7 is also synthesized and secreted by cancer cells as an inactive proenzyme. Aft er the activation, the MMP-7 is found in soluble active form or bound to the membrane of the tumour epithelium cells, which has also a proteolitic capability (17). MMP-9 was discovered for the fi rst time as an elastin binding protein, which is synthesized by the macrophages and the polymorphonuclears. In normal conditions, MMP-9 is expressed only in several cell types as trophoblasts, osteoclasts, leukocytes, and dendritic cells. It is also being expressed by several types of tumours, in the tumour cells and in the stromal cells. MMP-9 also degrades the components of the ECM, especially type IV, V, VII, X, XI, XIV collagen, fibronectin, elastin, osteonectin and entactin (18). Diverse results have been obtained from numerous examinations which have been made in order to determine the signifi cance of the MMPs in the diagnosing of malignancies and to determine their infl uence on the disease outcome (19).  (TIMP-1 and TIMP-2) in squamous head and neck cancer, showed that serum MMP-2 immunoreactive protein levels in healthy patients were higher than those in patients with cancer, while the MMP-9 and TIMP-1 levels were considerably higher in patients with squamous carcinoma. Th e authors determined an important correlation between the serum levels of MMP-9 and TIMP-1 with immunohistochemical expression of MMP-9 and TIMP-1 from tumour tissue (22 Th ey found out that patients with advanced cancer had considerably higher mean MMP-7 levels in comparison with those without metastases and in comparison with healthy subjects. Th ey discovered a signifi cant correlation between the MMP-7 levels and shorter survival time, which led them to the conclusion that the elevated MMP-7 serum levels are an independent prognostic factor for survival in patients with advanced CRC (24). We have observed (unpublished data) that in CRC patients with low MMP-7 but high LDH levels, MMP-7 values can increase during chemotherapy treatment, and would be therefore implicated in early acquired resistance, aft er initial response.
Th erefore, we speculate that MMP-7 would be implicated in primary chemoresistance in the subgroup of patients with well-known poor prognosis, to an even more aggressive phenotype, or both. Leelawat, Sakchinabut, Narog, and Wannaprasert analyzing the CEA, CA 19.9, MMP-7 and MMP-9 serum levels in patients with cholangiocarcinoma detected that only MMP-7 level was considerably higher in patients with cancer (25). Levels of total MMP-7 can be measured in human serum and it is feasible using a simple ELISA technique, as this has been recently shown in few other studies. Serum measurements of total MMP-2, MMP-7 and MMP-9 can be considered as an indirect estimation of tumour MMP-2, MMP-7 and MMP-9 expression. Other techniques, such as zymography, are useful to distinguish between activated MMP-2, MMP-7, MMP-9 and pro-forms and might be implemented in the near future for further analysis.

Conclusion
In our examination, we have determined that the MMP-2, MMP-7 and MMP-9 serum levels decrease considerably aft er the resection of the primary neoplasm, as well as the MMP-2 serum levels at 3th and 12th month postoperatively, than MMP-7 serum levels preoperatively and at 12th month and the MMP-9 serum levels at 3th, 9th and 12th month postoperatively are in correlation with the poor outcome of the CRC patients. Subsequently, detection of serum MMP-2, MMP-7 and MMP-9 is feasible and done through a noninvasive technique. Th ey could be potential serum markers which may be useful in the CRC detection and in monitoring of the disease progression.