Abstract
Purpose
Matrix metalloproteinases (MMPs) are known to play an important role in extracellular matrix remodeling during the process of tumor invasion and metastasis. However, little is known about their role in preinvasive lesions and early esophageal carcinomas.
Method
Immunohistochemical analysis of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression was carried out in paraffin-embedded sections of surgically resected esophageal squamous cell carcinoma (ESCC) (58 cases) and paired distal normal esophageal tissues (44 cases) and correlated with clinicopathological parameters.
Result
Overexpression of MMP-2 and MMP-9 proteins was observed in 39 (67%) and 32 (55%) of the 58 ESCCs, respectively localized in tumor cell cytoplasm and stromal elements. Histological evaluation of hematoxylin- and eosin-stained 44 matched distal normal esophageal tissue sections revealed that 26 comprised of normal epithelium, while 15 tissues showed evidence of dysplasia and three tissues showed hyperplasia. Interestingly, 12 (80%) and 13 (87%) of these 15 dysplasias showed immunostaining for MMP-2 and MMP-9 proteins, respectively. Low levels of MMP-2 and MMP-9 were observed in 10 (38%) and 6 (23%) of 26 matched histologically normal esophageal tissues, respectively. Higher MMP-2 immunopositivity was observed in well and moderately differentiated SCCs in comparison with poorly differentiated tumors. The expression of MMP-2 was significantly reduced with the progressive de-differentiation of esophageal SCCs (P =0.03). Overexpression of MMP-2 and MMP-9 in dysplasia as well as SCC suggests that these alterations occur in early stages of esophageal tumorigenesis.
Conclusion
Increased levels of MMP-2 and MMP-9 proteins in ESCCs as compared to normal esophageal tissues suggest their association with esophageal tumorigenesis. Increased levels of these MMPs are observed in majority of dysplasias analyzed herein, indicating that these alterations may be early events in esophageal tumorigenesis. In-depth studies are warranted to determine their role in development and progression of esophageal cancer.
Keywords: Matrix metalloproteinase-2, Matrix metalloproteinase-9, Esophageal squamous cell carcinoma, Dysplasia, Hyperplasia
Introduction
Esophageal cancer ranks amongst the ten most common cancers in the world with high incidence in developing countries (Landis et al. 1999). The mortality rates of the disease are often similar to the incidence rates, due to the relatively late stage of diagnosis, aggressive behavior, and paucity of effective treatment modalities (Kohn et al. 1995). Lymph node metastasis is a strong independent prognostic factor for esophageal carcinoma (Shimada et al. 1999). The spread of malignant tumors is a multistep process, involving rapid growth and invasion into lymphatics and blood vessels. Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The regulation of tissue remodeling is accomplished by complex control of the expression and activity of matrix metalloproteinases (MMPs). As tumor cells reach a metastatic site, MMPs mediate the degradation of basement membrane of vessels and the process of invasion is repeated (Hart et al. 1992; Stetler-Stevenson et al. 1993; Tryggvason et al. 1993; Kohn et al. 1995).
MMPs are a highly regulated super family of zinc-dependent endopeptidases, causally associated with the development and progression of tumors (Chambers et al. 1997). The proposed role of MMPs in tumor invasion is mainly based on their observed overexpression in invasive malignant tumors (Basset et al. 1997; Johnsen et al. 1998). Expression of MMP-1, MMP-2, MMP-3, and MMP-9 in esophageal squamous cell carcinoma (ESCC) has been reported though their relative levels in different types of superficial esophageal carcinomas are yet to be determined (Shima et al. 1992; Sato et al. 1999). Murray et al. (1998) suggested that MMP-1 expression in esophageal carcinoma cells could be associated with poor prognosis. Esophageal carcinoma in the Japanese population comprises largely of squamous cell carcinoma, wherein it has been demonstrated that MMP-2 and MMP-3 expression is positively correlated with the depth of invasion, lymph node metastasis, and vessel permeation (Shima et al.,1992). MMP-7, MMP-9, and MT1-MMP have also been shown to be closely associated with invasion depth and venous invasion in esophageal squamous cell carcinomas (Ohashi et al. 2000). The implication of MMP-7 expression at the invasive front has been substantiated by its correlation with disease recurrence and shorter disease-free and overall survival time in esophageal cancer (Yamamoto et al. 1999). On the other hand, MMP-1 mRNA levels in ESCC were shown to be inversely correlated with patient prognosis, suggesting that MMP-1 might play a crucial role in local invasion but not in systemic dissemination (Yamashita et al. 2001). These results suggest that MMPs may have diverse roles in tumor progression and invasion. Although these studies have emphasized the role of proteases in invasive carcinomas, little is known about their activities in premalignant squamous esophageal lesions. The aim of this study was to determine the expression and cellular localization of MMP-2 and MMP-9 in esophageal squamous cell carcinomas and matched histologically normal tissues, as well as in hyperplastic and dysplastic esophageal tissues, using immunohistochemistry. The clinical relevance of the data was determined by correlating the immunohistochemical findings with clinicopathological features of the patients.
Materials and methods
Fifty-eight esophageal carcinoma cases were enrolled in this study. These cases comprised of 44 surgically resected tissues and 14 endoscopic biopsy specimens. Biopsy as well as surgically resected specimens from human primary esophageal squamous cell carcinomas (ESCCs), and paired normal esophageal tissue specimens taken from a site distant from the cancerous lesion were obtained consecutively from the Department of Gastrointestinal Surgery, All India Institute of Medical Sciences, India. The tissue samples were fixed in 10% formalin and embedded in paraffin. Consecutive 5-μm sections were cut and mounted on glass slides for immunohistochemical analysis. The survival section of histopathologically confirmed ESCCs and paired normal esophageal tissues, having no contaminating tumor cells (26 cases) as determined by hematoxylin and eosin (H & E) staining, were used for analysis of MMP expression. H & E staining of three of these 44 matched distal normal esophageal tissues showed histological evidence of hyperplasia and 15 of 44 tissues showed dysplasia. These tissues were also used for analysis of MMP-2 and MMP-9 expression. Each patient’s clinical status was classified according to the pathological tumor, node, metastasis (pTNM) classification system (Sobin et al. 1997). The patients were grouped based on the tumor stage (pT1/pT2; pT3/pT4), nodal metastasis (pN0; pN1) and distal organ metastasis (pM0; pM1).
Immunohistochemistry
Paraffin-embedded sections of human ESCCs were stained with hematoxylin and eosin for histopathological analysis. The serial sections of ESCCs histologically confirmed normal hyperplastic as well as dysplastic esophageal tissues were used for immunostaining. Briefly, the sections were deparaffinized and preheated in microwave oven (20 min, 120 W, 3×5 min, 450 W) in citrate buffer [10 mM (pH 6.0)]. The sections were incubated in methanol containing hydrogen peroxide (0.3% v/v) for 20 min to quench the endogenous peroxidase activity, followed by incubation with 1% BSA for 1 h to block the non-specific binding. The sections were subsequently incubated with primary anti-MMP2 or anti-MMP9 mouse monoclonal antibody (Oncogene Research, Mass., USA) for 16 h at 4 °C and washed with PBS. Antibody was used at a dilution of 1:100 (1 µg/ml) in 1% BSA. The primary antibody (mouse monoclonal) was detected using biotinylated secondary antibody (rabbit anti-mouse IgG) and streptavidin, subsequently after PBS washings, tissue sections were incubated with horse-radish peroxidase streptavidin conjugate using LSAB+kit (Dako Labs, Denmark) and diaminobenzidine as chromogen. All incubations were performed at room temperature in a moist chamber. Slides were washed several times with PBS after each step. In negative controls, the primary antibody was replaced by PBS or non-immune mouse IgG of the same isotype to ensure specificity. MCF-7 human breast cancer cell line known to overexpress these proteins was used as positive controls. After hematoxylin counter staining all the slides were mounted with DPX and visualized under light microscope.
Positive criterion for immunohistochemical staining
The intensity of immunohistochemical staining was evaluated in five areas of the slide sections for correlation and confirmation of the tissue analysis. For MMP-2 and MMP-9 protein expression, specific staining in the tumor cell cytoplasm and/or stromal fibroblasts was defined as positive staining. The sections were counterstained with hematoxylin and the tumor differentiation status was graded as well (W), moderate (M), or poor (P). The histopathological investigation was blind, i.e., the slides were coded and pathologist did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the esophageal carcinomas while scoring the immunoreactivity.
Western blot analysis
The expression of MMP-2 and MMP-9 proteins in ESCCs was analyzed by western blot using specific antibodies. Briefly, the samples were electrophoresed and transferred to Hybond nitrocellulose membrane. Non-specific binding sites were blocked for 2 h with 5% non-fat milk in 20 mM Tris buffer pH 7.6 containing 137 mM NaCl and 0.05% Tween-20. The membranes were incubated with 5μg/ml of monoclonal antibodies against MMP-2 or MMP-9 (Oncogene Research, Mass., USA) overnight (12 h) then with secondary antibody rabbit anti-mouse IgG diluted 1:1,000 in 1% BSA for 2 h. After washing, reactive bands were visualized by Enhanced Chemiluminescence detection kit (Santa Cruz Biotechnology, California).
Analysis of MMP-2 and MMP-9 mRNA in esophageal normal and cancerous tissues
Reverse transcription of RNA isolated from cell lines and esophageal tissues was performed using 1μg total RNA (DNA free), 100 pmol/μl random hexamer primer, 1 mM dithiothreitol, 6 mM MgCl2, 500 μM of each deoxynucleoside triphosphate, and 200 U MMLV reverse transcriptase. The reverse transcription mixture was used directly as a template for polymerase chain reaction in a dilution of 1:20. Primers designed for MMP-2 and MMP-9 were: MMP-2 forward primer: 5’ GGG GCC TCT CCT GAC ATT 3’ Reverse primer: 5’ TCA CAG TCC GCC AAA TGA A 3’ and MMP-9 forward primer: 5’ TCC AAC CAC CAC CAC ACC GC 3’ and reverse primer: 5’ CAG AGA ATC GCC AGT ACT T 3’. The cycling parameters were initial denaturation at 94 °C for 2 min; each cycle consisted of 94 °C for I min, 55 °C for 1 min and 72 °C for 2 min. After 35 cycles another 72°C cycle was run for 7 min.
Follow-up
Of the 58 ESCC patients enrolled in this study, 35 patients could be followed up in regular follow-up clinic at Department of Gastrointestinal Surgery for a maximum period of 25 months for recurrence of the disease. Twenty-three cases were lost to follow-up due to poor patient compliance and socioeconomic restraints. During the follow up period ten patients died from the disease. Survival was expressed as the number of months from the date of surgery to the recurrence of the disease. Univariate analysis was carried out to investigate the prognostic relevance of MMP-2 and MMP-9 on clinical outcome.
Statistical evaluations
Statistical analyses of the data were performed with SPSS (Version 10.0) software. The statistical association between protein expression and various clinicopathological parameters was determined using Chi-square test. P-values less than 0.05 were considered to indicate statistical significance. Follow-up studies were analyzed by Kaplan-Meier test and overall survival was estimated. The univariate association between patient outcome and variables was assessed by log-rank test.
Results
Immunohistochemical analysis of MMP-2 and MMP-9 proteins in esophageal squamous cell carcinoma and normal esophageal tissues
Immunohistochemical analysis of MMP-2 and MMP-9 proteins was carried out in esophageal normal, hyperplastic, dysplastic, and malignant tissues. The results are summarized in Table 1. Basal or null expression of MMP-2 was observed in the normal esophageal epithelium. Ten of 26 (38%) normal tissues analyzed for MMP-2 protein expression showed basal immunoreactivity, while 16 (62%) cases did not show detectable immunostaining (Table 1). The representative histopathologically confirmed normal esophageal tissue section showing lack of MMP-2 immunoreactivity is shown in Fig. 1a. An interesting observation of this study was the detection of MMP-2 protein in 12 of 15 (80%) dysplastic esophageal epithelia and two of three hyperplastic epithelia. The photomicrograph in Fig. 1b shows positive immunostaining for MMP-2 protein detected in distal esophageal epithelia with histologically confirmed evidence of hyperplasia, although the intensity of the staining was considerably lesser than that observed in ESCCs. The immunostaining was localized in the cytoplasm as well as plasma membrane of epithelial cells in the proliferating zone. Among the matched distal esophageal tissues collected from ESCC patients, 15 specimens showed dysplastic epithelium on H & E staining. Interestingly, 12 of these 15 dysplastic tissues showed MMP-2 immunopositivity. The photomicrograph in Fig. 1c shows MMP-2 immunostaining localized predominantly in the cytoplasm of epithelial cells in the suprabasal layer, while the invading and the proliferative layers did not show immunopositivity.
Table 1.
Analysis of MMP-2 and MMP-9 expression in esophageal normal, hyperplastic, dysplastic, and malignant tissues
| Parameters | Total cases | MMP-2 Expression | MMP-9 Expression |
|---|---|---|---|
| Positive | Positive | ||
| n (%) | n (%) | ||
| ESCCs | 58 | 39 (67) | 32 (55) |
| Normal tissuesa | 26 | 10 (38) | 6 (23) |
| Age (years) | 58 | 39 (67) | 32 (55) |
| <40 | 5 | 3 (60) | 4 (80) |
| ≥40 | 53 | 36 (68) | 27 (51) |
| Gender | 58 | 39 (67) | 32 (55) |
| Male | 40 | 28 (70) | 20 (50) |
| Female | 18 | 10 (56) | 12 (67) |
| Histopathological grading | 58 | 39 (67) | 32 (55) |
| Well | 17 | 14 (82) | 8 (47) |
| Moderate | 32 | 22 69) | 18 (54) |
| Poor | 9 | 3 (33) | 6 (67) |
| Tumor location | 58 | 39 (67) | 32 (55) |
| Upper1/3/middle1/3 | 37 | 27 (73) | 19 (51) |
| Lower1/3 | 21 | 12 (57) | 13 (62) |
| Tumor stage | 43 | 27 (63) | 23 (53) |
| T1/T2 | 8 | 7 (88) | 4 (50) |
| T3/T4 | 35 | 20 (57) | 19 (54) |
| Lymphatic invasion | 43 | 27 (63) | 23 (53) |
| Negative (No) | 13 | 10 (77) | 5 (38) |
| Positive (N1) | 30 | 17 (57) | 18 (60) |
| Hyperplasiaa | 3 | 2 (67) | 2 (67) |
| Dysplasiaa | 15 | 12 (80) | 13 (87) |
aThe matched histologically normal, hyperplastic, and dysplastic esophageal tissues were obtained from site distant from site of esophageal carcinoma
Fig. 1a–l.
Immunohistochemical detection of MMP-2 and MMP-9 proteins in esophageal tissue sections using MMP-2 and MMP-9 monoclonal antibodies. Panels a–f show MMP-2 expression in a normal epithelium showing no detectable expression of MMP-2 protein; b hyperplastic epithelium showing intense cytoplasmic and membranous staining; c dysplastic epithelium showing predominantly cytoplasmic staining for MMP-2 protein in the suprabasal layer; d well-differentiated ESCC showing MMP-2 immunoreactivity in the cytoplasm and cell membrane of tumor cells; e moderately differentiated ESCC showing MMP-2 immunoreactivity in cytoplasm of tumor cells; f poorly differentiated ESCC showing MMP-2 immunoreactivity localized in the plasma membrane and stromal elements. Panels g–l show MMP-9 expression in g normal epithelium showing no detectable expression of MMP-2 protein; panel g–l show MMP-9 protein immunostaining; g normal epithelium showing no detectable level of MMP-9 protein; h hyperplastic epithelium showing cytoplasmic and membranous staining for MMP-9 protein; i dysplastic epithelium showing predominantly cytoplasmic and membranous staining for MMP-9 protein; j well differentiated esophageal SCC showing MMP-9 immunoreactivity in the cytoplasm and plasma membrane of tumor cells; k moderately differentiated esophageal SCC showing MMP-2 immunoreactivity in cytoplasm and nucleus of tumor cells; l poorly differentiated esophageal SCC showing MMP-9 immunoreactivity in cytoplasm of tumor cells. (Magnification ×100 a, b, c, f, h, j, l; magnification ×200 d, e, g, i, k)
Thirty-nine of 58 (67%) ESCCs showed overexpression of MMP-2 protein (P =0.017, Table 1). In ESCCs the MMP-2 immunoreactivity was localized in the cytoplasm and plasma membrane of tumor cells and the surrounding stromal elements.
A representative photomicrograph of a well-differentiated ESCC shown in Fig. 1d depicts MMP-2 immunoreactivity in epithelial cells and surrounding stromal elements in the tumor islands. The immunostaining was localized in the cytoplasm and plasma membrane, while the nuclei were not intensely stained. The moderately differentiated ESCCs showed MMP-2 immunostaining in epithelial cells predominantly localized in the cytoplasm (Fig. 1e). In the poorly differentiated SCCs, the MMP-2 immunoreactivity was localized in the plasma membrane of the epithelial cells as well as in the stromal elements (Fig. 1f). MCF 7, human breast cancer cell line was used as a positive control for MMP-2 expression (data not shown). In 24 of the 39 ESCCs showing MMP-2 immunopositivity, the staining was localized in both tumor cells and stromal where as in six cases only the tumor cells were immunostained, while in cases only stromal reactivity was observed.
MMP-2 expression on showed a significant inverse correlation with histopathological grade of tumor (P =0.03; Odds Ratio, OR=0.2). The expression of MMP-2 was significantly reduced with the progressive de-differentiation of the cancer Fig. 2. Higher MMP-2 immunopositivity was observed in well- and moderately differentiated SCCs than in poorly differentiated SCCs. Eighteen of 30 lymph node positive E SCCs showed MMP-2 immunoreactivity in tumor cells, whereas 23 tumors did not show stromal MMP2 immunostaining. However, MMP-2 protein level did not show any significant correlation nodal invasion as well as with other clinicopathological parameters such as age, gender tumor location, and tumor stage.
Fig. 2.
Histogram showing the correlation between MMP-2 protein expression and histopathological grade of ESCCs
MMP-9 overexpression was observed in 32 of 58 (55%) ESCCs (Table 1). Immunohistochemical analysis of MMP-9 protein expression in histologically normal esophageal tissues did not show detectable immunostaining for MMP-9 protein (Fig. 1g). The photomicrograph in Fig. 1h shows positive immunostaining for MMP-9 protein detected in some distal esophageal epithelia with histologically evidence of hyperplasia, although the intensity of the staining was considerably less than that observed in ESCCs. The immunostaining was localized in the cytoplasm as well as plasma membrane of epithelial cells. Among the matched distal esophageal tissues collected from ESCC patients, 15 specimens showed dysplastic epithelium on H and E. Interestingly, 13 of these 15 (87%) dysplastic tissues showed MMP-9 immunopositivity. The photomicrograph in Fig. 1i shows MMP-9 immunostaining localized predominantly in the cytoplasm and plasma membrane of the epithelial cells. Representative photomicrograph of a well-differentiated ESCC shown in Fig. 1j shows MMP-9 immunoreactivity in the cytoplasm and plasma membrane. The moderately differentiated ESCCs showed MMP-9 immunostaining predominantly localized in the cytoplasm and nucleus of the epithelial cells (Fig. 1k). In the poorly differentiated SCCs, the MMP-9 immunoreactivity was observed to be localized in the cytoplasm of the epithelial cells (Fig. 1l). Of the 32 ESCCs showing MMP9 overexpression, the immunoreactivity was localized in both tumor cells and stromal elements in 13 cases, where as in nine cases immunostaining was observed in the tumor cells only.
No significant correlation was observed between MMP-9 protein expression and clinicopathological parameters such as age, gender, tumor location, tumor stage, and nodal involvement.
Immunoblot analysis of MMP-2 and MMP-9 protein expression
Immunoblot analysis of MMP-2 and MMP-9 protein expression in esophageal squamous cell carcinoma, paired normal esophageal tissue, and human esophageal squamous cell carcinoma cell line (TE13) was carried out to corroborate the immunohistochemical analysis data Fig. 3. Panel A shows 2b band of 72 kDa and 69 kDa for MMP-2 in ESCC (lane 1) and TE13 cells (lane 3). The 72 kDa band corresponds to the latent form of the MMP-2 protein and the 69 kDa to the active form of the protein. Basal level of MMP-2 expression was also observed in some of the normal esophageal tissues as shown in lane 2.
Fig. 3.
Immunoblot analysis of MMP-2 and MMP-9 proteins in esophageal tissues. Panel A shows Western blot analysis of MMP-2 protein in showing in esophageal SCCs a 72 kDa band (lane 1) and TE13 esophageal cancer cell line (lane 3 used as a positive control). Basal level of MMP-2 protein expression was detected in normal esophageal mucosa (lane 2). A 43 kDa β actin band has been shown in the lower panel for equalization of protein. Panel B shows Western blot analysis of MMP-2 protein in esophageal SCC (lane 2) and TE13 cell line, used as a positive control (lane 1) depicting a 92 kDa band. Basel level of MMP-9 protein expression was observed in normal esophageal mucosa (lane 3). A 43 kDa β actin band has been shown in the lower panel for equalization of protein
Representative immunoblot for MMP-9 showed the presence of a 92 kDa band in TE13 cells (lane 1) and ESCC (lane 2) (Fig. 3, Panel B). The 92 kDa band corresponds to the latent form of the MMP-9 protein. Basal level of MMP-9 expression was also observed in some of the normal esophageal tissues as shown in lane 3.
Analysis of MMP-2 and MMP-9 mRNA expression in esophageal normal and cancerous tissues
The MMP-2 mRNA transcripts were analyzed in five ESCCs and matched non-cancerous esophageal tissues (for which adequate tissue specimens were available) and human esophageal squamous carcinoma cells (TE13) by RT-PCR. The PCR amplified cDNA (172 base pairs in size) was observed in five of five esophageal cancers (lanes 5, 7, and in 10 of the normal esophageal tissues (lane 10) and 11 in the TE13 cells (lane 12) (Fig. 4, Panel A).
Fig. 4.
RT-PCR analysis of MMP-2 and MMP-9 mRNA in esophageal tissues. Panel A: representative 1.5% agarose gel showing RT-PCR analysis of MMP-2 mRNA in five esophageal cancerous tissues, matched histologically normal esophageal tissues, and TE13 cell line (positive control). A 172 bp amplicon was observed in three ESCC tissues (lanes 5, 7, and 11) and one of the normal esophageal tissues (lane 10) and TE-13 cell line (lane 12); while MMP-2 transcript could not be observed in four out of five normal esophageal tissues (lanes 2, 4, 6, and 8) and two ESCC tissues (lanes 3 and 9). Lane 1 shows 100 bp DNA ladder. Panel B: representative 1.5% agarose gel showing RT-PCR analysis of MMP-9 mRNA in five matched histologically normal esophageal tissues, cancerous tissues, and TE13 cell line (positive control). A 272 bp amplicon was observed in ESCCs (lanes 3, 7, and 11) and TE13 esophageal cell line (lane 12), while MMP-9 transcript could not be observed in any of the normal esophageal tissues analyzed herein (lanes 2,4,6, 8 and 10) and two of ESCC tissues (lanes 5 and 9)
The amplified PCR cDNA band of 272 base pairs for MMP-9 was observed in three of five esophageal cancers (lanes 7, 11, and 12) as well as in the TE13 cancer cells (lane 3) in (Panel B, Fig. 4). No correlation was observed between expression of MMP-2 and MMP-9 and patient prognosis.
Discussion
To our knowledge the clinical significance of MMP-2 and MMP-9 expression in early stages of development of ESCC has not been demonstrated. MMP-2 and MMP-9 expression has been reported in a large number of human cancer cell lines and various human tumors including tumors of the ovary, breast, bladder, skin, head and neck, cervix, lung, uterus (Talvensaare et al. 1999; Chen et al. 2001), and pancreas (Davies et al. 1993; Naylor et al. 1994; Visscher et al. 1994; Hofmann et al. 2000; O-Charoenrat et al. 2001). MMP-2, 3, and 9 have been shown to be inversely correlated with nodal metastases in breast cancer (Remade et al. 1998). In this study, MMP-2 expression was evaluated in 58 ESCCs and 26 paired histologically normal esophageal tissues taken from a distal site of the same patients. The data demonstrated significant MMP-2 immunopositivity in ESCCs as compared to normal esophageal tissues corroborating the above-mentioned earlier reports of upregulation of MMP-2 in various human cancers including esophageal SCCs (Shima et al. 1992; Koyama et al. 2000). Taken together, with the previous reports our findings suggest that alterations in MMP-2 expression may be associated with esophageal tumorigenesis. It is important to determine the stage in development and progression of esophageal cancer in which the increase in MMP-2 expression occurs. In this study, 38% of the normal esophageal tissues analyzed showed low basal level of MMP-2 expression. A similar pattern of expression has been recently reported for MMP-1 in normal esophageal mucosa and proposed to be implicated in physiological processes such as growth of epithelial cells (Yamashita et al. 2001). Alternatively, the distant normal esophageal epithelia may show molecular alterations due to field cancerization resulting from exposure of the entire esophagus to the carcinogenic insults. The detection of MMP-2 protein in 12 of 15 dysplasias taken from a site distant from the tumor suggests that MMP-2 expression is an early event in esophageal tumorigenesis; however, its implication in the development of tumors remains to be ascertained.
The expression of MMP-2 protein has previously been reported to be localized in the cytoplasm of tumor cells and the stroma of ESCCs by in situ zymography and immunohistochemistry (Shima et al. 1992; Koyama et al. 2000). In this study, the immunostaining for MMP-2 protein was also observed in the cytoplasm and plasma membrane of tumor cells as well as in the stroma of ESCCs. However, no significance association was observed between MMP-2 immunopositivity and lymph node invasion in this study. In the Japanese population also the presence of MMP2 protein in ESCCs did not show significant correlation with the invasiveness of esophageal carcinomas (Koyama et al. 2000). However, the activation of pro MMP-2 was shown to positively correlate with lymph node metastasis, and lymphatic and vascular invasion. Increased MMP-2 immunopositivity was observed in well- and moderately differentiated esophageal carcinomas as compared to the poorly differentiated tumors. The current concept of the role of MMPs in tumor invasion is that in addition to their direct involvement in tumor invasion by facilitating extracellular matrix degradation, they also play important role in maintaining the tumor microenvironment thereby promoting tumor growth. In fact, MMPs have been reported to play direct role in other critical events in tumor evolution that occur at earlier stages; events such as tumor promotion, modulation of the growth of primary tumor, and angiogenesis (Sternlicht et al. 1999; Stetler-Stevenson et al. 1999 Yu et al. 2000). These studies are in accord with our observation of MMP-2 overexpression in well- and moderately differentiated ESCCs. The expression of MMP-3, MMP-7, MMP-9, and MMP-12 in peritumoral area has been suggested to form angiostatin from plasminogen, in turn limiting angiogenesis, thereby inhibiting tumor growth and invasion in vivo (Patterson et al. 1997; Dong et al. 1997; Lijnen et al. 1998).
The lack of a correlation between MMP-2 levels and disease prognosis observed in this follow up study further supports our proposition of a role for MMP-2 in early tumor development. Interestingly, MMP-2 expression has been suggested to be an early event during de-differentiation and malignant transformation in cervical neoplasia also. A similar lack of correlation between MMP-2 expression and clinical course or prognosis reported in cervical carcinoma (Talvensaare et al. 1999) corroborates our findings in esophageal cancer. Using zymography, elevated levels of MMP-2 and MMP-9 were observed in oral carcinomas in comparison with lichen planus or normal mucosa, suggesting that MMPs are upregulated in oral cancer (Sutinen et al. 1998). Using 3-methylcholanthrene and diethylnitrosamine-induced lung cancer model in Wistar rats, significant increase in MMP-2 levels was demonstrated in dysplasia and carcinoma in situ as well as early carcinoma in comparison with carcinoma in situ and metastatic carcinoma versus early carcinoma. The excess degradation and disruption of basement membrane by activated MMP-2 has been suggested to be a key event in inducing lung cancer invasion and metastasis (Chen et al. 2001). Immunohistochemical studies of MMP-2 and MMP-9 in preneoplastic lesions from cigarette smokers including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive SCC showed moderate and strong staining for MMP-2 in all preinvasive squamous lesions (Galateau-Salle et al. 2000). The reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma suggesting important roles for these MMPs in basement membrane remodeling in these lesions.
High MMP levels are associated with invasive lesions and poor prognosis in cancer patients (Uria et al. 2000). Using several esophageal cancer cell lines, MMP-9 has been shown to be implicated in in vitro invasion of esophageal cancer cells (Suzuki et al. 2001). In this study, significant MMP-9 immunopositivity was observed in ESCCs as compared to normal esophageal tissues, although no correlation of expression was observed with tumor type, grade or stage. The immuno-localization of the MMP-9 protein predominantly in the cytoplasm of cancer cells and the stroma with occasional staining in the tumor nuclei and in the invading mononuclear cells, corroborated the previous report in esophageal cancer by immunohistochemistry and in situ hybridization (Murray et al. 1998; Ohashi et al. 2000).
RT-PCR analysis demonstrated increased mRNA transcripts of MMP-2 and MMP-9 in esophageal SCCs in comparison with normal esophageal tissues suggesting that overexpression of MMP-2 and MMP-9 proteins may be due to enhanced transcription or increased stability of the transcripts indicating that regulation of their levels may mainly occur at the transcriptional level.
In conclusion, increased levels of MMP-2 and MMP-9 proteins in ESCCs as compared to normal esophageal tissues suggest their association with esophageal tumorigenesis. Interestingly, the increased levels of these MMPs are observed in majority of dysplasias analyzed herein, indicating that these alterations may be early events in esophageal tumorigenesis. In-depth studies are warranted to determine their role in development and progression of esophageal cancer
References
- 1.Basset P, Okada A, Chenard MP, Kannan R, Stoll I, Anglard P, Bellocq JP, Rio MC (1997) Matrix metalloproteinases as stromal effectors of human carcinoma progression therapeutical implications. Matrix Biol 15:535–541 [DOI] [PubMed] [Google Scholar]
- 2.Chambers AF, Matrisian LM (1997) Changing view of the role of matrix metalloproteinases in metastasis. J Natl. Cancer Inst 89:1260–1270 [DOI] [PubMed] [Google Scholar]
- 3.Chen PS, Zhai WR, Zhou XM, Zhang JS, Zhang YE, Ling YQ, Gu YH (2001) Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells. World J Gastroenterol 7:647–651 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Colby TV, Bennett W, Harris CC, Stetler-Stevenson WG, Liotta L, Ferrans VJ, Travis WD (2000) Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions. Hum Pathol 31:296–305 [DOI] [PubMed] [Google Scholar]
- 5.Davies B, Waxman J, Wasan H, Abel P, Williams G, Krausz T, Neal D, Thomas D, HAnby A, Balkwill F (1993) Levels of matrix metalloproteases in bladder cancer correlate with tumor grade and invasion. Cancer Res 53:5365–5369 [PubMed] [Google Scholar]
- 6.Dong Z, Kumar R, Yang X, Fidller IJ (1997) Macrophage derived metalloelastase is responsible for generation of angiostatin in Lewis lung carcinoma. Cell 88:801–810 [DOI] [PubMed] [Google Scholar]
- 7.Galateau-Salle FB, Luna RE, Horiba K, Sheppard MN, Hayashi T, Fleming MV, Colby TV, Bennett W, Harris CC, Stetler-Stevenson WG, Liotta L, Ferrans VJ, Travis WD (2000) Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions. Hum Pathol 3:296–305 [DOI] [PubMed] [Google Scholar]
- 8.Hart IR, Saini A (1992) Biology of tumour metastasis. Lancet 339:1453–1457 [DOI] [PubMed] [Google Scholar]
- 9.Hofmann UB, Westphal JR, Van Muijen GN, Ruiter DJ (2000) Matrix metalloproteinases in human melanoma. J Invest Dermatol 115:337–344 [DOI] [PubMed] [Google Scholar]
- 10.Johnsen M, Lund LR, Romer J, Almholt K, Dano K (1998) Cancer invasion and tissues remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol 10:667–671 [DOI] [PubMed] [Google Scholar]
- 11.Kohn EC, Liotta LA (1995) Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res 55:1856–1862 [PubMed] [Google Scholar]
- 12.Koyama H, Iwata H, Kuwabara Y, Iwase H, Kobayashi S, Fuji Y (2000) Gelatinolytic activity of matrix metalloproteinase-2 and −9 in oesophageal carcinoma; a study using in situ zymography. Eur J Cancer 36:2164–2170 [DOI] [PubMed] [Google Scholar]
- 13.Landis SH, Murray T, Bolden S, Wingo PA (1999) Cancer statistics. CA Cancer J Clin 49:8–31 [DOI] [PubMed] [Google Scholar]
- 14.Lijnen HR, Ugwu F, Bini A, Collen D (1998) Generation of angiostatin-like fragment from plasminogen by stromelysin-1. Biochemistry 37:4699–4702 [DOI] [PubMed] [Google Scholar]
- 15.Murray GI, Duncan ME, Arbuckle E, Melvin WT, Fothergill JE (1998) Matrix metalloproteinases and their inhibitors in gastric cancer. Gut 43:791–797 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Naylor MS, Stamp GW, Davies BD, Balkwill FR (1994) Expression and activity of MMPS and their regulators in ovarian cancer. Int J Cancer 58:50–56 [DOI] [PubMed] [Google Scholar]
- 17.O-Cheroenrat P, Rhys-Evans PH, Eccles SA (2001) Expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 127:813–820 [PubMed] [Google Scholar]
- 18.Ohashi K, Nemoto T, Nakamura K, Nemori R (2000) Increased expression of matrix metalloproteinase 7 and 9 and membrane type 1-matrix metalloproteinase in esophageal squamous cell carcinomas. Cancer 88:2201–2209 [PubMed] [Google Scholar]
- 19.Patterson BC, Sang QA (1997) Angiostatin conveting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 272:28823–28825 [DOI] [PubMed] [Google Scholar]
- 20.Remade AG, Neol A, Duggan C, McDermott E, Higgins NO, Foidart JM, Duffy MJ (1998) Assay of matrix metalloproteinases types 1, 2, 3 and 9 in breast cancer. Br J Cancer 77:926–931 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Sato F, Shimada Y, Watanabe G, Uchida S, Makino T, Imamura M (1999) Expression of vascular endothelial growth factor, matrix metalloproteinase-9 and E-cadherin in the process of lymph node metastasis in oesophageal cancer. Br J Cancer 80:1366–1372 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Shima I, Sasaguri Y, Kusukawa J, Yamana H, Fujita H, Kakegawa T, Morimatsu M (1992) Production of matrix metalloproteinase-2 and metalloproteinase-3 related to malignant behavior of esophageal carcinoma. A clinicopathologic study. Cancer 70:2747–2753 [DOI] [PubMed] [Google Scholar]
- 23.Shimada Y, Imamura M, Watanabe G, Uchida S, Harada H, Makino T, Kano M (1999) Prognostic factors of esophageal squamous cell carcinoma from the perspective of molecular biology. Br J Cancer 80:1281–1288 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sobin LH, Flemming ID (1997) TNM classification of malignant tumors, 5th edn. Union Internationale Centre le cancer and the American Joint Committee on Cancer. Cancer 80:1803–1804 [DOI] [PubMed] [Google Scholar]
- 25.Sternlicht MD, Lotcher A, Sympson CJ, Huey B, rougier JP, Gray JW, Pinkel D, Bissell MJ, Werb Z (1999) The stomal proteinase MMP3/Stromelysin-1 promotes mammary carcinogenesis. Cell 98:137–146 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Stetler-Stevenson WG, Liotta LA, Kliener DE (1993) Extracellular Matrix 6: role of matrix metalloproteinases in tumor invasion and metastasis. FASEB J 7:1434–1441 [DOI] [PubMed] [Google Scholar]
- 27.Sutinen M, Kainulainen T, Hurskainen T, Vesterlund E, Alexander JP, Overall CM, Sorsa T, Salo T (1998) Expression of matrix metalloproteinases (MMP-1 and −2) and their inhibitors (TIMP-1, -2 and −3) in oral lichen planus, dysplasia, squamous cell carcinoma, and lymph node metastasis. Br J Cancer 77:2239–2245 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Suzuki T, Kuwabara Y, Iwata H, Mitani M, Shinoda N, Sato A, Mitsui A, Sugiura M, Kato J, Fujii Y (2001) Role of matrix metalloproteinase-9 in in vitro invasion of esophageal carcinoma cells. J Surg Oncol 81:80–86 [DOI] [PubMed] [Google Scholar]
- 29.Talvensaari A, Apaja-Saekkinen M, Hoyhtya M, Westerlund A, Puistola U, Turpeenniemi T (1999) Matrix metalloproteinase 2 immunoreactive protein appears early in cervical epithelial dedifferentiation. Gynecol Oncol 72:306–311 [DOI] [PubMed] [Google Scholar]
- 30.Tryggvason K, Hoyhta M, Pyke M (1993) Type IV collagenases in invasive tumors. Breast Cancer Res Treat 24:209–218 [DOI] [PubMed] [Google Scholar]
- 31.Uria JA, Lopez-Otin C (2000) Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency, and activity. Cancer Res 60:4745–4751 [PubMed] [Google Scholar]
- 32.Visscher DW, Hoyhtya M, Ottosen SK, Liang CM, Sarkar FH, Crissman JD, Fridman R (1994) Enhanced expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the stroma of breast carcinomas correlates with tumor recurrence. Int J Cancer 59:339–344 [DOI] [PubMed] [Google Scholar]
- 33.Yamamoto H, Adachi Y, Itoh F, Iku S, Matsuno K, Kusano M, Arimura Y, Endo T, Hinoda Y, Hosokawa M, Imai K (1999) Association of matrilysin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma. Cancer Res 59:3313–3316 [PubMed] [Google Scholar]
- 34.Yamashita K, Mori M, Kataoka A, Inoue H, Sugimachi K (2001) The clinical significance of MMP-1 expression in oesophageal carcinoma. Br J Cancer 84:276–282 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Yu Q, Stamenkovic I (2000) Cell surface localized matrix metalloproteinase-9 proteolytically activatesTGF-β and promotes tumor invasion and angiogenesis. Genes Dev 15:163–176 [PMC free article] [PubMed] [Google Scholar]