Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2011  |  Volume : 54  |  Issue : 1  |  Page : 7--14

Matrix metalloproteinases 2 and 9 in situ mRNA expression in colorectal tumors from Iraqi patients


Mohanad M Ahmed, Suhad Hadi Mohammed 
 Department of Microbiology, College of Medicine, Kerbala University, Kerbala, Iraq

Correspondence Address:
Mohanad M Ahmed
Department of Microbiology, College of Medicine, Kerbala University, Kerbala
Iraq

Abstract

Context: Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) preferentially degrade the basement membrane, a key step in tumor invasion, metastases, and induction of vascularization of tumor tissue. Aim: To determine MMP-2 and MMP-9 in situ mRNA expressions in colorectal adenocarcinomas from Iraqi patients. Materials and Methods: Forty archived paraffin-embedded colorectal adenocarcinoma samples and their resection margins were enrolled in our study. Thin paraffin embedded sections (3-5 μm thick) of both tumor and resection margins were prepared for each respective biopsy and were used to detect MMP-2 and MMP-9 in situ mRNA expressions based on in situ hybridization technique. Statistical Analysis: Statistical analyses were conducted to describe different variables and parameters in this research, and to describe relationships with each other as well. For the comparisons, the t test of significance was used. The associations were found by chi-square (χ2 ) and analysis of variance (ANOVA) tests, or as appropriate, as well as 95% confidence interval. The correlations were calculated using correlation coefficient (r). Statistical significance was defined as P < 0.05. Results: Based on the current outcome, there were significant differences in MMP-2 and MMP-9 in situ mRNA expressions when each tumor sample were compared to its respective resection margin (P < 0.001 and P < 0.001, respectively). When tumor samples were analyzed based on their depth of invasion, means of in situ mRNA expressions of both MMP-2 and MMP-9 were significantly increased in the group in which tumor invaded submucosa into muscularis properia (SMP) compared to that of tumor in serosa (SE) group and tumor invading other organs (OR) group (P < 0.05 and P < 0.05, respectively). Furthermore, when tumor lymph node metastases were questioned, exclusively, MMP-2 in situ mRNA expression was significantly differentiated among N0, N1, and N2 groups (P < 0.05). Regarding the possible correlation between the two investigated parameters with respect to various histopathological variables, both MMP-2 and MMP-9 in situ mRNA expressions have significant positive correlation in tumor samples (r = 0.88), whereas in resection margins, this correlation was absent. Interestingly, MMP-2 and MMP-9 in situ mRNA expressions were found to correlate positively as well as significantly within tumor differentiation [well-differentiated (WD) adenocarcinoma: r = 0.78; moderately differentiated (MD) adenocarcinoma: r = 0.90; and poorly differentiated (PD) adenocarcinoma: r = 0.91], tumor stage (A-B: r = 0.70 and C-D: r = 0.95), depth of invasion (SMP: r = 0.77; SE: r = 0.87; and OR: r = 0.97), lymph node metastasis (N0: r = 0.82; N1: r = 0.92; and N2: r = 0.96), and tumor size (<3 mm 3 : r = 0.76 versus ≥3 mm3: r = 0.94). Conclusions: Overexpression of MMP-2 and MMP-9 are often associated with increased invasive metastatic potential of colorectal adenocarcinoma. However, their activities are essential during the early stages of tumor progression.



How to cite this article:
Ahmed MM, Mohammed SH. Matrix metalloproteinases 2 and 9 in situ mRNA expression in colorectal tumors from Iraqi patients.Indian J Pathol Microbiol 2011;54:7-14


How to cite this URL:
Ahmed MM, Mohammed SH. Matrix metalloproteinases 2 and 9 in situ mRNA expression in colorectal tumors from Iraqi patients. Indian J Pathol Microbiol [serial online] 2011 [cited 2022 Dec 6 ];54:7-14
Available from: https://www.ijpmonline.org/text.asp?2011/54/1/7/77316


Full Text

 Introduction



Colorectal cancer (CRC) is one of the most common malignancies among the populations in United States and Western Europe, and one of the leading causes of worldwide morbidity and mortality. In the United States alone, approximately 140,000 new cases are registered each year and the mortality caused by the condition exceeds 50,000. [1] In Europe, about 213,000 new cases and 110,000 deaths are reported each year. In addition, the lifetime CRC risk in the general population is 5%, but this figure rises dramatically with age; accordingly, by the age of 70, approximately half of the western population will have developed an adenoma. [2] Between January 1995 and December 1997, in Iraq, out of 24,900 registered malignant cases, colorectal tumors represented 4% of the cases. Only in 1995, 293 new cases were registered. Of these, 164 and 129 were the number of cancers of colon and rectum, respectively. However, in 1997, the number of registered cases increased to 354, of which 193 cases were restricted to the colon while 161 cases were those identified in the rectum. [3]

Nowadays, it has become apparent that the metastatic disease rather than the primary tumor itself is responsible for death in most solid tumors, including colorectal tumors. [4] The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs). [5] MMPs are a family of 25 [6] secreted and membrane associated zinc-dependent extracellular neutral [7],[8] endopeptidases which degrade ECM at physiological pH. [9] Together, the MMPs are able to process or degrade all ECM components. Each ECM element is cleaved by a specific MMP or MMP group. [10] Consistent with their role in tumor progression, high levels of a number of MMPs have been shown to correlate with poor prognosis in human cancers. [11] In line with this understanding, increased MMP activity has been detected and shown to correlate with invasion and metastatic potential in a wide range of cancers including ovary, lung, prostate, breast, colorectal and cervical cancer cells. [12],[13] MMPs are of great significance, not only due to their ability to degrade ECM component, but also, on the top of this, due to the modulation of tumor angiogenesis, growth as well as metastases by them. [14],[15] Cells within intact tissue usually do not store MMPs, and constitutive expression is minimal. Activity of MMPs is regulated at three levels including transcription, activation, and inhibition by natural inhibitors including tissue inhibitors of metalloproteinases (TIMPs) and nonspecific proteinase inhibitors. [10],[16]

Aim of this study was to assess possible correlations between MMP-2 and/or MMP-9 in situ mRNA expressions and various histopathological criteria of colorectal tumors.

 Materials and Methods



Archived paraffin-embedded tumor samples along with the histopathological report for each patient were used in this study. Collection of samples was concluded in the year 2003-2004.

Out of 40 patients from whom tumors, in addition to their resection margins (which are tumor free and considered as control group), were collected, 20 (50%) were males and 20 (50%) were females. Mean patient age was 54.75 years (between 28 and 82 years). H and E slides were prepared for the paraffin-embedded blocks and were examined again by a histopathologist to confirm the data.

Histologic Typing and Staging

Each tumor was assigned a histologic type according to the World Health Organization classification as follows: well-differentiated adenocarcinoma (comprising well-formed glands in which the nuclei were uniform in size and shape and retained a basal location), moderately differentiated adenocarcinoma (glands were less regular but remained easily recognizable and nuclei were large and lacked a basal location), and poorly differentiated adenocarcinoma (cells were arranged in irregular clusters, with little evidence of glandular differentiation). [17]

According to the tumor-node-metastases (TNM) staging system of the American Joint Commission on Cancer, the depth grading of tumor invasion in each of the carcinomas was classified into five groups as follows: Tis (carcinoma in situ or limited to mucosa), T1 (invading submucosa), T2 (invading muscularis propria), T3 (invading either subserosa or pericolic tissue), and T4 (through serosa or invading contiguous organs). [18] Tumor growth pattern was assessed as "expanding" (advancement of tumor occurred with a bulbous circumscribed pushing border) and "infiltrative" (tumor dissected sharply through the bowel wall). [19]

Based on Dukes' classification, pathological stages of colorectal carcinoma were classified into five groups as follows: A (tumor invading submucosa or muscularis propria), B (tumor extending beyond muscularis propria), C1 (with positive regional lymph nodes only), C2 (with a positive apical node), and D (with distant metastasis). [20],[21] Diagnosis was established by two independent pathologists, and cases of questionable diagnosis were omitted from the study.

In situ Hybridization

Thin, paraffin-embedded sections (3-5 μm thick) of both tumor and resection margin tissue were prepared on positively charged slides for the detection of MMP-2 and MMP-9 in situ mRNA expression by in situ hybridization.

In situ hybridization for the detection of MMP-2 and MMP-9 mRNAs was performed using an oligonucleotide probe complementary to a fragment of human MMP-2 and MMP-9 mRNAs. The sequence of each oligonucleotide probe does not cross hybridize with other known sequences, including mRNA for other types of MMPs. The probes were labeled with biotin (biotinylated) and purified by high performance liquid chromatography (Maxim Biotech, USA). Tissue sections (3-5 μm) of formalin-fixed, paraffin-embedded specimens were mounted on Fisherbrand Superfrost/Plus slides (Fisher Scientific, USA). The slides were dewaxed by backing overnight at 70 o C followed by dipping in xylene and rehydrated by serial dipping in decreasing ethanol concentration jars followed by dipping in deionized water as described previously. [14] The sections were treated with 0.2 N HCl for 20 min and digested with 10 mg/ml of proteinase K (Sigma, St. Louis, MO, USA) for 15 min at 37°C. After postfixation in 4% paraformaldehyde, each section was covered with 20 ml of denatured hybridization mixture containing 8% dextran sulfate, 125 mg/ml sonicated salmon sperm DNA, 40% deionized formamide, 250 mg/ml yeast tRNA, 13 Denhardt's solution, 1 mM ethylenediamine-tetra-acetic acid (EDTA, pH 7.4), 0.6 M NaCl, 10 mM Tris-HCl, and 2 mg/ml biotinylated oligonucleotide probe, placed in a moist chamber, and probes were allowed to react for 8-10 min at 70 o C and then were incubated at 37°C for 15 hours (in the case of housekeeping gene, slides were incubated at 95 o C). After hybridization, the slides were washed three times in 40% formamide in 23 SSC at 37°C for 1 hour each and incubated with RNase A solution for 30 min at 37 o C (this step was omitted for housekeeping gene containing slides). Following this, the slides were incubated with 150 ml of blocking solution for 30 min at room temperature and incubated with streptavidine-alkaline phosphotase conjugate (Maxim Biotech, USA) and counterstained with nuclear fast red stain. Finally, the slides were dehydrated, mounted with permanent mounting medium and examined. Both positive and negative controls were included for each run. The negative control was obtained by omitting the probe. The positive control was obtained by replacing the probe with housekeeping gene. Respective MMP mRNA expression was evaluated by comparing alkaline phosphatase staining using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) with the results obtained from the negative and positive controls.

Statistical Analysis

Statistical analyses were conducted to describe different variables and parameters in this research and to describe relationships with each other as well. For the comparison between tumor and resection margin regarding the investigated parameters, t test of significance was conducted. The association between MMP-2 and MMP-9 in situ mRNA expression along with tumor differentiation, depth of invasion, and lymph node metastasis was found using chi-square (χ2 ) and analysis of variance (ANOVA) tests as well as 95% confidence interval. On the other hand, the association between the investigated parameters and tumor stage as well as tumor size was determined using student t test. The correlations between the two investigated parameters with respect to various clinicopathological parameters were calculated using the correlation coefficient (r). Statistical significance was defined as P < 0.05.

 Results



Clinicopathological Data

Tumor samples, along with their respective resection margins, from 40 patients with colorectal adenocarcinoma were involved in our study. The mean age of colorectal patients was 54.75 years with a range of 28-82 years. The male:female ratio was 1:1 (M:F = 20:20). There were only two cases of recurrent colorectal carcinoma. According to the histologic differentiation, tumors were classified into three groups: well-differentiated adenocarcinoma (WD), moderately differentiated adenocarcinoma (MD), and poorly differentiated adenocarcinoma (PD). Among 40 cases, there were 7 cases with well-differentiated adenocarcinoma, 25 cases had moderately differentiated adenocarcinoma and 8 cases had poorly differentiated adenocarcinoma. On the other hand, patients were grouped based on different histopathological criteria including tumor stage A-B versus C-D, tumor depth of invasion [tumor invading submucosa into muscularis properia (SMP), tumor reaching serosa (SE), and tumors invading other organs (OR)], and tumor size (<3 mm 3 versus ≥3 mm 3 ). Other histopathological data are shown in [Table 1].{Table 1}

Matrix Metalloproteinases 2 and 9 in situ mRNA Expressions: Significant Correlations in mRNA in situ Expressions in Tumor and Resection Margins

Tumor samples biopsied from 40 cases were investigated for determining MMP-2 and MMP-9 mRNA in situ expression based on in situ hybridization technique. Their respective resection margins were analyzed as well. MMP-2 and MMP-9 typical staining pattern in tumor sample and resection margin are shown in [Figure 1] and [Figure 2], respectively. In general, the numbers of stained cells in resection margins were between 19-31% and 20-29%, whereas for tumor samples, the numbers of stained cells were higher and ranged between 30-99% and 30-96% for MMP-2 and MMP-9, respectively. The means ± standard error (SE) of mRNA in situ expressions in both resection margins and tumor samples were 25.3 ± 1.32 and 23.9 ± 0.97 versus 70.02 ± 3.42 and 73.7 ± 3.4 for MMP-2 and MMP-9, respectively [Table 2]. In order to categorize our patients into positive and negative cases, we calculated the 99% confidence interval for patients and used its lower limit as a borderline as described by Al-Murani et al. [22] Accordingly, all the values less than this line were considered negative. Inversely, the values more than this line were considered positive. Taking the lower limit as a cutoff value will ensure that the negative values will contain the minimum error in such a consideration. By defining our cutoff for the tumor values, it will be expected that all the resection margin values fall within the negative values and this was noticed in our resection margin data. For the purpose of comparison between tumor and resection margin means within each group of our parameters (MMP-2 and MMP-9), the t test of significance was conducted. Accordingly, there were significant differences between tumor samples versus their resection margins based on their respective MMP-2 and MMP-9 in situ expressions (P < 0.001 and P < 0.001, respectively) [Table 2].{Figure 1}{Figure 2}{Table 2}

Correlation Between mRNA in situ Expressions of Both Metalloproteinases 2 and 9 with Different Histopathological Variables

[Table 3] shows the correlation between the mRNA expression for both MMP-2 and MMP-9 with different histopathological variables. Our data analysis showed that there were no significant differences between the in situ mRNA expressions of both MMP-2 and MMP-9 with respect to tumor differentiation (P = 0.26 and P = 1.34, respectively), tumor stage (P = 0.13 and P = 0.12, respectively), and tumor size (P = 0.48 and P = 0.97, respectively), based on χ2 test and student t test of significance. However, means of the mRNA expression of stage A-B were higher than that of C-D for MMP-2 and MMP-9, respectively [Table 3]. Regarding tumor depth of invasion, mean levels of in situ mRNA expression of both MMP-2 and MMP-9 in the SMP group were significantly higher than that in SE and OR groups, on the basis of the 95% confidence interval and ANOVA test (P < 0.05 and P < 0.05, respectively) [Table 3]. As can be seen in [Table 3], patients were classified into three groups based on lymph node metastasis. Accordingly, both ANOVA and 95% confidence interval reflected no significant differences in mRNA in situ expression of MMP-9 (P > 0.05), but there were significant differences in mRNA in situ expression of MMP-2 with tumor lymph node metastasis (P < 0.05) [Table 3]. In addition, the means of in situ mRNA expression were found to be decreased in OR and N2 groups for both MMP-2 and MMP-9 [Table 3].{Table 3}

Correlations Between Metalloproteinases 2 and 9 with Respect to Different Histopathological Variables

The correlations between MMP-2 and MMP-9 mRNA in situ expression and in tumor and their resection margins and within different histopathological variables were analyzed using correlation coefficient (r). In the resection margins, all the correlations were weak, positive, and not significant. Another correlation was seen between MMP-2 and MMP-9 which was high, positive, and significant (r = 0.88) [Table 4].{Table 4}

Correlations between the two parameters within groups were also determined. Our results demonstrated that there was significant correlation between MMP-2 and MMP-9 within tumor differentiation (WD: r = 0.79; MD: r = 0.90; PD: r = 0.91) [Table 4], stage (A-B: r = 0.70; C-D: r = 0.95) [Table 4], tumor size (<3 mm 3 : r = 0.76 versus ≥3 mm 3 : r = 0.94) [Table 4], depth of invasion (SMP: r = 0.77; SE: r = 0.87; OR: r = 0.97) [Table 4], and with lymph node metastasis (N0: r = 0.82; N1: r = 0.96; N2: r = 0.92) [Table 4].

 Discussion



This study has shown that there is no or little expression of of MMP-2 and MMP-9 in apparently normal colonic mucosa (resection margin), however, there was a significant overexpression of both MMP-2 and MMP-9 in the investigated colorectal tumors (P < 0.001 and P < 0.001, respectively) [Table 2]. This result might possibly reflect the association between in situ mRNA expression of MMP-2 and MMP-9 with colorectal tumorigenesis. In addition, it may further support the hypothesis that high tumor MMP-2 and MMP-9 mRNA expression might be associated with increased metastatic potential, as postulated by Hofmann et al., [23] since the transition from in situ tumor growth to metastatic disease is defined by the ability of tumor cells at the primary site to invade local tissue and cross tissue barriers. [24] To initiate a metastatic process, neoplastic cells must first penetrate the basement membrane and then invade the interstitial stroma by active proteolysis by MMPs, especially MMP-2 and MMP-9. Subsequently, intravasation requires tumor cell invasion of the sub-endothelial basement membrane in a similar manner as tumor cell extravasation occurs in the distant organ. In addition to this, development of malignant phenotype is also associated with tumor-induced angiogenesis that not only allows tumor growth but also permits easy access to the vascular compartment, favoring hematogenous spread. [25] MMPs have been implicated in the promotion of angiogenesis and their action is proposed to be due to the release and potential activation of an inducer or possible degradation of an inhibitor of angiogenesis. [26] Endothelial cells at the tip of the growing neovessel synthesize and secrete a large array of metalloproteinases which allow the migration of neovessel in the connective tissue. [27] Therefore, elevated MMP-2 and MMP-9 mRNA expressions might be associated with tumor progression. However, our results are in contrast to that of Arii et al., [28] who found no correlation between MMP-2 mRNA level and invasion potential in hepatocellular carcinoma. Such a discrepancy might be due to differences in the type of tissue and subsequent differences in local microenvironment that may modulate the expression of certain gene(s) that code for MMPs.

Regarding the correlations between in situ mRNA expressions for both MMP-2 and MMP-9 with different histopathological variables, the current study demonstrated that there were no significant differences in mRNA in situ expressions of both MMP-2 and MMP-9 with tumor differentiation (P = 0.26 and P = 1.34, respectively), tumor stage (P = 0.13 and P = 0.12, respectively), and with tumor size (P = 0.48 and P = 0.97, respectively) [Table 3]. Although the differences between stages A-B versus C-D combined were not statistically significant, it constituted a sizable amount since the means of stage A-B were higher than that of C-D [Table 3]. These results are in contrast to previous data recorded by Murashige et al. [29] and Collins et al., [30] since they reported that MMP-2 mRNA in situ expression was overexpressed in CRC tissues and correlated with Duke's stage. These differences in the results might be due to the limited number of cases within each group enrolled in the current study, which might interfere with the documented results. Nevertheless, our results showed that the means of in situ mRNA expressions of both MMP-2 and MMP-9 within SMP group were significantly higher than that of SE and OR groups (P < 0.05 and P < 0.05, respectively) [Table 3]. This might possibly reflect the importance of MMP-2 and MMP-9 during the early stages of tumor malignancy to promote tumor cell invasion, and therefore, tumor progression. Interestingly and according to the current study, higher intensity of staining of MMP-2 and MMP-9 was seen in regions of invasion of the muscularis mucosa compared to superficial portions of the tumor, reflecting areas of active MMP-2 and MMP-9 at the deepest invasive margin of the tumor. Such an observation may further support and confirm the certain role for MMP-2 and MMP-9 during invasion and development of metastasis. Moreover, our results demonstrate that there were marginally significant differences among N0, N1, and N2 groups (P < 0.05) [Table 3] in mRNA expression of MMP-2 but not of MMP-9 (P > 0.05) [Table 3]. Here, we cannot speculate that MMP-2 in situ mRNA expression is more important than that of MMP-9 in association with lymph node metastasis, since the limited number of investigated cases did not reveal real significant differences in the mRNA in situ expression of MMP-2. It is noteworthy that the in situ mRNA expressions of MMP-2 and MMP-9 do not continuously increase during all stages of malignancy. Expression of MMP-2 and MMP-9 decreased with the tumor stage [Table 3]. Similarly the means of in situ mRNA expression of both MMP-2 and MMP-9 were often decreased along with tumor depth of invasion (78.36, 67.5, 68 and 80, 72.6, 67), and with N2 group (70.9, 75.9, 60.7 and 73.7, 77.3, 67.4, respectively) [Table 3]. This means that MMP-2 and MMP-9 activities are essential early on, when the tumor is being established and harnessing critical functions of the surrounding stroma. In addition, tumor microenvironment may possibly play a crucial role in modulation of certain gene(s) expression. [26]

The question that could be raised is: Where do MMP-2 and MMP-9 come from? In our study, in situ hybridization analysis showed that mRNA expression of MMP-2 and MMP-9 was observed in both tumor adenocarcinoma cells and stromal cells, especially in those within close contact with cancer cells. This might possibly reflect that the inductions of MMP-2 and MMP-9 mRNA expression in colorectal adenocarcinoma cells were stimulated by stromal cells regardless of their origin. This is in agreement with previous studies which indicated that the expression of MMPs was regulated by tumor-stromal interactions. This possibility was thought to contribute to tumor cell invasion and their further metastasis. [31] In addition to this, Dong et al. [32] showed that co-cultures of prostate cancer cells (PC) with stromal cells derived from various sources could specifically induce MMP-9 expression in prostate cancer cells at the transcriptional level, resulting in higher levels of pro-MMP-9 protein in the culture media. Chang and Werb [26] had shown that stromal cells (fibroblast, inflammatory cells and endothelial cells) secrete different types of MMPs in response to certain cytokines, chemokines, extracellular matrix metalloproteinase inducers (EMMPRIN) that are secreted from tumor cells (these tumor cells use secreted MMPs to disrupt basement membrane, invading nearby tissues and metastasize to different organs). By contrast, after the invasive events, TIMPs are expressed primarily by the tumor cells [33] and could serve as a regulatory mechanism for fine-tuning the activity of stromal MMPs so that tumor cells can have a role in determining where and when they can invade. [26] This indicates that all these studies mentioned above including our results stand on tumor-stromal interactions that may modulate the expression of certain gene(s) in both tumor and stromal cells. Therefore, it would be tempting to study possible role of certain type of cells and their secreted cytokines, growth factors, in addition to MMPs that could possibly affect tumor-stromal interaction in tumor progression.

How did MMP-2 and MMP-9 correlate in tumor tissues versus resection margins? The current study also focused on whether there were any correlations between MMP-2 and MMP-9 in situ mRNA expressions with respect to various histopathological variables. In general and at the resection margin level, the current data showed positive, weak, and statistically insignificant correlations between MMP-2 and MMP-9 (r = 0.185) [Table 4]. On the contrary, when tumor samples were under investigation, in situ mRNA expression for MMP-2 and MMP-9 demonstrated significant positive correlations (r = 0.88) [Table 4]. This might possibly be attributed to normal threshold of mRNA in situ expression for both MMP-2 and MMP-9. Since the resection margins are apparently normal tissues, there were no signs for tumorgenesis and vasculogenesis. Nevertheless, the current outcome failed to pointed out any correlation among the two parameters (MMP-2 and MMP-9) when they were analyzed together at the resection margins [Table 4].

Regarding the correlations between MMP-2 and MMP-9 along with different histopathological variables, our results demonstrated that there were positive, increasing and highly significant correlations between MMP-2 and MMP-9 along with different histopathological variables including tumor differentiation (WD: r = 0.79; MD: r = 0.90; and PD: r = 0.91) [Table 4], tumor stage (A-B: r = 0.70 and C-D: r = 0.95) [Table 4], tumor size (<3 mm 3 : r = 0.76 and ≥3 mm 3 : r = 0.94) [Table 4], depth of invasion (SMP: r = 0.77; SE: r = 0.82; and OR: r = 0.97) [Table 4], and lymph node metastasis (N0: r = 0.82; N1: r = 0.92; and N2: r = 0.96) [Table 4]. This might possibly reflect that MMP-2 and MMP-9 have the upper hand during tumor progression, keeping in mind the possible supportive role of other factors including other members of MMPs family, activation of certain oncogene(s), inactivation of tumor suppresser gene(s), and the constituent of the local microenvironment that all may further affect tumor progression. Furthermore, the significance of MMP-2 and MMP-9 in situ mRNA expression continuously correlating with different histopathological variables may support their synergistic effect during tumor progression.

To conclude, on the basis of our data, it can be said that overexpression of MMP-2 and MMP-9 is often associated with increased metastatic potential of colorectal adenocarcinoma tumors, since higher intensity of staining of MMP-2 and MMP-9 was seen in regions of invasion of the muscularis mucosa compared to superficial portions of the tumor, which shows possible areas of active MMP-2 and MMP-9 to be the deepest invasive margin of the tumor. In addition to this and according to our results, the decrease in local in situ mRNA expressions of MMP-2 and MMP-9 along with tumor stage and depth of invasion might possibly reflect that MMP-2 and MMP-9 activities are essential during the early stages of tumor progression, when the tumor is establishing and harnessing critical functions of the surrounding stroma, keeping in mind the fact that the local microenvironment (cytokine field) may further modulate the expression of certain gene(s) including those that code for MMP-2 and MMP-9. Furthermore, the in situ mRNA expressions of MMP-2 and MMP-9 in both stromal and adenocarcinoma cells may reflect that tumor progression is regulated by tumor-stromal interaction. Thus, MMP-2 and MMP-9 could be used as targets for timely therapeutic management of patients with primary colorectal adenocarcinoma.

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