Tissue Inhibitor of Matrix Metalloproteinase-2 in Nasopharyngeal Carcinoma

Abstract

By regulating matrix metalloproteinase (MMP) activity and controlling the breakdown of extracellular matrix components, tissue inhibitors of metalloproteinases (TIMPs) play an important role in the process of tumor invasion and metastasis. The present study was designed to clarify the role of TIMP-2 in nasopharyngeal carcinoma (NPC) patients and to evaluate its importance relative to clinicopathologic parameters. It was carried out in 30 patients with NPC and 20 controls. Tissue biopsies were studied and graded pathologically, and Western blot analysis was performed to assess TIMP-2 protein expression. Clinically, in accordance with TNM classification (T: tumor size, N: lymph node involvement, M: distant metastasis), 8 cases were diagnosed as stage II, 12 as stage III, and 10 cases as stage IV; however, pathologic typing with use of the World Health Organization (WHO) classification revealed the presence of 9 specimens of squamous cell carcinoma (WHO type 1), 6 cases of nonkeratinizing carcinoma (WHO type 2), and 15 cases of undifferentiated carcinoma (WHO type 3). The difference in percentage of TIMP-2 positivity between NPC patients (76.6%) and normal controls (30%) was statistically highly significant (P < .01). In addition, there was a significant positive correlation between TIMP-2 protein positivity and either the clinical staging or the histopathologic typing (P < .01) using Chi-square test (x²), suggesting that TIMP-2 can be used as a marker of the severity of NPC.

Accordingly, we can assume that TIMP-2 may play a role in regional lymph node and/or distant metastasis and in progression of squamous cell carcinoma. Further studies are needed to investigate the role of TIMP-2 as a marker for tumor progression and to evaluate its potential value in the follow-up of patients.

Introduction

Proteolytic degradation of the extracellular matrix (ECM) is a fundamental aspect of cancer development and a key event in the regulation of tumor proliferation and metastasis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are collectively capable of degrading most components of the basement membrane and ECM, facilitating cell migration.[1] Given their ubiquitous presence, MMPs play an important role in tissue breakdown and remodeling during both normal and pathologic conditions.[2] MMPs are secreted as inactive proenzymes and are transformed into active forms after cleavage of a propeptide domain of the molecule.[3] On the basis of their structure, cell localization, and substrate specificity, MMPs are categorized into several groups such as collagenases, gelatinases, stromelysins, and membrane-type MMPs.[4] MMPs are tightly regulated at various levels, including expression level, latent form activation, and the balance between enzyme levels and their inhibitors (TIMPs). TIMPs are the major endogenous regulators of MMPs and consist of 4 homologous members (TIMP 1-4). The balance between MMPs and TIMPs is critical in maintaining the integrity of ECM and its regulatory role in organ development, cell growth and differentiation.[5] Although each TIMP appears capable of inhibiting several MMPs, these proteins exhibit preferential inhibitory capacity (eg, TIMP-1 and -2 selectively inhibit MMP 9 and 2, respectively.[6]

Although early studies have shown TIMPs to have antitumor or antimetastatic effects, more recent reports indicate a dual function, with positive correlation between increased TIMP levels and poor outcome in some human malignancies. The mechanisms supporting the paradoxical positive effect of TIMPs in tumor progression are not completely understood and are the subject of intense investigation. This tumor-promoting activity may be attributable to either excessive proteolytic degradation of ECM, which leads to impairment of tumor cell adhesion and disruption of the cell-matrix interactions required for migration and invasion, or direct influence on cell survival and growth.[7]

The expressions of MMP family members in head and neck squamous cell carcinoma (SCC) tissues are reported widely; however, the correlation of these expressions with clinical features is still controversial.[8–15] Some studies demonstrated that the expressions of MMP family correlated with histologic grade,[9,11,14] tumor invasion,[13] clinical stage,[15] and/or lymph node involvement,[8–13,15] although their results were not uniform. Furthermore, a majority of previous studies analyzed these expressions in patients with a wide variety of tumor (T_1–4) and node (N_0–3) classifications. There is little known about the predictive value of expression of MMP family members for clinical outcomes and prognosis.

In this study, we used Western blot analysis to determine whether there is a correlation between TIMP-2 protein positivity and the incidence of nasopharyngeal carcinoma (NPC), clinical staging, and histopathologic typing. A better understanding of the role of TIMP-2 protein may enable us to design new therapeutic approaches for the management of NPC patients.

Subjects and Methods

Our study comprised 30 patients with NPC who attended the Kasr El-Aini ENT Clinic during the period 2004–2006 and 20 controls (during endoscopic sinus operation for sinusitis or allergic nasal polypi and well known to have no malignancy). NPC patients were subjected to full history taking, clinical examination with respect to nasopharyngeal region, computed tomography, magnetic resonance imaging, and nasopharyngoscopy to determine the full extent of the local spread of the tumor. Cases were categorized according to TNM classification.[16]

Sample Collection

Nasopharyngeal biopsies (from the NPC patients and the controls) were divided into 2 parts. The first specimen was studied and graded pathologically as one of the 3 predominant histologic types according to the World Health Organization (WHO) classification,[17] and the second was transferred into a liquid nitrogen container and washed with ice-cold saline. Fat and necrotic tissues were removed and tissues were stored at −80°C until used.

Detection of TIMP-2 Protein Expression

TIMP-2 protein expression was detected using immunoblotting technique (Western blot) according to the method of Sambrook.[18]

Sample preparation

Tissue samples were transferred into a clean Eppendorf tube and solubilized in 500 microliters ice-cold lysing buffer (HEPES 0.1mol/L, glycerol 10%, K2EDTA 1 mol/L, Triton-X-100 10 mol/L, and NaCl 0.5 mol/L). Protease inhibitors (benzamidine 10 mmol/L, beta-mercaptoethanol 10 mmol/L, aprotinin 5 mg/L, and PMSF 0.39 mmol/L) were freshly added to the lysing buffer before use. Solubilized tissues were sonicated on ice using an ultrasonic homogenizer (Ultra-Turax T25, IKA-Lab, Switzerland) for 3 bursts of 1 minute each, then centrifuged for 30 minutes at 18,000 rpm at 4°C. The supernatant was collected, aliquoted, and stored at −80°C until used.

Protein concentration was measured according to the method of Bradford.[19] SDS-polyacrylamide gel electrophoresis of proteins (SDS/PAGE): 1-dimensional slab gel eletrophoresis was done according to the standard protocol of Laemmli[20] with some modifications.

Electrophoretic separation of protein samples was followed by quantitative electrotransfer as described by Sambrook[18] using a mini transcellulose membrane (Hoefer Scientific Instruments, San Francisco, California).

Statistical Methods

Version 10 Statistical Package for Social Science software (SPSS Inc., Chicago, Illinois) was used to analyze the data. Univariate analysis was performed using the Chi-square method to find out the relation between various qualitative data. Comparisons of variables among subgroups were made using the Mann-Whitney test. Pearson's x² test with continuity correction and Mann-Whitney test were used to assess correlations between TIMP-2 expression and clinicopathologic parameters of nasopharyngeal carcinomas.

Results

The study comprised 20 controls and 30 cases of NPC that were staged clinically according to TNM classification. Eight cases were diagnosed as stage II, 12 cases as stage III, and 10 cases as stage IV; however, histopathologic examination of biopsies taken revealed the presence of 9 specimens classified as WHO type I, 6 specimens classified as WHO type 2, and 15 specimens classified as WHO type 3 (Table 1).

Table 1.

Clinical and Histopathologic Categorization of Nasopharyngeal Carcinoma Cases

Clinical Staging		Histopathologic Typing
Stage II	8 (26.7%)	WHO type 1	9 (30%)
Stage III	12 (40%)	WHO type 2	6 (30%)
Stage IV	10 (33.3%)	WHO type 3	51 (50%)

Stage II (T₂N₀M₀)

Stage III (T₃N₀M₀-T_1,2 or₃,N₁M₀)

Stage IV (T₄N₀M₀-any T,N₂ or N₃,.M₀-any T, any N,M₁)

T1: Tumor confined to 1 site or no tumor visible

T2: Involving 2 sites (both posterosuperior and lateral wall)

T3: Extension into nasal cavity or oropharynx

T4: Tumor invasion of skull or cranial nerve involvement (or both)

N1: Metastasis in a single ipsilateral lymph node < 3 cm

N2: Metastasis in contralateral, bilateral, multiple lymph nodes or > 3 cm but < 6 cm

N2: Metastasis in lymph nodes > 6 cm

M0: No distant metastasis

M1: Distant metastasis.

WHO type 1: Squamous cell carcinoma

WHO type 2: Nonkeratinizing carcinoma

WHO type 3: Undifferentiated carcinoma that is lymphoepithelioma and anaplastic carcinoma

Figure 1 shows the percent positivity of TIMP-2 protein in malignant cases. Figure 2 illustrates the comparison between the patients with malignancies and the controls.

Figure 1.

Detection of TIMP-2 protein by Western blot in NPC patients (Lanes 2–8). TIMP-2 protein (21 KDa) was detected at the expected molecular weight (Lanes 2, 3, 5–8). Recombinant TIMP-2 protein positive control (Lane 1) and negative control for the immunodetection system (Lane 9) were included.

Figure 2.

Detection of TIMP-2 protein by Western blot in NPC patients (Lane 2) and controls (Lanes 3–9). TIMP-2 protein (21 KDa) was detected at the expected molecular weight (Lanes 2, 5–7). Recombinant TIMP-2 protein (Lane 1) was included as a positive control.

The difference in percentage of TIMP-2 positivity between NPC patients and controls was statistically highly significant (Table 2), as determined by Chi-square test (x²). There was a significant positive correlation between TIMP-2 protein positivity and either the clinical staging or the histopathologic typing (P < .01) using Chi-square test (x²) (Table 3).

Table 2.

Percent Positivity of TIMP-2 Protein: Comparison Between Nasopharyngeal Carcinoma Patients and Controls

Positivity of TIMP-2	Malignant Cases	Controls
	23/30	6/20
	76.6%	30%
Pearson's Chi-squared test (x2)	9.1
P-value	< .01*

^*Highly significant

Table 3.

TIMP-2 Protein Positivity in Relation to Clinical Staging and Histopathologic Typing

TIMP-2	Histopathologic Typing				Clinical Staging
		Stage II	Stage III	Stage IV	WHO 1	WHO 2	WHO 3
	(−)	4	3	0	4	2	3
	(+)	4 (50%)	9 (75%)	10 (100%)	5 (44.4%)	3 (60%)	12 (80%)
Pearson's Chi-squared test (x2)	9.3				9.1
P* value	< .01				< .01

^*P < .01 = highly significant

Discussion

ECM turnover is an event that is tightly regulated. Much of the coordinate physiologic or discoordinate pathologic degradation of the ECM is catalyzed by class of proteases known as metalloproteinases. MMPs are a family of multidomain enzymes that are controlled by a class of specific tissue inhibitors of MMPs (TIMPs). TIMP-2 is an endogenous regulator and a potent inhibitor of MMPs.[21]

Recent in vitro studies[22–24] have clarified the mechanism of cell-mediated MMP-2 activation in tumor tissue, ie, pro-MMP-2 secreted by fibroblasts binds to TIMP-2 in combination with MT1-MMP on the cell surface, and the pro-MMP-2/TIMP-2/MT1-MMP ternary complex is formed. The pro-MMP-2 in the ternary complex is activated by adjacent MT1-MMP that is free from TIMP-2,[22–24] and the activated MMP-2 degrades ECM components. Therefore, on the basis of this theory, the significant association between MMP-2 expression and the expression of either MT1-MMP or TIMP-2 observed here is very reasonable. On the other hand, it is reported that different transcriptional regulation of MMP-2 and MMP-9 genes may be due to the presence of different promoter elements.[25]

In the present study, we attempted to demonstrate a correlation between TIMP-2 protein positivity and the incidence of NPC. We used Western blot analysis to evaluate the expression of TIMP-2 protein in cell lysate from tissue biopsies (from 30 NPC patients and 20 controls). The percent positivity of TIMP-2 protein was statistically highly significant (P < .01) in cancer patients (76.6%) compared with the controls (30%).

This result is consistent with that reported by Katayama and colleagues,[26] who found marked expression of MMP-2 and tissue inhibitor of MMP-2 in early-stage oral SCC. These MMP family members were mainly expressed on the cell surface and in the cytoplasm of tumor cells as well as on some endothelial cells or on the stromal fibroblasts surrounding tumor cells.

In addition, results of TIMP-2 expression in other malignant tumors confirmed the findings of the present study. The expression of TIMP-2 protein was found to be significantly higher in ovarian carcinoma,[27] prostatic adenocarcinoma,[28] thyroid cancer,[29] colorectal cancer,[30] and breast cancer.[31]

In general, TIMP-2 had been believed to suppress tumor invasion and metastasis by inhibiting MMP-2, because TIMP-2 inhibits collagenolysis activity of MMP-2 in vitro.[32] However, according to the model of cell-mediated MMP-2 activation, pro-MMP-2 bound to the TIMP-2/MT1-MMP complex is activated by MT1-MMP that is free from TIMP-2. The activated form of MMP-2 is inactivated by the attachment of TIMP-2.[22–24] Recently, high activation of MMP-2 in tongue SCC samples, detected by gelatin zymographic assay, has been reported to be associated with high expression of TIMP-2 in tumor cells.[15] Therefore, the immunoreactivity of TIMP-2 is likely to be useful for monitoring MMP-2 activation.

Moreover, in an attempt to determine the possible role of TIMP-2 protein expression in regional lymph node and/or distant metastasis and in progression of NPC, we tried to find a correlation between percent TIMP-2 protein positivity and either clinical staging or histopathologic typing. Indeed, we found a positive significant correlation with both clinical staging and histopathologic typing: the percentage of protein positivity increased with their gradual increase (P < .01). Our results are in accordance with those reported in recent immunohistologic studies,[14,33–36] indicating that TIMP-2 plays a positive role in tumor metastasis and that high expression of TIMP-2 correlates with lymphatic invasion and lymph node metastasis. Kallakury and colleagues[37] and Kamayama and colleagues[38] also found a significant correlation between TIMP-2 expression and advanced bladder cancer stage (P < .05).

By contrast, Grignon and colleagues,[39] and Ree and colleagues[40] found that TIMP-2 expression did not correlate with the stage and grade of bladder cancer and breast cancer, respectively, and Oberge and colleagues[41] reported that TIMP-2 protein expression had no significant association with the stage of colorectal cancer and therefore concluded that TIMP-2 protein may be of limited value in colorectal tumor staging.

In summary, the results of our study suggest that TIMP-2 may participate in regional lymph node and/or distant metastasis and may be a useful marker of severity of SCC, but further studies are needed to investigate the role of TIMP-2 in tumor progression and to evaluate its potential value in the follow-up of NPC patients.