Increased expression of MMP9 is correlated with poor prognosis of nasopharyngeal carcinoma

Introduction The aim of the present study was to analyze the expression of matrix metalloproteinase 9 (MMP9) in nasopharyngeal carcinoma (NPC) and its correlation with clinicopathologic features, including the survival of patients with NPC. Methods Using real-time PCR, we detected the mRNA expression of MMP9 in normal nasopharyngeal tissues and nasopharyngeal carcinoma (NPC) tissues. Using immunohistochemistry analysis, we analyzed MMP9 protein expression in clinicopathologically characterized 164 NPC cases (116 male and 48 female) with age ranging from 17 to 80 years (median = 48.4 years) and 32 normal nasopharyngeal tissues. Cases with greater than or equal to 6 and less than 6 of the score value of cytoplasmic MMP9 immunostaining were regarded as high expression and low expression, respectively. The relationship between the expression levels of MMP9 and clinical features was analyzed. Results The expression level of MMP9 mRNA was markedly greater in NPC tissues than that in the nasopharyngeal tissues. Immunohistochemical analysis revealed that the protein expression of MMP9 detected in NPC tissues was higher than that in the nasopharyngeal tissues (P = 0.004). In addition, high levels of MMP9 protein were positively correlated with the status of lymph node metastasis (N classification) (P = 0.002) and clinical stage (P < 0.001) of NPC patients. Patients with higher MMP9 expression had a significantly shorter overall survival time than did patients with low MMP9 expression. Multivariate analysis suggested that the level of MMP9 expression was an independent prognostic indicator (P = 0.008) for the survival of patients with NPC. Conclusion High level of MMP9 expression is a potential unfavorable prognostic factor for patients with NPC.


Background
Nasopharyngeal carcinoma (NPC) is one of the most common malignant diseases in the Chinese and other south-east Asians. Unfortunately, most NPC patients tend to present a more advanced stage of disease when first diagnosed due to its deep location and vague symptoms. Therefore, it is of great interest to search valuable factors for early diagnosis, prognosis prediction, and novel therapeutic strategies.
The formation and metastasis of NPC is a complex and continuous process with the participation of a number of key genes [1]. In a previous study, we used cDNA microarray to detect differentially expressed genes among NPC tissues and non-cancerous nasopharyngeal tissues. By means of the analysis of BRB-array tools, the expression of MMP9, a gene encoding matrix metalloproteinase 9, was shown to be markedly upregulated in NPC tissues, suggesting a possible role of MMP9 in promoting the pathogenesis of NPC [2].
MMP9, a member of the matrix metalloproteinases (MMPs), plays a critical role in breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as tumor metastasis [3]. MMP9 is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix, which promotes the metastasis of tumor cells [4].
In order to clarify the role of MMP9 in the pathogenesis of NPC, in the present study we investigated the correlation of MMP9 protein expression with clinicopathologic features, including the survival of patients. We found that the mRNA and protein expression levels of MMP9 were higher in NPC tissues than those in non-cancerous nasopharyngeal tissues. Furthermore, the relatively higher protein expression of MMP9 was associated with NPC progression and poor prognosis. Our results suggest that overexpressed MMP9 is an unfavorable prognostic factor for NPC patient's survival.

Sample collection
Seven primary fresh NPC samples and 5 non-cancerous fresh nasopharyngeal samples were collected from the People's Hospital of Zhongshan City, China, at the time of diagnosis before any therapy. All fresh samples were immediately preserved in liquid nitrogen. One hundred and sixty four undifferentiated NPC specimens and 32 non-cancerous nasopharyngeal specimens, both paraffinembedded, were obtained from the People's Hospital of Zhongshan City and the First Affiliated Hospital of Guangdong Medical School, Zhanjiang City, China. In the 164 NPC cases, there were 116 male and 48 female with age ranging from 17 to 80 years (median, 48.4 years). For the use of these clinical materials for research purposes, prior consents from the patients and approval from the Ethics Committees of these two hospitals were obtained. All specimens had confirmed pathological diagnosis and were staged according to the 1997 NPC staging system of the WHO.

Immunohistochemistry
Paraffin sections (3 μm) from samples of 164 NPC and 32 nasopharyngeal specimens were deparaffinized in 100% xylene and re-hydrated in descending ethanol series(100%, 90%,80%, 70% ethanol) and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. Endogenous peroxidase activity and non-specific antigen were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum, followed by incubation with goat anti-human MMP9 antibody (1:100) (Abcam, MA, USA) for 1 h at 37°C. After washing, the sections were incubated with biotin-labeled rabbit antigoat antibody for 10 min at room temperature, and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin Inc, China). The peroxidase reaction was developed using 3, 3-diaminobenzidine chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope.

Evaluation of staining
The immunohistochemically stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. The staining intensity was scored as previously described [6,7]. The extent of the staining, defined as the percentage of positive staining areas of tumor cells or normal nasopharyngeal epithelial cells in relation to the whole tissue area, was scored on a scale of 0 to 4 as the following: 0, < 10%; 1, 10-25%; 2, 26-50%; 3, 50-75%; and 4, >76%. The sum of the stainingintensity and staining-extent scores was used as the final staining score for MMP9 (0-7). For statistical analysis, a final staining scores of 0-5 and 6-7 were respectively considered to be low and high expression.

Statistical analyses
All statistical analyses were performed using SPSS 13.0 software. Data were presented as mean ± SD. The χ 2 test was used to analyze the relationship between the levels of MMP9 expression and clinicopathologic characteristics. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The significances of various variables in survival were analyzed using multivariate cox proportional hazards model. A P value of less than 0.05 was considered statistically significant.

MMP9 mRNA was highly expressed in NPC tissue
In order to assess the role of MMP9 in NPC, we performed real-time PCR to measure the expression of MMP9 mRNA transcripts in 7 freshly collected NPC tissues and 5 freshly collected normal nasopharyngeal tissues. Compared with normal nasopharyngeal tissues, NPC tissues showed higher expression levels of MMP9 mRNA with an average increase of 3.4-fold ( Figure 1).

Immunohistochemical analysis of MMP9 protein expression in NPC and nasopharyngeal tissues
We measured the expression levels and subcellular localization of MMP9 protein in 164 archived paraffinembedded NPC samples and 32 non-cancerous nasopharyngeal samples using immunohistochemical staining ( Figure 2A-E). Specific MMP9 protein staining was found in the cytoplasm of non-cancerous and malignant epithelial cells. Furthermore, we observed that in 77.4% (127/ 164) of NPC samples, MMP9 protein was highly expressed. In comparison, only 53.1% of non-cancerous nasopharyngeal samples had highly expressed MMP9 protein, significantly lower than that in the NPC samples (P = 0.008) ( Table 1).

Relationship between clinicopathological characteristics and MMP9 expression in NPC patients
The relationships between clinicopathological characteristics and MMP9 expression levels in individuals with NPC are summarized in Table 2. We did not find a significant association of MMP9 expression levels with patient's age, sex, smoking, tumor size (T classification), and status of distant metastasis (M classification) in 164 NPC cases. However, we observed that the expression level of MMP9 was positively correlated with the status of lymph node metastasis (N classification) (N0-N1 vs. N2-N3) (P = 0.002), and clinical stage (I-II vs. III-IV) (P < 0.000) in NPC patients (Table 3).

Survival analysis
To investigate the prognostic value of MMP9 expression for NPC, we assessed the association between the levels of MMP9 expression and patients' survival using Kaplan-Meier analysis with the log-rank test. In 164 NPC cases with prognosis information, we observed that the level of MMP9 protein expression was significantly correlated with the overall survival of NPC patients ( Figure 2F).
Patients with high level of MMP9 expression had poorer survival than those with lower level of MMP9 expression (P = 0.001). In addition, T, N, M classifications and clinical stages were also significantly correlated with patients' survival (P = 0.034, P < 0.001, P < 0.001, and P < 0.001 respectively). To determine whether MMP9 is an independent prognostic factor for NPC, we performed multivariate analysis of the levels of MMP9 protein expression adjusted for age, gender, smoking status, T classification, N classification, M classification, and clinical stages of NPC patients. The results showed that the level of MMP9 expression was an independent prognostic factor for NPC (Table 4).

Discussion
NPC is a malignant neoplasm arising from the mucosal epithelium of the nasopharynx, most often within the lateral nasopharyngeal recess and has been thought to be closely associated with Epstein-Barr virus infection, dietary, and genetic factors. The majority of the NPC deaths attribute to tumor metastases rather than primary tumors. However, the molecular mechanism of NPC invasion and metastasis remains incompletely understood.
In a recent microarray analysis, we found the significantly elevated level of MMP9 mRNA in NPC compared to non-cancerous nasopharyngeal tissues [2]. In this report, we found that MMP9 was expressed predominantly in the epithelial cells in both NPC and non-cancerous nasopharyngeal tissues by immunohistochemistry assay, which was consistent with Horikawa's result [8]. Similar to a report from Wong et al [9], we further presented the evidence that MMP9 was overexpressed at both mRNA and protein levels in NPC tissues compared to nasopharyngeal tissues, suggesting that MMP9 was involved in the pathogenesis of NPC.
MMP9 is a Zn 2+ dependent endopeptidase that mediates the degradation of extracellular matrix protein [10], and is associated with tumor invasion and metastasis [11,12]. It is synthesized and secreted in monomeric form as zymogen, and belongs to the gelatinase subgroup. Increased expression of MMP9 is usually seen in invasive and metastatic cancers such as colorectal cancer [13], gastric carcinoma [14], pancreatic carcinoma [15], breast cancer [16], and oral cancer [17]. The levels of MMP9 expression have also been found to be increased in nasal  NK/T-cell lymphoma [18], malignant astrocytomas, carcinomatous meningitis, and brain metastases [19]. In this study, we also found that MMP9 overexpression was significantly associated with T classification (tumor size), N classification (lymph node metastasis), and clinical stages of NPC patients. Overexpressed MMP9 in NPC may accelerate tumor growth by inducing angiogenesis and enhance local cell invasion and metastasis by degrading the extracellular matrix. Our results may indicate that MMP9 plays significant roles in NPC progression, including tumor invasion and metastasis. Similarly, Horikawa et al also showed that overexpressed MMP9 protein was positively correlated with lymph node metastasis of NPC [8]. Furthermore, the expression of MMP9 also showed a significant positive correlation with the expression of oncoprotein LMP1 in NPC tissues. Transfection of a LMP1 expression plasmid into C33A cell line could increase MMP9 expression [20]. These studies consistently suggest that overexpressed MMP9 may play an unfavorable role in NPC pathogenesis. However, the correlation between MMP9 expression and the survival of NPC patients has been seldom reported.
In the past few years, MMP9 overexpression in tumor cells has been shown to be an independent prognostic factor in several types of tumors, which has a favorable or unfavorable prognostic significance depending on tumor types [21][22][23][24]. In epithelial ovarian cancer, the higher the amount of MMP9 positive cancer cells, the longer was the 10-year disease-related survival (DRS) [21]. Interestingly, similar results were reported in breast cancer. Pellikainen found that in breast cancer, positive MMP9 expression in cancer cells favored patient's survival [22]. On the contrary, there was more evidence indicating that overexpression of MMP9 in cancer cells was not a favorable prognosis factor in non-small cell lung cancer(NSCLC) [23], colorectal cancer [24], and oesophageal carcinoma, etc [25]. In NSCLC and colorectal cancer, overexpressed MMP9 was markedly associated with shortened cancer-related survival. In oesophageal carcinoma, MMP9 overexpression was significantly correlated with the depth of tumor invasion, lymphatic permeation, nodal metastasis, and pathologic differentiation grade.
In the present study, we presented the evidence that MMP9 protein expression in NPC was inversely correlated with patient's overall survival. The patients with higher expression of MMP9 protein had shorter survival time. According to multivariate analyses, increased expression of MMP9 protein was a significant predictor of poor prognosis for NPC patients, especially for its patients at late-stage. These results were analogous to Li et al's report [26] but inconsistent with Wong et al's investigation [9]. Li et al used immunohistochemistry assay in NPC tissues to find that NPC patients with dual highexpression of MMP9/PAR-2 showed a significantly worse prognosis than those with single highly expressed protein or dual low or negatively expressed proteins, which strongly supported the reliability of our study. However, Wong et al reported that the increased level of plasma pro-MMP9 by enzyme linked immunosorbant assay did not correlate with NPC patients' clinical outcome. The discrepancy between our data and Wong et al's data would be most likely due to the different samples and method used.

Conclusion
In summary, our study demonstrated that the expression level of MMP9 was significantly increased in NPC and correlated with the malignant status of NPC. Furthermore, our data suggested that MMP9 was an important prognostic factor for NPC. Yet, due to the limited sample size of patients in our investigation, further studies would be needed to verify these findings and establish the role

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed